N OCIETY' - the Society for Reproductive Biology

NOCIETY'

(Front cover) Leadbeater's possum (Gymnobelideus leadbeateri) was though to have been extinct for more than 50 yearswhen it was rediscovered in the tall Mountain Ash (Eucalyptus delegatensis) forests of Victoria's Central Highlands in1961. This rare and beautiful possum is soft grey above with a prominent dark stripe along the midline, a creamy-whiteunder surface and a long dark furry tail. It is nocturnal and lives in small family groups or colonies which build their nestsin the hollows of large old eucalypts. It feeds mainly on insects and gum secreted from wattles, for example the Silverwattle (Acacia dealbata), in the understorey ofthe forest. Leadbeater's possum is one of the faunal emblems of the state ofVictoria and is endangered by the continued clearing and destruction of old growth forests, which has resulted in habitatfragmentation and subsequent population declines. They breed well in captivity and can be seen in the Nocturnal House atHealesville Sanctuary. Observing them in the wild is more challenging because of their rather shy and cryptic nature.Assistance in production of this publication was provided by the Medical lllustration Unit, MonashUniversity, and the Institute of Reproduction & Development, Monash University.Printed by Snap Printing, Notting Hill, Victoria1

The Australian Society forReproductive Biology (A.SRB)acknowledges the following for their support of the 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-SynthelaboSerono.. Hilton-on-the-Park, MelbourneCopyright © Australian Society for Reproductive Biology, 1999ISSN 0812·766223

The Australian Society forReproductive Biology (ASRB)Australian Society for Reproductive BiologyTABLE OF CONTENTS1999 ASRB Office Bearers 5Previous ASRB Office Bearers/Awardees 6Hilton-on-the-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 Society for Reproductive BiologyHILTON 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 the 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 the 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 for 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 the Lunchffrade ESA Meet the 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 the ESA Meet the1.00-1.30 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, Reproductive (Basic) Expressionthryoid, PTH Biology (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 the 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 for 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 the ductuli efferentes testisof the 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 themale Brush-tailed possum, Trichosurusvulpecula9.00 o 42 M Lin, X Zhang,Murdoch, RJ Aitken9.159.309.37oMo434445RIn vitro culture of brushtailed possumepididymal epithelium 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 thecapacitation status of fresh and frozenthawedram spermatozoaSecretory proteins from the femalereproductive tract of the brushtail possum(Trichosurus vulpecula): binding tosperm and effects on their survival invitroTimeFormat8.30 MPage46AuthorsTitleCY Feng, M Bhave, RJ mRNA editing of OTR: a new mechanismFaircloughfor 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 for menstruationA model for the study of uterine functionin the bitchG Nie, Y Li, H Minoura, J Mouse splicing factor SC35 mRNA is up­Findlay, L Salamonsen regulated at implantation sites in themouse 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 inthe endometrium of the 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 synthesis, plasma fattyacids and the onset of oestrous activity indairy cows Time Format PagePM Rhodes, BA Clark, SR Energy balance and the 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 the effectBlackberry, GB Martin of dietary fatty acids on LH pulsefrequencies in mature male sheep?PS Duggal, ND Hussey, RJNormanDetection of rat leptin receptor (isoformB) 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 the role of glucose in the 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 the 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 the mouseZ Zhang, MB Renfree, RV The effect of testicular cooling onShortspermatogenesis in the 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 the University of Nottingham before studyingwith Professor "Chris" Polge FRS at the University of Cambridge. His Ph.D. degree was awarded in 1971 forresearch on the Deep Freeze Preservation of Boar Semen. Subsequent research in Cambridge led to the birthof the 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 the procedures, which are now exploited by PPL Therapeutics at Roslin, for theproduction of proteins needed to treat human disease in the milk of farm animals.Over the past nine years, research has been focused on the factors regulating embryo development after nucleartransfer. This work led to the first birth of live lambs from embryo-derived cells and then to the 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 the current research are to develop biomedical applications of the nuclear transferprocedure. These include the provision of organs for xenotransplantation, the derivation of human cells fortherapy and the 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 the zonae pellucidae of severalmarsupial species9.009.07Mo7576RN Murdoch, MA Wade,MLinJK O'Brien, DA Oehler,SP Malowski, TL RothN-acetyl-D-glucosamine damages spermfrom the 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 for wildlifeEddscontrol: the 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 for protein synthesisWG James, 0 Lacham- Successful development of a 6-DMAPKaplan, AO Trounson replacement for bovine oocyte activationresearchCG Grupen, MB NottleChanges in intracellular calcium levels inporcine oocytes treated with ionomycinIn vitro maturation, fertilisation and earlyembryonic development in the dog9.23 M 77 WV Holt, SD Johnston Enhanced osmotic tolerance of Koalaspermatozoa following treatment with thecytoskeletal 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 the In vitrofertilisation of in vitro matured porcineoocytes by frozen epididymalspermatozoaS Galea, 0 Lacham- Confocal analysis of decondensation andKaplan, A Trounson pronuclear formation 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 Reproductive BiologySymposiumChair: 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 the 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 theMEE Jones, M Dyson, NG cyp 19 (aromatase) gene onWreford, ER Simpson, JK folliculogenesis in miceFindlaySN Reinke, 1M Gunn, MBRenfree, AO TrounsonPolyovular follicles in the 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 the 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 the 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 the 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 their interaction with nutrition in thecontrol 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 the 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 the uterine control of earlyembryonic development in the tammarwallaby, Macropus eugeniiTemporal and tissue specific expressionof mouse kallikrein (mKlk) genes andidentification of a novel mKlk mRNAtranscript during early development in themouse8.30 M 105 R Duckett, N Wreford, DTaggart8.378.529.079.229.37ooooo106107108109WV Ingman, SA RobertsonKL Loveland, L ChristyM Ricci, WG BreedSeasonal changes in the testicularmorphology of the Agile AnticinusMorphology of the testes and seminalvesicles of the male TGF~ 1 null mouseMAP2 in the testis: isoform 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 ofthe outer dense fibres from brush-tailedpossum spermatozoa110 X Zhang, F Molinia, M Lin A 49 kDa protein isolated from theacrosomal region of the 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 forhigh 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 the commercialisation ofAustralian R&D"11.00oo112 R SmithRegulation of parturition: the role of eRR11.3012.00o113 C McMillen114 E WallaceParturition and the HPA axis in the sheepfetus: unknown inhibitors and unlikelystimulatorsInhibins and activins: markers of maternaland fetal healthGraham Mitchell is a veterinary graduate and University gold medallist of the University of Sydney. Hestudied immunology with Sir Gustaf Nossal and Dr Jaq Miller at the Walter and Eliza Hall Institute of MedicalResearch (WEHI) where he obtained a PhD in 1969. After post-doctoral experience in California (Stanford),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 the global effort to develop newvaccines against parasitic diseases, and of the biotechnology revolution.In 1990 Mitchell was appointed Director of the historic and prestigious Royal Melbourne Zoological Gardenswhere he introduced a number of new initiatives in local and regional conservation and helped position themodern Melbourne Zoo as a key centre of recreation, education, conservation and research.In 1993 he returned to biomedical research as Director of Research in the R&D Division of CSL Limited,Australia's largest developer, manufacturer and marketer of ethical human biologicals and veterinary products.He was responsible for identifying and assessing new opportunities for CSL, nurturing external researchcollaborations of the company, and the research component of the 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 the academia-industry interface, has worked inmany overseas countries and been involved with the World Health Organisation for many years. He is anauthor of more than 350 publications, has received numerous awards for scientific achievements, and, in 1993,was appointed an Officer in the Order of Australia for services to science, in particular immunoparasitology.2829

Wednesday 29 th September(cont'd)SESSION 18: Reproductive 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 theShort, MB Renfree tammar wallaby, Maerapus eugeniiA Niasari-Naslaji, FSarhaddi, Y Damavandi, AAngurani, A NajiTitleA critical number of eggs stimulatesprolactin secretion in male emus duringthe 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 the form of oestradiolG Mylrea, S Morgan, R administration to yearling heifers at theHooperbeginning 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 the 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 the mostimportant role in the 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 the risk of contamination duringstorage in liquid nitrogenComparison of normal and methoxylatedethylene glycol for the 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 Society for Reproductive BiologyThirtieth 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) for thetreatment of infertility in the human and other mammalian species. The zona pellucida, surroundingthe mammalian oocyte is the major barrier to fertilization. Sperm must pass through this layer inorder to reach and fuse with the oocyte membrane and initiate embryonic development. The zonapellucida is also believed to protect the embryo during its preimplantation development in-vivo.Upon implantation in the uterus embryos at the blastocyst stage expand and exit from the 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 the zona pellucidaembryos may fail to hatch, hence reducing the chances of implantation and pregnancies. In thesecases, breaching or complete removal of the zona pellucida is necessary. Breaching of the zonapellucida is mainly done by micromanipulation procedures.To facilitate fertilization, a microneedle is used to penetrate the zona pellucida and leave the spermin close proximity to the plasma membrane or directly inside the oocyte cytoplasm in a processknown as intracytoplasmic sperm injection (lCS!). In this procedure, sharp glass capillaries areoperated manually to pierce the zona pellucida and the 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 inother mammalian species. These include mice, horses, cows, domestic cats and others and may bethe optimal procedure to be used for conservation of endangered species.Intracytoplasmic injection is also used to inject somatic cells into enucleated oocytes for the 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 the oocytecytoplasm.Assisted hatching and embryo biopsy for preimplantation diagnosis in the human are also done usingmicromanipulation procedures to partially remove the zona pellucida. Partial removal of the zonapellucida can be done chemically using low p~_ solutions, mechanically by a sharp microneedle, orby laser. The laser system for assisted hatching has been found to be easier to handle and morereproducible than the other two assisted hatching procedures. Using a laser beam directed on acertain point of the 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 the chemical and mechanical assisted hatching. The threeprocedures are found to enhance implantation and pregnancies in cases of failed IVF due to eitherthick or hardened zonae, suggesting that the techniques may be beneficial for 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 the regulation of intracellular, particularly intraorganellar, Ca 2 + and pH is of fundamentalimportance to the understanding of most cell systems. Two experimental strategies directly explore theinterorganellar 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 the mitochondria and while it has had somesuccess (Bowser et al., 1998), these fluorescence techniques are difficult and the attribution of results tospecific cell locations has not always been unambiguous. The second approach has also focussed on Ca 2 + andinvolves intracellular targeting of the luminescent, Ca 2 +-sensitive protein "aequorin", to specific organellesand has been in use for a number of years. Aequorin is a very sensitive Ca 2 + sensor and its properties havebeen thoroughly categorised well before 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 the light emission in terms of absolute [Ca 2 +J.Developments in molecular biological synthesis have recently provided Ca 2 +and pH sensors, that offer majoradvantages over chemically synthesized 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 the binding of Ca 2 +, calmodulin wraps around theM13 domain, thereby bringing the two flanking GFPs closer together. This proximity allows for Resonance.Energy Transfer (RET) of the emitted energy (from the "donor") directly into the excitation of the second"acceptor" GFP. The degree to which RET occurs represents a direct, ratiometric measure of free Ca 2 +concentration. The Ca 2 + affinity of the fusion protein is not fixed to that of the native calmodulin segment.Point mutations in this sequence with standard molecular genetic techniques have allowed fine-tuning of theCa 2 +affinities from as low as approximately 10- 8 M to up to 10- 2 M, suitable for different locations (i.e. highfor the cytosol, nucleus or mitochondria, and low for ER, Golgi or plasmalemma). The pH dependency ofvarious GFP mutants, in particular, that of enhanced yellow fluorescent protein (EYFP), with the highestpK'a of the mutants, provides exciting possibilities for 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 for infection of primary cultured adult cardiac myocytes.This system has already been used in our laboratory to create a recombinant aden0 viral plasmid containingthe SERCA2A gene and GFP for successful infection of adult cardiac myocytes. Protein-based sensors aredirected to their intended cellular location by 'tar~eting seque.nces' that are encoded within the sequence of aprotein. For nuclear localization the targeting sequence can be placed within a·protein or at the N or Cterminus and directs protein translocation to the nucleus of the cell, Similarly, for mitochondrial targeting aleader sequence directs the translocation of the protein to the mitochondria. ill the case of the endoplasmicreticulum (ER) the actions of a signal peptide and ER retention sequence together act to direct and thenretain a protein within the ER. Proteins lacking a targeting sequence are retained within the cytoplasm and,thus, 'targeted' to the cytoplasm. Our present work is characterising existing protein construct Ca 2 +-sensorsfor the ER and cytosol, and developing alternative versions with modified Ca 2 + affinities and differentlocalisation sequences, particularly for use in the mitochondria and at the 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 the 1970's. Heused TFM mice to demonstrate that regression of mouse mammary glands during embryogenesis, isan androgen regulated process. Since then, a number of studies in the mammary gland, prostate orvagina have used tissue recombinants to reveal. The nature of the cell - cell interactions between theepithelium and mesenchyme and the requirement for receptors on cell type. The purpose of thisworkshop is to describe the technique of constructing tissue recombinants using the prostate as amodel system. The development of the prostate is under androgenic control, but this talk will focuson the use of tissue recombinants to demonstrate a requirement for estrogens. Using the a estrogenreceptor knock-out (a ERKO) mouse model, tissue recombinations demonstrated that the presenceof ER a in the stroma and the epithelium 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 the interplay between the stroma and epithelium in mediatinghormonal responses by these organs. As the development of new mouse models of targeteddisruption of steroid receptors and steroidogenic enzymes became available, we can define thecellular mechanisms of all types of hormone responses.3637

MICROARRAYS: MICRO OR MACRO POTENTIAL?The Effect of Cyclic-Nucleotides on Fluid Reabsorptionin the Ductuli Efferentes Testis of the 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 the last few years has heralded a potentialfor providing new, sharp, tools for application to many questions in biology and medicine. At thisrelatively early stage, however, it is unclear as to what the 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 the strategies and common pitfalls which might be considered by thoseplanning microarray experiments. Other 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 the field of cancer, to which microarray analysis is beingapplied.IntroductionRecent work has shown that the ductuli efferentesare essential for male fertility (1). We have shownthat the ductuli reabsorb 96% of the fluid leavingthe testis and that the reabsorption is dependent onfluid flow rate and Na+ concentration of theperfusate(2,3). Our previous studies havedemonstrated that luminally adminstered cAMPsuppresses fluid reabsorption in the ductuliefferentis (4), indicating that the cAMP signaltransduction pathway is involved in controllingfluid reabsorption in the ductuli of the rat. In thehomologous proximal kidney tubule, cGMP hasalso been reported to reduce tubular reabsorption(5). The current study was undertaken to determinewhether cGMP has an effect on the fluidreabsorption in the ductuli efferentes.Materials and MethodsMicroperfusion experiments were performed onanaesthetized 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 the volume difference between theperfusate and collectate divided by the length ofthe perfused duct. Electrolyte composition in thecollectate was determined by energy-dispersivespectroscopy.ResultsTable 1 shows that the fluid reabsorption in theductuli efferentes is not significiantly changed by0.1-1.0mM dibutyryl-cGMP in the luminalperfusate. cGMP has no effect on reabsorption atconcentrations at which cAMP completelyabolishes reabsorption. Futhermore, there is nosignificant difference in the concentrations of Na+,K+ and cr , the major luminal ions, in thecollected fluid between the control and dibutyrylcGMP-treatedgroups (Table 2).DiscussionOur results strongly suggest that cGMP is not thesecond messenger mediating fluid regulation in theductuli efferentes. By contrast, both cGMP andcAMP are reported to mediate fluid reabsorptionin the proximal renal tubules (6,7). The Na+/H+exchanger has been reported as one of theprincipal transporters responsible for fluid andelectrolyte reabsorption in the ductuli efferentes(8) and its function could be regulated by cAMP asin the 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 inthe non-breeding season is of lower fertility than semenfrom the same animals in the autumn. These changes in thefertility 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 the possibility that certain seminalplasma proteins could influence the ability of sperm toretain their fertility after cryopreservation; thus, seasonalchanges in fertility of ram semen could be related toalterations in the composition of seminal plasma. Thepresent experiment was conducted to examine the 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 forvolume and density, then processed. Proteolytic inhibitors(benzamine and PMSF) were added to. a finalconcentration of 1 roM each and the seminal plasmaseparated by centrifugation (2x 10 min) at 1l,600g. Equalvolumes of seminal plasma from each ejaculate collectedon the same day for an individual ram were pooled,aliquoted and stored at -20°C until assay. Protein contentof the 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, the protein banding patterns were visuallyappraised. Samples from individual rams for each of the 12months were compared within the same gel and the proteinloading was normalised across all the lanes to correct forvariation in protein concentration between the samples.Protein identification. Five proteins that showed seasonalvariation were selected for 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, the bands of interestwere excised, and depending on the abundance of theprotein, either bands from 5 lanes or 10 lanes werecombined and the gel pieces washed extensively withwater, then 50 roM Tris-HCI, pH 8.5, containing 1 roMEDTA. The gel pieces were pulverized and suspended in0.4 mI of the 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 the supernatant was thenseparated from the gel slurry and volume reduced to < 100III by lyophillization. After acidication with trifluoroaceticacid to a final concentration of 1 % (v/v), the peptideswere resolved by reverse-phase HPLC. Individual peptidepeaks eluting from the column were collected and selected40peak samples were subjected to ~no acid sequencingusing the Applied Biosystems PrOCIse Sequencer (ABI).RESULTS: A seasonal change in the total proteinconcentration was detected in seminal plasma, with higherlevels (36.9 vs 23.0 mg/mI; P

In vitro culture of brushtailed possum epididymal epithelium 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 the epididymis to acquire motility and the ability forfertilisation. However, sperm maturation in marsupials is amuch longer and more complex process than that of theireutherian 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 eutherians duringthe past 2 decades, but not in marsupials species (2). Thispaper describes for the fITst time an in vitro cell culturesystem for a marsupial species, which supports epididymalepithelial cells from the brushtailed possum, and evidence ofsperm maturation induced by the cultured epididymal cells.Materials & MethodsThe epididymis from 5 possums were used to establish thecell culture system with a medium modified from eutheriancultures (3,4), and maintained at 36.5-37°C in 5% C02 in airwith humidity at 100%. Immunofluorescence ofcytokeratins was used as a marker for identification ofepithelial cells. Morphology of cultured cells was examinedby light and electron microscopy. Immature sperm obtainedfrom the proximal caput epididymis were co-cultured with7-day old cultured epithelial cells for the in vitro maturationstudy. Development of sperm motility and changes of spermhead orientation were examined as indicators of spermmaturation in the co-culture preparations.Results and DiscussionAll cell cultures of caput, corpus and cauda epididymisformed confluent epithelial monolayers with densely packedpolygonal cells. The cells remained viable for more than 2months in culture, and displayed typical epithelial characterspeculiar to each segment of the epididymis. EMobservations confIrmed that after 12 days in culture, thecells retained many structural features characteristic ofepithelial cells in the intact epididymis. These featuresincluded very slender and branched stereocilia andmicrovilli located on the apical surface of the 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 form the monolayers. This structuralintegrity suggested that the cultured epithelial cellsmaintained the secretory and resorptive functions of theepididymal principal.cells in intact organs.The maintenance of functional integrity of the cultured cellswas also evidenced by the 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 the maturestreamlined shape accompanied by the development of a fastflagellum beat with slow or medial progressive motility (Fig1 & Table 1).ConclusionsIt is envisaged that the present sperm co-culture system formarsupial species will greatly increase our understanding ofsperm-epididymal cell interactions in these species. It isanticipated that this system will also facilitate theidentification of specific epithelial factors, which may beexploited for the 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 the 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 the female reproductive tract of the brushtail possum (Trichosurus vulpecula):Binding to sperm and effects on their survival in vitroSidhu, K.S., Mate, K.E., Molinia*, F.e., Glazier*, A.M. and Rodger, J.e.Co-operative Research Centre for 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 for 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 thesame 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 then washed (600g, 6 min) in HSOF.The washed spermatozoa were resuspended in Dulbecco's phosphate-buffered saline (DPBS). TheDPBS contained either 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) for motility (light microscopy) and capacitation status (CTC fluorescence assay;3). Data for motility and proportion of F (uncapacitated), B (capacitated) and AR (acrosomereacted) pattern spermatozoa were analysed by analysis ofvariance and the results are presented inFigure 1.Figure 1. Motility and membrane status of spermatozoa (means ± s.e.m. pooled for 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 for fresh than frozen-thawed semen (85 and56 vs 42 and 13%, respectively; p < 0.001). Motility was always better in the presence of seminalplasma than in its absence and was highest for P and ram C (p< 0.001). There were more F- andless AR-pattern cells in the presence of seminal plasma from P and ram C than for the othertreatments or the control (p

mRNA editing of OTR: A new mechanism for 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 the late luteal phase of theoestrous cycle is thought to play an important role in regulating the time of luteolysis (1). In ruminants, thedevelopment of OTRs in the luminal epithelium is the 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 the rest ofendometrium such as caruncular stroma and deep glands. A large proportion of the endometrial OTR population incaruncular stroma and deep glands does not form until luteolysis is complete and the formation and activity of thesereceptors is probably oestrogen dependent. However, the basis for this difference in regulatory mechanisms remainsunclear. Our aim was to study the level of OTR mRNA expression in order to have better understanding of themechanisms 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 performed to amplify the 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 for further analysis. Three types of the clones from 600 bp products (designatedas 600A, 600B and 600C) were identified. Sequence analysis indicated eleven potential polymorphic sites in theirsequences, one in coding region and the others 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 the 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 the sequences of 600A, 600B and 600C, 1300D showed the presence of ACG­995 (Thr-331) and ACC-1I03 (Thr-368) codons. Instead, ATG-995 (Met-331) and ATC-1103 (Ile-368) codons werefound in the corresponding positions in the clones 600A, 600B and 600C. As OTR gene is a single-copy gene perhaploid genome, well conserved among vertebrate species (4), these results suggested the post-transcriptionalmodification existed at the positions, probably through mRNA editing.CONCLUSIONS Of particular interest is the 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 for the 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 further works nee~ to be done by searching whether mRNA editing occurred in the rest of the coding regionat different stage or from different type of cells, we have demonstrated that the difference of OTR populations inendometrium is due to mRNA editing and the existence of oxytocin receptor subtypes, resulting from mRNA editing.It is also suggested that the 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 other post transcriptional modification to coordinate.REFERENCES1. Flin~ ~PF, Lamming GE, Stew~ HJ and Abayasekara DRE 1994. The role of the endometrial oxytocin receptor indetermmmg the length of the stenle oestrous cycle and ensuring maintenance of luteal function in early pregnancy inruminants. Philosophical Transactions ofthe Royal Society (London) Series B 344 291-304.2. Wathes DC and Lamming GE 1995. The oxytocin receptor, luteolysis and the 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 the bovineoxtytocin receptor gene. DNA and Cell Biology 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 the proteases known to activate other MMPs:recently a family of membrane-associated (MT)­MMPs have been described that activate proMMP­2 in tumour cells. This study examined thelocalization ofMT1-MMP and MMP-2 in humanendometrium across the cycle and the 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.Northern analysis of cellular mRNA used an MT1­MMP probe from by Dr M Seiki (Tokyo).RESULTSImmunohistochemistry. MMP-2 wasimmunolocalized across the entire menstrual cycle,in 25/32 endometrial samples, but with greatestintensity in degrading menstrual tissue. It wasseen variously in the different cellularcompartments (luminal epithelium (4/22 samples)glands (18/32), stroma (24/32) endothelium(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 themenstrual phase but also in mid-proliferative andmid-secretory tissue, concomitant with tissueoedema. Epithelial staining was seen in glandsduring the 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 aroundthe periphery of the cells, indicative of its locationin the 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 the amount oflatent MMP-2 inthe 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 another class of protease.Immunostaining for MT1-MMP in the cells was reducedfollowing P treatment and became apparent in the cytoplasmrather than being cell membrane associated. Northernanalysis ofthe cells revealed a decrease in mRNA for 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. Northern 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 inthe presence ofP.• This activation is inhibited by a specific MMPinhibitor.• MT1-MMP mRNA expression in these cells is alsoinhibited by P.We postulate that MTI-MMP activates proMMP-2 inhuman endometrium, this activity being increased late in thecycle when P levels fall,. thus contributing to the 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 for the Study of Uterine Function in the BitchIntroductionIn human endometrium, matrix metalloproteinses(MMPs), enzymes involved in tissue degradationincrease dramatically prior to menstruation. MMPs areproduced by resident and migratory cells in the tissueand many factors contribute to their production andactivation. These include progesterone withdrawal andproducts of leukocytes (1). However, the 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 for menstruation (2,3) leukocyteinfiltration preceded tissue degradation suggesting thatthis model closely resembles human menstruation.AimThe aim of this study was to establish the mouse modelof menstruation in our laboratory and examine whetherMMPs are involved in the 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 they were subjected tohormonal treatment (estradiol 17 -~ (E) andprogesterone (P» and subsequently to a decidualstimulus (DS; 20 III of oil injected directly into theuterine lumen) (Fig.!).day48Leukocytes and matrix metalloproteinases are present at sites of tissuebreakdown in a mouse model for menstruationNakisa Malakooti and Lois A. SalamonsenPrince Henry's Institute of Medical Research and Education Program in Reproductive Biology,Monash University, Clayton, Victoria 3168, AustraliaFig. 1. Schematic representation of thetreatment protocol administered toovariectomized miceIn control animals, daily P injections were continueduntil autopsy. To see whether P withdrawal resulted intissue breakdown, 4 groups of animals were subjectedto hormonal treatment and the tissue was collected at52, 56 and 59 hours after the last P injection (Pwithdrawal) vs control animals. After autopsy uteriwere weighed, fixed and processed for histology (H&Estain) and immunohistochemistry (IHC) for desmin (amarker for 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 for 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 the 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 for menstruation in mice has been established.Leukocytes, MMP-7 & MMP-9 are present at the 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 the ovariectomised bitch, thatsimulated the 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 the uterus totrauma during dioestrus. Such trauma can beinduced by a silk suture placed in the lumenof the uterus (4).MATERIALS & METHODSMature bitches ( n=18 ) were ovariectomised andtreated with: oestradiol benzoate ( ODB, 0.6 - 4.8jlglkg, i.m., bj.d.) for 7 days (pro-oestrous group(n=2)); ODB for 11-12 days then progestagen (megestrol acetate ( MA, 2 mglkg, p.o., sj.d.) for4 days (oestrous group (n=2)); ODB then MAfor 35-37 days (dioestrous group (n=2))(l,2).These bitches were autopsied at the end of thetreatment period. Additional bitches receivedODB then 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 the same time as treated animals.In a further 3 bitches at ovariectomy a suturewas placed in the lumen of the uterus. Two ofthese bitches were treated with ODB (11 days)then MA (36-38 days) and were autopsied at theend 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 for 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 theoestrous cycle ( Figure 1,2 )(3). Markedendometrial exfoliation was observed at 3weeks after treatment. The endometrium hadrepaired by 9 weeks after treatment. Inhormone-treated bitches the silk suture resultedin CEH in one bitch and CEH with pyometrain the other 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 the normal oestrous cycleto facilitate studies of uterine function. Thismodel will be used to study further thepathogenesis of endometrial exfoliation andCEH.REFERENCES1) Concannon PW, et aI., (1975) Biology ofReproduction 13: 112-12l.2) Concannon PW, et al., (1979) Biology 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 the 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 the mostrelevant factor limiting successful pregnancy.During this process, the uterus undergoesmorphological and physiological changes totransform itself from a non-receptive to a receptivestate to accommodate a conceptus. The exactmolecular mechanisms underling the uterinedifferentiation critical for embryo implantation is notwell understood. The aims of our studies were toidentify and characterise genes specificallyregulated in the mouse uterus in association withembryo implantation.MATERIALS AND METHODSRNA differential display (DDPCR) was applied toidentify genes specifically regulated during thewindow of implantation in the mouse (1). InitiallyGenBank was screened to obtain the identity ofthe DDPCR-derived eDNA fragments. In the~bsence of a homologous sequence, a cDNAlibrary of mouse uterus was screened to obtain thefull cDNA sequence. Northern analysis was usedto ~etermine the uterine mRNA expression patterndunng early pregnancy. Pseudopregnant micewere compared with normal pregnant animals toexamine the effect of embryos on the expressionof uterine genes in relation to pregnancy.RESULTSSeveral genes were found to be either up- ordown-regulated at implantation sites during earlypregnancy in the mouse. One of them wasidentif!ed as the splicing factor SC35. Expression?f this .gene. was increased dramatically atI~plantatl.on sites, but not at interimplantationsites, dunng early pregnancy in the mouse uterus(Fig. 1). The up-regulation of splicing factor SC35in the uterus during early pregnancy was not seenin pseudopregnant animals (Fig.2).CONCLUSIONSOne of the genes identified by DOPCR as~pecifical~y related to the early phases of embryoImplantation was confirmed to be the SC35splicing factor. This gene was up-regulated atimplantation sites and its expression wasdependent on the presence of embryos in theuterus. The unique expression pattern of this gene~uggests an i~portant role for SC35 splicing factorIn pregnancy In the 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. Northern 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; the GAPDH probe was used toexamine the equal loading.+-3.0 kb+-1.8 kb+GAPDHFigure 2. Northern 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 the Rockefeller Foundation,WHO, Wellcome and NH&MRCIntroductionActivins and inhibins are members of the 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 the~A and ~B subunits. The type of mature dimer formedis dependant on availability of a subunits.Inhibins / activins have been widely implicated asparacrine agents in the ovary, and have importantimmunoregulatory functions. The placenta is a richsource of inhibin and activin during pregnancy, withthe potential for roles in regulating fetal-maternalinteractions. Whilst the 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 the 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 epithelium, although no cyclical pattern wasdetermined. ~A and ~B subunits were predominantlyexpr~ssed by glandular and surface epithelium, withmaxImal immunostaining in the late secretory andm~nstrual phases (Figure 1). Additionally, there waseVIdence of immunoreactivity for all subunits insubpopulations of leukocytes and in the vasculature ofsome samples.Figure 1 Immunostaining for f3B subunit in glandularepithelium in human endometrium!:::IiiEi---l1M P ES MS LSDEStage of CycleDeciduaAll inhibin subunits were strongly expressed by thepseudo-decidualised endometrium in the late secretoryphase and during early pregnancy. Intenseimmunostaining for ~A and ~B subunits was apparentin glands and stroma (Figure 2). In contrast, a subunitexpression was limited to the stroma, with prominentimmunostaining in the basement membrane of the largedecidualised cells (Table 2). ~ subunit expression wasalso apparent in vascular endothelium.T~le2Rgure2Differential expression oj f3A immunostaininginhibin / activin subunits in deciduain deciduaGlandsStromaex. - ++SA ++ ++BB ++ ++ConclusionsThese data confirm the expression of the a., ~A and ~Bsubunits by human endometrium. Further:• Glandular epithelium is the major site of synthesisin the non-pregnant endometrium.• Strong expression during decidualisation coincideswith extensive tissue remodeling and apoptosis.• Preferential expression of ~ subunits by glandularepithelium in the decidualised endometrium maydenote a bias towards activin production.• Expression of inhibin I activin subunits byleukocytes indicates a role in modulating theimmune 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 synthesis. PLA 2 catalysesthe hydrolysis of free fatty acids such as arachidonicacid from the sn-2 position of glycerophospatides inthe 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 the cytosol to the membrane(cPLA 2 ) and a 14kDa type IT secretory PLA 2 found inthe extracellular fluid (sPLA 2 ) (1). cPLA 2 requiresnanomolar calcium concentrations, is insensitive toreducing agents, but is inactivated by heat. sPLA 2 onthe other hand requires millimolar calciumconcentrations for activity and is sensitive toreducing agents and is not inactivated by heat (2).The onset and progression of labour is associatedwith increase prostaglandin formation (3). Thereforeactivation of PLA 2 activity could playa role in theonset of parturition in the tammar. The purpose ofthis study was to identify the presence of PLA 2activity within the reproductive tract of the tammarand characterise the 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 forhuman recombinant sPLA 2 and cPLA 2 were used tolocalise these enzymes in PFA fixed paraffinembedded uterine tissue from term wallabies, usingthe 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 the epitheliallining of the secretory glands in the 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 theamount of activity relative to the control. Heattreatment however reduced the enzymatic activity by72%. These data show that cPLA 2 is the predominantform 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 the more active form in the wallaby. As cPLA 2is the arachinoyl-specific enzyme this would suggestthat cPLA 2 plays an important role in liberating theprecursor for prostaglandin synthesis, arachidonicacid, at parturition in the 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 for 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 hypothesised that feeding certainrumen bypass FA will alter uterine prostaglandinsynthesis and influence ovarian function. Thisexperiment investigated the effect of supplementingdairy cows with rumen bypass FA during earlypostpartum (PP) on uterine prostaglandin synthesisand the onset of oestrous activity.Materials & MethodsFourteen recently calved cows were randomlyallocated to receive either bypass FA, in the form ofthe 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 the supplementation. The Rumentek feedhad a total fat content of 30%, of which 84% consistsof the 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 the jugular veinat 15 min intervals for 1 hr prior to and 1 hr after OTadministration, followed by 30 min intervals for anadditional 2 hr. These plasma samples were storedand the 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 for FAdetermination were collected on day 6 and day 32 PPand extracted using the 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 their involvement withprostaglandin synthesis. Results are expressed asmean±sem.ResultsFrom this study there was no effect of treatment onthe onset of ovarian activity. Two animals in theRumentek group had elevated plasma P4concentrations (>2 ng/ml) in the 3rd week PP. By day44 PP an additional 2 animals, both in the barleygroup, had elevated plasma P4 concentrations (>2.0ng/ml), evidence of corpus luteal formation. Basalplasma PGFM concentrations (R: 625±241 pglml; B:1076±265 pglml), were elevated during the first weekPP (day 6), as was the response to OT. At the latermeasurements basal PGFM concentrations, peakPGFM or the timing of peak PGFM concentrationsdid not vary across treatment groups. Differences inplasma 18:1 FAME content were at the p

Energy balance and the 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 the first 6 weeks of lactationcompared to cows in low body condition (1). The aims ofthe current study were to determine whether differencesin PPI are related to differences in energy balance and toexamine the relationship with pulsatile secretion ofluteinising hormone (LH) in the early postpartum period.Materials & MethodsTwo year old cows (13 Friesian, 8 Jersey) were managedduring the final trimester of pregnancy so that they 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 thereafter. Energy balance was calculated asthe difference between energy intake and energy requiredfor 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 the 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 themselves are added tothe diet, they also stimulate gonadotrophin secretion (1) suggesting that they may act via insulin orleptin to link feeding, metabolism and reproduction (2, 3). We tested these relationships bystudying the effects of dietary fatty acids on LH pulse frequency, CSF and plasma insulin, andplasma leptin concentrations.MethodsFifteen rams fitted with intracerebroventricular cannulae were acclimatized for 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 for 4 days, as a positive control. On Days -1 (beforetreatment), 4, 11 and 15, blood was sampled every 20 min for 24 h beginning at 07:00. On thesame days, CSF was sampled every 6 h. Plasma was assayed for LH, insulin and leptin, and CSFwas assayed for insulin.ResultsCompared to Day -1, LH pulse frequency increased in the fatty acid-fed group on Days 4 and 11(Fig. 1). There was no additional effect of the lupin supplement in the Fatty Acid group (Day 15).By contrast, the Control group showed a response only to the lupin supplement. In both groups,plasma and CSF insulin, and plasma leptin, levels were only increased by the 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 the Role of Glucose in the 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 for some time that the nutritional status of a ewe during the oestrous cycle caninfluence the number of ovulations (1), however the mechanism for 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 other systems including the reproductive system. In this study we have examined the uptake ofglucose and leptin by the sheep ovary and examined the 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 themhypogonadotrophic and then 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 the ovary andbrain.ovarian/carotid arterial blood, jugular venous blood and ovarian venousblood were collected over a 3 minute periods 48 hours after the start ofcombination LHIFSH treatment and stored frozen at -20°C until assayed.In another group of 6 ewes, oestrus was synchronised usingprogestagen sponges inserted for 12 days. At sponge removal the eweswere mated with entire rams. On day 12 of pregnancy bilateral jugularcannulae were inserted under local anaesthesia. The next day (day 13)groups of ewes (n = 3) were infused with either 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 theovary as shown in figure 1. This would suggest that the ovary like thebrain utilizes glucose rather 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 further insight intothe mechanism underlying ischemic/re-perfusiondamage in the testis. It has been proposed that themitochondria is the target for reactive oxygen speciesmediated damage (1). A rat model for male infertilitywas developed, using torsion followed by variousperiods of recovery. The mitochondrial "commondeletion" was measured in these testes as an indicatorofgeneral mitochondrial damage. We also hypothesizedthat this type ofdamage leads to the induction ofcertainfactors that playa vital role in apoptosis and protectionof the suviving cells, hence we carried out an RNAsubtractive hybridization between the control and thetorsioned 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 foreither 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 for this study (2). The effect oftorsion on the testes was detennined by the testicularbiopsy score count on representative tissues.Differentially expressed genes were isolated usingClontech PCR-select cDNA subtraction kit.ResultsBased on the biopsy scores it was clear that theexperimental model produced testicular damage thatwas more severe in the longer recovery groups (Tablel).However this did not correlated to the extent of the"common deletion " since no significant differenceswere seen between the control and torsioned testes (Fig1).Table 1. Testicular biopsy score counts in rat testestaking by counting 30 tubules in three rat testes. C isthe control testes and T is the torsioned testes. Scoresbelow 10 are considered subfertile. The values of theshort 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. Northern blots and sequecing are nowbeing undertaken to confirm which ones are truelydifferentially expressed and detennine their 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 the testis has been shown to reduce in vitro fertilization rates in sheep tlland whole-body heating causes some developmental retardation at the 8-cell stage and accumulation of morulae at 120 h in themouse (2). In this study, we have investigated the effects of whole body heat stress inflicted on male mice, on the developmentin vitro ofpreimplantation embryos sired with normal female mice up to 35 days after heating of the male.Materials and MethodsFemale C57/CBA Fl mice were superovulated using PMSGlhCG and mated naturally to a male of the same strain. Thesemales were either untreated (control) or had been exposed for 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 the 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 for up to 120 hours and scored in the 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 the Chi-squaretest.ResultsEmbryo development rates between control groups at D7 -D35 were not significantly different, and these data have beenpooled for analysis. During 24h - 120h of culture, the 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 further 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. Furthermore,whilst all nominated stages of embryo development were affected by paternal heat stress, the number of embryos at 2-C and Mto B stages appeared severely affected in all heat-sired groups. At D14, D21 and D35, other 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 theobservation that offspring may develop following transfer of a nucleus from the somatic cell of anadult animal to an enucleated oocyte. These include the provision of organs from animals fortransplantation to human patients, derivation of autologous cell therapy in human patients,development of new means to study the control of genetic diseases and enhanced animal breeding.The new procedures for 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, the 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 these are associated with increases in size of fetus or placenta, suggesting thatthere may be errors in imprinting. Similar abnormalities have been observed after culture ofruminant embryos.During cloning, the nucleus of a diploid somatic cell, with the DNA packed in appropriate nuclearproteins for that cell type, is transferred into an oocyte. The structure of the oocyte evolved in orderfor it to be able to accept a haploid nucleus, packed in protamines. It is perhaps surprising thatoffspring are ever born, rather than being disappointing that the procedure is so inefficient. Researchis required to define the mechanisms that act during reprogramming of gene expression in order toincrease the efficiency of the procedure.J.M. CumminsDivision of Veterinary and Biomedical Sciences, Murdoch University, Murdoch WA 6150Mitochondria were fIrst described over a century ago.However, the study of mitochondrial DNA (mtDNA)has exploded in the past two decades and is now one ofthe most rapidly growing areas of molecular geneticsand medicine. They almost certainly originated asendosymbiotic bacteria, and have retained their 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 the host cell cycle. However, it alsorequires close and species-specific congruence betweenmtDNA and mitochondrial control genes in the nucleus[1]. The importance of this harmony has implicationsfor cloning technology, for the study of imprinting andnuc1eo-cytoplasmic interactions, and for 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 thegenome, coupled with their importance in theproduction of ATP through oxidative phosphorylation,also means that many mutations and deletions in thegenome are associated with progressive bioenergeticdiseases [3]. Mitochondria are also one of the majorcellular sources of free radicals, which is probably whythey are sequestered away from the nuclear genome.However, the significance of deletions is probablyover-stated as most current PCR techniques grosslyoverestimate their prevalence [4].The path followed by mitochondria in the life cycle isextraordinary [5]. While sperm carry about 75 copies ofmtDNA into the oocyte at fertilization [6], these are ­almost universally - degraded by species-specific,ubiquitin-mediated proteolysis early in embryogenesis.This is probably to avoid compromising the embryowith degraded mtDNA, which is most likely toaccumulate in the sperm through exposure to freeradicals. The extent to which sperm rely onmitochondria for normal function is highly variablebetween speciesThere is an obligate delay in mtDNA replication in theembryo so that the pre-migratory primordial germ cellscontain only between 10 and 100 copies. Withcolonization of the gonads, mitochondria multiplyclonally. This restriction/amplification process acts as agenetic filter on copy number and ensures that in thefemale line the 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, the inexorable accumulation of deleteriousmutations in asexually reproducing entities [7]. Whilean attractive hypothesis, the 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 shortlybefore ovulation. This ensures the optimal start in lifefor the embryo. In the male line the spermmitochondria show reductions in mtDNA copy numberand acquire diminished ability to replicate. Duringspermiogenesis a unique haploid-encoded selenoproteincapsule forms on the midpiece and at some point themitochondria are tagged with ubiquitin to prepare themfor suicide after fertilization.There are a number of myths that have becomeestablished about mtDNA: that it does not enter the egg(false)[6]; that it cannot repair itself (also false); that itmutates much more rapidly than nuclear DNA (true butnot uniformly across the 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 beforeproteolytic destruction. This observation calls intoquestion the stability of the 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 thestan of pregnancy is enabled by an endocrinedialogue. The reliance on chorionicgonadotrophin (CG) as an embryonic signal ofviability, and capacity to support the corpusluteum, is apparently restricted to primates. Wequestioned the possible role of gonadotrophinreleasing hormone (GnRH) in regulating CGrelease during implantation. fu associated studies(with James Thomson, University of Wisconsin)we isolated and characterised the 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 the 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 the resulting change in CGsecretion and the differentiation stages ofembryos. In addition, embryonic stem cells wereisolated (4,5) and their secretion of CG monitored.Results:Control embryos (n=25) secreted a characteristicprofile of CG, with GnRH measurable in theculture 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 possibletherapeutic implications. Differentiatedembryonic stem cells also secreted CG andmaintained the developmental potential to formtrophoblast and all three embryonic germ layers.Discussion:Our results suggest a direct or indirect functionfor GnRH, secreted by the embryo during periimplantationstages of pregnancy, in theregulation 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 the mechanisms controllingprimitive streak formation in primates and theendocrine 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) OxfordReviews of Reproductive Biology 16: 1-323. Seshagiri, PB, Terasawa, E and Hearn, JP(1994Hum.Reprod.91300-1307.4. Thomson JA, Hearn JP et al. (1995)Proceedings of the 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 for 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 the 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 these 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 their 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 the dIfferentIatIng cells IS IndIcatIve ofembryonic stem cells.The potential value and use of embryonic stem cells are for the d~t~rminat~on of. the 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 these cells to be usedfor transplantation, be used for tissue engineering and gene therapy.The research with these putative embryonic stem cells is presently aimed to identify the factorsinvolved in the maintenance of stem cell phenotype and those involved in the check points directingearly differentiation events.References(1)Jones GM, Trounson AO, Lolatgis N, Wood C (1998) Factors affecting the 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 the zona pellucida (ZP). It is to thisstructure that sperm must fIrst bind, and then penetrate, before fertilisation can occur. Sperm-ZP binding is facilitated bythe glycoconjugates of the ZPs oligosaccharides (1). While the protein components of the mammalian ZP glycoproteinshave been shown to be highly homologous, there 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 theopossum to 8-10JllD in the koala (3), almost no work has been performed on characterising the glycoconjugatecomposition of marsupial ZPs. The aim of this study, therefore, was to determine and characterise, by the use ofdifferential lectin binding together with enzyme elimination and saponification, the saccharide components of the ZPsof follicular oocytes of several marsupial species each representing a different family within the 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% formalin). They were thenroutinely processed for light microscopy, embedded in paraffIn wax and sectioned. Sections were then 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 either desialylated (4) or saponified (5) respectively, prior to incubation with the lectins, as previouslydescribed. Specific controls involved incubating the lectins with 0.2-0AM of their inhibitory sugar prior to applicationto the 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 the fat-tailed dunnartsdisplayed the most deviation in their saccharides from those of other 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 the saponified ZPs of the fat-tailed dunnarts and no other species. The presence ofmannose may indicate, for the fIrst time, the presence ofN-linked oligosaccharides in the ZPs of dunnarts.ZPs of all species, except those of the opossum, appeared sialylated with increases in binding for f3-Gal(I-3)GaINAc(PNA) and a-D-Gal(NAc) (SBA) after removal of sialic acid with neuraminidase. Other glycoconjugates like thedisaccharides f3-Gal(1-4)GlcNAc and f3(1-4)-D-GlcNAc, as demonstrated by the lectins ECA and WGA respectively,appear to be common components within the ZPs of marsupials. The former, 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 speciesbefore 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 speciesbefore 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 eutherian mammalian ZPs (2), they also display considerable interspecific variation in their glycoconjugate content.Whether this variation relates to the species-specificity of sperm-ZP binding is unknown. Further 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 the landscape for agriculture andgrazing we have allowed .the numbers of. somespecies of macropods to Increase dramatIcally.This has lead to environmental degradation andcompetition for 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 performs essentialfunctions in fertilisation' (2). This study hasbeen conducted to provide proof of concept forZP molecules as potential target antigens in acontraceptive vaccine for macropods.Materials & Methods16 female Tammar wallabies (Macropuseugenii) were divided evenly into two treatmentgroups. The first group was immunised on 5occasions, at fortnightly intervals, with l00J.lgof whole porcine zona pellucida (PZP)emulsified in Freunds complete adjuvant for theinitial injection and incomplete Freunds for thesecond and subsequent injections. Thevaccinations were distributed over 4 sites, twosubcutaneous and two intramuscular. Thecontrol group received saline and Freundsadjuvant in the same manner. Blood sampleswere collected each time the animals wereimmunised and thereafter at monthly intervals.After the fifth injection the 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 the fertility of animals ineach group. Animals were sacrificed at the endof the breeding trial and the reproductive tractscollected for 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 the development ofcultured oocytes due to the absence of toxins which developmentally incompetent oocytes may produce.Brilliant Cresyl Blue (BCB) is reduced to a colourless compound by glucose 6-phosphate dehydrogenase inthe 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 the 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 for 90 minutes at 39°C in5 % CO 2 in air. Oocytes were then sorted into 2 groups based on their BCB staining (clear = no stain present,blue = BCB stain present). The oocytes were then matured for 44 hr, fertilised and cultured. A control groupof oocytes was matured without prior incubation. Oocytes were removed 17 h after insemination andassessed for maturation and fertilisation by staining with Hoechst 33342 and examining under UV light.Cultured embryos were examined on days 2 and 5 for 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 the 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 for 24 % of the total number of oocytes incubated with BCB it seems their removal fromculu:re ~~ould i~prove o~cyte and embryo development. However, there was a decrease in cleavage rate andno SIgnIfIcant dIfference In the maturation, fertilisation or development to morulae of blue compared withcontrol oocytes which suggests that removing the clear oocytes from culture did not necessarily improve theperformance of the remaining oocytes. It is possible that the extra handling of oocytes which occurs with theuse of the BCB test may adversely affect cleavage. Nevertheless, the percentage of cleaved embryos whichdeveloped to morulae was higher for blue than the 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 forpublication). .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 thewallaby was induced with 6 mg of porcine folliclestimulating hormone (FSH) (Folltropin V:Vetrapharm, Australia) administered i.m. twicedaily for 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 forprotein synthesis during in vitro maturation. Theeffect of inhibition of protein synthesis atdifferent stages of nuclear maturation wasexamined by die addition of 10 Jlg/ml CH to thematuration 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 beganthe process of chromatin condensation within 4 hof culture, at which stage the chromatin in mostKaren E. Mate & Janine BuistMarsupial CRC, School of Biological Sciences, Macquarie University, NSW, 2109.IntroductionOocytes from the tammar wallaby(Macropus eugenii) resemble those of eutherianmammals in their ability to complete meiosis invitro when cultured under suitable conditions.Recent work indicates that late maturationalevents in the marsupial egg, may be important notonly for developmental potential of the embryobut also for early fertilization events such assperm binding to the zona pellucida and to theplasmamembrane of the egg. In order to begin toaddress these issues, this study examines thetiming of maturation in vitro and its requirementsfor protein synthesis.eggs appeared as long homogeneous threads.After 8 hours of culture chromatin condensationhad progressed so that there 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 the 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 theprotein synthesis 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 for four hourswere able to progress to the 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 themost successful is the Ca++-ionophore A23187followed by 6-dimethylaminopurine (6-DMAP) (1).A similar success with oocyte activation has beenobserved in the sheep with ethanol exposure followedby 6-DMAP (2). However, concerns have been raisedto the effects the non-specific protein kinase inhibitor6-DMAP might have on embryo development (3,4).6-DMAP has been shown to accelerate thedisappearance of phosphorylated forms 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 the synthesis of proteins rather than theirphosphorylation. Cycloheximide treatment is capableof activating both young and aging oocytes at highrates. The majority of other artificial stimuli areunable to activate young oocytes adequately (5). Thishas often limited nuclear transfer procedures to theuse of aging oocytes, in which cytoskeletaldeterioration has been observed (5). The presentstudy investigated the effect of cycloheximidetreatment following ethanol exposure on IVM bovineoocytes.Materials & MethodsImmature oocytes were aspirated from abattoircollected ovaries and matured in-vitro for 24hr (6).Prior to activation, oocytes were denuded of allcumulus by vortexing for 3min in 50111 hyaluronidasesolution (lmg/ml). Mature oocytes that had extrudeda ~rst. polar body were used for the 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 withthe cycloheximide treatment. Treated oocytes werewashed through drops of equilibratedSOF120AAIBSA medium, and cultured for 3 days inSOF120AAIBSA at 39°C at 7% O 2, 5% CO , 288% N rOn day 3 (day 0 = day of activation) parthenoteswere transferred to fresh medium and cultured for afurther 4 days, after which blastocyst formation wasdetermined.ResultsNo significant difference in cleavage rates wasobserved between the activation treatments thatproduce diploid parthogenomes (Table 1). However,the cleavage rate of ethanol treated eggs, whichproduces haploid parthogenomes, was significantlylower than the diploid treatments. No significantdifference in blastocyst formation rates was observedbetween the 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 the 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 the peculiaritiesof canine reproductive biology compared to other domesticspecies. Canine oocytes are ovulated at the germinal vesiclestage and undergo maturation to metaphase II within theoviducts. Successful IVM and IVF to produce cleavedembryos has been documented only twice in the 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 other domestic species (3,4).The aim of this study was to explore a simplified method forIVM, 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 the 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 performedwhen 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 the ovary with a #22scalpel blade.All COCs were matured in synthetic oviductal fluid (SOF).Small antral COCs were supplemented with 5-10 % pooled,autologous follicular fluid. All maturation was performed 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, the COCs were washed once in SOF and thentransferred to 400 JlL of pre-equilibrated TALP containing5% pooled follicular fluid for fertilisation.('·IVF: Either 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 the first and second (sperm rich) fractions ofJ the 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 performed 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 the presumed zygotes were gentlypipetted to remove excess cumulus cells and ·sperm, thentransferred to 400 J..LL of pre-equilibrated SOP.74After 72 hours the embryos were denuded andexamined for polar body extrusion and blastomerenumber to determine stage ofdevelopment.Results: In-vitro maturation, fertilisation andembryonic development to the 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 the 8-cell stage ispossible. Yamada achieved 9/148 (6%) of embryosreaching the 5-8-cell stage.A number of other conclusions and comparisonsare made which describe the simplification of theprotocol. Unlike the previous studies (1,2), preincubationof sperm to enhance capacitation wasnot necessary to achieve fertilisation. Eight cellembryos were produced after only 24 hours invitromaturation rather than 72 hours (1,2).Oocytes recovered from small antral follicles aswell as from large preovulatory follicles arecapable of IVM, IVF and cleaving although thelow numbers preclude meaningful statisticalcomparisons. Oocytes recovered fromunstimulated, naturally cycling bitches were usedin this experiment which is in contrast to thebitches '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 for Conservation and Management ofMarsupials, Department ofBiological Sciences, The University ofNewcastle, NSW 2308, Australia.IntroductionThe semen of marsupials contains free N-acetyl-Dglucosamine(NAG) as the major sugar in seminalplasma (1,2). It exists in the ~emen of the tammar(Macropus eugenii) at a concentration of about 508mg/dl. Tammar epididymal sperm readily utilise NAGand accumulate the sugar intracellularly at a rate fourtimes faster than glucose (3). Since tammar spermnormally are only exposed to NAG in seminal plasmafollowing ejaculation, the response of ejaculated spermto the sugar was examined in the 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 the adverse effects that this recoveryprocess can exert on sperm.Materials & MethodsSemen was collected from six tammar wallabies byelectroejaculation. Motile sperm were recovered byswim-up for 30 min at 34°C into 5-10 ml of bufferedmedium placed over the 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, the 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 the suspension were either fixed forexamination by electron microscopy (EM) (5) orincubated in 1 ml of KRP for 2 h at 34°C in the presenceof no added substrate, 5 mM glucose, or 5 mM NAG.Motility was assessed microscopically and aliquots of thesuspension were fixed for examination by EM.ResultsSperm motility scores (scale 0-10) immediatelyfollowing swim-up into the various buffered mediawere generally high (mean = 8.4 ± 0.74) and nosignificant differences were detected between media.Following 2 h subsequent incubation in the presenceand absence of added glucose or NAG, motility scoresfell to between 5.0 ± 1.0 and 6.6 ± 1.8. However, in thecase of sperm recovered by swim-up into KRP andsubsequently incubated for 2 h in KRP containingNAG, the mean motility score fell to 1.2 ± 0.6 with nomotile sperm being detected 30 min later. Whensections were examined using the electron microscope,substantial membrane damage was detected in the postnuclearhead and mid-piece region in all sperm exposedto NAG following swim-up, irrespective of the mediumused for recovery. Less severe alterations were evidentin sperm subsequently exposed to glucose, and nomajor ultrastructural disturbances were detected insperm incubated in the absence of added substratesfollowing swim-up.ConclusionsThe results indicate that the 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 afforded some protection to the sperm duringswim-up, as reported for eutherian species (4), buttheireffects were only partial in this context. Thus, whilethese agents prevented the complete deterioration ofsperm motility, they failed to prevent NAG- and glucoseinducedultrastructural changes. The adverse effects ofNAG may be due to the excessive dilution of the spermby the swim-up procedures. This may alter thepermeability of the membrane with loss of enzymesthat process the NH 3 generated during the metabolismof NAG. The collection of viable sperm from ejaculatesis vital to research into the control of fertility ofmarsupials and is the 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 for Research ofEndangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA.IntroductionSeven of the 17 penguin species in existence areendangered (1). A better understanding of avianreproduction and the application of assisted breedingtechniques such as semen preservation and artificialinsemination (AI) may become important for captivepropagation and long-term survival of these species.However, information on ejaculate characteristics andsemen preservation is not available for any penguinspecies. The Magellanic penguin is not endangered, butits reproductive success in the wild has been decliningduring the past decade (2). The objectives of thepresent study were to: (i) develop a cooperative methodfor collecting Magellanic penguin semen; (ii)characterise the ejaculate parameters and semenproduction during a breeding season; (iii) compareholding temperatures for short-term storage; and (iv)compare cryoprotectants for long-term storage.Materials & MethodsSemen was obtained from a hand-reared adult maleusing a cooperative collection method. Ejaculates wereassessed for volume, pH and osmolality. Sperm qualitywas assessed by evaluating motility and forwardprogression (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 further diluted (1:1) with BPSE containingdimethylsulfoxide (DMSO) or ethylene glycol (EG),and equilibrated for 15 min (final cryoprotectantconcentration: 8%). Straws were frozen on dry ice for 5min and transferred to liquid nitrogen. Semen wasthawed (n=2 straws/sample) by holding straws for 5 sinair and then by placing them in a 50°C waterbath for 10s. Data were analysed by analysis of variance, andmeans were compared using Students t-tests or LSDtests. P0.05) in spermcharacteristics between treatments for short-termstorage, although there 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 for both treatments combined). SMI and percentviability decreased (P0.05) ascryoprotectants for 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 for cryoprotectant). Both SMIand viability remained relatively stable during the 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 ifthey are affected by the oxygenation state of the storagemedium. The study was extended to determine whetherprotein phosphorylation taking place during storageunder anaerobic and aerobic conditions are reversiblewhen spermatozoa are transferred from oneenvironment to another.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 the semen aged under anaerobic conditionswas transferred from an anaerobic chamber to anaerobic environment. Similarly, one half of the semenaged under aerobic conditions was transferred to theanaerobic chamber. Proteins were extracted from cellsharvested and washed on days 6, 9, and 12 of storageunder the 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 theaerobic 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 the presence of O 2 in the medium, butthe effect is the opposite of that which is documentedfor 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 the time under anaerobicconditions (lanes 2-4), semen aged all the 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 the blot.In conclusion, an increase in protein tyrosinephosphorylation during ageing is probably differentfrom the one observed during capacitation since, unlikethe latter, it is inhibited in the presence of O 2 and isreversed upon transfer of sperm cells from an anaerobicto aerobic environment. Furthermore, it occurs across amuch longer time span than capacitation. Theidentification of proteins that undergo tyrosinephosphorylation changes during ageing would enable abetter understanding of the role they 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 the more rapid loss of fertilitymay be due to the effects of reactive oxygenspecies (ROS) that may be generated in the courseof respiration.As part of a larger investigation into the effectsof storage in vitro on various biochemicalparameters of sperm mitochondria, the activity ofthree mitochondrial marker enzymes wasmonitored.The enzyme assays described in the literaturehave been applied almost exclusively to somaticmitochondria or purified enzymes (2-4). For thisreason, work was firstly carried out to determinewhether the 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 whetherthe activity of mitochondrial marker enzymes waschanging during the storage period and to assessthe effect of the storage conditions (aerobic oranaerobic) on any changes taking place.METHODSSemen was collected from mature Friesian bullsand diluted in 14G buffer (5). The semen wasstored either aerobically or anaerobically, at roomtemperature (20°C).Sperm extracts were prepared, for use in theenzyme 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 synthetase(4).RESULTSOver a period of 20 days the activity of bothcitrate synthase and ATP synthetase decreasedsignificantly (for both enzymes p < 0.001). Forcitrate synthase the difference in storageatmosphere had a significant effect on activity (p =0.005), with activity decreasing more in theaerobically stored semen than anaerobically stored.The third enzyme, cytochrome c oxidase, showedan increase in activity over time, with thedifference in storage atmosphere causing asignificant difference in activity (p = 0.001; Fig. 1).The increase in cytochrome c oxidase activity wassignificant for sperm stored anaerobically (p

Effect of calcium on the 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 for in vitro fertilisation can result in low fertilisation rates despite highmotility of sperm (1). Intracellular calcium increases during the 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 the vasdeferens, and then 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 for 44 hr. Oocytes werethen 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 for maturation and fertilisation under UV light. Six replicates were run and the 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 the 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 father their ownbiological children. The widespread adoption of ICSIin infertility clinics, however, has been questioneddue to the poor quality of sperm used to essentiallyforce 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 the inability in obtaining human oocytesfor 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 for analysis. Microinjection was conducted overthree consecutive days, with day 1 corresponding tothe day of sperm collection.Single morphologically normal and motile spermwere injected into morphologically normal andmature metaphase II oocytes. Injected oocytes wereleft in culture for 6-10 hours at which timefertilisation status was recorded. Capacitated spermsamples were stored in the dark at 20-23°C for up to72 hours.ResultsTable 1 represents fertilisation results obtained foreachcase 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 for each patientwith up to 100% fertilisation being achieved in Case2 (table 1).ConclusionsProlonged sperm incubation at room temperaturebefore 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. Therefore, sperm used over 72 hours postejaculation represent the 'fittest' sperm in the sample,being more likely to initiate fertilisation.However, the high fertilisation rates produced in thepresent study may also be caused by damage incurredto sperm chromatin by the production of reactiveoxygen species (ROS). ROS can be produced throughthe repeated centrifugation protocols established forsperm preparation also through exposure to oxygenas would occur with prolonged storage. ROS have thepotential to induce DNA fragmentation 2 producing aweakening of di-sulfide and protamine bonds withinthe sperm nucleus, resulting in partially decondensedsperm. When such sperm are injected into an oocyte,they rapidly decondense and form pronuclei.Although it may be beneficial to increase storage of asperm sample to increase fertilisation in severe malefactor patients, further 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, Bedford Park SA 5042# Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide SA 5006IntroductionProteoglycans (PGs) are major components of the 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 the species of PGs that they are associated with haveyet to be identified. The aim of this study was to isolate and characterise the 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 these follicles were randomly chosen and processed for lightmicroscopy to assess follicular health. Eighty five percent of these follicles were healthy, the remainder were in earlyatresia.Radiolabelling of Follicular Proteoglycans: A batch of follicles were incubated with 35S in an overnight culture, andthese added to the non radiolabelled follicles prior to extraction.Proteoglycan extraction: Sequential extractions were carried out on the 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 the remaining tissue.Chromatography: Anion-exchange chromatography on DEAE Sephacel columns was used to separate the PGs in theextracts for a large proportion of soluble proteins also present in the follicle. Size-exclusion chromatography of theurea/triton and sodium hydroxide extracts was performed on 90cm Sepharose CL2B and CUB columns.ELISA assays were used to detect the GAG's and PGs present in urea/triton extracts eluted from size exclusion columns.These detected the epitopes present on 4 or 6-sulphated chondroitin sulphate/dermatin sulphate (CSIDS), the CSIDSsmall leucine rich proteoglycan (SLRP) decorin, the SLRP biglycan, the large cartilage modular CSIDS aggrecan, thelarge modular heparan sulphate (HS) PG perlecan, and versican.GAG Characterisation: Nitrous acid was used to cleave HS side chains from PGs in the 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 either 4% paraformaldehyde (4-sulphateSCIDS or versican) or PLP fixative (perlecan). Sections for 4-sulphate SCIDS and versican immunolocalisation werepredigested with chondroitin ABC lyase to expose the antigen. ABC amplification and a PAP detection system withDAB was used.ResultsBiochemistry: After anion exchange chromatography the urea/triton extraction yielded 144.9~g of GAG and the NaOHextraction yielded 29.5 ~g. The radioactivity was highest in the NaOH extract (96.7% of total incorporated) indicatingthat the GAGs synthesized by follicles in culture were different from GAGs present in the uncultured follicles. 4­sulphated CSIDS and HS were detected in the NaOH extract. DS was the major GAG present, while CS and HS werealso observed, confIrming the presence of follicular GAGs documented in the literature 0,2).The major GAG present in the urea/triton extracts was 4-sulphated CSIDS. Three peaks of CSIDS reactivity wereobserved, indicating the presence of at least three species of CSIDS PGs within the follicle. The first peak (Kav 0.24­0.57) was of a similar size to a CSIDS PG previously reported in the follicular fluid of bovine (1) and porcine (2)ovaries. The second peak was Kav 0.57-0.81. Immunoreactivity to the 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 theantibody specific to the core protein of decorin.Immunohistochemistry: Perlecan was immunolocalised to the follicular basal lamina of antral follicles and thesubendothelial and smooth muscle basal laminas of vessels throughout the ovary. Immunoreactivity to 4-sulphatedCSIDS was detected in the connective tissue sheaths surrounding larger blood vessels. Antral follicles showed a variablestaining pattern, the theca interna was often immunopositive and in some follicles the basal lamina also appeared to belabelled. Versican was immunolocalised to the ovarian stroma and connective tissue surrounding blood vessels as wellas the theca interna and the basal lamina of large antral follicles.Discussion and ConlusionThese result for the 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, Bedford 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 before a smaller group of follicles continues to develop. The largest follicle develops into the dominant follicleand enlarges to a preovulatory size of> 10 nun and the subordinate follicles become atretic (1). Unfortunately thephysiological regulation of recruitment and dominance is not well understood. This study was undertaken to order toexamine if the two different morphological appearances of the bovine follicular basal lamina observed previously (2) arerelated to the physiological growth phase offollicles.Materials and MethodsAnimals All cows were 2 ~ years old, nulliparous and of composite breeding; 1.4 Angus, 1.4 Hereford, 1.4 Pinzgauer, and1/.1 Red Poll. Oestrous was synchronized by two injections of prostaglandin F2a (Lutalyse). During the first follicularwave of the 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 the synchronous emergence of a wave of growing follicles which were monitor by dailyultrasonography using a 7.5 MHz trans-rectal probe attached to the Aloka 500V machine. Ovaries were collected whenthe dominant follicle of the 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 for at least the 24 hours prior to ovary collection, but was still functionally dominant as determined by theabsence 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 performed 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 thelaboratory. Ovaries were cannulated and flushed with 20ml of the Hanks solution before being fixed with 50ml 2.5%glutaraldehyde for 5-10 min and then placed in fixative at 4°C overnight. The dominant follicle was identified, dissectedfrom the ovary, cut in half and the cross-sectional diameter measured. Small pieces of the follicle wall were taken andplaced in fixative and stored at 4°e. Further processing was conducted following established electron microscopicmethods.ResultsMorphology of the 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 the antrum.Plateau dominant follicles. All follicles had rounded granulosa cells, pyknotic nuclei were observed on the antral aspectof the 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, the membranagranulosa of the other 2 follicles consisted of flattened cells. Pyknotic nuclei were common and more frequentlydetected towards the antrum (3 follicles), scattered through the entire granulosa layer (3 follicles) or in basal cells (1follicle). Dead cells in the antrum were also a common feature offollicles at this stage.Morphology of the Basal LaminaAll follicles examined, irrespective of their stage of growth or atresia, had a basal lamina that was a single layer andclosely aligned to the 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 for the 2 days prior to follicle collection Values are mean ± SEMDifferent superscripts indicate significant differences (* p < 0.05, ANOVA, SNK)BasalLaminaalignedalignedalignedIn conclusion, the morphological appearance of the 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 the dominant follicle in cattle BiologyofReproduction 55: 1187-94(2) Rodgers HF and Rodgers RJ (1998) Ultrastructural changes in the follicular basal lamina ASRB Proceedings ofthe29th 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 themarmoset as a laboratory primate the 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 other species.Materials & Methods : Pairs of ovaries werecollected on day 4 of the 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 for 18h followed by afurther 6h pulse of 3H-thymidine to enableassessment of DNA synthesis and provide anindication of cell proliferation. MGC weretreated with 50 ng/ml IGF-I, 10nglml porcineFSH or a combination of the two hormones.At the 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 there was a largedifference in the DNA synthetic capacity ofMGC from periantral follicles and those fromthe next largest follicle class (average 3.7 foldhigher), there was less difference amongstMGC from the antral follicles classes. IGF-Iproved to be a potent stimulator of MGCDNA synthesis, increasing 3H countsapproximately equally in the different folliclegroups (average of 4.1 fold) above theirrespective 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 synthesis 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 rather than synergistic.Contrasting the trends in cell proliferation, progesteroneproduction was lowest from MOC from periantralfollicles and increased with increasing follicular size. Inthe 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 formation 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 Wreford 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 the femalereproductive tract, notably ovarian folliculogenesis.Aromatase, a cytochrome P450 (P450 arom ) encoded by thecyp19 gene, catalyses the conversion of C l9 androgens toestrogens. Estrogen exerts its actions via the estrogenreceptors (ER), ERa and ERp, which are prevalent ongranulosa cells [1]. An aromatase knockout mouse(ArKO), generated via disruption of exon 9 of the cyp 19gene [2], allowed us to examine the 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 for further analyses. Ovarieswere either fixed in formalin 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 the 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 the weight of the ArKO ovaries were< 50% of wildtype. By one year of age ovarian weighthad increased, possibly due to the increase in connectivetissue. Uterine mass was decreased in the 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 the number of primary follicleswas still greater than that observed in wildtype andheterozygote mice. By one year of age there were nosecondary or antral fo,llicles present in the ArKO ovary.At 21-23 weeks the 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 the ovaries was reflected in infertility at allages studied. Histological examination demonstrated thatthe ArKO ovary degenerated with age. Haemorrhagiccystic follicles appeared in the ovary at 21 weeks of ageand atresia, as measured by TUNEL assay, was observedto increase as a function of age. Coincident with the lossof follicles, extensive tissue remodelling, exemplified byan influx of macrophages and collagen deposition, wasobserved in the 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 the 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 the ArKOmice [2]. The impact of the 10% soy contained within themouse chow on the 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 thephenotype, with reduced ovarian weight, increased gonadal fatpads, and the appearance of haemorrhagic cysts at an earlierage (16 weeks). These results imply that phytoestrogenswithin the diet were acting as estrogens within the ovary andfat deposits of the 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 fortheir infertility. It is not clear whether the ovarian phenotypeobserved in the ArKO mouse is a direct result of the loss ofestrogen, or an indirect result of the 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 the 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 the human (1), dog (2),mouse (3), wild Norway rat (4) and goat (5). Generally,polyovular follicles occur more frequently in immatureovaries although they are also found in adults. Inmarsupials, the occurrence of polyovular follicles hasbeen observed in the American opossum, Didelphismarsupialis (6) and the Australian native cat, Dasyurusviverrinus (7). Similarly, it is noted that polyovularfollicles were first, and only, observed in the kangarooby van Beneden during the 1880's as previously cited(6). The aim of this study was to investigate whetherpolyovular 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 forlight microscopy and the top ten sections were examinedfor polyovular follicles. Polyovular follicles wereclassified according to the system used for 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 the exception of typeIII were observed. In the adult red kangaroo polyovularfollicles never contained more than two oocytes whilepolyovular follicles in the 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 the ages of adultswas not recorded, it is possible that some of theseindividuals were juveniles that had not yet completedovarian development and that the germ cell nests presentwere in the process of separation. Another explanationfor the presence of polyovular follicles may beenvironmental conditions which, as previously reported,?lays .a significant part in reproductive developmentmcludmg the delay of sexual maturity (8). Therefore,the 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 they appearedless common than in the 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 the oocyte which promotesgrowth and attenuates differentiation of follicular somatic 'cells (1). The aim of this study is todetermine the 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 Reproductive Medicine Unit, The University of Adelaide,Adelaide SA 5005.C:':,;kines are clearly now identified as pivotal mediators of the dynamic and transient cell-cellc(,!nmunication events underpinning the growth, remodelling and demise so characteristic ofreproductive tract tissues. The intrinsic capacity of these small glycoproteins to be producedtransiently, to act locally, and to be rapidly quenched is perfectly adapted to their roles as localmediators of endocrine hormone-governed cell proliferation, differentiation, activation and motility.\10st notably, cytokines regulate the interaction between somatic cells and infiltrating leukocytepopulations that are required to deal appropriately with the unique immunological challenges thataccompany ovulation, insemination, implantation and pregnancy.Our laboratory has focussed on the role of cytokines in endometrial biology, with a view toelucidating the cytokine pathways linking insemination with the cascade of molecular and cellularevents that culminate in implantation of the embryo. Our experiments in rodents and more recentlyin humans have demonstrated a role for seminal plamsa in 'priming' the female reproductive tractfor successful implantation. Specific factors in seminal plasma including transforming growth factor(TGF)-~ stimulate uterine and cervical epithelial cells to induce expression of an array of proinflammatorycytokines, which act subsequently to (1) initiate changes in the maternal immunecompartment facilitating induction of immunological tolerance to paternal transplantation antigens,and (2) promote the growth and development of the pre-implantation embryo.Complex cytokine networks also appear to operate in the ovary, most notably in regulating thetrafficking and activation phenotype of myelQid leukocytes implicated in the tissue remodellingaccompanying ovulation and in the development, function and demise of the 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 the 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 the 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 the recognition that Thl cytokines are required forsuccessful establishment of pregnancy. Many aspects of successful pregnancy can thus be viewed as a result ofa fine balance between these 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 the 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 the membranes in response to infection)can act at multiple foci in the biosynthetic pathway, exerting coordinated effects on intrauterine prostaglandinproduction. Enzymes involved in substrate release, conversion to active products, and inactivation throughmetabolism, are all targets for cytokine action. In addition, we have shown that at least one eicosanoid,thromboxane, is capable of stimulating cytokine production by amnion epithelial cells, demonstrating theexistence of a feed-forward loop which may act to amplify the 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 the membranes (but not placenta). We have interpreted this data as evidence of an inflammatory hyperresponsein the aetiology of a significant proportion of preterm deliveries.While Th2 cytokines might be expected to predominate during the majority of pregnancy, we postulated that aT h2 withdrawal could occur at time of delivery to allow the inflammatory processes of parturition to occurunhindered. Our recent data supports this hypothesis, with findings of decreased ~-l0 production by thechoriodecidua with normal term labour. More unexpected, however, was the finding that ~-10 exerted proinflammatoryeffects on the amnion at term, stimulating cytokine and prostaglandin production.-2 transcription in amnion.A better understanding of the 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 the 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 Biology, and lDepartment ofBiochemistry,University of Western Australia, Perth, Western Australia 6907..IntroductionThe MAPK signalling cascade has· been proposed ascritical to the events of cell survival and cell death,chiefly apoptosis, in a number of biological systems.Current evidence suggests that the 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 theapoptotic (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 the induction ofapoptosis within the CL (3). Although themechanisms of signal transduction in TNFa-mediatedapoptosis are largely unknown. Thus our aim in thispresent study was to determine whether MAP Kinases(p38, JNK, ERK) are present in the rat CL and whetherchanges in their expression correlate with the 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 either incubated without trophicsupport for 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 then incubated with the primary antibodies forERKI (Santa Cruz) and ERK2 (Santa Cruz)simultaneously, JNKI (Santa Cruz), or p38 (SantaCruz) diluted 1:1000 for Ih at room temperature. Thiswas followed by incubation with sheep anti-goat IgGsecondary antibody (Sanofi Diagnostics) for 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 performed as describedpreviously (4). A minimum of 3 animals was used foreach experimental variable.ResultsWestern blot analysis revealed the appropriatemolecular weight bands corresponding to ERKl&2(44 & 42 kDa) and p38 MAPK (38 kDa) (Fig. 1),~nder both culture conditions. No significant changeIn the amount of protein for either 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 the presence of JNKthrough Western blot analysis under either of theexperimental conditions.Time.__---AFig 2 (left)Autoradiographo C'l '

Progesterone Downregulation of Matrix Metalloproteinase-l (Ml\1P-l)• a role for 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 the steroid hormone receptorfamily, including androgens and progesterone (P). Themechanism for 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 the luciferase (Iuc) reporter in thepGL3 basic vector. Transfections were performed in theabsence 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 for transfection of pGL3 basic vector alone(background).Resultsto co-transfections of PR and 2002 bp or 1551 bp of 3'sequence had no further downregulatory effect. In cotransfectionsof :::;;1194 bp of MMP-1 promoter + Ets1 +PR, the addition of IO·7Pcaused a significantupregulation ofMMP-1 (p

Arom~tase and Sa.-reductase pathways and their interaction with nutrition in the 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 the control of LH secretion (1, 2). Wehave been using inhibitors of these enzymes to determine where they are located and with a view to testingwhether their 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 the 5a-reductase inhibitor, MK 434, has noeffect (3, 4). In this study, we used fadrozole again, and two other 5a-reductase inhibitors, to test whetheraromatase and 5a-reductase pathways interact with nutrition in the control ofLH secretion in mature Merinorams.Material and Methods: In Experiment 1, two groups of S rams were infused icv for 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 either 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) for 3 days starting on the 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 for 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 the High Diet and 3 the Low Diet. Two weekslater, the nutritional treatments were crossed over and the experiment was repeated. Blood was sampledevery 20 min for 24 h before and after inhibitor treatments and LH was measured in the plasma.Results: FCE 28260 did not affect LH pulse frequency (Fig. 1a ). When they were given iv, Finasteride +MK 386 increased LH pulse frequency in the High Diet group but not in the Low Diet group (Fig. Ib). Wheninfused icv, only fadrozole increased LH pulse frequency (Fig. 1c). No interactions between nutrition and thearomatase and 5a-reductase inhibitors were observed. In both Experiment 2 and 3, the effect of diet on LHpulse frequency was highly significant before start of inhibitor treatment.Discussion: The increase in LH pulse frequency after icv fadrozole treatment reinforces the conclusion thatcentral aromatization is an important step in the negative feedback effects of testosterone. All of ourattempts to increase LH pulse frequency using the most potent 5a-reductase inhibitors have failed,suggesting that this pathway is not important in the negative feedback system of intact rams. However, thepossibility that these inhibitors have a low affinity for the 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 etherphospholipid. It improves motility and inducesacrosome reaction in spermatozoa and acts as anautocrine growth/survival factor for the preimplantationembryo. The concentration of PAF in spermatozoa andembryos is inversely related t6 their quality. Amechanism of action is yet to be defined. In this study,we examined the 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 the late I-cell stage and 2-cell stageexpression was less consistent but was observedconsistently again from the 4-cell stage up until theblastocyst stage. IVF and culture delayed the expressionof PAF-R mRNA until the 8-cell compacted stage.This study investigated: (i) the presence of PAF-RmRNA and protein in the human and mousespermatozoa; (ii) the 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 thenseparated 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 performed on spermsamples under standard Perkin-Elmer conditions usingspecific primers for PAF-R.ImmunofluorescenceSpermatozoa or embryos at various stages ofdevelopmental were fixed in 70% ethanol or 2%paraformaldehyde (respectively), permeabilised in 1%Tween 20 (embryos) and non specific binding blockedwith 30% sheep heat inactivated serum. Immunotaggingwas performed 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 the midpiece and98distal head (Fig. 1). PAF-R location in mousespermatozoa was most prominent at the midpiece(Fig.!).Immunostaining of embryos collected fresh from thereproductive tract was significantly greater than isotypecontrols and the intensity was not different from the 1­cell up to blastocyst stage. During development thepattern of PAF-R localization changed from diffusecytoplasmic staining in zygotes to increasinglymembrane localized pat.tern of expression from the 2­cell stage onwards. There was no effect of IVF on thelevel 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 proteinfor the G-protein linked PAF-R is present in human andmouse spermatozoa and within the mousepreimplantation embryo. Unlike the expression ofmRNA in embryos, the 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 the 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 for 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 whether endogenous embryo-denved PAF can also Induce calCIUm tranSIents In the2-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 the oviducts of mice at the 2-cell stage of development 42h after eCG. Embryos wereincubated in Fura-2-AM (2uM) for 30min and then 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) therefore calcium measurements were made in media in the pres.en~eand absence of BSA. In the 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 the transient. These embryosstill responded to exogenous PAF with a calcium transient illustrating the specificity of the response. ~ PAF-r~cept~rantagonist (WEB 2086)' also blocked calcium transients. Depletion of Caj stores by -treatment WIth thapslgar~Ineliminated the 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 the PAF receptor for 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 the 2-cell stage.It is dependent on the presence of BSA. Previous studies have routinely excluded albumin and have therefore not beencapable of detecting the actions of embryo-derived hydrophobic compounds.Reference(1) Ammit, AJ. and O'Neill, C. (1997) Studies of the nature of the 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 2Centrefor Cell and Molecular Biology, 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, the murine homologue of fa!tJtfC!rcets inJ2!~Fafencodes a ubiquitinsp~-ubiquitinase required in the syncitialstage of Drosophila development (2). We soughtevidence of expression in mouse embryos andpursued the requirement for Fam expression inblastocyst formation and cell proliferati,on.Methods: Oocytes and preimplantation embryoswere collected from superovulated Quackenbushmice using standard techniques and media. Eggs andembryos were fixed and stained for confocalscanning laser microscopy using peptide specificantiserum. Western blotting used the sameantiserum. The antisense oligonucleotide (ODN)was designed to hybridise with the FAM initiationcodon and surrounding sequences, from nucleotides364-348 of the Genbank U67874 FAM sequence. Acomplementary sense ODN was used as control andcompared with no addition during culture for 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. Inthe unfertilised egg the Fam was localised to thespindle and chromosomes with some cytoplasmicstaining. Upon fertilisation Fam disappeared fromthe pronuclei and increased greatly in cytoplasmicintensity. Thereafter Fam was primarily found in thecytoplasm, particularly surrounding the nuclei, withno apparent change in distribution through toblastocyst formation.Western blotting confirmed the Fam knockout.The FAM antisense oligonucleotide prevented 98%of embryos from forming blastocysts (X 2 , P

humancowratsheepmouse102Ovarian steroid hormones and growth factors in the uterine control of early embryonicdevelopment in the 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 the uterine control of early embryonicdevelopment. Whilst about 60 species of eutherian, and30 species of marsupial exhibit diapause, the control isbetter understood in the tammar wallaby, Macropuseugenii, than in most other species. Under the influenceof a sucking young in the pouch, development of thetammar embryo in utero ceases completely at the 100­cell. blastocyst stage. Diapause is maintained for at least11 months, during which time there is no cell division orblastocyst growth. Despite the long period of arrest thereis no loss of embryonic viability. Most attention to datehas focused on the reactivation of corpus luteum andembryonic growth after the long arrest, with little focuson the mechanisms that control the onset of diapause.The aim of this study is to investigate the interactionsbetween ovarian hormones, uterine secretions and earlyembryonic growth as the blastocyst approaches andenters diapause.It is clear from work on eutherian species that there aremany growth factors, including. leukaemia-inhibitingfactor (LIF), epidermal-growth factor (EOF) and insulinlikegrowth factors (IOF-l & IOF-2), produced in theuterine secretions which may potentially regulateembryonic development. LIF is essential for growth ofthe early mammalian embryo (1). The expression of LIFin mice is maternally controlled by oestrogen andcoincides with blastocyst formation (2). Similarly, inpigs (3) and cattle (4) oestradiol upregulates uterine LIFproduction. In rabbits by contrast, progesterone, notoestradiol, regulates LIF expression (5). Neither steroidinduces uterine LIF production in the skunk, but LIFmRNA is absent during diapause and high around thetime of implantation (6).Our hypothesis is that in the tammar the release ofoestradiol from the ovary, around the time of ovulation,induces the production of several growth factors,including LIF, which enhance cleavage and are essentialfor formation of the early blastocyst. Over the next fewdays, without continued oestradiol stimulation and with atransient rise in progesterone post partum, growth factorproduction is downregulated, forcing the blastocyst intodiapause. Thus there 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 theperiod between conception and the onset of diapause hasbeen poorly defined. Daily plasma samples weretherefore collected immediately after parturition until day10 after birth from females that retained their young(n=8), those who had their 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 for the three experimental groupsdefined. The genetic sequence of LIP in the tammar wasdetermined by nested polymerase chain reaction (PCR)using degenerate primers developed from conservedregions of known eutherian species, and probesdeveloped from mouse and human LIP plasmids. The 38amino acid sequence of the peR product shows highhomology with previously described species (Table 1).The tammar LIP (tLIP) gene has a higher homology tohuman LIP than other eutherian species. Using part ofthe tLIP sequence as a probe the expression of LIP inreproductive tissues and the early embryo will beaccurately determined by in situ hybridisation.Acknowledgments:We thank Prof. RR Behringer (Department of MolecularGenetics, MD Anderson Cancer Centre, Houston, Texas)for providing his expertise and laboratory facilities incloning tLIF. We are also grateful to Dr. D Hilton(Walter & Eliza Hall Institute, Victoria) for kindlyproviding the 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 the 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 for 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 the uterine function of otherspecies (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 the non-pregnant mouse uterus(4). The aim of this study was to elucidate thetemporal and tissue specific expression pattern ofthese 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 byperforming reverse transcription (RT)-PCR, withgene specific primers, in the linear range to allowsemi-quantitative analysis. Southern hybridisationwith gene-specific probes and/or DNA sequencingconfirmed the specificity of the PCR.ResultsThe ubiquitous "house-keeping" gene, ~-actin, wasexpressed in all samples, confirming the integrityand quality of the cDNA (Figure la). Positivecontrols (salivary gland) yielded mKlk products ofthe predicted size. All mKlks displayed distinctexpression patterns. We observed the expression ofmKlk-1 (Figure Ib), mKlk-3, mKlk-9 and mKlk-21(Figure lc) in the fertilised egg to the 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 inthe embryo from day 9.5, with decreased levels byday 11 (Figure Ib). mKlk-5 was not detected in theembryo. In contrast, there was no consistentexpression in the uterus of mKlk-1 until day 7.5which then continued to day 11 (Figure Id). mKlk­21 expression in the 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 the 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 the embryo (Figures lc,e). mKlk-3, mKlk-5and mKlk-9 were not consistently expressed in theuterus ~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 for high or low pregnancy ratesSJ. Pearson, W.H. McMillan and AJ. PetersonAgResearch Ruakura, P.B. 3123, Hamilton New ZealandSeasonal Changes in the Testicular Morphology of the Agile AntechinusDuckett, R.*, Wreford, 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 either high (n =25,High) or low (n = 25, Low) pregnancy rates (1)creating a unique experimental resource to determinefactors involved in the successful establishment ofpregnancy in cattle. We report here the concentrationsof interferon-tau (IFN-'t), the conceptus-secreted factorcrucial for pregnancy signalling in ruminants, in theuterine luminal fluid (ULF) of the two sub-herds andtheir 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 the uterine hom ipsilateral to the corpus luteum.On day 17, the uterus of each cow was flushed in vivo,the number and length of each conceptus recorded, thevolume of the recovered fluid measured and a 50mlflush aliquot processed for 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 ofthe heteroscedasticity of the data, statistical analyseswere performed on rank transformed data whichinvolved ranking the observations in order from thelowest to the 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 the University of Cincinnati, Ohio,USA. Genotype was determined by diagnostic PCRusing three primers; two which flank the insertedsequence in exon 6 of the TGF~ 1 gene and one withinthe inserted sequence.The fertility of one homozygous null male offspringwas examined by caging with balb/c Fl females (n=6)for a total of4 weeks.7Jlm fresh frozen sections were prepared formorphometric analysis from two TGF~ 1 null males andone wildtype (TGF~1+/+) littermate by H & E staining,and for immunohistochemical analysis with rat antimouse monoclonal antibodies F4/80 (pan macrophageantigen) and TIB 120 (MHC Class II) and Till 128(CD lIb).ResultsMorphology of the testes and seminal vesicles of the male TGFf31 null mouseWV Ingman l and SA Robertson l . 2 .Department of Obstetrics and Gynaecologyl and Reproductive Medicine Unit 2 , University of Adelaide, SA 5005IntroductionTransforming growth factor ~ 1 (TGF~ 1) is a cytokinefound in murine seminal plasma which, afterinsemination, induces production of GM-CSF by theuterine epithelium of the oestrous female (1). Thissuggests TGF~ 1 may be involved in the inflammatorycascade which occurs in the 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 the physiological role of seminalTGF~1 in the induction of maternal tolerance topaternal antigens in semen. To this end we haveobtained heterozygote mice carrying a null mutation inthe TGF~ 1 gene (3) with the intent of establishing acolony which will provide male homozygous mice touse in mating experiments. Little is understood of thereproductive physiology of the TGF~ 1 null mouseexcept homozygous matings pairs are rarely fertile (IOrmsby, personal communication). Therefore an initialstudy was conducted to characterise the histology of thetestes and seminal vesicles of male TGF~1 null mice todetermine if there are any abnormalities in the malereproductive tract which may lead to infertility.MethodsOf 66 progeny produced from the original four females38% are wildtype, 48% are heterozygotes (TGF~I+/-)and 5% are homozygotes (TGF~1-/-). 6% died beforethe 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 thehomozygous male whereas the wildtype male matedfrequently over the same period.106Histology and immunohistochemistry revealed thatboth sperm production and macrophage number anddistribution in testes and seminal vesicles of the 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 the 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 the Testis: Isoform Expression PatternsSuggest Functional HeterogeneityKate L. Loveland and Liz ChristyInstitute for Reproduction and Development, Monash University, Clayton, Vic 3168Introduction and Aims:Microtubules playa central role in germ cell development, comprising the major structural element in mitotic andmeiotic spindles, in the manchette surrounding the developing sperm head, and in the sperm tail. Their assemblyand dissociation is governed by a heterogeneous family of microtubule-associated proteins (MAPs). To understand theregulation of microtubule formation and function in the testis, we have been studying microtubule-associated protein-2I(MAP2), which we have recently demonstrated can exist in a novel LMW isoform containing a nuclear localisationIsignal (1). To understand MAP2 function in the testis, we have used a panel of commercially available monoclonalantibodies, widely used in neuronal studies and known to bind to discrete re ions of the MAP2 ~otein. Immunohistochemistrywas used to assess the distribution PKA RII Calm0 dulm .pattern of MAP2 in fixed rat testis sections, and WesternTubulmblots were performed to determine antibody specificityHMWand MAP2 isoform size. MAP2 isoforms 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 the - Novel LMW·long projection arm encoded on exon 9. Protein (italics) AP-18 P MAP2sand antibody binding sites are indicated to the 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 the 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) for Western blot analysis.MAP2 proteins were detected using the antibodies (1:5,000 dilution) with the 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 then resuspended and boiled in immunoprecipitation buffer for 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 isoforms indicated with brackets.LMW isoforms indicated with arrowheads.Conclusions and Discussion:Our results indicate that MAP2 expression in the testis is developmentally rero.tlated, with discrete isoformsexpressed 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 the localisation patterns in adult rat testis, we hypothesise that MAP2 functionsin the meiotic spindle and in elongation of the sperm head. The nuclear isoform in spermatocytes may bepositioned to mediate rapid spindle assembly once the nucelar envelope has broken down, while thecytoplasmic form may coordinate the nucleation of microtubules at the 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 for 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 these abnormalities arise from mutations in genes criticallyrelated to the formation of the spermatozoa. To gain a better understanding of the 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 the possession of a putativephosphatidylethanolamine binding protein (PEBP). The aims of the present study were to clone the fulllength rat pebp2 transcript, its mouse orthologue and to initiate a functional characterization.Materials and MethodsThe remainder of the 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 their predicted encoded proteins were compared against databaseentries for homology and _sequence motifs, using the BLAST and Prosite programs respectively. Peptidesspecific for 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 antiserafor use in immunohistochemical techniques. Northern blotting and in situ hybridization were carried out todetermine the timing of pebp2 mRNA expression during spermatogenesis. RNA extracted from male andfemale Balb/c mice tissues were used for RT-PCR analysis oftissue distribution.Results5' RACE was successfully used to obtain an additional -1 kb of rat pebp2 cDNA sequence. Northernanalysis 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 the coding region with the 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 the 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 isoform was predicted to have a Mr of-21.2kDa and an isoelectric point (pI) of -8.43. The short isoform was predicted to have a Mr of -19.9 kDaand pI of -8.77. Both the predicted mouse and rat proteins contained a PEBP family signature, severalpotential phosphorylation sites and a nuclear localization signal. Tissue expression studies revealed thepresence of both the short and long isoforms 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 theEST database. The predicted protein sequences from all three species were novel and more closely related toone another than other previously identified members of the PEBP family.ConclusionWe have identified novel rat and mouse testis transcripts and proteins which we have designated pebp2 basedon the possession of a PEBP motif. Mouse pebp2 is predicted to encode two potentially phosphorylationregulatedisoforms of -21 and 20kDa which are expressed during spermiognesis. Further, pebp2 isofonnsappear to be integral component of several tissues throughout the body. The involvement of pebp2 in spermepididymal maturation, capacitation and fertilization is the subject of further research.IntroductionIn all therian mammals, nine prominent cytoskeletalstructures, the outer dense fibres, surround theaxoneme of the midpiece and principle piece of thesperm flagellum (Figure 1). The possible function(s)of the outer dense fibres may be to either maintain thepassive elastic recoil of the sperm flagellum (1)and/or to protect the flagella against shearing forcesduring epididymal transit (2). The isolation andcharacterisation of the outer dense fibres from rat,bull and human sperm have been previously reported(3, 4, 5), but as yet no study has been published forany marsupial species. The aim of the present study,which is part of a broader investigation of theevolution of eutherian and marsupial gametes, was toisolate and determine the protein composition of theouter dense fibres for the first time in a marsupialspecies, the brush-tailed possum, Trichosurusvulpecula-.Materials & MethodsSpermatozoa obtained from the 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 for 120 minutesat 4°C. Sperm flagella fractions were collected fromthe 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 for approximately 90minutes. The resultant suspension was layered over a20%, 40% and 60% (w/v) sucrose step gradient,centrifuged, and the outer dense fibres were collectedfrom the 40-60% interface. The isolated outer densefibres were either fixed for routine transmissionelectron microscopy and/or solubilised with 2% SDS­5% B-mercaptoethanol for 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 the sperm head bysonication and subsequent sucrose density gradientcentrifugation resulted in a high yield of flagella fromthe 40-60% interface. Incubation of the sperm flagellain SDS-DIT for 90 minutes resulted in the completesolubilisation of the axoneme, mitochondria andfibrous sheaths but the outer dense fibres remainedintact (Figure 1). SDS-polyacrylamide gels of theouter dense fibre fractions revealed 5 majorpolypeptide bands with molecular masses of 73, 57,52, 42 and 16 kDa, the last of which was the mostprominent band (Figure 2). In comparison, rat, bulland human sperm outer dense fibres contain 7, 3 and2 major polypeptides respectively (3, 4, 5), and themost 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 the proteins ofthe outer dense fibres has been described for the fIrsttime in a marsupial species, the brush-tailed possum.Polyc1onal antibodies are currently being raisedagainst the major possum outer dense fibrepolypeptides and these will be used toimmunocytochemically investigate the developmentand localisation of these cytoskeletal structures. Inaddition, techniques are currently being developed tosimilarly isolate and characterise the proteins of theother major cytoskeletal component of the possumsperm flagellum, the 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 for 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 the subacrosomal region ofmammalian sperm. These proteins, includingpre- and proacrosins, are suggested to beinvolved in the secondary binding of the spermand egg during fertilisation (1), and also in theattachment of the acrosome onto the nucleusduring sperm formation (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 eutherian proacrosin and causedsperm structural malformations in immunisedwallabies (4). Aim of this study was to identifyany similar proteins located in the subacrosomalregion of the brushtailed possum sperm using asimilar protocol for isolation of the 45 kDaprotein in the 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 for 30 minwith a swing rotor. Most of acrosomemembrane was extracted by 0.1 % Triton x-I00on ice for 1 hr. The sperm heads with thesubacrosome area were pelleted and incubatedwith 100mM NaOH on ice for 30 min. Thesupernatant of NaOH extraction was dialyzedagainst Milli-Q at 4°C for 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 the protein was analysed aftertransferring to the PVDF membrane with CAPSbuffer.ResultsThe SDS-PAGE gel (Fig 1) shows oneprominent protein with approximal 49kDa MWextracted from the subacrosomal region ofpossum sperm. Twenty-three residues of the N-terminal sequence of the. 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 withthe tarnmar wallaby. The function andsignificance of the 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 the 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 the parturient process. This isaccomplished via altered activities of enzymes of the prostaglandin biosynthetic cascade, in particular,prostaglandin H synthase (PGHS). The recent finding that two forms of this enzyme exist (constitutive,PGHS-1; inducible PGHS-2) has focussed interest on regulation of the'inducible form. Since amnion is amajor source of intrauterine prostaglandin production we have used the 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 the host response to an intrauterine infection are considered toplaya significant part in the mechanisms of preterm labour we have evaluated their actions on PGHS-2expression.In amnion AV3 cells, 50 ng/ml TNF-a. or 1 ng/ml ll..--1 ~ induces rapid but transient synthesis of PGHS-2mRNA while levels of PGHS-l mRNA remains constant. We have evaluated the mechanism of this effect.The -891/+9 fragment of the PGHS-2 promoter was subcloned upstream of the chloramphenicol acetyltransferase(CAT) gene. Sequential 5' deletion constructs containing regions -528/+9, -321/+9, -203/+9, and-52/+9 of the PGHS-2 promoter were synthesized by long-template PCR to define the regions required forTNF-a. and ll..--l ~ activity. Although the transcriptional effects of cytokines via the NF-KB promoterelement have attracted the most attention, recently we have found that the TNF-a. and ll..--1 ~ -responsiveregion resides within bases -52 and -203 of the PGHS-2 promoter, a region lacking this sequence. Sitedirectedmutagenesis of the NF-ll..-6 iliuclear factor of ll..--6) response element within the -203/+9 construct(p203ll..-M) reduced the basal and induced transcriptional activity to that of the -52/+9 construct and aconstruct containing only a TATA box (pminCAT). Mutational inactivation of the CRE (£AMP-Response~lement) to produce p203CRM yielded similar results. However, mutation of both elements within the ­203/+9 construct (p203ll..-CRM) further reduced basal and TNF-a. or ll..--I~ stimulated CAT productionbeyond that of the constructs containing the single site mutation. Electrophoretic mobility-shift assays(EMSA) using a double-stranded oligonucleotide derived from the 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 the unlabelled PGHS-2-derived CRE oligonucleotide completely eliminated the -132 complex while theNF-kB consensus oligonucleotide and the 203ll..-M oligonucleotide failed to reduce the intensity of thecomplex. Similarly, the CRE oligonucleotide, when used as a probe, formed a complex similar to that of theNF-ll..-6 probe. Antibodies to 3 isoforms of CIEBP (a., /3, and 8) were included in the EMSA reactions toidentify the protein which bound the NF-ll..-6 and CRE oligonucleotides. Higher molecular weightcomplexes (super-shifts) were only observed upon the addition of anti-CIEBP~. Therefore, there are 2elements within ·the PGHS-2 promoter, NF-IL6 and CRE, which convey the effects of TNF-a. and IL-l~.The interaction of these elements with CIEBP~ is then required for increased PGHS-2 transcription inamnion.110 111

REGULATION OF PARTURITION: THE ROLE OF eRHRoger SmithMothers and Babies Research Centre, Department OfEndocrinology, John Hunter Hospital, LockedBag No 1, Hunter Region Mail Centre NSW 2310Premature delivery is responsible for 70-80% of neonatal deaths in western countries and the ratesof preterm delivery have not changed in the last 20 years. The importance may be greater still if theBarker hypothesis linking low weight at birth with early onset vascular disease is confirmed. Whileour understanding of parturition in the sheep has progressed rapidly our knowledge of themechanisms 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 the sheep and the Tammar wallaby. Recent work in a number of laboratorieshas suggested that in humans and other primates the neuropeptide corticotrophin-releasing hormone(CRR) may playa key role.CRR is produced in the placentas of New and Old World monkeys and in the apes, includingourselves, and is secretedinto the maternal and fetal circulations. In humans the increase of maternalplasma CRH is exponential over the course of gestation, peaking at the time of delivery. In a largeprospective study we observed that the exponential increase was more rapid in those womendestined to deliver prematurely and slower in those destined to deliver late. While these dataindicated an association between CRH concentrations and the length of gestation they did notindicate that these events were causally related. However, CRR receptors are present on themyometrium and several studies have provided evidence for a direct myometrial action for CRR ininitiating human parturition. Our own work has taken a different line. Primate pregnancy isdistinguished from that of other mammals not only by the production of CRH but also in thatplacental production of estrogen is dependent on fetal adrenal formation of the estrogen precursorDHEA-S. We have shown that the fetal zone of the fetal adrenal gland possesses CRR receptors andthat stimulation of fetal adrenal cells in culture with CRR preferentially stimulates the formation ofthe enzymes required for DHEA-S SYnthesis and promotes the production of DHEA-S by thesecells. This provides a mechanism by which rising CRR concentrations can stimulate increasingestrogen production in the primate.In more recent work we have identified the region in the CRR promoter which appears to mediatethe exponential rise in CRR during pregnancy. Glucocorticoids stimulate placental CRR production(in contrast to the hypothalamus where they are inhibitory), this stimulation is mediated by aglucocorticoid interaction at the .cAMP response element. As an additional action of placental CRRis likely to be stimulation of cortisol synthesis a positive feedback mechanism has been proposed byMajzoub et al whereby CRR stimulates cortisol production and cortisol production stimulates CRRsynthesis-mathematically modelled, this interaction produces the 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 the past three decades, a number of elegant experimental studies have demonstrated that thefetal HPA axis plays a key role in the timing of parturition in the sheep. It is the case, however, thatthe precise events within the fetal HPA axis which result in the prepartum increase in cortisol andlead to delivery are unknown. There is controversy concerning the relative roles o~ the fet~lhypothalamus, pituitary and adrenal in the increase in adrenocortical growth. and sterOIdogenesIswhich occurs during the last 2-3 weeks of gestation. A range of hormones denved from the 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 the POMC derivedhormones on the 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 thefetal adrenal. This provides evidence for a role for this hormone in the cascade of events leading toactivation of the fetal adrenal cortex in late gestation.We have also demonstrated that there are separate subpopulations of CRF-responsive. and AVPresponsivecorticotrophs in the fetal anterior pituitary and that the CRF-responsive corticotrophs aresensitive to the negative feedback actions of cortisol. The heterogeneity of corticotrophic cell typesin the neurointermediate and anterior lobes of the fetal pituitary may underlie the heterogeneity ofPOMC derived peptide hormones present in the fetal circulation and the temporal changes in therelative concentrations of these hormones which stimulate the fetal adrenal.Finally, in the adult, acute or chronic hypoglycaemia potently stimulates the HPA axis and ACTHsecretion. Whilst the fetus is normally hypoglycaemic relative to the mother, the glucose-ACTHrelationship is poorly defined in the fetus. It is unclear whether the fetal HPA axis becomes moresensitive to the relative fetal hypoglycaemia in the prepartum period and whether this change isimplicated in the prepartum activation of the HPA axis. We have investigated the effects of acuteand chronic hypoglycaemia on the fetai HPA axis'and have speculated on the potential importance ofperipheral signals of fetal energy balance in the stimulation of the fetal HPA axis in late gestation.It is likely that the mechanism responsible for human parturition contains important redundanciesbut it seems that placental CRR is at least a player in the 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 the most important challenges in modern obstetricpractice. While maternal and perinatal mortality and morbidity have ~ignificantly declined thiscentury the incidence of pre-eclampsia has essentially remained unchanged. Further, while ourability to rescue the severely growth retarded and compromised infant in the neonatal period hasimproved beyond all expectations we remain disappointingly poor at detecting such pregnanciesearly enough to significantly alter antenatal events and prolong pregnancy.However, the recent development of sensitive and specific immunoassays for inhibins and activinsand the application of these assays to pregnancy has offered new possibilities for the prediction andearly detection of pre-eclampsia, preterm labour and fetal compromise - both acute and chronic.While originally purified from gonadal tissue and characterised by their ability to modulate FSHsecretion from the pituitary, in pregnancy inhibins and activins are of feto-placental origin withlargely undefined roles. Nonetheless, some years ago our observation that maternal serum inhibin Ais elevated in Down syndrome pregnancies heralded the first clinical application of these proteins.Subsequent to these 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 the 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 the earliest non-invasive marker of this disease yet described. If confirmed,then these findings offer new opportunities for early prediction and intervention.Equally exciting, maternal serum activin A is known to increase with advancing gestation,particularly in the last trimester, and in association with preterm delivery. While these observationsmay suggest a role for activin A in parturition our recent studies have been unable to confirm thisbut instead suggest that the association with labour may reflect fetal compromise and stress which inturn, probably via other mechanisms such as CRR, initiate labour. Thus, while activin may beuninformative 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 these human data, we have shown experimentally that induced acute fetal hyoxaemia in asheep model increases activin secretion and that levels fall rapidly with the return of normoxia.Further studies are now.. underway to further define the. J,ltility of _activin as a marker of fetalwellbeing in human pregnancy and to explore both what mechanisms underlie the increasedsecretion in a~s?cia~ion with f~topl~cental embarrassment and what advantages are conferred by thisresponse. EXCItIng tImes certaInly be ahead for the 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, theScience and Technology (S&T) landscape inAustralia is changing. One notable change over thepast couple of years has been in the level ofunderstanding across the S&T spectrum. of thecomponents of an "innovation culture" including theconstraints to commercialisation of Australia's R&Dendeavours. The number of conferences, workshopsand symposia on this general topic area is expandingrapidly with some already calling for less diagnosisand more therapy, radical if necessary. It is a fact,however, that these 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 theconversion of knowledge into wealth andemployment. This includes promotion of technologybasedenterprises as well as renewal of exisitingindustry. There is also increased understandingamongst the S&T practitioners of the differentimperatives, structures and functions of researchersand organisations across the spectrum - academiaand research institutes, manufacturing industry andthe financiaVlegal sector. However, as we approach agenuine knowledge-based economy, we are a longway from getting the settings right for all theingredients of innovation.Researchers in publicly-funded researchorganisations are increasingly "facing the 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 the present time isa rich landscape in which the diversity of private andpublic funding sources has been used to promotepluralism and to obviate the heavy hand ofcentralised control (see Stocker, J. Priority Matters,1998). Its diversity is its strength provided the issueof critical mass is kept in mind with opportunitiesgrasped for 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 the S&T spectrum ­scientists moving from academia into the 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 there are regrettablysome early suspicions in many such encounters in theAustralian scene with questions in each camp around:"...is this another case of a researcher wanting tosimply take the money and run..." with some CEOsbelieving that one of the 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 there will beconsiderable baggage deriving from past negativeexperiences on both sides. Moreover, theunderstanding of (and mutual respect for) the wayscience is done at either end of the S&T spectrum hashistorically been lacking. This is a major reason forthe discontinuity in Australia that is to our nationaldetriment and, besides preventing easy movement ofscientists back and forth between academia andindustry, impedes the 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 themajor impedance to the establishment and nurturingof technology-intensive start-up companies is thehostile capital gains tax environment in this country.It is hoped that the Ralph Review will address thiscomponent of the S&T spectrum as well as thevarious issues that need to be addressed at the "bigend of town" - i.e. established, tax paying businesses.Success in risk-taking ventures must be rewardedrather than penalised. Of course, there is no need toinvent a unique Australian solution to many of theseissues - unabashed plagiarism of systems proven towork in other jurisdictions should do just nicely!Also included in a listing of how we might dothings differently are additional means to promote theventure capital industry in Australia, increasing themobility of intellectual property, mentoring andassistance to entrepreneurs through technologyincubators and enhancing the 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 the dollar(including the research dollar) goes to where the bestvalue can be obtained.114115

--o121:.­.- ....:l~ S 8o b.O~54oI•IA critical number ofeggs stimulates prolactin secretion inmale emus during the 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 for prolactinsecretion is increasing daylength, but photoperiod does not appear to playa direct role in Winter.Since the increases in prolactin concentrations in Winter are associated with incubation, wehypothesise that the stimulus is the presence of eggs.We collected blood samples fortnightly in Autumn and twice weekly in Winter from 6 singlemales and from males of 10 mating pairs of emus. Serum was assayed for prolactin and testosterone.Data were analysed for effect of time, sex, status (paired v single), incubating status, and daysrelative to incubation when appropriate.There were no differences in eitherFigure 1: Serum concentrations of testosterone andtestosterone or prolactin concentrations betweenprolactin in male emus which incubated (.) or did notincubate (0) during the breeding season. paired non-incubator males (n=5) and singlemales, and no effect of time for these 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 these data are arranged relative to first dayof incubation (Fig. 2), testosterone was elevatedprior to incubation, and declined rapidly to nearthe limit of detection (0.06 ng/mL) within days ofthe start of incubation. Prolactin began toincrease prior to the start of incubation, and theprofile of prolactin secretion during clutchformation 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 clutchformation 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 ofthe mean concentration for 6 eggs or less. Once 8or more eggs were present, prolactin further increased to 6.6 ± 1.8 ng/mL, exceeding by 13 the value?f. t?e SI? for 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 the presence of eggs.However, a cntIcal number eggs m..ust fIrst ~e accumulated before the 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 the 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 synthetic progestin, which provideseffective contraception in women. It has several modesof action but primarily it inhibits the hypothalamicpituitaryaxis, which in turn prevents ovulation.Macropodid marsupials are highly fecund, so theirnumbers are capable of increasing rapidly, causingconsiderable management problems in public parks andwildlife sanctuaries. In these semi-urban areas alternatemanagement techniques to culling and baiting aredesirable.The aims of this study are to test the efficacy oflevonorgestrel as a possible anti-fertility agent formacropodid 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 the same length anddiameter as the levonorgestrel implants were placed infemale tammar wallabies carrying pouch young (n=12).Implants were inserted subcutaneously into the medialsubdermal connective tissue, between the shoulderblades. Pouch young were removed (RPY) 3 days afterinsertion, and the adult females given bromocriptine(25mg)(im) to synchronize reactivation of theirdiapausing blastocysts and initiate the 1 st cycle.Wallabies were checked daily 25-31 days followingpouch young removal, for birth and/or a mating plug.The new pouch young were removed 3-5 days postpartumto initiate the second cycle and bromocriptinewas given again to the adult females (25mg) (im).Blood samples (5m1) were collected from the lateral tailvein at specific times throughout pregnancy from 4levonorgestrel and 4 control implanted tammars.Plasma progesterone was measured using aradioimmunoassay validated for the tammar.Levonorgestrel implants were removed from anadditional 6 tammar wallabies one-month after theinitial implantation during the seasonal quiescenceperiod, reproduction in these animals was monitored forthe subsequent 9 months.RESULTSEleven of the twelve control implanted tammars gavebirth at the end of the frrst cycle and eight of theseanimals gave birth at the end of the second cycle (table1).~ 20e~~ 15e c-o~ 10Il..50Table 1: Birth in the tammar after treatment withcontrol or levonorgestrel implant.Control Levonorgestrel1 st Cycle 11/12 9/112 nd Cycle 8/11 0/8Nine of the twelve levonorgestrel treated animals gavebirth during the first pregnancy cycle, but none gavebirth at the end ofthe second pregnancy cycle (table 1).Birth was observed in 4 out of the 6 tammars that hadlevonorgestrel implants removed at the beginning of thebreeding season.Radioimmunoassay of progesterone showed nosignificant difference in circulating levels duringpregnancy for 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 the tammar. Normal progesteronelevels were observed throughout pregnancy inlevonorgestrel treated animals. Levonorgestrelsuccessfully inhibits conception at the post-partumoestrus in the tammar wallaby. The lack of a postpartummating at the end of the frrst cycle inexperimental tammars suggests that levonorgestrel issuppressing ovulation, but this is still to be confirmed.The contraceptive effects of levonorgestrel arereversible in the tammar upon implant removal.Levonorgestrel could provide an alternative method forpopulation control of other macropodid marsupials, likethe 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 these two species of cattle (1) usingovulating approach (2). Present study wasconducted to compare the 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) for ]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 thesame cnvironment and management and recorded inoestrus when they allowed mounting by otherheifers. On Day 3 of the ensuing cycle, heiferswere implanted with norgestomet and on Day 7 ofthis cycle (Day 0 of treatment) they 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 theprevious 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, there 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 onthe rate of progesterone released from the device (2). Itis not known whether environmental factors such asnutrition and housing can affect the profile ofprogesterone release from a CIDR device. This studywas conducted to determine the possible effect of someof these 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 for 4 weeks to monitorcomplete clearance of progesterone from the animaltissues following the operation. A CIDR device (1.9gprogesterone) was inserted into the vagina of each cowat least 4 weeks after ovariectomy and left in place for11 days. In the first II-day period of study cows werehoused in individual pens and fed with lucerne andoaten chaff (Table 1). In the second period, they weregrazed on mixed pasture (2 pastures, Table 1). Theinterval between the two periods was 4 weeks. Bloodsamples were taken daily for 11 days and progesteroneconcentrations were measured using RIA. A Soxhletextraction technique was used to determine the residualamount of progesterone in spent CIDR devicesfollowing insertion in the animal (2).Results and Discussion:The only difference in the diets fed in the two periodswas the higher dry matter content of the lucerne andoaten chaff in the first period (P = 0.001; Table 1).Plasma progesterone concentrations (1.18 ± 0.17 vs1.74 ± 0.34) and the amount of progesterone releasedfrom the CIDR devices (0.64 ± 0.04 vs 0.88 ± 0.04)were significantly lower during the 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 the amount ofprogesterone that is releasedfrom a device could include the rate of blood flow tothe anterior part of vagina, frequency of defaecation,composition and physical form of the diet. Althoughthere was not a significant difference in thecomposition of the diet, 'physical form and passage rateoffaeces in confined cows may have altered the contactbetween CrDR device and the vaginal wall therebyaltering the effective surface area for release. Bloodmetabolites and protein could also affect vaginalenvironment. However, the lack of physical activitywith confinement may also have altered the frequencyor pattern of contact of the device with the vaginalmucosa and consequently reduced the amount ofprogesterone released from a device.Table 1: Nutritional values of the 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 theInternational 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 the postpartumperiod have an important role in the dairy cattlefertility. The metabolic clearance rate (MCR) ofprogesterone also determines the 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 rather than to changes in hormoneproduction (2). It is proposed that the mechanism forcontrolling PP4 was MCR of progesterone rather thanproduction rate. In the present study, an external sourceof progesterone was administrated and plasmaprogesterone concentrations were measured toinvestigate the effect of the level of feeding on plasmaprogesterone (PP4) in cows which received twodifferent forms 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 the 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 for 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 the fITst period of the study,cows were fed a maintenance ration twice daily in themorning and afternoon, and offered on an individualbasis. In the second period, the entire procedure (partone) was repeated, but cows were fed half of the feedrelative to their requirements. Blood samples werecollected twice daily at 8.00h and 16.00h for 11 daysand progesterone concentrations were measured usingRIA.Results and Discussion:The level of feeding had a significant effect on plasmaprogesterone concentrations in the P4-s.c., but not inthe P4-p.v. group (Figure 1). Plasma progesterone wasnot significantly affected by day, time of sampling orany of the interactions (P > 0.05). Faecal outputsignificantly declined after reducing the maintenance(M) ration to V2 M (Table 1). There was no significantdifference in the mass of progesterone released from aCIDR device during the M or Yz M periods (0.66 vs0.63 g). Other 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 there is a directrelationship between the level of feed intake and rate ofblood flow to the liver and gut, and consequentlyprogesterone clearance rate (3). Studies in cattle (5)also showed that the frequency of feeding had asignificant role on peripheral progesteroneconcentrations. Several factors may affect plasmaprogesterone concentration, including the difference inprogesterone release rate. The difference in response tothe level of feeding between two groups may be due tothe form that progesterone was administrated. The dailydose of progesterone injection and release rate ofprogesterone in the P4-s.c. group was greater than inthe P4-p.v. group. This may have influenced the MCRrate of progesterone. These data demonstrate that thelevel of feeding and the route of progesteroneadministration have a key role in controlling plasmaprogesterone levels.Table 1: Mean (± SE) of feed intake and faecal outputduring the 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 the form of oestradiol administration to yearling heifers at the 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 the beginning of an 8-dayCIDR treatment achieved a high submission rate of94% with comparable conception rate of 62% on thefirst day of insemination (d1). The standard Genermateprogramme recommended by LIC (NZ) for use insynchronising yealing heifers uses the 10mg ODB(CIDIROLTM, InterAg, Hamilton, NZ) capsules instead.AimTo investigate whether there were differences in themode of action of ODB under the 2 different synchronysystems, and whether heifers could be set-timeinseminated with comparable conception rates.Materials & MethodsYearling heifers from 8 participating herds wererandomly allocated into 2 treatment groups; treatedeither with the standard Genermate programme (OM)or the new synchrony system (VKT)The GM programme involved the insertion of anintravaginal progesterone releasing insert (1.43gCIDRTM, InterAg, Hamilton, NZ) with a 10mg ODBcapsule (CIDIROLTM) placed in the groove of the insertinto each of the 358 animals for 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), the animals werescored on tailpaint removal as described by Macmillanand Taufa et ai, and set-time inseminated with semenfrom high fertility bulls from LIC. All the returns to 1 51inseminations were synchronised with the 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 the uterine contents at6-8 weeks from 1 51 insemination, and again 6-8 weeksfrom when the bulls were removed from the herdResultsThere were no significant differences in conceptionrates to 1 51 and 2 nd inseminations between the twotreatments (Table 1). However, there were herddifferences in conception rate to 2 ndinsemination andthe final pregnancy rate (p = .037; p = 0.001respectively). The mean conception rate to 1Slinsemination for both treatments was 56% compared to62% for inseminating on detection of oestrus (2). Thefinal pregnancy rates were 96% and 94% for the OMand the VKT systems respectively.Of the animals which were definitely in oestrus, 59%and 58% conceived to that insemination for the OMand VKT systems respectively; of those for which therewas some doubt about being in oestrus, 54% conceivedfor 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 the 2 herdsConclusionsThe results from this trial demonstrate that satisfactorypregnancy rates could be achieved by set-timeinsemination of heifers. However, it is difficult tointerpret the results. It is quite possible that the oestrousonset is much earlier in the VKT system because of theimmediate effects of the ODB injection, andconsequently, insemination should start earlier.Both system may be useful to herdowners who wish tograze their heifers in situations which require minimalhandling and involving set-time insemination.AcknowledgementsThe authors thank the farmers for the use of theiranimals, InterAg for their support with CIDR andCIDIROL, LIC for inseminating and ParnellLaboratories for POP.References1.Macmillan, K.L., Taufa, V.K. (1997). Proc. of theNZ Soc. Anim. Prod. 47:238.2. Taufa, V.K., Macmillan, K.L. et al. (1998) Proc. ofthe 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 performance indairy cattle (1). Suckling is a powerful inhibitor of the hypothalamic-pituitary axis in postpartum cattle, and may serveas a natural physiological protective mechanism that prevents the contaminated postpartum bovine uterus frompremature exposure to progesterone. High plasma progesterone levels attenuate the 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 the 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 either a single deslorelin implant (n=41) or no treatment (n=24). Each implant was inserted subcutaneously onthe outer surface of the ear withing 7 days of calving. Implants were removed 28 days later through a small incisionover the surface of the implant. Blood samples were collected 3 times weekly for 4 weeks while an implant was in placeand then weekly for a further 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 for 28 days resulted in a prolonged postpartum anovulatory anoestrus that persisted for 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 the major proteins secreted by the ovine uterus during pregnancy.The secretion of UTMP by the 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 these proteins are found in abundance during most of pregnancy and increase with the progressionof gestation (2). Little is known as to the function of UTMP although these proteins have been shown toinhibit immune function (3). The endometrium is the3500..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 these 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 the gestationalprofile of UTMP in ovine allantoic fluid and ovineamniotic fluid with the resultant assay developed.Material and MethodsUTMP, purified from ovine uterine secretions,were used to raise antisera in chickens (JMCKI7) whichwere then 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 logtransformation.ResultsThe following values are presented as meanconcentration of immunoactive UTMP ± SE. As shownin figure 1 the 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 for research into gene function, gene regulation and developmental biology. Amajor advantage of ES cells is the capacity of ES cells to be precisely manipulated at themolecular level in vitro and to integrate into the host embryo in vivo there by giving rise tochimaeric offspring. These offspring can produce ES cell-derived germ cells enabling thebreeding of mouse strains homozygous for a gene alteration (germline chimaeras). An importantaspect of gene targeting using ES cells is the efficient production of germline chimaeras. Wereport here the factors affecting the production of germline chimaeras, which we have measuredin our gene targeting studies.Materials and Methods 1. Effect of the source of host embryos on the chimaera production:Blastocysts from BALB/c and C57BIIKa mice which were either 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 the 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 they 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 forchimaera production. Morula aggregation of ES cells can produce chimaeras but the yield islower when compared to blastocyst injection ofES cells. When an ES cell line is used for genetargeting, it is always advisable to test the production of germline chimaeras of the cell lines,although these 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 for 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 for an allele of the major histocompatibility locus (MHC) (1). We maintain asmall colony of inbred SLA miniature pigs in quarantine to allow studies in the field ofxenotransplantation.To allow expansion of the herd and removal of quarantine restrictions, a new miniature pig herd wasinitiated via embryo transfer using first and second generation offspring bred from the 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 anaesthesia using a Foley catheter(16-18 Fr) from the 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 further injection of 500 iu hCG (Chorulon, Intervet (Aust) Pty.Ltd.) 73 h after eCG. Recipients were selected for embryo transfer following detection of standingheat (24 h post hCG). Embryos were surgically transferred to recipients two days after heatdetection.One hundred and forty 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 the 21piglets born 16 are currently being used to further expand the 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 the International Embryo Transfer Society (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 the1st day of insemination by extending the 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 condensethe 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 the efficacy of a whole herd synchronyprogramme for 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) for either 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 for the 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 the previously usedCIDR inserts. These were reinserted at d13 for 7 days,with half of the 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 further i.m.injection of Img ODB on d35, and were inseminated ifdetected in oestrus.Bulls joined the herds from around d46. Individualconception dates and pregnancy were confirmed by rectalpalpation of the uterine contents 8 weeks after the firstround of AI, and again, 8 weeks after the bulls wereremoved from the herd.Results and DiscussionThere were no differences in submission (SR) andconception (CR) rates over the first 4 days for either the7-d CC or ilie 8-d CC cows but the submission patternwas significantly different (Table 1). On dO, only 13% ofthe 7-d CC cows were inseminated compared to 81 % forthe 8-d CC cows (p < 0.001). The cumulative figureswere 91 % and 97%, 95% and 97%, for the 7-d and 8-dCC cows respectively, for d1and d4.128The PR of cows inseminated on dO also differed (5%vs 41 %; p < 0.001, for the 7-d CC and 8-d CC cowsrespectively), but the cumulative PR were similar by d4.Of the cows that had their returns synchronised to 2 ndinseminations, 84% were inseminated from d22-d24. TheODB at CIDR reinsertion improved the 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 the CRof cows with returns synchronised to till inseminations(53% vs 55% for 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 the cows to be inseminated on dO but the oestrous.s:ynchrony was not significantly precise for set-timeinsemination of all cows. The modifications applied towhole herd synchrony programme enabled 80% of thecows to be pregnant to AI over 3 synchrony periods.AcknowledgmentsThe authors thank the farmers for the use of their animals,InterAg for their support with CIDR and CIDIROL,Intervet for Oestradiol Benzoate, Parnell Laboratories forPGF and DRC staff for their 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 for long-term maintenance of geneticresources, such as selection and/or control lines, raregenotypes, insurance against loss for unique stocks,as well as provide accurate evaluation of selectionresponse, free from genetic drift. It has not generallybeen used for these purposes because of a lack ofconfidence in the technology and demonstration of itssuccess for long term programs. This paper reportson the survival to term of transferred sheep embryosthat had been frozen for 13 years. Survival ofrecently vitrified embryos and the 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 for 12years from sires of the 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 the insemination groupthan the 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 further 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 for 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 the 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 the uteri of 91synchronised BLxMerino ewes on day 6. Plasmasamples were taken on day 18-19 and assayed forprogesterone; ewes with plasma concentrations >1nglml were judged pregnant. Ultrasonic pregnancydiagnosis was also performed on day 54-55, and theday of lambing and number of lambs born werealso recorded. For comparison, the performance 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 ateither stage of pregnancy. However, at d18-19 thepregnancy rates for NP embryos at d6 and d7 weregreater than for 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 there are a number of highly effectivecryopreservation (freezing) protocols for mouse oocytes andembryos, it remains almost impossible to freeze oocytes andearly embryos for 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 the 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 for cryopreservation of kidney(1), it was possible that they would suit lipid rich embryosbetter than conventional EG, even though these compoundsare more toxic than EG. This study evaluated the 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 the hCG injection. Blastocysts forthe 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 the EG component (1/4 or1/2) was substituted for Ethylene Glycol Dimethyl Ether("DE", 1,2-Di methoxyethane, Sigma Cat. E 1129) orEthylene' Glycol Monomethyl Ether ("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 for 72 h todetermine the proportion developing intoblastocysts. Meanwhile the embryos vitrified at theblastocyst stage were in vitro cultured for 24 h todetermine their viability (re-expanding andhatching).ResultsThe development of 2-cell and blastocyst stageembryos vitrified in the 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 the proportion of 2-cell embryosdeveloping into blastocysts.ConclusionUp to 10% of the EO can be replaced by either ofthese methoxylated compounds, but some toxicitywas evident at the highest concentration (10%).This indicates that further studies on lipid richembryos should be performed.Table 1. Composition ofthe 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 for the Conservation and Management of MarsupialsLandcare Research, PO Box 69, Lincoln 8152, New ZealandCryopreservation of spermatozoa as a gene-banking strategy, together with relevant assisted-breedingtechniques, is likely to become an integral component of marsupial conservation biology (1). Recovery ofmotile spermatozoa has been achieved for the brushtail possum in diluents containing 17.5% glycerol (2,3).Recently, laparoscopic artificial insemination (AI) technology has been established for superovulated tammarwallabies and brushtail possums (4). In this study, the 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, TheNetherlands) followed 78 h later by 4 mg porcine LH (Vetrepharm, Australia). On each day of insemination,spermatozoa were flushed from the 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 for the fresh sperm inseminations, the other was diluted 1: 1 withcryoprotectant media (35% glycerol in EMEM media), then frozen in pellet-form on dry ice and stored inliquid nitrogen (3), prior to thawing on the 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 performed with the 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 for 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 the first time that fertile conceptions have been achieved with frozen-thawed sperm for 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 either in the 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 the "Cook" catheter procedure in Exp.l resulted into traditional methods of ex situ management and breeding of the birth of 2 pouch young from 5 attempts; thecaptive mammals. Although these techniques are increasingly urogenitoscopic procedure resulted in 3 offspring from 4being used in the captive propagation of eutherian 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 the 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 the birth of one pouch young from 2donors. Semen was collected by means of an artificial vagina attempts. Although the injection of hCG in F48 appeared to(1). The mean (± s.e.m.) concentration, % forward motility result in the 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 the 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 the administration of hCG in this animal, notrespectively. Undiluted semen was maintained at roomtemperature' for 15 to 30 min prior to insemination.only induced a luteal phase, but presumably resulted inovulation. This Koala is the only marsupial to be bornOvulation was induced in 9 oestrous females by the means offollowing both hormonal induction of presumed ovulationIC and two methods of insemination were used (Exp.1). Theand AI. Further investigations are under way to determine thefIrst involved a custom designed insemination catheterefficacy 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 the dorsal portion of the urogenital sinusimmediately caudal to the vaginal ostia (2). Five female hCG.Koalas were gently restrained and inseininated using thismethod. The second insemination method involvedvisualisation of the 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 the femalein ventral recumbency on a tilting table set at an incline ofapproximately 30°. This procedure was accomplished afteranaesthetising the female (3). In total approximately 1 ml ofundiluted semen was either deposited into the most cranialportion of the urogenital sinus immediately caudal to thevaginal ostia or directly into the vaginae using a 3.5 Fr."Tom-cat" catheter (Becton Dickinson Labware, U.S.A).Four Koalas were inseminated using this method.Figure 1 : Cook insemination catheter (Scale: O. 5 x)Table I : Progestogen concentration (ng/ml) in the peripheralplasma of Koalas on the 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 the 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 the "Cook" catheter, the other (F32) using theF42 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 the upper 99.95% confidence limit of basalF48 hCG I Cook 0.5 5.3 6.8 NOoestrous progestogen concentration noted on the 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 the development of anartificial insemination procedure in the 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 inhibitsthe uptake of this technology into the 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 hypothesis to explainthis is that differentiation of the 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 the 1 51stageof a sequential embryo culture system, termed SOF­99 (AgResearch Ruakura, Hamilton NZ), for ~ 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 the uterine horn ipsilateral tothe CL of a suitably synchronised recipient. Asecond cohort of embryos were cultured for a further48 h in the 2 nd stage of SOF-99. At the 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 the tracts either flushed with salineor dissected to recover the fetuses. Quantitative datarecorded included crown-rump (CRL) and allantoislength. Qualitative data recorded included degree ofvascularity of the fetus, yolk sac and allantois. Thesewere collectively used to determine if the conceptusappeared morphologically normal or abnormal.Quantitative data was analysed using Least Squaresprocedure. Data presented are the 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 the three days (48, 48and 62% for Day 23, 26 & 29, respectively).Furthermore, the proportion of fetuses recovered didnot differ between the 2 treatments (Day 5 = 36/75,48% vs. Day 7 = 42/75, 56%). From the 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 100Wreford, 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 the title) for 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

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