N OCIETY' - the Society for Reproductive Biology
N OCIETY' - the Society for Reproductive Biology
N OCIETY' - the Society for Reproductive Biology
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
N<strong>OCIETY'</strong>
(Front cover) Leadbeater's possum (Gymnobelideus leadbeateri) was though to have been extinct <strong>for</strong> more than 50 yearswhen it was rediscovered in <strong>the</strong> tall Mountain Ash (Eucalyptus delegatensis) <strong>for</strong>ests of Victoria's Central Highlands in1961. This rare and beautiful possum is soft grey above with a prominent dark stripe along <strong>the</strong> midline, a creamy-whiteunder surface and a long dark furry tail. It is nocturnal and lives in small family groups or colonies which build <strong>the</strong>ir nestsin <strong>the</strong> hollows of large old eucalypts. It feeds mainly on insects and gum secreted from wattles, <strong>for</strong> example <strong>the</strong> Silverwattle (Acacia dealbata), in <strong>the</strong> understorey of<strong>the</strong> <strong>for</strong>est. Leadbeater's possum is one of <strong>the</strong> faunal emblems of <strong>the</strong> state ofVictoria and is endangered by <strong>the</strong> continued clearing and destruction of old growth <strong>for</strong>ests, which has resulted in habitatfragmentation and subsequent population declines. They breed well in captivity and can be seen in <strong>the</strong> Nocturnal House atHealesville Sanctuary. Observing <strong>the</strong>m in <strong>the</strong> wild is more challenging because of <strong>the</strong>ir ra<strong>the</strong>r shy and cryptic nature.Assistance in production of this publication was provided by <strong>the</strong> Medical lllustration Unit, MonashUniversity, and <strong>the</strong> Institute of Reproduction & Development, Monash University.Printed by Snap Printing, Notting Hill, Victoria1
The Australian <strong>Society</strong> <strong>for</strong><strong>Reproductive</strong> <strong>Biology</strong> (A.SRB)acknowledges <strong>the</strong> following <strong>for</strong> <strong>the</strong>ir support of <strong>the</strong> 1999 Conference:MAJOR MEETING SPONSORS:[ Parke-DaViS) (Seron~Parke DavisSeronoSPEAKER/SYMPOSIUM SPONSORS:Astrazeneca4 l.f) NOVARTISrfdAstraiZeneca Novartis Reproduction, Fertility& DevelopmentS.L~TCHELL SPONSOR:IIAZA ResearchAD:An Australiancompllny supportingAustralian ResearchTRADE EXHIBITORS:AUSTRALIAN SOCIETY FORREPRODUCTIVE BIOLOGYPROGRAM AND MINIPOSTERSTHIRTIETHAnnual Conference26 th - 29 th September 1999AZA ResearchCryologicImmuno DiagnosticsLife TechnologiesMerck, Sharp & DohneNovartisOrganon (Aust) Pty LtdParke DavisPhannacia & UpjohnPfizer Pty LtdSanofi-Syn<strong>the</strong>laboSerono.. Hilton-on-<strong>the</strong>-Park, MelbourneCopyright © Australian <strong>Society</strong> <strong>for</strong> <strong>Reproductive</strong> <strong>Biology</strong>, 1999ISSN 0812·766223
The Australian <strong>Society</strong> <strong>for</strong><strong>Reproductive</strong> <strong>Biology</strong> (ASRB)Australian <strong>Society</strong> <strong>for</strong> <strong>Reproductive</strong> <strong>Biology</strong>TABLE OF CONTENTS1999 ASRB Office Bearers 5Previous ASRB Office Bearers/Awardees 6Hilton-on-<strong>the</strong>-Park Floorplan 7Location map of conference venues 8Joint ASRB and ESA Program Schedule 9ASRB Session details 13Abstracts 33Author Index 139Subject Index 1431999 Office BearersChairmanDeputy ChairmanHonorary SecretaryHonorary TreasurerCommittee MembersClinical RepresentativePostgraduate RepresentativeCommunications OfficerChair, JSA CommitteeMarilyn RenfreeLyn HindsJeremy ThompsonMark HarveyJohn CurlewisMark HedgerDavid PhillipsJill ShawRob NormanAnthony PreshawJim CumminsGraeme MartinASRB Program Organising Committee 1999ChairMembersDavid PhillipsTeija Peura .Peter Temple-SmithAlan TilbrookMaryWlodekPatrick WrightLocal Organising Committee (ESA/ASRB) 1999ChairSecretaryMembersMark HedgerPatricia Tart (ACTS)Ben CannyPam Mamers (ENSA)Jock MacmillanSarah MeachemLiza O'DonnellDavid Phillips45
Australian <strong>Society</strong> <strong>for</strong> <strong>Reproductive</strong> <strong>Biology</strong>HILTON ON THE PARK FLOORPLANPast Office Bearers & AwardeesYEAR1969-701970-711971-721972-731973-741974-751975-761976-771977-781978-791979-801980-811981-821982-831983-841984-851985-861986-871987-881988-891989-901990-911991-921992-931993-941994-951995-961996-971997-981998-99CHAIRMANTJ RobinsonCWEmmensDM de KretserNWMooreJK FindlayBMBindonBP SetchellJC RodgerRJ ScarammuzziAOTrounsonRF SeamarkMB RenfreeTREASURERRG WalesIG WhiteGM StoneGHMcDowellBG MillerCD NancarrowLA HindsGB MartinCGTsonisLJ WiltonJD CurlewisMB HarveySECRETARYJR GodingBMBindonIACumming.GODING/FOUNDERSLECTUREIACumminWHanselDT BairdTD GloverCH T ndale-BiscoeGMH ThwaitesRJ Scaramuzzi r-_KP:::..;.:;;;"...:;.;,M.::.;:c.:;..N:.::.at:::.L-----lBKFollett PJ Lut'enRJ Rodger &J WilsonTKRobertsCBGowBMBindon SP FlahertJMCumminsG EvansA BellveWHanselC O'NeillLJ WiltonBP SetchellHG Bur erBJ WaddellA Sto"anoffJM SheltonFWBazer MB HarveGD Thorburn AHTornePL KayeRMMoor HMassaCR Austin DK GardnerJK Findlay SW WalkdenLABrownSalamonsenGCLi ns CMMarkeMPHedgerI Huhtaniemi MJ Hotzel & SMcDou allRF Seamark Ivan WezelNo Lecture S RobinsonJGThompson r--;:::--;-::..::::::.::.:::.....---+-~~~---JFBronson M Jas erDM de Kretser M Panteleon. ci·············§·················r·i···· ~~... . ..~ i• •·t:;\I Bi!., )00)1· fZ'~"• •••6 7
1210345678911121314OTHER CONFERENCE VENUESA B C D E F G H I JABC DE F G HI JVenuesCon:krence:o Hilton on <strong>the</strong> Park, 192 Wellington Parade, East Melbourne (Map ref06), Tel: 9419 2000Dinner:@The Powerhouse Function Centre, Lakeside Drive, Melbourne (Map refF13), Tel: 95103644Student Evening:@ Bridie O'Reilly's Pub, 462 Chapel Street, South Yarra (Map refIll), Tel: 98277788Accommodation:IHilton on <strong>the</strong> Park, 192 Wellington Parade, East Melbourne (Map ref06), Tel: 94192000II East Melbourne Apartment Hotel, 25 Hotham St, East Melbourne (Map refH6), Tel: 94122555Birches Serviced Apartments, 160 Simpson St, East Melbourne (Map refH5), Tel: 94172344. • Albert Heights Serviced Apartments, 83 Albert St, East Melbourne (Map ref05), Tel: 9419 0955Melbourne Sheraton, 13 Spring Street, Melbourne (Map refE6), Tel: 9205 9999: St. Hilda's College, College Crescent, Parkville (Map refD2), Tel: 9248 84001234567891011121314Joint ASRBIESA Program <strong>for</strong> Sunday, September 26, 1999.:a0 0=~~
~=TIME BALLROOM BALLROOM STRADBROKE HUNTINGFIELD DELACOMBE LATROBE BALLROOM2 3 18.30-9.00ESA Plenary Lecture: ASRB Orals: ASRB Orals:9.00-9.30 Sheppard Sperm maturation Endometrial ENSA& viability I Function9.30-10.00 ESA Servier Award10.00-10.30 Morning Tea Morning Tea Morning Tea Morning Tea Morning Tea10.30-11.00ASRB Orals: ASRB Orals:11.00-11.30 ESA Novartis Junior Nutrition & Testicular InjuryScientist FinalistsReproduction11.30-12.00 ENSA12.00-12.30ASRB Founders Lecture:12.30-1.00 Wilmut1.00-1.30Lunchffrade ESAMeet <strong>the</strong> Lunchffrade ESA Meet <strong>the</strong> Lunchffrade1.30-2.00 Expert: Array Expert: HRT ESA PosterTechnologyviewing session2.00-2.30ESAOrals: ESA Orals: ESAOrals:2.30-3.00 HRTFemale& ASRB GH, GH-R, IGFs Comparative ENSAMale (Clinical) Symposium: (Basic) Endocrinology ESA Poster3.00-3.30 From Gamete to (Basic) discussion:EmbryoHypothalamus-3.30-4.00 Afternoon tea Afternoon tea Afternoon tea Afternoon tea Afternoon tea pituitary4.00-4.30 Afternoon teaESA Orals: ASRB Orals: ESA Orals: Leptin, ESA Orals: ESAPoster4.30-5.00 Pituitary- Oocyte body homeostasis Male discussion:adrenal Maturation (Basic) Reproduction Thyoid and5.00-5.30 (Clinical) (Basic) ENSA molecularendocrinology5.30-6.00ESAAnnual6.00-6.30 GeneralMeeting~o 1-'-a>r.n~ r.n>~~o~~=e~~~o=~~ ....00("D~~~("D~N~....~\C\C\C •~...~TIME BALLROOM BALLROOM STRADBROKE HUNTINGFIELD DELACOMBE LATROBE BALLROOM2 3 18.00-8.30ESA Plenary Lecture:8.30-9.00 Simpson9.00-9.30ASRB Orals:ASRB Orals:9.30-10.00 Sperm maturation ESA Orals: ESAOrals: Ovarian ESA Orals:ESA Symposium: & viability II Fetus & Placenta Case Reports development Endocrine10.00-10.30 Hormones and Sport (Basic) (Clinical) & function Cancer (Basic)10.30-11.0011.00-11.30 Morning tea Morning tea Morning tea Morning tea Morning tea Morning tea11.30-12.00ESA Harrison Plenary Lecture:12.00-12.30 Burger12.30-1.00Lunchffrade ESAMeet <strong>the</strong> ESA Meet <strong>the</strong>1.00-1.30 Lunchffrade expert (Clinical): Expert (Basic):TSH Confocal Lunchffrade1.30-2.00 Lunchffrade LunchfTrade2.00-2.30 ASRB ESAPosterSymposium:viewing session2.30-3.00 ESAOrals: Immune ESA Orals: ESAOrals:Pot pouri, Regulators in IGFIIGFBPs Gene3.00-3.30 osteoporosis, <strong>Reproductive</strong> (Basic) Expressionthryoid, PTH <strong>Biology</strong> (Basic) ESAPoster3.30-4.00 (Clinical) Afternoon tea discussion:Reproduction,4.00-4.30 Afternoon tea Afternoon tea Afternoon tea Clinical & BasicASRB Orals:4.30-5.00 Junior ScientistFinalistsESA meet <strong>the</strong> POC5.00-5.305.30-6.006.00-6.30ASRBAGMo 1-'-=flO+.> en~ en>~~o~~S8'~~=("Dtf)Q..~ ...ṙ.n~~~~~N00 ....~\C\C'= •
Joint ASRBIESA Program <strong>for</strong> Wednesday, September 29, 1999.::E0 0~?""l...:l...:l-. 0e3 ,2 U '.;;:j .r::0 eo Uc:: ~=:§'t: c::~'s ~5'So~ .30Cf.l ;>.e~~ ~ - .-'sU 't: ,~•• U-
Monday 27 th SeptemberSESSION 2:Sperm MaturationIFunction ITime: 8.30 - 10.00 amOralslMinipostersVenue: StradbrokeMonday 27 th September (cont.)Chairs: Russell Jones and Ramakrishnan VishwanathSESSION 3:Endometrial FunctionOralslMiniposterTime: 8.30 - 10.00 amVenue: HuntingfieldTime Format Page Authors Title Chairs: Rebecca Jones and Sarah Robertson8.30 o 39 SY Man, J Clulow, RCJonesThe effect of cyclic-nucleotides on fluidresorption in <strong>the</strong> ductuli efferentes testisof <strong>the</strong> rat8.45 M 40 JF Smith, RS-F Lee, RM Identification of proteins with seasonalMcDonald, GR Murray, J patterns of secretion in ram seminalParrplasma8.53 M 41 REA Knowling, CM Leigh, A preliminary characterisation ofWG Breedglycosidases in epididymal fluid of <strong>the</strong>male Brush-tailed possum, Trichosurusvulpecula9.00 o 42 M Lin, X Zhang,Murdoch, RJ Aitken9.159.309.37oMo434445RIn vitro culture of brushtailed possumepididymal epi<strong>the</strong>lium and induction ofepididymal sperm maturation in cocultureES Gellatly, G Evans, The addition of seminal plasma to frozenWMC Maxwell, ST thawed ram semen affects sperm-mucusMortimerinteractionCA McPhie, G Evans,WMCMaxwellKS Sidhu, KE Mate, FCMolinia, AM Glazier, JCRodgerThe effect of seminal plasma on <strong>the</strong>capacitation status of fresh and frozenthawedram spermatozoaSecretory proteins from <strong>the</strong> femalereproductive tract of <strong>the</strong> brushtail possum(Trichosurus vulpecula): binding tosperm and effects on <strong>the</strong>ir survival invitroTimeFormat8.30 MPage46AuthorsTitleCY Feng, M Bhave, RJ mRNA editing of OTR: a new mechanismFairclough<strong>for</strong> regulation8.37 0 47 J Zhang, AL Hampton. G Progesterone inhibits activation of latentNie, LA Salamonsen matrix metalloproteinase (MMP)-2 inhuman endometrium by regulatingexpression of membrane type 1-MMP8.53 M 48 N Malakooti, LASalamonsen9.009.159.309.45oooo49505152P-y Chu, PJ Wright, CSLeeLeukocytes and matrix metalloproteinasesare present at sites of tissue breakdown ina mouse model <strong>for</strong> menstruationA model <strong>for</strong> <strong>the</strong> study of uterine functionin <strong>the</strong> bitchG Nie, Y Li, H Minoura, J Mouse splicing factor SC35 mRNA is upFindlay, L Salamonsen regulated at implantation sites in <strong>the</strong>mouse uterus during early pregnancyRL Jones. LA Salamonsen, Differential expression of inhibin andJK Findlay activin subunits in non-pregnant andpregnant human endometriumLT Sebastian, G Shaw, MB Identification and functionalRenfree, GE Rice characterisation of phospholipase A 2 in<strong>the</strong> endometrium of <strong>the</strong> tammar wallaby,Macropus eugenii1415
Monday 27 th September(cont.)SESSION 4:Nutrition & ReproductionTime: 10.30 - 12.00 amOrals/MlnipostersVenue: StradbrokeMonday 27 th September(cont.)Chairs: Chris Scott and Alan TilbrookSESSION 5: Testicular Injury Orals/MinipostersTime Format Page10.30 o 5310.4511.0011.0711.2311.30oM 55oMo54565758AuthorsS Meier, BA Clark, SRMorgan, GA VerkerkTitleThe effects of feeding rumen bypass fattyacids during early postpartum on uterineprostaglandin syn<strong>the</strong>sis, plasma fattyacids and <strong>the</strong> onset of oestrous activity indairy cows Time Format PagePM Rhodes, BA Clark, SR Energy balance and <strong>the</strong> postpartumMorgan, GA Verkerk interval in pasture fed dairy cowsD Blache, MA Blackberry,R Tellam, PE Vercoe, GBMartinInteractions between photoperiod, foodsupply and breed affect plasma leptinconcentrations in mature male sheepLM Chagas, D Blache, MA Does insulin or leptin mediate <strong>the</strong> effectBlackberry, GB Martin of dietary fatty acids on LH pulsefrequencies in mature male sheep?PS Duggal, ND Hussey, RJNormanDetection of rat leptin receptor (iso<strong>for</strong>mB) rnRNA in rat ovary using a semiquantitativereverse transcriptionpolymerasechain reaction (RT-PCR)assayJ McFarlane, K Kauter, B Ovarian uptake of leptin and glucose andCampbell, D Baird, A <strong>the</strong> role of glucose in <strong>the</strong> regulation ofLeigh, R Scaramuzzi leptin levels in sheep10.3010.3710.5211.0011.07MooMM5960616263Time: 10.30 - 11.30 amChairs: Jim Cummins and Moira O'BryanAuthorsVenue: HuntingfieldTitleD Mehmet, JM Cummins, Quantitation of <strong>the</strong> commonF Ahmed, R Martin, J mitochondrial DNA deletion in humanWhelan testicular tissues by competitive PCRusing a chimaeric competitor constructFA Ahmed, J Whelan, AMJequier, JM CumminsJ Yaeram, BP Setchell, SMaddocksThe role of mitochondria in ischaemic/reperfusioninjury in rat testesThe effects of whole body heat-stress ofmale mice on aspects of paternal fertilityB-k Zhu, SK Walker, BP The effect of paternal heat stress onSetchell, S Maddocks development in-vitro of preimplantationembyos in <strong>the</strong> mouseZ Zhang, MB Renfree, RV The effect of testicular cooling onShortspermatogenesis in <strong>the</strong> rat1617
Monday 27 th September (cont.)SESSION 6:Founders LectureTime: 12.00 - 1.00 pmVenue: Ballroom 2 + 3Chair: Marilyn RenfreeProfessor Ian WilmutRoslin Institute, EdinburghSESSION 7:Monday 27 th September(cont.)Symposium: From Gamete to Embryo - New HorizonsTime: 2.00-4.00 pm Venue: Ballroom 3Chair: Ian Wilmut"Cloning in biology and medicine"Time Format Page Authors Title2.00 0 65 JMCummins Mitochondrial DNA: our alternativegenome2.40 0 66 IPHearn Primate stem cellsProfessor Wilmut obtained a B.Sc. in Agricultural Science at <strong>the</strong> University of Nottingham be<strong>for</strong>e studyingwith Professor "Chris" Polge FRS at <strong>the</strong> University of Cambridge. His Ph.D. degree was awarded in 1971 <strong>for</strong>research on <strong>the</strong> Deep Freeze Preservation of Boar Semen. Subsequent research in Cambridge led to <strong>the</strong> birthof <strong>the</strong> first calf from a frozen embryo - "Frosty" - in 1973.3.20 0 67 B Reubinoff, M Pera, C Human embryonic stem cellsFong, AO TrounsonHe moved to Edinburgh in 1973. Initially, studies at Animal Breeding Research Organisation were focused ondevelopmental and physiological studies of prenatal mortality in sheep and pigs. Subsequent research wasconcerned with developing techniques of multiple ovulation/embryo transfer (MOET) in sheep and cattle.This research established <strong>the</strong> procedures, which are now exploited by PPL Therapeutics at Roslin, <strong>for</strong> <strong>the</strong>production of proteins needed to treat human disease in <strong>the</strong> milk of farm animals.Over <strong>the</strong> past nine years, research has been focused on <strong>the</strong> factors regulating embryo development after nucleartransfer. This work led to <strong>the</strong> first birth of live lambs from embryo-derived cells and <strong>the</strong>n to <strong>the</strong> birth of lambsderived from foetal and adult cells, including Dolly. Recently, genetic changes were introduced into sheep bynuclear transfer from cultured modified cells.As well as being Principal Investigator in Roslin Institute he also serves as Chief Scientific Officer of GeronBio-Med. The objectives of <strong>the</strong> current research are to develop biomedical applications of <strong>the</strong> nuclear transferprocedure. These include <strong>the</strong> provision of organs <strong>for</strong> xenotransplantation, <strong>the</strong> derivation of human cells <strong>for</strong><strong>the</strong>rapy and <strong>the</strong> cloning and modification of animals in agriculture.This symposium is sponsored by:rfdReproduction, Fertility and Development1819
Tuesday 28 th SeptemberMonday 27 th September(cont.)SESSION 8:Sperm maturation/viability IIOralslMinipostersSESSION 8:Oocyte maturationOralslMinipostersTime: 9.00-10.30 am Venue: StradbrokeChairs: Gareth Evans and John RodgerTime: 4.00-5.30 pmVenue: StradbrokeChairs: Chris Grupen and Jeremy Thompson Time Format Page Authors TitleTime4.00FonnatoPage68AuthorsJA Chapman, OWWiebkin, WG BreedTitleInterspecific variation in glycoconjugatesof <strong>the</strong> zonae pellucidae of severalmarsupial species9.009.07Mo7576RN Murdoch, MA Wade,MLinJK O'Brien, DA Oehler,SP Malowski, TL RothN-acetyl-D-glucosamine damages spermfrom <strong>the</strong> tammar when recovered fromejaculates by swim-up proceduresSemen collection, characterisation andcryopreservation in a Magellanic penguin(Spheniscus magellanicus)4.15 o 69 AL Kitchener, DJ Kay, LM Immunocontraception <strong>for</strong> wildlifeEddscontrol: <strong>the</strong> zona pellucida as a target inmacropods4.304.374.525.005.15MoMoo7071727374A Preshaw, WMCMaxwell, G EvansKE Mate, J BuistSS Metcalfe, 1M Gunn, JMShawSelection of porcine oocytes prior to invitro maturation and in vitro fertilisationusing Brilliant Cresyl BlueThe timetable of nuclear maturation oftammar wallaby oocytes in vitro andrequirements <strong>for</strong> protein syn<strong>the</strong>sisWG James, 0 Lacham- Successful development of a 6-DMAPKaplan, AO Trounson replacement <strong>for</strong> bovine oocyte activationresearchCG Grupen, MB NottleChanges in intracellular calcium levels inporcine oocytes treated with ionomycinIn vitro maturation, fertilisation and earlyembryonic development in <strong>the</strong> dog9.23 M 77 WV Holt, SD Johnston Enhanced osmotic tolerance of Koalaspermatozoa following treatment with <strong>the</strong>cytoskeletal disrupting agent,cytochalasin D9.309.459.5210.07oMoM78798081J Krzyzosiak, G McMillan, Regulation of protein tyrosineR Vishwanath, P Molan phosphorylation, during in vitro aging ofejaculated bovine spermatozoaDVR Bullen, P Molan, R The effect of in vitro storage onVishwanathmitochondrial marker enzyme activity inbovine spermatozoaA Preshaw, WMCMaxwell, G EvansEffect of calcium on <strong>the</strong> In vitrofertilisation of in vitro matured porcineoocytes by frozen epididymalspermatozoaS Galea, 0 Lacham- Confocal analysis of decondensation andKaplan, A Trounson pronuclear <strong>for</strong>mation in human sperminjected into murine oocytes10.15 o 82 S Galea, 0 Lacham- Preliminary study: high fertilisation ratesKaplan, A Trounson produced with prolonged storage of malefactor patient sperm following ICSI intomurine oocytes2021
Tuesday 28 th September (cont'd)Tuesday 28 th September (cont'd)SESSION 10:Ovarian development/functionTime: 9.00-10.15 amOralslMinipostersVenue: LatrobeSESSION 11:Immune Regulators in <strong>Reproductive</strong> <strong>Biology</strong>SymposiumChair: Ann DrummondTime: 1.30-3.00 pm Venue: Ballroom 3Time Format Page Authors TitleChair: Brian Setchell9.00 o 83 ME McArthur, S Byers, HF Proteoglycans and glycosaminoglycans ofIrving-Rodgers, RJ small bovine ovarian folliclesRodgers Time Format Page Authors Title9.159.229.309.45MMoM84HF Irving-Rodgers, ML Morphological changes in <strong>the</strong> membranaMussard, RJ Rodgers, JE granulosa and basal lamina during growthKinder and regression of bovine dominantfollicles85 RB Gilchrist, LJ Ritter, RJNorman, DT Armstrong8687Changes in marmoset granulosa cellresponsiveness to FSH and IGF-I withfollicular growthKL Britt, AE Drummond, The effects of targeted disruption of <strong>the</strong>MEE Jones, M Dyson, NG cyp 19 (aromatase) gene onWre<strong>for</strong>d, ER Simpson, JK folliculogenesis in miceFindlaySN Reinke, 1M Gunn, MBRenfree, AO TrounsonPolyovular follicles in <strong>the</strong> pouch youngand adult kangaroo1.302.002.30ooo899091MP Hedger, 0 Gerdprasert,MKO'BryanSA RobertsonMD Mitchell, JA KeelanThis symposium is sponsored by:Testicular macrophages, inflammationand infertilityCytokine networks in <strong>the</strong> endometriumand ovary during early pregnancyCytokine production and actions inhuman pregnancy and parturitionrfdReproduction, Fertility and Development9.53 o 88 RB Gilchrist, LJ Ritter, DTArmstrongGrowth-promoting activity of oocytes isattained concomitant with <strong>the</strong> acquisitionof meiotic competence2223
Tuesday 28 th September (cont'd)SESSION 12:Junior Scientist FinalistsTime: 3.30-5.00 pmOralsVenue: Ballroom 3Chair: Marilyn RenfreeTuesday 28 th September (cont.)Time Format Page Authors Title3.30 o 92 MAAbdo,MABogoyevitch, AMDharmarajan3.454.004.15ooo939495S Hudson, S RobertsonLM Kilpatrick, LASalamonsenT Meehan, C Print, DM deKretser, K LovelandExistance of extracellular signal-relatedkinase (ERK) and p38 mitogen activatedprotein kinase (MAPK) in <strong>the</strong> rat corpusluteum (CL)Uterine antigen-presenting cells aredysregulated in GM-CSF deficient miceProgesterone downregulation of matrixmetalloproteinase-l (MMP-l) - a role ofEts transcription factors?Bcl-2 family member expression patternsin mouse testis germ cellsSESSION 13:ASRB Annual General MeetingTime: 5.00-6.00 pm Venue: Ballroom 34.30o96TP Sharma, D Blache, GM Aromatase and 5a.-reductase pathwaysMartinand <strong>the</strong>ir interaction with nutrition in <strong>the</strong>control of pulsatile secretion of LH inmale sheep4.45 o 97 EJ Whiteside, MMJackson, AC Herington,MB HarveyTissue inhibitor of metalloproteinase-3(TIM:P-3) is a key regulator ofextracellular matrix (ECM) degradationduring mouse embryo implantation242S
Wednesday 29 th SeptemberWednesday 29 th September(cont'd)SESSION 14:Embryo development/implantation Miniposters/OralsSESSION 15:TestisMiniposters/OralsTime: 8.30-10.00 amVenue: StradbrokeTime: 8.30-10.00 am'Venue: HuntingfieldChairs: Mark Harvey and Graham JenkinChairs: Simon Madqocks and Peter Temple-SmithTime Format Page Authors Title Time Format Page Authors Title8.30 M 98 T Stojanov, C Wu, CO'Neill8.378.459.009.159.22MooMM99100101R Bathgate, M Emerson, A Embryo-derived PAP induces Ca 2 +Travis, C O'Neilltransients in <strong>the</strong> mouse preimplantationembryoKJ Boucaut, EJ Whiteside, Expression of ADAM 10, ADAM 17 andMM Jackson, A Teh, J TNFa in peri-implantation mouseGarcia-Aragon, AC embryosHerington, MB Harvey102 CM Hearn, MB Renfree103M Pantaleon, M KanaiAzum, J Mattick, KKaibuchi, P Kaye, S WoodCS Chan, MB Harvey, JAClementsPlatelet-activating factor receptor inhuman and mouse spermatozoa andmouse preimplantation embryosExpression of FAM, a ubiquitin specificprotease in mouse embryosOvarian steroid hormones and growthfactors in <strong>the</strong> uterine control of earlyembryonic development in <strong>the</strong> tammarwallaby, Macropus eugeniiTemporal and tissue specific expressionof mouse kallikrein (mKlk) genes andidentification of a novel mKlk mRNAtranscript during early development in <strong>the</strong>mouse8.30 M 105 R Duckett, N Wre<strong>for</strong>d, DTaggart8.378.529.079.229.37ooooo106107108109WV Ingman, SA RobertsonKL Loveland, L ChristyM Ricci, WG BreedSeasonal changes in <strong>the</strong> testicularmorphology of <strong>the</strong> Agile AnticinusMorphology of <strong>the</strong> testes and seminalvesicles of <strong>the</strong> male TGF~ 1 null mouseMAP2 in <strong>the</strong> testis: iso<strong>for</strong>m expressionpatterns suggest functional heterogeneityDM Hickox, JR Morrison, Cloning and characterisation of a novelKL Sebire, H-H Keah, testis transcript with homology toMTW Heam, DM de phosphatidylethanolamine bindingKretser, MK O'Bryan proteins - pebp2Isolation and partial characterisation of<strong>the</strong> outer dense fibres from brush-tailedpossum spermatozoa110 X Zhang, F Molinia, M Lin A 49 kDa protein isolated from <strong>the</strong>acrosomal region of <strong>the</strong> brushtailedpossum (Trichosurus vulpecula)spermatozoa9.30 o 104 SJ Pearson, WH McMillan,AJ PetersonThe relationship between IFN-'tconcentrations and embryo developmentin a herd of recipient cattle selected <strong>for</strong>high or low pregnancy rates26 27
Wednesday 29 th September (cont.)SESSION 17:ASRB Plenary Lecturekindly sponsored by: AstraZeneca4Wednesday 29 th September(cont'd)Time: 12.30-1.30 pmChair: Alan TilbrookVenue: Ballroom 3SESSION 16:PregnancylParturition Joint ASRBIESA SymposiumTime: 10.30am-12.30 pmVenue: DelacombeChairs: Mary Wlodek and Brendan WaddellDr Graham MitchellFoursight AssociatesTime Format Page Authors10.30 0 III M MitchellTitleRegulation of prostaglandin H synthase-2expression in human intrauterine tissues"Constraints and opportunities in <strong>the</strong> commercialisation ofAustralian R&D"11.00oo112 R SmithRegulation of parturition: <strong>the</strong> role of eRR11.3012.00o113 C McMillen114 E WallaceParturition and <strong>the</strong> HPA axis in <strong>the</strong> sheepfetus: unknown inhibitors and unlikelystimulatorsInhibins and activins: markers of maternaland fetal healthGraham Mitchell is a veterinary graduate and University gold medallist of <strong>the</strong> University of Sydney. Hestudied immunology with Sir Gustaf Nossal and Dr Jaq Miller at <strong>the</strong> Walter and Eliza Hall Institute of MedicalResearch (WEHI) where he obtained a PhD in 1969. After post-doctoral experience in Cali<strong>for</strong>nia (Stan<strong>for</strong>d),England (Mill Hill) and Switzerland (Basel) he returned to Australia in 1973 and established a new program inimmunoparasitology at WEill. This program became a maj or component of <strong>the</strong> global ef<strong>for</strong>t to develop newvaccines against parasitic diseases, and of <strong>the</strong> biotechnology revolution.In 1990 Mitchell was appointed Director of <strong>the</strong> historic and prestigious Royal Melbourne Zoological Gardenswhere he introduced a number of new initiatives in local and regional conservation and helped position <strong>the</strong>modern Melbourne Zoo as a key centre of recreation, education, conservation and research.In 1993 he returned to biomedical research as Director of Research in <strong>the</strong> R&D Division of CSL Limited,Australia's largest developer, manufacturer and marketer of ethical human biologicals and veterinary products.He was responsible <strong>for</strong> identifying and assessing new opportunities <strong>for</strong> CSL, nurturing external researchcollaborations of <strong>the</strong> company, and <strong>the</strong> research component of <strong>the</strong> development of new products includingvaccines.Dr Mitchell is recognised as one of Australia's leading biological scientists. His expertise extends over a widerange of science and technology. He has detailed knowledge of <strong>the</strong> academia-industry interface, has worked inmany overseas countries and been involved with <strong>the</strong> World Health Organisation <strong>for</strong> many years. He is anauthor of more than 350 publications, has received numerous awards <strong>for</strong> scientific achievements, and, in 1993,was appointed an Officer in <strong>the</strong> Order of Australia <strong>for</strong> services to science, in particular immunoparasitology.2829
Wednesday 29 th September(cont'd)SESSION 18: <strong>Reproductive</strong> Endocrinology Miniposters/OralsTime: 1.30-3.00 pm Venue: StradbrokeChairs: Jon Curlewis and Anne TurnerWednesday 29 th September(cont'd)SESSION 19: Gamete Technology Miniposters/OralsTime: 1.30-3.00 pm Venue: HuntingfieldChairs: Chis Maxwell and Teija PeuraTime Format Page Authors TitleTime1.301.371.522.072.152.222.302.372.452.52FormatMooMMMMMMMPage116117118119120121122123124125AuthorsJ VanCleef, E Prendergast,D Blache, GB MartinC Nave, G Shaw, RV Effects of levonorgestrel on fertility in <strong>the</strong>Short, MB Renfree tammar wallaby, Maerapus eugeniiA Niasari-Naslaji, FSarhaddi, Y Damavandi, AAngurani, A NajiTitleA critical number of eggs stimulatesprolactin secretion in male emus during<strong>the</strong> breeding seasonEffect of follicle regressing agents,oestrogen and progesterone, on ovarianfollicular dynamics in Bas taurus and Basindieus heifersAR Rabiee, KL Macmillan, Faecal progesterone metabolites InF Schwarzenberger ovariectomised dairy cows treated withprogesteroneAR Rabiee, KL Macmillan, Effect of feeding managemen onMJ Rathboneprogesterone release from CIDR devicesin ovariectomised dairy cowsAR Rabiee, KL Macmillan Effect of nutrition on plasmaprogesterone concentrations inovariectomised dairy cows treated withprogesteroneVK Taufa, KL Macmillan, Varying <strong>the</strong> <strong>for</strong>m of oestradiolG Mylrea, S Morgan, R administration to yearling heifers at <strong>the</strong>Hooperbeginning of treatmentAM Padula,Macmillan, TE TriggDR Williams,McFarlaneJRY Xia, J McFarlane, TO'SheaKL Deslorelin implants prevent earlypostpartum ovulations in Holstein dairycattleThe gestational profile of uterine milkproteins in ovine allantoic fluid andamniotic fluidConcentrations of progesterone, FSH andfollistatin in maternal plasma acrossgestation in <strong>the</strong> Merino ewes that arehomozygous or non-carriers of Booroolagene (Fee B )1.301.371.451.522.072.152.222.372.523.003.15MMMoMMooMoo126127128129130131132133134135136RLi, I Lyons, RHarvey, LRobbJJ Arlaud, L Baker, RLWilliams, CP Cunningham,1M Lewis, WG Brown, MBorg, D Kerton, AJ FrenchVK Taufa, KL Macmillan,G Verkerk, SR Morgan,AM Day, R HooperNM Fogarty, WMCMaxwell, J Eppleston, GEvansG Evans, KM Rao, NMFogarty, WMC MaxwellLL Kuleshova, JM Shaw,AO TrounsonM Pangestu, JM ShawBS Choo, J Shaw, LKuleshova, A TrounsonFC Molinia, JV MyersEmbryonic stem cell line plays <strong>the</strong> mostimportant role in <strong>the</strong> production ofgermline chimaerasEstablishment of a SLA inbred miniaturepig herd free of quarantine restrictionsControlled breeding of lactating cyclingdairy cowsSheep embryos viable after 13 yearsstorageBirth of lambs following vitrification ofin vitro produced ovine embryosRapid cooling of embryos in protein-freesolutions within a straw-in-straw packageto reduce <strong>the</strong> risk of contamination duringstorage in liquid nitrogenComparison of normal and methoxylatedethylene glycol <strong>for</strong> <strong>the</strong> cryopreservationof 2-cell and blastocyst stages mouseembryosThe viability of bovine primordialfollicles cryopreserved within pieces ofovarian tissue using slow and rapidcooling protocolsFertility of frozen-thawed possumspermatozoa after laparoscopic artificialinseminationSD Johnston, MR Birth of Koala pouch young followingMcGowan, PO'Callaghan, artificial inseminationR Cox, B Houlden, S Haig,GTaddeoJG Thompson, S Cox, WH Fetal abnormality is more pronouncedMcMillan, AJ Peterson, M following transfer to synchronisedDonnison, LT McGowan, recipients of day 5 bovine IVF embryosA Ledgardcompared to day 7 embryos30 31
Australian <strong>Society</strong> <strong>for</strong> <strong>Reproductive</strong> <strong>Biology</strong>Thirtieth Annual ConferenceAbstracts3233
MICROMANIPULATION PROCEDURES FOR GAMETES AND EMBRYOSO. Lacham-Kaplan, G. M. Jones, A. TrounsonCentre For Early Human Development, Institute of Reproduction and Development, MonashUniversity Melbourne, Victoria, AustraliaMicromanipulation procedures have been used as an artificial reproductive technology (ART) <strong>for</strong> <strong>the</strong>treatment of infertility in <strong>the</strong> human and o<strong>the</strong>r mammalian species. The zona pellucida, surrounding<strong>the</strong> mammalian oocyte is <strong>the</strong> major barrier to fertilization. Sperm must pass through this layer inorder to reach and fuse with <strong>the</strong> oocyte membrane and initiate embryonic development. The zonapellucida is also believed to protect <strong>the</strong> embryo during its preimplantation development in-vivo.Upon implantation in <strong>the</strong> uterus embryos at <strong>the</strong> blastocyst stage expand and exit from <strong>the</strong> zonapellucida in a process known as 'hatching'. The zona pellucida may prevent fertilization of oocytesby subfertile sperm. Moreover, due to abnormal thickening and hardening of <strong>the</strong> zona pellucidaembryos may fail to hatch, hence reducing <strong>the</strong> chances of implantation and pregnancies. In <strong>the</strong>secases, breaching or complete removal of <strong>the</strong> zona pellucida is necessary. Breaching of <strong>the</strong> zonapellucida is mainly done by micromanipulation procedures.To facilitate fertilization, a microneedle is used to penetrate <strong>the</strong> zona pellucida and leave <strong>the</strong> spermin close proximity to <strong>the</strong> plasma membrane or directly inside <strong>the</strong> oocyte cytoplasm in a processknown as intracytoplasmic sperm injection (lCS!). In this procedure, sharp glass capillaries areoperated manually to pierce <strong>the</strong> zona pellucida and <strong>the</strong> plasma membrane. Using lCSl, fertilizationwith very low numbers of normal sperm, immotile sperm, epididymal and testicular sperm, and germcells such as round spermatids and secondary spermatocytes have been achieved in humans and ino<strong>the</strong>r mammalian species. These include mice, horses, cows, domestic cats and o<strong>the</strong>rs and may be<strong>the</strong> optimal procedure to be used <strong>for</strong> conservation of endangered species.Intracytoplasmic injection is also used to inject somatic cells into enucleated oocytes <strong>for</strong> <strong>the</strong> purposeof cloning farm and laboratory animals. A recent use of non-sharp microinjection capillaries attachedto a Piezo system, which is operated by electromagnetic pulses, has been shown to be less invasiveand more effective when germ cells and somatic cells were injected directly into <strong>the</strong> oocytecytoplasm.Assisted hatching and embryo biopsy <strong>for</strong> preimplantation diagnosis in <strong>the</strong> human are also done usingmicromanipulation procedures to partially remove <strong>the</strong> zona pellucida. Partial removal of <strong>the</strong> zonapellucida can be done chemically using low p~_ solutions, mechanically by a sharp microneedle, orby laser. The laser system <strong>for</strong> assisted hatching has been found to be easier to handle and morereproducible than <strong>the</strong> o<strong>the</strong>r two assisted hatching procedures. Using a laser beam directed on acertain point of <strong>the</strong> zona pellucida, accurate and repeatable holes are created. The procedure is safeand does not interfere with embryo development. The laser system offers a significantmethodological improvement over <strong>the</strong> chemical and mechanical assisted hatching. The threeprocedures are found to enhance implantation and pregnancies in cases of failed IVF due to ei<strong>the</strong>rthick or hardened zonae, suggesting that <strong>the</strong> techniques may be beneficial <strong>for</strong> all IVF producedembryos.3435
NEW GENETICALLY TARGETTED SENSORS FOR THE CONFOCAL IMAGING OFINTRACELLULAR Ca 2 + AND pH IN LIVING CELL SYSTEMSDavid A. Williams, David N. Bowser, Angela M. Reilly, Rekha Panchal, Megan L. Smart, RobertNicholls and Steven PetrouConfocal and Fluorescence Imaging Group, Department of Physiology, The University ofMelbourne, Parkville, 3052, Australia.The majority of cellular processes are sensitive to changes in Ca 2 + and/or W ion concentration. As such,study of <strong>the</strong> regulation of intracellular, particularly intraorganellar, Ca 2 + and pH is of fundamentalimportance to <strong>the</strong> understanding of most cell systems. Two experimental strategies directly explore <strong>the</strong>interorganellar ion concentrations in living cells. The first involves loading of intact cells with membranepermeantfluorophores, with subsequent quenching or removal of potentially confounding cytosolicfluorescence. This strategy has largely focussed on Ca 2 + and <strong>the</strong> mitochondria and while it has had somesuccess (Bowser et al., 1998), <strong>the</strong>se fluorescence techniques are difficult and <strong>the</strong> attribution of results tospecific cell locations has not always been unambiguous. The second approach has also focussed on Ca 2 + andinvolves intracellular targeting of <strong>the</strong> luminescent, Ca 2 +-sensitive protein "aequorin", to specific organellesand has been in use <strong>for</strong> a number of years. Aequorin is a very sensitive Ca 2 + sensor and its properties havebeen thoroughly categorised well be<strong>for</strong>e its use in "targeted mode". However, it has always been difficult toimage (its luminescence producing less that one photon per molecule) and has suffered from difficultiesinvolved in calibrating <strong>the</strong> light emission in terms of absolute [Ca 2 +J.Developments in molecular biological syn<strong>the</strong>sis have recently provided Ca 2 +and pH sensors, that offer majoradvantages over chemically syn<strong>the</strong>sized Ca 2 + sensors and targeted Ca 2 + sensors with sub-optimalcharacteristics. "Cameleons" are genetically engineered protein sensors of free Ca 2 +comprised of tandemfusions of a blue or cyan mutant of fluorescent protein (GFP), calmodulin, calmodulin-binding protein M13and an enhanced green or yellow emitting GFP. Following <strong>the</strong> binding of Ca 2 +, calmodulin wraps around <strong>the</strong>M13 domain, <strong>the</strong>reby bringing <strong>the</strong> two flanking GFPs closer toge<strong>the</strong>r. This proximity allows <strong>for</strong> Resonance.Energy Transfer (RET) of <strong>the</strong> emitted energy (from <strong>the</strong> "donor") directly into <strong>the</strong> excitation of <strong>the</strong> second"acceptor" GFP. The degree to which RET occurs represents a direct, ratiometric measure of free Ca 2 +concentration. The Ca 2 + affinity of <strong>the</strong> fusion protein is not fixed to that of <strong>the</strong> native calmodulin segment.Point mutations in this sequence with standard molecular genetic techniques have allowed fine-tuning of <strong>the</strong>Ca 2 +affinities from as low as approximately 10- 8 M to up to 10- 2 M, suitable <strong>for</strong> different locations (i.e. high<strong>for</strong> <strong>the</strong> cytosol, nucleus or mitochondria, and low <strong>for</strong> ER, Golgi or plasmalemma). The pH dependency ofvarious GFP mutants, in particular, that of enhanced yellow fluorescent protein (EYFP), with <strong>the</strong> highestpK'a of <strong>the</strong> mutants, provides exciting possibilities <strong>for</strong> targeted pH measurements.These sensors can be produced in situ by gene transfer of plasmid DNA into a number of cell types with avariety of well-established transfection techniques. Targeted chameleons can also be packaged intorecombinant adenoviruses (Adeasy system) to allow <strong>for</strong> infection of primary cultured adult cardiac myocytes.This system has already been used in our laboratory to create a recombinant aden0 viral plasmid containing<strong>the</strong> SERCA2A gene and GFP <strong>for</strong> successful infection of adult cardiac myocytes. Protein-based sensors aredirected to <strong>the</strong>ir intended cellular location by 'tar~eting seque.nces' that are encoded within <strong>the</strong> sequence of aprotein. For nuclear localization <strong>the</strong> targeting sequence can be placed within a·protein or at <strong>the</strong> N or Cterminus and directs protein translocation to <strong>the</strong> nucleus of <strong>the</strong> cell, Similarly, <strong>for</strong> mitochondrial targeting aleader sequence directs <strong>the</strong> translocation of <strong>the</strong> protein to <strong>the</strong> mitochondria. ill <strong>the</strong> case of <strong>the</strong> endoplasmicreticulum (ER) <strong>the</strong> actions of a signal peptide and ER retention sequence toge<strong>the</strong>r act to direct and <strong>the</strong>nretain a protein within <strong>the</strong> ER. Proteins lacking a targeting sequence are retained within <strong>the</strong> cytoplasm and,thus, 'targeted' to <strong>the</strong> cytoplasm. Our present work is characterising existing protein construct Ca 2 +-sensors<strong>for</strong> <strong>the</strong> ER and cytosol, and developing alternative versions with modified Ca 2 + affinities and differentlocalisation sequences, particularly <strong>for</strong> use in <strong>the</strong> mitochondria and at <strong>the</strong> plasma membrane.Bowser, D.. Minamikawa, T., Nagley, P & Williams, D. (1998) Biophysical Journal. 75: 2004-2014.TISSUE RECOMBINANT TECHNOLOGYGail P. RisbridgerMonash Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road,Clayton, Victoria, 3168.The technique of constructing tissue recombinants was pioneered by Kratochwil in <strong>the</strong> 1970's. Heused TFM mice to demonstrate that regression of mouse mammary glands during embryogenesis, isan androgen regulated process. Since <strong>the</strong>n, a number of studies in <strong>the</strong> mammary gland, prostate orvagina have used tissue recombinants to reveal. The nature of <strong>the</strong> cell - cell interactions between <strong>the</strong>epi<strong>the</strong>lium and mesenchyme and <strong>the</strong> requirement <strong>for</strong> receptors on cell type. The purpose of thisworkshop is to describe <strong>the</strong> technique of constructing tissue recombinants using <strong>the</strong> prostate as amodel system. The development of <strong>the</strong> prostate is under androgenic control, but this talk will focuson <strong>the</strong> use of tissue recombinants to demonstrate a requirement <strong>for</strong> estrogens. Using <strong>the</strong> a estrogenreceptor knock-out (a ERKO) mouse model, tissue recombinations demonstrated that <strong>the</strong> presenceof ER a in <strong>the</strong> stroma and <strong>the</strong> epi<strong>the</strong>lium is essential to obtain a full response to estrogeniccompounds such as diethylstilbestrol. The preparation and use of tissue recombinants is a uniqueand powerful method of dissecting <strong>the</strong> interplay between <strong>the</strong> stroma and epi<strong>the</strong>lium in mediatinghormonal responses by <strong>the</strong>se organs. As <strong>the</strong> development of new mouse models of targeteddisruption of steroid receptors and steroidogenic enzymes became available, we can define <strong>the</strong>cellular mechanisms of all types of hormone responses.3637
MICROARRAYS: MICRO OR MACRO POTENTIAL?The Effect of Cyclic-Nucleotides on Fluid Reabsorptionin <strong>the</strong> Ductuli Efferentes Testis of <strong>the</strong> Rat.Suet Y. Man, John Clulow and Russell C. JonesDepartment of Biological Sciences, University of Newcastle, NSW2308, Australia.Michael J McKayPeter MacCallum Cancer Institute, St Andrews Place, Melbourne, AustraliaThe development of DNA microarray technologies over <strong>the</strong> last few years has heralded a potential<strong>for</strong> providing new, sharp, tools <strong>for</strong> application to many questions in biology and medicine. At thisrelatively early stage, however, it is unclear as to what <strong>the</strong> true impact of microarrays, or DNAChips, will be. Various types of microarrays have been developed, including normal tissue- andtumour- derived arrays (generated from pathological specimens) and proteomic approaches. Thispresentation will focus on mRNA-based systems, with a particular emphasis on RNA expressionprofiling and some of <strong>the</strong> strategies and common pitfalls which might be considered by thoseplanning microarray experiments. O<strong>the</strong>r applications of RNA microarrays will be brieflyconsidered.Two main RNA microarray strategies are now in relatively common use. These are oligonucleotidearrays, typically based on photolithographic deposition of DNA on silicon, and arrayed cDNAs,derived from known genes or expressed sequence tags (ETSs). eDNA arrays can comprise completeor partial cDNA fragments.Some work towards developing mRNA-based microarrays in Australia will be discussed, illustratedby specific research questions, mainly in <strong>the</strong> field of cancer, to which microarray analysis is beingapplied.IntroductionRecent work has shown that <strong>the</strong> ductuli efferentesare essential <strong>for</strong> male fertility (1). We have shownthat <strong>the</strong> ductuli reabsorb 96% of <strong>the</strong> fluid leaving<strong>the</strong> testis and that <strong>the</strong> reabsorption is dependent onfluid flow rate and Na+ concentration of <strong>the</strong>perfusate(2,3). Our previous studies havedemonstrated that luminally adminstered cAMPsuppresses fluid reabsorption in <strong>the</strong> ductuliefferentis (4), indicating that <strong>the</strong> cAMP signaltransduction pathway is involved in controllingfluid reabsorption in <strong>the</strong> ductuli of <strong>the</strong> rat. In <strong>the</strong>homologous proximal kidney tubule, cGMP hasalso been reported to reduce tubular reabsorption(5). The current study was undertaken to determinewhe<strong>the</strong>r cGMP has an effect on <strong>the</strong> fluidreabsorption in <strong>the</strong> ductuli efferentes.Materials and MethodsMicroperfusion experiments were per<strong>for</strong>med onanaes<strong>the</strong>tized male Wister rats. A length ofefferent duct was isolated and perfused withKrebs-ringer bicarbonate solution containingdifferent concentrations of dibutyryl-cGMP at arate of 0.1 JlJ.min- 1 • The reabsorption rate wascalculated from <strong>the</strong> volume difference between <strong>the</strong>perfusate and collectate divided by <strong>the</strong> length of<strong>the</strong> perfused duct. Electrolyte composition in <strong>the</strong>collectate was determined by energy-dispersivespectroscopy.ResultsTable 1 shows that <strong>the</strong> fluid reabsorption in <strong>the</strong>ductuli efferentes is not significiantly changed by0.1-1.0mM dibutyryl-cGMP in <strong>the</strong> luminalperfusate. cGMP has no effect on reabsorption atconcentrations at which cAMP completelyabolishes reabsorption. Fu<strong>the</strong>rmore, <strong>the</strong>re is nosignificant difference in <strong>the</strong> concentrations of Na+,K+ and cr , <strong>the</strong> major luminal ions, in <strong>the</strong>collected fluid between <strong>the</strong> control and dibutyrylcGMP-treatedgroups (Table 2).DiscussionOur results strongly suggest that cGMP is not <strong>the</strong>second messenger mediating fluid regulation in <strong>the</strong>ductuli efferentes. By contrast, both cGMP andcAMP are reported to mediate fluid reabsorptionin <strong>the</strong> proximal renal tubules (6,7). The Na+/H+exchanger has been reported as one of <strong>the</strong>principal transporters responsible <strong>for</strong> fluid andelectrolyte reabsorption in <strong>the</strong> ductuli efferentes(8) and its function could be regulated by cAMP asin <strong>the</strong> proximal tubules.Table 1. Effect of Dibutyryl-cGMP and DibutyrylcAMPon Fluid Reabsorption in Perfused RatDuctuli Efferentis. Mean + S.. E M ., * P
Identification of proteins with seasonal patterns of secretion in ram seminal plasma.J. F. Smith, R. S-F. Lee, R. M. McDonald, G. R. Murray, J. Parr.AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand.INTRODUCTION: Ram semen collected and frozen in<strong>the</strong> non-breeding season is of lower fertility than semenfrom <strong>the</strong> same animals in <strong>the</strong> autumn. These changes in <strong>the</strong>fertility of frozen semen cannot be readily explained bychanges in sperm numbers alone, although associatedalterations in maintenance of sperm motility after freezinghave been reported (1). Differences in field fertility afterinsemination with frozen bull semen have been linked toconcentrations of specific seminal plasma proteins (2).This observation raises <strong>the</strong> possibility that certain seminalplasma proteins could influence <strong>the</strong> ability of sperm toretain <strong>the</strong>ir fertility after cryopreservation; thus, seasonalchanges in fertility of ram semen could be related toalterations in <strong>the</strong> composition of seminal plasma. Thepresent experiment was conducted to examine <strong>the</strong> seasonalvariations in protein content and composition of ramseminal plasma.METHODS: Ten rams of proven fertility were used in thisstudy. On one day each month two or three ejaculates werecollected by artificial vagina from each ram. Semensamples were kept on ice after collection and evaluated <strong>for</strong>volume and density, <strong>the</strong>n processed. Proteolytic inhibitors(benzamine and PMSF) were added to. a finalconcentration of 1 roM each and <strong>the</strong> seminal plasmaseparated by centrifugation (2x 10 min) at 1l,600g. Equalvolumes of seminal plasma from each ejaculate collectedon <strong>the</strong> same day <strong>for</strong> an individual ram were pooled,aliquoted and stored at -20°C until assay. Protein contentof <strong>the</strong> seminal plasma was determined using a modifiedLowry method (3). Seminal plasma proteins were analysedby electrophoresis in 12.5 % denaturing acrylamide gelsafter 5-fold dilution in 10 rnM Tris-Cl, pH 8.0, containing1 mM EDTA. After electrophoresis and staining withCoomassie-blue, <strong>the</strong> protein banding patterns were visuallyappraised. Samples from individual rams <strong>for</strong> each of <strong>the</strong> 12months were compared within <strong>the</strong> same gel and <strong>the</strong> proteinloading was normalised across all <strong>the</strong> lanes to correct <strong>for</strong>variation in protein concentration between <strong>the</strong> samples.Protein identification. Five proteins that showed seasonalvariation were selected <strong>for</strong> identification by peptidesequencing. Briefly, seminal plasma proteins (40 Ilg perlane) were electrophoresed in denaturing 12.5 %polyacrylamide gels (20 x 20 x 0.075 cm). After stainingwith Coomassie-blue and destaining, <strong>the</strong> bands of interestwere excised, and depending on <strong>the</strong> abundance of <strong>the</strong>protein, ei<strong>the</strong>r bands from 5 lanes or 10 lanes werecombined and <strong>the</strong> gel pieces washed extensively withwater, <strong>the</strong>n 50 roM Tris-HCI, pH 8.5, containing 1 roMEDTA. The gel pieces were pulverized and suspended in0.4 mI of <strong>the</strong> same buffer and Endoproteinase Lys C(sequencing grade, Promega), added to a finalconcentration of 0.1 mg/mI. The mixture was incubated at37°C overnight with shaking and <strong>the</strong> supernatant was <strong>the</strong>nseparated from <strong>the</strong> gel slurry and volume reduced to < 100III by lyophillization. After acidication with trifluoroaceticacid to a final concentration of 1 % (v/v), <strong>the</strong> peptideswere resolved by reverse-phase HPLC. Individual peptidepeaks eluting from <strong>the</strong> column were collected and selected40peak samples were subjected to ~no acid sequencingusing <strong>the</strong> Applied Biosystems PrOCIse Sequencer (ABI).RESULTS: A seasonal change in <strong>the</strong> total proteinconcentration was detected in seminal plasma, with higherlevels (36.9 vs 23.0 mg/mI; P
In vitro culture of brushtailed possum epididymal epi<strong>the</strong>lium and induction of epididymal spermmaturation in co-cultureMinjie Lin, Xiyi Zhang, Ray Murdoch and R. John AitkenMarsupial CRC, Dept of Biological Sciences, The University of Newcastle, NSW 2308, AustraliaIntroductionIn mammals, testicular sperm undergo a maturation processin <strong>the</strong> epididymis to acquire motility and <strong>the</strong> ability <strong>for</strong>fertilisation. However, sperm maturation in marsupials is amuch longer and more complex process than that of <strong>the</strong>ireu<strong>the</strong>rian counterparts (1). This process is poorlyunderstood, mainly because it cannot as yet be investigatedin vivo. Techniques of in vitro co-culture of sperm andepididymal cells have been developed in eu<strong>the</strong>rians during<strong>the</strong> past 2 decades, but not in marsupials species (2). Thispaper describes <strong>for</strong> <strong>the</strong> fITst time an in vitro cell culturesystem <strong>for</strong> a marsupial species, which supports epididymalepi<strong>the</strong>lial cells from <strong>the</strong> brushtailed possum, and evidence ofsperm maturation induced by <strong>the</strong> cultured epididymal cells.Materials & MethodsThe epididymis from 5 possums were used to establish <strong>the</strong>cell culture system with a medium modified from eu<strong>the</strong>riancultures (3,4), and maintained at 36.5-37°C in 5% C02 in airwith humidity at 100%. Immunofluorescence ofcytokeratins was used as a marker <strong>for</strong> identification ofepi<strong>the</strong>lial cells. Morphology of cultured cells was examinedby light and electron microscopy. Immature sperm obtainedfrom <strong>the</strong> proximal caput epididymis were co-cultured with7-day old cultured epi<strong>the</strong>lial cells <strong>for</strong> <strong>the</strong> in vitro maturationstudy. Development of sperm motility and changes of spermhead orientation were examined as indicators of spermmaturation in <strong>the</strong> co-culture preparations.Results and DiscussionAll cell cultures of caput, corpus and cauda epididymis<strong>for</strong>med confluent epi<strong>the</strong>lial monolayers with densely packedpolygonal cells. The cells remained viable <strong>for</strong> more than 2months in culture, and displayed typical epi<strong>the</strong>lial characterspeculiar to each segment of <strong>the</strong> epididymis. EMobservations confIrmed that after 12 days in culture, <strong>the</strong>cells retained many structural features characteristic ofepi<strong>the</strong>lial cells in <strong>the</strong> intact epididymis. These featuresincluded very slender and branched stereocilia andmicrovilli located on <strong>the</strong> apical surface of <strong>the</strong> cells, welldevelopedrough endocytoplasmic reticulum network,bundles of 10-nm filaments, Golgi apparatus, mitochondria,numerous secretory and pinocytosis vesicles coalescing withlysosomes, desmosome and tight junctions holdingneighbours cells to <strong>for</strong>m <strong>the</strong> monolayers. This structuralintegrity suggested that <strong>the</strong> cultured epi<strong>the</strong>lial cellsmaintained <strong>the</strong> secretory and resorptive functions of <strong>the</strong>epididymal principal.cells in intact organs.The maintenance of functional integrity of <strong>the</strong> cultured cellswas also evidenced by <strong>the</strong> in vitro maturation study. After 2days in coculture with caput, corpus and cauda epididymalcell monolayers, a proportion of sperm (significantly higherthan that in control incubations) exhibited changes of headorientation from immature T shape to <strong>the</strong> maturestreamlined shape accompanied by <strong>the</strong> development of a fastflagellum beat with slow or medial progressive motility (Fig1 & Table 1).ConclusionsIt is envisaged that <strong>the</strong> present sperm co-culture system <strong>for</strong>marsupial species will greatly increase our understanding ofsperm-epididymal cell interactions in <strong>the</strong>se species. It isanticipated that this system will also facilitate <strong>the</strong>identification of specific epi<strong>the</strong>lial factors, which may beexploited <strong>for</strong> <strong>the</strong> manipulation of male fertility.References(1) Lin M, RodgerJC (1999) J Anat 194: 223-232(2) Moore HPM, Pacey AA (1997) J Reprod Fert Sup 19: s3(3) Moore et aI. (1992) Fert Steril58: 776-783(4) Carballada R, Saling PM (1997) J Reprod Fert 110: 171Fig. 1. The population of streamlined-shape spermatozoa was higher(proximately 5-8 times) in Day 4 coculture of epididymal cells than thatofsmall intestine cells. * p~ 0.05; **, p~ 0.001.~ 252.s 20III~!. 15•" CP.5 10eI 5U)30..---------------~**_ OaY2/*EliEI Oay-41*IIISmallIntealMIIcaput Corpus caudaEpldlclymla Epldldymla EplcllclymlsCell Types In Day 2 and Day 4 Co-culturesTable 1: Motility development in coculture preparations.Cultured c:ell types Coc:ulturetime Sperm motility c:haracter(days)1 Twitching and slow tail beatingCaput epididymis 3 Fast tail beating with progression
The effect of seminal plasma on <strong>the</strong> capacitation status offresh and frozen-thawed ramspermatozoa.C.A. McPhie, G. Evans and W.M.C. MaxwellDepartment ofAnimal Science, University of Sydney, NSW 2006 AustraliaSecretory proteins from <strong>the</strong> female reproductive tract of <strong>the</strong> brushtail possum (Trichosurus vulpecula):Binding to sperm and effects on <strong>the</strong>ir survival in vitroSidhu, K.S., Mate, K.E., Molinia*, F.e., Glazier*, A.M. and Rodger, J.e.Co-operative Research Centre <strong>for</strong> Conservation and Management of Marsupials, Macquarie University, NSW2109 Australia *Landcare Research, PO Box 69, Lincoln 8152 New ZealandIntroductionCryopreservation causes capacitation-like changes in ram spermatozoa (1) perhaps because seminalplasma, which is believed to contain decapacitation factors, is removed during preparation ofspermatozoa <strong>for</strong> freezing. In this experiment, fresh and frozen-thawed ram spermatozoa weresupplemented with seminal plasma in an attempt to limit capacitation-like membrane changes.Materials and MethodsSeminal plasma was prepared from semen collected from three mature rams (A, B and C) bycentrifugation (12000g, 15 minutes) followed by filtration (0.2).!; Milex). Pooled semen from <strong>the</strong>same three rams, fresh or frozen-thawed (as described by Evans and Maxwell; 2), was centrifuged '(600g, 15 min) through a Percoll gradient (90:45%, v/v) and <strong>the</strong>n washed (600g, 6 min) in HSOF.The washed spermatozoa were resuspended in Dulbecco's phosphate-buffered saline (DPBS). TheDPBS contained ei<strong>the</strong>r no seminal plasma (control; Con), 20% seminal plasma from rams A, B, orC orpooled from all three rams (P). Spermatozoa were assessed immediately and after 6 hrincubation (37 0 C) <strong>for</strong> motility (light microscopy) and capacitation status (CTC fluorescence assay;3). Data <strong>for</strong> motility and proportion of F (uncapacitated), B (capacitated) and AR (acrosomereacted) pattern spermatozoa were analysed by analysis ofvariance and <strong>the</strong> results are presented inFigure 1.Figure 1. Motility and membrane status of spermatozoa (means ± s.e.m. pooled <strong>for</strong> fresh and frozensemen and incubation period) exposed to no seminal plasma (Con: control), seminal plasma fromrams A, B or C, or pooled from rams A, Band C (P).=QN 080.-= e 60 0Fa...~c..l;I.)..... 400~eIJ= 20ClJtJ• BII AR.- c:~ Motility....0~Q.lCon A B C PSource of Seminal PlasmaResults and DiscussionMotility and percentage ofF-pattern cells was higher <strong>for</strong> fresh than frozen-thawed semen (85 and56 vs 42 and 13%, respectively; p < 0.001). Motility was always better in <strong>the</strong> presence of seminalplasma than in its absence and was highest <strong>for</strong> P and ram C (p< 0.001). There were more F- andless AR-pattern cells in <strong>the</strong> presence of seminal plasma from P and ram C than <strong>for</strong> <strong>the</strong> o<strong>the</strong>rtreatments or <strong>the</strong> control (p
mRNA editing of OTR: A new mechanism <strong>for</strong> regulationIC.Y. Feng, 2M. Bhave, 2R.J. FaircloughlCenter For Bioprocessing and Food Technology, 2School of Life Sciences and Technology, Victoria UniversityofTechnology, PO Box 14428, MCMC Melbourne 8001, Victoria, AustraliaINTRODUCTION The increase of uterine oxytocin receptor (OTR) concentrations over <strong>the</strong> late luteal phase of <strong>the</strong>oestrous cycle is thought to play an important role in regulating <strong>the</strong> time of luteolysis (1). In ruminants, <strong>the</strong>development of OTRs in <strong>the</strong> luminal epi<strong>the</strong>lium is <strong>the</strong> key point to achieve a· maximal prostaglandin F2ex. (PGF2ex.)response to an oxytocin challenge (2). The regulation of this OTR population is different from that in <strong>the</strong> rest ofendometrium such as caruncular stroma and deep glands. A large proportion of <strong>the</strong> endometrial OTR population incaruncular stroma and deep glands does not <strong>for</strong>m until luteolysis is complete and <strong>the</strong> <strong>for</strong>mation and activity of <strong>the</strong>sereceptors is probably oestrogen dependent. However, <strong>the</strong> basis <strong>for</strong> this difference in regulatory mechanisms remainsunclear. Our aim was to study <strong>the</strong> level of OTR mRNA expression in order to have better understanding of <strong>the</strong>mechanisms underlying uterine OTR regulation.METHODS mRNA samples were used from sheep uterine endometrium at day 15 of oestrous cycle. Two 3'RACE(Rapid Amplification of eDNA Ends) (Life Technologies and Clontech) were per<strong>for</strong>med to amplify <strong>the</strong> 3'end of OTReDNA.RESULTS Two bands were generated by both 3'RACE systems, a major band of size 600 bp and a minor band of size1300 bp. These products were cloned <strong>for</strong> fur<strong>the</strong>r analysis. Three types of <strong>the</strong> clones from 600 bp products (designatedas 600A, 600B and 600C) were identified. Sequence analysis indicated eleven potential polymorphic sites in <strong>the</strong>irsequences, one in coding region and <strong>the</strong> o<strong>the</strong>rs in 3'untranslated region. These consisted of TIC conversions (positions1128, 1193, 1206, 1208 and 1465); AlG conversions (positions 1183, 1185, 1218, 1468); a T/A conversion at position1252 and an AC insertion/deletion at position 1259. The TIC conversion at position 1128 (600B) in <strong>the</strong> codingsequence which did not cause an amino acid change. One positive clone from 1300 bp of RACE products was obtained(designated as l300D). In comparison to <strong>the</strong> sequences of 600A, 600B and 600C, 1300D showed <strong>the</strong> presence of ACG995 (Thr-331) and ACC-1I03 (Thr-368) codons. Instead, ATG-995 (Met-331) and ATC-1103 (Ile-368) codons werefound in <strong>the</strong> corresponding positions in <strong>the</strong> clones 600A, 600B and 600C. As OTR gene is a single-copy gene perhaploid genome, well conserved among vertebrate species (4), <strong>the</strong>se results suggested <strong>the</strong> post-transcriptionalmodification existed at <strong>the</strong> positions, probably through mRNA editing.CONCLUSIONS Of particular interest is <strong>the</strong> presence of two amino acid changes, from Met to Thr and lIe to Thr incarboxyl terminus. Serine and threonine existing in carboxyl terminus are well known to be potential phosphorylationsites (2) which has been identified as an important mechanism <strong>for</strong> <strong>the</strong> regulation of G-protein coupling of somereceptors. It is suggested that editing occurring in OTR mRNAs in carboxyl terminus may effect G-protein coupling.Alt~ough fur<strong>the</strong>r works nee~ to be done by searching whe<strong>the</strong>r mRNA editing occurred in <strong>the</strong> rest of <strong>the</strong> coding regionat different stage or from different type of cells, we have demonstrated that <strong>the</strong> difference of OTR populations inendometrium is due to mRNA editing and <strong>the</strong> existence of oxytocin receptor subtypes, resulting from mRNA editing.It is also suggested that <strong>the</strong> regulation can be achieved by RNA editing to activate G-protein coupling. Thesignificance of mRNA editing in untranslated region remains unclear. As gene expression can be regulatedundergoing alternative splicing of pre-mRNAs and alternative polyadenylation site choice, mRNA editing inuntranslated region might be an o<strong>the</strong>r post transcriptional modification to coordinate.REFERENCES1. Flin~ ~PF, Lamming GE, Stew~ HJ and Abayasekara DRE 1994. The role of <strong>the</strong> endometrial oxytocin receptor indetermmmg <strong>the</strong> length of <strong>the</strong> stenle oestrous cycle and ensuring maintenance of luteal function in early pregnancy inruminants. Philosophical Transactions of<strong>the</strong> Royal <strong>Society</strong> (London) Series B 344 291-304.2. Wa<strong>the</strong>s DC and Lamming GE 1995. The oxytocin receptor, luteolysis and <strong>the</strong> maintenance of pregnancy. J. Reprod.Fertil., Suppl. 4953-67.3. Hargrave PA, Mcdowell JR, Siemiatkowski JEC, Kuhn H, Wang JK, Curtis DR, Mohana RJK, Argos P andFeldmann RJ 1982. The carboxyl-terminal one-third of bovine rhodopsin: Its structure and function. Vision Res. 221429-1438.4. Bathgate R, Rust W, Balvers M, Hartung S, Morley Sand Ivell R 1995. Structure and expression of <strong>the</strong> bovineoxtytocin receptor gene. DNA and Cell <strong>Biology</strong> 14 1037-1048.Progesterone Inhibits Activation of Latent Matrix Metalloproteinase (Ml\1P)-2 in Human Endometriumby Regulating Expression of Membrane Type I-Ml\1P.Jin Zhang, Anne L. Hampton, Guiying Nie and Lois A. SalamonsenPrince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168INTRODUCTIONMatrix metalloproteinases (MMPs) playa criticalrole in endometrial remodelling and menstruation(1). They secreted as latent enzymes requiringactivation. MMP-2 is unusual as it is not activatedby <strong>the</strong> proteases known to activate o<strong>the</strong>r MMPs:recently a family of membrane-associated (MT)MMPs have been described that activate proMMP2 in tumour cells. This study examined <strong>the</strong>localization ofMT1-MMP and MMP-2 in humanendometrium across <strong>the</strong> cycle and <strong>the</strong> control of itsactivation in cultured endometrial stromal cells.MATERIALS AND METHODSEndometrial tissue was from women with regularmenstrual cycles and no apparent endometrialdysfunction. Immunohistochemistry used antiseraagainst MMP-2 and MT1-MMP (Oncogene).Endometrial stromal cells (ESC) were preparedand cultured in serum free conditions with addedestradiol 17~(E, 10- 8 M) and without or withprogesterone (P, 10- 7 M) (2). Gelatin zymographydetected latent and active MMP-2 in medium.Nor<strong>the</strong>rn analysis of cellular mRNA used an MT1MMP probe from by Dr M Seiki (Tokyo).RESULTSImmunohistochemistry. MMP-2 wasimmunolocalized across <strong>the</strong> entire menstrual cycle,in 25/32 endometrial samples, but with greatestintensity in degrading menstrual tissue. It wasseen variously in <strong>the</strong> different cellularcompartments (luminal epi<strong>the</strong>lium (4/22 samples)glands (18/32), stroma (24/32) endo<strong>the</strong>lium(20/32), vascular smooth muscle (16/32), alldecidual cells (9 samples) and in leukocytes(menstrual phase). MT1-MMP was most stronglystained in leukocytes, predominently during <strong>the</strong>menstrual phase but also in mid-proliferative andmid-secretory tissue, concomitant with tissueoedema. Epi<strong>the</strong>lial staining was seen in glandsduring <strong>the</strong> mid-late secretory phase and decidualcells were also stained. The leukocytes expressingMT-MMP were subsets of monocytelmacrophages,neutrophils and granular lymphocytes but noteosinophils or mast cells and staining was around<strong>the</strong> periphery of <strong>the</strong> cells, indicative of its locationin <strong>the</strong> cell membrane.Cell culture studies. Latent MMP-2 was releasedinto culture medium by all ESC. Active MMP-2was also detectable and was significantlydecreased by P treatment (Fig. 1). There was nodifference between <strong>the</strong> amount oflatent MMP-2 in<strong>the</strong> two sets of cultures. Addition of a specificinhibitor ofMMPs (Ro 31-4724) to E-treated cellsdemonstrated that activation was due to an MMP,not to ano<strong>the</strong>r class of protease.Immunostaining <strong>for</strong> MT1-MMP in <strong>the</strong> cells was reducedfollowing P treatment and became apparent in <strong>the</strong> cytoplasmra<strong>the</strong>r than being cell membrane associated. Nor<strong>the</strong>rnanalysis of<strong>the</strong> cells revealed a decrease in mRNA <strong>for</strong> MTMMP following P-treatment (Fig. 2).proMMP-2MMP-21:Q)ENm_150c..: 100~I~WQ)-:.~g~ 8 50'0C------_EE+PI ...........*EFig.i. Zymogram and densitometric analysis showingMMP-2 activation, in ESC without (E) and with P (E+P).I E E+P 1I E E+P 11 2, --=-:...----.. Mr x1 03Fig.2. Nor<strong>the</strong>rn analysis ofMTi-MMP mRNA in ESCcultured without (E) or with P (E+P)*E+P-3.9kbGAPDHCONCLUSIONS• MMP-2 is expressed without cyclical variation and byall cell types in human endometrium.• MT1-MMP is also expressed in this tissue,predominently in specific subsets of leukocytes but alsoin decidual cells.• Activation of latent MMP-2 occurs in cultures ofendometrial stromal cells and is significantly reduced in<strong>the</strong> presence ofP.• This activation is inhibited by a specific MMPinhibitor.• MT1-MMP mRNA expression in <strong>the</strong>se cells is alsoinhibited by P.We postulate that MTI-MMP activates proMMP-2 inhuman endometrium, this activity being increased late in <strong>the</strong>cycle when P levels fall,. thus contributing to <strong>the</strong> tissuebreakdown at menstruation.References.1. Salamonsen 1998 Trends Endocrino!. Metab. 9(8) 305-309.2. Salamonsen et al., 1997 J Clin Endo. Metab. 82:1409-1415.Supported by NH&MRC.46 477262
A Model <strong>for</strong> <strong>the</strong> Study of Uterine Function in <strong>the</strong> BitchIntroductionIn human endometrium, matrix metalloproteinses(MMPs), enzymes involved in tissue degradationincrease dramatically prior to menstruation. MMPs areproduced by resident and migratory cells in <strong>the</strong> tissueand many factors contribute to <strong>the</strong>ir production andactivation. These include progesterone withdrawal andproducts of leukocytes (1). However, <strong>the</strong> precisesequence of events leading to menstruation is notknown. As it is not possible to manipulate menstruationin women, an animal model is required. In a publishedmouse model <strong>for</strong> menstruation (2,3) leukocyteinfiltration preceded tissue degradation suggesting thatthis model closely resembles human menstruation.AimThe aim of this study was to establish <strong>the</strong> mouse modelof menstruation in our laboratory and examine whe<strong>the</strong>rMMPs are involved in <strong>the</strong> tissue degradation.Materials and MethodsThe model was established in two stages:1. Induction of decidualisation, 2. Withdrawal ofprogesterone. Eight week old Swiss out-bred mice wereovariectomised. One week later <strong>the</strong>y were subjected tohormonal treatment (estradiol 17 -~ (E) andprogesterone (P» and subsequently to a decidualstimulus (DS; 20 III of oil injected directly into <strong>the</strong>uterine lumen) (Fig.!).day48Leukocytes and matrix metalloproteinases are present at sites of tissuebreakdown in a mouse model <strong>for</strong> menstruationNakisa Malakooti and Lois A. SalamonsenPrince Henry's Institute of Medical Research and Education Program in <strong>Reproductive</strong> <strong>Biology</strong>,Monash University, Clayton, Victoria 3168, AustraliaFig. 1. Schematic representation of <strong>the</strong>treatment protocol administered toovariectomized miceIn control animals, daily P injections were continueduntil autopsy. To see whe<strong>the</strong>r P withdrawal resulted intissue breakdown, 4 groups of animals were subjectedto hormonal treatment and <strong>the</strong> tissue was collected at52, 56 and 59 hours after <strong>the</strong> last P injection (Pwithdrawal) vs control animals. After autopsy uteriwere weighed, fixed and processed <strong>for</strong> histology (H&Estain) and immunohistochemistry (IHC) <strong>for</strong> desmin (amarker <strong>for</strong> decidualisation), leukocytes (leukocytecommon antigen, LCA) and MMP-7 & MMP-9.Morphometric analysis was used to determine numbersof leukocytes staining per 1000 stromal cells.ResultsPositive staining <strong>for</strong> desmin confirmed decidualisationand examination of H&E sections identified areas oftissue breakdown (Table 1).Table 1. Number of animals with decidualised andbreaking down tIssue.Group (n) Decidualisation BreakdownControl (8) 7/8 0/852 hrs. (4) 2/4 2/456 hrs. (6) 4/6 2/659 hrs. (9) 8/9 6/9Morphometric analysis of LCA showed <strong>the</strong> number ofleukocytes at a site of tissue breakdown is increasedcompared with a site where no breakdown is occurring(142 vs 93 cells/1oo0 stromal cells).MMP-7 and MMP-9 were irnmunolocalized withinmany leukocytes. Extracellular MMP-9 was apparent atsites of tissue breakdown, suggesting activation ofleukocytes and release ofMMP-9.ConclusionsA model <strong>for</strong> menstruation in mice has been established.Leukocytes, MMP-7 & MMP-9 are present at <strong>the</strong> siteof tissue breakdown.References1. Salamonsen, L.A. and Woolley, D.E. (1996) ReprodMed Rev 5:185-203.2. Finn, C.A. and Pope, M. (1985) J Endocrinol 1:20519.3. Finn, C.A. (1984) J EndocrinoI100:295-3oo,Po-yin Chu, P.I. Wright and C.S. LeeDepartment of Veterinary Science, The University of Melbourne,Werribee, Victoria 3030, Au·straliaEmail: p.chu2@pgrad.unimelb.edu.auINTRODUCTIONThe aim of this study was to establish amodel, in <strong>the</strong> ovariectomised bitch, thatsimulated <strong>the</strong> normal oestrous cycle. Thismodel will be used to study uterine functionwith special reference to endometrialexfoliation and cystic endometrial hyperplasia(CEH). CEH is a response of <strong>the</strong> uterus totrauma during dioestrus. Such trauma can beinduced by a silk suture placed in <strong>the</strong> lumenof <strong>the</strong> uterus (4).MATERIALS & METHODSMature bitches ( n=18 ) were ovariectomised andtreated with: oestradiol benzoate ( ODB, 0.6 - 4.8jlglkg, i.m., bj.d.) <strong>for</strong> 7 days (pro-oestrous group(n=2)); ODB <strong>for</strong> 11-12 days <strong>the</strong>n progestagen (megestrol acetate ( MA, 2 mglkg, p.o., sj.d.) <strong>for</strong>4 days (oestrous group (n=2)); ODB <strong>the</strong>n MA<strong>for</strong> 35-37 days (dioestrous group (n=2))(l,2).These bitches were autopsied at <strong>the</strong> end of <strong>the</strong>treatment period. Additional bitches receivedODB <strong>the</strong>n MA and were autopsied 3 weeks (mid-anoestrous group (n=2)) and 9 weeks ( lateanoestrousgroup (n=2)) after treatment.Untreated control bitches (n=1 per group) wereautopsied at <strong>the</strong> same time as treated animals.In a fur<strong>the</strong>r 3 bitches at ovariectomy a suturewas placed in <strong>the</strong> lumen of <strong>the</strong> uterus. Two of<strong>the</strong>se bitches were treated with ODB (11 days)<strong>the</strong>n MA (36-38 days) and were autopsied at <strong>the</strong>end of treatment. One bitch with suture butnot treated with hormones served as control.During all treatments observations were madeof vulval size, vulval discharge, vaginalcytology and oestrous behaviour, and bloodsamples were taken to determine plasmaconcentrations of oestradiol. At autopsy uterinetissues were taken <strong>for</strong> histology.RESULTSThe treatments induced oestrous behaviour,vulvar swelling, vulval discharge, vaginalsmears, plasma oestradiol concentrations anduterine histology similar to that observed inovary-intact bitches at each stage of <strong>the</strong>oestrous cycle ( Figure 1,2 )(3). Markedendometrial exfoliation was observed at 3weeks after treatment. The endometrium hadrepaired by 9 weeks after treatment. Inhormone-treated bitches <strong>the</strong> silk suture resultedin CEH in one bitch and CEH with pyometrain <strong>the</strong> o<strong>the</strong>r bitch. The silk suture inducedCEH was similar to that observed in studiesof ovary-intact bitches (4). The control bitchshowed no CEH or pyometra.,..."_iO-~~~~~~~~Q-~~~~~~~O----....---.... C'lMa Oestradiol................. SCI.!"2 ."....... 40 -r------~~~-..... 100_- 30 80~j• Vulvar swelling................ Haemorrhagic vulval discharge·_··oe_... A ce m ti100% r~iNii-==~=;:;;ii;;;ii180%60%40%20%O%-H...........~......'P""P'.....................= 60 .......S 20 400~ 10 "0 v.l-.= ..!5 OoQ«;~~P-r-.,..,..,..,.'TT..,.;:JitIr~iW1-J-O'" u O-C'l~~~~~~-N~~~~~O.... ~~~~;................NColDayDayFigure 1 Vulvar swelling, haemorrhagicvulval discharge and acceptance ofmating in ovariectomised bitches(n=10-6) treated with ODB (day I·11/12) and MA (day 12/13-20)Figure 2 Plasma oestradiol concentrations andsuperficial cell index (SCI) inovariectomised bitches (n=10-6) treatedwith ODB (day 1-11/12) and MA(day 12/13-20)CONCLUSIONWe have established in ovariectomised bitchesa model simulating <strong>the</strong> normal oestrous cycleto facilitate studies of uterine function. Thismodel will be used to study fur<strong>the</strong>r <strong>the</strong>pathogenesis of endometrial exfoliation andCEH.REFERENCES1) Concannon PW, et aI., (1975) <strong>Biology</strong> ofReproduction 13: 112-12l.2) Concannon PW, et al., (1979) <strong>Biology</strong> ofReproduction 20: 799-809.3) Watts JR(1998) PhD Thesis,Melboume Uni.4) Nomura K (1995) J. Vet. Med. Sci. 57(1):9-16.49
Mouse splicing factor SC35 mRNA is up-regulated at implantation sites in <strong>the</strong> mouseuterus during early pregnancyGuiying Nie, Ying Li, Hiroyuki Minoura, Jock Findlay & Lois SalamonsenPrince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168DIFFERENTIAL EXPRESSION OF INHmIN AND ACTIVIN SUBUNITS IN NON-PREGNANT AND. PREGNANT HUMAN ENDOMETRIUMRebecca L. Jones, Lois A. Salamonsen and Jock K. FindlayPrince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, AustraliaINTRODUCTIONThe process of embryo implantation is <strong>the</strong> mostrelevant factor limiting successful pregnancy.During this process, <strong>the</strong> uterus undergoesmorphological and physiological changes totrans<strong>for</strong>m itself from a non-receptive to a receptivestate to accommodate a conceptus. The exactmolecular mechanisms underling <strong>the</strong> uterinedifferentiation critical <strong>for</strong> embryo implantation is notwell understood. The aims of our studies were toidentify and characterise genes specificallyregulated in <strong>the</strong> mouse uterus in association wi<strong>the</strong>mbryo implantation.MATERIALS AND METHODSRNA differential display (DDPCR) was applied toidentify genes specifically regulated during <strong>the</strong>window of implantation in <strong>the</strong> mouse (1). InitiallyGenBank was screened to obtain <strong>the</strong> identity of<strong>the</strong> DDPCR-derived eDNA fragments. In <strong>the</strong>~bsence of a homologous sequence, a cDNAlibrary of mouse uterus was screened to obtain <strong>the</strong>full cDNA sequence. Nor<strong>the</strong>rn analysis was usedto ~etermine <strong>the</strong> uterine mRNA expression patterndunng early pregnancy. Pseudopregnant micewere compared with normal pregnant animals toexamine <strong>the</strong> effect of embryos on <strong>the</strong> expressionof uterine genes in relation to pregnancy.RESULTSSeveral genes were found to be ei<strong>the</strong>r up- ordown-regulated at implantation sites during earlypregnancy in <strong>the</strong> mouse. One of <strong>the</strong>m wasidentif!ed as <strong>the</strong> splicing factor SC35. Expression?f this .gene. was increased dramatically atI~plantatl.on sites, but not at interimplantationsites, dunng early pregnancy in <strong>the</strong> mouse uterus(Fig. 1). The up-regulation of splicing factor SC35in <strong>the</strong> uterus during early pregnancy was not seenin pseudopregnant animals (Fig.2).CONCLUSIONSOne of <strong>the</strong> genes identified by DOPCR as~pecifical~y related to <strong>the</strong> early phases of embryoImplantation was confirmed to be <strong>the</strong> SC35splicing factor. This gene was up-regulated atimplantation sites and its expression wasdependent on <strong>the</strong> presence of embryos in <strong>the</strong>uterus. The unique expression pattern of this gene~uggests an i~portant role <strong>for</strong> SC35 splicing factorIn pregnancy In <strong>the</strong> mouse.a.zd4.5 d5.5r----ll10 "- ...0- Q) 0-S.5 - .5ewi~ .E .Epregd4.5I pseudo~p10 "-a. Q) 10 10ewi.5 - C":i ~~ E: ~ ~+-3.0 kb+-1.8 kb+GAPDHFigure 1. Nor<strong>the</strong>rn blot analysis of mRNA detected by a642 bp eDNA probe of mouse SC35 splicing factor(1066-1708 bp). Total RNA (15 jJg) was isolated fromwhole uterus of non-pregnant mice (NP) and 3.5 daypregnant (d3.5) mice, from implantation sites (Imp) andinterimplantation sites (Inter) of day 4.5 (d4.5) and 5.5(d5.5) pregnant mice; <strong>the</strong> GAPDH probe was used toexamine <strong>the</strong> equal loading.+-3.0 kb+-1.8 kb+GAPDHFigure 2. Nor<strong>the</strong>rn blot analysis of total RNA (15 pg)isolated from whole uterus of non-pregnant (NP) andd~y (d) 3.5 pr~gnant (preg) mice, from implantationsItes (Imp) and mterimplantation sites {Inter) of day 4.5pregnant mice and from whole uterus of day 3.5 and4.5 pseudopregnant (pseudo preg) mice. The loadingwas checked by GAPDH probe.(1) Nie et al. (1997). Proceedings of ASRB, P54.*Supported by <strong>the</strong> Rockefeller Foundation,WHO, Wellcome and NH&MRCIntroductionActivins and inhibins are members of <strong>the</strong> TGF-~superfamily and have diverse functions in cellularproliferation, differentiation and apoptosis. Inhibins areheterodimeric proteins, composed of an a and ~subunit, whilst activins· comprise two ~ subunits.Multiple ~ subunits have been identified, including <strong>the</strong>~A and ~B subunits. The type of mature dimer <strong>for</strong>medis dependant on availability of a subunits.Inhibins / activins have been widely implicated asparacrine agents in <strong>the</strong> ovary, and have importantimmunoregulatory functions. The placenta is a richsource of inhibin and activin during pregnancy, with<strong>the</strong> potential <strong>for</strong> roles in regulating fetal-maternalinteractions. Whilst <strong>the</strong> expression of individualsubunits has been described in endometrium, somecontroversy exists (1, 2, 3). A definitive description ofdifferential subunit expression is necessary to clarifypotential functions.MethodsImmunohistochemistry was conducted to localiseinhibin I activin subunits in endometrium (Table 1),using antibodies raised against <strong>the</strong> a, ~A and ~Bsubunits (kind gift from Salk Institute).Semi-quantitative analysis of staining distribution andintensity was conducted. Control tissues included wererat ovary, third trimester decidua and placenta.Table 1 Biopsy detailsStae:e of cycle DavRanl!e NumberMenstrual (M) dl-4 6Proliferative (P) d5-13 5Earlv SecretorY (ES) d14-18 5Mid Secretory (MS) d19-23 5Late Secretory (LS) d24-28 8Decidua (DE) 8-10 weeks 5ResultsNon-pregnant endometriumVariable expression of a subunit was detected inglandular epi<strong>the</strong>lium, although no cyclical pattern wasdetermined. ~A and ~B subunits were predominantlyexpr~ssed by glandular and surface epi<strong>the</strong>lium, withmaxImal immunostaining in <strong>the</strong> late secretory andm~nstrual phases (Figure 1). Additionally, <strong>the</strong>re waseVIdence of immunoreactivity <strong>for</strong> all subunits insubpopulations of leukocytes and in <strong>the</strong> vasculature ofsome samples.Figure 1 Immunostaining <strong>for</strong> f3B subunit in glandularepi<strong>the</strong>lium in human endometrium!:::IiiEi---l1M P ES MS LSDEStage of CycleDeciduaAll inhibin subunits were strongly expressed by <strong>the</strong>pseudo-decidualised endometrium in <strong>the</strong> late secretoryphase and during early pregnancy. Intenseimmunostaining <strong>for</strong> ~A and ~B subunits was apparentin glands and stroma (Figure 2). In contrast, a subunitexpression was limited to <strong>the</strong> stroma, with prominentimmunostaining in <strong>the</strong> basement membrane of <strong>the</strong> largedecidualised cells (Table 2). ~ subunit expression wasalso apparent in vascular endo<strong>the</strong>lium.T~le2Rgure2Differential expression oj f3A immunostaininginhibin / activin subunits in deciduain deciduaGlandsStromaex. - ++SA ++ ++BB ++ ++ConclusionsThese data confirm <strong>the</strong> expression of <strong>the</strong> a., ~A and ~Bsubunits by human endometrium. Fur<strong>the</strong>r:• Glandular epi<strong>the</strong>lium is <strong>the</strong> major site of syn<strong>the</strong>sisin <strong>the</strong> non-pregnant endometrium.• Strong expression during decidualisation coincideswith extensive tissue remodeling and apoptosis.• Preferential expression of ~ subunits by glandularepi<strong>the</strong>lium in <strong>the</strong> decidualised endometrium maydenote a bias towards activin production.• Expression of inhibin I activin subunits byleukocytes indicates a role in modulating <strong>the</strong>immune response during trophoblast invasion andI or menstruation.References1. Leung et al (1998) Hum Reprod 13 : 3469-3477.2. Otani et al (1998) Obstet Gynaecol 91: 685-692.3. Petraglia et at (1998) J Clin Endocrinol Metab 83: 1194-1200.50 51
IDENTIFICATION AND FUNCTIONAL CHARACTERISATION OF PHOSPHOLIPASE A2 INTHE ENDOMETRIUM OF THE TAMMAR WALLABY, MACROPUS EUGENIIL.T Sebastian, a.Shaw, M.B. Renfree and G.E. Rice lDepartment of Zoology, The University of Melbourne, Parkville~ 3052lperinatal Research Centre, Royal Women's Hospital, Carlton, 3053THE EFFECTS OF FEEDING RUMEN BYPASS FATTY ACIDS DURING EARLYPOSTPARTUM ON UTERINE'PROSTGLANDIN SYNTHESIS, PLASMA FATTY ACIDS ANDTHE ONSET OF OESTROUS ACTIVITY IN DAIRY COWSS Meier, BA Clark, SR Morgan, GA VerkerkDairying Research Corporation, Private Bag 3123, Hamilton, New ZealandIntroductionPhospholipase A 2 (PLA 2 ) can be a rate-limitingenzyme in prostaglandin syn<strong>the</strong>sis. PLA 2 catalyses<strong>the</strong> hydrolysis of free fatty acids such as arachidonicacid from <strong>the</strong> sn-2 position of glycerophospatides in<strong>the</strong> cell membrane. Two major types of calciumdependentnon-pancreatic PLA 2 have beencharacterised. An 85 kDa arachidonoyl specific PLA 2which is activated by calcium-dependenttranslocation from <strong>the</strong> cytosol to <strong>the</strong> membrane(cPLA 2 ) and a 14kDa type IT secretory PLA 2 found in<strong>the</strong> extracellular fluid (sPLA 2 ) (1). cPLA 2 requiresnanomolar calcium concentrations, is insensitive toreducing agents, but is inactivated by heat. sPLA 2 on<strong>the</strong> o<strong>the</strong>r hand requires millimolar calciumconcentrations <strong>for</strong> activity and is sensitive toreducing agents and is not inactivated by heat (2).The onset and progression of labour is associatedwith increase prostaglandin <strong>for</strong>mation (3). There<strong>for</strong>eactivation of PLA 2 activity could playa role in <strong>the</strong>onset of parturition in <strong>the</strong> tammar. The purpose ofthis study was to identify <strong>the</strong> presence of PLA 2activity within <strong>the</strong> reproductive tract of <strong>the</strong> tammarand characterise <strong>the</strong> relative contribution of sPLA 2and cPLA 2 to total PLA 2 activity.Materials & MethodsAssay ofPLA 2 Activity: 25 J.lL of substrate, L-a-palmitoyl,2-[arachidonoyl-l- l4 C] phosphatidyl ethanolamine(NEN Research Products) in Tris buffer(100 mM, pH 8.0, 10 mM CaCl 2 and 300 roM NaCl)was incubated with diluted endometrial sampleshomogenised in 1 M NaCI-20 roM Hepes (pH 7.4).Samples were treated with 12.5 mM of EGTA ordithiothreitol (DTT) or heat (57°C, 5 min).Immunohistochemistry: Two antisera specific <strong>for</strong>human recombinant sPLA 2 and cPLA 2 were used tolocalise <strong>the</strong>se enzymes in PFA fixed paraffinembedded uterine tissue from term wallabies, using<strong>the</strong> TSATM Indirect Kit (NEN Life Sciences, Boston,USA) and dilutions of 1:300. Binding was visualisedusing DAB with Methyl Green counter staining.ResultssPLA 2 and cPLA 2 were localised within <strong>the</strong> epi<strong>the</strong>liallining of <strong>the</strong> secretory glands in <strong>the</strong> endometrium..,..,Figure 1. Section of tammar endometrium showinglocalisation of cPLA 2 in endometrial glands (photo digitallyfiltered to highlight brown staining)Incubation of endometrial tissue homogenates withEGTA or DTT did not significantly reduce <strong>the</strong>amount of activity relative to <strong>the</strong> control. Heattreatment however reduced <strong>the</strong> enzymatic activity by72%. These data show that cPLA 2 is <strong>the</strong> predominant<strong>for</strong>m ofPLA 2 •~ 2500:§ec.2000ro1500E.!::~ 1000">~~ a..500ControlEGTATreatmentHEATFigure 2. PLA 2 activity in control, EGTA treated, DTTtreated and heated term endometrium samples (mean ±SEM).ConclusionsThis study has successfully characterised PLA 2activity in a marsupial. While both secretory andcytosolic PLA 2 have been identified, cPLA 2 appearsto be <strong>the</strong> more active <strong>for</strong>m in <strong>the</strong> wallaby. As cPLA 2is <strong>the</strong> arachinoyl-specific enzyme this would suggestthat cPLA 2 plays an important role in liberating <strong>the</strong>precursor <strong>for</strong> prostaglandin syn<strong>the</strong>sis, arachidonicacid, at parturition in <strong>the</strong> tammar.References(1) Mayer, R.J and Marshall, L.A., (1993) FASEB.J7: 339-348.(2)Farrugia W.F., Rice G.E., Wong M.H., Scott K.,(1997) J. Endocrin. 153: 151-157.(3) Shaw, G., Gehring H.M., and Bell E.C., (1999)Biol. Reprod. 60: 611-614.onIntroductionThe current treatment regime <strong>for</strong> postpartumanovulatory anoestrous cows involving hormonaltreatment is cost effective but does not restoresubfertile animals to full reproductive potential. Analternative approach may be strategic feedsupplementation with fatty acids (FA). The feedingof rumen bypass FA has been shown to affect bothovarian and uterine function (1, 2, Bill Thatcher pers.corron.). It has been hypo<strong>the</strong>sised that feeding certainrumen bypass FA will alter uterine prostaglandinsyn<strong>the</strong>sis and influence ovarian function. Thisexperiment investigated <strong>the</strong> effect of supplementingdairy cows with rumen bypass FA during earlypostpartum (PP) on uterine prostaglandin syn<strong>the</strong>sisand <strong>the</strong> onset of oestrous activity.Materials & MethodsFourteen recently calved cows were randomlyallocated to receive ei<strong>the</strong>r bypass FA, in <strong>the</strong> <strong>for</strong>m of<strong>the</strong> Rumentek supplement (R; 2 kg/head/day;Rumentek Industries Pty. Ltd., Moree, NSW) or anisocaloric ration of barley (B), from day 7 to day 44PP. All cows grazed ryegrass/clover pasture inaddition to <strong>the</strong> supplementation. The Rumentek feedhad a total fat content of 30%, of which 84% consistsof <strong>the</strong> FA's, 18:1, 18:2 and 18:3 (43.2%, 24.6% and16.3% of total fat content respectively). An oxytocinchallenge (aT), by Lv. injection of 100 i.u. OT(Butocin®, BOMAC Laboratories Ltd., ManukauCity, NZ), were given on days 6, 18, 25, 32 and 39PP. Plasma samples were taken from <strong>the</strong> jugular veinat 15 min intervals <strong>for</strong> 1 hr prior to and 1 hr after OTadministration, followed by 30 min intervals <strong>for</strong> anadditional 2 hr. These plasma samples were storedand <strong>the</strong> PGF 2et metabolite, 13,14-dihydro-15-ketoprostaglandin F 2et (PGFM) was measured. Plasmaprogesterone (P4) concentrations were measured insamples taken by coccygeal venipuncture on alternatedays from day 0 to 44 PP. Plasma samples <strong>for</strong> FAdetermination were collected on day 6 and day 32 PPand extracted using <strong>the</strong> method of Hamilton & Comai(3). Fatty acid methyl esters (FAME) were preparedand identified using gas chromatography andexpressed as a percentage of total FAME. Changes in18:1, 18:2n6, 18:3n6, 20:3 and 20:4n6 wereexamined due to <strong>the</strong>ir involvement withprostaglandin syn<strong>the</strong>sis. Results are expressed asmean±sem.ResultsFrom this study <strong>the</strong>re was no effect of treatment on<strong>the</strong> onset of ovarian activity. Two animals in <strong>the</strong>Rumentek group had elevated plasma P4concentrations (>2 ng/ml) in <strong>the</strong> 3rd week PP. By day44 PP an additional 2 animals, both in <strong>the</strong> barleygroup, had elevated plasma P4 concentrations (>2.0ng/ml), evidence of corpus luteal <strong>for</strong>mation. Basalplasma PGFM concentrations (R: 625±241 pglml; B:1076±265 pglml), were elevated during <strong>the</strong> first weekPP (day 6), as was <strong>the</strong> response to OT. At <strong>the</strong> latermeasurements basal PGFM concentrations, peakPGFM or <strong>the</strong> timing of peak PGFM concentrationsdid not vary across treatment groups. Differences inplasma 18:1 FAME content were at <strong>the</strong> p
Energy balance and <strong>the</strong> postpartum interval in pasture fed dairy cows.FM Rhodes, BA Clark, SR Morgan and GA VerkerkDairying Research Corporation, Private Bag 3123, Hamilton, New Zealand.IntroductionPrevious work carried out in New Zealand has shown thatinterval to first postpartum ovulation (PPI) is related tobody condition score at calving (1,2). Cows calving inhigh body condition had a shorter PPI, but produced moremilk and lost weight during <strong>the</strong> first 6 weeks of lactationcompared to cows in low body condition (1). The aims of<strong>the</strong> current study were to determine whe<strong>the</strong>r differencesin PPI are related to differences in energy balance and toexamine <strong>the</strong> relationship with pulsatile secretion ofluteinising hormone (LH) in <strong>the</strong> early postpartum period.Materials & MethodsTwo year old cows (13 Friesian, 8 Jersey) were managedduring <strong>the</strong> final trimester of pregnancy so that <strong>the</strong>y calvedin low (n=ll) or high (n=lO) body condition (BCS).Following calving all animals were offered a generouspasture allowance. Time of first postpartum ovulationwas determined from weekly ultrasound examination ofovaries and from measurement of progesteroneconcentrations in milk samples collected 3 times a week.Individual daily yields of milk solids (MS) and pastureintakes (DMI) were measured over 5 days, 20 days aftercalving. Liveweight and BCS were recorded at calvingand weekly <strong>the</strong>reafter. Energy balance was calculated as<strong>the</strong> difference between energy intake and energy required<strong>for</strong> maintenance and lactation (MJ metabolisableenergy/day). Pulsatile secretion ofLH was measured over16 h, 11 days after calving. Concentrations of glucose, 13OR butyrate (BOHB), non-esterified fatty acids (NEFA)and insulin in blood were also measured at this time.Differences between cows ovulating Early (PPI
Does insulin or leptin mediate <strong>the</strong> effect of dietary fatty acids onLH pulse frequency in mature male sheep?Lucia M. Chagas, Dominique Blache, Margaret A. Blackberry and Graeme B. MartinFaculty of Agriculture, The University of Western Australia, Nedlands, Western Australia 6907IntroductionRams supplemented with lupin grain show an increase in LH pulse frequency that is accompaniedby increases in insulin and leptin concentrations in plasma, insulin concentrations in CSF, and alsoby increases in peripheral concentrations of fatty acids. When fatty acids <strong>the</strong>mselves are added to<strong>the</strong> diet, <strong>the</strong>y also stimulate gonadotrophin secretion (1) suggesting that <strong>the</strong>y may act via insulin orleptin to link feeding, metabolism and reproduction (2, 3). We tested <strong>the</strong>se relationships bystudying <strong>the</strong> effects of dietary fatty acids on LH pulse frequency, CSF and plasma insulin, andplasma leptin concentrations.MethodsFifteen rams fitted with intracerebroventricular cannulae were acclimatized <strong>for</strong> 4 weeks to amaintenance diet (MD) consisting of 700 g of oaten chaff + 50 g of lupins. They were thanallocated to 2 nutritional treatments: Control (MD, n =7) or Fatty Acids (MD + 127 g sodiumacetate + 64 g sodium propionate + 60 g vegetable oil, daily; n = 8). After 11 days of treatment,all rams were given 750 g lupins daily <strong>for</strong> 4 days, as a positive control. On Days -1 (be<strong>for</strong>etreatment), 4, 11 and 15, blood was sampled every 20 min <strong>for</strong> 24 h beginning at 07:00. On <strong>the</strong>same days, CSF was sampled every 6 h. Plasma was assayed <strong>for</strong> LH, insulin and leptin, and CSFwas assayed <strong>for</strong> insulin.ResultsCompared to Day -1, LH pulse frequency increased in <strong>the</strong> fatty acid-fed group on Days 4 and 11(Fig. 1). There was no additional effect of <strong>the</strong> lupin supplement in <strong>the</strong> Fatty Acid group (Day 15).By contrast, <strong>the</strong> Control group showed a response only to <strong>the</strong> lupin supplement. In both groups,plasma and CSF insulin, and plasma leptin, levels were only increased by <strong>the</strong> lupin supplement onDay 15.Plasma LH (pulses/24 h)10 I j *~ ~*i7.552.5CSF insulin (IlU/ml)Plasma insulin (IlU/ml)___*--.?Is ?It *~i~ i~l~~ rLi 25 rot.;oControl Fatty acids Control Fatty acids Control Fatty acids ControlPlasma leptin (ng/ml)?Is *~j~O. r-Li ~Figure 1: Effects of dietary fatty acids on LH pulse frequency, plasma and CSF insulin and plasma leptinconcentrations (mean ± s.e.m.) on Day -1 (D), Day 4 (.), Day 11 (1), and after lupin supplementation on Day 15 (.).* P
Ovarian Uptake of Leptin and Glucose and <strong>the</strong> Role of Glucose in <strong>the</strong> Regulation of Leptin Levels inSheepJim McFarlane, Kathleen Kauter, Broce Campbell), David Baird 2 , Amande Leigh 2 and Rex Scaramuzzi 2Animal Physiology, University of New England, Armidale NSW 2351; IDepartment of Obstetrics &Gynaecology, University of Nottingham, Nottingham UK; 2Royal Veterinary College, Royal College Street,London NWl OTU, UK.IntroductionIt has been known <strong>for</strong> some time that <strong>the</strong> nutritional status of a ewe during <strong>the</strong> oestrous cycle caninfluence <strong>the</strong> number of ovulations (1), however <strong>the</strong> mechanism <strong>for</strong> this remains unknown. Leptin is arecently reported hormone released predominately by adipose tissue (2) and was originally proposed as aregulator of satiety and energy expenditure. Recent studies have shown that it is likely to play an importantrole in many o<strong>the</strong>r systems including <strong>the</strong> reproductive system. In this study we have examined <strong>the</strong> uptake ofglucose and leptin by <strong>the</strong> sheep ovary and examined <strong>the</strong> role of glucose as a regulator of blood plasma leptinlevels.Materials and MethodsTwelve ewes with an ovarian autotranplants were treated with a GnRH antagonist to make <strong>the</strong>mhypogonadotrophic and <strong>the</strong>n follicular development was induced using a combination of pulsatile LHtreatment and continuous FSH infusion (3). Samples (5ml) of58706050~ 30::I 20c;10Carotid Ovarian JugUlarCarotid Ovarian JugUlarFigure 1: Uptake ofglucoseand leptin by <strong>the</strong> ovary andbrain.ovarian/carotid arterial blood, jugular venous blood and ovarian venousblood were collected over a 3 minute periods 48 hours after <strong>the</strong> start ofcombination LHIFSH treatment and stored frozen at -20°C until assayed.In ano<strong>the</strong>r group of 6 ewes, oestrus was synchronised usingprogestagen sponges inserted <strong>for</strong> 12 days. At sponge removal <strong>the</strong> eweswere mated with entire rams. On day 12 of pregnancy bilateral jugularcannulae were inserted under local anaes<strong>the</strong>sia. The next day (day 13)groups of ewes (n = 3) were infused with ei<strong>the</strong>r saline or glucose(50mMlh) until day 18. Pregnancy was confirmed by ultrasound on day22. Blood was collected by jugular venepuncture once a day from day 8after mating until day 25.The concentration of glucose was determined in triplicate using afluorimetric glucose oxidase based assay. Plasma leptin levels weredetermined in duplicate using a discontinuous indirect competitiveELISA, which uses an antiserum raised against bovine recombinantleptin (br-Ieptin).Results & DiscussionA significant (P< 0.05) uptake of glucose was observed by <strong>the</strong>ovary as shown in figure 1. This would suggest that <strong>the</strong> ovary like <strong>the</strong>brain utilizes glucose ra<strong>the</strong>r than fatty acids as its primary source ofenergy. A significant (P
THE EFFECTS OF WHOLE BODY HEAT-STRESS OF MALE MICEONASPECTSOFPATERNALFERTaITY· The Role of Mitochondriain Ischaemic/ Re-Perfusion Injury in Rat Testes·F. A. Ahmed l , J. Whelan l , A.M. Jequier2, and 1. M. Cummins 3lDepartment ofBioc~emistr;, ~~~ersity of ':'estemAus~alia,~edla~ds 6907 WA, 2perthAndrology, WesternAustralIa and DIVISIOn ofVetennary and BIOmedIcal SCIence, Murdoch UniversityIntroductionThe aim ofthis study was to gain fur<strong>the</strong>r insight into<strong>the</strong> mechanism underlying ischemic/re-perfusiondamage in <strong>the</strong> testis. It has been proposed that <strong>the</strong>mitochondria is <strong>the</strong> target <strong>for</strong> reactive oxygen speciesmediated damage (1). A rat model <strong>for</strong> male infertilitywas developed, using torsion followed by variousperiods of recovery. The mitochondrial "commondeletion" was measured in <strong>the</strong>se testes as an indicatorofgeneral mitochondrial damage. We also hypo<strong>the</strong>sizedthat this type ofdamage leads to <strong>the</strong> induction ofcertainfactors that playa vital role in apoptosis and protectionof <strong>the</strong> suviving cells, hence we carried out an RNAsubtractive hybridization between <strong>the</strong> control and <strong>the</strong>torsioned testes to identify differentially expressedgenes.Materials & MethodsThe study was comprised ofeight groups of3 rats each.Groups 1-3 were controls, groups 4-8 subjected tounilateral testicular torsion and allowed to recover <strong>for</strong>ei<strong>the</strong>r 0, 1 hour, I day, 1 wk or 4 weeks respectively.The mitochondrial common deletion was estimatedusing a competitive quantitative PCR assay that wasspecifically developed <strong>for</strong> this study (2). The effect oftorsion on <strong>the</strong> testes was detennined by <strong>the</strong> testicularbiopsy score count on representative tissues.Differentially expressed genes were isolated usingClontech PCR-select cDNA subtraction kit.ResultsBased on <strong>the</strong> biopsy scores it was clear that <strong>the</strong>experimental model produced testicular damage thatwas more severe in <strong>the</strong> longer recovery groups (Tablel).However this did not correlated to <strong>the</strong> extent of <strong>the</strong>"common deletion " since no significant differenceswere seen between <strong>the</strong> control and torsioned testes (Fig1).Table 1. Testicular biopsy score counts in rat testestaking by counting 30 tubules in three rat testes. C is<strong>the</strong> control testes and T is <strong>the</strong> torsioned testes. Scoresbelow 10 are considered subfertile. The values of <strong>the</strong>short and long recovery groups were averaged.Groupcontrolnon-recovery-Cnon-recovery-Tshort recovery-Cshort recovery-Tlong recovery··Clong recovery-T60Average Score9.9 ± 0.189.9 ± 0.189.8 ± 0.379.8 ± 0.378.3 ± 1.99.7 ± 0.52.4 ± 1.2918 clones were found to be differentially expressed bytorsioned testes. Nor<strong>the</strong>rn blots and sequecing are nowbeing undertaken to confirm which ones are truelydifferentially expressed and detennine <strong>the</strong>ir identity.0.05
THE EFFECT OF PATERNAL HEAT STRESS ON DEVELOPMENTIN-VITRO OF PREIMPLANTATION EMBRYOS IN THE MOUSEZhu Bi-ke, Walker, S.K.*, Setchell, B.P. and Maddocks, S.Department of Animal Science, University of Adelaide, Waite Campus, Glen Osmond, SA 5064and *Turretfield Research Centre, SARDI, Rosedale SA 5350IntroductionPaternal heat stress induced by localised heating of <strong>the</strong> testis has been shown to reduce in vitro fertilization rates in sheep tlland whole-body heating causes some developmental retardation at <strong>the</strong> 8-cell stage and accumulation of morulae at 120 h in <strong>the</strong>mouse (2). In this study, we have investigated <strong>the</strong> effects of whole body heat stress inflicted on male mice, on <strong>the</strong> developmentin vitro ofpreimplantation embryos sired with normal female mice up to 35 days after heating of <strong>the</strong> male.Materials and MethodsFemale C57/CBA Fl mice were superovulated using PMSGlhCG and mated naturally to a male of <strong>the</strong> same strain. Thesemales were ei<strong>the</strong>r untreated (control) or had been exposed <strong>for</strong> 24 hours to an elevated ambient temperature of 36°C and 62%humidity, at 7, 14, 21 or 35 days previously. One-cell embryos were collected from <strong>the</strong> oviducts of mated females, 24-26 hoursafter hCG injection, and cultured in a defined medium (CZB(3» in an atmosphere of 5% C02/5%02/90%N2 at 37°C. Embryodevelopment was monitored at 24 hr intervals <strong>for</strong> up to 120 hours and scored in <strong>the</strong> categories of 2cell (2C), 4 cell (4C) tomorula (M), 8 cell (8C) to early blastocyst (EB), M to blastocyst (B), and finally B. Results were analysed using <strong>the</strong> Chi-squaretest.ResultsEmbryo development rates between control groups at D7 -D35 were not significantly different, and <strong>the</strong>se data have beenpooled <strong>for</strong> analysis. During 24h - 120h of culture, <strong>the</strong> proportion of 2-C, 4-C to M, 8-C to EB, M to Band B wassignificantly (P < 0.001) reduced in heat-sired groups compared to control-sired groups (table 1). The proportion of l-eembryos undergoing fur<strong>the</strong>r development was significantly reduced by D7 after heating, and had not recovered to controllevels by D35 after heating. Maximum impairment to development was recorded by D14 and D21 after heating. Fur<strong>the</strong>rmore,whilst all nominated stages of embryo development were affected by paternal heat stress, <strong>the</strong> number of embryos at 2-C and Mto B stages appeared severely affected in all heat-sired groups. At D14, D21 and D35, o<strong>the</strong>r stages also demonstratedsignificant impairment.Table 1. Theeffiect 0 f paternalheat stress on in VItro development of embI) osC D7 D14 D21 D35No.I-C 185 136 1 114 1 108 1 185 12-C (24h) 183(98.9t 99(72.8)b.2 14(12.3)c.2 21(19.4/,2 68(36.8)d,24-C to M (48h) 182(98.4t 97(71.3)b.2 37(32.5/,3 9(8.3)e.3 55(29.7)d.38-C to EB (72h) 184(99.5)a 95(69.9)b,2 23(20.2)C,2 19(17.6t,2 75(40.5)d,2M to B (96h) 181(97.8t 71(52.2)b.3 21 (18.4t.4 12(11.1)C,4 55(29.7)d.4B (l20h) 178(96.2t 64(47.l)b.4 5(4.4t·5 1(0.9)c,5 36(19.5)d.5Values WIth dIfferent alphabetIcal superscnpts dIffer slgmficantly (P
MITOCHONDRIAL DNA: OUR ALTERNATIVE GENOME.CLONING IN BIOLOGY AND MEDICINEIan WilmutRoslin Institute, and Geron Bio-Med, Roslin, Midlothian, EH25 9PS. UKRevolutionary new opportunities in biology, medicine and agriculture were created by <strong>the</strong>observation that offspring may develop following transfer of a nucleus from <strong>the</strong> somatic cell of anadult animal to an enucleated oocyte. These include <strong>the</strong> provision of organs from animals <strong>for</strong>transplantation to human patients, derivation of autologous cell <strong>the</strong>rapy in human patients,development of new means to study <strong>the</strong> control of genetic diseases and enhanced animal breeding.The new procedures <strong>for</strong> nuclear transfer have proved to be repeatable as many laboratories haveobtained offspring using a variety of different nuclear donor cell types in four species. However, allgroups also report significant limitations. First, <strong>the</strong> proportion of embryos developing to term is lowin all large experiments. Secondly, prenatal death occurs at all stages of gestation. Thirdly, postnataldeath is increased in many reports. Finally, developmental abnormalities are described in mostreports. In some cases <strong>the</strong>se are associated with increases in size of fetus or placenta, suggesting that<strong>the</strong>re may be errors in imprinting. Similar abnormalities have been observed after culture ofruminant embryos.During cloning, <strong>the</strong> nucleus of a diploid somatic cell, with <strong>the</strong> DNA packed in appropriate nuclearproteins <strong>for</strong> that cell type, is transferred into an oocyte. The structure of <strong>the</strong> oocyte evolved in order<strong>for</strong> it to be able to accept a haploid nucleus, packed in protamines. It is perhaps surprising thatoffspring are ever born, ra<strong>the</strong>r than being disappointing that <strong>the</strong> procedure is so inefficient. Researchis required to define <strong>the</strong> mechanisms that act during reprogramming of gene expression in order toincrease <strong>the</strong> efficiency of <strong>the</strong> procedure.J.M. CumminsDivision of Veterinary and Biomedical Sciences, Murdoch University, Murdoch WA 6150Mitochondria were fIrst described over a century ago.However, <strong>the</strong> study of mitochondrial DNA (mtDNA)has exploded in <strong>the</strong> past two decades and is now one of<strong>the</strong> most rapidly growing areas of molecular geneticsand medicine. They almost certainly originated asendosymbiotic bacteria, and have retained <strong>the</strong>ir ownhighly truncated genome, lacking introns. This is acircular, double-stranded DNA molecule of c 16Kblacking histones, using a unique genetic code.Replication is in response to local needs and isuncoupled from <strong>the</strong> host cell cycle. However, it alsorequires close and species-specific congruence betweenmtDNA and mitochondrial control genes in <strong>the</strong> nucleus[1]. The importance of this harmony has implications<strong>for</strong> cloning technology, <strong>for</strong> <strong>the</strong> study of imprinting andnuc1eo-cytoplasmic interactions, and <strong>for</strong> attempts to"rescue" defective human oocytes by cytoplasmictransfer [2]. Mitochondria play a central role inmammalian tissue bioenergetics, in growth, in ageingand in apoptosis. The lack of redundancy in <strong>the</strong>genome, coupled with <strong>the</strong>ir importance in <strong>the</strong>production of ATP through oxidative phosphorylation,also means that many mutations and deletions in <strong>the</strong>genome are associated with progressive bioenergeticdiseases [3]. Mitochondria are also one of <strong>the</strong> majorcellular sources of free radicals, which is probably why<strong>the</strong>y are sequestered away from <strong>the</strong> nuclear genome.However, <strong>the</strong> significance of deletions is probablyover-stated as most current PCR techniques grosslyoverestimate <strong>the</strong>ir prevalence [4].The path followed by mitochondria in <strong>the</strong> life cycle isextraordinary [5]. While sperm carry about 75 copies ofmtDNA into <strong>the</strong> oocyte at fertilization [6], <strong>the</strong>se are almost universally - degraded by species-specific,ubiquitin-mediated proteolysis early in embryogenesis.This is probably to avoid compromising <strong>the</strong> embryowith degraded mtDNA, which is most likely toaccumulate in <strong>the</strong> sperm through exposure to freeradicals. The extent to which sperm rely onmitochondria <strong>for</strong> normal function is highly variablebetween speciesThere is an obligate delay in mtDNA replication in <strong>the</strong>embryo so that <strong>the</strong> pre-migratory primordial germ cellscontain only between 10 and 100 copies. Withcolonization of <strong>the</strong> gonads, mitochondria multiplyclonally. This restriction/amplification process acts as agenetic filter on copy number and ensures that in <strong>the</strong>female line <strong>the</strong> oocytes are remarkably homoplasmic(ie only one type of mtDNA is found). This numericalbottleneck means that mitochondrial genotype canchange very rapidly between successive generations. Itis also a stringent means of combatting Muller'sratchet, <strong>the</strong> inexorable accumulation of deleteriousmutations in asexually reproducing entities [7]. Whilean attractive hypo<strong>the</strong>sis, <strong>the</strong> notion that oocyte atresiamight selectively eliminate defective mtDNA[8] lacksevidence. The mitochondria in oocytes are almostdormant but reach a total of around 1-3 x 10 5 shortlybe<strong>for</strong>e ovulation. This ensures <strong>the</strong> optimal start in life<strong>for</strong> <strong>the</strong> embryo. In <strong>the</strong> male line <strong>the</strong> spermmitochondria show reductions in mtDNA copy numberand acquire diminished ability to replicate. Duringspermiogenesis a unique haploid-encoded selenoproteincapsule <strong>for</strong>ms on <strong>the</strong> midpiece and at some point <strong>the</strong>mitochondria are tagged with ubiquitin to prepare <strong>the</strong>m<strong>for</strong> suicide after fertilization.There are a number of myths that have becomeestablished about mtDNA: that it does not enter <strong>the</strong> egg(false)[6]; that it cannot repair itself (also false); that itmutates much more rapidly than nuclear DNA (true butnot uni<strong>for</strong>mly across <strong>the</strong> genome) and that it does notrecombine. Recent evidence based on rare mutationssuggests that recombination has indeed occurred inhistorical human populations [9,10]. This could ariseby fusion of sperm and egg mitochondria be<strong>for</strong>eproteolytic destruction. This observation calls intoquestion <strong>the</strong> stability of <strong>the</strong> molecular "clock" used toreconstruct human populations based on maternalinheritance of mtDNA.References1. Shadel GS Clayton DA, Ann Rev Biochem,1997,66:409-352. Cohen J, et al., Mol Human Reprod, 1998,4:269-2803. Wallace DC, Sci Amer, 1997,277:40-474. Ahmed F, et al., Biotech, 1999,26:290-2965. Cummins JM, Rev Reprod, 1998,3:172-1826. Ankel-Simons F and JM Cummins, PNAS,'1996,93:13859-138637. Bergstrom CT and J Pritchard, Genetics, 1998,149:2135-468. Short RV, Journal of Experimental Zoology,1998,281:359-3619. Eyre-Walker A, et ai., Proc R Soc Lond B,1999,266:477-48410. Hagelberg E, et ai., Proc R Soc Lond B, 1999,266:485-4926465
Implantation and transplantation: endocrine signals in cell lineage choiceJohn P. HearnResearch School of Biological Sciences, Australian National UniversityPO Box 475, Canberra, ACT 2601, AustraliaTHE DEVELOPMENT AND POTENTIAL USE OF HUMAN EMBRYONIC STEM CELLSIntroduction:The rapid establishment of embryo-maternalatr~;;:'hment, implantation and differentiation at <strong>the</strong>stan of pregnancy is enabled by an endocrinedialogue. The reliance on chorionicgonadotrophin (CG) as an embryonic signal ofviability, and capacity to support <strong>the</strong> corpusluteum, is apparently restricted to primates. Wequestioned <strong>the</strong> possible role of gonadotrophinreleasing hormone (GnRH) in regulating CGrelease during implantation. fu associated studies(with James Thomson, University of Wisconsin)we isolated and characterised <strong>the</strong> similarities inendocrine secretion from totipotent or pluripotentembryonic stem cells in marmoset and rhesusmonkeys.Materials and Methods:The development of non-surgical embryorecovery, microassays to measure GnRH and CG,and embryo culture to <strong>the</strong> in vitro equivalent ofday 12 after embryo attachment, were describedearlier (1,2,3). fu current studies we incubatedsingle embryos with exogenous GnRH, agonist orantagonist, measuring <strong>the</strong> resulting change in CGsecretion and <strong>the</strong> differentiation stages ofembryos. In addition, embryonic stem cells wereisolated (4,5) and <strong>the</strong>ir secretion of CG monitored.Results:Control embryos (n=25) secreted a characteristicprofile of CG, with GnRH measurable in <strong>the</strong>culture fluid by mid blastocyt stages ofdevelopment. fucubation with antagonist (n=15)reduced CG secretion and slowed differentiation.fucubation with agonist (n=15) enhanced CGsecretion but drastically inhibited attachment anddifferentiation. fucubation with exogenous GnRH(n=10) greatly enhanced both CG secretion andembryo differentiation, suggesting an unexpectedstimulus to embryo survival, with possible<strong>the</strong>rapeutic implications. Differentiatedembryonic stem cells also secreted CG andmaintained <strong>the</strong> developmental potential to <strong>for</strong>mtrophoblast and all three embryonic germ layers.Discussion:Our results suggest a direct or indirect function<strong>for</strong> GnRH, secreted by <strong>the</strong> embryo during periimplantationstages of pregnancy, in <strong>the</strong>regulation of CG secretion. There may also bedirect effects on cell growth anddifferentiation. The ability to enhance ordiminish embryo attachment, outgrowth anddifferentiation may provide a novel (andcontrolled) approach to study intra-embryoniccell signalling by stimulating or inhibitingperi-implantation events (6,7). In embryonicstem cell preparations, using a similarapproach, it may be possible to dissectgenetically in vitro <strong>the</strong> mechanisms controllingprimitive streak <strong>for</strong>mation in primates and <strong>the</strong>endocrine signals that influence choice in celllineage.References:1. Hearn JP et al (1994) In: Marshall'sPhysiology of Reproduction, 4 th Ed. Pp.535-676, Chapman and Hall, London2. Webley, GE and Hearn JP (1994) Ox<strong>for</strong>dReviews of <strong>Reproductive</strong> <strong>Biology</strong> 16: 1-323. Seshagiri, PB, Terasawa, E and Hearn, JP(1994Hum.Reprod.91300-1307.4. Thomson JA, Hearn JP et al. (1995)Proceedings of <strong>the</strong> National Academy ofSciences 92: 7844-7848.5. Thomson, JA, Hearn, JP et al (1996) Biol.Reprod. 55, 254-259.6. Hearn, JP (1997) Endocrine Signals inembryo implantation In Marsupial biology,recent research, new perspectives, pp 22-30UNSW Press, Sydney7. Stouffer, RL and Hearn, JP (1998) In: TheEndocrinology of Pregnancy. Pp 35-57Humana Press, NJ. t-\rfiJ\\ C.,.!t'l{ 'j\ ,'." . /~I'i,~(2)(3)Ben Reubinoff l ,2, Martin Pera l , AriffBo~gs03, Chui Fong 3 and Alan Trounson lICentre <strong>for</strong> Early Human Development, fustitute of Reproduction and Development, MonashUniversi~, 2Department of Obstetrics and Gynecology, H~dassah l!niv~rsity H?spital, Israel andDepartment of Obstetrics and Gynaecology, NatIonal UnIverSIty of SIngaporeHuman embryos can be grown to <strong>the</strong> blastocyst s.tage efficie~tly in ~itro (1) and. will ~ttach t~ andoutgrow in plastic culture dishes in vitro (2). It IS also pOSSIble to Immunosurgically Isolate ~nnercell mass cells (ICM) of human blastocysts and to maintain <strong>the</strong>se cells in long-term culture 10 anundifferentiated state. The isolation and passage of undifferentiated human embryonic cells waspublished by Thomson et al. (3). We have independently derived undiffere~tiat~d human embryoniccells that maintain <strong>the</strong>ir phenotype through extended culture and passage In VItro. These cells aremultipotential and are able to differentiate in vitro and when ~enotra~s~lanted in~o ,a ~ery. widerange of cell and tissue types. The multipotentiality of <strong>the</strong> dIfferentIatIng cells IS IndIcatIve ofembryonic stem cells.The potential value and use of embryonic stem cells are <strong>for</strong> <strong>the</strong> d~t~rminat~on of. <strong>the</strong> int~in.sic andextrinsic factors that direct lineage specific differentiation. The abIlIty to dIrect dIfferentIation andto maintain stem cell phenotype during large scale multiplication may enable <strong>the</strong>se cells to be used<strong>for</strong> transplantation, be used <strong>for</strong> tissue engineering and gene <strong>the</strong>rapy.The research with <strong>the</strong>se putative embryonic stem cells is presently aimed to identify <strong>the</strong> factorsinvolved in <strong>the</strong> maintenance of stem cell phenotype and those involved in <strong>the</strong> check points directingearly differentiation events.References(1)Jones GM, Trounson AO, Lolatgis N, Wood C (1998) Factors affecting <strong>the</strong> success ofhuman blastocyst development and pregnancy following IVF and embryo transfer. FertH.Steril. 70: 1022-29.Bongso A, Fong C-Y, Ng SC, Ratnam S (1994) Isolation and culture of inner cell mass cellsfrom human blastocysts. Human Reprod. 9: 2110-17.Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, JonesJM (1998) Embryonic stem cell lines -derived from human blastocysts. Science 282 (5391):1061-62.66""""-~G67
. INTERSPECIFIC VARIATION IN GLYCOCONJUGATES OF THE ZONAE PELLUCIDAE OF SEVERALMARSUPIAL SPECIESLA. Chapman, O.W. Wiebkin l and.W.G. BreedDepartment of Anatomical Sciences, University of Adelaide, SA 5005 and IDepartment ofMedicine, University ofAdelaide,.SA 5005'I "-" :Jl' ,"'; C' 1.-...-"!\v J ,.:,> .:..y_.J\Introduction .jThe eggs of all vertebrates are surrounded by a glycoprotein coat known as <strong>the</strong> zona pellucida (ZP). It is to thisstructure that sperm must fIrst bind, and <strong>the</strong>n penetrate, be<strong>for</strong>e fertilisation can occur. Sperm-ZP binding is facilitated by<strong>the</strong> glycoconjugates of <strong>the</strong> ZPs oligosaccharides (1). While <strong>the</strong> protein components of <strong>the</strong> mammalian ZP glycoproteinshave been shown to be highly homologous, <strong>the</strong>re appear to be interspecifIc differences in glycoconjugate content (2).While we have previously shown interspecific variation in thicknesses of marsupial ZPs, ranging from IJllD in <strong>the</strong>opossum to 8-10JllD in <strong>the</strong> koala (3), almost no work has been per<strong>for</strong>med on characterising <strong>the</strong> glycoconjugatecomposition of marsupial ZPs. The aim of this study, <strong>the</strong>re<strong>for</strong>e, was to determine and characterise, by <strong>the</strong> use ofdifferential lectin binding toge<strong>the</strong>r with enzyme elimination and saponification, <strong>the</strong> saccharide components of <strong>the</strong> ZPsof follicular oocytes of several marsupial species each representing a different family within <strong>the</strong> marsupial infrac1ass.Materials and MethodsOvaries of brushtail possums (Trichosurus vuIpecuIa), fat-tailed dunnarts (Sminthopsis crassicaudata), koalas(Phascolarctos cinereus), eastern grey kangaroos (Macropus giganteus) and grey short-tailed opossums (Monodelphisdomestica) were fixed in Rossman's fluid (90% ethanol saturated with picric acid and 10% <strong>for</strong>malin). They were <strong>the</strong>nroutinely processed <strong>for</strong> light microscopy, embedded in paraffIn wax and sectioned. Sections were <strong>the</strong>n deparaffinised,rehydrated, blocked with 1% BSA in Tris-buffered saline and incubated with one of 7 FITC-conjugated lectins andviewed under a fluorescent microscope. To detect sugar residues masked by sialic acid or O-acetyl groups, sectionswere first ei<strong>the</strong>r desialylated (4) or saponified (5) respectively, prior to incubation with <strong>the</strong> lectins, as previouslydescribed. Specific controls involved incubating <strong>the</strong> lectins with 0.2-0AM of <strong>the</strong>ir inhibitory sugar prior to applicationto <strong>the</strong> ovary sections. Results were recorded as positive (+) or negative (-) binding, with positive binding beingsubjectively categorised into mild (+), strong (++) and intense (+++).Results and DiscussionThere is considerable variation in glycoconjugate content of marsupial ZPs. The ZPs of <strong>the</strong> fat-tailed dunnartsdisplayed <strong>the</strong> most deviation in <strong>the</strong>ir saccharides from those of o<strong>the</strong>r marsupial ZPs, with positive reactivity to alliectinsoccurring after desialyation (Table 1) and saponification (Table 2). Most signifIcantly, a-D-mannose, as demonstratedby Con A, was found only in <strong>the</strong> saponified ZPs of <strong>the</strong> fat-tailed dunnarts and no o<strong>the</strong>r species. The presence ofmannose may indicate, <strong>for</strong> <strong>the</strong> fIrst time, <strong>the</strong> presence ofN-linked oligosaccharides in <strong>the</strong> ZPs of dunnarts.ZPs of all species, except those of <strong>the</strong> opossum, appeared sialylated with increases in binding <strong>for</strong> f3-Gal(I-3)GaINAc(PNA) and a-D-Gal(NAc) (SBA) after removal of sialic acid with neuraminidase. O<strong>the</strong>r glycoconjugates like <strong>the</strong>disaccharides f3-Gal(1-4)GlcNAc and f3(1-4)-D-GlcNAc, as demonstrated by <strong>the</strong> lectins ECA and WGA respectively,appear to be common components within <strong>the</strong> ZPs of marsupials. The <strong>for</strong>mer, however, also appears to be masked tosome extent by O-acetyl groups, with increases in binding to ECA after saponification.Table 1. Lectin binding to ZPs of 5 marsupial speciesbe<strong>for</strong>e and, in brackets, after desialyationBrushtall Fat-tailedPossum Dunnart Koala Kane:aroo OoossumPNA ++(+++) - (++) ++(+++) ++(+++) +(+)RCA·I1 -(-) +(++) +(+) - (+) -(-)WGA +++(+++) ++(+++) +++(+++) +++(+++) ++(++)SNA 0(+) +(++) ++(++) 0(+) -(-)SBA - (+) 0(+) - (++) -C++) -(-)Table 2. Lectin binding to ZPs of 5 marsupial speciesbe<strong>for</strong>e and, in brackets, after saponificationBrushtail Fat-tailedPossum Dunnart Koala Kanl!aroo OoossumPNA ++(+++) - (+) ++(++) ++(+++) +(++)ECA - (+++) - (+) +(++) +(++) +(++)Con A 0(_) - (+++) -(-) - (0) -(-)RCA-II 0(_) +(+) +(+) - (+) -(-). WGA +++(+++) ++(++) +++(+++) +++(+++) ++(+++)SNA - (0) +(+) ++(++) - (++) - (++)SBA -(-) - (++) -(-) 0(0) -(-)ConclusionWhile marsupial ZPs contain common saccharide components such as GlcNAc and GalNAc, which are also commonin eu<strong>the</strong>rian mammalian ZPs (2), <strong>the</strong>y also display considerable interspecific variation in <strong>the</strong>ir glycoconjugate content.Whe<strong>the</strong>r this variation relates to <strong>the</strong> species-specificity of sperm-ZP binding is unknown. Fur<strong>the</strong>r work is required toelucidate this.References1. Florman, H.M., and Wassarman, P.M. (1985) Ce1141:313-324.2. Skutelsky, E., et al., (1994) J Reprod Fertill00:35-41.3. Chapman, lA., and Breed, W.G. (1998) Aust Mamm Soc conference proceedings, Perth Australia:23.4. Riley, C.B., and Elhay, MJ. (1994) Ch. 3. In I Laboratory Histopathology: A Complete Reference'68IMMUNOCONTRACEPTION FOR WILDLIFE CONTROL:THE ZONA PELLUCIDA AS A TARGET IN MACROPODS.A.L. Kitchener, DJ. Kay and L.M. EddsMarsupial CRC, Unive:sity of Newcastle, Callaghan NSW 2308, Australia.IntroductionBy altering <strong>the</strong> landscape <strong>for</strong> agriculture andgrazing we have allowed .<strong>the</strong> numbers of. somespecies of macropods to Increase dramatIcally.This has lead to environmental degradation andcompetition <strong>for</strong> resources. Contraceptivevaccines are being investigated as a means ofcontrolling problem population of macropods(1). The zona pellucida (ZP) is beingconsidered as a target as it per<strong>for</strong>ms essentialfunctions in fertilisation' (2). This study hasbeen conducted to provide proof of concept <strong>for</strong>ZP molecules as potential target antigens in acontraceptive vaccine <strong>for</strong> macropods.Materials & Methods16 female Tammar wallabies (Macropuseugenii) were divided evenly into two treatmentgroups. The first group was immunised on 5occasions, at <strong>for</strong>tnightly intervals, with l00J.lgof whole porcine zona pellucida (PZP)emulsified in Freunds complete adjuvant <strong>for</strong> <strong>the</strong>initial injection and incomplete Freunds <strong>for</strong> <strong>the</strong>second and subsequent injections. Thevaccinations were distributed over 4 sites, twosubcutaneous and two intramuscular. Thecontrol group received saline and Freundsadjuvant in <strong>the</strong> same manner. Blood sampleswere collected each time <strong>the</strong> animals wereimmunised and <strong>the</strong>reafter at monthly intervals.After <strong>the</strong> fifth injection <strong>the</strong> animals were placedin a breeding trial with two PZP and two controlanimals placed with two stud males in eachbreeding group. Success of natural mating andartificial insemination after super-ovulation (3)were used to assess <strong>the</strong> fertility of animals ineach group. Animals were sacrificed at <strong>the</strong> endof <strong>the</strong> breeding trial and <strong>the</strong> reproductive tractscollected <strong>for</strong> immunohistology. Immuneresponses were evaluated using ELISAs andwestern blots (4).ResultsImmunised wallabies produced strong andsustained immune responses to PZP (Figure 1).67% of control animals produced offspring bynormal mating. PZP immunised animals failedto produce any offspring. Fertilised ova wererecovered from all control animals that had beenartificially inseminated, showing that 100% ofcontrol animals were fertile. Artificial ~.nsemination demonstrated that PZP immunised\~allabies failed to ovulate. The ovaries of PZP .. )_immunised super-ovulated wallabies were...'1\smaller, 0.239g +/- 0.15,3g co~pared to 0.781g.! +/_ 0.275g, and had fewer folhcles
Selection of porcine oocytes prior to in vitro maturationand in vitro fertilisation using Brilliant Cresyl BlueA. Preshaw, W.M.C. Maxwell and G. EvansDepartment of Animal Science, University of Sydney, NSW 2006, AustraliaIntroductionDetection and removal of oocytes unable to undergo maturation in vitro may improve <strong>the</strong> development ofcultured oocytes due to <strong>the</strong> absence of toxins which developmentally incompetent oocytes may produce.Brilliant Cresyl Blue (BCB) is reduced to a colourless compound by glucose 6-phosphate dehydrogenase in<strong>the</strong> porcine oocyte undergoing oogenesis. In this experiment BCB was used to detect oocytes unable toundergo in vitro maturation (1,2). Oocyte maturation, fertilisation, cleavage and development to morulae wasexamined after removal of oocytes from <strong>the</strong> culture system.Methods and MaterialsOocytes were aspirated from 2-4 mm follicles on pre-pubertal slaughterhouse ovaries. The oocytes wereincubated in 13 11M BCB in BSA-free NCSU 37 (3) + 10 % porcine follicular fluid <strong>for</strong> 90 minutes at 39°C in5 % CO 2 in air. Oocytes were <strong>the</strong>n sorted into 2 groups based on <strong>the</strong>ir BCB staining (clear = no stain present,blue = BCB stain present). The oocytes were <strong>the</strong>n matured <strong>for</strong> 44 hr, fertilised and cultured. A control groupof oocytes was matured without prior incubation. Oocytes were removed 17 h after insemination andassessed <strong>for</strong> maturation and fertilisation by staining with Hoechst 33342 and examining under UV light.Cultured embryos were examined on days 2 and 5 <strong>for</strong> cleavage and development to morula stage. Data wereanalysed by chi square and are presented in Table 1.Table 1. Maturation, fertilisation, cleavage and development to morulae of oocytes after incubation withBCB compared' WIth umncu . bated oocytes (control).Treatment Control Stained (Blue) Unstained (Clear)Number fixed 57 60 57Maturation (%) 42 (74t 49 (81t 4 (7)bFertilisation (%) 26 (46)a 25 (42)a 2 (3.5)bNumber cultured 157 455 105Cleaved (%) 73 (46.5)a 147 (32.3)b 3 (2.9)CMorulae (% of cultured) 28 (17.8)a 89 (19.6)a 1 (1)bMorulae (% of cleaved) 38.4 % a 60.5 % b 33.3 % aValues WIth dIfferent superscnpts In <strong>the</strong> same row are significantly different (P < 0.05).Results and DiscussionClear oocytes had lower maturation and fertilisation rates than both control and blue oocytes. As clearoocytes accounted <strong>for</strong> 24 % of <strong>the</strong> total number of oocytes incubated with BCB it seems <strong>the</strong>ir removal fromculu:re ~~ould i~prove o~cyte and embryo development. However, <strong>the</strong>re was a decrease in cleavage rate andno SIgnIfIcant dIfference In <strong>the</strong> maturation, fertilisation or development to morulae of blue compared withcontrol oocytes which suggests that removing <strong>the</strong> clear oocytes from culture did not necessarily improve <strong>the</strong>per<strong>for</strong>mance of <strong>the</strong> remaining oocytes. It is possible that <strong>the</strong> extra handling of oocytes which occurs with <strong>the</strong>use of <strong>the</strong> BCB test may adversely affect cleavage. Never<strong>the</strong>less, <strong>the</strong> percentage of cleaved embryos whichdeveloped to morulae was higher <strong>for</strong> blue than <strong>the</strong> control oocytes. These results confirm that BCB stainingcan be used to identify oocytes with potential to undergo in vitro maturation and fertilisation and that suchoocytes, when cleaved, have greater potential to develop to morulae.References1. Ericsson, S.A., Boice, M.L., Funahashi, H. and Day, B.N. (1993). Therio. 39: 214 (Abstr).2. Roca, J., Martinez, E., Vazquez, J.M. and Lucas, X. (1999). Reprod. Ferti!. Dev. (accepted <strong>for</strong>publication). .3. Petters, R.M. and Wells, K.D. (1993). J. Reprod. Fertil. Supp. 48: 61-73.THE TIMETABLE OF NUCLEAR MATURATION OF TAMMAR WALLABY OOCYTESIN VITRO AND REQUIREMENTS FOR PROTEIN SYNTHESISMethodsDevelopment of multiple ovarian follicles in <strong>the</strong>wallaby was induced with 6 mg of porcine folliclestimulating hormone (FSH) (Folltropin V:Vetrapharm, Australia) administered i.m. twicedaily <strong>for</strong> four days. The following day, oocyteswere collected from follicles greater than 2 mm indiameter. Oocytes were cultured in Eagle'smmImum essential medium (EMEM)supplemented with 10% fetal calf serum (FCS),10 I1g/ml FSH, 10 Jlg/ml pLH, 100 IU/mlpenicillin and 100 Jlg/ml streptomycin at 36°C in5% CO 2 in air.Cycloheximide (CH) (Sigma Chemical Co., StLouis, MO) was added at final concentrations of1, 10 and 100 Jlg/ml, to examine requirements <strong>for</strong>protein syn<strong>the</strong>sis during in vitro maturation. Theeffect of inhibition of protein syn<strong>the</strong>sis atdifferent stages of nuclear maturation wasexamined by die addition of 10 Jlg/ml CH to <strong>the</strong>maturation media after 0 and 24 hr of culture.Each experiment consisted of at least threereplicates with at least 10 oocytes per treatment.ResultsTammar wallaby eggs cultured in vitro began<strong>the</strong> process of chromatin condensation within 4 hof culture, at which stage <strong>the</strong> chromatin in mostKaren E. Mate & Janine BuistMarsupial CRC, School of Biological Sciences, Macquarie University, NSW, 2109.IntroductionOocytes from <strong>the</strong> tammar wallaby(Macropus eugenii) resemble those of eu<strong>the</strong>rianmammals in <strong>the</strong>ir ability to complete meiosis invitro when cultured under suitable conditions.Recent work indicates that late maturationalevents in <strong>the</strong> marsupial egg, may be important notonly <strong>for</strong> developmental potential of <strong>the</strong> embryobut also <strong>for</strong> early fertilization events such assperm binding to <strong>the</strong> zona pellucida and to <strong>the</strong>plasmamembrane of <strong>the</strong> egg. In order to begin toaddress <strong>the</strong>se issues, this study examines <strong>the</strong>timing of maturation in vitro and its requirements<strong>for</strong> protein syn<strong>the</strong>sis.eggs appeared as long homogeneous threads.After 8 hours of culture chromatin condensationhad progressed so that <strong>the</strong>re were regions oftightly condensed chromatin interspersed withmore threadlike chromatin. Germinal vesiclebreakdown (GVBD) was complete by 18 h ofculture, and 85 % of eggs had reached metaphase 1.The majority of eggs (68%) were at <strong>the</strong> metaphaseI stage at 24 h, and 11 % had already progressedto anaphase I. Approximately 20% of eggs hadcompleted maturation (to metaphase mby 36 h,and 86% of eggs had completed maturation after42 h of culture.All concentrations (1, 10 and 100 Jlg/ml) of <strong>the</strong>protein syn<strong>the</strong>sis inhibitor, cycloheximide (CH)significantly inhibited GVBD (p < 0.01), howevercondensation of chromatin did occur in a smallnumber of oocytes. The inhibitory effects of CHon oocyte maturation was reversible; eggs that hadbeen pre-incubated in 10 Jlg/ml CH <strong>for</strong> four hourswere able to progress to <strong>the</strong> metaphase IT stage ofmeiosis after 44 h in vitro culture. The addition ofCH to wallaby eggs already at MI of meiosisprevented normal progression to MIT (p
SUCCESSFUL DEVELOPMENT OF A 6·DMAP REPLACEMENT FOR BOVINE OOCYTEACTIVATION RESEARCH.W.G. James, O.La~ham-Kaplan and A.a. TrounsonInstitute ofReproduction and Development, Monash University, Melbourne, Australia.IntroductionAn efficient method of activation following nucleartransfer and sperm injection in bovine is necessary toinduce successful preimplantation development. Theactivation treatment currently considered to be <strong>the</strong>most successful is <strong>the</strong> Ca++-ionophore A23187followed by 6-dimethylaminopurine (6-DMAP) (1).A similar success with oocyte activation has beenobserved in <strong>the</strong> sheep with ethanol exposure followedby 6-DMAP (2). However, concerns have been raisedto <strong>the</strong> effects <strong>the</strong> non-specific protein kinase inhibitor6-DMAP might have on embryo development (3,4).6-DMAP has been shown to accelerate <strong>the</strong>disappearance of phosphorylated <strong>for</strong>ms of proteins.Development of 6-DMAP treated mouse embryosresulted in severely compromised post-implantationdevelopment with no live pups born (3).The agentcycloheximide can be used to replace 6-DMAP,targetting <strong>the</strong> syn<strong>the</strong>sis of proteins ra<strong>the</strong>r than <strong>the</strong>irphosphorylation. Cycloheximide treatment is capableof activating both young and aging oocytes at highrates. The majority of o<strong>the</strong>r artificial stimuli areunable to activate young oocytes adequately (5). Thishas often limited nuclear transfer procedures to <strong>the</strong>use of aging oocytes, in which cytoskeletaldeterioration has been observed (5). The presentstudy investigated <strong>the</strong> effect of cycloheximidetreatment following ethanol exposure on IVM bovineoocytes.Materials & MethodsImmature oocytes were aspirated from abattoircollected ovaries and matured in-vitro <strong>for</strong> 24hr (6).Prior to activation, oocytes were denuded of allcumulus by vortexing <strong>for</strong> 3min in 50111 hyaluronidasesolution (lmg/ml). Mature oocytes that had extrudeda ~rst. polar body were used <strong>for</strong> <strong>the</strong> study. TheactIvatIOn treatments which were examined are: [1]A23187 (38IlM; 10min) followed by 6-DMAP (2JlM;5hr); [2] ethanol (7%;5min) followed by 6-DMAP(2IlM ; 5hr); [3] ethanol (7%;5min) followed bycycloheximide (IOllg/ml) and cytochalasin-B(5Ilg/ml) (5hr); [4] ethanol alone (7%;5min). 6DMA~ treatment results in diploid parthogenomes,so to SImulate this cytochalasin-B was combined with<strong>the</strong> cycloheximide treatment. Treated oocytes werewashed through drops of equilibratedSOF120AAIBSA medium, and cultured <strong>for</strong> 3 days inSOF120AAIBSA at 39°C at 7% O 2, 5% CO , 288% N rOn day 3 (day 0 = day of activation) par<strong>the</strong>noteswere transferred to fresh medium and cultured <strong>for</strong> afur<strong>the</strong>r 4 days, after which blastocyst <strong>for</strong>mation wasdetermined.ResultsNo significant difference in cleavage rates wasobserved between <strong>the</strong> activation treatments thatproduce diploid parthogenomes (Table 1). However,<strong>the</strong> cleavage rate of ethanol treated eggs, whichproduces haploid parthogenomes, was significantlylower than <strong>the</strong> diploid treatments. No significantdifference in blastocyst <strong>for</strong>mation rates was observedbetween <strong>the</strong> A23187/6-DMAP and Eth/6-DMAPtreatments, however, a significantly higher rate wasobserved between Eth/cyclo/cytoB and Eth/6-DMAP(P
,S'\V:Y;n-vitro maturation, fertilisatio.n and early embryonic development in <strong>the</strong> dog'/ ~. Steven S. Metcalfe, Ian M. Gunn, Jillian M. ShawInstitute of Reproduction and Development, Monash Medical Centre, 246 Clayton Rd Clayton Vic 3168Introduction: In-vitro maturation (IVM) of canine oocytesis an underdeveloped procedure because of <strong>the</strong> peculiaritiesof canine reproductive biology compared to o<strong>the</strong>r domesticspecies. Canine oocytes are ovulated at <strong>the</strong> germinal vesiclestage and undergo maturation to metaphase II within <strong>the</strong>oviducts. Successful IVM and IVF to produce cleavedembryos has been documented only twice in <strong>the</strong> dog (1,2).The most advanced embryo produced was an 8-cell (1).Supplementation of media with follicular fluid during IVMand IVF has been beneficial in o<strong>the</strong>r domestic species (3,4).The aim of this study was to explore a simplified method <strong>for</strong>IVM, IVF and IVP of early cleavage dog embryos usingfollicular fluid supplementation during IVM and IVF.Materials and Methods: Nine unstimulated LabradorRetriever bitches were used as ovary donors. Starting within7 days from <strong>the</strong> onset of pro-oestrus, serum progesteronewas measured at 24-hour intervals by chemiluminescentimmunoassay that had been previously validated in canines.Ovaries were recovered by ovariohysterectomy per<strong>for</strong>medwhen serum progesterone levels were 9.1-24.4 nmol L- 1 •Cumulus oocyte complexes (COCs) were aspirated fromlarge preovulatory follicles (>5mm) using a short-bevel, 16G needle. COCs from small antral follicles «5mm) wererecovered after repetitive slicing of <strong>the</strong> ovary with a #22scalpel blade.All COCs were matured in syn<strong>the</strong>tic oviductal fluid (SOF).Small antral COCs were supplemented with 5-10 % pooled,autologous follicular fluid. All maturation was per<strong>for</strong>med in400 JlL pre-equilibrated media in humidified 4 well Nuncplates, without mineral oil, at 39°C in modular incubatorsgassed with 5% CO 2 , 7% 0 2, 88% N 2 • After 24 hours ofmaturation, <strong>the</strong> COCs were washed once in SOF and <strong>the</strong>ntransferred to 400 JlL of pre-equilibrated TALP containing5% pooled follicular fluid <strong>for</strong> fertilisation.('·IVF: Ei<strong>the</strong>r of two Labrador Retrievers with recent histories')! of successfully siring litters were used as semen donors.,::y Semen was collected by masturbation using an artificial)~ vagina. Only <strong>the</strong> first and second (sperm rich) fractions ofJ <strong>the</strong> ejaculate with sperm motility> 90% were used. Sementr' was diluted 1:1 in Tris-citrate-fructose extender and chilled\ ..j . to 5°C until use. All inseminations were per<strong>for</strong>med within"1 24 hours of collection. Semen was prepared bycentrifugation and 10-min. swim-up in pre-equilibratedTALP. Insemination used 9.9 to 20.5 x 10 5 total motilesperm.IVP: After 18-24 hours <strong>the</strong> presumed zygotes were gentlypipetted to remove excess cumulus cells and ·sperm, <strong>the</strong>ntransferred to 400 J..LL of pre-equilibrated SOP.74After 72 hours <strong>the</strong> embryos were denuded andexamined <strong>for</strong> polar body extrusion and blastomerenumber to determine stage ofdevelopment.Results: In-vitro maturation, fertilisation andembryonic development to <strong>the</strong> 8-cell stage didoccur (Table 1).Table 1.COC sourceI(2.2) II7 5 4 5 5(5.3) (3.8) (3.0) (3.8) (3.8)PB =polar body, c =blastomere numberConclusion: This experiment confirmed Yamada1sresults that IVM, IVF and IVP to <strong>the</strong> 8-cell stage ispossible. Yamada achieved 9/148 (6%) of embryosreaching <strong>the</strong> 5-8-cell stage.A number of o<strong>the</strong>r conclusions and comparisonsare made which describe <strong>the</strong> simplification of <strong>the</strong>protocol. Unlike <strong>the</strong> previous studies (1,2), preincubationof sperm to enhance capacitation wasnot necessary to achieve fertilisation. Eight cellembryos were produced after only 24 hours invitromaturation ra<strong>the</strong>r than 72 hours (1,2).Oocytes recovered from small antral follicles aswell as from large preovulatory follicles arecapable of IVM, IVF and cleaving although <strong>the</strong>low numbers preclude meaningful statisticalcomparisons. Oocytes recovered fromunstimulated, naturally cycling bitches were usedin this experiment which is in contrast to <strong>the</strong>bitches 'superovulated' with gonadotrophins usedby Yamada et al.References:1. Yamada S et at (1992). Bioi Reprod 46:853-858.2. Hewitt D (1997). PhD Thesis. Univ London3. Sun et at (1994). Therio 41:981-988.4. Choi et at (1998). Therio 49: 1103-1112.Research funding provided by Guide DogsAssociations ofVictoria.N_ACETYL-D-GLUCOSAMINE DAMAGES SPERM FROM THE TAMMAR WHEN RECOVERED FROMEJACULATES BY SWIM-UP PROCEDURESR.N. Murdoch, M.A. Wade, and M. LinCRC <strong>for</strong> Conservation and Management ofMarsupials, Department ofBiological Sciences, The University ofNewcastle, NSW 2308, Australia.IntroductionThe semen of marsupials contains free N-acetyl-Dglucosamine(NAG) as <strong>the</strong> major sugar in seminalplasma (1,2). It exists in <strong>the</strong> ~emen of <strong>the</strong> tammar(Macropus eugenii) at a concentration of about 508mg/dl. Tammar epididymal sperm readily utilise NAGand accumulate <strong>the</strong> sugar intracellularly at a rate fourtimes faster than glucose (3). Since tammar spermnormally are only exposed to NAG in seminal plasmafollowing ejaculation, <strong>the</strong> response of ejaculated spermto <strong>the</strong> sugar was examined in <strong>the</strong> present study. Sincetammar semen rapidly coagulates on ejaculation, it wasnecessary to recover motile sperm from ejaculatesusing swim-up procedures. Various swim-up mediawere tested in an attempt to define conditions thatwould minimise <strong>the</strong> adverse effects that this recoveryprocess can exert on sperm.Materials & MethodsSemen was collected from six tammar wallabies byelectroejaculation. Motile sperm were recovered byswim-up <strong>for</strong> 30 min at 34°C into 5-10 ml of bufferedmedium placed over <strong>the</strong> ejaculate. Three media wereused. Medium 1 was Krebs-Ringer phosphate (KRP)buffer (3). Medium 2 was KRP buffer containing 1mglml sodium hyaluronate, 1 mM Desferal, and 0.0003%(w/v) catalase (4). Medium 3 was EMEM containing10% (v/v) fetal calf serum and 25 mM HEPES. Allmedia (pH 7.4) contained penicillin (l00 IU/ml),streptomycin (50 !J.g/ml) and 0.23 mM pyruvate.Following swim up, <strong>the</strong> sperm-rich medium was removedand centrifuged (500 g, 5 min) at room temperature. Thesperm plug was dispersed in 1.5 ml of pyruvate-free KRPbuffer. Aliquots of <strong>the</strong> suspension were ei<strong>the</strong>r fixed <strong>for</strong>examination by electron microscopy (EM) (5) orincubated in 1 ml of KRP <strong>for</strong> 2 h at 34°C in <strong>the</strong> presenceof no added substrate, 5 mM glucose, or 5 mM NAG.Motility was assessed microscopically and aliquots of <strong>the</strong>suspension were fixed <strong>for</strong> examination by EM.ResultsSperm motility scores (scale 0-10) immediatelyfollowing swim-up into <strong>the</strong> various buffered mediawere generally high (mean = 8.4 ± 0.74) and nosignificant differences were detected between media.Following 2 h subsequent incubation in <strong>the</strong> presenceand absence of added glucose or NAG, motility scoresfell to between 5.0 ± 1.0 and 6.6 ± 1.8. However, in <strong>the</strong>case of sperm recovered by swim-up into KRP andsubsequently incubated <strong>for</strong> 2 h in KRP containingNAG, <strong>the</strong> mean motility score fell to 1.2 ± 0.6 with nomotile sperm being detected 30 min later. Whensections were examined using <strong>the</strong> electron microscope,substantial membrane damage was detected in <strong>the</strong> postnuclearhead and mid-piece region in all sperm exposedto NAG following swim-up, irrespective of <strong>the</strong> mediumused <strong>for</strong> recovery. Less severe alterations were evidentin sperm subsequently exposed to glucose, and nomajor ultrastructural disturbances were detected insperm incubated in <strong>the</strong> absence of added substratesfollowing swim-up.ConclusionsThe results indicate that <strong>the</strong> ultrastructure and motilityof tammar sperm recovered from ejaculates by variousswim-up procedures is impaired by subsequentexposure to NAG. The inclusion in media of agentssuch as ·hyaluronate, serum albumin, catalase andDesferal af<strong>for</strong>ded some protection to <strong>the</strong> sperm duringswim-up, as reported <strong>for</strong> eu<strong>the</strong>rian species (4), but<strong>the</strong>ireffects were only partial in this context. Thus, while<strong>the</strong>se agents prevented <strong>the</strong> complete deterioration ofsperm motility, <strong>the</strong>y failed to prevent NAG- and glucoseinducedultrastructural changes. The adverse effects ofNAG may be due to <strong>the</strong> excessive dilution of <strong>the</strong> spermby <strong>the</strong> swim-up procedures. This may alter <strong>the</strong>permeability of <strong>the</strong> membrane with loss of enzymesthat process <strong>the</strong> NH 3 generated during <strong>the</strong> metabolismof NAG. The collection of viable sperm from ejaculatesis vital to research into <strong>the</strong> control of fertility ofmarsupials and is <strong>the</strong> subject of our continuinginvestigations.References1. Rodger JC, White IG (1974) J Reprod Fertil39: 383-386.2. Rodger JC, White LG. (1976) J Reprod Fertil46: 467-469.3. Murdoch RN, Jones RC (1998) Molec Reprod Devel 49:92-99.4. Vishwanath R, Shannon P (1997) Reprod Fertil Dev 9:321-331.5. Lin M, Harman A, Rodger JC (1997) J Anat 190: 377-395.75
SEMEN COLLECTION, CHARACTERISATION AND CRYOPRESERVATION IN A MAGELLANICPENGUIN (Spheniscus magellanicus).J. K. O'Brien, D. A. Oehler, S. P. Malowski and T. L. RothCenter <strong>for</strong> Research ofEndangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA.IntroductionSeven of <strong>the</strong> 17 penguin species in existence areendangered (1). A better understanding of avianreproduction and <strong>the</strong> application of assisted breedingtechniques such as semen preservation and artificialinsemination (AI) may become important <strong>for</strong> captivepropagation and long-term survival of <strong>the</strong>se species.However, in<strong>for</strong>mation on ejaculate characteristics andsemen preservation is not available <strong>for</strong> any penguinspecies. The Magellanic penguin is not endangered, butits reproductive success in <strong>the</strong> wild has been decliningduring <strong>the</strong> past decade (2). The objectives of <strong>the</strong>present study were to: (i) develop a cooperative method<strong>for</strong> collecting Magellanic penguin semen; (ii)characterise <strong>the</strong> ejaculate parameters and semenproduction during a breeding season; (iii) compareholding temperatures <strong>for</strong> short-term storage; and (iv)compare cryoprotectants <strong>for</strong> long-term storage.Materials & MethodsSemen was obtained from a hand-reared adult maleusing a cooperative collection method. Ejaculates wereassessed <strong>for</strong> volume, pH and osmolality. Sperm qualitywas assessed by evaluating motility and <strong>for</strong>wardprogression (sperm motility index, SMI), viability(Hoechst 33258) and morphology.For short-term storage (n=3 replicates), semen wasdiluted (1:3; semen:diluent) with Beltsville PoultrySemen Extender (BPSE; 3) within 10 min of collection.BPSE pH and osmolarity were adjusted to simulateMagellanic penguin semen pH (7.4 ± 0.5) andosmolarity (430 ± 10 mosm/kg). The diluted semen wasdivided into 2 aliquots and held at 4°C or 21°C. Spermquality assessments were made at 0, 1 and 3 h postdilution.For long-term storage (n=3 replicates), diluted (l:1)semen was fur<strong>the</strong>r diluted (1:1) with BPSE containingdimethylsulfoxide (DMSO) or ethylene glycol (EG),and equilibrated <strong>for</strong> 15 min (final cryoprotectantconcentration: 8%). Straws were frozen on dry ice <strong>for</strong> 5min and transferred to liquid nitrogen. Semen wasthawed (n=2 straws/sample) by holding straws <strong>for</strong> 5 sinair and <strong>the</strong>n by placing <strong>the</strong>m in a 50°C waterbath <strong>for</strong> 10s. Data were analysed by analysis of variance, andmeans were compared using Students t-tests or LSDtests. P0.05) in spermcharacteristics between treatments <strong>for</strong> short-termstorage, although <strong>the</strong>re appeared to be a slightadvantage to maintaining semen at 4°C. Semen samplesmaintained high numbers of viable (77.8 ± 5.4%) andmorphologically normal (67.9 ± 2.5%) sperm at 3 hours(data <strong>for</strong> both treatments combined). SMI and percentviability decreased (P0.05) ascryoprotectants <strong>for</strong> penguin spermatozoa (data notshown). Frozen-thawed semen maintained 68.5 ± 4.6%and 78.4 ± 3.2% of its pre-freeze SMI and viability,respectively (data pooled <strong>for</strong> cryoprotectant). Both SMIand viability remained relatively stable during <strong>the</strong> 3 hstorage interval post-thaw (Table 1). Similar to freshlycollected ejaculates, frozen-thawed semen exhibited anincrease in bent cells (P
REGULATION OF PROTEIN TYROSINE PHOSPHORYLATION, DURING IN VITRO AGING OFEJACULATED BOVINE SPERMATOZOA.J. Krzyzosiak1,2*, G. McMillan 1 , R. Vishwanath l and P. Molan 2lUvestock Improvement Corporation, Private Bag 3016, Hamilton, New Zealand, 2Department of Biological Sciences,University ofWaikato, Private Bag 3105, Hamilton, New ZealandINTRODUCTIONTyrosine phosphorylation of various sperm proteinsplays a role in regulation of motility, capacitation, andzona pellucida binding of sperm (1). Reactive oxygenspecies promote tyrosine phosphorylation of spermproteins during capacitation (2). The objective was todetermine if changes in protein-tyrosinephosphorylation occur during in vitro storage, and if<strong>the</strong>y are affected by <strong>the</strong> oxygenation state of <strong>the</strong> storagemedium. The study was extended to determine whe<strong>the</strong>rprotein phosphorylation taking place during storageunder anaerobic and aerobic conditions are reversiblewhen spermatozoa are transferred from oneenvironment to ano<strong>the</strong>r.METHODSSemen collected from three bulls, was diluted in acommercial diluent (CAPROGEN®) (3), divided andstored under anaerobic or aerobic conditions at ambienttemperature (18-20°C). On day 5 of in vitro storage,one half of <strong>the</strong> semen aged under anaerobic conditionswas transferred from an anaerobic chamber to anaerobic environment. Similarly, one half of <strong>the</strong> semenaged under aerobic conditions was transferred to <strong>the</strong>anaerobic chamber. Proteins were extracted from cellsharvested and washed on days 6, 9, and 12 of storageunder <strong>the</strong> four conditions. Tyrosine-phosphorylatedproteins were separated by SDS-PAGE and imrnunodetectedon a Western blot with 4G10 antiphosphotyrosineantibody using an ECL detectionsystem.RESULTSWe observed a time dependent increase in tyrosinephosphorylation of number of proteins under anaerobicbut not under aerobic conditions. When sperm agedunder anaerobic conditions were transferred to <strong>the</strong>aerobic environment, proteins that were phosphorylatedup to this point were dephosphorylated duringsubsequent aerobic incubation. Conversely when sperminitially incubated under aerobic conditions weretransferred to an anaerobic environment, proteins whichdid not display increased protein tyrosinephosphorylation until this point became progressivelyphosphorylated on tyrosine residues (Fig. 1).DISCUSSIONBoth protein tyrosine kinase and phosphatase activitiesare affected by <strong>the</strong> presence of O 2 in <strong>the</strong> medium, but<strong>the</strong> effect is <strong>the</strong> opposite of that which is documented<strong>for</strong> sperm undergoing capacitation (4). Changes intyrosine phosphorylation are very slow (12 days) when78compared to typical receptor mediated tyrosinephosphorylation (15 s), or those observed in ejaculatedbovine, sperm incubated under capacitating conditions(4 h) (1).o 6 9 12 6 9 12 6 9 12 6 9 12Fig. 1 Western blot showing tyrosine-phosphorylatedproteinsfrom: semen aged all <strong>the</strong> time under anaerobicconditions (lanes 2-4), semen aged all <strong>the</strong> time underaerobic conditions (lanes 5-7), semen transferred onday five from an anaerobic to an aerobic environment(lanes 8-10), semen transferred on day five from anaerobic to an anaerobic environment (lanes 11-13).Storage time (days) is displayed under <strong>the</strong> blot.In conclusion, an increase in protein tyrosinephosphorylation during ageing is probably differentfrom <strong>the</strong> one observed during capacitation since, unlike<strong>the</strong> latter, it is inhibited in <strong>the</strong> presence of O 2 and isreversed upon transfer of sperm cells from an anaerobicto aerobic environment. Fur<strong>the</strong>rmore, it occurs across amuch longer time span than capacitation. Theidentification of proteins that undergo tyrosinephosphorylation changes during ageing would enable abetter understanding of <strong>the</strong> role <strong>the</strong>y play in thisprocess.REFERENCESl.Galantino-Homer HL, Visc~nti PE, Kopf GS,BiolofReprod, 1997; 56: 707-719.2.Aitken RJ, Paterson M, Fisher H, Buckingham DW,van Duin M J Cell Sci, 1995; 108: 2017-2025.3.Shannon P, J Dairy Sci, 1965; 48: 1357-1361.4.Aitken R, Buckingham D, Harkiss D, Paterson M,Fisher H, Irvine D, Mol Cell Endocrinol, 1996; 17:8393.THE EFFECT OF IN VITRO STORAGE ON MITOCHONDRIAL MARKER ENZYME ACTIVITYIN BOVINE SPERMATOZOAtD. V. R. Bullen I . 2 , P. MolanI, and R. Vishwanath 2lUniversity of \Vaikato, Department ofBiological Sciences, Private Bag 3105, Hamilton 2001, New Zealand, 2LivestockImprovement Corporation Ltd., Private Bag 3016, Hamilton 2001, New ZealandINTRODUCTIONBovine sperm lose fertility rapidly during in vitrostorage at room temperature. Motility is alsogradually lost but at a notably lower rate (1). It hasbeen proposed that <strong>the</strong> more rapid loss of fertilitymay be due to <strong>the</strong> effects of reactive oxygenspecies (ROS) that may be generated in <strong>the</strong> courseof respiration.As part of a larger investigation into <strong>the</strong> effectsof storage in vitro on various biochemicalparameters of sperm mitochondria, <strong>the</strong> activity ofthree mitochondrial marker enzymes wasmonitored.The enzyme assays described in <strong>the</strong> literaturehave been applied almost exclusively to somaticmitochondria or purified enzymes (2-4). For thisreason, work was firstly carried out to determinewhe<strong>the</strong>r <strong>the</strong> assays could be reliably applied to anenzyme preparation extracted from spermmitochondria. Then, enzyme activity wasmonitored at regular intervals, throughout a periodof storage.The aim of this study was to determine whe<strong>the</strong>r<strong>the</strong> activity of mitochondrial marker enzymes waschanging during <strong>the</strong> storage period and to assess<strong>the</strong> effect of <strong>the</strong> storage conditions (aerobic oranaerobic) on any changes taking place.METHODSSemen was collected from mature Friesian bullsand diluted in 14G buffer (5). The semen wasstored ei<strong>the</strong>r aerobically or anaerobically, at roomtemperature (20°C).Sperm extracts were prepared, <strong>for</strong> use in <strong>the</strong>enzyme assays, by sonicating sperm samples in anextraction buffer containing 200mM Tris, 2%sodium citrate, and 0.1% Triton X-100. Theenzyme activity of sperm extracts were analysed at0,6, 12, and 20 days after collection.Three enzymes were assayed: citrate synthase(2); cytochrome c oxidase (3); ATP syn<strong>the</strong>tase(4).RESULTSOver a period of 20 days <strong>the</strong> activity of bothcitrate synthase and ATP syn<strong>the</strong>tase decreasedsignificantly (<strong>for</strong> both enzymes p < 0.001). Forcitrate synthase <strong>the</strong> difference in storageatmosphere had a significant effect on activity (p =0.005), with activity decreasing more in <strong>the</strong>aerobically stored semen than anaerobically stored.The third enzyme, cytochrome c oxidase, showedan increase in activity over time, with <strong>the</strong>difference in storage atmosphere causing asignificant difference in activity (p = 0.001; Fig. 1).The increase in cytochrome c oxidase activity wassignificant <strong>for</strong> sperm stored anaerobically (p
Effect of calcium on <strong>the</strong> in vitro fertilisation of in vitro maturedporcine oocytes by frozen epididymal spermatozoa.A. Preshaw, W.M.C. Maxwell and G. EvansDepartment of Animal Science, University of Sydney, NSW 2006, AustraliaCONFOCAL ANALYSIS OF DECONDENSATION AND PRONUCLEAR FORMATION IN HUMANSPERM INJECTED INTO MURINE OOCYTESSandra Galea, Orly Lacham Kaplan and Alan Trounson.Institute of Reproduction and Development, Monash University, 27-32 Wright street, Clayton Victoria Australia.IntroductionThe use of frozen epididymal sperm <strong>for</strong> in vitro fertilisation can result in low fertilisation rates despite highmotility of sperm (1). Intracellular calcium increases during <strong>the</strong> acrosome reaction of sperm but its role incapacitation is not fully understood (2). This study was undertaken to determine if doubling calciumconcentration and increasing sperm concentration could increase fertilisation rates of frozen-thawedepididymal sperm.Methods and MaterialsSperm were aspirated from slaughterhouse epididymides, by retrograde injection of air through <strong>the</strong> vasdeferens, and <strong>the</strong>n frozen in a TFE diluent (3) at a concentration of 500 x 10 6 sperm/ml. Oocytes wereaspirated from 2-4 mm follicles on pre-pubertal slaughterhouse ovaries and matured <strong>for</strong> 44 hr. Oocytes were<strong>the</strong>n fertilised in 100 III droplets of TALP-PVA (4) + 4 roM caffeine containing 4.66 or 9.32 roM CaCh and62.5 x 10 3 , 125 X 10 3 , 250 X 10 3 or 500 x 10 3 sperm/ml. Oocyte maturation and fertilisation were carried outat 39°C in 5% CO 2 in air. Oocytes were removed 17 h after insemination, stained with Hoechst 33342 andassessed <strong>for</strong> maturation and fertilisation under UV light. Six replicates were run and <strong>the</strong> data were analysedby ANOVA and are presented in Tables 1 and 2.Table 1. Mean monospermic, polyspermic and total fertilisation rates of oocytes in media with differingCaCI 2 content ( a11 f our sperm concentratIOns . )Calcium No of Mean monospermic Mean polyspermic Mean totalconcentration (roM) oocytes fertilisation fertilisation fertilisation(% ±SE) (% + SE) (% + SE)4.66 650 20.8 + 2.2 a 6.4 + 2.0 a 27.3 + 2.8 a9.32 717 35.0 ± 2.2 b 15.2 + 2.0 b 50.2 + 2.8 bMeans WIth dIfferent superscnpts in <strong>the</strong> same columns are significantly different (P
PRELIMINARY STUDY: HIGH FERTILISATION RATES PRODUCED WITH PROLONGEDSTORAGE OF MALE FACTOR PATIENT SPERM FOLLOWING ICSI INTO MURINE OOCYTES.Sandra Galea, Orly Lacham Kaplan and Alan Trounson.Institute of Reproduction and Development, Monash University, 27-32 Wright street, Clayton Victoria Australia.IntroductionThe advent of intracytoplasmic sperm injection hasenable previously infertile males to fa<strong>the</strong>r <strong>the</strong>ir ownbiological children. The widespread adoption of ICSIin infertility clinics, however, has been questioneddue to <strong>the</strong> poor quality of sperm used to essentially<strong>for</strong>ce fertilisation. Of major concern are highfertilisation failure rates of up to 50% with ICSI.The fertilising competency of a sperm sample is notassessed prior to its use in ICSI treatment. This is inpart due to <strong>the</strong> inability in obtaining human oocytes<strong>for</strong> research purposes. Direct injection of humansperm into mouse oocytes provides a reliable andaccurate model of human fertilisation 1.Materials & MethodsSperm samples from two males presenting withrepeated fertilisation failure with ICSI treatment wereused <strong>for</strong> analysis. Microinjection was conducted overthree consecutive days, with day 1 corresponding to<strong>the</strong> day of sperm collection.Single morphologically normal and motile spermwere injected into morphologically normal andmature metaphase II oocytes. Injected oocytes wereleft in culture <strong>for</strong> 6-10 hours at which timefertilisation status was recorded. Capacitated spermsamples were stored in <strong>the</strong> dark at 20-23°C <strong>for</strong> up to72 hours.ResultsTable 1 represents fertilisation results obtained <strong>for</strong>eachcase studlY.leASE 1 I#INJ SUR. (%) #FERT (%):OAyT-TI28 4 (14) 0(0)iDAY2 I 29 4 (13.8) 2 (50)*DAY 3 I 40 8 (20) 6 (75)*gAS§.~J#INJ SUR. (%) #FERT (%)DAY 1 I 30 10 (33) 0(0)DAY 2 I 35 17 (48) 12 (53)*,DAY3 I 36 19 (53) 19 (100)*. .* mdicates slgmflcance at p«0.05).Legend:INJ - number of injected oocytes.SUR - number of oocytes surviving injection.FERT - number of oocytes with 2PBs & 2 PNs82A significant difference in fertilisation rate wasobtained between day 1 and day 2-3 <strong>for</strong> each patientwith up to 100% fertilisation being achieved in Case2 (table 1).ConclusionsProlonged sperm incubation at room temperaturebe<strong>for</strong>e ICSI may enhance fertilisation in some ICSIpatients.Increased fertilisation produced as a result ofprolonged sperm storage may be improved due toimproved sperm selection. The swim-up procedureproduces a concentration of motile sperm thatdecreases progressively as sperm begin to weakenand die. There<strong>for</strong>e, sperm used over 72 hours postejaculation represent <strong>the</strong> 'fittest' sperm in <strong>the</strong> sample,being more likely to initiate fertilisation.However, <strong>the</strong> high fertilisation rates produced in <strong>the</strong>present study may also be caused by damage incurredto sperm chromatin by <strong>the</strong> production of reactiveoxygen species (ROS). ROS can be produced through<strong>the</strong> repeated centrifugation protocols established <strong>for</strong>sperm preparation also through exposure to oxygenas would occur with prolonged storage. ROS have <strong>the</strong>potential to induce DNA fragmentation 2 producing aweakening of di-sulfide and protamine bonds within<strong>the</strong> sperm nucleus, resulting in partially decondensedsperm. When such sperm are injected into an oocyte,<strong>the</strong>y rapidly decondense and <strong>for</strong>m pronuclei.Although it may be beneficial to increase storage of asperm sample to increase fertilisation in severe malefactor patients, fur<strong>the</strong>r studies on post fertilisationand embryonic development are required todetermine embryo viability.References(1) Yanagimuchi, R. (1998) Hum. Reprod. 13(Suppl. 1):87-89.(2) Twigg, 1., Irvine, D.S., Houston, P. et al (1998) Mol.Hum. Reprod. 4: 439-45.Grant SupportMonash-IVF.PROTEOGLYCANSANDGLYCOSANUNOGLYCANSOF SMALL BOVINE OVARIAN FOLLICLESME McArthur*#, S Byers#, HF Irving-Rodgers* and RJ Rodgers**Department of Medicine, Flinders University of South Australia, Bed<strong>for</strong>d Park SA 5042# Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide SA 5006IntroductionProteoglycans (PGs) are major components of <strong>the</strong> extracellular matrix and have been implicated as having roles incellular proliferation, differentiation, growth factor regulation and tissue architecture. Ovarian follicles of many speciesare known to contain glycosaminoglycans (GAGs) 0,2). However <strong>the</strong> species of PGs that <strong>the</strong>y are associated with haveyet to be identified. The aim of this study was to isolate and characterise <strong>the</strong> PGs present within bovine ovarian follicles.Materials and MethodsFollicles: Ovaries were collected from non-pregnant, cycling heifers from a local abattoir within 20 min of slaughter,placed in hepes-buffered Earle's balanced-salt solution (EBSS) containing protease inhibitors. Follicles of between 1-3mm were dissected from whole ovaries. Ten percent of <strong>the</strong>se follicles were randomly chosen and processed <strong>for</strong> lightmicroscopy to assess follicular health. Eighty five percent of <strong>the</strong>se follicles were healthy, <strong>the</strong> remainder were in earlyatresia.Radiolabelling of Follicular Proteoglycans: A batch of follicles were incubated with 35S in an overnight culture, and<strong>the</strong>se added to <strong>the</strong> non radiolabelled follicles prior to extraction.Proteoglycan extraction: Sequential extractions were carried out on <strong>the</strong> pooled follicles (total weight = 5.43g).1 Initial extractions in 6M urea followed by 6M urea plus 0.1 % triton XI00 were pooled and treated as one extract.2 Extraction with O.IM NaOH to solubilise <strong>the</strong> remaining tissue.Chromatography: Anion-exchange chromatography on DEAE Sephacel columns was used to separate <strong>the</strong> PGs in <strong>the</strong>extracts <strong>for</strong> a large proportion of soluble proteins also present in <strong>the</strong> follicle. Size-exclusion chromatography of <strong>the</strong>urea/triton and sodium hydroxide extracts was per<strong>for</strong>med on 90cm Sepharose CL2B and CUB columns.ELISA assays were used to detect <strong>the</strong> GAG's and PGs present in urea/triton extracts eluted from size exclusion columns.These detected <strong>the</strong> epitopes present on 4 or 6-sulphated chondroitin sulphate/dermatin sulphate (CSIDS), <strong>the</strong> CSIDSsmall leucine rich proteoglycan (SLRP) decorin, <strong>the</strong> SLRP biglycan, <strong>the</strong> large cartilage modular CSIDS aggrecan, <strong>the</strong>large modular heparan sulphate (HS) PG perlecan, and versican.GAG Characterisation: Nitrous acid was used to cleave HS side chains from PGs in <strong>the</strong> samples of NaOH extracts whichhad been dialysed, lysophilised and resuspended in H 2 0. Digestion with chondroitinase ACII lyase was used to digestchondroitin-4-sulphate side chains, or chondroitinase ABC lyase to digest both CS and DS side chains.Immunohistochemisty: Cyostat secions of fresh frozen ovaries were fixed in ei<strong>the</strong>r 4% para<strong>for</strong>maldehyde (4-sulphateSCIDS or versican) or PLP fixative (perlecan). Sections <strong>for</strong> 4-sulphate SCIDS and versican immunolocalisation werepredigested with chondroitin ABC lyase to expose <strong>the</strong> antigen. ABC amplification and a PAP detection system withDAB was used.ResultsBiochemistry: After anion exchange chromatography <strong>the</strong> urea/triton extraction yielded 144.9~g of GAG and <strong>the</strong> NaOHextraction yielded 29.5 ~g. The radioactivity was highest in <strong>the</strong> NaOH extract (96.7% of total incorporated) indicatingthat <strong>the</strong> GAGs syn<strong>the</strong>sized by follicles in culture were different from GAGs present in <strong>the</strong> uncultured follicles. 4sulphated CSIDS and HS were detected in <strong>the</strong> NaOH extract. DS was <strong>the</strong> major GAG present, while CS and HS werealso observed, confIrming <strong>the</strong> presence of follicular GAGs documented in <strong>the</strong> literature 0,2).The major GAG present in <strong>the</strong> urea/triton extracts was 4-sulphated CSIDS. Three peaks of CSIDS reactivity wereobserved, indicating <strong>the</strong> presence of at least three species of CSIDS PGs within <strong>the</strong> follicle. The first peak (Kav 0.240.57) was of a similar size to a CSIDS PG previously reported in <strong>the</strong> follicular fluid of bovine (1) and porcine (2)ovaries. The second peak was Kav 0.57-0.81. Immunoreactivity to <strong>the</strong> core protein of bovine perlecan andimmunoreactivity to versican overlapped this peak. Peak 3 (Kav 0.81-1.0) corresponded to a peak of reactivity to <strong>the</strong>antibody specific to <strong>the</strong> core protein of decorin.Immunohistochemistry: Perlecan was immunolocalised to <strong>the</strong> follicular basal lamina of antral follicles and <strong>the</strong>subendo<strong>the</strong>lial and smooth muscle basal laminas of vessels throughout <strong>the</strong> ovary. Immunoreactivity to 4-sulphatedCSIDS was detected in <strong>the</strong> connective tissue sheaths surrounding larger blood vessels. Antral follicles showed a variablestaining pattern, <strong>the</strong> <strong>the</strong>ca interna was often immunopositive and in some follicles <strong>the</strong> basal lamina also appeared to belabelled. Versican was immunolocalised to <strong>the</strong> ovarian stroma and connective tissue surrounding blood vessels as wellas <strong>the</strong> <strong>the</strong>ca interna and <strong>the</strong> basal lamina of large antral follicles.Discussion and ConlusionThese result <strong>for</strong> <strong>the</strong> first time identify three proteoglycans in ovarian follicles - decorin, perlecan and versican.1. Grimek HJ, Bellin ME, Ax RL (984) Chromatographic comparison of chondroitin-containing proteoglycans fromsmall and large bovine ovarian follicles. Biochem. Biophys. Res: Comm. 104, 1401-1406.2. Yanagishita M, Rodbard D, Hascall VC (1979) Isolation and characterization ofproteoglycans from porcine ovarianfollicular fluid. J. BioI. Chern. 254, 911-920.83
MORPHOLOGICAL CHANGES IN THE ME:MBRANA GRANULOSA AND BASAL LAMINADURING GROWTH AND REGRESSION OF BOVINE DOMINANT FOLLICLESHF Irving-Rodgers, ML Mussard*, RJ Rodgers, and JE Kinder*Department of Medicine, Flinders University of South Australia, Bed<strong>for</strong>d Park SA 5042, AustraFa*Departrnent of Animal Sciences, University of Nebraska, Lincoln, Nebraska, 68583, USAIntroductionTwo or three waves of follicles emerge in each bovine oestrous cycle. Follicles in each wave grow to about 5 mm indiameter be<strong>for</strong>e a smaller group of follicles continues to develop. The largest follicle develops into <strong>the</strong> dominant follicleand enlarges to a preovulatory size of> 10 nun and <strong>the</strong> subordinate follicles become atretic (1). Un<strong>for</strong>tunately <strong>the</strong>physiological regulation of recruitment and dominance is not well understood. This study was undertaken to order toexamine if <strong>the</strong> two different morphological appearances of <strong>the</strong> bovine follicular basal lamina observed previously (2) arerelated to <strong>the</strong> physiological growth phase offollicles.Materials and MethodsAnimals All cows were 2 ~ years old, nulliparous and of composite breeding; 1.4 Angus, 1.4 Here<strong>for</strong>d, 1.4 Pinzgauer, and1/.1 Red Poll. Oestrous was synchronized by two injections of prostaglandin F2a (Lutalyse). During <strong>the</strong> first follicularwave of <strong>the</strong> next oestrus cycle (between days 7-10, day 0 = oestrous), and once follicular dominance had been identified,all follicles 2: 5 nun were aspirated using an Aloka 500V ultrasound machine attached with a 5 MHz trans-vaginalaspiration probe. This initiated <strong>the</strong> synchronous emergence of a wave of growing follicles which were monitor by dailyultrasonography using a 7.5 MHz trans-rectal probe attached to <strong>the</strong> Aloka 500V machine. Ovaries were collected when<strong>the</strong> dominant follicle of <strong>the</strong> ensuing wave was in one of three development stages: 1) Growing; had reached dominance,was greater than 8 mm and had not reached plateau stage. 2) Plateau; had reached dominance, >8 mm and had staticgrowth <strong>for</strong> at least <strong>the</strong> 24 hours prior to ovary collection, but was still functionally dominant as determined by <strong>the</strong>absence of new growth of follicular development. 3) Regression; had reached dominance, but was starting to regress insize, and emergence of a new wave of follicular development was evident. Ovariectomies were per<strong>for</strong>med bylaparotomy via para-lumbar incision and if possible both ovaries were collected.Microscopy Following ovariectomies ovaries were placed into cold Hanks BSS and immediately transported to <strong>the</strong>laboratory. Ovaries were cannulated and flushed with 20ml of <strong>the</strong> Hanks solution be<strong>for</strong>e being fixed with 50ml 2.5%glutaraldehyde <strong>for</strong> 5-10 min and <strong>the</strong>n placed in fixative at 4°C overnight. The dominant follicle was identified, dissectedfrom <strong>the</strong> ovary, cut in half and <strong>the</strong> cross-sectional diameter measured. Small pieces of <strong>the</strong> follicle wall were taken andplaced in fixative and stored at 4°e. Fur<strong>the</strong>r processing was conducted following established electron microscopicmethods.ResultsMorphology of <strong>the</strong> membrana granulosaGrowing dominant follicles. Five follicles had a healthy granulosa layer and no or very few pyknotic nuclei and basalgranulosa cells that were rounded in shape. One follicle had cells with pyknotic nuclei located near <strong>the</strong> antrum.Plateau dominant follicles. All follicles had rounded granulosa cells, pyknotic nuclei were observed on <strong>the</strong> antral aspectof <strong>the</strong> membrana granulosa (2 of 4 follicles) or amongst basal cells (1 follicle).Regressing dominant follicles. These follicles were atretic,S follicles had rounded granulosa cells, <strong>the</strong> membranagranulosa of <strong>the</strong> o<strong>the</strong>r 2 follicles consisted of flattened cells. Pyknotic nuclei were common and more frequentlydetected towards <strong>the</strong> antrum (3 follicles), scattered through <strong>the</strong> entire granulosa layer (3 follicles) or in basal cells (1follicle). Dead cells in <strong>the</strong> antrum were also a common feature offollicles at this stage.Morphology of <strong>the</strong> Basal LaminaAll follicles examined, irrespective of <strong>the</strong>ir stage of growth or atresia, had a basal lamina that was a single layer andclosely aligned to <strong>the</strong> granulosa.GranulosaDominant Diameter Days Growth Rate # Thickness CellFollicle (nun) > 5 nun (mm1day) * {Jlm} * LayersGrowing (n=6) 9.1 ± 0.6 3.7 ± 0.7 1.2 + 0.4 a 57.5 ± 3.2 a 6.7 ± 0.7Plateau (n=4) 9.9 ± 0.5 7.5 ± 1.7 0.0±0.0 b 38.0 ± 22.2 ab 4.8 + 1.4Regressing (n=7) 9.0 ± 0.9 8.3 ± 0.9 -0.6 ± 0.1 c 27.6 + 5.6 b 4.0±0.7# Follicle growth rate <strong>for</strong> <strong>the</strong> 2 days prior to follicle collection Values are mean ± SEMDifferent superscripts indicate significant differences (* p < 0.05, ANOVA, SNK)BasalLaminaalignedalignedalignedIn conclusion, <strong>the</strong> morphological appearance of <strong>the</strong> basal lamina of bovine dominant follicles is not related to growth oratresia.References(1) Ginter OJ, Wiltbank Me, Fricke PM, Gibbons JR, Kot K (1996) Selection of <strong>the</strong> dominant follicle in cattle <strong>Biology</strong>ofReproduction 55: 1187-94(2) Rodgers HF and Rodgers RJ (1998) Ultrastructural changes in <strong>the</strong> follicular basal lamina ASRB Proceedings of<strong>the</strong>29th Annual Conference, 1584Changes in Marmoset Granulosa Cell Responsivness to FSH and IGF-IWith Follicular Growth.RB Gilchrist, LJ Ritter, RJ Norman and DT Armstrong.Department of Obstetrics & Gynaecology, The University of Adelaide, The Queen ElizabethHospital, Adelaide.Introduction : Despite common use of <strong>the</strong>marmoset as a laboratory primate <strong>the</strong> processand regulation of folliculogenesis' in thisspecies is poorly understood. The aims of thisstudy were to characterise changes inmarmoset mural granulosa cells (MGC)function with follicular growth, in terms ofresponsiveness to hormones know to beimportant in o<strong>the</strong>r species.Materials & Methods : Pairs of ovaries werecollected on day 4 of <strong>the</strong> follicular phase from7 adult marmosets primed with FSH. Allantral follicles were manually excised andsorted .into 5 classes according to size;periantral (420-640llm), small antral (>6401OOOllm), large antral (>1.0-1.4mm), verylarge antral (>lA-2mm) and preovulatory(>2mm). MGC were collected from puncturedfollicles and cultured <strong>for</strong> 18h followed by afur<strong>the</strong>r 6h pulse of 3H-thymidine to enableassessment of DNA syn<strong>the</strong>sis and provide anindication of cell proliferation. MGC weretreated with 50 ng/ml IGF-I, 10nglml porcineFSH or a combination of <strong>the</strong> two hormones.At <strong>the</strong> completion of culture, supernatantswere collected to allow assessment ofprogesterone production.Results : Regardless of treatment,incorporation of 3H-thymidine was very highin MGC from periantral follicles and generallyprogressively declined with increasing folliclesize (Figure 1). Whilst <strong>the</strong>re was a largedifference in <strong>the</strong> DNA syn<strong>the</strong>tic capacity ofMGC from periantral follicles and those from<strong>the</strong> next largest follicle class (average 3.7 foldhigher), <strong>the</strong>re was less difference amongstMGC from <strong>the</strong> antral follicles classes. IGF-Iproved to be a potent stimulator of MGCDNA syn<strong>the</strong>sis, increasing 3H countsapproximately equally in <strong>the</strong> different folliclegroups (average of 4.1 fold) above <strong>the</strong>irrespective control levels. FSH increased 3Hincorporation to a lesser extent; -2.8 fold. Thehighest levels of 3H incorporation were16....---------------------,1514.§ 13e 12o E' 11o~ 10~~ 9:§ 8isoS 7~ 6....=Q,I~:; 4~ 3Controlc:::::J Periantralcz:z:a Small AntralI2S2S2l Large Antral.... V. Large Antral_ PreovulatoryIGF-ITreatmentFSHFigure 1 : DNA syn<strong>the</strong>sis in marmoset muralgranulosa cells as influenced by follicle sizeand hormones in vitro.IGF+FSHobserved with FSH and IGF-I in combination, althoughthis effect was additive ra<strong>the</strong>r than synergistic.Contrasting <strong>the</strong> trends in cell proliferation, progesteroneproduction was lowest from MOC from periantralfollicles and increased with increasing follicular size. In<strong>the</strong> absence of hormones, mean progesterone levelsincreased 5.5 fold from 0.15 to 0.85 pmoles/1oo0 cellsin MOC from periantral and preovulatory folliclesrespectively. IOF-I had no effect on progesteroneproduction. In contrast, porcine FSH dramaticallyincreased progesterone secretion on average lOA foldabove control levels, with maximal levels of 5.30pmoles/1000 MOC from preovulatory follicles. No FSHx IGF-I interaction was observed in progesterone levels.Conclusions : At antrum <strong>for</strong>mation marmosetMGCundergo a dramatic transfonnation from rapidly growingto more differentiated cells. With increasing antralfollicle size, MOC retain some proliferativeresponsiveness to IOF-I however respond markedly toheterologous FSH with increasing progesteronesecretion.(Supported by The NH&MRC).85
THE EFFECTS OF TARGETED DISRUPTION OF THE CYP 19 (AROMATASE) GENE ONFOLLICULOGENESIS IN MICEKara L Britt, Ann E Drummond, Margaret E E Jones, Mitzilee Dyson, Nigel G Wre<strong>for</strong>d 1 ,Evan R Simpson and Joc.k K FindlayPrince Henry's Institute ofMedical Research, PO Box 5152, Clayton, Victoria 3168 andIDepartment of Anatomy, Monash University, Clayton, Victoria 3168, Australia.IntroductionA primary function of estrogen is to maintain <strong>the</strong> femalereproductive tract, notably ovarian folliculogenesis.Aromatase, a cytochrome P450 (P450 arom ) encoded by <strong>the</strong>cyp19 gene, catalyses <strong>the</strong> conversion of C l9 androgens toestrogens. Estrogen exerts its actions via <strong>the</strong> estrogenreceptors (ER), ERa and ERp, which are prevalent ongranulosa cells [1]. An aromatase knockout mouse(ArKO), generated via disruption of exon 9 of <strong>the</strong> cyp 19gene [2], allowed us to examine <strong>the</strong> local role of estrogenin ovarian folliculogenesis.Materials and MethodsBody, ovarian, gonadal fat and uterine weights fromwildtype, heterozygous and ArKO mice at 10-12, 16-18,21-26, weeks and one year of age were measured. Serumand ovaries were utilised <strong>for</strong> fur<strong>the</strong>r analyses. Ovarieswere ei<strong>the</strong>r fixed in <strong>for</strong>malin and paraffin embedded, orfrozen in OCT compound. Paraffin sections of wildtype,heterozygote and ArKO ovaries were histologically· andstereologically assessed. The numbers of primary,secondary and antral follicles were calculated using avariation of <strong>the</strong> fractionator method [3].Results and DiscussionThe ovarian weights of ArKO mice were reducedcompared to wildtype littermates up to 21-26 weeks ofage, at which time <strong>the</strong> weight of <strong>the</strong> ArKO ovaries were< 50% of wildtype. By one year of age ovarian weighthad increased, possibly due to <strong>the</strong> increase in connectivetissue. Uterine mass was decreased in <strong>the</strong> ArKO, to 50%of wildtype at 10-12 weeks and< 25% by one year. Gonadal fat pad weights of ArKOmice were increased by 2-fold compared to wildtype at 2126 weeks. ArKO ovaries at 10-12 weeks contained anincreased number of primary follicles compared toheterozygote littermates, and less secondary follicles (Fig1). At 21-23 weeks of age <strong>the</strong> number of primary follicleswas still greater than that observed in wildtype andheterozygote mice. By one year of age <strong>the</strong>re were nosecondary or antral fo,llicles present in <strong>the</strong> ArKO ovary.At 21-23 weeks <strong>the</strong> oocytes of heterozygote primaryfollicles had greater nuclear diameters than wildtype (Het111lm vs WT 9Ilm). Secondary and antral follicles showeda similar difference when compared to ArKO (Het 151lmvs ArKO llllm; Het l61lm vs ArKO 121lm respectively).A block in follicle development and a lack of corpora lutea(CL) within <strong>the</strong> ovaries was reflected in infertility at allages studied. Histological examination demonstrated that<strong>the</strong> ArKO ovary degenerated with age. Haemorrhagiccystic follicles appeared in <strong>the</strong> ovary at 21 weeks of ageand atresia, as measured by TUNEL assay, was observedto increase as a function of age. Coincident with <strong>the</strong> lossof follicles, extensive tissue remodelling, exemplified byan influx of macrophages and collagen deposition, wasobserved in <strong>the</strong> ArKO ovaries.8620015010050o~~ 250oi. 200j § 150c~ 100;:f 50o20015010050oo WT• HET1 0 -1 2 wee ks• KOFig 1: Effect ofage (a,b)and genotype (X,y) onfollicle number.Different letters denoting significance (* no follicles at thisstage ofdevelopment observed).Serum gonadotrophin (FSH and LH) levels measured bvradioimmunoassay were found to be elevated in <strong>the</strong> ArKOmice, and continued to increase with age. Interestingly at 1012 weeks heterozygote animals had increased FSH and LHlevels compared to wildtype, whilst remaining lower thanArKO levels. Earlier studies showed an increase intestosterone and undetectable estradiol levels in <strong>the</strong> ArKOmice [2]. The impact of <strong>the</strong> 10% soy contained within <strong>the</strong>mouse chow on <strong>the</strong> ovarian phenotype was investigated usingmice raised from birth on soy-free mouse chow. ArKO micemaintained on a soy-free diet displayed an earlier onset of <strong>the</strong>phenotype, with reduced ovarian weight, increased gonadal fatpads, and <strong>the</strong> appearance of haemorrhagic cysts at an earlierage (16 weeks). These results imply that phytoestrogenswithin <strong>the</strong> diet were acting as estrogens within <strong>the</strong> ovary andfat deposits of <strong>the</strong> mice. Thus mice on a soy-free diet providea more comprehensive model of estrogen deprivation.In summary, age-dependent disruption of folliculogenesis andovulation was observed in ArKO female mice, accounting <strong>for</strong><strong>the</strong>ir infertility. It is not clear whe<strong>the</strong>r <strong>the</strong> ovarian phenotypeobserved in <strong>the</strong> ArKO mouse is a direct result of <strong>the</strong> loss ofestrogen, or an indirect result of <strong>the</strong> elevated gonadotrophinsand testosterone or both.[1] Drummond AE et al., Mol. Cell. Endo. 1999 149:153[2] Fisher CR et al., PNAS. (USA). 1998; 95: 6965-6970.[3] Gundersen HJG et al., APMIS 1988; 96:857-881.Supported by NH&MRC of Australia (Regkey 983212)Polyovular Follicles in <strong>the</strong> Pouch Young and Adult KangarooSusan N. Reinke l , Ian M. Gunn!, Marilyn B. Renfree 2 , Alan O. Trounson l1Institute ofReproduction and Development, Monash University, Clayton, Victoria, 3168, Australia; 2 Department ofZoology, University ofMelbourne, Parkville, Victoria, 3052, Australia.Introduction. Polyovular follicles (follicles containingmore than one oocyte)-have been observed in a numberof mammalian species including <strong>the</strong> human (1), dog (2),mouse (3), wild Norway rat (4) and goat (5). Generally,polyovular follicles occur more frequently in immatureovaries although <strong>the</strong>y are also found in adults. Inmarsupials, <strong>the</strong> occurrence of polyovular follicles hasbeen observed in <strong>the</strong> American opossum, Didelphismarsupialis (6) and <strong>the</strong> Australian native cat, Dasyurusviverrinus (7). Similarly, it is noted that polyovularfollicles were first, and only, observed in <strong>the</strong> kangarooby van Beneden during <strong>the</strong> 1880's as previously cited(6). The aim of this study was to investigate whe<strong>the</strong>rpolyovular follicles occurred in two species of sexuallyimmature and mature kangaroos.Materials and Methods. Both eastern grey kangaroos(Macropus giganteus) and red kangaroos (Macropusrufus) were shot at Dubbo and Gunbar in NSW. Redkangaroos were also collected at Nymagee (NSW) andeastern grey kangaroos at Dunkeld (Vic) and Narromineand Warren in NSW. One ovary was scavenged fromeach female pouch young (PY) (n=6, ages 120 to 261days) and adult (n=28) eastern grey kangaroos, and fromfemale PY (n=3, ages 67 to 135 days) and adult (n=14)red kangaroos. The age of adult kangaroos was notdetermined. Whole, uncut ovarian tissue was fixed <strong>for</strong>light microscopy and <strong>the</strong> top ten sections were examined<strong>for</strong> polyovular follicles. Polyovular follicles wereclassified according to <strong>the</strong> system used <strong>for</strong> opossumovaries (6): Germ cell nests: oocytes in mutual contactand surrounded by a layer of flattened granulosa cells(Fig. 1); Polyovular follicles type I: oocytes separated bygranulosa cells but still within a single follicle (Fig. 2);Polyovular follicles type II: oocytes in mutual contact(Fig. 3); Polyovular follicles type III: oocytes withinfollicle found in a linear arrangement.Results and Discussion. The follicle populationconsisted of monovular follicles although polyovularfollicles were also found in PY and adult material. Alltypes of polyovular follicles with <strong>the</strong> exception of typeIII were observed. In <strong>the</strong> adult red kangaroo polyovularfollicles never contained more than two oocytes whilepolyovular follicles in <strong>the</strong> adult eastern grey kangaroofrequently had more than two oocytes. The mostcommonly observed polyovular follicle type was that ofgerm cell nests which were found in both species of PYand only in eastern grey adults. As <strong>the</strong> ages of adultswas not recorded, it is possible that some of <strong>the</strong>seindividuals were juveniles that had not yet completedovarian development and that <strong>the</strong> germ cell nests presentwere in <strong>the</strong> process of separation. Ano<strong>the</strong>r explanation<strong>for</strong> <strong>the</strong> presence of polyovular follicles may beenvironmental conditions which, as previously reported,?lays .a significant part in reproductive developmentmcludmg <strong>the</strong> delay of sexual maturity (8). There<strong>for</strong>e,<strong>the</strong> incidence of polyovular follicles observed in thisstudy could be a result of environmental factors, such aschemical sprays and toxins, which have been known toeffect reproductive organs.Conclusions. Polyovular follicles occur in both red andeastern grey kangaroos. Adult eastern grey kangarooshad three types of polyovular follicles but <strong>the</strong>y appearedless common than in <strong>the</strong> eastern grey pouch young.Fig. 1. Germ cell nest from adult female eastern greykangaroo. H & E, x 250. Bar =33J.l.m.Fig. 2. An atretic Graafian follicle containing two ova fromadult female eastern grey kangaroo. H & E, x 63. Bar =135J.l.m.Fig. 3. Biovular follicle from adult female eastern greykangaroo. H & E, x 160. Bar=53 J.l.m.References1. Arnold L (1912) Anat Rec. 6: 413-422.2. McDougall K, Hay MA, Goodrowe KL, Gartley CJ, King WA(1997) J Reprod Fert. 51: 25-31.3. Fekete E (1950) Anat Rec. 108: 699-707.4. Davis DE, Hall 0 (1950) Anat Rec. 107: 187-192.5. Joshi C, Nanda BS, Saigal RP (1976) Anat Anz. 139: 299-304.6. Hartman CO (1926) Am]Anat. 37: 1-51.7. O'Donoghue CH (1912) AnatAnz. 41: 353-368.8. Newsome A E (1965) Aust J 'hJo. 13: 735-759.87
GROWTH-PROMOTING ACTIVITY OF OOCYTES Is ATTAINED CONCOMITANT WITHTHE ACQUISITION OF MEIOTIC CO:MPETENCERB Gilchrist, LJ Ritter and DT Armstrong. -Department of Obstetrics & Gynaecology, The University of Adelaide, The Queen ElizabethHospital, Adelaide.Introduction : Cumulus cells are markedly less differentiated than mural granulosa cells (MGC),reflected in a higher proliferative capacity and in a reduced capacity to secrete progesterone. Wehave recently shown that this is due to a soluble factor(s) secreted by <strong>the</strong> oocyte which promotesgrowth and attenuates differentiation of follicular somatic 'cells (1). The aim of this study is todetermine <strong>the</strong> developmental expression throughout oogenesis of this oocyte-secreted growthfactor(s).",. :', r\Jtt)f' l~'~ c.~. . .............~i~f- i p"1:"-:'~ J',~. i-h t.~\r....L P
CYTOKINE NETWORKS IN THE ENDOMETRIUM AND OVARY DURINGEARLY PREGNANCYSarah A Robertson, Ph.D.Department of Obstetrics and Gynaecology and <strong>Reproductive</strong> Medicine Unit, The University of Adelaide,Adelaide SA 5005.C:':,;kines are clearly now identified as pivotal mediators of <strong>the</strong> dynamic and transient cell-cellc(,!nmunication events underpinning <strong>the</strong> growth, remodelling and demise so characteristic ofreproductive tract tissues. The intrinsic capacity of <strong>the</strong>se small glycoproteins to be producedtransiently, to act locally, and to be rapidly quenched is perfectly adapted to <strong>the</strong>ir roles as localmediators of endocrine hormone-governed cell proliferation, differentiation, activation and motility.\10st notably, cytokines regulate <strong>the</strong> interaction between somatic cells and infiltrating leukocytepopulations that are required to deal appropriately with <strong>the</strong> unique immunological challenges thataccompany ovulation, insemination, implantation and pregnancy.Our laboratory has focussed on <strong>the</strong> role of cytokines in endometrial biology, with a view toelucidating <strong>the</strong> cytokine pathways linking insemination with <strong>the</strong> cascade of molecular and cellularevents that culminate in implantation of <strong>the</strong> embryo. Our experiments in rodents and more recentlyin humans have demonstrated a role <strong>for</strong> seminal plamsa in 'priming' <strong>the</strong> female reproductive tract<strong>for</strong> successful implantation. Specific factors in seminal plasma including trans<strong>for</strong>ming growth factor(TGF)-~ stimulate uterine and cervical epi<strong>the</strong>lial cells to induce expression of an array of proinflammatorycytokines, which act subsequently to (1) initiate changes in <strong>the</strong> maternal immunecompartment facilitating induction of immunological tolerance to paternal transplantation antigens,and (2) promote <strong>the</strong> growth and development of <strong>the</strong> pre-implantation embryo.Complex cytokine networks also appear to operate in <strong>the</strong> ovary, most notably in regulating <strong>the</strong>trafficking and activation phenotype of myelQid leukocytes implicated in <strong>the</strong> tissue remodellingaccompanying ovulation and in <strong>the</strong> development, function and demise of <strong>the</strong> corpus luteum.CYTOKINE PRODUCTION AND ACTIONS IN HUMAN PREGNANCY AND PARTURITIONM.D. Mitchell and J.A. KeelanDept of Pharmacology and Clinical Pharmacology, University of Auckland, Private Bag 92019, Auckland,New ZealandThe importance of cytokines and modulation of immune cell function in successful pregnancy and parturitionhas become increasingly appreciated. The concept of pregnancy as a 'Th2 phenomenon', while not without itsflaws, provides a useful framework within which <strong>the</strong> roles and significance of cytokines in pregnancy can beinterpreted sensibly. The model views pregnancy as a condition requiring a bias towards humoral (T h 2)immunity and away from cell mediated (Thl) immunity: T h 2 cytokines (ie. ~-4, ~-10, TGF-(3) are viewed ashaving a protective role against <strong>the</strong> effects of pro-inflammatory (Thl-like) cytokines such as TNF-a, IL-l, ~2, rr...-6 and y-interferon, which are potentially harmful to placental (trophoblast) survival and pregnancymaintenance. The interesting dichotomy of this concept is <strong>the</strong> recognition that Thl cytokines are required <strong>for</strong>successful establishment of pregnancy. Many aspects of successful pregnancy can thus be viewed as a result ofa fine balance between <strong>the</strong>se two cytokine expression phenotypes. Conversely, several gestational pathologies(recurrent miscarriage, preeclampsia and preterm birth) have been found associated with a dominant Thlcytokine profile.Leukocytosis of <strong>the</strong> decidua and fetal membranes, accompanied by increased cytokine and eicosanoidproduction, is now known to be associated with preterm labour and delivery resulting from intrauterineinfection. Pro-inflammatory cytokines such as TNF-a (released from <strong>the</strong> membranes in response to infection)can act at multiple foci in <strong>the</strong> biosyn<strong>the</strong>tic pathway, exerting coordinated effects on intrauterine prostaglandinproduction. Enzymes involved in substrate release, conversion to active products, and inactivation throughmetabolism, are all targets <strong>for</strong> cytokine action. In addition, we have shown that at least one eicosanoid,thromboxane, is capable of stimulating cytokine production by amnion epi<strong>the</strong>lial cells, demonstrating <strong>the</strong>existence of a feed-<strong>for</strong>ward loop which may act to amplify <strong>the</strong> inflammatory cascade once initiated. Studieshave shown greatly elevated concentrations of cytokines (particularly amnion-derived cytokines, ~-6 and ~8) in amniotic fluid in pregnancies with confirmed chorioamnionitis, but also in preterm deliveries with nosigns of microbial invasion. Our studies of cytokine concentrations in amnion, choriodecidual and placentaltissues suggest that a large proportion of preterm deliveries are associated with increased cytokine productionin <strong>the</strong> membranes (but not placenta). We have interpreted this data as evidence of an inflammatory hyperresponsein <strong>the</strong> aetiology of a significant proportion of preterm deliveries.While Th2 cytokines might be expected to predominate during <strong>the</strong> majority of pregnancy, we postulated that aT h2 withdrawal could occur at time of delivery to allow <strong>the</strong> inflammatory processes of parturition to occurunhindered. Our recent data supports this hypo<strong>the</strong>sis, with findings of decreased ~-l0 production by <strong>the</strong>choriodecidua with normal term labour. More unexpected, however, was <strong>the</strong> finding that ~-10 exerted proinflammatoryeffects on <strong>the</strong> amnion at term, stimulating cytokine and prostaglandin production.-2 transcription in amnion.A better understanding of <strong>the</strong> precise roles of cytokine-mediated events during early pregnancy isnecessary to improve reproductive outcomes in humans and livestock or threatened species, whereimplantation failure is still <strong>the</strong> major cause of pregnancy loss.9091
EXISTENCE OF EXTRACELLULAR SIGNAL-RELATED KINASE (ERK) AND p38 MITOGENACTIVATED PROTEIN KINASE (MAPK) IN THE RAT CORPUS LUTEUM (CL).M.A. Abdo, M.A. Bogoyevitch l and A.M. DharmarajanDepartment of Anatomy & Human <strong>Biology</strong>, and lDepartment ofBiochemistry,University of Western Australia, Perth, Western Australia 6907..IntroductionThe MAPK signalling cascade has· been proposed ascritical to <strong>the</strong> events of cell survival and cell death,chiefly apoptosis, in a number of biological systems.Current evidence suggests that <strong>the</strong> selective activationof MAPK family members may be important in'determining a cell's fate. MAPK activation occurs inresponse to growth factors, heat shock, and cytokines(1). Tumor necrosis factor - alpha (TNFa) is acytokine that has been shown to activate both <strong>the</strong>apoptotic (p38 and c-JUN NH2-terminal protein kinase(JNK)), and anti-apoptotic (ERK) MAPK pathways indifferent cell systems (2). As reported previously,TNFa has been implicated in <strong>the</strong> induction ofapoptosis within <strong>the</strong> CL (3). Although <strong>the</strong>mechanisms of signal transduction in TNFa-mediatedapoptosis are largely unknown. Thus our aim in thispresent study was to determine whe<strong>the</strong>r MAP Kinases(p38, JNK, ERK) are present in <strong>the</strong> rat CL and whe<strong>the</strong>rchanges in <strong>the</strong>ir expression correlate with <strong>the</strong> onset ofspontaneous apoptosis and TNFa-induced apoptosis.Materials & MethodsTotal protein and DNA was isolated from pregnant rat(Day 16, Wistar) CL cultured under two experimentalconditions. CL were ei<strong>the</strong>r incubated without trophicsupport <strong>for</strong> up to 8h in minimal essential medium(MEM: Gibco BRL) to induce spontaneous apoptosis,or 6h in MEM supplemented with 30% fetal bovineserum (FBS: Trace Biosciences) and increasingconcentrations of recombinant rat TNFa (Genzyme).Proteins were resolved by SDS-PAGE and transferredto nitrocellulose membrane (0.45Ilm). Membraneswere <strong>the</strong>n incubated with <strong>the</strong> primary antibodies <strong>for</strong>ERKI (Santa Cruz) and ERK2 (Santa Cruz)simultaneously, JNKI (Santa Cruz), or p38 (SantaCruz) diluted 1:1000 <strong>for</strong> Ih at room temperature. Thiswas followed by incubation with sheep anti-goat IgGsecondary antibody (Sanofi Diagnostics) <strong>for</strong> Ih atroom temperature. The reaction was visualized usingECL detection (Pierce). All washes were in Trisbuffered saline supplemented with 0.1 % Tween 20. 3'end DNA labelling was per<strong>for</strong>med as describedpreviously (4). A minimum of 3 animals was used <strong>for</strong>each experimental variable.ResultsWestern blot analysis revealed <strong>the</strong> appropriatemolecular weight bands corresponding to ERKl&2(44 & 42 kDa) and p38 MAPK (38 kDa) (Fig. 1),~nder both culture conditions. No significant changeIn <strong>the</strong> amount of protein <strong>for</strong> ei<strong>the</strong>r p38 MAPK orERKI&2 was detected in response92to a 7-fold increase in DNA fragmentation after 8hincubation (Fig. 2) or to increasing concentrations of TNFa(2-fold increase in DNA fragmentation at 125ng/mL) (Fig.3). We were unable to demonstrate <strong>the</strong> presence of JNKthrough Western blot analysis under ei<strong>the</strong>r of <strong>the</strong>experimental conditions.Time.__---AFig 2 (left)Autoradiographo C'l '
Progesterone Downregulation of Matrix Metalloproteinase-l (Ml\1P-l)• a role <strong>for</strong> Ets transcription factors?L.M. Kilpatrick and L.A. SalamonsenPrince Henry's Institute ofMedical Research, P.O. Box 5152, Clayton, Vic, 3168.Bcl-2 family member expression patterns in mouse ~estisTerri Meehan\ Cris Print 2 , David M. de Kretser\ Kate Lovela.r:td .J Institute ofReproduction and Development, Monash Uni~ersity, Cl~yton, Ylct?na 3168,~ Walter and Eliza Hall Institute, Royal Melbourne HOSPital, ParkYdle, Vlctona 3050germ cellsIntroductionMatrix metalloproteinases (MMPs) are criticalenzymatic mediators of human endometrialremodelling. MMP expression is downregulated byseveral members of <strong>the</strong> steroid hormone receptorfamily, including androgens and progesterone (P). Themechanism <strong>for</strong> downregulation by P is unclear.However we postulate that, like androgens (1), Pdownregulates MMP-l expression through aninteraction with its receptor (PR) and Ets transcriptionfactors.Materials & MethodsThe human endometrial carcinoma cell line, Ishikawa,was transiently transfected with a full-length (4372bp)MMP-1 promoter construct or one of seven 5'-deletionconstructs linked to <strong>the</strong> luciferase (Iuc) reporter in <strong>the</strong>pGL3 basic vector. Transfections were per<strong>for</strong>med in <strong>the</strong>absence of endogenous P or estrogen. The effect onMMP-1 expression of co-transfection withcombinations of hPR and Ets1 and exposure of cells to1O. 7 M P was determined by luciferase assay at 26hours. Transfection efficiency was monitored by ~_galactosidase (~-gal ) transfection. Results werecorrected <strong>for</strong> transfection of pGL3 basic vector alone(background).Resultsto co-transfections of PR and 2002 bp or 1551 bp of 3'sequence had no fur<strong>the</strong>r downregulatory effect. In cotransfectionsof :::;;1194 bp of MMP-1 promoter + Ets1 +PR, <strong>the</strong> addition of IO·7Pcaused a significantupregulation ofMMP-1 (p
Arom~tase and Sa.-reductase pathways and <strong>the</strong>ir interaction with nutrition in <strong>the</strong> control ofpulsatile secretion of LH in male sheepT.P. Sharma, D. Blache and G.B. MartinFaculty of Agriculture (Animal Science), The University of Western Australia, Nedlands 6907, AustraliaIntroduction: Testosterone and its metabolites produced by 5a-reductase (DHT) and aromatase (oestradiol)inhibit LH secretion in male sheep and also interact with nutrition in <strong>the</strong> control of LH secretion (1, 2). Wehave been using inhibitors of <strong>the</strong>se enzymes to determine where <strong>the</strong>y are located and with a view to testingwhe<strong>the</strong>r <strong>the</strong>ir regulation plays a role in responses to nutrition in intact Merino rams. The aromatase inhibitor,fadrozole, given iv or icv, incr~ases LH pulse frequency but <strong>the</strong> 5a-reductase inhibitor, MK 434, has noeffect (3, 4). In this study, we used fadrozole again, and two o<strong>the</strong>r 5a-reductase inhibitors, to test whe<strong>the</strong>raromatase and 5a-reductase pathways interact with nutrition in <strong>the</strong> control ofLH secretion in mature Merinorams.Material and Methods: In Experiment 1, two groups of S rams were infused icv <strong>for</strong> 24 h with 0.1 and 0.01mg/kg of 5a-reductase inhibitor (FCE 28260, a 4-azasteroid) while 5 control rams received vehicle. InExperiment 2, two groups of 12 rams were fed ei<strong>the</strong>r a Low Diet (400 g wheaten chaff + 10% lupins and 2%minerals) or a High Diet (800 g wheaten chaff + 800 g lupins + 2% minerals). Six animals from each groupwere infused iv (jugular) with a combination of 5a-reductase inhibitors (finasteride + MK386; 1 mg/kgdaily; Merck Sharp and Dohme, NJ, USA) <strong>for</strong> 3 days starting on <strong>the</strong> 12th day of dietary treatment. Controls(n=6) from each group received vehicle only. In Experiment 3, 4 groups of 5 rams were infused icv <strong>for</strong> 48 hwith vehicle, or fadrozole (20 ~g/kg daily), or finasteride + MK386 (10 ~g/kg daily), or fadrozole +finasteride + :tvlK386. Two rams from each group received <strong>the</strong> High Diet and 3 <strong>the</strong> Low Diet. Two weekslater, <strong>the</strong> nutritional treatments were crossed over and <strong>the</strong> experiment was repeated. Blood was sampledevery 20 min <strong>for</strong> 24 h be<strong>for</strong>e and after inhibitor treatments and LH was measured in <strong>the</strong> plasma.Results: FCE 28260 did not affect LH pulse frequency (Fig. 1a ). When <strong>the</strong>y were given iv, Finasteride +MK 386 increased LH pulse frequency in <strong>the</strong> High Diet group but not in <strong>the</strong> Low Diet group (Fig. Ib). Wheninfused icv, only fadrozole increased LH pulse frequency (Fig. 1c). No interactions between nutrition and <strong>the</strong>aromatase and 5a-reductase inhibitors were observed. In both Experiment 2 and 3, <strong>the</strong> effect of diet on LHpulse frequency was highly significant be<strong>for</strong>e start of inhibitor treatment.Discussion: The increase in LH pulse frequency after icv fadrozole treatment rein<strong>for</strong>ces <strong>the</strong> conclusion thatcentral aromatization is an important step in <strong>the</strong> negative feedback effects of testosterone. All of ourattempts to increase LH pulse frequency using <strong>the</strong> most potent 5a-reductase inhibitors have failed,suggesting that this pathway is not important in <strong>the</strong> negative feedback system of intact rams. However, <strong>the</strong>possibility that <strong>the</strong>se inhibitors have a low affinity <strong>for</strong> <strong>the</strong> 5a-reductase of sheep can not be ruled out.p
Plateletaactivating factor receptor in human and mouse spermatozoa and mouse preimplantation\.(~--. v~~J'"~e~~k- + h..............~~ j·v..... j..Ji.. t+T. Stojanov, C. Wu & C. 0 'NeillHuman Reproduction Unit, RNSH; Department ofPhysiology, University ofSydneyIntroductionPlatelet activating factor (PAF, 1-0-alkyl-2-acetyl-snglyceryl-3-phosphocholine)is a potent e<strong>the</strong>rphospholipid. It improves motility and inducesacrosome reaction in spermatozoa and acts as anautocrine growth/survival factor <strong>for</strong> <strong>the</strong> preimplantationembryo. The concentration of PAF in spermatozoa andembryos is inversely related t6 <strong>the</strong>ir quality. Amechanism of action is yet to be defined. In this study,we examined <strong>the</strong> expression of a G-protein linkedreceptor to PAF (PAF-R).We have previously shown (1) that PAF-R mRNAtranscripts were detected in freshly fertilized mousezygotes. By <strong>the</strong> late I-cell stage and 2-cell stageexpression was less consistent but was observedconsistently again from <strong>the</strong> 4-cell stage up until <strong>the</strong>blastocyst stage. IVF and culture delayed <strong>the</strong> expressionof PAF-R mRNA until <strong>the</strong> 8-cell compacted stage.This study investigated: (i) <strong>the</strong> presence of PAF-RmRNA and protein in <strong>the</strong> human and mousespermatozoa; (ii) <strong>the</strong> presence of PAF-R protein inmouse preimplantation embryos and its pattern ofexpression following in vitro fertilisation and culture(IVF).Materials and methodsGamete collection and cultureSpermatozoa were obtained from three fertile donors(human) or were aspirated from cauda epididymis' offertile males (mouse). Motile spermatozoa were <strong>the</strong>nseparated by swim up into modified-RTF.Mouse embryos were collected fresh from reproductivetract (fresh) at various stages during preimplantationdevelopment. IVF embryos were cultured in modifiedRTF. Embryos were immunostained 17, 27, 42, 66 and90 hours post hCG.RNA extraction and RT-PCRRNA was extracted from spermatozoa with TRIzolReagent (Gibco). RT-PCR was per<strong>for</strong>med on spermsamples under standard Perkin-Elmer conditions usingspecific primers <strong>for</strong> PAF-R.ImmunofluorescenceSpermatozoa or embryos at various stages ofdevelopmental were fixed in 70% ethanol or 2%para<strong>for</strong>maldehyde (respectively), permeabilised in 1%Tween 20 (embryos) and non specific binding blockedwith 30% sheep heat inactivated serum. Immunotaggingwas per<strong>for</strong>med by incubation of gametes withmonoclonal antibody to PAF-R (human) (Alexis Corp.)followed by incubation in secondary fluorescent FITCanti-mouseIgG (Santa Cruz Biotechnology).Spermatozoa and embryos were viewed with anepifluorescent microscope (Nikon).ResultsPAF-R mRNA and protein were present in all samplesof human and mouse spermatozoa tested.Immunostained human spermatozoa showed specificstaining which was most prevalent at <strong>the</strong> midpiece and98distal head (Fig. 1). PAF-R location in mousespermatozoa was most prominent at <strong>the</strong> midpiece(Fig.!).Immunostaining of embryos collected fresh from <strong>the</strong>reproductive tract was significantly greater than isotypecontrols and <strong>the</strong> intensity was not different from <strong>the</strong> 1cell up to blastocyst stage. During development <strong>the</strong>pattern of PAF-R localization changed from diffusecytoplasmic staining in zygotes to increasinglymembrane localized pat.tern of expression from <strong>the</strong> 2cell stage onwards. There was no effect of IVF on <strong>the</strong>level and pattern ofprotein expression.Figure 1: PAF-R protein in human and mouse spennatozoa.i) human spennatozoa, ii) mouse spennatozoa, iii) mousespennatozoa incubated in non-immune IgGFigure 2: PAF-R protein in mouse preimplantation embryos.i) i-cell zygote, ii) 2-cell embryo, iii) 2-cell embryo incubatedin non-immune IgG iv) 4-cell embryo v) 8-cell embryo vi)blastocyst.ConclusionsThe results of this study showed that mRNA and protein<strong>for</strong> <strong>the</strong> G-protein linked PAF-R is present in human andmouse spermatozoa and within <strong>the</strong> mousepreimplantation embryo. Unlike <strong>the</strong> expression ofmRNA in embryos, <strong>the</strong> onset of PAF-R proteinexpression was not delayed following IVF and culture.(1) Stojanov T. & O'Neill C. (1999). Bioi Reprod 60(3):674-82Figure 1325~300.s 275o6 250u as 225u :u 200~ 175~ 150.5 125100o2 4Time (min)medium containing albumin (-, n=50) or no albumin(., n=50).8.- ~~ --b ~~~i- ~~f ", ~ ...;....JL-.\.d- ...Embryo-derived PAF induces Ca 2 + transients in <strong>the</strong> mouse preimplantation embryoR. Bathgate, M. Emerson, A. Travis and C. O'NeillRuman Reproduction, Department ofPhysiology, University of Sydney, Royal North Shore Hospital,St Leonards, NSW, 2065Introduction' .Platelet-activating factor (PAF) is an autocrine factor involved in regulation of embryo development. Its mecha~lsm ofaction remains to be defined. Transient increases in intracellular calcium are universal cues <strong>for</strong> cell-cycle progressIOn andregulation of differentiation and development. Exogenous PAF was S?own to induce a ~ansient rel.ease of i~trac~llularcalcium. In this study we investigate whe<strong>the</strong>r endogenous embryo-denved PAF can also Induce calCIUm tranSIents In <strong>the</strong>2-cell embryo.Meilio~ .Female mice were superovulated by Lp. injection of 10 IU eCG followed 48h l~ter by 10 ill hCG and mated overmght.Embryos were flushed from <strong>the</strong> oviducts of mice at <strong>the</strong> 2-cell stage of development 42h after eCG. Embryos wereincubated in Fura-2-AM (2uM) <strong>for</strong> 30min and <strong>the</strong>n mounted on a coverslip coated with Cell-Tak and sealed onto aperspex perfusion chamber. Embryos were monitored with Ion Vision software package and changes in Fura-2 ratio(calculated using excitation wavelengths of 340 and 380 nm) recorded.Results and DiscussionRelease ofPAF from embryos requires albumin (1) <strong>the</strong>re<strong>for</strong>e calcium measurements were made in media in <strong>the</strong> pres.en~eand absence of BSA. In <strong>the</strong> presence of albumin embryos showed characteristic calcium transients but did not do so In Itsabsence (Figure 1). It was determined that this transient was due to embryo-derived PAF by showing that addition ofrecombinant PAF:acetylhydrolase (it degrades PAF to an inactive metabolite) prevented <strong>the</strong> transient. These embryosstill responded to exogenous PAF with a calcium transient illustrating <strong>the</strong> specificity of <strong>the</strong> response. ~ PAF-r~cept~rantagonist (WEB 2086)' also blocked calcium transients. Depletion of Caj stores by -treatment WIth thapslgar~Ineliminated <strong>the</strong> observed calcium transient, suggesting that it is reliant on Caj stores (Figure 2). Embryos superfused WIthmedium containing 3ug/ml BSA responded more quickly and with a greater amplitude than those exposed to 3mg/mlBSA, suggesting that BSA competes with <strong>the</strong> PAF receptor <strong>for</strong> PAF.Figure 1.The change in [Ca 2 +]j in response to perfusion Figure 2.The [Ca 2 +]j response of 2-cell embryos to PAFFigure 2550~ 500.s450§U 400asU 350:s :03001 250:E 200••••••SHS&BB••eeBB B ••1 5 0 ~--r--.----r-..-----'2 4 6 8 10Tim e (m in)after depletion of internal stores by thapsigargin.Thapsigargin treated (-, n=63), control Ce, n=49).Embryos exhibit a transient release of internal calcium stores in response to embryo derived PAF during <strong>the</strong> 2-cell stage.It is dependent on <strong>the</strong> presence of BSA. Previous studies have routinely excluded albumin and have <strong>the</strong>re<strong>for</strong>e not beencapable of detecting <strong>the</strong> actions of embryo-derived hydrophobic compounds.Reference(1) Ammit, AJ. and O'Neill, C. (1997) Studies of <strong>the</strong> nature of <strong>the</strong> binding by albumin of platelet-activating factorreleased from cells. Journal ofBiological Chemistry. 272: 18772-78.99
Expression of FAM, a ubiquitin specific protease in mouse embryosM. Pantaleon l , M.Kanai-Azum 2 , J. Mattick 2 , K. Kaibuchi 3 , P. Kaye l and S.Wood 4lDepartment ofPhysiology and Pharmacology and 2Centre<strong>for</strong> Cell and Molecular <strong>Biology</strong>, The UniversityofQueensland, Brisbane, Qld, 4072; 3NARA Institute ofofScience and Technology, Takayama, Ikoma, Japanand 4Department ofBiochemistry, University ofAdelaide, Adelaide SA 5001.The ubiquitin pathway is a key regulator of cell cycleprogression, differentiation, signal transduction andapoptosis; events which all occur during mammalianpreimplantation development. Using a gene trap, werecently cloned Fam, <strong>the</strong> murine homologue of fa!tJtfC!rcets inJ2!~Fafencodes a ubiquitinsp~-ubiquitinase required in <strong>the</strong> syncitialstage of Drosophila development (2). We soughtevidence of expression in mouse embryos andpursued <strong>the</strong> requirement <strong>for</strong> Fam expression inblastocyst <strong>for</strong>mation and cell proliferati,on.Methods: Oocytes and preimplantation embryoswere collected from superovulated Quackenbushmice using standard techniques and media. Eggs andembryos were fixed and stained <strong>for</strong> confocalscanning laser microscopy using peptide specificantiserum. Western blotting used <strong>the</strong> sameantiserum. The antisense oligonucleotide (ODN)was designed to hybridise with <strong>the</strong> FAM initiationcodon and surrounding sequences, from nucleotides364-348 of <strong>the</strong> Genbank U67874 FAM sequence. Acomplementary sense ODN was used as control andcompared with no addition during culture <strong>for</strong> 72hours from late 2-cell stage.1"\IFig. 1. Fam staining: A, fertilised egg; B, blastocyst.100Results: Fam was found in fertilised eggs, andembryos of all subsequent preimplantation stages. In<strong>the</strong> unfertilised egg <strong>the</strong> Fam was localised to <strong>the</strong>spindle and chromosomes with some cytoplasmicstaining. Upon fertilisation Fam disappeared from<strong>the</strong> pronuclei and increased greatly in cytoplasmicintensity. Thereafter Fam was primarily found in <strong>the</strong>cytoplasm, particularly surrounding <strong>the</strong> nuclei, withno apparent change in distribution through toblastocyst <strong>for</strong>mation.Western blotting confirmed <strong>the</strong> Fam knockout.The FAM antisense oligonucleotide prevented 98%of embryos from <strong>for</strong>ming blastocysts (X 2 , P
humancowratsheepmouse102Ovarian steroid hormones and growth factors in <strong>the</strong> uterine control of early embryonicdevelopment in <strong>the</strong> tammar wallaby, Macropus eugenii.rat92%Cyrma M Hearn -and Marilyn B RenfreeDepartment of Zoology, University ofMelbourne, Parkville, Victoria 3052Embryonic diapause provides a powerful tool tounderstand <strong>the</strong> uterine control of early embryonicdevelopment. Whilst about 60 species of eu<strong>the</strong>rian, and30 species of marsupial exhibit diapause, <strong>the</strong> control isbetter understood in <strong>the</strong> tammar wallaby, Macropuseugenii, than in most o<strong>the</strong>r species. Under <strong>the</strong> influenceof a sucking young in <strong>the</strong> pouch, development of <strong>the</strong>tammar embryo in utero ceases completely at <strong>the</strong> 100cell. blastocyst stage. Diapause is maintained <strong>for</strong> at least11 months, during which time <strong>the</strong>re is no cell division orblastocyst growth. Despite <strong>the</strong> long period of arrest <strong>the</strong>reis no loss of embryonic viability. Most attention to datehas focused on <strong>the</strong> reactivation of corpus luteum andembryonic growth after <strong>the</strong> long arrest, with little focuson <strong>the</strong> mechanisms that control <strong>the</strong> onset of diapause.The aim of this study is to investigate <strong>the</strong> interactionsbetween ovarian hormones, uterine secretions and earlyembryonic growth as <strong>the</strong> blastocyst approaches andenters diapause.It is clear from work on eu<strong>the</strong>rian species that <strong>the</strong>re aremany growth factors, including. leukaemia-inhibitingfactor (LIF), epidermal-growth factor (EOF) and insulinlikegrowth factors (IOF-l & IOF-2), produced in <strong>the</strong>uterine secretions which may potentially regulateembryonic development. LIF is essential <strong>for</strong> growth of<strong>the</strong> early mammalian embryo (1). The expression of LIFin mice is maternally controlled by oestrogen andcoincides with blastocyst <strong>for</strong>mation (2). Similarly, inpigs (3) and cattle (4) oestradiol upregulates uterine LIFproduction. In rabbits by contrast, progesterone, notoestradiol, regulates LIF expression (5). Nei<strong>the</strong>r steroidinduces uterine LIF production in <strong>the</strong> skunk, but LIFmRNA is absent during diapause and high around <strong>the</strong>time of implantation (6).Our hypo<strong>the</strong>sis is that in <strong>the</strong> tammar <strong>the</strong> release ofoestradiol from <strong>the</strong> ovary, around <strong>the</strong> time of ovulation,induces <strong>the</strong> production of several growth factors,including LIF, which enhance cleavage and are essential<strong>for</strong> <strong>for</strong>mation of <strong>the</strong> early blastocyst. Over <strong>the</strong> next fewdays, without continued oestradiol stimulation and with atransient rise in progesterone post partum, growth factorproduction is downregulated, <strong>for</strong>cing <strong>the</strong> blastocyst intodiapause. Thus <strong>the</strong>re may be a direct interaction betweenovarian steroid' hormones and uterine" growth factorsecretion in initiation of diapause.human81%79%The endocrine changes and endometrial responses in <strong>the</strong>period between conception and <strong>the</strong> onset of diapause hasbeen poorly defined. Daily plasma samples were<strong>the</strong>re<strong>for</strong>e collected immediately after parturition until day10 after birth from females that retained <strong>the</strong>ir young(n=8), those who had <strong>the</strong>ir young removed at birth (n=8)and a group that carried a blastocyst in diapause (n=8).Plasma progesterone and oestradiol was measured byradioimmunoassay (RIA) as previously described (7),and a hormone profile <strong>for</strong> <strong>the</strong> three experimental groupsdefined. The genetic sequence of LIP in <strong>the</strong> tammar wasdetermined by nested polymerase chain reaction (PCR)using degenerate primers developed from conservedregions of known eu<strong>the</strong>rian species, and probesdeveloped from mouse and human LIP plasmids. The 38amino acid sequence of <strong>the</strong> peR product shows highhomology with previously described species (Table 1).The tammar LIP (tLIP) gene has a higher homology tohuman LIP than o<strong>the</strong>r eu<strong>the</strong>rian species. Using part of<strong>the</strong> tLIP sequence as a probe <strong>the</strong> expression of LIP inreproductive tissues and <strong>the</strong> early embryo will beaccurately determined by in situ hybridisation.Acknowledgments:We thank Prof. RR Behringer (Department of MolecularGenetics, MD Anderson Cancer Centre, Houston, Texas)<strong>for</strong> providing his expertise and laboratory facilities incloning tLIF. We are also grateful to Dr. D Hilton(Walter & Eliza Hall Institute, Victoria) <strong>for</strong> kindlyproviding <strong>the</strong> human and mouse LIF plasmids.References:1. Stewart CL (1994) Mol Reprod Dev 39:233-82. Bhatt H et al. (1991) Proc Natl Acad Sci 88:11408123. Yelich IV et al. (1997) Biol Reprod 57:1256-654. Reinhart KC et al. (1998) Mol Hum Reprod 4:301-3085. Yang ZM et al. (1996) Mol Reprod Dev 43:470-66. Hirzel DJ et al. (1999) Biol Reprod 60:484-927. Fletcher TP & Renfree MB (1988) JRF 83:193-200Table 1. A cross species comparison of homology between <strong>the</strong> tLlF protein to known sequencessheep pig tammar87% 92% 88%84%74% 82% 790/076%71% 76% 73%TEMPORAL AND TISSUE SPECIFIC EXPRESSION OF MOUSE KALLIKREIN (mKlk) GENES ANDIDENTIFICATION OF A NOVEL mKlk mRNA TRANSCRIPT DURING EARLY DEVELOPMENT INTHE MOUSE.C.S. Chan, M.B. Harvey and J.A. Clements .Centre <strong>for</strong> Molecular Biotechnology, School ofLife Sciences, Queensland University ofTechnology, Bnsbane 400l.IntroductionThe tissue kallikreins are a highly conservedmultigene family of serine proteases that act on adiverse range of substrates including growth factors,extracellular matrix (ECM) glycoproteins andproteinases and are important regulators of ECMdegradation and cell proliferation (1). They havebeen implicated in <strong>the</strong> uterine function of o<strong>the</strong>rspecies (2,3). The expression of five mKlk genesmKlk-1(tissue kallikrein), mKlk-3, mKlk-5, mKlk-9(epidermal growth factor binding protein) and mKlk21- was detected in <strong>the</strong> non-pregnant mouse uterus(4). The aim of this study was to elucidate <strong>the</strong>temporal and tissue specific expression pattern of<strong>the</strong>se five mKlks during early development.Materials & MethodsFertilised eggs, two-cell embryos, blastocysts, day7.5, day 9.5 and day 11 embryos were collected fromsix-week old, superovulated, mated female BCBFlmice. Uterine tissue was collected at correspondingdevelopmental stages. Total RNA was extracted andexpression of each mKlk and ~-actin was detected byper<strong>for</strong>ming reverse transcription (RT)-PCR, withgene specific primers, in <strong>the</strong> linear range to allowsemi-quantitative analysis. Sou<strong>the</strong>rn hybridisationwith gene-specific probes and/or DNA sequencingconfirmed <strong>the</strong> specificity of <strong>the</strong> PCR.ResultsThe ubiquitous "house-keeping" gene, ~-actin, wasexpressed in all samples, confirming <strong>the</strong> integrityand quality of <strong>the</strong> cDNA (Figure la). Positivecontrols (salivary gland) yielded mKlk products of<strong>the</strong> predicted size. All mKlks displayed distinctexpression patterns. We observed <strong>the</strong> expression ofmKlk-1 (Figure Ib), mKlk-3, mKlk-9 and mKlk-21(Figure lc) in <strong>the</strong> fertilised egg to <strong>the</strong> two-cell stage.Only mKlk-21 continued to be expressed until day7.5 of pregnancy. mKlk-9 expression reappeared atday 7.5 and was consistently detected until day 11(data not shown). mKlk-1 was again expressed in<strong>the</strong> embryo from day 9.5, with decreased levels byday 11 (Figure Ib). mKlk-5 was not detected in <strong>the</strong>embryo. In contrast, <strong>the</strong>re was no consistentexpression in <strong>the</strong> uterus of mKlk-1 until day 7.5which <strong>the</strong>n continued to day 11 (Figure Id). mKlk21 expression in <strong>the</strong> uterus displayed a similarpattern but was detected at much lower levels(Figure Ie). Of interest, a novel mKlk-21-likemRNA·I3 bp larger was detected in <strong>the</strong> uterus, buta.310243234b.6 ......3 : -~~~-~ ~.-~3c. M - + f.e. 2-cell blast d 7.5 d 9.5 i..ll.31 • : ~ _ __~ .,~w ->20194d.603383310e. 31. .lG4~~tG\.;~not in <strong>the</strong> embryo (Figures lc,e). mKlk-3, mKlk-5and mKlk-9 were not consistently expressed in <strong>the</strong>uterus ~e! th~ tiT~: 2-cell blast d 7.5 d 9.5 ~2019- _st.iU-_ _llllliIU1l1l'l- _ i'$!
The relationship between uterine IFN-'t concentrations and embryo developmentin a herd ofrecipient cattle selected <strong>for</strong> high or low pregnancy ratesSJ. Pearson, W.H. McMillan and AJ. PetersonAgResearch Ruakura, P.B. 3123, Hamilton New ZealandSeasonal Changes in <strong>the</strong> Testicular Morphology of <strong>the</strong> Agile AntechinusDuckett, R.*, Wre<strong>for</strong>d, N.* and Taggart, D.#*Institution ofReproduction and Development and Department of Anatomy. Monash University, Clayton 3168.#Department of Zoology, University ofMelbourne, Parkville 3052.INTRODUCTIONFollowing repeated twin embryo transfer, we haveestablished a herd of cattle with ei<strong>the</strong>r high (n =25,High) or low (n = 25, Low) pregnancy rates (1)creating a unique experimental resource to determinefactors involved in <strong>the</strong> successful establishment ofpregnancy in cattle. We report here <strong>the</strong> concentrationsof interferon-tau (IFN-'t), <strong>the</strong> conceptus-secreted factorcrucial <strong>for</strong> pregnancy signalling in ruminants, in <strong>the</strong>uterine luminal fluid (ULF) of <strong>the</strong> two sub-herds and<strong>the</strong>ir relationship to embryo development and potentialpregnancy rates.MATERIALS AND METHODSEstrus was synchronised using standard CIDR-BTMdevice protocols. On day '7 each cow received by nonsurgicaltransfer two in vitro derived day 7 blastocystsinto <strong>the</strong> uterine hom ipsilateral to <strong>the</strong> corpus luteum.On day 17, <strong>the</strong> uterus of each cow was flushed in vivo,<strong>the</strong> number and length of each conceptus recorded, <strong>the</strong>volume of <strong>the</strong> recovered fluid measured and a 50mlflush aliquot processed <strong>for</strong> analysis. Aliquots of 100llg of ULF protein were loaded into 15% SDSpolyacrylamide gels and Western-immunoblottedusing a bovine IFN-'t antibody kindly provided by DrRM Roberts, University of Missouri. The intensity ofeach band was analysed by densistometry. Because of<strong>the</strong> heteroscedasticity of <strong>the</strong> data, statistical analyseswere per<strong>for</strong>med on rank trans<strong>for</strong>med data whichinvolved ranking <strong>the</strong> observations in order from <strong>the</strong>lowest to <strong>the</strong> highest (2)RESULTSThe embryo survival rate (ES) and mean conceptuslength is shown in Table 1.Table 1. ES (%) and conceptus length (CL, cm,+ _S.e.m.)on day 17 ofgestatIOnSubherd ES CLLow 44 a 4.77 ± 0.7 cHigh 64 b 6.52 + 0.6 dMeans wIth diffenng superscripts are significantlydifferent (P
TGF~1 heterozygote mice on a SCID background (4)were imported from <strong>the</strong> University of Cincinnati, Ohio,USA. Genotype was determined by diagnostic PCRusing three primers; two which flank <strong>the</strong> insertedsequence in exon 6 of <strong>the</strong> TGF~ 1 gene and one within<strong>the</strong> inserted sequence.The fertility of one homozygous null male offspringwas examined by caging with balb/c Fl females (n=6)<strong>for</strong> a total of4 weeks.7Jlm fresh frozen sections were prepared <strong>for</strong>morphometric analysis from two TGF~ 1 null males andone wildtype (TGF~1+/+) littermate by H & E staining,and <strong>for</strong> immunohistochemical analysis with rat antimouse monoclonal antibodies F4/80 (pan macrophageantigen) and TIB 120 (MHC Class II) and Till 128(CD lIb).ResultsMorphology of <strong>the</strong> testes and seminal vesicles of <strong>the</strong> male TGFf31 null mouseWV Ingman l and SA Robertson l . 2 .Department of Obstetrics and Gynaecologyl and <strong>Reproductive</strong> Medicine Unit 2 , University of Adelaide, SA 5005IntroductionTrans<strong>for</strong>ming growth factor ~ 1 (TGF~ 1) is a cytokinefound in murine seminal plasma which, afterinsemination, induces production of GM-CSF by <strong>the</strong>uterine epi<strong>the</strong>lium of <strong>the</strong> oestrous female (1). Thissuggests TGF~ 1 may be involved in <strong>the</strong> inflammatorycascade which occurs in <strong>the</strong> female reproductive tractduring early pregnancy. TGF~ is also recognised as apotent immune deviating molecule which inducestolerance to antigens at several musocal sites (2). It isour aim to investigate <strong>the</strong> physiological role of seminalTGF~1 in <strong>the</strong> induction of maternal tolerance topaternal antigens in semen. To this end we haveobtained heterozygote mice carrying a null mutation in<strong>the</strong> TGF~ 1 gene (3) with <strong>the</strong> intent of establishing acolony which will provide male homozygous mice touse in mating experiments. Little is understood of <strong>the</strong>reproductive physiology of <strong>the</strong> TGF~ 1 null mouseexcept homozygous matings pairs are rarely fertile (IOrmsby, personal communication). There<strong>for</strong>e an initialstudy was conducted to characterise <strong>the</strong> histology of <strong>the</strong>testes and seminal vesicles of male TGF~1 null mice todetermine if <strong>the</strong>re are any abnormalities in <strong>the</strong> malereproductive tract which may lead to infertility.MethodsOf 66 progeny produced from <strong>the</strong> original four females38% are wildtype, 48% are heterozygotes (TGF~I+/-)and 5% are homozygotes (TGF~1-/-). 6% died be<strong>for</strong>e<strong>the</strong> age of weening and were not genotyped. Theseratios are in accordance with those reported elsewhere(5).There were no mating plugs or sperm positive vaginalsmears recovered from normal females caged with <strong>the</strong>homozygous male whereas <strong>the</strong> wildtype male matedfrequently over <strong>the</strong> same period.106Histology and immunohistochemistry revealed thatboth sperm production and macrophage number anddistribution in testes and seminal vesicles of <strong>the</strong> TGF~1null mouseFig 1: H & E stain oftestis from TGF~I-/- maleFig 2: H & E stain of seminal vesicle from TGF~ 1-/- malewas comparable to <strong>the</strong> control (Fig 1 and 2 showmorphology of testis and seminal vesicle of TGF~1 nullmale respectively).ConclusionThus far we have been unsuccessful in mating TGF~1null males with normal females. Those males we haveproduced appear to have normal testicular and seminalvesicle structure. Currently, mating of heterozygotes isunderway to provide more TGF~1 null mice to study.References1. Temellen, Seamark and Robertson (1998) BioIReprod 58:1217-12252. Letterio and Roberts (1998) Annu Rev Immunol16:137-1613. Shull, Ormsby, Kier, Pawlowski, Diebold, Yin,Allen, Sidman, Proetzel, Calvin, Annunziata andDoetschman (1992) Nature 359:693-6994. Diebold, Eis, Yin, Ormsby, Boivin, Darrow,Saffitz and Doetschman (1995) Proc Nat! Acad SciUSA 92:12215-122195. Shull and Doetschman (1994) MRD 39:239-246HM2AP18'AP20MAP2 in <strong>the</strong> Testis: Iso<strong>for</strong>m Expression PatternsSuggest Functional HeterogeneityKate L. Loveland and Liz ChristyInstitute <strong>for</strong> Reproduction and Development, Monash University, Clayton, Vic 3168Introduction and Aims:Microtubules playa central role in germ cell development, comprising <strong>the</strong> major structural element in mitotic andmeiotic spindles, in <strong>the</strong> manchette surrounding <strong>the</strong> developing sperm head, and in <strong>the</strong> sperm tail. Their assemblyand dissociation is governed by a heterogeneous family of microtubule-associated proteins (MAPs). To understand <strong>the</strong>regulation of microtubule <strong>for</strong>mation and function in <strong>the</strong> testis, we have been studying microtubule-associated protein-2I(MAP2), which we have recently demonstrated can exist in a novel LMW iso<strong>for</strong>m containing a nuclear localisationIsignal (1). To understand MAP2 function in <strong>the</strong> testis, we have used a panel of commercially available monoclonalantibodies, widely used in neuronal studies and known to bind to discrete re ions of <strong>the</strong> MAP2 ~otein. Immunohistochemistrywas used to assess <strong>the</strong> distribution PKA RII Calm0 dulm .pattern of MAP2 in fixed rat testis sections, and WesternTubulmblots were per<strong>for</strong>med to determine antibody specificityHMWand MAP2 iso<strong>for</strong>m size. MAP2 iso<strong>for</strong>ms are broadlyMAP2a and bcharacterised as high molecular weight (HMW: > 200r::-r::-::lkDa) and low molecular weight (LMW; -75 kDa) due~LMWMAP2cHM 2 AP-20Ito alternative splicing that includes or excludes <strong>the</strong> - Novel LMW·long projection arm encoded on exon 9. Protein (italics) AP-18 P MAP2sand antibody binding sites are indicated to <strong>the</strong> right.IMaterials and Methods:Monoclonal antibodies (HM-2: Sigma; AP-18, AP-20: Neomarkers) recognising MAP2 were used (1:200 dilution) onBouin's fixed, paraffin-embedded rat testis sections and detected by immunohistochemistry using <strong>the</strong> Catalysed SignalAmplification kit (DAKO). Tissue lysates were prepared by homogenisation in immunoprecipitaion buffer (1), boiled,and ressolved by 10% SDS-PAGE, and transferred to Immobilon P membranes (Millipore) <strong>for</strong> Western blot analysis.MAP2 proteins were detected using <strong>the</strong> antibodies (1:5,000 dilution) with <strong>the</strong> ECL Plus Chemiluminescence system.(Amersham). Germ cell lines, gc-1 and gc-2 (2), and Leydig (TM3) and Sertoli (TM4) cell lines (all isolated fromimmature mouse testes), were maintained in 10% fcs in DMEM (gc-l and gc-2) or DMEM:FI2. Cell pellets were snapfrozen and <strong>the</strong>n resuspended and boiled in immunoprecipitation buffer <strong>for</strong> Western blot analysis.Results 12 3 4 5 12 3 4 5Nucleus and cytoplasm of:::;~:'t:(~g~1eearly round spermatids (RS).Cytoplasm of spermatocytesimmediately prior to and atmetaphase of meiosis, andin spermatogonia.Elongating sperm head.[ •..r..">"~M12345["".•...~.:-_ ..~~~.. AP18 ..123451: day 2 brain2: adult brain.. ~. ""'.....~ ~. 3: sperm> ..*-'11 ••• ~ 4: day 7 testis.. AP2D;~ .N()l~Ab,. 5: adult testisHMW iso<strong>for</strong>ms indicated with brackets.LMW iso<strong>for</strong>ms indicated with arrowheads.Conclusions and Discussion:Our results indicate that MAP2 expression in <strong>the</strong> testis is developmentally rero.tlated, with discrete iso<strong>for</strong>msexpressed at particular stages of germ cell maturation that are localised to different intracellular compartments.The germ cell lines, gc-l and gc-2, may serve as useful models to study MAP2 structure and functionin spermatogonia. Based on <strong>the</strong> localisation patterns in adult rat testis, we hypo<strong>the</strong>sise that MAP2 functionsin <strong>the</strong> meiotic spindle and in elongation of <strong>the</strong> sperm head. The nuclear iso<strong>for</strong>m in spermatocytes may bepositioned to mediate rapid spindle assembly once <strong>the</strong> nucelar envelope has broken down, while <strong>the</strong>cytoplasmic <strong>for</strong>m may coordinate <strong>the</strong> nucleation of microtubules at <strong>the</strong> onset of meiotic metaphase.References: (1) Loveland et al (1999) J BioI Chern. In press.(2) Hoffman et al (1992) Exp Cell Res. 201: 417.107
Cloning and Characterisation ofa Novel Testis Transcript with Homology toPhosphatidylethanolamine Binding Proteins - pebp2ISOLATION AND PARTIAL CHARACTERISATION OF THE OUTER DENSE FmRES FROM BRUSH·TAILED POSSUM SPERMATOZOAM. RiccI and W.O. BreedDepartment of Anatomical Sciences, The University of Adelaide, S.A., 5005Deborah M. Hickox l , John R. Morrison l , Kimberley L. Sebire l , Hooi-Hong Keah 2 , Milton T.W. Hearn 2 ,David M. de Kretser 1 , Moira K. O'BryanIllnstitute of Reproduction and Development and The Centre <strong>for</strong> Bioprocess Technology,2Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia.IntroductionMany infertile men show an increased percentage of abnormally shaped and immotile sperm arising fromunknown factors. It is postulated that some of <strong>the</strong>se abnormalities arise from mutations in genes criticallyrelated to <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> spermatozoa. To gain a better understanding of <strong>the</strong> mechanisms underlyingsperm development and function we set out to clone rat cDNAs involved in spermiogenesis. A previouslyunreported partial cDNA transcript was isolated and designated pebp2 based on <strong>the</strong> possession of a putativephosphatidylethanolamine binding protein (PEBP). The aims of <strong>the</strong> present study were to clone <strong>the</strong> fulllength rat pebp2 transcript, its mouse orthologue and to initiate a functional characterization.Materials and MethodsThe remainder of <strong>the</strong> rat pebp2 cDNA was obtained using a 5' rapid amplification of cDNA ends (RACE)method. The orthologous mouse sequence was obtained by screening a mouse testis lambda expressionlibrary. Isolated clone sequences and <strong>the</strong>ir predicted encoded proteins were compared against databaseentries <strong>for</strong> homology and _sequence motifs, using <strong>the</strong> BLAST and Prosite programs respectively. Peptidesspecific <strong>for</strong> pebp2 were produced using Fmoc-based chemistry and were linked to keyhole limpethaemocyanin (KLH). Peptide-KLH conjugates were immunized into rabbits to raise pebp2-specific antisera<strong>for</strong> use in immunohistochemical techniques. Nor<strong>the</strong>rn blotting and in situ hybridization were carried out todetermine <strong>the</strong> timing of pebp2 mRNA expression during spermatogenesis. RNA extracted from male andfemale Balb/c mice tissues were used <strong>for</strong> RT-PCR analysis oftissue distribution.Results5' RACE was successfully used to obtain an additional -1 kb of rat pebp2 cDNA sequence. Nor<strong>the</strong>rnanalysis revealed 2 encoded transcripts of -1.6kb and -3.0kb which were up-regulated at day 25 and day 35post-partum, respectively. Screening of a mouse testis expression library identified a novel cDNA sequencewhich shared 93% homology in <strong>the</strong> coding region with <strong>the</strong> rat pebp2 sequence. In agreement with rat pebp2expression, mouse pebp2 mRNA was expressed in late pachytene spermatocytes and round spermatids. Thisresult was supported by immunohistochemical staining data which showed pebp2 protein in round andelongating spermatids and on sperm heads at spermiation. One of <strong>the</strong> six mouse pebp2 clones sequenced wasthought to represent an alternative splice variant which would result in an in-frame deletion of 14 aminoacids. This was supported by RT-PCR analyses. The long mouse iso<strong>for</strong>m was predicted to have a Mr of-21.2kDa and an isoelectric point (pI) of -8.43. The short iso<strong>for</strong>m was predicted to have a Mr of -19.9 kDaand pI of -8.77. Both <strong>the</strong> predicted mouse and rat proteins contained a PEBP family signature, severalpotential phosphorylation sites and a nuclear localization signal. Tissue expression studies revealed <strong>the</strong>presence of both <strong>the</strong> short and long iso<strong>for</strong>ms of mouse pebp2 in many tissues including brain, heart, kidney,liver, lung, skeletal muscle. In addition a putative human orthologue has been identified by searching <strong>the</strong>EST database. The predicted protein sequences from all three species were novel and more closely related toone ano<strong>the</strong>r than o<strong>the</strong>r previously identified members of <strong>the</strong> PEBP family.ConclusionWe have identified novel rat and mouse testis transcripts and proteins which we have designated pebp2 basedon <strong>the</strong> possession of a PEBP motif. Mouse pebp2 is predicted to encode two potentially phosphorylationregulatediso<strong>for</strong>ms of -21 and 20kDa which are expressed during spermiognesis. Fur<strong>the</strong>r, pebp2 isofonnsappear to be integral component of several tissues throughout <strong>the</strong> body. The involvement of pebp2 in spermepididymal maturation, capacitation and fertilization is <strong>the</strong> subject of fur<strong>the</strong>r research.IntroductionIn all <strong>the</strong>rian mammals, nine prominent cytoskeletalstructures, <strong>the</strong> outer dense fibres, surround <strong>the</strong>axoneme of <strong>the</strong> midpiece and principle piece of <strong>the</strong>sperm flagellum (Figure 1). The possible function(s)of <strong>the</strong> outer dense fibres may be to ei<strong>the</strong>r maintain <strong>the</strong>passive elastic recoil of <strong>the</strong> sperm flagellum (1)and/or to protect <strong>the</strong> flagella against shearing <strong>for</strong>cesduring epididymal transit (2). The isolation andcharacterisation of <strong>the</strong> outer dense fibres from rat,bull and human sperm have been previously reported(3, 4, 5), but as yet no study has been published <strong>for</strong>any marsupial species. The aim of <strong>the</strong> present study,which is part of a broader investigation of <strong>the</strong>evolution of eu<strong>the</strong>rian and marsupial gametes, was toisolate and determine <strong>the</strong> protein composition of <strong>the</strong>outer dense fibres <strong>for</strong> <strong>the</strong> first time in a marsupialspecies, <strong>the</strong> brush-tailed possum, Trichosurusvulpecula-.Materials & MethodsSpermatozoa obtained from <strong>the</strong> cauda epididymidesof 10 adult possums were decapitated by sonication,layered over a 20%,40% and 60% (w/v) sucrose stepgradient and centrifuged at 2500 x g <strong>for</strong> 120 minutesat 4°C. Sperm flagella fractions were collected from<strong>the</strong> 40-60% interface and purity was assessed byNomarski microscopy. Sperm flagella were pelletedand incubated in 1% sodium dodecyl sulfate (SDS),2mM dithiothrietol (DIT), 25mM Tris-HCI (pH 8.0),and shaken at room temperature <strong>for</strong> approximately 90minutes. The resultant suspension was layered over a20%, 40% and 60% (w/v) sucrose step gradient,centrifuged, and <strong>the</strong> outer dense fibres were collectedfrom <strong>the</strong> 40-60% interface. The isolated outer densefibres were ei<strong>the</strong>r fixed <strong>for</strong> routine transmissionelectron microscopy and/or solubilised with 2% SDS5% B-mercaptoethanol <strong>for</strong> 5 minutes at 100°C andrun on linear gradient (7.5-15%) SDS polyacrylamidegels which were stained with Coomassie.Results and DiscussionSeparation of flagella from <strong>the</strong> sperm head bysonication and subsequent sucrose density gradientcentrifugation resulted in a high yield of flagella from<strong>the</strong> 40-60% interface. Incubation of <strong>the</strong> sperm flagellain SDS-DIT <strong>for</strong> 90 minutes resulted in <strong>the</strong> completesolubilisation of <strong>the</strong> axoneme, mitochondria andfibrous sheaths but <strong>the</strong> outer dense fibres remainedintact (Figure 1). SDS-polyacrylamide gels of <strong>the</strong>outer dense fibre fractions revealed 5 majorpolypeptide bands with molecular masses of 73, 57,52, 42 and 16 kDa, <strong>the</strong> last of which was <strong>the</strong> mostprominent band (Figure 2). In comparison, rat, bulland human sperm outer dense fibres contain 7, 3 and2 major polypeptides respectively (3, 4, 5), and <strong>the</strong>most prominent bands in rat and bull outer densefibres are 27 kDa and 33 kDa in mass (3, 4).f'~8·"'.tt." I'.., '•. '.·.,.< ~:;1 ;Figure 1. Electron micrograph of isolated possum outer dense fibres; in~et:transverse section of principle piece of possum sperm flagellum showmgouter dense fibres (arrows).Figure 2. SDS-PAGE of isolated possum outer dense fibres (ODF) andmolecular weight standards (STD).ConclusionsThe isolation and molecular weights of <strong>the</strong> proteins of<strong>the</strong> outer dense fibres has been described <strong>for</strong> <strong>the</strong> fIrsttime in a marsupial species, <strong>the</strong> brush-tailed possum.Polyc1onal antibodies are currently being raisedagainst <strong>the</strong> major possum outer dense fibrepolypeptides and <strong>the</strong>se will be used toimmunocytochemically investigate <strong>the</strong> developmentand localisation of <strong>the</strong>se cytoskeletal structures. Inaddition, techniques are currently being developed tosimilarly isolate and characterise <strong>the</strong> proteins of <strong>the</strong>o<strong>the</strong>r major cytoskeletal component of <strong>the</strong> possumsperm flagellum, <strong>the</strong> fibrous sheath.References(1) Phillips DM (1972) J. Cell Biol.53: 561-573.(2) Baltz JM et al. (1990). Biol. Reprod. 43: 485491.(3) Oko R (1988) BioI. Reprod. 39: 169-182.(4) Brito et ai. (1986) Gamete Res. 15: 327-336.(5) Henkel et al. (1992). BioI. Chern. Hoppe-Seyler373: 685-689.2108109
A 49 KDA PROTEIN ISOLATED FROM THE ACROSOMAL REGIONOF THE BRUSHTAILED POSSUM (Trichosurus vulpecula) SPERMATOZOAXiyi Zhang, *Frank Molinia and Minjie LinCooperative Research Centre <strong>for</strong> Conservation and Management of MarsupialsDepartment of Biological, University of Newcastle, NSW 2308, Australia; *Manaaki Whenua-LancareResearch, Lincoln 8152, New ZealandREGULATION OF PROSTAGLANDIN H SYNTHASE·2 EXPRESSION IN HUMANINTRAUTERINE TISSUESM.D. Mitchell, S. Potter and W.R. HansenIntroductionAfter acrosome reaction, some proteins areretained in <strong>the</strong> subacrosomal region ofmammalian sperm. These proteins, includingpre- and proacrosins, are suggested to beinvolved in <strong>the</strong> secondary binding of <strong>the</strong> spermand egg during fertilisation (1), and also in <strong>the</strong>attachment of <strong>the</strong> acrosome onto <strong>the</strong> nucleusduring sperm <strong>for</strong>mation (2). Recently, a 45 kDsubacrosomal protein has been isolated fromtammar wallaby sperm in our lab (3). The 45 kDprotein had N-terminal amino acid sequencehomologous to eu<strong>the</strong>rian proacrosin and causedsperm structural mal<strong>for</strong>mations in immunisedwallabies (4). Aim of this study was to identifyany similar proteins located in <strong>the</strong> subacrosomalregion of <strong>the</strong> brushtailed possum sperm using asimilar protocol <strong>for</strong> isolation of <strong>the</strong> 45 kDaprotein in <strong>the</strong> tammar wallaby.Materials & MethodsPossum sperm were flushed from caudaepididmis and vas efferents. The sperm werefrozen, stored and transported in liquid nitrogen.The sperm were washed in 4°C TBS after it wasthawed at 40°C. The sperm were decapitated bya Broswell 450 sonicator (2x5s, 0.6s pulse, at25W). Sperm heads were purified on a 3 stepsucrose gradient (10ml 75%, 10ml 68%, and4ml 35% sucrose in TBS with Protease InhibitorCocktail, and centrifuged at 6000g <strong>for</strong> 30 minwith a swing rotor. Most of acrosomemembrane was extracted by 0.1 % Triton x-I00on ice <strong>for</strong> 1 hr. The sperm heads with <strong>the</strong>subacrosome area were pelleted and incubatedwith 100mM NaOH on ice <strong>for</strong> 30 min. Thesupernatant of NaOH extraction was dialyzedagainst Milli-Q at 4°C <strong>for</strong> overnight and run onSDS-page with 6% stacking gel and 12%separating gel. The sperm morphology wasexamined by electron microscope followingextraction. The N-terminal amino acidsequencing of <strong>the</strong> protein was analysed aftertransferring to <strong>the</strong> PVDF membrane with CAPSbuffer.ResultsThe SDS-PAGE gel (Fig 1) shows oneprominent protein with approximal 49kDa MWextracted from <strong>the</strong> subacrosomal region ofpossum sperm. Twenty-three residues of <strong>the</strong> N-terminal sequence of <strong>the</strong>. 49 kD protein weredetermined, and showed were more than 60 %homology with porcine preproacrosin and 75 %with tammar wallaby preproacrosin.Fig 1. SDS-PAGE gel shows one prominentband of possum subacrosomal protein with anapproximate molecular weight of 49 kD. LanesStd, and PAP show molecular weight standardsand I protein bands extracted by NaOHrespectively......................',..',"~.,~:.:::'.~..~'~:-f~;-:~~~~;x~~~'~:~:~~!F~:'~~~?'i~;~~'Std,'PAPPAP > PAP~49kDConclusionsA putative possum proacrosin was successfullyisolated using a similar protocol was used with<strong>the</strong> tarnmar wallaby. The function andsignificance of <strong>the</strong> protein are currently beingdetermined in our laboratory.References(1).Oko R (1995)Reprod Ferti! Develop 7: 777797(2) Jones R, Parry R, Lo, Leggio L, Nickel, P(1996) Molecu. Human Reprod 8:597-605.(3)Lin M, Zhang X, Wade M, Harris M andNickel M (1998) J Reprod Fert 113, 257-267.(4) Lin M, Zhang X (1998) 10 th Europeanworkshop on Molecular and CellularEndocrinology of <strong>the</strong> Testis B3, Capri, Italy.Dept of Pharmacology and Clinical Pharmacology, University of Auckland, Private Bag 92019, Auckland,New ZealandAn increase in intrauterine prostaglandin production is a critical component of <strong>the</strong> parturient process. This isaccomplished via altered activities of enzymes of <strong>the</strong> prostaglandin biosyn<strong>the</strong>tic cascade, in particular,prostaglandin H synthase (PGHS). The recent finding that two <strong>for</strong>ms of this enzyme exist (constitutive,PGHS-1; inducible PGHS-2) has focussed interest on regulation of <strong>the</strong>'inducible <strong>for</strong>m. Since amnion is amajor source of intrauterine prostaglandin production we have used <strong>the</strong> amnion-derived cell lines (WISH andAV3) in our studies. Moreover, since proinflammatory cytokines such as interleukin-l ~ ( n..-~) and tumournecrosis factor alpha (TNF-a.) released during <strong>the</strong> host response to an intrauterine infection are considered toplaya significant part in <strong>the</strong> mechanisms of preterm labour we have evaluated <strong>the</strong>ir actions on PGHS-2expression.In amnion AV3 cells, 50 ng/ml TNF-a. or 1 ng/ml ll..--1 ~ induces rapid but transient syn<strong>the</strong>sis of PGHS-2mRNA while levels of PGHS-l mRNA remains constant. We have evaluated <strong>the</strong> mechanism of this effect.The -891/+9 fragment of <strong>the</strong> PGHS-2 promoter was subcloned upstream of <strong>the</strong> chloramphenicol acetyltransferase(CAT) gene. Sequential 5' deletion constructs containing regions -528/+9, -321/+9, -203/+9, and-52/+9 of <strong>the</strong> PGHS-2 promoter were syn<strong>the</strong>sized by long-template PCR to define <strong>the</strong> regions required <strong>for</strong>TNF-a. and ll..--l ~ activity. Although <strong>the</strong> transcriptional effects of cytokines via <strong>the</strong> NF-KB promoterelement have attracted <strong>the</strong> most attention, recently we have found that <strong>the</strong> TNF-a. and ll..--1 ~ -responsiveregion resides within bases -52 and -203 of <strong>the</strong> PGHS-2 promoter, a region lacking this sequence. Sitedirectedmutagenesis of <strong>the</strong> NF-ll..-6 iliuclear factor of ll..--6) response element within <strong>the</strong> -203/+9 construct(p203ll..-M) reduced <strong>the</strong> basal and induced transcriptional activity to that of <strong>the</strong> -52/+9 construct and aconstruct containing only a TATA box (pminCAT). Mutational inactivation of <strong>the</strong> CRE (£AMP-Response~lement) to produce p203CRM yielded similar results. However, mutation of both elements within <strong>the</strong> 203/+9 construct (p203ll..-CRM) fur<strong>the</strong>r reduced basal and TNF-a. or ll..--I~ stimulated CAT productionbeyond that of <strong>the</strong> constructs containing <strong>the</strong> single site mutation. Electrophoretic mobility-shift assays(EMSA) using a double-stranded oligonucleotide derived from <strong>the</strong> NF-ll..-6 element at -132 and AV3 nuclearextracts from control or TNF-a. or ll..--1/3 treated cells identified a DNA-protein complex whose specificitywas determined by competition with sequence-specific oligonucleotides. Interestingly, 50-fold molar excessof <strong>the</strong> unlabelled PGHS-2-derived CRE oligonucleotide completely eliminated <strong>the</strong> -132 complex while <strong>the</strong>NF-kB consensus oligonucleotide and <strong>the</strong> 203ll..-M oligonucleotide failed to reduce <strong>the</strong> intensity of <strong>the</strong>complex. Similarly, <strong>the</strong> CRE oligonucleotide, when used as a probe, <strong>for</strong>med a complex similar to that of <strong>the</strong>NF-ll..-6 probe. Antibodies to 3 iso<strong>for</strong>ms of CIEBP (a., /3, and 8) were included in <strong>the</strong> EMSA reactions toidentify <strong>the</strong> protein which bound <strong>the</strong> NF-ll..-6 and CRE oligonucleotides. Higher molecular weightcomplexes (super-shifts) were only observed upon <strong>the</strong> addition of anti-CIEBP~. There<strong>for</strong>e, <strong>the</strong>re are 2elements within ·<strong>the</strong> PGHS-2 promoter, NF-IL6 and CRE, which convey <strong>the</strong> effects of TNF-a. and IL-l~.The interaction of <strong>the</strong>se elements with CIEBP~ is <strong>the</strong>n required <strong>for</strong> increased PGHS-2 transcription inamnion.110 111
REGULATION OF PARTURITION: THE ROLE OF eRHRoger SmithMo<strong>the</strong>rs and Babies Research Centre, Department OfEndocrinology, John Hunter Hospital, LockedBag No 1, Hunter Region Mail Centre NSW 2310Premature delivery is responsible <strong>for</strong> 70-80% of neonatal deaths in western countries and <strong>the</strong> ratesof preterm delivery have not changed in <strong>the</strong> last 20 years. The importance may be greater still if <strong>the</strong>Barker hypo<strong>the</strong>sis linking low weight at birth with early onset vascular disease is confirmed. Whileour understanding of parturition in <strong>the</strong> sheep has progressed rapidly our knowledge of <strong>the</strong>mechanisms of human parturition has lagged behind. Human parturition is not associated with achange in estrogen to progesterone ratios and is not precipitated by glucocorticoids as observed inspecies as disparate as <strong>the</strong> sheep and <strong>the</strong> Tammar wallaby. Recent work in a number of laboratorieshas suggested that in humans and o<strong>the</strong>r primates <strong>the</strong> neuropeptide corticotrophin-releasing hormone(CRR) may playa key role.CRR is produced in <strong>the</strong> placentas of New and Old World monkeys and in <strong>the</strong> apes, includingourselves, and is secretedinto <strong>the</strong> maternal and fetal circulations. In humans <strong>the</strong> increase of maternalplasma CRH is exponential over <strong>the</strong> course of gestation, peaking at <strong>the</strong> time of delivery. In a largeprospective study we observed that <strong>the</strong> exponential increase was more rapid in those womendestined to deliver prematurely and slower in those destined to deliver late. While <strong>the</strong>se dataindicated an association between CRH concentrations and <strong>the</strong> length of gestation <strong>the</strong>y did notindicate that <strong>the</strong>se events were causally related. However, CRR receptors are present on <strong>the</strong>myometrium and several studies have provided evidence <strong>for</strong> a direct myometrial action <strong>for</strong> CRR ininitiating human parturition. Our own work has taken a different line. Primate pregnancy isdistinguished from that of o<strong>the</strong>r mammals not only by <strong>the</strong> production of CRH but also in thatplacental production of estrogen is dependent on fetal adrenal <strong>for</strong>mation of <strong>the</strong> estrogen precursorDHEA-S. We have shown that <strong>the</strong> fetal zone of <strong>the</strong> fetal adrenal gland possesses CRR receptors andthat stimulation of fetal adrenal cells in culture with CRR preferentially stimulates <strong>the</strong> <strong>for</strong>mation of<strong>the</strong> enzymes required <strong>for</strong> DHEA-S SYn<strong>the</strong>sis and promotes <strong>the</strong> production of DHEA-S by <strong>the</strong>secells. This provides a mechanism by which rising CRR concentrations can stimulate increasingestrogen production in <strong>the</strong> primate.In more recent work we have identified <strong>the</strong> region in <strong>the</strong> CRR promoter which appears to mediate<strong>the</strong> exponential rise in CRR during pregnancy. Glucocorticoids stimulate placental CRR production(in contrast to <strong>the</strong> hypothalamus where <strong>the</strong>y are inhibitory), this stimulation is mediated by aglucocorticoid interaction at <strong>the</strong> .cAMP response element. As an additional action of placental CRRis likely to be stimulation of cortisol syn<strong>the</strong>sis a positive feedback mechanism has been proposed byMajzoub et al whereby CRR stimulates cortisol production and cortisol production stimulates CRRsyn<strong>the</strong>sis-ma<strong>the</strong>matically modelled, this interaction produces <strong>the</strong> exponential rise observed inhuman pregnancy.PARTURITION AND THE HYPOTHALAMO.PITUITARY.ADRENOCORTICAL (HPA)AXIS IN THE SHEEP FETUS: UNKNOWN INIDBITORS AND UNLIKELYSTIMULATORSIC McMillen, JT Ross, TG Butler, MB Adams, L Edwards, J Schwartz,A Jurisevic, KL Wames and ME SYmondsDepartment of Physiology, University of Adelaide, Adelaide, SA 5000, Australia, Department ofChild Health, Queens Medical Centre, University Hospital, Nottingham, UK.During <strong>the</strong> past three decades, a number of elegant experimental studies have demonstrated that <strong>the</strong>fetal HPA axis plays a key role in <strong>the</strong> timing of parturition in <strong>the</strong> sheep. It is <strong>the</strong> case, however, that<strong>the</strong> precise events within <strong>the</strong> fetal HPA axis which result in <strong>the</strong> prepartum increase in cortisol andlead to delivery are unknown. There is controversy concerning <strong>the</strong> relative roles o~ <strong>the</strong> fet~lhypothalamus, pituitary and adrenal in <strong>the</strong> increase in adrenocortical growth. and sterOIdogenesIswhich occurs during <strong>the</strong> last 2-3 weeks of gestation. A range of hormones denved from <strong>the</strong> ACTHprecursor, proopiomelanocortin (POMC), have been postulated to act as inhibitors (POMC, proACTH) or' stimulators (N-POMC 1-77, ACTH 1-39) of fetal adrenocortical growth andsteroidogenesis. The source, relative concentrations and modes of action of <strong>the</strong> POMC derivedhormones on <strong>the</strong> fetal adrenal are not clear. We have recently shown, however, that infusion of NPOMC 1-77 into fetal sheep at 136-138 days gestation results in an increased fetal adrenal: bodyweight ratio and a differential activation of P450 17 hydroxylase (CYP17) mRNA expression in <strong>the</strong>fetal adrenal. This provides evidence <strong>for</strong> a role <strong>for</strong> this hormone in <strong>the</strong> cascade of events leading toactivation of <strong>the</strong> fetal adrenal cortex in late gestation.We have also demonstrated that <strong>the</strong>re are separate subpopulations of CRF-responsive. and AVPresponsivecorticotrophs in <strong>the</strong> fetal anterior pituitary and that <strong>the</strong> CRF-responsive corticotrophs aresensitive to <strong>the</strong> negative feedback actions of cortisol. The heterogeneity of corticotrophic cell typesin <strong>the</strong> neurointermediate and anterior lobes of <strong>the</strong> fetal pituitary may underlie <strong>the</strong> heterogeneity ofPOMC derived peptide hormones present in <strong>the</strong> fetal circulation and <strong>the</strong> temporal changes in <strong>the</strong>relative concentrations of <strong>the</strong>se hormones which stimulate <strong>the</strong> fetal adrenal.Finally, in <strong>the</strong> adult, acute or chronic hypoglycaemia potently stimulates <strong>the</strong> HPA axis and ACTHsecretion. Whilst <strong>the</strong> fetus is normally hypoglycaemic relative to <strong>the</strong> mo<strong>the</strong>r, <strong>the</strong> glucose-ACTHrelationship is poorly defined in <strong>the</strong> fetus. It is unclear whe<strong>the</strong>r <strong>the</strong> fetal HPA axis becomes moresensitive to <strong>the</strong> relative fetal hypoglycaemia in <strong>the</strong> prepartum period and whe<strong>the</strong>r this change isimplicated in <strong>the</strong> prepartum activation of <strong>the</strong> HPA axis. We have investigated <strong>the</strong> effects of acuteand chronic hypoglycaemia on <strong>the</strong> fetai HPA axis'and have speculated on <strong>the</strong> potential importance ofperipheral signals of fetal energy balance in <strong>the</strong> stimulation of <strong>the</strong> fetal HPA axis in late gestation.It is likely that <strong>the</strong> mechanism responsible <strong>for</strong> human parturition contains important redundanciesbut it seems that placental CRR is at least a player in <strong>the</strong> game.112113
INffiBINS AND ACTIVINS: MARKERS OF MATERNAL AND FETAL HEALTHEuan M WallaceDepartment of Obstetrics and Gynaecology, Monash University, Clayton, VictoriaThe prediction, detection and management of pre-eclampsia, fetal growth restriction and latepregnancy fetal compromise remain three of <strong>the</strong> most important challenges in modern obstetricpractice. While maternal and perinatal mortality and morbidity have ~ignificantly declined thiscentury <strong>the</strong> incidence of pre-eclampsia has essentially remained unchanged. Fur<strong>the</strong>r, while ourability to rescue <strong>the</strong> severely growth retarded and compromised infant in <strong>the</strong> neonatal period hasimproved beyond all expectations we remain disappointingly poor at detecting such pregnanciesearly enough to significantly alter antenatal events and prolong pregnancy.However, <strong>the</strong> recent development of sensitive and specific immunoassays <strong>for</strong> inhibins and activinsand <strong>the</strong> application of <strong>the</strong>se assays to pregnancy has offered new possibilities <strong>for</strong> <strong>the</strong> prediction andearly detection of pre-eclampsia, preterm labour and fetal compromise - both acute and chronic.While originally purified from gonadal tissue and characterised by <strong>the</strong>ir ability to modulate FSHsecretion from <strong>the</strong> pituitary, in pregnancy inhibins and activins are of feto-placental origin withlargely undefined roles. None<strong>the</strong>less, some years ago our observation that maternal serum inhibin Ais elevated in Down syndrome pregnancies heralded <strong>the</strong> first clinical application of <strong>the</strong>se proteins.Subsequent to <strong>the</strong>se studies, several groups have now shown that maternal serum inhibin A andactivin A are also elevated in women with established pre-eclampsia and that inhibin A is elevatedin <strong>the</strong> second trimester of pregnancy in women who subsequently develop pre-eclampsia. Mostrecently, we have shown that inhibin A may be elevated in such women as early as 10 weeks ofpregnancy, representing <strong>the</strong> earliest non-invasive marker of this disease yet described. If confirmed,<strong>the</strong>n <strong>the</strong>se findings offer new opportunities <strong>for</strong> early prediction and intervention.Equally exciting, maternal serum activin A is known to increase with advancing gestation,particularly in <strong>the</strong> last trimester, and in association with preterm delivery. While <strong>the</strong>se observationsmay suggest a role <strong>for</strong> activin A in parturition our recent studies have been unable to confirm thisbut instead suggest that <strong>the</strong> association with labour may reflect fetal compromise and stress which inturn, probably via o<strong>the</strong>r mechanisms such as CRR, initiate labour. Thus, while activin may beunin<strong>for</strong>mative in predicting labour onset it may be a useful marker of fetal wellbeing. Maternalserum activin levels are significantly increased in association with fetal growth restriction and inpregnancies with increased placental vascular resistance, indicative of placental insufficiency. Insupport of <strong>the</strong>se human data, we have shown experimentally that induced acute fetal hyoxaemia in asheep model increases activin secretion and that levels fall rapidly with <strong>the</strong> return of normoxia.Fur<strong>the</strong>r studies are now.. underway to fur<strong>the</strong>r define <strong>the</strong>. J,ltility of _activin as a marker of fetalwellbeing in human pregnancy and to explore both what mechanisms underlie <strong>the</strong> increasedsecretion in a~s?cia~ion with f~topl~cental embarrassment and what advantages are conferred by thisresponse. EXCItIng tImes certaInly be ahead <strong>for</strong> <strong>the</strong> obstetric endocrinology.CONSTRAINTS AND OPPORTUNITIES IN THECOMMERCIALISATION OF AUSTRALIAN R&DGraham F. MitchellFoursight Associates Pty Ltd, Levell, Richard Allen Building, 164 Flinders Lane, Melbourne, Vic 3000In response to various pressures and imperatives, <strong>the</strong>Science and Technology (S&T) landscape inAustralia is changing. One notable change over <strong>the</strong>past couple of years has been in <strong>the</strong> level ofunderstanding across <strong>the</strong> S&T spectrum. of <strong>the</strong>components of an "innovation culture" including <strong>the</strong>constraints to commercialisation of Australia's R&Dendeavours. The number of conferences, workshopsand symposia on this general topic area is expandingrapidly with some already calling <strong>for</strong> less diagnosisand more <strong>the</strong>rapy, radical if necessary. It is a fact,however, that <strong>the</strong>se meetings still attract largenumbers of participants particularly if models ofcommercialisation are presented.In terms of government policy, we see evidence ofgenuine attempts to promote entrepreneurship and <strong>the</strong>conversion of knowledge into wealth andemployment. This includes promotion of technologybasedenterprises as well as renewal of exisitingindustry. There is also increased understandingamongst <strong>the</strong> S&T practitioners of <strong>the</strong> differentimperatives, structures and functions of researchersand organisations across <strong>the</strong> spectrum - academiaand research institutes, manufacturing industry and<strong>the</strong> financiaVlegal sector. However, as we approach agenuine knowledge-based economy, we are a longway from getting <strong>the</strong> settings right <strong>for</strong> all <strong>the</strong>ingredients of innovation.Researchers in publicly-funded researchorganisations are increasingly "facing <strong>the</strong> market aswell as Government" and adopting a portfolioapproach to fund raising (see Mitchell and Nossal,1999, Int. J. Parasitol., in press).The S&T scene in Australia at <strong>the</strong> present time isa rich landscape in which <strong>the</strong> diversity of private andpublic funding sources has been used to promotepluralism and to obviate <strong>the</strong> heavy hand ofcentralised control (see Stocker, J. Priority Matters,1998). Its diversity is its strength provided <strong>the</strong> issueof critical mass is kept in mind with opportunitiesgrasped <strong>for</strong> co-location and establishment ofnetworks - genuine long-term collaborative andproductive networks and not just an administrative orfund-raising tool.One of our real persistent problems is a lack ofmobility of individuals across <strong>the</strong> S&T spectrum scientists moving from academia into <strong>the</strong> financialsector, industry or Government and back again. Theacademia-industry interface has always been difficultin Australia. As we have stated recently (Mitchell andNossal, loco cit):-"It has to be remembered that <strong>the</strong>re are regrettablysome early suspicions in many such encounters in <strong>the</strong>Australian scene with questions in each camp around:"...is this ano<strong>the</strong>r case of a researcher wanting tosimply take <strong>the</strong> money and run..." with some CEOsbelieving that one of <strong>the</strong> most dangerous places onearth is to stand between a scientist and a bucket ofmoney! Or alternatively "...does this company haveanyone remotely capable of appreciating science atall let alone my brilliant discovery that is potentiallyworth billions...". The overstatements areacknowledged! The point is that <strong>the</strong>re will beconsiderable baggage deriving from past negativeexperiences on both sides. Moreover, <strong>the</strong>understanding of (and mutual respect <strong>for</strong>) <strong>the</strong> wayscience is done at ei<strong>the</strong>r end of <strong>the</strong> S&T spectrum hashistorically been lacking. This is a major reason <strong>for</strong><strong>the</strong> discontinuity in Australia that is to our nationaldetriment and, besides preventing easy movement ofscientists back and <strong>for</strong>th between academia andindustry, impedes <strong>the</strong> development of bothintellectual property (IP) and products, and thusnational wealth, and thus tax dollars, and thus morepublicly-funded research, etc."There seems to be universal agreement that <strong>the</strong>major impedance to <strong>the</strong> establishment and nurturingof technology-intensive start-up companies is <strong>the</strong>hostile capital gains tax environment in this country.It is hoped that <strong>the</strong> Ralph Review will address thiscomponent of <strong>the</strong> S&T spectrum as well as <strong>the</strong>various issues that need to be addressed at <strong>the</strong> "bigend of town" - i.e. established, tax paying businesses.Success in risk-taking ventures must be rewardedra<strong>the</strong>r than penalised. Of course, <strong>the</strong>re is no need toinvent a unique Australian solution to many of <strong>the</strong>seissues - unabashed plagiarism of systems proven towork in o<strong>the</strong>r jurisdictions should do just nicely!Also included in a listing of how we might dothings differently are additional means to promote <strong>the</strong>venture capital industry in Australia, increasing <strong>the</strong>mobility of intellectual property, mentoring andassistance to entrepreneurs through technologyincubators and enhancing <strong>the</strong> skills. base in R&D&C&M - research, development, commercialisation andmanagement. Some have predicted that we probablyonly have 3-5 years to get things right if we are toexpand and capitalize on our S&T capabilities andcompete in a changing world where <strong>the</strong> dollar(including <strong>the</strong> research dollar) goes to where <strong>the</strong> bestvalue can be obtained.114115
--o121:..- ....:l~ S 8o b.O~54oI•IA critical number ofeggs stimulates prolactin secretion inmale emus during <strong>the</strong> breeding seasonJ. Van eleeff, E. Prendergast, D. Blache and G.B. Martin.Faculty of Agriculture (Animal Science), The University of Westem Australia, Nedlands 6907.Increasing concentrations of prolactin are seen in Winter in male emus which incubate a clutch ofeggs and in all male emus during seasonal fattening in Spring. The Spring stimulus <strong>for</strong> prolactinsecretion is increasing daylength, but photoperiod does not appear to playa direct role in Winter.Since <strong>the</strong> increases in prolactin concentrations in Winter are associated with incubation, wehypo<strong>the</strong>sise that <strong>the</strong> stimulus is <strong>the</strong> presence of eggs.We collected blood samples <strong>for</strong>tnightly in Autumn and twice weekly in Winter from 6 singlemales and from males of 10 mating pairs of emus. Serum was assayed <strong>for</strong> prolactin and testosterone.Data were analysed <strong>for</strong> effect of time, sex, status (paired v single), incubating status, and daysrelative to incubation when appropriate.There were no differences in ei<strong>the</strong>rFigure 1: Serum concentrations of testosterone andtestosterone or prolactin concentrations betweenprolactin in male emus which incubated (.) or did notincubate (0) during <strong>the</strong> breeding season. paired non-incubator males (n=5) and singlemales, and no effect of time <strong>for</strong> <strong>the</strong>se groups(Fig. 1). In contrast, in incubator males (n=5),•I••e• • o105 119 133 147 161 175 189 203Julian Dayoboth hormones changed over time, withtestosterone decreasing and prolactin increasing.When <strong>the</strong>se data are arranged relative to first dayof incubation (Fig. 2), testosterone was elevatedprior to incubation, and declined rapidly to near<strong>the</strong> limit of detection (0.06 ng/mL) within days of<strong>the</strong> start of incubation. Prolactin began toincrease prior to <strong>the</strong> start of incubation, and <strong>the</strong>profile of prolactin secretion during clutch<strong>for</strong>mation followed a sigmoidal curve describinga cubic function (Fig. 3). Increases were notevident until at least 6 eggs were present;prolactin concentration at this stage of clutch<strong>for</strong>mation was 1.2 ± 0.5 ng/mL (mean ± SD).When 7 eggs were present, prolactin increased to3.1 ± 1.3 ng/mL (mean ± SD), exceeding 6 SD of<strong>the</strong> mean concentration <strong>for</strong> 6 eggs or less. Once 8or more eggs were present, prolactin fur<strong>the</strong>r increased to 6.6 ± 1.8 ng/mL, exceeding by 13 <strong>the</strong> value?f. t?e SI? <strong>for</strong> 6.eggs or less. Thereafter, increasing number of eggs did not affect prolactin. EmusInItiated IncubatIon at clutch sizes 7 (n = 1), 8 (n = 3) or 12 (n = 1) eggs.For m~~ emus, stimulation of prolactin secretion in Winter requires <strong>the</strong> presence of eggs.However, a cntIcal number eggs m..ust fIrst ~e accumulated be<strong>for</strong>e <strong>the</strong> cue becomes effective.Figure 2: Serum concentrations of testosterone ( 0 ) andprolactin (. ) in individual incubator emus, relativeto day incubation began.Testosterone(nglmL)4o 0 ••~ oo~ooooo oo~Jt""".• •Prolactin(ng/mL)o ~ i ~ ~'rrsr'Ofiii i-84 -70 -56 -42 -28 -14 0 14 28 42 56Day relative to start of incubation1284oEffects of levonorgestrel on fertility in <strong>the</strong> tammar wallaby, Macropus eugenii.C. Nave, G. Shaw, R.V. Short, and M.B. RenfreeDepartment of Zoology, Melbourne University, Parkville 3052. Australia.INTRODUCTIONLevonorgestrel is a syn<strong>the</strong>tic progestin, which provideseffective contraception in women. It has several modesof action but primarily it inhibits <strong>the</strong> hypothalamicpituitaryaxis, which in turn prevents ovulation.Macropodid marsupials are highly fecund, so <strong>the</strong>irnumbers are capable of increasing rapidly, causingconsiderable management problems in public parks andwildlife sanctuaries. In <strong>the</strong>se semi-urban areas alternatemanagement techniques to culling and baiting aredesirable.The aims of this study are to test <strong>the</strong> efficacy oflevonorgestrel as a possible anti-fertility agent <strong>for</strong>macropodid marsupials, using tammar wallabies as amodel species.MATERIALS AND METHODSFemale tammar wallabies carrying pouch young (n=12)received a single - levonorgestrel implant (Leiras,Finland). Implants composed of thin-walled siliconetubing, sealed at each end with silastic adhesive were2.5mm in diameter, 4.3cm in length and contained75mg of levonorgestrel. Control implants consisting ofa hollow silicone tubing of <strong>the</strong> same length anddiameter as <strong>the</strong> levonorgestrel implants were placed infemale tammar wallabies carrying pouch young (n=12).Implants were inserted subcutaneously into <strong>the</strong> medialsubdermal connective tissue, between <strong>the</strong> shoulderblades. Pouch young were removed (RPY) 3 days afterinsertion, and <strong>the</strong> adult females given bromocriptine(25mg)(im) to synchronize reactivation of <strong>the</strong>irdiapausing blastocysts and initiate <strong>the</strong> 1 st cycle.Wallabies were checked daily 25-31 days followingpouch young removal, <strong>for</strong> birth and/or a mating plug.The new pouch young were removed 3-5 days postpartumto initiate <strong>the</strong> second cycle and bromocriptinewas given again to <strong>the</strong> adult females (25mg) (im).Blood samples (5m1) were collected from <strong>the</strong> lateral tailvein at specific times throughout pregnancy from 4levonorgestrel and 4 control implanted tammars.Plasma progesterone was measured using aradioimmunoassay validated <strong>for</strong> <strong>the</strong> tammar.Levonorgestrel implants were removed from anadditional 6 tammar wallabies one-month after <strong>the</strong>initial implantation during <strong>the</strong> seasonal quiescenceperiod, reproduction in <strong>the</strong>se animals was monitored <strong>for</strong><strong>the</strong> subsequent 9 months.RESULTSEleven of <strong>the</strong> twelve control implanted tammars gavebirth at <strong>the</strong> end of <strong>the</strong> frrst cycle and eight of <strong>the</strong>seanimals gave birth at <strong>the</strong> end of <strong>the</strong> second cycle (table1).~ 20e~~ 15e c-o~ 10Il..50Table 1: Birth in <strong>the</strong> tammar after treatment withcontrol or levonorgestrel implant.Control Levonorgestrel1 st Cycle 11/12 9/112 nd Cycle 8/11 0/8Nine of <strong>the</strong> twelve levonorgestrel treated animals gavebirth during <strong>the</strong> first pregnancy cycle, but none gavebirth at <strong>the</strong> end of<strong>the</strong> second pregnancy cycle (table 1).Birth was observed in 4 out of <strong>the</strong> 6 tammars that hadlevonorgestrel implants removed at <strong>the</strong> beginning of <strong>the</strong>breeding season.Radioimmunoassay of progesterone showed nosignificant difference in circulating levels duringpregnancy <strong>for</strong> both control and experimental tammars(fig. 1).o--- Control-+-Levonorgestret5 10 15 20 25Days since pouch young removal30Fig 1: Plasma progesterone levels throughoutpregnancy in one control and one levonorgestrel treatedtammar.DISCUSSIONLevonorgestrel does not interfere with a pregnancyfollowing diapause in <strong>the</strong> tammar. Normal progesteronelevels were observed throughout pregnancy inlevonorgestrel treated animals. Levonorgestrelsuccessfully inhibits conception at <strong>the</strong> post-partumoestrus in <strong>the</strong> tammar wallaby. The lack of a postpartummating at <strong>the</strong> end of <strong>the</strong> frrst cycle inexperimental tammars suggests that levonorgestrel issuppressing ovulation, but this is still to be confirmed.The contraceptive effects of levonorgestrel arereversible in <strong>the</strong> tammar upon implant removal.Levonorgestrel could provide an alternative method <strong>for</strong>population control of o<strong>the</strong>r macropodid marsupials, like<strong>the</strong> eastern grey kangaroo, Macropus giganteus, insmall, selected wildlife parks and sanctuaries.116 117
EFFECT OF FOLLICLE REGRESSING AGENTS, OESTROGEN AND PROGESTERONEON OVARIAN FOLLICULAR DYNAMICS IN BOS TAURUS AND BOS INDICUS HEIFERSA. Niasari-Naslaii ·u~ F. Sarhaddi'~~ Y: Damavandi', A. Angurani~ and A. Naji'~':Dept. Clinic. Sci., Vet. Med. F~c., Te~lran Unive.rsity, P.O.Box: 14155-6453, Tehran, IRAN..Dept. Cattle and Buffaloes, Anim. SCI. Res. InstItute, P. O. Box: 31585-1483, Karaj, IRAN.Faecal progesterone metabolites in ovariectomised dairy cows treated with progesteroneA.R. Rabiee 1 , K.L. Macmillan 1 and F. Schwarzenberger 2IDepartment of Veterinary Science, University ofMelboume, 250 Princess HWY, Werribee, VIC, Australia2Inst. of Biochemistry and L.B ohzmann Inst. of vet. med. Endocrinology, University of Veterinary Medicine,Veterinarplatz 1, A-1210, Vienna, AustriaIntroductionMost studies on follicular dynamics of cattle havebecn conducted in Bas taurus breeds and resultshave bcen extended to Bos indicus cattle withoutconsideration of possible differences. More recentlywe have addressed differences in follicular growthbetween <strong>the</strong>se two species of cattle (1) usingovulating approach (2). Present study wasconducted to compare <strong>the</strong> response of ovarianfolliclcs to follicle regressing agents in Bas taurusand Bas indicus heifers.Materials and MethodsStage of oestrous cycle was synchronised in 35month-old Holstein heifers (n=6; 500±14 kg LW)and 3) -month-old Sistani heifers (n=6; 313±8 kgLW) using norgestomet implant (3 mg; Crestar®;Intervet, Holland) <strong>for</strong> ]2 days in conjunction withhCG (500 IU; Chonrlon®; Intervet, Holland) onDay 5 and prostaglandin analogue (] 5 mg,Prosolvin®; Intervet, Holland) on Day ]] afterimplant insertion (] ,2). Heifers were kept in <strong>the</strong>same cnvironment and management and recorded inoestrus when <strong>the</strong>y allowed mounting by o<strong>the</strong>rheifers. On Day 3 of <strong>the</strong> ensuing cycle, heiferswere implanted with norgestomet and on Day 7 ofthis cycle (Day 0 of treatment) <strong>the</strong>y were given anintramuscular injection of oestradiol valerate (5 mg)and norgestomet (3 mg). Implant was removed onDay )2 of treatment. Ovarian follicle growth wasmonitored daily by ultrasonography using a 5 MHzrectal probe. Ovulation was confirmed bydisappearance of ovulatory follicle present <strong>the</strong>previous day, and appearance of antral follicles ~5mm was defined as emergence of new follicles.Data were analysed by One-way and repeatedmeasures ANOYA and Kruscal-Wallis test ofSAS/STAT and are presented as means ± SEM.ResultsOn Day 0 of treatment, <strong>the</strong>re was no difference(P>0.05) in follicle diameter between Holstein(12.3±0.99 mm) and Sistani (]0.8±0.48 mm)heifers (Figure 1). Following injection of oestradiolvalerate and norgestomet, existing follicle regressedbut this process started earlier (P
Effect offeeding management on progesterone release from CIDR devices inovariectomised dairy cowsA.R. Rabieet, K.L. Macmillan! and MJ. Rathbone 2IDepartment of Veterinary Science, University of Melbourne, 250 Princess HWY, Werribee, VIC, Australia2InterAg, 558 Te rapa Road, P.O. Box 20055, Hamilton, New ZealandIntroduction:The effects of nutrition on progesterone production andclearance rates in dairy cattle have not been intensivelystudied. The early postpartum period in cattle ischaracterized by anovulatory anoestrus. The duration ofanoestrus can be affected by body condition andnutritional intake. Progesterone administrated pervagina (CIDR device) has been widely used to treat thiscondition in dairy cattle (1). The efficacy of thistreatment when using a CIDR device partly depends on<strong>the</strong> rate of progesterone released from <strong>the</strong> device (2). Itis not known whe<strong>the</strong>r environmental factors such asnutrition and housing can affect <strong>the</strong> profile ofprogesterone release from a CIDR device. This studywas conducted to determine <strong>the</strong> possible effect of someof <strong>the</strong>se factors on progesterone release from a CrDRdevice in dairy cows.Materials and Methods:Six non-lactating Holstein-Friesian cows wereovariectomised. Plasma and faecal progesteroneconcentrations were measured <strong>for</strong> 4 weeks to monitorcomplete clearance of progesterone from <strong>the</strong> animaltissues following <strong>the</strong> operation. A CIDR device (1.9gprogesterone) was inserted into <strong>the</strong> vagina of each cowat least 4 weeks after ovariectomy and left in place <strong>for</strong>11 days. In <strong>the</strong> first II-day period of study cows werehoused in individual pens and fed with lucerne andoaten chaff (Table 1). In <strong>the</strong> second period, <strong>the</strong>y weregrazed on mixed pasture (2 pastures, Table 1). Theinterval between <strong>the</strong> two periods was 4 weeks. Bloodsamples were taken daily <strong>for</strong> 11 days and progesteroneconcentrations were measured using RIA. A Soxhletextraction technique was used to determine <strong>the</strong> residualamount of progesterone in spent CIDR devicesfollowing insertion in <strong>the</strong> animal (2).Results and Discussion:The only difference in <strong>the</strong> diets fed in <strong>the</strong> two periodswas <strong>the</strong> higher dry matter content of <strong>the</strong> lucerne andoaten chaff in <strong>the</strong> first period (P = 0.001; Table 1).Plasma progesterone concentrations (1.18 ± 0.17 vs1.74 ± 0.34) and <strong>the</strong> amount of progesterone releasedfrom <strong>the</strong> CIDR devices (0.64 ± 0.04 vs 0.88 ± 0.04)were significantly lower during <strong>the</strong> period where cowswere confined and fed with lucerne and oaten chaff (P= 0.02, Fig. 1). These differences may have beenassociated with diet or with housing. Factors whichmay affect <strong>the</strong> amount ofprogesterone that is releasedfrom a device could include <strong>the</strong> rate of blood flow to<strong>the</strong> anterior part of vagina, frequency of defaecation,composition and physical <strong>for</strong>m of <strong>the</strong> diet. Although<strong>the</strong>re was not a significant difference in <strong>the</strong>composition of <strong>the</strong> diet, 'physical <strong>for</strong>m and passage rateoffaeces in confined cows may have altered <strong>the</strong> contactbetween CrDR device and <strong>the</strong> vaginal wall <strong>the</strong>rebyaltering <strong>the</strong> effective surface area <strong>for</strong> release. Bloodmetabolites and protein could also affect vaginalenvironment. However, <strong>the</strong> lack of physical activitywith confinement may also have altered <strong>the</strong> frequencyor pattern of contact of <strong>the</strong> device with <strong>the</strong> vaginalmucosa and consequently reduced <strong>the</strong> amount ofprogesterone released from a device.Table 1: Nutritional values of <strong>the</strong> dietsDM 1 CP 2 Digestibility ME 3Lucerne & 86.4 12.5 64.2 8.9oaten ChaffMixed 41.4 12 61.0 8.5pasture1 Dry Matter 2 Crude Protein 3 Metabolisable Energy2.5GICE 1.5.!!~e 1Il..0.5ChaffPastureFigure 1: Plasma P4 and progesterone released fromCIDR devicesReferences:1) Macmillan KL and Burke CR (1996) Anim. Reprod.Sci. 42: 307-3202) Rathbone MI, et al. (1998) Proceedings of <strong>the</strong>International Symposium on Controlled Release ofBioactive Materials, 25: 249-250.Effect of nutrition on plasma progesterone concentrations in ovariectomiseddairy cows treated with progesteroneA.R. Rabiee and K.L. Macmillan1Department of Veterinary Science, University of Melbourne, 250 Princess HWY, Werribee, VIC, AustraliaIntroduction:Regulatory mechanism controlling luteal function andplasma progesterone (PP4) levels during <strong>the</strong> postpartumperiod have an important role in <strong>the</strong> dairy cattlefertility. The metabolic clearance rate (MCR) ofprogesterone also determines <strong>the</strong> relationship betweenprogesterone production rate and plasma concentration(1). The inverse relationship between feeding level andconcentrations of plasma progesterone (PP4) wasattributed to differences in clearance rate ofprogesterone ra<strong>the</strong>r than to changes in hormoneproduction (2). It is proposed that <strong>the</strong> mechanism <strong>for</strong>controlling PP4 was MCR of progesterone ra<strong>the</strong>r thanproduction rate. In <strong>the</strong> present study, an external sourceof progesterone was administrated and plasmaprogesterone concentrations were measured toinvestigate <strong>the</strong> effect of <strong>the</strong> level of feeding on plasmaprogesterone (PP4) in cows which received twodifferent <strong>for</strong>ms ofprogesterone treatment.Materials and Methods:Six non-lactating Holstein-Friesian cows, which hadbeen previously ovariectomised, were allocatedrandomly into two groups: P4 by subcutaneousinjection (P4-s.c.) and P4 administration per vagina(P4.;p.v.). Cows in <strong>the</strong> P4-s.c. group received a dose of200mg progesterone Iday as in a single injection. Thosein P4-p.v. group, each had a CIDR device containing1.9g progesterone inserted per vagina <strong>for</strong> 11 days. Eachdevice was replaced with a new one after 11 days.Cows were housed in individual pens and fed withlucerne and oaten chaff. In <strong>the</strong> fITst period of <strong>the</strong> study,cows were fed a maintenance ration twice daily in <strong>the</strong>morning and afternoon, and offered on an individualbasis. In <strong>the</strong> second period, <strong>the</strong> entire procedure (partone) was repeated, but cows were fed half of <strong>the</strong> feedrelative to <strong>the</strong>ir requirements. Blood samples werecollected twice daily at 8.00h and 16.00h <strong>for</strong> 11 daysand progesterone concentrations were measured usingRIA.Results and Discussion:The level of feeding had a significant effect on plasmaprogesterone concentrations in <strong>the</strong> P4-s.c., but not in<strong>the</strong> P4-p.v. group (Figure 1). Plasma progesterone wasnot significantly affected by day, time of sampling orany of <strong>the</strong> interactions (P > 0.05). Faecal outputsignificantly declined after reducing <strong>the</strong> maintenance(M) ration to V2 M (Table 1). There was no significantdifference in <strong>the</strong> mass of progesterone released from aCIDR device during <strong>the</strong> M or Yz M periods (0.66 vs0.63 g). O<strong>the</strong>r studies (2,4) have also shown thatprogesterone MCR significantly increased with anincrease in feed intake from Yz M to 2M, or from % Mto M; but not from Yz M to M in intact or CIDR-treatedewes. It has been suggested that <strong>the</strong>re is a directrelationship between <strong>the</strong> level of feed intake and rate ofblood flow to <strong>the</strong> liver and gut, and consequentlyprogesterone clearance rate (3). Studies in cattle (5)also showed that <strong>the</strong> frequency of feeding had asignificant role on peripheral progesteroneconcentrations. Several factors may affect plasmaprogesterone concentration, including <strong>the</strong> difference inprogesterone release rate. The difference in response to<strong>the</strong> level of feeding between two groups may be due to<strong>the</strong> <strong>for</strong>m that progesterone was administrated. The dailydose of progesterone injection and release rate ofprogesterone in <strong>the</strong> P4-s.c. group was greater than in<strong>the</strong> P4-p.v. group. This may have influenced <strong>the</strong> MCRrate of progesterone. These data demonstrate that <strong>the</strong>level of feeding and <strong>the</strong> route of progesteroneadministration have a key role in controlling plasmaprogesterone levels.Table 1: Mean (± SE) of feed intake and faecal outputduring <strong>the</strong> periods of M and Y2 M in both groups ofcows7~6~ 5 m:l1/2 M.sc:eQ) 43*Ql2~a. 10P 4 'pvP4-s.c.P 4 -s cP4-p.v.Feed intake (KgDM) (KgDM)M 11.86 ± 0.43 11.62 ± 1.30V2M 5.91+ 0.24 5.80 ± 0.62Faecal outputM 4.64± 0.09 4.9 ± 0.18Y2M 2.65 + 0.07 2.56 ±0.19Figure 1: plasma progesterone concentrations in P4-s.cand P4-p.v cowsReferences:1) Tait JF (1963) J. Clin. Endocr. 23: 1285.2) Parr RA et al. (1987). J. Reprod. Ferti!. 80: 317320.3) Parr RA (1992) Reprod. Ferti!. Dev. 4: 297-300.4) Williams AH and Cumming IA (1982). J. Agric.Sci. 98: 517-522.5) Vasconcelos JLM et al. (1998) ADSA-ASAS jointmeeting.120 121
Varying <strong>the</strong> <strong>for</strong>m of oestradiol administration to yearling heifers at <strong>the</strong> beginning oftreatment.VK Taufa, KL Macmillan!, G Mylrea 2 , S Morgan, R HooperDairying Research Corporation, Private Bag 3123, Hamilton, New ZealandIntroductionPrevious heifer trials with an Lm. injection of 1mgoestradiol benzoate (ODB) at <strong>the</strong> beginning of an 8-dayCIDR treatment achieved a high submission rate of94% with comparable conception rate of 62% on <strong>the</strong>first day of insemination (d1). The standard Genermateprogramme recommended by LIC (NZ) <strong>for</strong> use insynchronising yealing heifers uses <strong>the</strong> 10mg ODB(CIDIROLTM, InterAg, Hamilton, NZ) capsules instead.AimTo investigate whe<strong>the</strong>r <strong>the</strong>re were differences in <strong>the</strong>mode of action of ODB under <strong>the</strong> 2 different synchronysystems, and whe<strong>the</strong>r heifers could be set-timeinseminated with comparable conception rates.Materials & MethodsYearling heifers from 8 participating herds wererandomly allocated into 2 treatment groups; treatedei<strong>the</strong>r with <strong>the</strong> standard Genermate programme (OM)or <strong>the</strong> new synchrony system (VKT)The GM programme involved <strong>the</strong> insertion of anintravaginal progesterone releasing insert (1.43gCIDRTM, InterAg, Hamilton, NZ) with a 10mg ODBcapsule (CIDIROLTM) placed in <strong>the</strong> groove of <strong>the</strong> insertinto each of <strong>the</strong> 358 animals <strong>for</strong> 10 days (-dI2=day ofinsert insertion). At -d6 from device insertion eachanimal was given an Lm. injection of prostaglandin(250~g PGF; cloprostenol, Estroplan, ParnellLaboratories, Auckland, NZ). The inserts wereremoved on -d2.!he V.KT s~stem had CIDR inserts placedmtravagmally mto each of 368 animals, with aninjec~ion of 1mg ODB (CIDIROLTM, InterAg,HamIlton, NZ) at device insertion (-d10). The deviceswere removed on -d2 and each animal was injectedLm. with 250~g PGF. At 24h after device removal (d1) each animal was injected with 0.75 mg ODB.At 48h after insert removal (dO), <strong>the</strong> animals werescored on tailpaint removal as described by Macmillanand Taufa et ai, and set-time inseminated with semenfrom high fertility bulls from LIC. All <strong>the</strong> returns to 1 51inseminations were synchronised with <strong>the</strong> previouslyused-CIDR inserts; inserted on d16, removed on d21and inseminated on detection of oestrus from d23.Individual dates of conception and pregnancy wereconfirmed by rectal palpation of <strong>the</strong> uterine contents at6-8 weeks from 1 51 insemination, and again 6-8 weeksfrom when <strong>the</strong> bulls were removed from <strong>the</strong> herdResultsThere were no significant differences in conceptionrates to 1 51 and 2 nd inseminations between <strong>the</strong> twotreatments (Table 1). However, <strong>the</strong>re were herddifferences in conception rate to 2 ndinsemination and<strong>the</strong> final pregnancy rate (p = .037; p = 0.001respectively). The mean conception rate to 1Slinsemination <strong>for</strong> both treatments was 56% compared to62% <strong>for</strong> inseminating on detection of oestrus (2). Thefinal pregnancy rates were 96% and 94% <strong>for</strong> <strong>the</strong> OMand <strong>the</strong> VKT systems respectively.Of <strong>the</strong> animals which were definitely in oestrus, 59%and 58% conceived to that insemination <strong>for</strong> <strong>the</strong> OMand VKT systems respectively; of those <strong>for</strong> which <strong>the</strong>rewas some doubt about being in oestrus, 54% conceived<strong>for</strong> both treatments.Table 1: Conception rates to ISland 2 nd inseminations.OM Programme VKT Sync SystemFarms 1st insem 2nd 1st 2nd(%) insem insem insem(%) (%) (%)1 50 44 76 1002 68 58 56 823* 32 73 56 674 58 50 50 505 62 73 53 556 60 50 50 477 61 60 69 788* 55 39 47 42Range 32 - 68 39 -73 47 -76 42 -100Ave. 58 57 54 58* Delayed start to InSemmatIOn by 4h In 1 of <strong>the</strong> 2 herdsConclusionsThe results from this trial demonstrate that satisfactorypregnancy rates could be achieved by set-timeinsemination of heifers. However, it is difficult tointerpret <strong>the</strong> results. It is quite possible that <strong>the</strong> oestrousonset is much earlier in <strong>the</strong> VKT system because of <strong>the</strong>immediate effects of <strong>the</strong> ODB injection, andconsequently, insemination should start earlier.Both system may be useful to herdowners who wish tograze <strong>the</strong>ir heifers in situations which require minimalhandling and involving set-time insemination.AcknowledgementsThe authors thank <strong>the</strong> farmers <strong>for</strong> <strong>the</strong> use of <strong>the</strong>iranimals, InterAg <strong>for</strong> <strong>the</strong>ir support with CIDR andCIDIROL, LIC <strong>for</strong> inseminating and ParnellLaboratories <strong>for</strong> POP.References1.Macmillan, K.L., Taufa, V.K. (1997). Proc. of <strong>the</strong>NZ Soc. Anim. Prod. 47:238.2. Taufa, V.K., Macmillan, K.L. et al. (1998) Proc. of<strong>the</strong> 29 m ASRB Conf.: School ofVeter~nary Sciece, University ofMelbourne, 250 Princes Highway, Werribee, Victoria, australia 3030.Vet Products, LIvestock Improvement Corp., Hamilton, NZ.122Conclusions-e- Control (n=24).80-+- Deslorelin (n=41)~ooDeslorelin implants prevent early postpartum ovulations in Holstein dairy cattle.5A.M. Padula!, K.L. Macmillan!, T.E.Trigg 2lUniversity ofMelbourne, Veterinary Clinical Centre, Princes Hwy, Werribee, 30302peptech Animal Health, Pty Ltd, Sydney, Australia.IntroductionThe occurrence of ovulation within 3 weeks of calving has been associated with decreased reproductive per<strong>for</strong>mance indairy cattle (1). Suckling is a powerful inhibitor of <strong>the</strong> hypothalamic-pituitary axis in postpartum cattle, and may serveas a natural physiological protective mechanism that prevents <strong>the</strong> contaminated postpartum bovine uterus frompremature exposure to progesterone. High plasma progesterone levels attenuate <strong>the</strong> cellular uterine defencemechanisms. Prolonged luteal function has been reported in a high proportion of cattle that ovulate within 25 days ofcalving (2). Deslorelin implants have successfully been used in cattle to induce reversible oestrous cycle suppression inbeef heifers (3) and provide a method of achieving short term downregulation of pituitary responsiveness. Theobjective of this study was to investigate <strong>the</strong> potential of a potent OnRH agonist, deslorelin, to prevent ovulation in earlypostpartum dairy cattle.Materials & MethodsA total of 65 Holstein-Friesian cows on a commercial dairy farm in East Gippsland were used in this study. Cattlereceived ei<strong>the</strong>r a single deslorelin implant (n=41) or no treatment (n=24). Each implant was inserted subcutaneously on<strong>the</strong> outer surface of <strong>the</strong> ear withing 7 days of calving. Implants were removed 28 days later through a small incisionover <strong>the</strong> surface of <strong>the</strong> implant. Blood samples were collected 3 times weekly <strong>for</strong> 4 weeks while an implant was in placeand <strong>the</strong>n weekly <strong>for</strong> a fur<strong>the</strong>r 6 weeks. Plasma progesterone was measured using a commercial kit (Coat-a-Count®,DPC, Los Angeles) and levels greater than 1.0 ng/ml were considered indicative ofluteal activity. Data was analysed bysurvival analysis using Minitab.ResultsTreatment <strong>for</strong> 28 days resulted in a prolonged postpartum anovulatory anoestrus that persisted <strong>for</strong> at least 14 days afterimplant removal. The median interval from calving to 1st ovulation was extended by 23.5 days (57.5 vs 34.0; p
The Gestational Profile of Uterine Milk Proteins in Ovine Allantoic Fluid and Amniotic Fluid.D.R. Williams and l.R. McFarlaneDivision of Animal Physiology, University ofNew England, Armidale NSW 2351.IntroductionUterine milk proteins (UTMP) are <strong>the</strong> major proteins secreted by <strong>the</strong> ovine uterus during pregnancy.The secretion of UTMP by <strong>the</strong> ovine uterus is stimulated by long term exposure to progesterone (1).Accordingly only trace quantities of UTMP can be detected in uterine secretions during early pregnancyhowever <strong>the</strong>se proteins are found in abundance during most of pregnancy and increase with <strong>the</strong> progressionof gestation (2). Little is known as to <strong>the</strong> function of UTMP although <strong>the</strong>se proteins have been shown toinhibit immune function (3). The endometrium is <strong>the</strong>3500..4 3000~ 2500ge 2000'-'t:l... 1500~E--1000::J 500o50BAllantoic Fluid75 110 125 130Gestational Age (days)140Figure 1. Gestational profile of UTMP in ovineallantoic fluid.6AABBAmniotic FluidABo-tJ--.LtJ---4L---4-L---4J.--4----t50 75 125 130 140Gestational Age (days)Figure 2. Gestational profile of UTMP in ovineamniotic fluid.50Aonly known source of UTMP, however <strong>the</strong>se proteinshave also been identified in ovine allantoic fluid andovine amniotic fluid (1). The aims of this study were todevelop a competitive ELISA capable of quantitativelymeasuring UTMP and to' determine <strong>the</strong> gestationalprofile of UTMP in ovine allantoic fluid and ovineamniotic fluid with <strong>the</strong> resultant assay developed.Material and MethodsUTMP, purified from ovine uterine secretions,were used to raise antisera in chickens (JMCKI7) whichwere <strong>the</strong>n used to develop a competitive ELISA.Allantoic fluid and amniotic fluid samples werecollected from thirty-three merino ewes across gestationand were stored at -20°C until assayed. The datacollected were organised into gestational age groups andanalysed by one way analysis of variance after logtrans<strong>for</strong>mation.ResultsThe following values are presented as meanconcentration of immunoactive UTMP ± SE. As shownin figure 1 <strong>the</strong> concentration of UTMP in allantoic fluidat day 50 of gestation was 367.8 ± 12.7 Jlg/mL. UTMPlevels increased rapidly and significantly (p
EMBRYONIC STEM CELL LINE PLAYS THE MOST IMPORTANT ROLE IN THEPRODUCfION OFGERMLINE CIDMAERAS .RuiIi Li, Ian Lyons, Richard Harvey, Lorraine Robb.The Walter and Eliza Hall Institute ofMedical Research, P.O, Royal Melbourne HospitalVictoria 3050 .Introduction The isolation of embryonfc stem (ES) cells from mouse blastocysts has proved avaluable tool <strong>for</strong> research into gene function, gene regulation and developmental biology. Amajor advantage of ES cells is <strong>the</strong> capacity of ES cells to be precisely manipulated at <strong>the</strong>molecular level in vitro and to integrate into <strong>the</strong> host embryo in vivo <strong>the</strong>re by giving rise tochimaeric offspring. These offspring can produce ES cell-derived germ cells enabling <strong>the</strong>breeding of mouse strains homozygous <strong>for</strong> a gene alteration (germline chimaeras). An importantaspect of gene targeting using ES cells is <strong>the</strong> efficient production of germline chimaeras. Wereport here <strong>the</strong> factors affecting <strong>the</strong> production of germline chimaeras, which we have measuredin our gene targeting studies.Materials and Methods 1. Effect of <strong>the</strong> source of host embryos on <strong>the</strong> chimaera production:Blastocysts from BALB/c and C57BIIKa mice which were ei<strong>the</strong>r superovulated or naturallymated were used as host embryos. Ten to 15 ES cells (CCE) were inj ected into blastocysts whichwere transferred to pseudopregnant mice. 2. Chimaera production by morulae aggregation orblastocyst injection of ES cells. Embryos (F2: C57B1I6 x C57Bl/10) at morula stage and atblastocyst stage were aggregated and injected with ES cells (E14) respectively. Subsequentchimaera production was examined. 3. The effect of different ES cell lines on <strong>the</strong> production ofgermline chimaeras. Six well established ES cell lines were cultured and inj ected into blastocysts(F2). The production of germline chimaeras was examined by GPI (glucose phosphateisomerase) analysis of sperm (Mann et.a!., 1993).Results A higher number of chimaeras wasproduced when host embryos were fromsuperovulated C57BIIKa compared withBALB/c mice. Superovulation did not affectchimaera production ofbIastocysts injected withES cells, instead, it was superior than naturalmating in certain mouse strains. More pupswere born and a higher percentage of chimaeraswas produced in blastocyst inj ection comparedto morula aggregation (70% vs 17 % ) of EScells. Different ES cell lines gave rise todifferent production of germline chimaeraswhen <strong>the</strong>y were injected into blastocyst (Fig. 1).Fig. 1: Germllne Chimaeras of ES Cell Lines• CCE2d E141m W9.S~ R1[:J D3II J1.1Discussion. These studies show that superovulation is an effective way to supply embryos <strong>for</strong>chimaera production. Morula aggregation of ES cells can produce chimaeras but <strong>the</strong> yield islower when compared to blastocyst injection ofES cells. When an ES cell line is used <strong>for</strong> genetargeting, it is always advisable to test <strong>the</strong> production of germline chimaeras of <strong>the</strong> cell lines,although <strong>the</strong>se ES cell lines had demonstrated production ofgermline chimaeras elsewhere.0/010080604020o L-......J~~..JIllL.__~a...L-=-Cb Im a eLa s Germlin e Chimaer asESTABLISHMENT OF A SLA :(NBRED MINIATURE PIG HERD FREE OFQUARANTINE RESTRICTIONSJ.J. Arlaud a , L. Baker a , R.L. Williams a , C. P. Cunningham b , I.M. Lewis a , W.G. Browne,M. BorgC, D. Kerton C and AJ. French aaCentre <strong>for</strong> Early Human Development, Institute of Reproduction and Development, Monash University,VIC, 3168 .bSchool of Veterinary Science, University of Melbourne, Parkville, 3052CPig Research and Training Centre, Victorian Institute of Animal Science, Werribee, 3030The inbred SLA miniature pig was developed by selective breeding to create a genetically definedanimal, homozygous <strong>for</strong> an allele of <strong>the</strong> major histocompatibility locus (MHC) (1). We maintain asmall colony of inbred SLA miniature pigs in quarantine to allow studies in <strong>the</strong> field ofxenotransplantation.To allow expansion of <strong>the</strong> herd and removal of quarantine restrictions, a new miniature pig herd wasinitiated via embryo transfer using first and second generation offspring bred from <strong>the</strong> originalsixteen imported founding animals. Estrous synchronization and superovulation of donor sows wasattained using a previously described protocol (2). Briefly, donor sows (>190 days old), mated ondetected estrus and confirmed pregnant by non return to estrus (days 18-22) and ultrasound (days21-35), were aborted by two prostaglandin (PG, Juramate, Jurox Pty. Ltd.) injections (1.0 & 0.5 mg)24 h apart, and superovulated with 1000 iu eCG (Folligon, Intervet (Aust) Pty. Ltd.). The eCG wasgiven simultaneously with second PG treatment. Ovulation was synchronized 72 h after eCG with500 iu hCG (Chorulon, Intervet (Aust) Pty. Ltd.). Donors were mated twice approximately 32 h and49 h after hCG. Day 3 (4-8 cell embryos) were collected under anaes<strong>the</strong>sia using a Foley ca<strong>the</strong>ter(16-18 Fr) from <strong>the</strong> upper two thirds of each uterine hom. Zona intact embryos were repeatedlywashed (lOx) in fresh media and treated with 0.25% trypsin (60-90 s) to remove infectious agentsaccording to lETS guidelines (3.). Outbred commercial recipients (Large WhitelLandrace) weresynchronized 24 h behind that of donors by injection of 1000 iu of eCG (Folligon, Intervet (Aust)Pty. Ltd.) 24 h after weaning and a fur<strong>the</strong>r injection of 500 iu hCG (Chorulon, Intervet (Aust) Pty.Ltd.) 73 h after eCG. Recipients were selected <strong>for</strong> embryo transfer following detection of standingheat (24 h post hCG). Embryos were surgically transferred to recipients two days after heatdetection.One hundred and <strong>for</strong>ty four embryos were collected from 21 donors (Avg. 6.9 embryos/donor) andtransferred to nine outbred commercial recipients. Five recipients farrowed 21 live piglets. Of <strong>the</strong> 21piglets born 16 are currently being used to fur<strong>the</strong>r expand <strong>the</strong> new herd. Comparison between birthweights of SLA miniature pigs born and suckled on SLA miniature sows and recipients (LargeWhitelLandrace) are currently being examined.1. Sach, D.H.,Leight, G., Cone, J., Schwarz, S., Stuart, L., and Rosenberg, S., (1976) Transplantation22(6):559-5672. Arlaud, J., Williams, R.L., Michalska, A.E., and French, AJ., (1998) Proc. Aust. Soc. Reprod. Biol. 29:19(abst.)3. Manual of <strong>the</strong> International Embryo Transfer <strong>Society</strong> (1990) (Eds. Stringfellow, D.A. and Seidel, S.M.,)Champaign IL, USAReferencesMann GB, Fowler KJ, Grail D, Dunn AR. (1993). Identification of germ-line chimaeras by polymerasechain reaction and isoenzyme analysis ofmouse spermatozoa.). Reprod. Fertil. 99: 505-512.126127
Controlled Breeding of Lactating Cycling Dairy CowsVK Taufa, KL Macmillan!, G Verkerk, SR Morgan,AM Day, R Hooper .Dairying Research Corporation, Private Bag 3123, Hamilton, NZ. I School of Veterinary Science, University of Melbourne, 250Princes Highway, Werribee, Victoria, Australia 3030.IntroductionPrevious trials in heifers achieved a high submission rateof 94% with comparable conception rate of 62% on <strong>the</strong>1st day of insemination by extending <strong>the</strong> CIDR insertionperiod from 7 to 8 days, with an oestradiol benzoate(ODB) injection at 24h after CIDR removal (1, 2). Theapplication of this system to cycling dairy cows withincorporation of two resynchrony treatments to condense<strong>the</strong> submission of cows returning to 1st or 2 ndinseminations could reduce whole herd inseminations to aperiod ofless than 14 days over 3 synchrony periods.AimTo improve <strong>the</strong> efficacy of a whole herd synchronyprogramme <strong>for</strong> use in seasonally-bred dairy herds.Materials & MethodsPre-mating heat records and/or ovarian palpation wereused to identify cycling cows (CC) in 10 commercialdairy herds. The CC cows were all treated with aprogesterone releasing intravaginal insert (CIDRTM,InterAg, Hamilton, NZ) <strong>for</strong> ei<strong>the</strong>r 7 days or8 days (7-dCC; 8-d CC) with 2mg ODB injected i.m (OestradiolBenzoate, Intervet, Auckland, NZ) at insertion (-d9).At insert removal (-d2 and -d1 <strong>for</strong> <strong>the</strong> 7-d CC and 8-d CCtreatments, respectively), each cow was given an i.m.injection of 500~g prostaglandin (PGF; cloprostenol,Estroplan, Parnell Laboratories, Auckland, NZ).At dO, any cow that had not been seen in oestrus, asdetermined by tailpaint removal, was given an Lm.injection of 1mg ODB (CIDIROLTM, InterAg, Hamilton,NZ). The cows were inseminated on detection of oestrusfrom dO. Those cows which had been inseminated fromdO-d2 were resynchronised with <strong>the</strong> previously usedCIDR inserts. These were reinserted at d13 <strong>for</strong> 7 days,with half of <strong>the</strong> animals allocated randomly at reinsertionto receive an i.m. injection of 1mg ODB. Any cowreturning to oestrus was inseminated on detection from48h after insert removal (d22). Cows which wereinseminated from d22-d24 received a fur<strong>the</strong>r i.m.injection of Img ODB on d35, and were inseminated ifdetected in oestrus.Bulls joined <strong>the</strong> herds from around d46. Individualconception dates and pregnancy were confirmed by rectalpalpation of <strong>the</strong> uterine contents 8 weeks after <strong>the</strong> firstround of AI, and again, 8 weeks after <strong>the</strong> bulls wereremoved from <strong>the</strong> herd.Results and DiscussionThere were no differences in submission (SR) andconception (CR) rates over <strong>the</strong> first 4 days <strong>for</strong> ei<strong>the</strong>r <strong>the</strong>7-d CC or ilie 8-d CC cows but <strong>the</strong> submission patternwas significantly different (Table 1). On dO, only 13% of<strong>the</strong> 7-d CC cows were inseminated compared to 81 % <strong>for</strong><strong>the</strong> 8-d CC cows (p < 0.001). The cumulative figureswere 91 % and 97%, 95% and 97%, <strong>for</strong> <strong>the</strong> 7-d and 8-dCC cows respectively, <strong>for</strong> d1and d4.128The PR of cows inseminated on dO also differed (5%vs 41 %; p < 0.001, <strong>for</strong> <strong>the</strong> 7-d CC and 8-d CC cowsrespectively), but <strong>the</strong> cumulative PR were similar by d4.Of <strong>the</strong> cows that had <strong>the</strong>ir returns synchronised to 2 ndinseminations, 84% were inseminated from d22-d24. TheODB at CIDR reinsertion improved <strong>the</strong> submission rate(57%) compared to those cows that received No ODB(43%). Similarly, injecting ODB atd35 gave a partialsynchrony, with 90% of those cows returning to 3rt!inseminations, bred from d41-d46.There were no effects of ODB treatment on <strong>the</strong> CRof cows with returns synchronised to till inseminations(53% vs 55% <strong>for</strong> ODB, No ODB treatments,respectively). The CR of cows which received ODB atd35 was 55%.Table 1: SR, CR and PR on dO and cumulatively to d4,d24 or d46, and mean interval from planned start ofmating (PSM) to 1st inseminations and to day ofconceptIOn.Variable 7-dCC 8-d CCn 973 923% %dOSR 13 81IdSR 91 954dSR 97 97dOCR 38 491dCR 55 484dCR 51 4822d-24d CR 48 5841d-46d CR 48 55OdPR 5 414dPR 50 4724dPR 68 6846dPR 79 81Final PR 88 91Intervals (d)PSM-1 sl Insem. 1 2PSM-Concept. 17 18ConclusionsThe extra day of progesterone pnmmg and reducedinterval from CIDR removal to ODB injection, allowed80% of <strong>the</strong> cows to be inseminated on dO but <strong>the</strong> oestrous.s:ynchrony was not significantly precise <strong>for</strong> set-timeinsemination of all cows. The modifications applied towhole herd synchrony programme enabled 80% of <strong>the</strong>cows to be pregnant to AI over 3 synchrony periods.AcknowledgmentsThe authors thank <strong>the</strong> farmers <strong>for</strong> <strong>the</strong> use of <strong>the</strong>ir animals,InterAg <strong>for</strong> <strong>the</strong>ir support with CIDR and CIDIROL,Intervet <strong>for</strong> Oestradiol Benzoate, Parnell Laboratories <strong>for</strong>PGF and DRC staff <strong>for</strong> <strong>the</strong>ir generous help.References1. Day, M.L., et al. (1997). Proc. Aust. Soc. Reprod. BioI.28:101.2. Taufa, V.K., Macmillan, K.L., et al. (1998). Proc. Aust.Soc. Reprod. BioI. 29:18.SHEEP EMBRYOS VIABLE AFTER 13 YEARS STORAGEN. M. Fogarty, W.M.C. Maxwell l , J. Eppleston 2 and G. Evans lAgricultural Institute, Orange NSW 2800, lUniversity of Sydney, NSW 2006 and 2PO Box 20,Bathurst NSW 2795IntroductionFrozen embryo technology has been used to importnew genetic material with embryos successfullytransferred up to a year after freezing (1). It can alsobe used <strong>for</strong> long-term maintenance of geneticresources, such as selection and/or control lines, raregenotypes, insurance against loss <strong>for</strong> unique stocks,as well as provide accurate evaluation of selectionresponse, free from genetic drift. It has not generallybeen used <strong>for</strong> <strong>the</strong>se purposes because of a lack ofconfidence in <strong>the</strong> technology and demonstration of itssuccess <strong>for</strong> long term programs. This paper reportson <strong>the</strong> survival to term of transferred sheep embryosthat had been frozen <strong>for</strong> 13 years. Survival ofrecently vitrified embryos and <strong>the</strong> success oflaparoscopic insemination with long-term frozensemen was assessed concurrently. Genotype andenvironmental effects (at collection and transfer),including stage and grade of embryos, on survivalwere examined.Materials & MethodsEmbryos (n=414), conventionally frozen 13 yearspreviously, were transferred (2/ewe) to maturecrossbred ewes by modified laparoscopic minilaparotomyprocedure. Two control treatmentsinvolved: transfer of recently collected and vitrifiedembryos produced in vivo (n=48) or in vitro (n=134)and intrauterine insemination of ewes (n=51) usingfrozen-thawed semen that had been frozen <strong>for</strong> 12years from sires of <strong>the</strong> long-term embryos. Pregnancywas assessed by progesterone assay (18d), ultrasoundscanning (54d) and lambing (term). Data wereanalysed by maximum likelihood procedures.ResultsMore ewes were pregnant in <strong>the</strong> insemination groupthan <strong>the</strong> embryo transfer groups at 18d (P
Birth of lambs following vitrification of in vitro produced ovine embryosG Evans, KM Rao, NM Fogarty* & WMC MaxwellDepartment of Animal Science, The University of Sydney, NSW 2006 &*NSW Agriculture, Agricultural Institute, Orange, NSW 2800IntroductionVitrification is potentially a cheap and simplemethod of cryopreservation of embryos. Thetechnique has been applied to ovine embryosproduced in vivo but not extensively to thoseproduced in vitro. This experiment was designedto compare pregnancy rates and development toterm of in vitro and in vivo produced vitrifiedembryos. For comparison, conventionally frozenembryos were contemporaneously transferred tosurrogate ewes and a fur<strong>the</strong>r group of ewes wasinseminated with frozen semen (Fogarty et al;1999).Materials & MethodsOocytes were sourced from adult Merino ewes at alocal abattoir, and matured and fertilised in vitro(O'Brien et aI, 1996). Presumptive IVP zygoteswere cultured in SOF-BSA <strong>for</strong> up to 8 days.Blastocysts were removed from culture on days 6, 7and 8, and any remaining late morula stageembryos were also removed on day 8. All embryoswere immediately vitrified in 0.25 ml straws inVS3a according to <strong>the</strong> method of Rall & Wood(1994). Embryos were also vitrified aftercollection from superovulated Merino ewes on day6 (day 0 = oestrus). All such embryos were devitrifiedand transferred in pairs to <strong>the</strong> uteri of 91synchronised BLxMerino ewes on day 6. Plasmasamples were taken on day 18-19 and assayed <strong>for</strong>progesterone; ewes with plasma concentrations >1nglml were judged pregnant. Ultrasonic pregnancydiagnosis was also per<strong>for</strong>med on day 54-55, and <strong>the</strong>day of lambing and number of lambs born werealso recorded. For comparison, <strong>the</strong> per<strong>for</strong>mance ofewes carrying long-term' conventionally frozen'embryos (n=207) and ewes inseminated with frozensemen on day 0 (n=51) was assessedcontemporaneously (Fogarty et al; 1999). Datawere analysed by X 2 and maximum likelihoodprocedures.ResultsThe results of pregnancy diagnosis at days 18-19and 54-55 are presented in Table 1Table 1. Pregnancy rates of ewes with vitrifiedembryos (n=2 embryos per ewe)Group n % % numberewes Pregnant Pregnant fetusesd18 d54-55IVPd6 27 74.1 44.4 16IVPd7 20 68.4 50.0 11IVPd8 20 50.0 25.0 7in vivo 24 50.0 33.3 10There were no significant differences in pregnancyrates between vitrification treatment groups atei<strong>the</strong>r stage of pregnancy. However, at d18-19 <strong>the</strong>pregnancy rates <strong>for</strong> NP embryos at d6 and d7 weregreater than <strong>for</strong> frozen embryos (49.5%; P
COMPARISON OF NORMAL AND METHOXYLATED ETHYLENE GLYCOL FOR THECRYOPRESERVATION OF 2-CELL AND BLASTOCYST STAGES MOUSE EMBRYOSM. Pangestu1.2 and J.M. Shawl1)lnstitute ofReproduction and Development, Monash University, Clayton 3168 Australia,2)Faculty ofAnimal Husbandry, Jenderal Soedirman University, Purwokerto 53122 IndonesiaIntroductionAlthough <strong>the</strong>re are a number of highly effectivecryopreservation (freezing) protocols <strong>for</strong> mouse oocytes andembryos, it remains almost impossible to freeze oocytes andearly embryos <strong>for</strong> most domestic species. The problemappears to be common to all species with "dark" (supposedlylipid rich) cytoplasm. It has recently been shown thatmethoxylation Le. replacing one or more OH group(s) byOCH3 group(s) of <strong>the</strong> cryoprotectant ethylene glycol (EG)increases its permeability, reduces its viscosity and increasesits capacity to bind lipids. As methoxylated ethylene glycol(s)have been successfully used <strong>for</strong> cryopreservation of kidney(1), it was possible that <strong>the</strong>y would suit lipid rich embryosbetter than conventional EG, even though <strong>the</strong>se compoundsare more toxic than EG. This study evaluated <strong>the</strong> efficacyand toxicity of cryoprotectants containing EG ormethoxylated EG's on early (2-cell) and late (blastocyst)stage mouse embryos.Materials and MethodsMouse embryos were generated by superovulating C57xCBAF1 females with PMSG (1OIU/ml), and hCG (1OIU/ml) 48 hlater followed by mating to Fl males. Two cell embryoswere collected 48 h after <strong>the</strong> hCG injection. Blastocysts <strong>for</strong><strong>the</strong> vitrification studies were derived from in vitro cultured 2cell embryos. The baseline cryoprotectant solution contained20% dimethyl sulfoxide (DMSO), 20% ethylene glycol (EG)and 0.6 M sucrose (2). Part of <strong>the</strong> EG component (1/4 or1/2) was substituted <strong>for</strong> Ethylene Glycol Dimethyl E<strong>the</strong>r("DE", 1,2-Di methoxyethane, Sigma Cat. E 1129) orEthylene' Glycol Monomethyl E<strong>the</strong>r ("ME", 2Methoxyethanol, Sigma Cat. E 5378) (Table 1).All embryos were cryopreserved and thawed inopen pulled straws (2). After thawing and 2 stepdilution embryos were washed and cultured inMTF medium and incubated at 37°C, in 5% CO 2 inair. Two cell embryos were cultured <strong>for</strong> 72 h todetermine <strong>the</strong> proportion developing intoblastocysts. Meanwhile <strong>the</strong> embryos vitrified at <strong>the</strong>blastocyst stage were in vitro cultured <strong>for</strong> 24 h todetermine <strong>the</strong>ir viability (re-expanding andhatching).ResultsThe development of 2-cell and blastocyst stageembryos vitrified in <strong>the</strong> baseline vitrificationsolution was comparable to that of controls andembryos vitrified in solutions containing 5% DE orME (Table 1). At a concentration of 10% both DEor ME reduced <strong>the</strong> proportion of 2-cell embryosdeveloping into blastocysts.ConclusionUp to 10% of <strong>the</strong> EO can be replaced by ei<strong>the</strong>r of<strong>the</strong>se methoxylated compounds, but some toxicitywas evident at <strong>the</strong> highest concentration (10%).This indicates that fur<strong>the</strong>r studies on lipid richembryos should be per<strong>for</strong>med.Table 1. Composition of<strong>the</strong> vitrification solutions and in vitro development of vitrified embryosVitrification solution compositionStage vitrified2 cell embryos BlastocystsVitrification Methoxylated EthyleneBlastocyst(%)(%)DMSO%Hatchingsolution compoundNglycol %N ViableNon-treatedBaselineDE5DEIOME5ME1005%10%5%10%20%15%10%15%10%2020202020222424262425100888869*9276485049505049100100100100100100444649444651*Statistically different compared to controls =P
INTRODUCTIONFERTILITY OF FROZEN-THAWED POSSUM SPERMATOZOA AFTERLAPAROSCOPIC ARTIFICIAL INSEMINATIONMATERIALS AND METHODSRESULTS AND DISCUSSIONFemales inseminatedProportion pregnantTotal embryos (up to 4-cell)/eggsFC Molinia' and JV MyersCooperative Research Centre <strong>for</strong> <strong>the</strong> Conservation and Management of MarsupialsLandcare Research, PO Box 69, Lincoln 8152, New ZealandCryopreservation of spermatozoa as a gene-banking strategy, toge<strong>the</strong>r with relevant assisted-breedingtechniques, is likely to become an integral component of marsupial conservation biology (1). Recovery ofmotile spermatozoa has been achieved <strong>for</strong> <strong>the</strong> brushtail possum in diluents containing 17.5% glycerol (2,3).Recently, laparoscopic artificial insemination (AI) technology has been established <strong>for</strong> superovulated tammarwallabies and brushtail possums (4). In this study, <strong>the</strong> fertility of frozen-thawed possum spermatozoa wasexamined after laparoscopic AI.Female possums (n = 21) were superovulated with a single injection of 15 iu PMSG (Intervet, TheNe<strong>the</strong>rlands) followed 78 h later by 4 mg porcine LH (Vetrepharm, Australia). On each day of insemination,spermatozoa were flushed from <strong>the</strong> cauda epididymides of 2 males with EMEM media (EMEM with nonessential amino acids, 20 roM Hepes, 0.23 roM sodium pyruvate, 2.2 gIL sodium bicarbonate, 100 iu/mlpenicillin, 100 •g/ml streptomycin all from Sigma, USA, pH 7.4) (5). Samples were pooled and split into twoaliquots: one aliquot was stored at 4·C <strong>for</strong> <strong>the</strong> fresh sperm inseminations, <strong>the</strong> o<strong>the</strong>r was diluted 1: 1 withcryoprotectant media (35% glycerol in EMEM media), <strong>the</strong>n frozen in pellet-<strong>for</strong>m on dry ice and stored inliquid nitrogen (3), prior to thawing on <strong>the</strong> same day. Uterine insemination with fresh (n = 9) or frozenthawed(n = 9) spermatozoa (5 x 10 6 motile sperm/uterus in a 0.2 ml dose) was per<strong>for</strong>med with <strong>the</strong> aid of alaparoscope at 26-29 h post LH treatment. As a control, an additional 3 females were inseminated withEMEM media alone. Ovaries were examined and eggs recovered by flushing oviducts and uteri withheparinised (12.5 iu/ml) PBS, 2 days after AI. Eggs retrieved were stained with Hoechst 33342 (Sigma, USA)and assessed <strong>for</strong> fertility by fluorescence microscopy. Proportions of embryos recovered were compared by.2 analysis of contingency tables.Table 1: Fertility offresh and frozen-thawed spermatozoa after AI of PMSGILH superovulated possumsControl Fresh Frozen-thawed30/30/697/912/20This is <strong>the</strong> first time that fertile conceptions have been achieved with frozen-thawed sperm <strong>for</strong> any marsupial.Similar proportions of eggs were fertilized following AI with fresh or frozen-thawed spermatozoa and 1-2embryos per female were recovered as previously reported (4). Work is currently focussed on generatingviable young using AI with frozen-thawed spermatozoa, by allowing embryos to continue development invivo ei<strong>the</strong>r in <strong>the</strong> inseminated female or by using embryo transfer technology to surrogate recipients.REFERENCES1. Mate KE, Molinia FC, Rodger JC (1998) Anim Reprod Sci 53:65-76.2. Rodger JC, Cousins SJ, Mate KE (1991)Reprod Fertil Dev 3:119-125.3. Molinia FC, Rodger JC (1996) Reprod Fertil Dev 8:681-684.4. Molinia FC, Gibson RJ, Brown AM, Glazier AM, Rodger JC (1998) J Reprod Fertill13:9-17.5. Sistina Y, Lin M, Mate KE, Robinson ES, Rodger JC (1993) Reprod Fertil Dev 5:1-14.13495/911/28Birth of Koala pouch young following artificial inseminationSD Johnston a , MR McGowan a , P O'Callaghan\ R Coxc, B Houlden d , S Haig 2 and G Taddeo eaThe University ofQueensland, 4072;bLone Pine Koala Sanctuary (LPKS), Fig Tree Pocket, 4069; CBioquest Ltd., NorthRyde, 2113; dTaronga Zoo, PO Box 20, Mosman, 2088 and eCook Australia Pty I:..td, Eight Mile Plains, 4122.INTRODUCTIONRESULTS AND DISCUSSIONAssisted breeding technology offers exciting new alternatives AI using <strong>the</strong> "Cook" ca<strong>the</strong>ter procedure in Exp.l resulted into traditional methods of ex situ management and breeding of <strong>the</strong> birth of 2 pouch young from 5 attempts; <strong>the</strong>captive mammals. Although <strong>the</strong>se techniques are increasingly urogenitoscopic procedure resulted in 3 offspring from 4being used in <strong>the</strong> captive propagation of eu<strong>the</strong>rian wildlife, attempts. Results of progestogen estimations from Koalas onproduction of marsupials by means of artificial insemination day 14 and 28 post IC are shown in Table 1 and confIrm that(AI) has yet to be accomplished.a luteal phase was successfully induced in all 9 females.MATERIALS AND METHODSPaternity analysis revealed that all teaser males could beEleven mature female Koalas held at LPKS were artificially excluded as possible sires. The Koala pouch young producedinseminated between December 1997 and March 1998. in Exp.l represent <strong>the</strong> first marsupials conceived and bornFourteen mature males were used as "teasers" to induce following AI.ovulation by means of interrupted coitus (IC) (2), or as sperm In Exp. 2, AI resulted in <strong>the</strong> birth of one pouch young from 2donors. Semen was collected by means of an artificial vagina attempts. Although <strong>the</strong> injection of hCG in F48 appeared to(1). The mean (± s.e.m.) concentration, % <strong>for</strong>ward motility result in <strong>the</strong> subsequent induction of a luteal phase (Table 1),and rate of movement of Koala spermatozoa used in this no pouch young was produced. However, AI of F326 culminated in <strong>the</strong> birth of an offspring, providing strongstudy was 162.2 ± 33.9 x 10 I ml, 76.1 ± 2.6 and 3.9 ± 0.2, evidence that <strong>the</strong> administration of hCG in this animal, notrespectively. Undiluted semen was maintained at roomtemperature' <strong>for</strong> 15 to 30 min prior to insemination.only induced a luteal phase, but presumably resulted inovulation. This Koala is <strong>the</strong> only marsupial to be bornOvulation was induced in 9 oestrous females by <strong>the</strong> means offollowing both hormonal induction of presumed ovulationIC and two methods of insemination were used (Exp.1). Theand AI. Fur<strong>the</strong>r investigations are under way to determine <strong>the</strong>fIrst involved a custom designed insemination ca<strong>the</strong>terefficacy of this ovulation induction treatment, particularly in(Figure 1) used to deposit approximately 1 ml of undilutedregards to optimal dose rates and timing of administration ofsemen into <strong>the</strong> dorsal portion of <strong>the</strong> urogenital sinusimmediately caudal to <strong>the</strong> vaginal ostia (2). Five female hCG.Koalas were gently restrained and inseininated using thismethod. The second insemination method involvedvisualisation of <strong>the</strong> vaginal ostia by means of an otoscopefitted with a 70 mm x 10 mm speculum (Welch Allyn, Model# 20200, USA) and was facilitated by positioning <strong>the</strong> femalein ventral recumbency on a tilting table set at an incline ofapproximately 30°. This procedure was accomplished afteranaes<strong>the</strong>tising <strong>the</strong> female (3). In total approximately 1 ml ofundiluted semen was ei<strong>the</strong>r deposited into <strong>the</strong> most cranialportion of <strong>the</strong> urogenital sinus immediately caudal to <strong>the</strong>vaginal ostia or directly into <strong>the</strong> vaginae using a 3.5 Fr."Tom-cat" ca<strong>the</strong>ter (Becton Dickinson Labware, U.S.A).Four Koalas were inseminated using this method.Figure 1 : Cook insemination ca<strong>the</strong>ter (Scale: O. 5 x)Table I : Progestogen concentration (ng/ml) in <strong>the</strong> peripheralplasma of Koalas on <strong>the</strong> day of insemination (dO), 14 and 28days fo llowing attempted induction ofovulation.Induction of ovulation in 2 oestrous Koalas was attempted by Koala Induction I dO d14 d28 Pouchintra-muscular administration of 250 LV. of hCG AI methodyoung(Chorulon®, Intervet, Castle-Hill, Australia) on <strong>the</strong> day ofF8 IC I Cook 0.5 6.4 13.3 NOinsemination (Exp.2). One female (F48) was inseminatedF20 IC / Cook 1.0 4.9 9.8 YESusing <strong>the</strong> "Cook" ca<strong>the</strong>ter, <strong>the</strong> o<strong>the</strong>r (F32) using <strong>the</strong>F42 IC I Cook 0.2 3.1 12.0 NOurogenitoscopic procedure described above. All femaleF43 IC I Cook 0.2 4.9 7.1 NOKoalas were kept isolated from males following AI.F44 IC / Cook 0.3 9.4 26.0 YESSuccessful induction of a luteal phase in non-parturientF36 IC I Tomcat 0.9 5.4 11.2 NOKoalas was confirmed by demonstrating an elevatedF2 IC I Tomcat 0.1 3.4 20.8 YESprogestogen concentration on day 14 and 28 post IC orF28 Ie I Tomcat 0.1 8.7 11.9 YESfollowing injection of hCG. A progestogen concentrationF45 IC I Tomcat 0.5 4.8 10.0 YESabove that of <strong>the</strong> upper 99.95% confidence limit of basalF48 hCG I Cook 0.5 5.3 6.8 NOoestrous progestogen concentration noted on <strong>the</strong> day ofF32 hCGI Tomcat 0.2 7.5 15.5 YESinsemination (day 0) and derived from all 11 females wasconsidered confinnatory evidence that a luteal phase had beeninduced. This value was determined to be 0.7 ng I ml. In Exp.I, paternity analysis of resulting offspring (4) was alsoconducted in order to exclude teaser males as possible sires.REFERENCES1. Johnston SD (1999) Studies toward <strong>the</strong> development of anartificial insemination procedure in <strong>the</strong> Koala Phascolarctoscinereus. PhD Thesis, The University ofQueensland.2. Johnston SD et al., (1997) J Reprod Fert 109:319-323.3. McGowan MR et aI., (1995) Aust Vet J 72:472-473.4. Houlden Bet aI., (1996) Mol Ecol 5:269-281.135
Fetal abnormality is more pronounced following transfer to synchronised r~cipientsofDay 5bovine IVP embryos compared to Day 7 embryos.J.G. Thompson, S. Cox, W.H. McMillan, AJ. Peterson, M. Donnison, L.T. McGowan and A. Ledgard.AgResearch Ruakura Research Centre, P.B. 3123, Hamilton, N.Z.IntroductionEarly fetal loss following transfer of in vitroproduced (IVP) bovine embryos significantly inhibits<strong>the</strong> uptake of this technology into <strong>the</strong> dairy and beefindustries. Pregnancy rates at Day 35 are ~ 60%, butdrop to 35-40% at term. Recently, this laboratory hasdescribed that some of this loss is due to abnormalallantois development (1). One hypo<strong>the</strong>sis to explainthis is that differentiation of <strong>the</strong> ICM andtrophectoderm may be compromised by in vitroculture. Transferring Day 5 embryos (Le. earlycompaction) may overcome this loss.Materials & MethodsBovine immature oocytes were obtained fromabattoir-derived ovaries and matured and fertilised invitro. Putative zygotes were cultured in <strong>the</strong> 1 51stageof a sequential embryo culture system, termed SOF99 (AgResearch Ruakura, Hamilton NZ), <strong>for</strong> ~ 120 hunder 7% 0" 5% CO, , 88% N, at 39°C. Earlycompacting morula (N=75) were washed once inemCare (ICP, Auckland, NZ) and transferred(I5/recipient, N=5) into <strong>the</strong> uterine horn ipsilateral to<strong>the</strong> CL of a suitably synchronised recipient. Asecond cohort of embryos were cultured <strong>for</strong> a fur<strong>the</strong>r48 h in <strong>the</strong> 2 nd stage of SOF-99. At <strong>the</strong> cessation ofculture, embryos were graded and transferable qualityblastocyst stage embryos (N=75) were similarlytransferred (I5/recipient, N=5) into synchronisedrecipients. On Days 23 (N=2/treatment), 26(2/treatment) and 29 (lltreatment), recipients wereslaughtered and <strong>the</strong> tracts ei<strong>the</strong>r flushed with salineor dissected to recover <strong>the</strong> fetuses. Quantitative datarecorded included crown-rump (CRL) and allantoislength. Qualitative data recorded included degree ofvascularity of <strong>the</strong> fetus, yolk sac and allantois. Thesewere collectively used to determine if <strong>the</strong> conceptusappeared morphologically normal or abnormal.Quantitative data was analysed using Least Squaresprocedure. Data presented are <strong>the</strong> predicted meanvalues. The proportion of normal embryos wereanalysed using X'!..ResultsIndividual conceptuses were relatively easy torecover from all three days of development, howeverit was noted that allantois anastomosis was initiatedby Day 29, making it difficult to fully dissect outindividual conceptuses. The proportion of recoveredfetuses did not differ between <strong>the</strong> three days (48, 48and 62% <strong>for</strong> Day 23, 26 & 29, respectively).Fur<strong>the</strong>rmore, <strong>the</strong> proportion of fetuses recovered didnot differ between <strong>the</strong> 2 treatments (Day 5 = 36/75,48% vs. Day 7 = 42/75, 56%). From <strong>the</strong> criteriaused, fewer fetuses recovered from Day 5 transfers(22/36, 61 %) were morphologically normalcompared to Day 7 transfers (36/42, 86%, P
AUTHOR INDEX138Abdo,MA 92Adams, MB 113Ahmed, FA 59,60Aitken, RJ 42Angurani, A 118Arlaud, JJ 127Armstrong, DT 85,88Baird, D 58Baker, L 127Bathgate, R .- 99Bhave, M 46Blache, D 55,56,96,116Blackberry, MA 55,56Bogoyevitch, MA 92Bongso, A 67Borg, M 127Boucaut, KJ 101Bowser, DN 36Breed, WG .41,68,109Britt, KL : 86Brown, WG 127Buist, J 71Bullen, DVR 79Butler, TG 113Byers, S 83Campbell, B 58Chagas, LM 56Chan, CS 103Chapman, JA 68Choo, BS 133Christy, L 107Chu, P-Y 49Clark, BA 53,54Clements, JA 103Clulow, J 39Cox, R 135Cox, S 136Cummins, JM 59,60,65Cunningham, CP 127Damavandi, Y 118Day, AM 128de Kretser, DM 95,108Dharmarajan, AM 92Donnison, M 136Drummond, AE 86Duckett, R 105Duggal, PS 57Dyson, M 86Edds, LM 69Edwards, L 113Emerson, M 99Eppleston, J : 129Evans, G .43,44,70,80,129,130Fairclough, RJ 46Feng, CY 46Findlay, JK 50,51,86Fogarty, NM .129,130Fong, C 67French, AJ ~ 127Galea, S 81,82Garcia-Aragon, J 101Gellatly, ES 43Gerdprasert, 0 89Gilchrist, RB 85,88Glazier, AM 45Grupen, CG 73Gunn, 1M 74,87Haig, S 135Hampton, AL 47Hansen, WR 111Harvey, MB 97,101,103Harvey, R 126Hearn, CM 102Hearn, JP 66Hearn, MTW 108Hedger, MP 89Herington, AC 97,101Hickox, DM 108Holt, WV : 77Hooper, R 122,128Houlden, B 135Hudson, S 93Hussey, ND 57fugman, WVI06Irving-Rodgers, HF 83,84Jackson, MM 97,101James, WG 72Jequier, AM 60Johnston, SD 77,135Jones, GM 35Jones, MEE 86Jones, RC 39Jones, RL 51Jurisevic, A 113Kaibuchi, K 100Kanai-Azum, M 100Kauter, K 58Kay, DJ 69Kaye, P 100Keah, H-H 108139
AUTHOR INDEX (cont'd)AUTHOR INDEX (cont'd)Keelan, JA 91Kerton, D 127Kilpatrick, LM 94Kinder, JE 84Kitchener, AL 69Knowling, REA 41Krzyzosiak, J 78Kuleshova, L 131,133Lacham-Kaplan, 0 35,72,81,82Ledgard, A 136Lee, CS 49Lee, RS-F 40Leigh, A 41,58Leigh, CM 41Lewis, lM 127Li, R 126Li, Y 50Lin, M 42,75,110Loveland, KL 95,107Lyons, I 126Macmillan, KL .119,120,121,122,123,128Maddocks, S 61,62Malakooti, N 48Malowski, SP 76Man, SY 39Martin, GB 55,56,96,116Martin, R 59Mate, KE 45,71Mattick, J 100Maxwell, WMC .43,44,70,80,129,130McArthur, ME 83McDonald, RM 40McFarlane, JR 58,124,125McGowan, LT 136McGowan, MR 135McKay, MJ 39McMillan, G 78McMillan, WH 104,136McMillen, IC 113McPhie, CA 44Meehan, T 95Mehmet, D 59Meier, S 53Metcalfe, SS 74Minoura, H 50Mitchell, GF 115Mitchell, MD 91,111Molan, P 78,79Molinia, FC .45,110,134Morgan, SR 53,54,122,128140Morrison, JR 108Mortimer, ST 43Murdoch, RN .42,75Murray, GR 40Mussard, ML 84Myers, JV 134Mylrea, G 122Naji, A ; 118Nave, C : 117Niasari-Naslaji, A 118Nicholls, R ~ 36Nie, G 47,50Norman, RJ 57,85Nottle, MB 73O'Brien, JK 76O'Bryan, MK 89,108O'Callaghan, P 135Oehler, DA 76O'Neill, C 98,99O'Shea, T 125Padula, AM 123Panchal, R 36Pangestu, M 132Pantaleon, M 100Parr, J 40Pearson, SJ 104Pera, M 67Peterson, AJ 104,136Petrou, S 36Potter, S 111Prendergast, E 116Preshaw, A 70,80Print, C 95Rabiee, AR 119,120,121Rao, KM 130Rathbone, MJ 120Reilly, AM 36Reinke, SN 87Renfree, MB 52,63,87,102,117Reubinoff, B 67Ricci, M 109Rice, GE 52Risbridger, GP 37Ritter, U 85,88Rhodes, FM 54Robb, L 126Robertson, SA 90,93,106Rodger, JC 45Rodgers, RJ 83,84Ross, JT · 113Roth, TL 76Salamonsen, LA .47,48,50,51,94Sarhaddi, F 118Scaramuzzi, R 58Schwartz, J 113Schwarzenberger, F 119Sebastian, LT 52Sebire, KL 108Setchell, BP 61,62Sharma, TP 96Shaw, G 52,117Shaw, JM 74,131,132,133Short, RV 63,117Sidhu, KS 45Simpson, ER 86Smart, ML 36Smith, JF 40Smith, R 112Stojanov,T 98Symonds, ME 113Taddeo, G 135Taggart, D 105Taufa, VK 122,128Teh, A 101Tellam, R 55Thompson, JG 136Travis, A 99Trigg, TE 123Trounson, AO .35,67,72,81,82,87,131,133Van Cleef, J 116Vercoe, PE 55Verkerk, GA 53,54,128Vishwanath, R 78,79Wade, MA 75Walker, SK 62Wallace, EM 114Wames, KL 113Whelan, J 59,60Whiteside, EJ 97,101Wiebkin, OW 68Williams, DA 36Williams, DR 124Williams, RL 127Wilmut, I 64Wood, S 100Wre<strong>for</strong>d, NG 86,105Wright, PJ : 49Wu, C 98Yaeram, J 61Yin, X 125Zhang, J 47Zhang, X 42,110Zhang, Z 63Zhu, B-k 62141
SUBJECT INDEXThis index was compiled using up to six key words(primarily from <strong>the</strong> title) <strong>for</strong> each abstract.Index TermPageAAcrosome..... 110ACTH 113Activin 51,114Adrenal gland : 112,113Ageing 59,78,79AI 134,135Apoptosis 92,95Aromatase 37,86,96Array technology 38Atresia 84,87,118BBirds 76,116Blastocyst 35,72,97,101,132Booroola 125Bull. 78,79cCalcium 36,73,80Capacitation ,. 44,45Cervical mucus 43Cloning.................................................................. 64,65Confocal 36,81Corpus luteum 92Cow 53,54,72,83,84,104,118,119,120,121,122,123,128,133,136CRR 112Cryopreservation 76,77,129,130,131,132,133,134cyclic AMP 39,99Cytokines 89,90,91,92,93,101,106,111Cytoskeleton 77,109DDifferential display 50Differentiation 64,66,67DNA 38,59,64,65Dog 49,74EEfferent duct 39142143
· SUBJECT INDEXSUBJECT INDEXIndex TermPageIndex Term (Cont'd)PageE (cont'd)LElectrolytes , 39Embryo 35, 62,64,66,67,72,74,98,99,100,101,103,104,126,127,129,130,131,132,136Endometrium : 47,48,49,51,52,90,94Energy balance 54,55,56,96,113,119,120,121Epididymis 41,42,80Extracellular matrix............ 83,97,103Leukocytes 48,51,90Leptin 55,56,57,58Leydig cells 95,105LIF' 102LH 54,56,57,86,96Luteolysis 46FFatty acids 53,56Fetus 113,114,136Follicles 83,84,85,86,87,88,118,133Follistatin........................................................................................................................................ 125FSH 85,86,125GGerm cells 63,87,95,107Glucocorticoids 112Glucose 58Glycosidases 41GnRH agonist. 123Granulosa cells....................................................................................................................... 84,85,88HIIIJHuman 47,51,59,67,81,82,91,112,114ICSI 35,81,82IGF 85,102Immunocontraception.............................................................................................................. 69Implantation 50,66,90,97,101Infertility 61,62,89,106,117Inflammation 49,89,90,91,93Inhibin 51,114Insulin 56Interferon 104In vitro maturation 70,71,72,73,74,82,130,136NF 70,74,80,81KKangaroo..................................................................................................................................... 68,87Koala 68,77,135Knockouts 37,86,93,106144MMacrophages 89Marsupial mice................................................................. 68,105Micromanipulation.. 35Meiosis 71,88,107Menstruation 47,48Metalloproteinases '" , 47,48,94,97Microtubule-associated proteins 107Mitochondria 59,60,65,79mRNA 38,46,47,50,51,57,113Mouse 37,48,50,61,62,86,88,93,95,97,98,99,100,101,103,106,108,126,132NNuclear transfer 35,64,65,72Nucleotides 39Nutrition 54,55,96,119,120,121oOestrogen 37,49,86,111,118,122,128Oestrus 53,123,128Oligosaccharides 41,68,75Oocyte 35,64,70,71,72,73,74,88,100Ovary 57,58,83,84,85,86,87,90,102,118,133Oviduct proteins ! 45Oxytocin 46pPAP 98,99Parturition 52,91,112,113,114PCR 57,59,60,98,101,103pH 35,36,41Photoperiod 55Pig 70,73,80,127Phospholipases ,. 52Possum 41,42,45,68,109,110,134145
SUBJECT INDEXNOTESIndex Term (cont'd)PagepPostpartum interval.............................................................................................................. 53,54,123Pregnancy 50,51,90,91,104,114,117,122,124,125,128,129,136Primate 66,85,112Progesterone 47,58,85,94,117,118,119,120,121,125,128Prolactin 116Prostaglandin 52,53,91,111Prostate.. 37Proteases 100,101,103Proteoglycans 83QIRRat. 39,57,60,63,92,96,107Ram 40,43,44,55,56,108Research funding...... 115sSeasonal changes 40,55,105,116Seminal plasma 40,43,44,75Seminal vesicles. 106Sertoli cells 63,95,105Sheep 40,43,44,46,55,56,58,96,114,124,125,129,130Signal transduction 92,94,95,100,111Spermatogenesis 61,62,63,105,106,108Spermatozoa 35,42,43,44,45,75,76,77,78,79,80,81,82,98,108,109,110,134Spermatoza structure 107,108,109,110Sperm storage medium 78,79,134Stem cells 64,66,67,126Steroids 37,86,102,117,118,119,120,121,122TTesticular injury 60,61,62,63,89Testosterone....... 116Testis 59,60,61,62,63,89,95,105,106Tissue recombinants 37UNIW!X!Y/ZUterus 45,46,47,48,49,50,93,104,111,124Uterine milk protein 124Wallaby, Tammar 52,69,71,75,102,117Zona pellucida....................................................................................................................... 35,68,69146 147
NOTES&df)~kw-.JJJ) s~('I? ~(/'JV\\V\._1- Gl,v"'-1< C'vt1:j' ~6h-_:"148