announcement.. - the Society for Reproductive Biology

announcement .. ....PROF. 8. P. SETCHElLAUSTRALIAN. SOCIETY FOR REPRODUCTIVE BIOLOGYSIXTEEN'l'H ANNUAL CONFERENCEMeLbouPne~AU8t~LiaANNUAL CONFERENCE 1985August/September, 1985at University of Adelaide.AUGUST 26-29, 1984PROGRAMMEANDABS'rRACTS OF PAPERSCoplfI"ight AU8t~Uan Society fo7' Rep7'oductive BioLogy~ 1984Abstracts should be prepared by May 1985.Local Organiser: Pat Quinn (08) 243 6709CONTENTSPersonnelAcknowledgementsPr.ogramme GuideProgrammeAuthorsAbstractsCampus MapMinutes of 15th AGM 1983Member~hip Application Formiiiiiivv-xvxvi-xx1-108

AUSTRALIAN SOCIE'lY FOR REPRODUCTIVE BIOLOGYAugust, 1984THE AUSTRALIAN SOCIETY FOR REPRODOC'TIVE BIOLOGYOFFICE BEARERSUJish to thank the foUowing forotheiro BUpp07't; of the 1984 meet;ingChairnnnSe~roetary and EditoroT7"easuroeroDR J. K. FINDLAYDR T.K. ROBERTS ANSETT AIRLINESDR. B.G. MILLER MILES LABORATORIES PTY LTDCorrurt:ittee Memberos DR J. CUMMINS ORGANON AUSTRALIADR. J. FAlCONER SANDOZ AUSTRALIADR P. QUINNDR J. RODGERDR.A.o. TROONSONMR. P. LUTJENThe cont;ent;s of these Pr'oceedings have not been edited by theSOciety oro its Editoro and aroe roeprooduced as submitted.RssponsibiZity foro accuroacy of the comnuni~ations roests withthe authops.PROGRAMMECOMMITTEEMateroia~ in these Pr'o~eedings rrrzy not be roepY'odu~ed withoutpeY'mission of the So~iety.Chai7'nnnCorrurt:iUee MemberosPROF. D.M. DE KRETSERDR. W. CHAM:,EYPr'i~e of PT'0~eeding8 to non-memberos: $Al o. 00 ptus postage.DR.DR.G.P. RISBRIDGERL. ST1\PLESLOCAL ORGANI SIN GJ. 0' SHEA (CONVENOR)I. CLARKEP. LUTJENG. RISBRIDGERR. CARSONCO MMITTEE11. de LUISER. SIMPSONL. CUSWORTHP. TARTB. STEWART!:iI~':'::';;QC~ i [posters...""""- ~ --""'~'---. OralsGRATTAN ROADr~t~"':.'::"!;i iiii

HourMonday, Augus't: 27PROGRl\.MME GUIDE'l\1esday, August 28Wednesday, August 29AUSTRALIAN SOCIETY' FOR REPRODUCTIVE BIOLOGY0830Session 1: OVARIANHORMONES & LUTEQLYSISsession 5: MALESymposiumPROGRAMME0900100010301100Invited Lecture:Dr C WangAbst. 1-4Oral presentationsTEASession 2: COMPARATIVEREPRODUCTIVE BIOLOGYAbst. 5-10Abst. 40-45Oral presentationsTEAsession:6 INFERTILITYAbst: 46-50BIOCHEMI.STRY INREPRODUCTIONTEAsession 9: (A) MALE(B) SEASONALITY &Abst. 81-1031400 - 16001600 - 1800SUnday. Auqust 26thRegistrationsymposilJl\:Biology of ReproductionThe seminiferous growth factor; acandidate for regulating cellproliferation during development ofthe mammalian testis.The mechanism of LH and FSH secretion.The physiology of inhibin.Union Theat:r>eUnion Theat:r>eA Bellve5'. IabrieH.G. Burger1200130014001530h16001730Oral presentationsGODING LECTUREDr. A. BellveSession J(A) OVARIANHORIDNES & LUTEOLYSIS;INHIBIN & INHIBITORS(B) COMPARATIVEREPRODUCTIVE BIOLOGY;EARLY EMBRYONICo E.VELOPMENTAbst. 11-34oral presentationsHARRISON LECTUREDr. F, IabrieLUNCHSession 7(A): IVF &INFERTILITY(B) OVARY & OVULATIONRATEAbst: 51-75poster discussionSession 10: EARLYEMBRYONIC DEVELOPMENTAbst. 104-108Poster discussion Poster discussion Oral presentationssession 4: IVFAbst. 35-39Oral presentationsTEAsession 8: SEASONALITY& FERTILITYAbst. 76-80Oral presentationsASRBAGMJunior Scientist AwardPresented.Posters located throughout Meeting Eor viewing and discussions in Pathology MU::lmJlll.Oral presentations in Theatre 2, Medical 'centre.Goding and Harrison Lectures in Union Theatre.08300900Jl:>nday. August 27thsession 1::

1030104511001115Session 2:5678COWARATIVE REPRODUCTIVE BIOLOGY. Oral presentationsOJ.airman: Dr. A. BlackshawTheatr>e 2Seasonal reproductive 1uiescencein the tammar wallaby(MZepopus eugenii)Concentrations of oestradiol inplasma and ovarian tissue throughoutpregnancy in the tammar ·,.,allaby(Macpopus eugeniilAttenuated embryogenesis andunilateral OVUlation in theCalifornian leaf-nosed bat Macpotu8califoPnicus: a morphometric analysisof ovarian dynamicsPeripheral plasma proges terone andoestradiol-17S concentrations duringthe breeding season andpregnancy in the grey headed frui t ba t(Ptepopus poliocephalus)S .J. McOJnnellC.H. Tyndale­BiscoeG. ShawM.B. !'enfreeE.G. CrichtonP. H. KrutzschP.A. TowersL. M3.rtin14151617Monoclonal antibodies to pig relaxinFenprostalene as a luteolysin incattleIncreased FSH in merino ewesimnunized with an inhibin-enrichedfraction from bovine follicularfluidDepression of FSH in ovariectomizedewes by bovine follicular fluid:neutralization by serum from ewesimnunized against partially purifiedbovine inhibinJ. PattersonJ.D.O'S1eaC.S. LeeM.R. BrandonS.R. McPheeP.A. WhylieI.G. PearsonL. D. StaplesI.E'. DavisT.J. &::{uiresI •.J. ClarkeL.P. cahillT. O'SheaS.A.R. Al-ObaidiB.M. BindonM.A. HillardJ. K. FindlayS.A.R. AI-QbaidiB.M. BindonM. A • HillardT. 0' SheaL.R. Piper11309One bat:four endometriaL. M3.rtinP.A. Towers18In vivo evidence that thepituitary gland is the main sitefor the action of inhibinI.J. ClarkeJ. K. FindlayJ.T. Cummins114510Hormonal control of parturition inthe vivparous lizard, TiZiqua pugosaJAMES GODING HEKJRIA!. LECTUREB. FergussonS.D. Bradshaw19Ovarian and circulating inhibinlevels during sexual developmentof the female ratV.W.K. LeeJ. McMasterN. OJlvinH. Quigg1200-13001400-1530111213Transformation of the nuclear matrixduring mouse spermatogenesis, fertilisationand embryogenesisSession 3A: OVARIAN HORKlNES Ii LO'rEOLYSISINHIBIN «INHIBITORS. Poster diScussionOJ.airman: Prof. H.G. BurgerCharacterization of two differentandrogefr-binding sites within theovine ovarian follicleInterstitial space and transport inrat corpora luteaComparison of ovarian ~ndorphinlikeimmunoreactivity l.n immatureand adult Sprague-Dawley rats!Jr. A.BellvePathology MuseumS. CampoR. CarsonJ. FindlayA.M. DharmarajanN.W. BruceG.T. MeyerS.J. IolaitD.J. AutelitanoA. I. SmithA.T. LimB.H. 'lOhJ.W. Funder20211400 - 153022Control of inhibin secretion byrat granulosa cells in vit7'oDirect inhibition of folliculargrO,o1th by steroid-free follicularfluidSession 3B: COMPARATIVE REPRODOCTIVE BIOLOGYEARLY EMBRYONIC DEI7ELOPHENT Poster di.sCQssionChairman: Dr. R.,F. Seamark PathoZogy Museum'!he effect of presence of femaleson seasonal testosterone concentrationsin male Kowari 's , DasyupoidesbypneiV.W.K. LeeN. ColvinJ. McM3.sterH. Q.1iggL' P. cahillI.J. ClarkeJ.T. CunminsJ.K. FindlayT.P. Fletchervivii

160023242526272829303132333435Testiollar regression after thebreeding season in the male fruitbat, Ptepopus potiocephatusThe age of sexual maturity in thefemale bandicoot Isoodon Nacpoupus:the effect of seasonUterine and embryonic reactivationafter diapause in the tammar wallabyEffects of carbon monoxide of theplacental barrier of the rat: Amorpholle tric studyThe effects of oestrogen andprogesterone on protein synthesisand secretion by isolated epithelialcells from sheep endometriumEndometrial function in the pregnantewe: Characterization of secretedproteinsSteroid hormone levels in histotrophand plasma of SCMS during the preimplantationperiodPregnancy in rats follaoringsympathetic denervation of ovary,oviduct and upper uterine hornEffects of early ovariectomy onpre- and post-implantationdevelopnent of mouse embryosProgesterone priming and earlyembryo survival in the eweMaternal blocJcing antibodies arenot essential for survival of thefetal graftDiagnosis of early pregnancy incattle using real time imagingsession 4: IVF oral PresentationsoreJ.C. RodgerJ.F. WilkinsD.G. FowlerTheatre 2D.J. JessupA.O. Trounson161516301645170008300845090009150930094536373839404142434445Re lationship between pregnancyassociatedplasma protein a (PAPP-A)in human peri-ovulatory follicularfluid and the collection andfertilization of human ova in· vitpoProstaglandins and steroids infollicular fluid as prognosticatorfor human IVF resultsThe prognostic value of betahCG,oestradiol 1713 and progesteronein early human pregnanciesEmbryo viability and uterinereceptivity in human IVFsession 5:Chairman:1hesday August 28thMALE. Ot:"al presentationsDr J. CUmminsThe effects of hypothyroidism orhyperthyroidism on testis andpituitary function in prepubertalramsUltrastructural localisation ofhuman chorionic gonadotrophin onisolated rat Leydig cellsProteoglycan production by culturedmyoid and Sertoli cells of theimmature rat testisThe effects of excurrent ductobstruction upon the developingrat testisHuman acrosomal hyaluronidase andseminal plasma arylsulphataseEffect of pH on the uptake of thecalcium uptake and respiration ofram and boar sperm in the presence ofionophore A23187J.L. YovichJ.D. StangerJ.G. GrudzinskasH.O. lbppenM. SesankoA.O. TrounsonJ.L. YovichS.C. McCl:>lmJ.D. StangerA. TuvikJ.G. GrudzinskasP.A.W. lbgersB.J. MilneA.O. TrounsonTheatre :1Y. ChandrasekharB. SetchellA.E •. JacksonE.F. GlasgowD. M.. de KretserE.W. 'D1ompsonJ.S.H. ElkingtonA.W. BlackshawC.A. YatesP.O. Temple­SmithA. StojanoffA.O. TrounsonA.M.. SimpsonI.G. Whitevii ii X

Session 6: INFERTILITY. lEal presentationsChairman: Prof. DooM.. de Kretser Theatre 255The effect of maternal age on thesuccess rate of in vitpofertilization (IVY)A. TrounsonC.M. C3.roD. Jessup103010451100111546474849Evidence that clomiphene citrate(CC) acts primarily at ahypothalamic site in womenCilia and sperm tail ultrastructureand function in normal SUbjectsand patients with Young's,syndromeIs conventional bacteriology ofsemen useful in the managementof male infertility?Increased frequency ofautoantibodies in men withsperm antibodiesJ.F. KerinJ. LiuS.S.C. YenG. PhillipouL.J. WiltonH. TeichtahlP.O. TemplesmithD.M. de KretserM.G. JenningsH. W.G. BakerM.P. McGowanH.W.G. BakerG.N. ClarkeS.H. KohM.N. cauchi5657585960Studies on the cryopreservationof 2-cell mouse embryosEvaluation of frozen-thawed humanembryosGlycosyltransferase levels inhuman seminal plasmaSialic acid deficiency in cervicalmucus hostilityEffects of surgical lubricantson semen analysisL.R. M:lhrL FreemannL.R. M:lhrL. FreemanA. O. TrounsonM.T. de WittP.J. LutjenJ. HoyA.O. 'IrounsonP.J. tlltjenM. de wittJ.C. McBainA.O. TrounsonJ.P.P. TylerM.N. SchoelMn113050Pharmacology of the erectiletissue of the penisC .J. C3.ratiR.G. GoldieE.J. KeoghS. Wisneiwski61Acute cellular and hunoralresponses to equine streptococcalendometritiss. Munyuap. williamsonJ. PenhaleJ. Murray1200 - 13001400 - 1530515253KEITH HARRISON M.EK>RIAL LECTUREEndocrine therapy for prostaticcancerSession 7A: IVF & INFERTILITY. Poster discussionChairman: Dr. B. Hudson Pat;hoZogy !l,JusewnWithdrawnPlasma progesterone changes insuperovulated women before, duringand immediately after laparoscopyfor IVF and their relation to theinitiation of pregnancy afterembryo transferEndocrine changes around the timeof oocyte recovery in womensupero\'Ulated for in vit-rsofertilizationUnion TheatreDr. F. LabrieP. TaylorA. 'lrounsonMoo BesankoV.B. MacLachlanH.O. tbppenA. Trounson621400 - 153063646566Pregnancy associated plasmaprotein-A (PAPP-A) in humanseminal plasmaSession 7B; 0I1ARY & OVULATION RATE.Chainaan: Dr. 'ii.A. ChaaleyCompensatory hypertrophy andovulation in sympatheticallydenervated rat ovaries afterunilateral ovariectoI1¥Olange of ovulation rate withtime in ewes imnunized againstacdrostenedioneThe effect of· monensin supplementon ovarian activity and responseto FSH challenge in ewe lambsOvulation rate of ewes subsequentto FSH administration early in theoestrous cycleM••J.SinosichPoster discussionPathology MuseumS.N. WylieW.R. GibsonR.I. CoxP.A. WilsonM.S.F. ~ngF.P. SumbungP. E. WilliamsonM.M. RalphR.F. SeamarkP.E. Geytenbeek54Effect of follicle stinulation andand follicle number on the rise inearly luteal phase pregesteroneconcentration following follicleaspiration in womenJ.D. StangerJ.L. YovichD.L. Willcoxxxi

l'676869707172737475Effects on LH profiles of selectionfor shorter intervals between eggsin australorp and white leghornhens maintained under continuouslightEffect of FSH dose and treatmentregime on ovulatol;y response insheepInhibition of mitosis by ovinefollicular fluid in vitpoSuperovulation and embl;yo recovel;yin the merinoOvulation rate responses to longlong term weaning weightselection in merinosThe effect of plasma progesteroneconcentration during the lutealphase on subsequent Ovulationrate in sheepThe effect of epidermal growthfactor on reproductive functionof ewesMtibody levels in the ewe andits progeny follOo1ing steroidimmunizationEffect of high levels ofandrostenedione immunity inpregnant ewes on the sexualactivity displayed by their maleprogeny. C.B. GowR.J. ScararnuzziN.B. CarterB.L. SleldonP.J. SharpJ. ~plestonR.J. BiltonN.W. /oboreR. CarsonM. de GarisM.B. NottleD. T. ArmstrongM.K. HollandP. H. SlarpeG.N. HinchT.N. EdeyC•J. 'n

84858687888990Amino acids in the genital ductsof the male Japanese Q..1ailThe mammalian epididymis:structural differentiationand functions of the ductuliefferentesTestosterone and the immaturebovine epididymispattens of protein secretionalong the length of the mature ovineepididymis in vit~oProduction and output of spermatozoaand epididymal sperm reserves ofsexually rested or frequentlyejaculated dogsA bicarbonate and calcium dependentinduction of rapid guinea-pi.g spermacrosome reactions by monovalentionophoresATP induced activation ofdemembranated ram spermJ. ClulowR.C. JonesR.C. JonesK.M. JurdM.K. HollandD. E. BrooksM.J.J. CarabottD.E. BrooksP.R. DaviesI.C.A. MartinRoss V.HyneI.G. WhiteJ.K. voglmayr96979899100101GnRH in continuous low dose inducesovulation in both post-partum andseasonally anoestrus ewesThe use of rams, GnRH, andimmunization against androstenedioneand oestrone for the induction offertile oestrus in acyclicpost-partum ewesEffects of an analOg.le of GnRH onpregnancy rates in dairy cattleThe use of PMSG to prevent "sunmerintertility" in pigsReduced oestrogenic effect ofyarloop pasture sprayed with benomylEffect of sperm dose on conceptionrate following uterine A.I. in ewesA.H. WilliamsP.J. WrightI.J. ClarkeP.J. WrightP.E. GeytenbeekI.J. ClarkeR.M. HoskinsonK.L. MacmillanV.K. 'IaufaA.M. DayD. P. rennessyN. E. JohnstonN.R. AdamsK.P. CrokerI.F. DavisD.J. KertonS.R. McPheeI. GrantL.P. Cahill91 Changes in guinea-'pig sperm intra-'cellular sodium and potassiumcontent during capacitation92 Effects of malonic, maleic, citricand caffeic acids on the motilityof human sperm and penetration ofcervical mucusK.P. EdwardsR. V. HyneP. Brown-~odmanI.G. WhiteP. Y. W. 010101E.J. Post102 Developnental changes in themetabolism of progesterone anddihydrotestosterone by the bloodof newborn lambs103 Insulin in ovine mi lk: pre-deliveryand post-weaningC.D. NancarrowP.J. ConnellJ. FalconerR.F. Sleldra,keJ.S. Robinson93941100 - 1300The effect of (S) - Ct chloroh~rinand 6-chloro-6-deoxyglucose onthe matabolism of guinea pig spermatozoain vitrooEffects of non:-dialysable substancesin ram seminal plasma on ramspermatozoa during dilution, coolingand deep-freezingsession 98: SEASONALI"l'Y .& FERTILI"l'Y.Chairman: Dr. 8.M. BindonA.R. JonesS.A. FordP. S1tumorangI.C.A. Martin1:'Oster discussionPathology Museum140014151430session 10:EARLY EMBRYONIC DEVJn,OPMENT.O1airlDan: Dr. J. Shelton104 Maternal recognition of pregnancyprior to implantation105 125I _ BSA uptake bypre-implantation mouse embryos106Effect of proteitl-'free culturemedia on mouse and human embryodevelopment invit7'oOI:'al presentationsTheatre TwoC. O'NeillL. B. Pembl.eP.L. KayeC.M. CaroA.O. Trounson95 Reduction of T.H pulse frequencyby steroids: differences betweenmerino and suffolk ewesxivG. B. ThomasD.T. PearceC.M. OldhamG.B. Martin14451500107108Effects of insulin onpreim~lantationmouse embryosMicroinjection of a chickenhistone gene into mouse zygotesxvH.G. GardnerP.L. KayeA. MichalskaA.J. R:>binsP. QuinnJ.R.E. wellsR.F. seamark

NAMEABSTRACT NO.Adams, N.R••••••••••••••••••••••••••••••••••••••.•••••••• 100Al-Obaidi, S.A.R····· ••••••••••••••••••••••••••••••••• 1 6, 17Armstrong, D. T••••••••••••••••••••••••••••••••••••••••••••70Atkinson, S •••••••••••••••••••••••••••••••••••••••••••••••80Autelitano, D·J······· ••• • •••••••••••••••••••••••••••••••• 1 3Baker, H.W.G••••••••••••••••••••••••••••••••••••••••••48, 49Besanko, M··········· •••••••••••••••••••••••••••••••••• 3 7 ,52Bilton, R.J.•••••••••••••••••••••••••••••••••••••••••••••••68Bindon, B.M·········· ••• • ••••••••••••••••••••••••• 16 , 17,71Blackshaw, A.W c ••••••23, 42Bradshaw, 5.0••••••••••••••••••••••••••••••••••••••••••••• 10Brandon, M.R•••••••••••••••••••••••••••••••••••••••••••••• 14Brooks, O. E•••••••••••••••••••••••••••••••••••••••83, 86, 87BrCMn,B.W••••••••••••••••••••••••••••••••••••••••••••••••75Brown-Woodman, P············ 92Browne, C.A•••••••••••••••••••••••••••••••••••••••••••••••81Bruce, N.W••••••••••••••••••••••••••••••••••••••••12, 26, 77cahill, L.P···········.··· ••••••••••••••••••••••••15, 21, 101Cameron, A.W. N••••••••••••••••••••••••••••••••••••••••••••79Campo, S ••••••••••••••••••••••••••••••••••••••••••••••••••11carabott, M.J.J•••••••••••••••••••••••••••••••••••••••••••87Carati, C.J•••••••••••••••••••••••••••••••••••••••••••••••50Caro, 'C,.M••••••••••••••••••••••••••••••••••••••••••••55, 106Carruthers, T.D•••••••••••••••••••••••••••••••••••••••••••78carson, R•••••••••••••••••••••••••••••••••••••••••••••11, 69Carter, N. B•••••••••••••••••••••••••••••••••••••••••••••••67cauchi, M. N•••••••••••••••••••••••••••••••••••••••••••••••49Chandrasekhar, '(···········•••••••••••••••••••••••••••••••4 0Chow, P. Y.W•••••••••••••••••••••••••••••••••••••••••••••••92Clarke, G.N•••••••••••••••••••••••••••••••••••••••••••••••49Clarke, I.J•••••••••••••••••••••••••••••••15, 18, 21, 96, 97ClulCM, J •••••••••••••••••••••••••••••••••••••••••••••••••84Colvin, N•••••••••••••••••••••••••••••••••••••••••••••19, 20Connell, P.J•••••••••••••••••••••••••••••••••••••••••••••• 102Cox, R.I••••••••••••••••••••••••••••••••••••••••••64, 74, 75Crichton, E.G••••••••••••••••••••••••••••••••••••••••••••••7Croker, K.P••••••••••••••••••••••••••••••••••••••••••••••100Ct:Dnmins, J.T••••••••••••••••••••••••••••••••••••••••••18, 21Cummins, L. J ••••••••••••••••••••••••••••••••••••••••••••••72CurnCM, O.H•••••••••••••••••••••••••••••••••••••••••••••••79Davies, P. R•••••••••••••••••••••••••••••••••••••••.••••••••88Oavis, I. F•••••••••••••••••••••'••••'••••••••••••••••••1 5, 101Cay, A.M•••••••••••••• ~•••••••••••••••••••••••••••••••••• ~8de Garis, M••••••••••••••••••.•••••••••••••••••••••••••••••69de Kretser, D.M·········· ••••••••••••••••••••• 41 , 47, 81,82de Witt, M.T••••••••••••••••••••••••••••••••••••••••••58, 59Dharmarajan, A.M••••••••••••••••••••••••••••.••••••.••••••••12Doughton, B·············.·•••••••.••••••••••••••••••••••••• 27Edey, T.N•••••••••••••••••••••••••••••••••••••••••••••••••71Edwards, K.P••••••••••••••••••••••••••••••••••••••••••••••91Elkington, J. S. H••••••••••••••••••••••••••••••••••••••••••42Eppleston, J •••••••••••••••••••••••••••••••••••••••••••••• 68Fairclough, R.J ~ ••••••••••;~Fairnie, I.J•••••••••;·.··.· •• • ••• • ••• • ••·• •••••••••••••••;03Falconer, J ••••••••••••••••• ~ •••••••••••••••••••••••••••• 10Fergusson, B•••••••••• •• ••• •• •••• • •••• • •• •• •• • •• ~••;;·.;;••,1Findlay, J.K••••••••••••••••••••••••••••••16, " , , 22Fletcher, T.P••• ~ •••••••••••••••••••••••••••••••••••••••• '93FOrd, S.A '~34Fowler, 1).G"•••••••••••••••••••••••• ~ .~ •.' ••••••• ~ ••••••• ; ~ •5 7Freemann, L••••••••• ~ •••.•••••••••••••••••••••••::::::::•• :13Funder, J.W•••••••••••••••••• ~................. 107Gardner, H.G•••••••••••••••.•••••••••••••••••••••••••••••• 24Gemmell, R~T•••••••• ~ •••••••••••••••••••.••••••••_•••••• ~~. '97Geytenbeek t p.• E••••••••••••••••••••••••••.•••••••••••••30' 63GibS'on, W~R.. • • • • • • •• •• • • • • • •• • • • •••• •• • •• • •• • • •• • • • • • , 41GlasgClli, E.F••••.•••••.•••••••••••••••••••••••••••••••••••• '50Gol4ie, R. G•.•••••••••••••••••••••••••••••••• '.'.:'•••: ••••••'.: '.: :S7Gow, C.B...................................... . .101Grant, I···························.. ·····:·.::·.:·.:·.:·::.;6: 38Grudz inskas, J.G. • • • • • • • • • • • • • • • • • • • • • • • • '. 79:~~~,R~:;::::'0'.:'.::'. '.'.'.::: ~.'.'.:.............•..........' '.:' .,..'. :: ~Hedger, M. P••••••••••••••••••••••.•••••••••••••••••••••••• :99Hennessy, D.P•••••••••••••••••••••·····················6·· 17••••••••••••• ••1 ,Hillard, M.A••••••••••••••• ~.......•••• • •••••••••••71Hinch, G.N••••••••••••••• 0 • • • • • ••• • • •• •• • • • •• • . • 1Hooper, S.B·············································;~··~6Holland, M.K••••••••••••••••••••••••••••••••••••••••• '37' 33Happen, H.O........................................... , 97Hoskinson, R.M••••••••••••••••••••••••••••••••••••••••••• '58Boy, J •••••••••••••••••••••••••.•••••••••••••••••••••• ~;••91Hyne, R.V••••••••• , ••••••••••••••••••••••••••••••••••• , 82Ishida, H•••••••••••••••••••••••••••••••••••••••••••••••••41Jackson, A..E ••••••••••••••••••••••••••••••••••••••••••••• '48Jennings, M.G••••••••••••••••••••••••••••••••••••••••• ;;;••55Jessup, D.J... •••••••••• ••••••••••• •••••••••• ••••••••• , 99Johnston, N. E•••••••••••••••••••••••••••••••••••••••••••• '93Jones, A.R•••••••••••••••••••••••••••••••••••••••••••••~~ '85Jones, R.C............................................. ,73Jorgensen, G.r••••••••••••••••••••••••••••••••••••••••••••Jurd, K. M••••••••••••••••••••••••••••••••••••••••••• ~ ~5'.;07 85Kaye, P.L........................................... 4~ 79Keogh, E.J............................................ , 46Kerin, J.F•••••••••••••••••••••••••• ~.~ ••••••••••••••••••;OlKerton, D.J••••••••••••••••••••••••••••••••••••••.•••••••• 71Kinghorn, B.P•••••••••••••••••••••••••••••••••••••••••••• '49Koh, S.H•••••••••••••••••••••••••••••••••••••••••••••••••• 7Krutzsch, P.H•••••••••••••••••••••••••••••••••••••••••••••;4Lee, c.s..............................••••...••....... ~;. '20Lee, V.W.K.. •••••• •••••••••••• •••••••••• •••••••• •••••• , 13Lim, A.T••••••••••••••••••••••••••••••••••••••••••••••••••79Lindsay, D.R•••••••••••••••••••••••••••••••••••••••••••• "46Liu, J ••••••••••••••••••••••••••••••••••••.•••••••••••• : •••13Lola.it, s.J••••••••••••••••.•••••••••••••••••••••••••••;~.••59Lutjen,' P.J....... ••••• ••••• ••.••••••••••• ••••••••••••• ,xvixvii

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F •••••••••••••••••••••••••••••••••••••29, 66, 108setchell, B•••••••••••••••••••••••••••• •••••••••••••••••• .40Sharp, P.J•••••• ••••••• •••••••••••••••••••••••••••••••••••~~Sharpe, P.H••••••••••••••• •••••••••••••••••••••••••••• 70 ,Shaw, G••••••••••••••••••••••••••• •••••••••• •••••••6, 25, 7~Sheldon, B. L••••••••••••••••••••••••••••••••••••••••••••••6Sheldrake, R.F••••••••••••••••••••••••• •••••••••••••••••• 1 03Simpson, A.M•••••••••••••••••••••••••••• ••••••••••••••••• .45Sinosich, M.J••••••••••••••••••••••• • •• •• ••••••••••••••••• 6 2siturnorang, P•••••••••••••••••••••• •••••••••••••••••••••• .94Smith, A..I .•.........................•....................1;SquiresT.J••••••••••••••••••••••••••••••••••••••••••••••1Stanger: J.D•••••••••••••••••••• • •••• •••••••••••• .36, 38, 54Staples, L. '0•••••••••••••••••••••••••••••••••••••••••••15, 4Stojanoff, A••••••••••••••••••••••••••••••• ••••••••••••••.4:stone, B. A.••••••••••••••••••••••••••••••••••••••••••••••••2Stone, G. M. ••••••••••••••••••••••••••••••••••••••••••••••••28Sumbung, F.P••••••••••••••••••••••••••• •••••••••••••••••• .65Taufa, V.K••••••••••••••••••••••••••••••••• •••••••••••••• .98Taylor, P ··.············52Teichtahl, H••••••••••••• ••••••••••••••••••••••••••••••••.:;Temple-Smith, P. D•••••••••••••••••••••••••••••••••••••43,Tennison, M 73Thomas, G. B•••••••••••••••••••••••••••••••••••••••••••• o. • .9 5Thonp son, E. W•••••••••••••••••••••••••••••••••••••••••••••4 ~Thorburn, G. D••••••••••••••••••••••••••••••••••••••••••• 1 ,Thwaites, c.J•••••••••••••••••••••••••• • ••••••••••••••••• .71Toh, B. H••••••••••••••••••••••••••••••••••••• •••••••••••• .13Towers P.A•••••••••••••••••• ~ ••••••••••••••••••••••••••8, 9Trouns~n, A.O••••35, 37,39,44,52,53,55,57,58,59,106Tuvik, A••••••••••••••••••••••••••••••••••••• •••••••••••• .38Tweedale, R••••••••••••••••••••••••••••••••••••• ••••••••• .73Tyler, J.P.P••••••••••••••••••••••••••••••• •••••••••••• •••60Tyndale-Biscoe C. H., '.' .590voglmayr, J.K••••••••••• ••••••••••••••••••••••••••••••••••·Vinijsanun, A•••••••••••••••••••••••••••••••••• •• •• •••••• .31Waddell, B.J •••••••• .77wastie, C.J••••• ••••••74Waters, M.J•••••••••••••••••••••••••••••••••••• •••••••••• .73watkins, W.B ••••••••• 1 , 3wells J.R.E•••••••••••••••••••••••••••••••••••••••••••••108White; I.G••••••••••••••••••••••••••••••••••••••••45, 90, 92whylie, P.A. •••••••• 1 5, 4Wilkins, J. F 54Willcox, D.L••••••••••••••••••••••••••••••••• •••••••••••• .58Williams, A.R•••••••••••••••••••••••••••••••••••••••••••••96Williamson P.E•••••••••••••••••••••••••••••••••••61, 65, aDwilson . P.~•••••••• ; ••••••••••••••••••••••••••••••64, 74, 75Wilton: L.J••••••••••••••••••• • •• ••••••••••••••••••••••••• 41W isneiwski s s 0, 64 75wong, M.S.F••••••••••• ,••••••••••••••••••••••••••••••••• ' 97wright, P.J•••••••••••••••••••••••••••••••••• •••• .76, 96,xViiixix

Whylie, P. A••••••••••"•••••••••••••• '" ••••••••••••••••••4,1 5Wylie, S.N••••••••••••••••••••••••••••••••••••••••••••••••63Ya tes, C.A·····•• •• •• •• ••••••••••••••••••••••••••••••••••• 4 3Yen, s.S.C 46Yovich, J.L•••••••••••••••••••••••••••••••••••••••36,· 38, 54Zhang, X••••••••••••••••••••••••••••••••••••••••••• ~ ••••••281COINCIDENTAL RELEASE OF OXYTOCIN, NEOROPHYSIN AND PROSTAGLANDIN F 2aINTO THE UTERO-OVARIAN VEIN OF SHEEP DURING LUTEAL REGRESSIONS.B. Hooper, W.B. Watkins* and G.D. 'lborburnDepartment of Physiology Monash University, Clayton, Australia and*postgraduate School of Obstetrics and Gynaecology,University ot Auckland, New ZealandMeasurement of oxytocin (OXT) concentrations in the peripheralplasma of sheep has revealed that concentrations are high during theluteal phase of the cycle and low during luteolysis (1). Since thecorpus luteum (C.L.) secretes OXT into the ovarian vein (2), measurementof OXT in the peripheral plasma may not adequately reflect OXT releasefrom the ovary. Similarly, prostaglandin F 2a(PGF 2a) is metabolisedrapidly by the lung, it is therefore necessary to measure PGF 2ainuterine venous effluent in order to establish the pattern by which it isreleased from the uterus. 'lbe aim of the present study was to assessthe temporal relationship between ovarian OXT release and uterine PGF 2arelease into the utero-ovarian vein (Uov) of cycling and early pregnantsheep.Six Border-Leicestor x merino ewes were mated with either avasectomized (3 ewes) or an intact (3 ewes) ram. All ewes wereimplanted with UOV, jugular vein and carotid artery catheters between 6to 8 days after mating (Days 6-8). From the day after surgery, sampleswere collected daily from each catheter, except on days 13-16 whensamples were collected hourly for 12 consecutive hours. UOV' plasmasamples were analysed by RIA for OXT, oxytocin-associated neurophysin(ONP), progesterone and PGF 2a• Jugular vein and carotid artery sampleswere analysed for OXT and ONP.The concentrations of OXT and ONP in the UOV draining a C.L.foll~wed a similar pattern to that reported for the jugular vein (1).During the luteal phase, UW OXT and ONP concentrations were high (400to 10,000 pg/ml and 4 to 20 ng/ml, respectively) and declined on aboutday 12, approximately 2 days before the decline in progesterone. Ondays 13 to 16, large pulses of OXT and ONP (>300 pg/ml and >4 ng/mlrespectively) coincided very closely with large pulses of PGF 2a(>5ng/rol) in the UOV. It was not possible to discriminate temporallybetween these pulses. In addition the pulses of OXT and ONP in the UOVwere represented by much smaller pulses in the peripheral plasma.In early pregnant animals, UW OXT and ONP concentrations alsodeclined on about day 12. On days 13 to 16, large PGF 2apUlses wereabsent in the UW, however some large pulses of OXT and ONP (>200 pg/mland 3 ng/ml respectively) did occur. 'lhis suggests that oxytocinrelease may occur in the absence of a PGF2a stimulus during days 13-16.The existence of coincident pulses of OXT, ONP and PGF 2ain theUW, during luteolysis, provides further evidence to suggest that OXT isinvolved in the luteolytic process in sheep. 'lbe cyclical pattern ofovarian oxytocin release, however, remains an enigma, particularly sinceit occurs in both cycling and early pregnant sheep.xx(1) SChams, D., Lahlou-Kassi, A. and Glatzel, P. J. Endo. (1982) 92:9-13.(2) Fl.int, A.P.F. and Sheldrick, E.L. Nature (1982) 2:22.: 587-588.

2CHARACTERIZATION OF OXYTOCIN-CONTAINING PARTICLES WITHIN THE OVINE CORPUSLUTEtM.G.E. Rice and G.D. Thorburn, Department of Phys iology, Monash University,Clayton, Victoria. 3168.Immunoreactive oxytocin has been identified in corpora lutea frompregnant and non-pregnant Women and cowes (1,2,3,4) and non-pregnant sheep(Wathes and Swan, 1982) and there is now conSiderable evidence consistent withan ovarian site of oxytocin production (5,6). At present the subcellularlocus of storage and mechanism of release of luteal oxytocin remains to beelucidated. The aim of the present study was to characterize the subcellularlocalization of oxytocin within corpora lutea of non-pregnant ewes.Specifically, we have addressed the question whether oxytocin is compartmentalizedwithin storage granules similar to the neurohypophysial secretorygranules which contain oxytocin and vasopressin.Corpora lutea were obtained on day 7 or 8 of the oestrous cycle fromanaesthet ized Border-Leicester cross ewes. The tissue was homogeniZed in0.32M sucrose, 0.05% bactracin and subjected to differential centrifugation(900 x g. 10,000 x g, 20,000 x g.30,OOO x g and 100,000 x g). 82% of totaloxytocin in corpora lutea was recovered in particulate fractions and 57% wasassociated with the 10,000 x g pellet. To further characterize particulateoxytocin, the 10,000 x g pellet was subjected to Percoll density gradientcentrifugation (30r. Percoll, 40,000 x g for 30 min at 4°C). Oxytocin wasassociated with a particulate fraction which sedimented to a density of 1.054- 1.061g/ml.3 H-Oxytocin, included in the homogenization buffer, was not incorporatedinto the particulated-oxytocin fraction prepared by Percoll dens itycentrifugation. This result is indicative that the particle-bound oxytocin,observed in this study, represents endogenous compartmentalization of thepeptide and negates the possibility that it represents adsorption of solublepeptide to particulate matter in the homogenate. Particulate oxytocin,incubated in buffered medium at 37°C, was stable for upto Ih and the releaseof oxytocin was not affected by the pH of the incubation medium, over therange 5.5 to 8.5. Oxytocin release, however, was stimulated by incubatingparticle-bound oxytocin in buffered medium of low osmolality « 20QnOsm).These data are similar to the physico-chemical properties reported forpeptide-containing neurohypophysial secretory granules. Ultrastructuralanalysis of oxytocin-containing fractions revealed the presence of electrondensegranules (dia 200-25Onm). These data are suggest ive that oxytocin, inthe corpus luteum of sheep, is contained within a population of secretorygranules which occur in high numbers during the mid-luteal phase of theoestrous cycle.1. Wathes, D. C., Swann, R.W., Birkett, S.D.. Porter, D.G. and Pickering,B.T. (1983). Endocrinology, 113: 693-698.2. Wathes, D.C., Swann, R.W., Pickering, B.T., Porter, D.G., Hull, M.G.R.and Drife. J.O. (1982). Lancet, ii: 410.3. Fields, P.A., Eldridge, R.K., Fuchs, A-R, Roberts, R.F. and Fields, M.J.4.(1983). Endocrinology, 112: 1544-1546.Khan-Dawood, F.S. and Dawood, M.Y. (1983).1129-1132.J. Clin. Endocrin. Metab. , .2!..:5. Flint, A.P.F. and Sheldrick, E.L. (1983). J.Reprod.Fert, 67: 215-225.6. Sheldrick, E.L. and Flint, A.P.F. (1981). Prostaglandins, 22: 631-636.PROSTAGLANDIN F 2AND OVARIAN OXYTOCIN SECRETIONDURING THE OESTROUS 8YCLE AND EARLY PREGNANCY IN THE EWER.J. Fairclough, A.J. Peterson, L.G. Moore* and W.B. Watkins*Ruakura Research Station, Hamilton, N.Z.,*National Womens Hospital, Epsom, Auckland, N.Z.3Oxytocin, produced in relatively large amounts by the ovine corpusluteum, is thought to play a major role in luteolysis in the ewe bystimulating uterine prostaglandin (PG) F production during Days 12-15of the cycle (1,2). However in early pregnancy there is a diminutionof surges of both the PGF 2metabolite 13, 14-dihydro-15-keto-PGF andthe oxytocin associated neffrophysin (OT-N) (3). The present paperexamines whether PG's may be involved in stimulating ovarian oxytocinrelease On Days 13-15 in non-pregnant and pregnant ewes. PGfl1 andOT-N concentrations were used as an indirect index of uterine PGF andovarian oxytocin release respectively (1,4).Experiment I: Nine Romney ewes were given im injections of indomethacin(n=4) at a dose rate of 4mg/kg bodyweight or indomethacin vehicle(n=5) at 0700, 1100 and 2400h over Days 13-15 of the oestrous cycle.Blood samples were collected at hourly intervals from 0700-1800h overDays 13-15 of the oestrous cycle. Plasma OT-N concentrations weremeasured by radioimmunoassay (RIA). Experiment 2: Nine additional eweswere given 20mg indomethasone (n=4) or vehicle (n=5) twice daily intothe uterine horn over Days 12-14 of the cycle. Blood samples, ~ollectedat hourly intervals from 1300h On Day 12 to 2100h on Day 14, wereassayed for PGFM and OT-N content by RIA. Experiment 3: Twenty ewes(10 pregnant (P) and 10 non-pregnant (NP)) were given 2mg PGF im On 2aDay 13 (n=5P, 5NP) or Day 15 (n=5, 5NP) post estrus. Blood sampleswere collected at -30, -15, 0, 5, 10, 15, 30 and 60 min. fro~ the PGF 2ainjection and the samples assayed for plasma OT-N cOncentratIons.In ewes given indomethacin systemically (Experiment 1) there wascomplete suppression of OT-N surges. Results from experiment 2 showedthat intra uterine injections of indomethacin did not alter the magnitudeor number of surges in OT-N concentrations compared with controlewes. It is concluded that an extra uterine source of PG's is involvedin the regu~ation of ovarian oxytocin secretion in ewes at luteolysis.Results obtained from experiment 3 showed a marked surge in plasma OT­N concentrations in 8 of the 10 pregnant ewes and in all non-pregnantewes On Day 13 of the cycle. On Day 15 of the estrous cycle there wasnO marked increase in OT-N levels in response to the PGF 2stimulus.It would appear that the conceptus does not inhibit the o~arian oxytocinresponse to a PGF stimulus during early pregnancy in the ewe.2a(1) Fairclough, R.J., Moore, L.G., McGowan, L.T., Peterson, A.J.,Smith, J.F., Tervit, H.R. and Watkins, W.B. (1980). Prostaglandins20, 199.(2) Flint, A.P.F. and Sheldrick, E.L. (1983). J.Reprod.Ferti.~, 215.(3) Moore, L.G., Peterson, A.J., Tervit, H.R., Fairclough, RaJ.,McGowan, L.T., Smith, J.F. and Watkins, W.B. (1982). Prostaglandins24, 79.(4) Moore, L.G., Watkins, W.B., Choy, R. and Elliott, R. (1984). Proc.N.Z.End.Soc. Abst. 9.

INFUSION OF4PGF2a INTO UTERINE LYMPHATIC DUCTS CAUSES LOCALLUTEOLYSIS IN THE EWE.P.A. Whylie*, J.D. O'Sheat and L.D. Staples.Animal Research Institute, D.A.V., Werribee, Victoria, 3030., *Schoolof Agriculture and tveterinary School, University of Melbourne.Recent studies in sheep have demonstrated pulsatile release ofprostaglandin F2a (PGF2a) into utero-ovarian (UO) lymph at the expectedtime of luteolysis (I). Thus it has been postulated that theselymphatics may participate in the local control of corpus luteum (CL)function (2). This study tested the effect of PGF2a infusions into aUO lymphatic duct On luteal function in Corriedale ewes.In regularly cycling ewes bearing unilateral ovulations, the CLwere marked with india ink at laparotomy under fluothane anaesthesia onDay 7 or 8 of the cycle. A major uterine lymphatic duct ipsilateral tothe marked CL was identified and PGF2a (Sigma tris salt) in physiologicalsaline was infused (2.2 ml/h) for 3h at dose rates of 0 (n=3 ewes,control), O. I (n=4) , 2 (n=3), 20 (n=6) and lao ~g/h (n=S). Lutealfunction was assessed by detenaining jugular venous progesterone (P)levels by R.I.A. before, during and after infusion. These ewes werealso checked twice daily for oestrus and ovaries were collected andexamined histologically 1-2 days after observed oestrus. In 4 ewesbearing bilateral ovulations, PGF2a was infused unilaterally at O. I, 2,20 and lao ~g/h (one ewe per dose) to test for peripheral effects.Their ovaries were examined histologically 72h after infusion.AilS ewes which received 100 ~g/h re-ovulated with regression ofmarked CL and 3 of 5 showed oestrus within 72h. In 6 ewes infused at20 ~g/h P levels declined (co < 15% of pre-infusion level) in 4 within72h and 2 of these had re-ovulated by this time (Table I).TABLE I.TreatmentPGF2a(YB/h)o0.1220100P & Oestrous Patterns of Ewes After Infusion of PGF2aEwes with decline Interval to Ewes with Newin Pat:Oestrus (h) Ovulations withinn 22-72h 168-264h72h3 o 3 240,240,264o4 o 4 216,240,240,264 o3 1 2 192,216,240o6 4 I 72,144,168,216,-,­ 25 5 0 48,64,72,-,-5P level declined sharply at 22-30h post-infusion for one ~we at 2 ~g/h,but along with all other ewes infused at 2, 0.1 and 0 ~g/h, this eweexhibited normal P, oestrus and ovulation patterns between day 16-18 ofthe cycle. The immediate luteolytic effect of lOa and 20 ~g/h doses ofPGF2a was confirmed in ewes bearing bilateral CL and the effect wasconfined to the CL ipsilateral to the infused lymphatic thus demonstratinga local effect. We conclude that UO lymphatic ducts can participatein local control of CL function in the ewe.(I) Staples, L.D., O'Shea, J.D., Harper, C•• Howse, A. and Thorburn, G.(1983). Proc.Aust.Soc.Reprod.Biol. 15th Ann. ConE. p8.(2) Staples. L.D., Fleet, I.R. and Heap, R.B. (1982). J.Reprod.Fert.~ : 409-420.P.A.W. was supported by The Australian Development Assistance Bureau.S2ASONAL REPRODUCTIVE QUIESCENCE IN THE TAMMAR WALLABY (!44CROPu~~0GENII): TEP~INATION BY INCREASING THE DUPATION OF THE NOCTURNALRISE OF PLASMA t>lELATONIN.58.J. MCConnell and C.H.Tyndale-Biscoe*.Deparu~ent of Behavioural Biology, Research School of BiologicalSciences, Australian National University, GPO Box 4, Canberra City,ACT, 2601 and *Division of Wildlife and Rangelands Research, CSIROAustralia, PO Box 84, Lyneham, ACT, 2602.A characteristic feature of pineal physiology is a marked dailypattern in the secretion of melatonin, with the highest plasma levelsdetectable during the dark phase. It appears to be a component ofthis daily profile that mediates the effects of photoperiod onreproduction in eutherian mammals (1). Although manipulation ofphotoperiod has been shown to influence reproduction in marsupials (2)hew this information reaches the neuroendocrine-reproductive axis ofthese mammals is not known. However, the pineal i~ implicated in atleast one species, the tammar (3).The corpus luteum and blastocyst of tamrnars are held in~Jiescence during the period of natural increasing daylength(seasonal quiescence). Both reactivate shortly after the change todecreasing daylength in December, and birth occurs 26-27 days later.The pineal gland may mediate the effects of decreasing photoperiod asthe duration of the nocturnal melatonin rise increased by 2 hoursafter a change from 15L:9D to 12L:12D, and births occurred 32 + 0.4days later (mean + s.e.m.) (4). -To test this~ twelve tammars were injected subcutaneously witheither melatonin (Sigma - 400ng/kg, N=6) or the arachis oil vehicle(N=6), 2.5 hours before dark each day for 15 days whilst maintainedon 15L:9D. They were then exposed to 12.5L:ll.5D. The melatonininjections mimicked the endogenous melatonin rise of 12L:12D. Themelatonin injected group gave birth 32 + 0.8 days after the start ofinjections, which was significantly different (P < 0.005) from theinterval in the control group of 46.0 ~ 1.1 days. This study showsthat exogenous melatonin given 2.5 hours in advance of the endogenousrise fully mimics the tammar's response to photoperiod change, butthe events that occur during the first 6 days after the commencementof melatonin treatment or photoperiod change remain unresolved.(1) Lincoln, G. (1983) Na~ure 302, 755.(2) Tyndale-Biscoe, C.H. (1980lIn "Endocrinology 1980", Cumming, I.A., Funder, J.W. and Mendelsohn, F.A.O. (Eds.), pp. 277-282.Australian Academy of Science, Canberra.(3) Renfree, M.B. et al. (1981) Nature 293, 138-139.(4) McConnell, S.J. (1983) Proc.Int.UniO;-Physiol. Sci. 15, 438.147ti

6CONCENTRATIONS OF OESTRADIOL IN PLASMA AND OVARIAN TISSU~THROUGHOUT PREGNANCY IN THE TAMMAR WALLABY (Macpopus eugenii)G. Shaw & M.B. RenfreeDept physiology & pharmacology, university of Qld, st Lucia, Qld, andDepartment of Anatomy, Monash University, Clayton, Victoria.Lactating tammars carry a diapausing blastocyst, which willreactivate if the young is removed prematurely. Metabolic reactivationof the embryo and uterus (1,2,3) apparently occur coincident with orprior to the first significant rise in plasma progesterone concentrationson dS (4) suggesting that another hormone may be involved. oestradiol isa likely candidate since injection of quiescent tammars with oestradiolinduced mitoses in the blastocysts (5). In this study oestradiolconcentrations were measured by RIA in peripheral blood samples from 10tammars after their pouch young were removed to terminate diapause.oestradiol concentrations rose from 8.3:1.2 pg/ml on days 3 and 4to a peak of 1S.8±2.9 pg/ml on dS, and then fell to 10.5:2.7 pg/ml on d7(Fig. 1). Five females mated post-partum and these all had elevatedoestradiol concentrations at this time, whilst no peak was seen infemales which did not mate (Fig. 1). parturition caused no apparentchange in oestradiol concentrations.Fig.Peripheral plasma oestradiol in tammars after RPYSince the corpus luteum may be a significant source of oestradiol in thetammar(6,7), ovarian tissue extracts were assayed after LH-20 chromatographY.Corporalutea contained 20-100pg!CL oestradiol during pregnancy.The highest ovarian oestradiol content (>1200 pg) was measured in wholeovaries containing Graafian follicles from full term pregnant females.The rise in oestradiol concentrations at dS may be important inthe termination of diapause, although, since it Occurs at the same timeas the progesterone peak but after uterine reactivation an earlier changein another hormone may be responsible. The peri-partum increase inplasma oestradiol concentrations coincides with oestrus. The source ofthis oestrogen appears to be the preovulatory follicle.(1) Moore GPM (1978) ~ Cell. Physiol.94, 99-107.(2) Thornber EJ, Renfree ME & Wallac~GI (1981)~ Emb. Exp. Morph. 62,325-38.(3) Shaw G & Renfree MB (1984) Abstract - these proceedings. ---(4) Hinds LA & Tyndale-Biscoe CH (1982) ~ Endocr. 93, 99-107.(5) Smith MJ & Sharman GB (1969) Aust.~ Biol. sci.:2:2, 171-180.(6) Evans SM, Tyndale-Biscoe CH & Sutherland RL Ci980) J. Endocr. 86,13-23.(7) Renfree MB, Wallace GI & Young IR (1982) ~ Endocr.~, 397-40~7ATTENUATED EMBRYOGENESIS AND UNILATERAL OVULATION IN THE CALIFORNIANLEAF-NOSED BAT MACROTUS CALIFORNICUS: A MORPHOMETRIC ANALYSIS OFOVARIAN DYNAMICSE.G.Crichton and P.H. KrutzschDepartments of Anatomy, University of Adelaide, S.A. and Universityof Arizona, Tucson, U.S.A.In Macrotus californicus (Phyllostomatidae), normal embryogenesis(March to June) is preceded by a period of delayeddevelopment (October to March) characterized by implantation and slowgrowth of the embryo to the primitive streak stage (1). A corpusluteum (CL) is present in the right ovary (RO); it enlarges attransition (March) when uterine proliferation is enhanced and ahemodichorial placenta forms (2). Plasma oestrogens (3), progesterone(4) and immunoreactive pituitary prolactin cells (5) aredepressed during delay.This study aimed to follow the dynamics of ovarian functionthroughout the annual reproductive cycle. A computerized analysis ofcounts of normal and atretic follicles in each growth stage andmeasurements of healthy multilaminar follicles (MLF) and CL in serialsections of both ovaries (n= 3 to 5 bats of estimated age of 1-3years/month) revealed that waves of follicular growth were initiatedin June and January. Increased MLF resulting from the first wave ofrecruitment appeared from August to October during which periodhealthy MLF also attained a greater size but atresia was high. Thesecond wave of growth resulted in increased percentages of healthyMLF in March. Vesiculation of follicles was seen bilaterally fromJuly to January though twice as many vesicular MLF were present inthe RO than in the left at ovulation (Oct./Nov.). Although thesingle Graafian follicle formed only in the RO, left ovarianovulation could be induced by right ovariectomy.The data suggest that in conjunction with reduced CL activity ahigh rate of atresia up to and following ovulation may bring about analteration in steroid levels appropriate to the provision of anadequate uterine environment for normal embryogenesis. The lattermight be attained at the termination of the second wave of follicular"rowth which results in an increase in healthy MLF in March and~herefore presumably an increase in oestrogens. The reason why theleft ovary is anovulatory is unclear since, apart from lower numbersof follicles, it appears to function normally.1. Bradshaw, G.V.R. Science 136: 645-646 (1962).2. Bodley, H.D. Anat. Rec. 180: 351-367 (1974).3. Burns, J.M. and Easley, R.G. Gen. Compo Endocrinol 32: 163­166 (1977).4. Burns, J.M. and Wallace, W.E. Gen. Compo Endocrinol 25: 529­533 (1975).5. Richardson, B.A. J. Reprod. Fert. 2i: 379-389 (1979).

89PERIPHERAL PLASMA PROGESTERONE AND OESTRADIOL-178 CONCENTRATIONSDURING THE BREEDING SEASON AND PREGNANCY IN THE GREY HEADEDFRUIT BAT (Pte~opus po~iocephalus)P.A. TOwers and L. MartinDepartment of physiology and pharmacology,university of QUeensland, St. Lucia 4067, Australia.P. poliocephalus is a seasonal breeder and is thought to be areflex ovulator. Females give birth to a single young in OCtoberafter 6 months gestation. plasma samples were collected via cardiacpuncture, throughout the year, from captives and bats shot in thewild. Progesterone and oestradiol-17S concentrations were measured byRIA. Reproductive tracts were fixed and examined histologically.In non-pregant females, progesterone levels showed little changethrough the year (13.3:2.1* ng/ml, n=30). oestradiol levels alsosh;wed little change (6.9±1.1 pg/ml, n=36), even at the height of thebreeding season. However few samples have been collected from thisperiod, and most ovaries remain to be sectioned. Thus bats with largepreovulatory follicles may have been missed. Pte~ovid ovaries areencapSUlated, follicles and the co~pus luteum (CL) ~re not visibleexternally.During the first 3 months of pregnancy, foetal growth is slow andplasma progesterone levels increase little above those of non-pregnantbats (May/June pregnant, 19.6:2.9 ng/ml, n=9). At this stage theovary adjacent to the gravid uterine horn contains a single largeapparently healthy, secretory CL. After June foetal growth is rapidand plasma progesterone levels rise to high values at the end ofpregnancy (Sept/oct, 298±64 ng/ml, n=13). oestradiol levels showcomparable increases (June pregnant, 15.5±5.8 pg/ml, n=6; sept/OCtpregnant, 184±35 pg/ml, n=6). Over the second half of pregnancy, theCL does not enlarge, but shows increasing connective tissue/leuocycteinfiltration. No antral follicles are seen in the ovary with the CL;contralaterally, small antral follicles are seen throughoutpregnancy. It is likely that in the second half of pregnancy theplacenta is the major source of progesterone (and probably also ofoestradiol); preliminary results indicate placental progesteronelevels in late pregnancy of 5.2±1.1 ~g/g, n=10).The failure to detect large differences in progesterone levelsbetween non-pregnant bats without CLs, and 2 month-pregnant bats withapparently healthy functional CLS, is intriguing. In Pte~ovidspreimplantation progestational development of the endometri~ isrestricted to the uterine horn ipsilateral to the CL and to that partimmediately beside the CL (1). It seems likely that pregnancy isestablished with minimal secretion of progesterone from the CL, embryoimplantation/early development requiring high levels only locally, andperipheral values remaining unchanged.* mean ± SEM for all values(1) Marshall, A.J. (1953) J. Endocrinol. 9: 42-44.ONE BAT: FOUR ENDOMETRIALen Martin and P.A.TOwersDepartment of physiology & pharmacologyUniversity of QUeensland, St. Lucia 4067, QUeensland.The bicornuate rodent uterus responds to systemic sex steroids asa unit; endometrial proliferative responses to oestrogens andprogestins vary widely from anima~ to animal but in anI indiv~d';lalboth uterine horns show near-ident~cal responses [e.g. [ H]-thym:l.d:l.nelabelling indices]. Attempts to demonstrate local effects of ovariansteroids by hemiovariectomy, give negative or equivocal results.In contrast, fruit bats (genus Pte~opus) have bicornuate uteriwhich respond locally to hormones from the ipsilateral ovary.Endometrial progestation, limited to the horn ipsilateral to thesingle C07"pUS Zuteum (CL) of early pregnancy, was first described inthe Indian Fruit Bat, P. giganteus by Marshall (1). we now show thatin Australian Pte~opids:-(a) the endometrium responds locally to the ipsilateral prevulatoryfollicle by glandular epithelial hypertrophy and hyperplasia(b) endometrial r~sponses vary with increasing distance from theipsilateral follicle/CL.The paradigm of variation in endometrial function within oneanimal is a P.alecto treated with gonadotrophins (25 iou. PMS; 25Lu., hCG) to induce ovulation. One ovary contained a large healthyeL, the other, 3 large antral follicles with proliferating granulosacells. The endometrium immediately proximal to the CL was clearlyprogestational with stromal nuclear hypertrophy/mitoses, plusepithelial nuclear hypotrophy/cytoplasmic eosinophilia (2). Thedistal ipsilateral endometrium was atrophic; the intermediate regionshowed massive cell death. The contralateral endometrium immediatelyproximal to the growing follicles was non-progestational and showeddistinct signs of oestrogenic stimulation [epithelial nuclearhypertrophy/cytoplasmic basophilia plus stromal oedoema (2)]. Thedistal region of this endometrium was atrophic.In another gonadotrophin treated bat with luteal tissue in bothovaries, both uterine horns showed patterns of development asdescribed above for the horn ipsilateral to the CL; i.e. proximalprogestation, distal atrophy plus intermediate regions of cell death.The mechanisms producing such diversity of endometrial responsewithin one bat remain unknown. Experiments with [3H]-oestradriol-1713applied directly to the uterus indicate that concentration andaffinity of oestrogen receptors may differ in the endometria proximalto and distal from the ovary. It also seems likely that a specialovarian-uterine circulation is involved.(1) Marshall, A.J. (1953) J. Endocrinol. 9, 42-44(2) Martin, L., Finn, C.A. and Trinder, G. (1973). J. Endocr.2.§.,133-144, 303-307.

10HORMONAL CONTROL OF PARTURITION IN THE VIVIPAROUS LIZARD, TILIQUA RUGOSAB. Fergusson and S.D. BradshawZoology Department, University of Western Australia, Nedlands, W.A. 6009The viviparous lizard TiZiqua rugosa breeds in October/November andfertilized females carry developing embryos in their uteri through thesummer until they are born fully-developed in late March or early April.A:ginine vasotocin has both antidiuretic and uterotonic activity in rept~les(1). It is of interest to determine how pregnant female T.rugosa maintain gestation through the summer when the concentration ofAVT in blood plasma should be relatively high in order to maintain waterbalance.Measurements of total body water content and plasma osmotic pressureover two seasons indicate that T. rugosa experience chronic dehydrationas Summer progresses and plasma levels of AVT are significantlyhigher in late summer (January-March, i ± SE = 17.6 ± 1.7 pg/ml) than inwater-loaded individuals (8.4 ± 0.8 pg/ml). Pregnant females in Marchwere found to be more dehydrated than non-pregnant females and males.This could be interpreted as (a) a facultative dehydration, which wouldcause elevation of AVT levels in pregnant females in preparation forparturition, or (b) a consequence of suppression of AVT secretion inorder to prevent premature abortion. However, neither of these argumentsis supported by measurements of plasma AVT which show no significantdifference between pregnant females and other T. rugosa.Plasma progesterone concentrations are elevated in pregnant females(323 ± 55 pg/ml) compared with non-pregnant females (27 ± 8 pg/ml) andmales (22 ± 6 pg/ml). The levels in pregnant females decline to aa100 pg/ml about 10 days prior to parturition. Pre-treatment of ovariectomizednon-pregnant females with injections of both progesterone and17B-oestradiol almost entirely inhibits AVT-induced in vitro contractionsof uterine tissue. However, the sensitivity of the uterus to AVTis not reduced in pregnant females when compared with non-pregnantfemales; and ovariectomy also had no effect on sensitivity. Spontaneouscontractions are only present in pregnant females.This research indicates that the dual role played by AVT in thisviviparous species, i.e., that of an antidiuretic as well as a parturienthormone, is accommodated by a tonic secretion of AVT which appearsessential for the maintenance of water balance, associated with a withdrawalof progesterone secretion by the ovary/placenta (?) just prior toparturition. Other factors than progesterone alone, however, appear todetermine uterine sensitivity to AVT.(1) Bentley, P.J. (1974). In "Handbook of Physiology", Section 7:Endocrinology, Vol. IV,-Part 1, pp. 545-563. Am. Physiol. Soc.,Washington, D.C.11CHARACTERIZATION OF TWO DIFFERENT ANDROGEN-BINDING SITES WITHINTHE OVINE OVARIAN FOLLICLEStella Campo,Ronald Carson and Jock FindlayMedical Research Centre, Prince Henry's Hospital, Melbourne 3004.Androgens can have both positive and negative effects on folliculargrowth and preliminary studies (1) demonstrated the presence.ofandrogen binding sites in several follicular compartments: In.thlsstudy, two specific androgen bin~ing sit:s were.char~cterlsed ln theovine follicle using different llgands, lncubatlon tlmes, and charcoalseparaCion steps (3HDHT and 3HT, 15 min, 20 s; 3HR1881 18h, 10 min).The first site with characteristics very similar to testicglar ABP interms of its capacity, affinity, association (15 mins at 4 C) anddissociation races (t range = 2 to 30 mins) and specificity fornatural and synthetic~androgens, was found in serum, follicular fluidand the 27000 xg particulate and cytosol fractions of granulosa cells(Tab Ie 1).TABLE l. The characteristics of androgen-binding sites in sheepserum and ovarian follicle fractions determined by Scatchard analysis.Fraction Tracer No. Androgen Bindingof *k **Capacitydobs.xl0-9M fmole/mg proteinSerum DHT 2 0.91 (0.9-1.09) 571 ± 98T 2 2.78 (5.05-1.54) 626 ± 92Follicular DHT 3 0.96 (1.09-0.86) 507 ± 189Fluid T 3 1.71 (0.94-3.12) 499 ± 106Granulosa Cell DHT 2 1.50 (1.80-1.30) 316 ± 31Particulate T 1.33 (1.17-1.58) 471 ± 25Granulosa Cell DHT 3 3.28 (2.7-4.0) 1032 ± 57CytosolT*Geometric mean (range), ** Arithmetic mean ± s.d.The second, classic androgen receptor was found in the cytosolwith high affinity and low capacity for the synthetic androgen R l881:k 2.8 (2.7 - 3.0) 10-9M (geometric mean, range); 81 ± 4 fmoles{mgprgtein (mean ± s.d.); and a very slow steroid-protein rate of dissociation: t = 24h. The presence of the ABP-like component in granulosacell cytos~l was confirmed from Scatchard analysis, dissociationrates, and specificity studies in presence of the androgen receptorpre-incubated with 100 nM non-labelled R • 1881The complex actions which androgens exert on granulosa cellfunction may be mediated by interactions in vivo between these extraand intra-cellular specific androgen binding sites.(1) Campo, S., Carson, R., and Findlay, J.K. Proe. Aust. Soc.Reprod. BioI. 15: Abstr. 27 (1983).

12INTERSTITIAL SPACE AND TRANSPORT IN RAT CORPORA LUTEAA.M. Dharmarajan, N.W. Bruce and G.T. MeyerDepartment of Anatomy and Human Biology,The University of Western Australia, Nedlands 6009.The corpus luteum (CL) of the rat has a high rate of blood flow,an extensive surface area of fenestrated capillaries and numeroussurface projections on the luteal cell membranes (1). These featuressuggest a well developed and complex set of transport mechanisms. Alltransport must proceed across the interstitial space but thesignificance of this element has not been considered. Qualitativereports suggest that it is extensive in the corpus luteum (2) butquantitative accounts are not available and it is not known whether itcorrelates with rate of secretory activity. Here we presentquantitative data on its volume in rats at day 16 and 22 of gestation:periods of high and low rates of progesterone secretion respectively.Eight rats at each stage were anaesthetized with ether,heparinized and one ovary from each rat was fixed by close perfusionat approximately physiological pressure and flow rate. Three CL fromeach ovary were blocked in araldite and examined at the lightmicroscopic (LM) level. In addition two CL from each ovary of Day 16pregnant rats were cut into small sections, blocked in araldite andexamined at the electron microscopic (EM) level and analysed withstandard morphometric techniques (3).At the LM level the percentage of CL occupiedspace was calculated to be 7.1 ± 0.4% (mean ± SEM)0.3% at Day 22: the 37% difference was significanttest). At the EM level, interstitial space at Day0.3% of the CL which was less than that estimatedpresumably due to fluid loss on sectioning.magnification, however, it was possiblecompartments in the space, that between adjacent0.1% of the' total CLl and that between luteal(3.7 ± 0.4%).by the interstitialat Day 16 and 4.4 ±(P(O.Ol, unpaired t16 occupied 5.2 ±at the LM level andWith the higherto distinguish twoluteal cells (1.5 ±cells and capillariesThese results quantitatively confirm that the interstitial spaceis extensive in the rat CL and suggest that it depends on secretoryactivity. An extensive space increases diffusion distance betweenblood and luteal cell cytoplasm but preliminary data we have on rateof lymph drainage from the rat CL suggests a volume turnover of aboutfive times a minute. Whether percolations of fluid within the spaceassists transport is yet to be determined.(1) Dharmarajan, A.M., Bruce, N.W and Meyer, G.T. (1984). Aust. Conf.Electron Microsc. 8: 24(2) Enders, A.C. (1973). BioI Reprod~: 158(3) Weibel, E.R. (1979). Stereological Methods. Vol. 1. PracticalMethods For Biological Morphometry. London: Academic Press.COMPARISON OF OVARIAN ~ENDORPHIN-LIKE IMMUNOREACTIVITYIN IMMATURE AND ADULT SPRAGUE-DAWLEY RATSS.l. Lolait, D.l. Autelitano, A.I. Smith, A.T. LimB.H. Toh* and l.W. FunderMedical Research Centre, Prince Henry's Hospital and *MonashUniversity Dept. Pathology and Immunology, Alfred Hospital,. Melbourne, Victoria.In the brain and pituitary, adrenocorticotropic hormone andB-endorphin (6EP) are derived from a common precursor, pro-opiomelanocortin(POMC). Recently, POMC-derived peptides have been localized inperipheral tissues (1). In studies from our own l~bo:atory, we ~aveshown that ovaries from both the ewe and rat contaln lmmunoreactlve(ir)-BEP, and that ovarian ir-6EP levels appear related to theoestrous cycle (1,2).In the present study, we have determined the ovarian localizationof BEP by immunocytochemistry, and the levels of ovarian eEP by radioimmunoassay(RIA) (2), in immature (18-21 day) and four-day cyclingadult Sprague-Dawley rats. The molecular species of ir-eEP presentwere determined by subjecting ovarian extracts to Sephadex GSO chromatographyand RP-HPLC; eluate fractions were assayed for eEP by RIA.In immature rats pre-antral, and to a lesser extent, antralfollicular granulosa cells contain ir-eEP whereas in adult rats thepredominant localization is in the corpora lutea. Levels of ovarianir-BEP in immature rats are ~four to six-fold lower than those foundin adult rats (e.g. immature 1.2 ± 0.12 ng/gm (mean ± SEM, n=12)compared with pro-oestrous adult 8.4 ± 0.96 ng/gm (mean ± SEM, n=14)).We have also shown that in ovarian extracts from both immature andadult rats there are immunoreactive species co-chromatographing withBEP, e-lipotropin and POMC. The ir-eEP profile patterns differ,however, with BEP and the putative high M precursor as the majorimmunoreactive components in immature andradult rat ovarian extracts,respectively. In addition, the ir-BEP peak in immature rats isbroader than the equivalent fraction in adult rat ovarian extracts,suggesting the possibility of multiple forms of ir-eEP of similar butnot identical M. RP-HPLC of this peak from a Sephadex GSO columnindicated that BEP _ was the major immunoreactive speci~s pres~nt.l 31The differences In ovarian levels, and chromatographlc profllesof ir-BEP between immature and adult rats suggests that there arebasic changes in ovarian POMC expression and processing duringdevelopment, perhaps reflecting the activity of circulating gonadotropinsand/or sex steroids. The low levels of ir-BEP in the ovarycompared with those in the pituitary (~2-3 fold less) suggests thepossibility of a paracrine rather than endocrine role in ovarianfunction.(1) Lolait, S.J., Autelitano, DoJ., Lim, A.T., Toh, B.H., and FunderJ.W. Proc. Opioid Peptides in Periphery, Rome 1984 (in press).(2) Lim, A.To, Lolait, S., Barlow JoW., 0, W.S., Zois, I., Toh, B.H.,and Funder, l.W. Nature 303, 709-711 (1983).13

u14MONOCIDNAL ANl'1BODIES 'ID PIG RELAXINJ. Patterson, J.D. O'Shea, C.S. Lee and M.R. BrandonDepartment of Veterinary Preclinical Sciences,University of Melbourne, Parkville, Victoria, 3052.Polyclonal antisera to relaxin of varying specificity havebeen widely used in research for immunohistochemical localization,assays and receptor studies on relaxin in numerous species includingthe pig. A recently published method of purification has allowed thepreparation of relaxin of high purity (1) and has provided anopportunity to use this material to prepare monoclonal antibodies.The preparation of monoclonal antibodies to porcine relaxin, thelrvalidation and use in the localization of relaxin in the corpus luteumof the pig by immunohistochemistry is the subject of this report.Using relaxin supplied by Dr. W.A. Charnley (prepared by theacetone!HCl technique) Balb/C mice were immunized and subsequentlytheir spleen cells fused with NS-l myeloma cells to produce hybridomassecreting antibodies to pig relaxin (2). Screening of hybridomasupernatants revealed six cell lines producing antibodies whichreacted with the original immunogen (crude pig relaxin), partiallypurified human placental relaxin and purified pig relaxin (1).After purification from ascites fluid (3) one monoclonalantibody was conjugated with F1TC and used for directimmunofluorescent localization of relaxin in pig tissues. Otherstudies utilized a HRP labelled second antibody. Uterus, placenta,liver, pancreas and corpus luteum from ten pregnant sows from 30 daysto 112 days gestation were examined along with ovarian and uterinetissues from three non-pregnant sows and testicular tissue from oneadult boar. Other tissues from pregnant guinea pigs, rats, sheep andcows expected to contain relaxin were examined for cross-reactivity.Only the corpus luteum of the pregnant sow showed a positivereaction. The reaction product was present mainly in the large lutealcells. SUbjectively, the intensity of the staining increased from day30 to 110 of gestation. By day 112 (i.e. within approximately 48 h ofterm) the intensity had declined. With HRP staining the positivematerial was granular in its distribution. The monoclonal antibodieswere shown to be specific to relaxin by immuno-Western blotting. Inaddition they have been used to purify biologically active relaxin bymonoclonal antibody affinity chromatography (4).(1) Walsh, J.R. and Niall, H.D. Endocrinology, 107: 1258-1260 (1980).(2) Patterson, J. PhD Thesis, University of Melbourne (1983).(3) Hsuing, Li-min, Barclay, A.N., Brandon, M.R., Siro, E. andPorter, R. Biochem.J. 203: 293-298 (1982).(4) Patterson, J. and Brandon, M.R. Proc. Endocrine Society of Aust.(1984) .FENPROSTALENE AS15A LUTEOLYSIN IN CATTLES.R. McPhee. P.A. Whylie, I.G. Pe~rson*, L.D. Staples, I.F. DavisT.J. Squires, 1.J. Clarket and L.P. CahillDepartment of Agriculture, Animal Research Institute, Werribee*Syntex Agribusiness, Pennant Hills, Sydney andtMedical Research Centre, Prince Henry's Hospital, Melbourne.A new analogue of prostaglandin F2a, fenprostalene (FP, Syntex)has been registered for use in Australia. Field studies (1) found thatthe efficacy of FP was variable when injected s.c. into the caudal fold.Experiment I. (Batch comparison) The luteolytic effectiveness offour different commercial batches of FP, one of which was used in (I),were compared with cloprostenol (CP, r.c.r.) and a placebo in 36 heifersand 40 parous cows. The animals were observed for oestrus twice dailyfor two cycles. On Day 12 of the third cycle each animal was injecteds.c. at time 0 h in the neck with the recommended dose rates of 1 mg ofFP, 0.5 mg of CP or 2 ml of placebo. Luteolysis was assessed by determinationof progesterone concentration in jugular venous blood collectedat -2, 0, 6, 12, 24, 48, 72, and96 h. Animals were also observed twicedaily for subsequent oestrus.Animals in all groups except the control group showed oestrusbetween 3 and 4 days after the injection. Progesterone levels 2 daysafter PG injections were < 17% of pre-injection levels for all ani~~~~except those in the control group. These results demonstrated that-CP-­and all batches of FP were equally luteolytic in both heifers and cowswheninjected s.c. in the neck.Experiment 2. (Injection site comparison) Three groups each offive parous cows were injected s.c. with 1 mg of one batch of FP onDay 12 of the cycle into the caudal fold. escutcheon or the neck.Blood sampling and oestrus detection were conducted as before. Theonset of oestrus pattern was satiSfactory only after injections to theneck (Table I).TABLE I. No. of cows in oestrus after FP injection at different sites.Injection Days after FP injectionSite n 4 5 6 7 8 >8Caudal fold 5 1 1 3Escutcheon 5 1 2 INeck 5 4 1At 72 h after injection, mean progesterone levels for cows injectedin the caudal fold, escutcheon or neck were 42, 19 and 4% respectivelyof mean pre-injection levels.The results indicate that luteolysis occurs at a similar time afters.c. injection in the neck with either cloprostenol or fenprostalene butthat site of injection may alter the efficacy of FP as a luteolysin inmature cows.(I) Davis, I.F., McPhee, S.R. and Clarke, I.J. Proc. Aust. Soc.Anim. Prod. 15: 313 (1984).P.A.W. was supported by the Australian Development Assistance Bureau.The project was funded in part by Syntex Agribusiness.

16INCREASED FSH IN MERINO EWES IMMUNIZED WITH AN INHIBIN-ENRICHED FRACTIONFROM BOVINE FOLLICULAR FLUIDT. O'Shea, S.A.R. Al-Obaidi, B.M. Bindon*, M.A. Hillard and J.K. Findlay+Department of Physiology, University of New England, Armidale, N.S.W.,*C.S.I.R.O., Division of Animal Production, Armidale, N.S.W. and ~1edicalResearch Centre, Prince Henry's Hospital, Melbourne, VIC.The immunogen involved in the increased ovulation race seen whenewes were immunized (1) with an inhibin-enriched fraction from bovinefollicular fluid (bFF, Peak 2) has not been accurately characterized.The control (C) and immunized (I) ewes described previously (1)were reimmunized (bFF, Peak 2 or b.s.a.) and synchronized with theprostaglandin analogue Cloprostenol. After the synchronized oestrus,6 x 1 hr plasma samples were obtained from 0900 hr, on days 8, 12, 14of the oestrous cycle and on day 2 of the subsequent cycle. Oestruswas recorded and progesterone was measured at day 14. FSH was determinedusing the NIAMDD oFSH RIA system.The I ewes had significantly elevated (P < 0.01) FSH (Table 1) ondays 14 and 2 (subsequent cycle).TABLE 1: Plasma FSH in Control (C) and Immunized (I) ewes during theoestrous cycle. Values are the daily means ± S.E. for 11 ewes expressedas In ng/ml; NIAMDD-oFSH-RPl.DAY 8 12 14 2C ewesI ewes0.71 ± 0.110.90 ± 0.130.74 ± 0.100.97 :!: 0.120.671.11± 0.08± 0.110.69 :!: 0.081.03 ± 0.08FSH at days 12, 14 and 2 was significantly correlated with theovulation rate (O.R.) determined immediately after day 2 (Table 2).FSH at days 14 and 2 was significantly correlated with the mean O.R.as determined in the previous months. There was no correlation withO.R. of the next cycle.TABLE 2: Correlation coefficient of FSH (In ng/ml) with ovulation·rate (n = 21-22).DAY 8 12 14Experimental cycle 0.382 0.483* 0.485* 0.458*Subsequent cycle 0.315 0.177 0.344 0.383Mean from previous 4laparoscopies 0.215 0.305 0.541** 0.462**p < 0.05 **p < 0.01These results were what would be expected if inhibin is theimmunogen. The same plasma samples are being assayed for FSH in aheterologous RIA in another laboratory. FSH is also being measuredin late luteal phase in a different group of C and I animals.(1) O'Shea, T., Cummins, L.J., Bindon, B.M. and Findlay, J.K. Proc.Aust. Soc. Reprod. BioI. 14: 85 (1982).17DEPRESSION OF FSH IN OVARIECTOMIZED EWES BY BOVINE FOLLICULAR FLUID:NEUTRALIZATION BY SERUM FRO~1 EWES IMMUNIZED AGAINST PARTIALLY PURIFIEDBOVINE INHIBINS.A.R. Al-Obaidi, B.M.Bindon*, M.A. Hillard, T. O'Shea and L.R. Piper*Department of Physiology, University of New England, Armidale, N.S.W.and *C.S.I.R.O., Division of Animal Production, Armidale, N.S.W.Ewes immunized against an inhibin-enriched fraction of bovine follicularfluid (bFF) show increased ovulation rates (1). The serum of suchewes contains precipitating antibodies against the antigen used forimmunization (1). The present report presents further evidence that thisserum is able to neutralize the FSH depressing effects of bFF in ewes.Merino ewes previously immunized against the bFF fraction (I ewes)or bovine serum albumin tc ewes) were ovariectomized two years afterprimary immunization. Eight months later 8 ewes of each type were injectedsubcutaneously four time during two days with 4 ml of the bFF fractioncontaining 2191 u inhibin/ml. FSH was measured by NIAMDD-oFSH-RIA oncomposite samples drawn at 4 x 2 h intervals just preceding and for 5followir.g the first injection of the bFF fraction. The results in Table 1show that FSH was significantly depressed in the C ewes (day 1: 16.5 ±1.8 ng/ml) but unaltered in the I ewes (day 1; 18.2 ± 1.9 ng/ml).TABLE 1: Mean ± S. E. daily incremental changes from day 1 of plasma FSH(ng/ml: NIAMDD-oFSH-RP1) in ovariectomized control (C) and immunized (I)ewes injected with an inhibin-enriched fraction of bFF.GROUP DAY 2346C (n=8)I (n=8)-1.8 :!: 0.50.3 ± 0.4-6.9 :!: 0.8-1.3 :t 0.3-6.3 ± 0.8-1.1 ± 0.5-2.7-0.2:!: 0.7:t 0.4-1.1 :!: 0.6-0.3 ± 0.7Long-term ovariectomized Merinos were also injected four times overtwo days with 2 ml bFF containing 1252 u inhibin per mI. At the times ofbFF inj ection the ewes were treated as shown in Table 2 with pooled charcoal-dextrantreated serum from the C and I ewes described above. FSHwas again measured in 4 x 2 hr plasma samples taken just before and for5 days after the first bFF injections. (Mean FSH at day 1 for the threegroups were 26.5 ± 2.9; 18.1 ± 2.9; 21.5 ± 2.9 ng/ml).TABLE 2: Mean ± S.E. daily incremental changes from day 1 of plasma FSH(ng/ml: NIAMDD-oFSH-RPI) in ewes treated with 2 ml bFF and either control(C) or bFF-immune (I) serum.TREATMENT n DAY 2 3 4 5 6bFF+20 ml C serum 7-10.6:!:1.7 -13.2±2.4 -11.1±2.2 -6.3 ± 1.3 -4.3 ± 1. 5bFF+10 ml I serum 7-6.4:t1.1 - 7.7:t1.5 -4.3:t1.4 -2.9±0.7 -2.4±1.4bFF+20 ml I serum 8- 5.1:!:0.8 - 5.1±0.5 -4.2±0.8 -3.9:!:0.8 -2.8 ± 0.8The degree of depression of FSH by bFF was significantly (P < 0.02)reduced by treatment with 20 ml (I) serum. These two experiments provideevidence that serum of bFF-immunized ewes contains substances, presumablyantibodies, that neutralize the FSH-depressing action of bFF in vivo.1. O'Shea, T., Cummins, L.J., Bindon, B.M. and Findlay, J.K. Froc.Aust. Soc. Reprod. BioI. 14: 85 (1982).

(1)(3)(4)I.J. Clarke,18IN VIVO EVIDENCE THAT THE PITUITARY GLAND IS THE MAIN------ SITE FOR THE ACTION OF INHIBINJ.K. Findlay and J.T. CumminsMedical Research Centre, Prince Henry's Hospital, Melbourne 3004.To regulate gonadotrophin secretion, inhibin could act at thehypothalamus, to inhibit GnRH secretion, or at the pituitary level.The aim of this study was to determine the site of action of inhibinin vivo. Our inhibin source was ovine follicular fluid (oFF) that hadbeen stripped with charcoal, had a potency of 22 KU/ml as determinedby the method of Lee et al (1) and contained 1.14 nmol/l oestradiol­178. Two experiments were performed in ovariectomized (OVX) ewes.Expt. 1: Three OVX ewes were given s.c. injections of 2 ml oFF on theevening of day 1 and then b.i.d. on days 2. 3 and 4 of the. experiment.Three OVX control ewes were given 2 ml ch2rcoal treated hypophysectomizedsheep serum. Blood samples were collected from these sheep ondays 1 and 4 at 10 min intervals for 6 h. Expt. 2: Hypothalamopituitarydisconnection (HPD) (2) was performed on 3 OVX ewes that thenreceived GnRH replacement (250 ng pulses i.v. every 2 h); after 1 weekthese sheep were then treated with oFF (as above) and jugular venoussamples were collected over 3 GnRH pulses on days 1 to 4 of theexperiment. There is no daily variation in LH and FSH responses in OVXHPD, GnRH pulsed ewes (3).In experiment 1, oFF caused plasma FSH levels to fall from 43±2ng/ml (mean ± SEM) on day 1 to

20CONTROL OF INHIBIN SECRETION BY RAT GRANULOSA CELLS IN VITRO----V.W.K. Lee, N. Colvin, J. McMaster, and H. QuiggMedical Research Centre, Prince Henry's Hospital, Melbourne, 3004.Pregnant mare serum gonadotrophin (PMSG) treatment of immaturefemale rats leads to elevated levels of inhibin (INB) in the ovary andperipheral blood (1). Ovariectomy in PMSG-primed rats rapidly reducedcirculatory levels of INB indicating that the ovaries, in particularthe granulosa cells, are the source of blood INB. This study examinesrat granulosa cells in culture with respect to stimulation of INBsecretion by PMSG and the effect of gonadal steroids on the PMSGstimulatedrelease of INB.Immature female rats (D22-30) were implanted s.c. with stilboestrol(10 mg tablet, Hamilton Labs., Adelaide) and 3 days later theovaries were removed and placed in McCoy's Sa medium containing 6 roMethylene bis(oxyethylenenitrile) tetraacetic acid (EGTA). Granulosacells were obtained by puncturing the follicles and subsequent centrifug~tion.The cells were washed 3 times and resuspended in McCoy's Sa6med1um. Granulosa cells (1-1.6xl0 viable cells) in a total volume of1.8 ml medium were incubated for 24 or 48 h periods. The media wereremoved, extracted with charcoal (1 mg/ml) and assayed for inhibinbioactivity (2).E....::lio~lit)GO-\0:::0oI"STEflOIO(lO"Ml ­PMSG (mIU!ml)c"-P-;?,!,,;

22THE EFFECT OF PRESENCE OF FEMALES ON SEASONAL TESTOSTERONECONCENTRATIONS IN MALE KOWARIS, DASYUROIDES BYRNEI.T.P. FletcherZoology Dept., La Trobe University, Bundoora, Viet. 3083. Present Address,CSIRO, Division of Wildlife and Rangelands Research, A.C.T. 2602The presence of females is known to effect testosterone (T) levelsin many eutherian species (1) and in one marsupial species, Macropuseugenii (2). Dasyuroides byrnei, a small carnivorous marsupial, isa seasonal breeder like the tammar and an effect of the presence offemales on males might be expected.Five adult male kowaris were caged with females and four werecaged alone in a constant temperature room under the natural Melbournephotoregimen. From January to December 1981, the males were weighed,~crotal width measured, urine checked for presence of sperm and ablood sample taken for T determination by radioimmunoassay (RIA). Thefemales were also weighed, pouches checked o.:d any newborn youngremoved, urine examined for presence of cornified epithelial cells anda blood sample taken for progesterone (F) determination by RIA.The oestrus cycle of the kowari is of 60 days duration and theremay be up to 3 periods of oestrous in May/June, July/August andSeptember/October. The P profile of the oestrous cycle is bimodal witha rise (3-4ng/ml) and fall in P concentration about 7 days prior tooestrus. After oestrus there is another rise (10-12ng/ml) and fallassociated with the luteal phase.In males housed alone there was no significant seasonal change inT concentration which fluctuated but did not exceed 1.8ng/ml (Table 1).All males in this group showed sperm in the urine by April despite Tvalues being low. In males housed with females maximum mean T valuesof 3-4ng/ml measured in May and again in August and October weresignificantly higher (P < 0.5) than those measured in males housedalone but in the non-breeding season there was no significant difference.Sperm were present in samples by April in all males in this group.Table 1. Mean testosterone concentration (ng/ml + SD) in male kowarishoused alone (n=4) or with females (n=5).Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov DecMales alone 1.1 1.1 1.0 0.9 1.1 1.1 1.0 0.6 1.3 1.3 1.4 1.0+ 0.3 0.3 0.2 0.2 0.1 0.4 0.1 0.1 0.2 0.5 0.1 0.1Males with 1.2 1.1 1.1 1.2 2.6 1.9 2.2 3.1 1.8 2.0 0.9 1.2females 2:. 0.5 0.2 0.2 0.2 0.7 0.7 0.4 0.7 0.5 0.9 0.3 0.4The i~crease in T concentration occurred about the time of the preoestrous r~se and fall in P in the associated female. No significantd~fference in mean scrotal width and body weight was apparent betweene~ther group of males but scrotal width showed a seasonal variation(P < 0.5).Seasonal changes in scrotal width and sperm production in malekowaris is not correlated with T concentration but there is a correlationbetween onset of oestrus in the female and T values in the male. Thissuggests the male is responding to a cue from pre-oestrus females. The~ata indicate that T levels in males is influenced by progesterone levelslon the female.(1) Lincoln, G.A. (1981) The Testis. pp 255-302. Raven Press, New York.(2) Catling, P.C. and Sutherland, R.L. (1980). J. Endocr. 86: 25-33.23TESTICULAR REGRESSION AFTER THE BREEDING SEASON IN THE MALEFRUIT BAT, PTEROPUS POLIOCEPHALUS.M.A.McGuckin and A.W.BlackshawDepartment of Physiology and Pharmacology, University of Queensland,St Lucia, Qld., 4067.Testis size alters seasonally in Pteropus poliocephalus reaching amaximum weight in March (3.62 "!:- 0.11 g/kg) and being fully regressed inthe months August/September (1.81 ~ 0.15 g/kg).This study results from analysis of 5 urn tissue sections takenfrom testes sampled from wild adult male bats during the breeding (n=5)and non-breeding (n=5) seasons. Eight stages of spermatogenesis basedon cellular associations have been described in the fruit bat (1).All germ cells within two tubular cross-sections of each of the eightstages of spermatogenesis from each testis were counted. Cell countswere corrected by an adaptation of Abercrombie's formula (2).Peripheral plasma testosterone levels were determined by RIA.After the breeding season both the interstitial and seminiferoustissue of the fruit bat testis exhibit regression. During the breedingseason Leydig cells show a larger nucleus and more abundant cytoplasm,at which time peripheral plasma testosterone levels are very high(54.2 ~ 13.5 ng/ml), than after. the breeding season when the Leydigcells regress and peripheral testosterone levels are low (2.25 ~ 1.4ng/ml). Although spermatozoa are still produced by the fruit battestis after the breeding season an increased germ cell loss isobserved at this time (see Table 1). Increased cell loss appears toocc~r throughout the cycle but particularly auring the final spermatogonialdivision to form the primary spermatocytes and during meioticdivision to form round spermatids.TABLE 1: Germ cell loss in the fruit bat testis. Actual values aremean corrected cell counts per tubular cross-section. Expected valuesare predicted from the number of intermediate spermatogonia in stage 6.Cell Type Stage Breeding Non-breedingActual Expected Actual ExpectedI 6 8.25 ±. 0.60 8.25 5.36 ±. 0.38 5.36B 8 15.65 ~ 0.72 16.50 10.67 ~ 0.96 10.72P 3 27.81~1.47 33.00 14.17:!:-0.66 21.44RS 5 91.37 ~ 7.74 132.00 36.28 "!:- 3.68 85.76LS 6 73.92 + 4.22 132.00 26.81 + 3.79 85.76Key: I= intermediate spermatogonia, B= B spermatogonia, P= pachytenespermatocytes, RS= round spermatids, LS= elongated spermatids.All numerical values are means ±. S.E. 's.(1) McGuckin, M.A. and Blackshaw, A.W. (1983). Proc. Aust. Soc.Reprod. BioI. 15: 39.(2) Swierstra, E.E~and Foote, R.H. (1963). J. Reprod. Fert. 5: 309.

24THE AGE OF SEXUAL MATURITY IN THE FEMALE BANDICOOT ISOODON !~CROURUS:THE EFFECT OF SEASONR.T. GemmellDepartment of Anatomy, University of Queensland,Queensland.In the southern regions of Australia, the breeding season for thebandicoot commences in July and continues until February; there is anon-breeding period during the months April to June. In Queensland,the bandicoot breeds throughout the year, however there is a decreasein the breeding rate from March to June. In this study, the influenceof seasonality in breeding activity on the age at which the femalebandicoot gives birth to her first litter was examined.Juvenile bandicoots of known age were housed with adult males inan outside enclosure 900 sq. metres in area. The bandicoots weretrapped and examined weekly for the presence of young in the pouch.Female bandicoots produced their first litter between 161 and 356days of age and weighed between 680 and 1220 g. (n = 27). Thesebandicoots could be divided into two groups on the basis of their ageat sexual maturity. Animals born from February to August gave birthto their first litter at 201.8 ± 8.6 days of age and at a weight of798.5 ± 22.1 g (n = 14). Birth occurred in bandicoots born fromAugust to December at 260.5 ± 13.9 days of age, when they weighed964.6 ± 37.2 g (n = 13). (Animals born in August were found inboth groups).The litter size of the faster maturing animals was 2.57 ± 0.31young per litter, which was similar to that of the slower maturinganimals, 2.69 ± 0.34 young per litter.Bandicoots born early in the breeding season were capable ofproducing a litter in the same season. Similarly, sexual maturity wasnot delayed in bandicoots which were born late in the season and hadtheir early development during the months April to June. However,bandicoots born in the months August to December, which normally wouldreach sexual maturity in the months from April to June, delayed theirfirst litter until the start of the new season.Bandicoots which delay their sexual maturity, are greater inweight when they produce their first litter than the faster developinganimals. Therefore it is unlikely that a decrease in the rate ofgrowth and development during April to June prevents the onset ofsexual maturity. It is probable that the environmental cues whichcause the inhibition of breeding in the adult female, also affect thetransition of the juvenile female into adulthood.UTERINE AND EMBRYONIC REACTIVATIpN AFTER DIAPAUSE IN THE TAMMAR WALLABYTable 1 uptake and incorporation of [3H1Table 2 Endometrial leucine incorporation after RPY in tammars25G. Shaw & M.B. RenfreeDept Physiology & pharmacology, University of Qld, St Lucia, Qld, andDepartment of Anatomy, Monash university, Clayton, Victoria.During lactation tammars usually carry a diapausing embryo. Atd5 after removing the pouch young (RPY) to initiate embryonic developmentthere is a transient peak in peripheral plasma progesteroneconcentrations (1). The first visible sign of reactivation of theblastocyst is expansion on d8 (2). So embryonic reactivation may betriggered by progesterone or by a change in uterine secretions. In thisstudy in vit~o incorporation of [3Hl uridine by embryos and of [3 Hlleucine by endometrial explants were measured after reactivation. Tammarshave 2 separate uteri but only a single embryo so it is possible to makecomparisons between the gravid and adjacent non-gravid endometrium.Day 0 (quiescent) embryos had low uptake and incorporation ofuridine (into acid soluble and insoluble material respectively) (Table1). At d5 3/4 embryos had substantially higher uptake and incorporation,and this high activity continued to d9 when the embryos were expanding.uridine by tammar blastocystsStage N Blastocyst. [3H1 uridine (cpm/embryo)days RPY diam. (~m) Uptake Incorporationo 2 261,276 38,25 20,375 4 270±2 284±100 229±527 3 272±16 504±227 135±529 3 396±96 1310±176 250±48Incorporat~on of leucine by the endometrium into secreted protein(acid insoluble material from conditioned medium) and tissue protein wassignificantly higher at d4 than dO (Table 2). Incorporation increased inboth gravid and non-gravid uteri up to d12. At d26 gravid uteri incorporatedfar more leucine than the adjacent non-gravid uteri (Table 2).stage N Incorp. by tissue Incorp. secretiondays RPY(dpm/~g DNA) (dpm/~g DNA)oGravid Non-grav. Gravid Non-grav.3 167±4 124±11 1.24±0.28 1.18±O.1643 184±9 213±20 3.69±0.32 4.01±0.1163 238±36 268±25 4.70±0.12 8.25±3.0483 272±44 240±29 7.02±1.29 6.12±0.56122 331,397 285,325 10.3,10.2 6.83,8.75262 273,195 207,182 15.5,23.8 3.85,4.18These studies confirm previous observations (3,4) that tammarblastocysts reactivate metabolically before or about d5 RPY, and showthat uterine reactivation is measurable by d4. This apparently precedesthe first significant rise in plasma progesterone between d4 and d5 (1)suggesting that uterine (and hence embryonic) reactivation may betriggered by an earlier change in another hormone.(1) Hinds LA & Tyndale-Biscoe CH (1982) J. Endocr. 93, 99-107.(2) Tyndale-Biscoe CH (1979) Ciba Found. Symp. 64,11-73-185.(3) Thornber EJ, Renfree MB & Wallace GI (19811f~ Emb. Ex~ Morph.62,325-38.(4) Moore GPM (1978) ~ Cell. Physiol.~, 31-36. ---

26EFFECTS OF CARBON MONOXIDE ON THE PLACENTAL BARRIER OF THE RAT:A MORPHOMETRIC STUDY.N.W.BruceDepartment of Anatomy and Human Biology,The University of Western Australia, Nedlands, W.A. 6009.Babies of women who smoke during pregnancy are generally lighterat birth. Placental weight, however, is maintained or even increasedand so birth weight:placental weight ratios are substantially lower insmokers. The maintenance or increase in placental weight could be tocompensate for the reduction in oxygen carrying capacity of blood dueto the carbon monoxide (CO) content of cigarette smoke. If suchcompensation occurs, it might be more evident in the gas exchange areaof the placenta. This possibility was examined in pregnant rats sincetheir placenta is well suited to quantitative histological analysis.Eleven rats were mated and kept throughout pregnancy in a chamberprovided with air flow to which CO was added to a concentration of 100ppm. This level had been shown to generate carboxyhaemoglobin levelssimilar to that found in women smoking 20 to 40 cigarettes daily. Tencontrol rats were kept in a similar chamber provided with room airalone. Rats were anaesthetized on day 22 of gestation, the day beforeexpected parturition, their uterine contents were fixed by perfusionand their placentas removed and blocked in araldite. Sections, lumthick, were cut and examined by standard morphometric techniques (1).Carbon monoxide did not affect fetal survival and no grossmalformations were observed. Fetal weight in co treated rats was 10%lighter (P(O.OS, unpaired t test) than in the controls but placentalweight was 16% heavier (P

28STEROID HORMJNE LEVELS IN HIsrorROPH AND PLASMA. OF SOVl3THE PRE-IMPIANI'ATION PERIOD29OORINGENDOHETRIAL FUNCTION IN THE PREGNANT EWE:CHARACTERIZATION OF SECRETED PROTEINSG.M. Stone, X.Zhang and B.G. HillerDepartments of Veterinary Physiology and Animal Husbandry,University of Sydney, Sydney, NSW, 2006.Protein secretion by intercotyledonary endometrium increases dramaticallybetween days 20 and 100 of pregnancy (1). On days 84-112,fractionation on Sephadex G-200 of the proteins secreted in vitro bvendometrial explants in the presence of L-[4,5- J H] leucine-revealed· asingle dominant peak of 'pregnancy protein' (PP) with a molecular weight(~M) of about 60,000 (2). Proteins in this peak were fractionated onDEAE-cellulose, using a continuous NaCl gradient in 10 roM Tris-HC1, pH8.3, to remove albumin. This partially purified PP was then subjected tofurther characterization.Electrophoresis in 7.5% SDS-acrylamide gels with ~ markers yieldeda single peak of 3H at 60,000 daltons, indicating a lack of sub-unitstructure in PP. Density gradient centrifugation on 5-20% sucrose at150,000 g (ave) showed a major peak at S = 4.36, confirming that PP is aprotein or group of proteins with a MW of about 60,000. Aliquots ofpartially purified PP were incubated in the presence and absence ofpronase (from Streptomyces griseus). Comparisons of the behaviour oftreated and untreated samples upon subsequent electrophoresis in 1% agargels or density gradient centrifugation confirmed the proteinaceousnature of PP. The glycoprotein nature of PP was shown by its behaviouron ~f~inity chromatography on heparin-Sepharose. Samples of partiallypur~f~ed PP were eluted with a stepwise salt gradient. Whereas some 3 Hand essentially all of the material absorbing at 280 nm eluted with0.15 M NaCl, the majority of 3H eluted with 0.4 M NaCl.In order to harvest more PP in vivo we have collected the luminalfluid (uterine milk) from the non-pregnant uterine horn (NPH) of unilaterallypregnant ewes. A ligature of umbilical tape was placed aroundthe horn contralateral to the corpus luteum at 5 days after joining (3).From 4 ewes killed on days 91-110 of pregnancy (fetal weight = 880 ±257 g, mean ± SE), 135 ± 19 ml of uterine milk containing 2.15 ± 0.10 gof protein were recovered. Rates of PP secretion in vitro (2) fromendometrial explants from NPH and PH were contrasted.~results (NPH95 ± 27 3H dpm/~g DNA; PH, 112 ± 5 ~ dpm/~g DNA) suggest that PP secr~tionis regulated systemically, presumably by progesterone (2). Fractionationsof uterine milk spiked with 3H_PP on Sephadex G-200 andheparin-Sepharose suggest that PP comprises a significant portion of theprotein in uterine milk. Biochemical and immunological studies to furthercharacterize secreted endometrial proteins are in progress.(1) Miller, B.G., Tassell, R. and Stone, G.M. (1983). J. Endocr. 96:137-146.(2) Miller, B.G., Tassell, R. and Stone, G.M. (1983). Proc. Aust. Soc.Reprod. Biol. 15: EII-E12.(3) Bazer, F.W., Roberts, R.M., Basha, S.M.M., Zavy, M.T., Caton, D.and Barron, D.H. (1979). J. Anim. Sci. 49: 1522-1527.B.A. stone and R.F. SearrarkDepartrrent of Obstetrics and Gynaecology, University of Adelaide,The Queen Elizabeth Hospital, W:Jodville, South Australia, 5001.Bet~en days 8 and l5post-coiturn, viable pig blastocysts transformfran spheres to filarrents. As those blastocysts which fail to elongatewill not inplant successfully, it is proposed that failure by sorreblastocysts to elongate accounts for a proportion of the high level ofembryonic loss which occurs irmediately prior to inplantation in thepig. As part of a study on identification of potential blastocystgrowth stinlUlants in uterine fluid, ~ have detennined levels ofsteroids in histotroph and in plasrra of mated (pregnant) and of unmatedsows betv.een days 8 and 15 post-oestrus (p.o.).Histotroph was recovered fran uteri :intrediately after slaughter ofthe an:i.m3.ls, by flushing each uterine horn with 10 ml normal saline.Steroid levels in plasma and in uterine washings ~re assayed usingvalidated RIA procedures, after chranatography of extracts on Lipidex.The results are sumnarized. in Table 1.TABLE 1. Concentrations of steroids in histotroph and in plasma ofpregnant and non-pregnant sows between days 8 and 15 p.o. (nM).STEROID HISTOlROPH PLASMA.Pregnant Non-pregnant Diff. Pregnant Non-pregnant Diff.**** ****NS*- not significant; * - P

30PREGNANCY IN RATS FOLLOWING SYMPATHETIC DENERVATION OF OVARY, OVIDUCTAND UPPER UTERINE HORNP.J. Roche,H.C. Parkington and W.R GibsonDepartment of Physiology, Monash University, Clayton, Vic., 3168.Sympathetic nerves supply the ovary and reproductive tract, but itremains unclear what role they play in establishing and maintainingpregnancy. They could support progesterone production by corpora luteaor regulate oviducal and uterine motility which would in turn influenceoocyte transport, embryo spacing and parturition. We decided to testwhether denervation of the ovary and oviduct would prevent pregnancy andwhether destruction of the rich innervation at the utero-tubal junction,the site of initiation of uterine contractions, would interfere withparturition.The ovarian vascular pedicle and ovarian suspensory ligament on oneor both sides were briefly frozen in order to destroy sympathetic nerves(1) • The rats were then allowed to rna teo Most were found to bepregnant when they were killed 7 days after mating. In six of theunilaterally operated rats the ovary on the operated side had no nervesdetectable by fluorescence histochemistry. It was found thatnoradrenaline had been eliminated from the upper part of the uterinehorn (HPLC:

f32PROGRESTERONE PRIMING AND EARLY EMBRYO SURVIVAL IN THE EWEB.G. Miller and N.W. MooreDepartment of Animal Husbandry, University of Sydney, Camden, 2570.Ovulations induced in seasonally anoestrous ewes by the introdu t'~ft:~s are not accompanied by oestrus and the resultant corpora lut~a~oni t I reg:ess premat~ely. A period of progesterone (P) priming immedae y pr~or to ram ~ntroduction stimulates overt oestrus and mainteo~ corpora lutea (1) and conception rates at the induced oestru n~nceh~gh (2). It is unclear whether the priming P also regulates s may eembryo development in these ewes and if so ho " " earlyA high proportion of Day 4 (Day 0 = ~ay of ~est~um~ch pr~m~ng is required.~e:elopment following transfer to the uterus of o:ar:~~;:~z:~o:w:~r:~l~ p4~h day after ~e~trus induced by oestradiol (E) given after 12 day~ reatment (Pr~~ng P) with a further period of P treatment f 11 s~ng oestrus (Maintenance P) (3,4). 0 ow-In the present experiment one or two Da 4synchronously to the uterus of each of 63 ov~rie~~:r~:dw:~:st~~~sferredrecovered 17-18 days later (i.e. 21-22 da s after inwerePew~s.received the standard E and Mainten~ce P treat~~~~= 7;~tru~~ Allr~m~ng P treatments we d 2 . ~xbe~r4 8 d re use: 1 or 24 mg P were given daily for 1thays , such that the last or only treatment was always given 32'hore e commencement of E treatment.TABLE 1. Proportion of ewes with normal and (normal + abnormal) embryos.84112*0*8/10 7/12 9/10 10/128/10 6/11 9/10 11/112/10 4/10 8/10 9/1029/40 33/406/36 19/36sig 'f' In thet case (2 of ewes 2 with normal erobryos, the effect of duration wasn~ ~can d.f. X = 7.75, P

34 35IMAGING PREGNANCY STATUS IN SHEEP AND CATTLE WITH INTRARECTAL PROBESJ. F. Wilkins and D. G. FowlerAgricultural Research Centre, RMB 944, Tamworth, N.S.W., 2340Real-time ultrasound imaging (R.U.I.) has great potential to improvelivestock production by its many uses in reproductive research and management.We have developed scanning techniques with R.U.I. usingexternal transducers for pregnancy and litte:. size diagnosis in sheep(1). Alternative techniques using internal probes per re~tum (2) havepossible applications which are being evaluated. Internal probes areused in the mare for early confirmation of pregnancy and detection ofmultiple embryos, as well as imaging ovarian follicle development (3).Such techniques in the cow need development and evaluation. Thisreportdeals with 2 separate studies; firstly, imaging the pregnant tract incattle at early stages of gestation; secondly, the use of internalprobes in sheep at early and later stages.The cattle used were 32 Fresian heifers (2 y.o.) given oestrus synchronisationtreatment and artificially inseminated (A.I.). They werescanned while standing in a crush at 9, 17, 23, 32, 37, 47, 58 and 75days after A.I. using a Vetscan imager (Fischer Ultrasound) fitted with3. 5 MHz transducer inserted pel' rectum. The ewes examined ranged inpregnancy from 25-100 days. They were scanned while standing and whileon their back. Scanners used were Toshiba SAL32A with IVB-505S intracorporealprobe (broad band frequency 2-9MHz) and Aloka SSD-210DX withUST-58l3-5 transrectal probe (5 MHz) .In all pregnant heifers, the fluid diagnostic of pregnancy wasclearly visible by 32 days. In others it was seen at 23 days, withsuggestions even earlier. Thus, reliable confirmation of pregnancy by25-30 days is a conservative expectation. The embryo was often seen at32 days and in most cases by 37. There appeared to be large variationbetween animals in fluid development at early stages. There was alsoevidence of embryonic loss which should be clarified by further studie~These observations are preliminary to the characterisation of the echogenicfeatures of the non pregnant tract throughout the oestrus cycleand the development of the conceptus through pregnancy.The internal probes examined with sheep had problems with depth ofinsertion and limited manoeuvrability. Image clarity was worse withboth machines with the ewe standing, probably due to the uterus fallingfurther away from the probe and having a less continuous contact withinthe abdomen. The fluid of the conceptus was easily visualized in theearly stages (25 days). Embryos were clearly defined at 40 days. Laterstages (80-100 days) gave problems in loss of image clarity with depthof scan and in limited ability to orient the structures imaged, whichmay affect the accuracy of multiple diagnosis. Speed and safety ofthe procedure are other aspects that will affect its use. Scanning ewesper rectum should be highly accurate for pregnancy diagnosis but is yetto be fully examined for multiples at various stages.(1) Fowler, D.G. and Wilkins, J.F. (1984). Livestock Prod.Sci. (In press).(2) Botero-Herrera, a., Gonzalez-Stagnaro, C., Poulin, N. and Cognie, Y.(1983). Proc. 34th Ann. Meeting of Federation Europeene deZootechnie, Madrid. p. 702.(3) Palmer, E. and Driancourt, M.A. (1980). Theriogenology. 13 : 203-216.GroupTHE EFFECT OF INCREASING DOSE OF EXOGENOUS GONADOTROPHIN (hMG)ON SUCCESS RATE OF IVFID.J. Jessup and A.a. 'TrounsonDepartment of Obstetrics and Gynaecology, Monash U~iversitYIQueen Victoria Medical Centre, Melbourne, Austral~a, 3000.Clomi hene citrate and human Menopausal Gonado~rophin (hMG) are used tostimula~e multiple follicular growth and maturat~on (1). Pregnancy rateincreases proportionally with the number of mature oocytes obtained atlaparoscopy and the n~er ~f embryos transferred (2). This studyinvestigates the relat~onsh~p between the total dose of hMG used andsuccess of IVF. . / f' dpatients were treated with 100-1SOmg clo~phene day for ~ve aysbeginning ten days before the expected day of their LH surge (3). hMGwas given at 75-225 IU/day beginning on the second to f~urth day ~f .clomi hene treatment and cont.inued until ~lasma oestrad~ol began mcre~s~ngaboVe P 800pg/ml. There was no difference ~ pregnancy rate between patientsiven 100 or 150mg clomiphene. Patients w~r~ allocated to four groups~epending on the total quantity of hMG adm~~stered. These groups andthe pregnancy rates observed are shown in Table 1.TABLE 1Pregnancy rates for treatment groupsII III IVTreatment Clomiphene Clomiphene+hMG Clomiphene+hMG Clomiphene+hMG(75-450 IU) (525-975 IU) (1050-2400 IU)Pregnancies/Patients 33/454(7.3%) 19/215(8.8%) 66/438(15.1%) 10/111 (9 .1%)TreatedPregnancy rate was significantly higher in Group III (X 2 =15.1, p=0.0001)than in the other groups. The age of patients ,was distr~uted equallyThe proportion of patients hav~ng laparoscopy and embryoacross grouos.. Itransfer wa~ similar for all groups. Maximum plasma oestrad10concentrations were significantly higher in Groups II an~ ~II.Wh~~'cantlYcompared to Groups I and IV. The patients in Group III a s~gn~ ~thmore follicles at laparoscopy and more oocytes recovered than the 0 errou s patients in Group I had fewer embryos transferred then the other~rou~s: Patients in Group III had signficantly more embryos transferredthan Group IV but had a similar number to Group II. .We conclude that increasing the quantity of hMG g~ven.to women w~ll, th f IVF However there is a group of women (Group~ncrease e success0.' hM . d'cative ofIV) who fail to respond as expected.to hi~h doses of G, 1n ~some resistance to gonadotrophin stLmulat10n.(1) Trounson, A.a. In: CUrrent Topics in ExperimentalEndocrinology, vol. 5, p. 43, Academic Press, (1983).(2) Speirs, A.L., et ale Fertil. Steril. 39: 468-471 (1983).(3) McIntosh, J.E.A., et ale Fertil. Sterile 34: 125-130 (1980).

36 37RELATIONSHIP BETWEEN PREGNANCY-ASSOCIATED PLASMA PROTEIN A(PAPP-A) IN HUMAN PERI-OVULATORY FOLLICULAR FLUIDAND THE COLLECTION AND FERTILIZATION OF HUMAN OVA IN VITROJ.L. Yovich, J.D.Stanger* a~d J.G. Grudzinskas***~~~~~tment of Obstetric~ and Gynaecology, University of WA,**D Laboratory, Cambr~dge Hospital Wembley Perth W A Perth WA.(~~~~~~~:;ef~,O~~~~~~~C~.~~d Gynaecology, Th~ Londo~ H~s;italThe concentration of PAPP-A ' .was estimated a d .~n per~-ovulatory follicular fluidfertilization o~ hcompared ~o v~r~ous parameters associated with thefollicles in 42 ~an ova ~n v~tro. In all, the fluid from 86stimulated 'th p~th~ents wa~ collected. Follicle growth wasw~ e~t er clom~phene or a clomi h hgonadotrophin (hMG) combination a d l' P ~ne- uman menopausalcho' . n ovu at~on ~nduced with huma.r~on~c gonadotrophin (hCG). The follicle f 5' nst~mulated with cl . h s rom pat~entswithout hCG Th om~~ ene-hMG were aspirated on a natural LH surge~:~~~~~~o~~:~o~~~~:!~~~!~i~i~~~~l;~~ee~~;;~l~~i~:;e t:~~n;~~::q~;ntPAPP-~ was detected in 83 of the 86 folliclesranges vary~ng from undetect bl t 1 044 / aspirated inPAPP-A was si n' , a eo, ug 1. The concentration ofl7B Oestr d' Ig ~f~cantly ~orrelated with increasing follicle sizea ~o concentrat~on and oocyte recovery There 'correlation b t P P • was noto be fertili:e~e=~d ~= ~;v~~~c~~t~ation and t~e abi~ity of the eggnon pregnant c cles T ~tw~e~ foll~cles trom pregnant andPAPP-A y, . ,here was no s~gn~f~cant difference in theconcentrat~on ~n follicular fl'd 11 meanstimulated with 1 . Ul co ected from patientsHowever in th c oml~hene to t~ose stimulated with clomiphene-hMG, ose pat~ents recelving clomiph hMG' •concentration of P~PP A '. , ene- stlmulation, thepatients sur ed . - was s~gn~flcantly greater in fluid where(P < O.OOl).g spontaneously to those patients receiving hCGThis study suggests PAPP-A rna b ' .pregnancy potential of hum bY e a poor lndlcator of the~egree of maturity of the ~~l~~~leu~nC~~ld be,u~e~ 1 0 i~vestigate thelnduction. This may have rele e perlO ,0 lowlng ovulationappropriate interval between ~~~c~, for example, ~n de~ermining theoptimal in-vitro fertilization. y e recovery and lnsem~nation for(1)Yovich, J.L., Stanger, J.D., TUV1'k, A IM d•• and Yovich J.M.e • J. Aust. ~:645-649 (1984).PROSTAGLANDINS AND STEROIDS IN FOLLICULAR FLUID AS PROGNOSTICATORFOR HUMAN IVF RESULTSH.O. Hoppen, M.Besanko and A.O. TrounsonDepartment of Obstetrics and Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Victoria, 3000.Follicular growth and oocyte maturation seem to depend on the constituentsof the follicular fluid. Prostaglandin levels increase in response tothe preovulatory gonadotrophin rise and have been associated with the biochemicalevents immediately preceding ovulation in experimental animals.Limited information indicates elevated PGF;aa levels around mid cycle inhumans. We have analysed PGE;a' PGF 2' oestradiol (E;a)' progesterone (P)and androstenedione (A) levels in fo~licular fluid using specific radioimmunoassays,the performance of which have been confirmed by GLC-MS andHPLC.FolliCular contents were aspirated during laparoscopy 24-26h afterendogenous LH surge or 34-36h after HCG injection from patients in theIvF programme after induction of multiple follicular development withclomiphene/HMG. The follicles were classified into different groupsaccording to Whether or not an egg was collected, fertilised, developednormally and resulted in successful pregnancy after transfer, or accordingto follicular size. Part of the results are summarised in Table 1.Table 1.PG and Steroid Concentrations Related to Follicle Quality andEgg Performance (Mean ± SD)PGE PGF E;a PROG ADIONN (NG/ML) (NG/ML) (UG/MI.) (UG/ML) (uG!ML)l. Immature Follicles 24 1.0± 0.4 1.1±O.4 2.2 ±0.8 9.9±S.3 0.27±0.182. No. Eggs Collected 37 2.1± 1.3 2.2±1.3 O.87±0.6 10.l±4.6 0.34±0.393. Not Fertilised 42 5.4;: 9.8 4.3±7.2 1.0 iO.S 13.7i7.1 0.30iO.lS4. Abnormal EmbryoDevelopment 27 6.6±12.7 4.5i5.9 1.0 ±0.4 13.6i4.0 0.27iO.14'5. Normal DevelopmentNon Pregnant 76 4.6± 8.6 3.2±5.3 1.1 iO.5 11. 2i6. 5 0.33±O.336. P:r::egnant 4S 3.3± 3.6 3.2i2.3 1.1 ±0.4 11.4±4.8 0.23±0.117. Other 6 2.l± 1.3 2.2±1.0 1.2 ±0.4 11.9±4.7 O.19±O.06Entire Population 257 4.0± 8.0 3.1i4.7 1.1 ±0.5 11.7±5.8 0.30±O.26Statistical analysis of the data indicates that PG levels are significantlylower in immature follicles than in any other group (Mann-Whitney, p

THE PROGNOSTIC VALUE OF BETA HCG, PAPP-A, OESTRADIOL 17 BETAAND PROGESTERONE IN EARLY HUMAN PREGNANCIESJ.L. Yovich, S.C. McCo1m*, J.D. Stanger*, A. Tuvik* andJ.G. Grudzinskas#Dept. of Obstetrics & Gynaecology, University of W.A., Perth W.A.* PIVET Laboratory, Cambridge Hospital, Wembl8y, Perth W.A.#Dept. of Obstetrics &Gynaecology, The London Hospital, London UK.Various hormonal parameters have been studied to predict earlyhuman pregnancy failure. PAPP-A (pregnancy associated plasma protein,A) is a macromolecular glycoprotein present in the maternalcirculation during pregnancy and which has recently been reported topredict compromised pregnancies in which fetal life was previouslydemonstrated ultrasonically, by depressed circulating levels.Of 82 pregnancies following treatment for infertility, 65developed viable embryos which proceeded bey~-i 20 weeks gestation.The other 17 pregnancies did not proceed beyond 13 weeks gestationand comprised 2 ectopic gestations, 2 spontaneous abortions (viableembryos confirmed on ultrasound) and 13 cases of blighted ova(failure to develop ultrasound evidence of fetal development withinthe gestational sac). In this study, we have measured beta hCG,PAPP-A, oestradiol-17 beta (E2) and progesterone (P4) concentrationseach week prospectively beginning within 2-3 weeks of the knownovulation date.We compared the data from the 13 anembryonic pregnanciesto the 65 ongoing pregnancies at each week. Serum levels were notsignificantly different between the 2 groups of women for any of theparameters studied at week 5. At 6 weeks only beta hCG38levels weresignificantly reduced in the anembryonic group (P < 0.05).Thereafter there was a continuing and significant wide disparitybetween the beta hCG levels of the 2 groups. The levels of PAPP-Aand E2 were significantly depressed in the anembryonic group(P < O. 05) by weeks 7 and 8 respectively. P4 did not show asignificant difference between the groups until the final weekprior to abortion or when uterine curettage was undertaken. Asimilar proportion of women from each group were treated withmedroxyprogesterone acetate as part of a study assessing progestagensupport therapy in early pregnancy but there was no influence on theabove findings.These results indicate that quantitative beta hCGconcentrations provide an earlier indication of pregnancy failurefor anembryonic gestations than PAPP-A, E2 or P4. The differencebetween these results and those obtained for aborting pregnancieswhere fetal development was demonstrated may reflect the differentcompartments from which beta hCG and PAPP-A are generated. PAPP-Ahas been shown to be released in vitro by trophoblast and deciduawhilst beta hCG is derived only from trophoblast. In thepathophysiology of blighted ova the decidua-trophoblast relationshipmay be preserved until a later stage whilst spontaneous abortionsmay occur as a consequence of poor decidual integrity.UTERINE RECEPTIVITY IN HUMAN IVFEMBRYO VIABILITY ANDNo. ofEmbryosTransferredNo. ofPatientsPatientNotPregnant39P.A.W. RogerS, B.J. Milne* and A.O. Trounsont' and Gynaecology and computing*,tm ts of Obste r~CSDepar en Clayton,'8Austral~a, 316 .Monash University,all probability of an embryoIt is possible to express the over f the probability that the uterus is, t s as a product 0 .llilplanting(E) (1)~n a u eru , 't that the embryo is v~able . ,t' e (U) and the probab~l~ Y . .t' possible to estllilaterecep ~v . . 1 embryo implantat~ons, ~ ~sUsing data from m~lt~P ~ ihood methods. In the present study a ,E and U using maxllilum l~kel d to derive estimates for E and U, w~thcomputer programme waS d~velope . based on combined data for 821their corresponding conf~dence reg~on,embryo transfers.f observed and expectedh IVF results summary 0TABLE 1 Monash-QVMC-E~wort U=O 362 ± 0.094 and E=0264 ± 0.062rates of implantat~on where . .'( ) - . te 95% confidence lllil~tS)(These are approxllil aOneNumber of ImplantationsTwo Three Four192 179 (173.7) 13 (18.3)16 (6.5)257 214 (214.4) 37 (36.1)222 (18.9) 356 (52.8)(2.3)340 259 (266.1)1 (3.4) 1 (0.8) o (0.1)342 34 (31.3) 6 (6.4)4, 's based on the assumptions: U and E areThis model for implantat~on ~ f t s influencing implantation, and oneh the only ac or mbindependent, t ey are . fl ce the chance of other e ryosembryo implanting does not ~n uensurviving.and E the probabilities of pregnancyUsing these derived val~e~~~o~ are' 1=9.53%, 2=16.58%, 3=21.74%,after transfer of 1 to ~s once 3 or 4 embryos have b~en4=25.48%, 5=28.38% and 6=30.44% .. Th , nancy rate will only be ach~evedgt~ansferred a dramatic increase ~n pre should be directed to this problem,... Wh'l future researc h - .by increasing U. ~ e tes can already be achieved by treez~n~a major increase in pregnancy ra d t rus For example, the probab~l~tyd donation of embryos to a sec~n u e .. 28 38% but if the patientan th t nsfer ot 5 embryoS ~s ., bab'lityof pregnancy from e ra f subsequent replacement, the pro ~has 3 transferred and 2 frozen or Th's is an overall increase in the, s to 34.72%. ~of pregnancy1 t' s~ncrease ? In conclusion, these calcu a ~on .probability of pregnancy of 2~%'f the chance of pregnancy and the r~sksenable the accurate assessm~n °d 'th multiple embryo transfers.'1 gnancy assoc~ate w~of mult~P e pre 11 G N and Johnston,A L Lopata A., Gronow, M.J., Ke ow, ..(1) Speirs, ." St '1' 39.468-471 (1983).W.I.H. Fert. er~. __·

40THE EFFECTS OF HYPOTHYROIDISM OR HYPERTHYROIDISM ON TESTIS ANDPITUITARY FUNCTION IN PREPUBERTAL RA}1SY. Chandrasekhar and B.P. SetchellDepartment of Animal Scie~ces, University of Adelaide, Waite AgriculturalResearch Inst1tute, Glen Osmond, South Australia S064.The :ffects of ~hyroid hormones on the testes and pituitary of ramlambs durlog maturat10n has beeo investigated. Twelve Merino 1 b(16 week old: 23.0±0.7 kg initial body weight(BW» d ram am 5h th ·d () . were ma eYPOs yrol 0 w1th daily oral doses of 35 mg methyl thiouracil/kg BWfor ~eeks; hyperthyroid (ER) by daily sic injections of 2S ~gthyr~xlne (T4)/kg BW or left as controls(C). Measurements of T4 1 1conf1rmed the effectiveness of these treatments.were obtainedThe fall. :ve soWlng retiU l tsC a ERTestis vol increase (cm 3 )(initial vol = 9.2±1.3)Basal T levels (ng/mUl7.0±'~.Oa 24.0±6.0 a 2.3±l.Ob(S-S wk of treatment) l.14±0.2S a 0.33±O.OSb O.23±0.08 bBasal LH levels (ng/ml)(5-8 wk of treatment) 0.32±0.lSa 0.06±O.01bNo. O.12±0.04LH pulses/animal/12h b6.25 a 3.75 a l.obResponses to ~H(ng/ml) 2.S± 1.3 a l. S±O .3 a l. 3±O. 3aLHRH injections T II1.3±O.6 a O.7±0.3 a O.l±O.04Basal T productionb(ng/min) 67.5±24 a IS .6±S.~T increase5 .6± l.Oc(ng/mOl PVC 5.2±1.0a 3 .6± 1.8 a 2 .O±O. 7Ih after hCG bISV 263±31 a l47±4gbT l79±21COne ratio ISV/PVC b62.7±22 a 48.7±2l a 1l0±23lb after hCGbTesticular growth was almost arrested in the ER group b t t·the 0 group. Circulating LH levels were low in both 0 and E~ no 1nfrom 5th week of treatment, and testosterone (T) levels were 19ro~psboth ER and i 0 1 b Th ow 1n. n am s. ere were no differences in FSH levels.Dur1ng the 7th week of treatment basal LH levels as well 1frequency (measured in samples collected every 20 min for 12 h)s pu severy much reduced in ER lambs. The T response to LHRH (5 ng/k ;~)elower io ER a~though the decrease in LH response was not signi~icantwasThe T productlon and the response to hCG injection (15 IU/kg BW) .measured under pentobar~itone anaesthesia at the end of 8 weeks oftreatment ~ere reduced ln both 0 and ER groups. The ratio of Tconceo(trat1on in the internal spermatic vein (ISV) and post "cava PVC) was h" h . ER 1 b erlor venarate of T. 19 er ln am s, suggesting an increased clearanceThese results suggest that the testicular development during the~~oc~ssd~f puberty was arrested in hyperthyroidism and the capacity fe ey 19 cells to secrete T was reduced in both hyperthyroid1"sm and ohypothyroidism.ULTRASTRUCTURAL LOCALISATION OF HUMAN CHORIONIC GONADOTROPHINON ISOLATED RAT LEYDIG CELLS.A.E.Dept. of Anatomy,41Jackson, E.F. Glasgow & D.M. de Kretser.Monash University, Clayton, Victoria.The production of testosterone by rat Leydig cells is stimulated bythe binding of LH or hCG to specific cell surface receptors.Preliminary studies to visualise this binding in vivo, after injectionof hCG, made use of antibodies to the hormone and an immunoperoxidasestain. Light microscopy results indicated that binding occurred to manycells in the intertubular space, inclUding those which also labelledwith an anti - macrophage marker. In order to elucidate the precisenature of the cells which bound the hormone, an electron microscopicmethod of immunoperoxidase staining was used on preparations of isolatedintertubular cells.Intertubular cells of adult male rat testes were isolated bycollagenase dispersal at 34°C for 10 minutes and the cells wereseparated by centrifugation at aoOg through 0 - 77% Percoll gradients.prior studies using 125-I labelled hCG had indicated two peaks ofhormonal binding corresponding to cells of two distinct buoyantdensities (1). These two peaks were retained and the germ cellsdiscarded. The resulting preparation, which included cells identifiedmorphologically as Leydig cells, macrophages and mesenchymal cells, wasincubated with hCG for one hour at 4°C, to allow hormonal binding tooccur without internalisation. The cells were washed to remove unboundhormone and heated to 34°C for intervals of 5 to 60 minutes, in order toinvestigate the uptake of the hCG. '!he cells were then fixed in 1 %glutaraldehyde for 15 minutes and stained with a four layer peroxidase ­anti - peroxidase method (2). The primary antibodies were monoclonalantibodies to free hCG and to the beta subunit and these showed no crossreactivity with each other and did not cross react with human LH, FSH orTSH. An antibody to the alpha subunit also recognised the alpha subunitof the other gonadotrophins. Following the staining procedure, thecells were osmicated and processed for electron microscopy.Results indicated that hCG bound to the surface of Leydig cells ina fine granular pattern at 4°C, and that aggregation of the hormoneresulted in the formation of clumps on the surface of the cells as theywere heated. Surprisingly, no internalisation of the hormone wasseen. In contrast, binding was seen on cells which were identifiedmorphologically as macrophages, and these cells showed extensiveinternalisation of the hormone into lysosomes. 'l11e results of thisstudy question the accepted view that LHfhCG is internalised andfurthermore demonstrates binding to testicular macrophages. Furtherstudies are in progress to elucidate the functional role of thishormonal binding to the testicular macrophages.(1). Laws A.a., Kerr J.B., de Kretser D.M. Froc. Endoc. Soc. Aus.Abstract No. 75 (1982).(2). Hancock W.W. & Atkins R.C., Aust. N.Z. J. Med. 11:594-598 (1981).

42 43-, TCI,::;:SC.:!!..N PRODUCTION BY CuLTURED t-1YOE', ANI:'::£R'~"'::LI ;:ELLS OF THE I!1.f>.lATURE Rll.T TESTE THE EFFECTS OF EXCURRENT DUCT OBSTRUr.T1m~ UPON 7HI DEVELOPINGRAT TESTIS.E.W. Thompson, J.S.H. Elkington and A.W. Blackshaw~chool of Science, Griffith University, Nathan, Qld. and Department ofC.A.Yates & P.D.Temple-SmithPhysiology and Pharmacology, Ur.iversity of Queensland, St. Lucia. Qld.Department of Anatomy, Monash University, Melbourne, Victoria.We have demonstrated the incorporation of 35 504 into sulfoprotein,predominantly proteoglycan, by Sertoli cells, and its stimulation bv d~butY~llcyclic cAMP (BU2cM1P) and FSH + IB~~ (1). This study extendsconsist of the efferent ducts, epididymis and vas deferens. Studies haveThe excurrent ducts, which transport sperm from the testisthe work to include a similar analysis of cultured peritubular myoidshown in adult rats that obstructions of the excurrent ducts causedcells and Sertoli-myoid co-cultures (cultured in the presence or'absenceextensive testicular damage. The effects of obstructions on theof BU2CAMP after isolation from immature rats). Cell cultures were preparedand treated as described previously (2,3).the testis and excurrent ducts to the effects of obstruction.developing rat testis were studied to observe if there was adaptacion byThe six treatment groups (myoid cells (M), Sertoli cells (S),Obstructions were made unilaterally by dipolar coagulation at themyoid/Sertoli cell co-culture (MS) ± BU2cAMP (0.5mM)) were incubatedwith Na3~s04 (10,Ci/ml) for 72 hours at 37°C. 35 S04 incorporation intcglycosam1noglycans (GAG) and the analysis of species of GAG were estimatedas described previously (1). Androgen binding protein (ABP) wasestimated by equilibrium dialysis (3). BU2CAMP stimulated an increasein ABP levels, 2.5 fold (S) and 1.75 fold (MS). In the absence or presen~~of BU2CAMP MS produced 1.9 and 1.3 times the ABP of S respec::ive;\'.~YOld cell cultures produced a background level of ABP.Myoid cell and Sertoli cell cultures incorporated 35 S04 intosecreted and cel~~lar proteoglycans. BU2cAMP stimulated (2.3 fold M,1:5 fold 5) the '·S04 incorporation into secreted proteoglycans but;nere was no synergistic effect of coculture. BU2CAMP did not eleva~e_5S0~ incorporation into cellular proteoglycans of Sertoli cells or coculturesand reduced the level in myoid cells by 25%. There was asynergistic effect of coculture in the absence (2.3 fold stimulation;or presence (2.5 fold stimulation) of BU2CAMP.The proportion of chondroitin sulphate in secreted GAG was88.6% (myoid cells), 82.4% (Sertoli cells) and 91% (coculture). BU2C~~Phad no effect on this ratio. Heparin was the major cellular GAG; 69%(Sertoli cells), 59% (myoid cells) and 50.4% (co-culture). BuzcAMP haclittle effect on the production of heparin in any of the cultures.The results indicate that mechanisms of control of secreted prcter:;­glycan production are different to those for cellular GAGS. Thestimulated production of secreted proteoglycans with BU2CAMP ~n bu~r,myoid and Sertoli cultures indicates that this parameter mav be underpeptide hormone control in both cell types in vivo. These ~oleculesmay well be incorporated into the basement membrane at the mvoid-Ser1:Gl,cell interrace. -Elkington, J.S.H. and Fritz, I.B. (1980) Endocr~nology 107, 970.Thompson, E.W., Elkington, J.S.H. and Blackshaw, A.W. (1983)Aust. Soc. Reprod. BioI. Abstracts p. 35.Tung, P.S., ;'ina /Fritz, LB. (1980) Biol. Reprod. 27. 207.levels of the efferent ducts and caput epididymidis (proximal excurrentduct) and corpus and cauda epididymidis (distal excurrent duct)respectively in 15 day old (D15) anaesthetized racs. The rats weresacrificed at D45, D60, D120 and DlSO and the testes removed. Body andtesticular weights were measured, daily sperm production (DSP) wasestimated (1) and tissues were processed for light microscopy.From D45, testes with proximal excurrent duct obstructions showedreduced DSP compared with the control (Table 1). Testicular weightdecreased by D60 and continued to remain below control levels and wasassociated with germ cell loss from the seminiferous epithelium.TABLE 1: DAILY SPERM PRODUCTION ( mi11ion sperm'gm.of I testisAGE:D45 D60 D120 D180Control 6.2 + 3.0 12.2 ± 3.8 16.4±-1.7 17.4±1.5Eft. duct 0.6 ± 0.3a 0.3 ±- 0.4b 0.5 + a.Sb 0.8 ± a.7bCaput 2.5 ± 1.4a 0.5 ± 0.3b 0.5 + 0.9b 1. 2 ± 1. 6bCorpus 6.3 ± 1.5 5.4 ± 4.6b 2.4 ± 5.7b 3.3 + 6.3bCauda 6.2 ± 3.0 6.3 + 4.5 11.3 + 9.2 14.7 + 6.7All values mean ± SD (a = p~O.Ol; b = p

44 45HUMAN ACROSOMAL HYALURONIDASE AND SEMINAL PLASMA ARYLSULPHATASEA. Stojanoff and A.O. TrounsonDepartment of Obstetrics and Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Australia, 3000.Human semen contains hydrolytic enzymes, such as hyaluronidase andarylsulphatase which facilitate the passage of spermatozoa through theouter layers of the egg. The conventional hyaluronidase assay is basedon the Reissig spectrophotometric method of liberating terminalN-acetylglucosamine from hyaluronic acid. Arylsulphatase activity wasdetermined by measuring p-nitrocatecol, the product of the action ofarylsulphatase on p-nitrocatecol sulphate. We report here on theoptimization of the hyaluronidase and arylsulphatase assays. Apurification procedure is described and some properties of hyaluronidaseare presented. Furthermore, the effect of divalent cations and carbonylreagents on the colour reaction and on enzyme activity were investigated.It has been shown (1) that sperm extracts contain a greater amount ofhyaluronidase than seminal plasma and this was used as a source ofpurification. Acrosomal hyaluronidase and arylsulphatase were extractedfrom wahsed sperm using 0.1% Triton X-100 in 0.1% Hyamine 2389.Purification of acrosomal hyaluronidase was performed using SephacrylS-200 chromatography, Concanavalin A-sepharose and Heparin-sepharosechromatography. Elution of hyaluronidase from the Heparin-sepharosecolumn (1.2 x 10cm) at 4°C, resolved the enzyme into two discrete peaksof approximately equal activity, indicating that the enzyme may exist asmultiple forms. Hyaluronidase showed a substrate concentration of5mg/ml and a pH op~imum of 5.~. Aryl2~lpha~ise exhibited a pH range of5.0 to 5.5. The d~valent cat~ons, Ca , Fe , tested for their effect~n ~~ ~Olorimetric determination2~f N-~~etYlglucosamineshowed no~nhib~t~on at 0.05M. However, Cu , Hg , completel¥ eliminated allevidence of a colour reaction at 0.05M. At 1.OM, Ca + showed a 10% dropin absorbance. Semicarbazide had no effect on the colour conversion butcompletely inhibited hyaluronidase activity at 0.05M.The Reissig test consists of two distinct steps. The inhibitory pointof action by the cations was determined (Table 1).TABLE 1 Percentage absorbance at 585nrn depending on the time of additionof the interfering agentAb 585nm (% of Control)Interfering Final Addition before the Addition after theAgent Concentration 3 min Heating Period 3 min Heating Periodcu 2 +H 2+g2+Ca20mM10mM5M1197100In conclusion, the characterization and purification of humanhyaluronidase will help to identify the actual location and function ofthe enzyme.(1) Stojanoff, A., Mahadevan, M.M. and Trounson, A.O. Proc. Aust.Soc. Reprod. Biol. 15: 62 (1983).19238EFFECT OF pH ON THE CALCIUM UPTAKE AND RESPIRA~ION OF RAMAND BOAR SPERM IN THE PRESENCE OF IONOPHORE A23187A.M.Simpson and LG. WhiteDepartment of Veterinary Physiology, University of Sydney,N.S.W., 2006This investigation ~~s prompted by an apparent discrepancy betweenthe ionophore induced Ca-' uptake of ram and boar sperm.Ram sperm were collected by electrical ejaculation and boar spermfr~m t~e cauda. epididymis after sl2gghl~r. The sperm were washedtw~ce l.n modl.f~ed-R~nger buffers. Ca solution was added and thesperm suspension incubated at 30°C. Aliquots were filtered atintervals under low vacuum, dried and counted by liquid scintillation(1). Sperm motility was scored by microscopic examination. Therespiratory activity of the sperm suspensions was determined at 37°Cusing a Rank oxygen electrode. The effect of the additio~ of 3.2 ~mA23187, in three experiments, on the uptake of 1.0mM 5caCl (10~Ci/ml) by washed ram and boar sperm incubated at six pH leveis isshown in Table 1.TABLE 1. ca 2 + uptake (mean :: SE) of ram and boar sperm (nmoles/10 8sperm!20 mins) at varlOUS pH levels in the presence of A23187.Sperm A23187 pH(~M) 6.0 7.0 7.5 8.0 8.5 9.0Ram 0.00 1. 40::0.2 1.41!0.7 1.48+0.2 1.48::0.7 1.08::0.6 1.56+0.43.20 0.29!0.1 0.68::0.4 2. 73~0.9 11.90::0.9 14.86::1.9 15. 09!1. 8Boar 0.00 0.86::0.2 1.42!1.0 2.24!0.9 2.03::0.01 2.40::1.° 2.85!0.93.20 0.77::0.1 3.90::1.2 3.39::1.1 3.51::0.9 4.47::0.2 8.80+1.2Calcium accumulation by ram sperm in the presence of ionophoreA23187 was elevated when the pH of the medium was increased f 0m pH7.52to 9. 0. There was a particularly steep increase in the Ca + uptakebetween pH 7.5 and ,8. 0, and this was paralleled by increased 0consumption. In three experiments the overall mean + SE values for 0 28uptake (~1/hr/10 sperm) were for the controls: pH 7.5, 21.50 + 1.20~pH' 8. 0, 21. OS :: 1. 29 and ionophore: pH 7.5, 21. 25 :: 2.7; pH 8.0,28.00 :: 1.2.The ionophore induced Ca 2+ uptake of boar sperm showed quite adifferent pattern with increase in pH of the medium (Table 1). Theincrease commenced at a slightly lower pH of 7.° and plateaued at amuch lower level than in the case of ram sperm. At pH 9.0 there wasan abrupt increase in Ca 2+; this did not occur with ram sperm and isprobably of doubtful physiological significance as most of the spermwere immotile.These observations suggest a sP2cies difference in the influenceof pH on the operation of membrane Ca + pumps in ram and boar sperm.(1) Petersen, R.M., Seyler, D., Bundman, D. and Freund, M.J. Reprod.Fert. 55:385-390 (1979).

~o=c-------46EVIDEN:E THAT c::LaoITPHENE CITRATE (CC) ACTS PRI.MAAIT.Y AT AHYPOI'HALAMIC SITE IN WJMEN'J.P. Kerin*, J. Liu+, S.S.C. Yen+ and G. Phillipou**Dewrt. Obstet, Gynaecol, univ. Mel., Queen Eliz. Hosp., Sth. Aust.and +nepart. Reprod. Med., Univ. of california, San Diego, LaJolla, DSA.Despite the widespread use of claniphene citrate during the last 25years to induce ovulation in v.aoen the precise rcechanism and primarysite of its action has not teen detennined. Eight nornaJ. WOIIEnvolunteers rratched for age, w=ight, cycle length and ovulatory statusv;ere subjected to identical regirres except that 6 .-,;ere given CC (clanidMerrell) 15Dm3'/day on the evening of days 2, 3 and 4 of their rrenstrualcycle and 2 who served as controls w=re given identical placerotablets. At the 8 hour p:>int each w:Jl1lal1 received a bolus of GnRH (10ugIV) on day 2 and 5 to assess the change in pituitary responsivenessfollowing exposure to CC. Under controlled clinical laboratoryconditions foreann venous blocd was sarrpled 10 minutely for 10 hoursfran 0830 hrs in each ~ on day 2 (prior to CC or placero) and on day5 (3 days after exposure to CC or placeto). These 960 sanples v;ereanalysed by RIA for IR, FSH and prolactin (PRL). Pulse anplitude andfrequency of these horrrones .-,;ere analysed by a conputer program based ondata of Clifton and Steiner (Endocrinology 112:1057, 1983)TABLE 1. Conparison of plasrra ill, FSH and PRL pulse frequency, cycleperiod, pulse arcplitude and rrean concentration tefore (day 2) and after3 days exposure to CC (day 5).Cycle day Day 2 Day 5 PPulse frequency IR/8hrs 3.2±l.9 7.3±2.0

48IS CONVENTIONAL BACTERIOLOGY USEFUL IN THEMANAGEMENT OF MALE INFERTILITY?M.G Jennings, H.W.G.Baker and M.P. McGowanMedical Research Centre Prince Henry's Hospital and FloreyInstitute: Melbourne, Vic. 3004.An aetiological role for non-specific genital tract infectionmaie infertility has long been postulated and has led to th (GT~) incu ture of semen in some laboratories Th e rout~nenation depends in part upon the . . e value of such routine examiityand that the diagnosis of GTir~mLsef~~a7 GTI causes male infertiltowarrant the time and mon s su LCLently accurate and frequentcriteria for diagnosis of G;r ;~~~~S~:dit. ~he most comprehensiveet al. (1), which combine clinical micr~~io~~:,ar~ th~se of Comhairevariables. To assess the value f ' It ~ca an cytologicalGTI h 0 cu ure and examination of f, t e frequency of bacterial infect' d b" semen orComhaire's dia . Lon an com LnatLons ofI f 4~n20stic crLteria (Table) was determined in 1 396samp es rom men over a two year period. ' semenmens. TABLE: Comhaire's Diagnostic Criteria (No., %) in 1 ' 396 Semen Speci-~l-lNil a bCRITERION~tLI(i) (ii)E-t1-1~ Nil 734(52.6) n(0.8) 437(31.3) 6(0.4) 5(0.4)c..:>a63 (4.5) 20(1.4) 5(0.4)b72(5.2) 43(3.1)Comhaire ' s criteria (1): a. leucocytes > 10 6 / l' 'sory sex gland function (e hi h H 0 . m ~ b. dLsturbed accesc.bacteriospermia (i) non~~~tho:en~ >1~4/~~co(77~' mu~us streak~ng);Any combination of a+b a+c or b .' LL pat ogens >10 /ml., , +c constLtutes a positive diagnosis.The frequency of GTI accordin . .than 15%. Bacteriospermia wa 1 g.to Comhaire's crLterLa was lesss a so Ln f requent being 1 th 11~'t he f ew pathogens cultured (E coli Kl .' ess an ,•• Ofunspecified Gram negative bac~lli a~d sebSLella), Pro;eus, S. faecalis,a possible cont i • aureus , 53% were S. aureusth 106 am.nant. Furthermore, only 1 specimen (OJJ7%) h d 'an pathogenLc bacteria/ml also S . a morecorrelation in the presence of ~ t' d,aureus~ The:e was a lack oftion (e.g. criterion b) sheddingerdaL~t Lagnth°stLc c:L~eria and infeccriteria.' ou on e signLfLcance of thet' These findings indicate that the routine bacteriological exam;na-Lon of semen specimens is not merited.•(1) Comhaire, F., Verschraegen G. and VAndrol. ~' 32-45. ' ermeulen, L. (1980) Int. J.C49INCREASED FREQUENCY OF AUTOANTIBODIES IN MEN WITH SPERM ANTIBODIESH.W.G.Baker, G.N. Clarke, M.P. McGowan, S.H. Koh and M.N. CauchiDepartment of Pathology and Reproductive Biology, Royal Women'sHospital, Medical Research Centre, Prince Henry t s Hospital andHoward Florey Institute, Melbourne, Australia.Sperm antibodies appear in many men with obstructive azoospermiapresumably as a result of immunization via systemic absorption of spermantigens. Sperm autoimmunity also occurs without genital tract obstructionsfor unknown reasons: approximately 6% of infertile men havepositive serum sperm immobilization tests (SIT). Because we foundthese men had family histories of autoimmune diseases more often thanother infertile men (1), we determined the frequency of ~utoantibodiesin the sera of 102 infertile men with positive SIT (flspontaneousflgroup)and compared them with those in 277 sperm antibody negative patientsof comparable ages and semen quality (control group) and 57 men withpositive SIT and continuing or past genital tract obstruction, e.g.vasec tomy or vasovasostomy (flobstructive" group). SIT was performedaccording to WHO recommendaLions and all positive tests were confirmedin additional serum samples. The autoantibodies: antinuclear (ANA),antimitochondrial (AM), antismooth muscle (ASM) and antigastric parietal(AGPC), were detected by indirect immunofluorescence. The thyroid autoantibodies:antimicrosomal(ATM) and antithyroglobulin (ATG) weretested using the Thymune M and Thymune T kits and rheumatoid factor(RF) by the RapiTex-RF kit. There was a significantly higher frequencyof ATM in the men with spontaneous autoimmunity than in the controlgroup (Table 1) and organ-specific autoantibodies (AGPC, ATG, ATM)together were also more frequent than in the control group (18.6 versus8.7%, p

50PHARMACOLOGY OF THE ERECTILE TISSUE OF THE PENIS*Carati C J, Goldie R G ,Keogh E J, Wisneiwski S51WITHDRAWNReproductive Medicine Research Institute, *Department of Pharmacology,Queen Elizabeth II Medical Centre, Nedlands, W.A., 6009Erection results from an engorgment of the penis due to increasedblood flow into the paired erectile bodies known as corpora cavernosa.Contraction of the smooth muscle in these bodies restricts blood flowand ensures the flaccid state, while its relaxation results in erection.This process is under the control of the autonomic nervous system, amalfunction of which may result in impotence. The final neuroeffectorof erection is unknown, but cholinergic, adrenergic and peptidergicsystems have all been implicated (1). This study examined thepharmacology of the erectile tissue of the dog and man, with a view tothe elucidation of the autonomic control of erection and the developmentof a pharmacological treatment for impotence.Strips of cavernosal tissue were placed in organ baths inKrebs-Henseleit buffer at 37 o C, aerated with 5% CO in 02' Changes in2its contractile status (isometric tension) were measured, with a restingtension of 500 mg. There was a significant contractile response to thealpha-adrenoceptor agonist phenylepherine. This contraction was onlypartially relaxed by the beta-adrenoceptor agonist, isoprenaline,indicating the dominance of an alpha effect. Isoprenaline (beta and lbeta agonist) and rimiterol (beta agonist) relaxed precontracted2 2preparations with equal potency, indicating that relaxa~ion was mediatedby beta -adrenoceptors. Noradrenaline failed to relax precontracted,2alpha-blocked (phentolamine l5mM, 1 hr) preparations, while adrenalineand vasoactive intestinal polypeptide (VIP) relaxed them in aconcentration-dependent manner. Carbachol, a cholinoceptor agonist, andhistamine had no effect on either resting or contracted preparations.In summary, contraction of erectile tissue was mediated byalpha-adrenoceptors which dominated the beta-adrenoceptors responsiblefor its relaxation. The beta-adrenoceptors were of the beta SUbtype,2upon which noradrenaline has little effect. Thus, the funct~onalsignificance of beta -adrenoceptors in erectile tissue is questionable2since adrenaline is a minor neurotransmitter in penile tissue. Thisdata is consistent with the suggestion that VIP is the probable effectorof penile erection (1). Potential pharmacological treatments forimpotence include local alpha-adrenoceptor-blockade and VIP injection.(1) Ottesen B, Wagner G, Virag R, Fahrenkrug J (1984)Penile erection: posssible role for vasoactive intestinalpolypeptide as a neurotransmitter.Brit. Med. J. Vol. 288, p 9-11

52PLASMA PROGESTERONE CHANGES IN SUPEROVULATED WOMEN BEFORE, DURING ANDIMMEDIATELY AFTER LAPAROSCOPY FOR IVF AND THEIR RELATION TO THEINITIATION OF PREGNANCY AFTER EMBRYO TRANSFERP. Taylor, A. Trounson and M. BesankoDepartment of Obstetrics and Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Australia, 3000.The present study was undertaken to determine the nature of the accutechanges that occur in serum progesterone (P) values immediately prior to,during and after laparoscopic oocyte recovery.Twenty patients were randomly selected from the Monash IVF/ET programme.All underwent stimulation of ovulation with clomiphene citrate (Clomid,William S. Merrell, USA) and human menopausal gonadotrophin (Humegon,Organon, Holland). Oocyte recovery was timed for 32-36h after the onsetof the plasma luteinizing hormone surge or the HCG injection. In 19patients, laparoscopy was carried out under general anaesthesia and inone patient epidural anaesthesia was used.Serum P values were measured using a direct double antibody radioimmunoassay.Blood sampling was performed 48, 24 hours and 60, 30 and 10 minsprior to the induction of anaesthesia and then at 10 minute intervalsfor the next two hours. Further sampling occurred at 4, 12, 24 and 48hours after the induction of anaesthesia. Eighteen of the 20 patientshad an embryo transfer and three became pregnant. There was no differencein P values between the pregnant and non-pregnant group at 48 and 24 hoursprior to the induction of anaesthesia.TABLE 1 Mean changes in P during laparoscopy for oocyte recovery (ngP/ml)Time (mins) from induction of anaesthesia (S.E.)Patients -60 0 30 60 90 120 720A(n=lS) 5.4(1.3) 6.7(1.6) 4.4(0.9) 3.1(0.6) 1.9(0.3) 2.4(0.6) 7.0(1.7)B(n=3) 12.0 (1.6) 15.1(0.6) 5.6(0.6) 5.3(0.9) 7.4(1.4) 6.9(1.9) 7.0(3.0)A = Not Pregnant,B = PregnantPlasma P levels decreased immediately after the induction of anaesthesiain all patients (Table 1), in response to, either or both, anaesthesia orr~moval.of the follicular contents. However! P levels were significantlyh~gher ~n the pregnant patients immediately prior to (-60 to -30 mins;P

EFFECT OF FOLLICLE STIMULATION AND FOLLICLE NUMBER ON THE RISEIN EARLY LUTEAL PHASE PROGESTERONE CONCENTRATION FOLLOWINGFOLLICLE ASPIRATION IN WOMEN.JAMES D. STANGER, JOHN L. YOVICH II AND DAVIV L. WILLCOX *Pivet Laboratory, Cambridge Hospital, Wembley, Perth,# Department of Obstetrics and Gynaecology and * Departmentof Anatomy, University of Western Australia, Perth, W.A.The rate of rise of serum progesterone was monitored from the~ay of ovulatio~ (day 12) to day 18 of the menstrual cycle~n 58 women adIllltted for in vitro fertilization and embryotr~nsfer. (IVF/ET) at Perth, Western Australia. Folliclest~mulat~on was achieved with 100 mg clomiphene citrate on~ays 2 to 6 with or without human menopausal gonadotrophin~G) f:om day 6 . Even though the level of progesterone wash~gh:r ~n.those patients receiving clomiphene/hMG, there wasno s~gnif~can~ difference over the early luteal phase. Onereason for ~h~s was the variation in the number of folliclesgenerated ~~th both stimulation regimes. When analysed by onewa~ analys~s of variance on each day, a significant correlationefx~sted between progesterone concentration and follicle numberrom day 15 to day 18.Five patients. conceived during the course of this study. Four ofthese were st~mulated with clomiphene and one with clomiphene/hMG. The number of follicles and the number of embrvos transferred~anged from 2 ta 5. The progesterone concentration in the early~teal phas: ~f the patients stimulated with clomiphene was int.e u~per l~m~t for that group. It is suggested that a precocious~~s~ ~n.progesterone caused by the presence of multiple numbersoll~cles could create a uterine environment more favourableto early embryo growth and that the relationship between embryonumber and pregnancy in IVF/ET may be facilitated by elevatedPfrogesterone concentrationin the early luteal phase resultingrom follicle stimulation.5455THE EFFECT OF MATERNAL AGE ON THE SUCCESS RATEOF IN VITRO, FERTILIZATION (IVF)A. Trounson, C.M. Caro and D. JessupDepartment of Obstetrics and Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Australia, 3000.In women approaching the age of menopause, menstrual cycles becomevariable in length, oestrogen levels in the follicular phase are lower,baseline levels of LH and FSH may increase and fluctuate, there is areduced number of primary ovarian follicles and there are on occasionsno secondary follicles or copora lutea present in their ovaries. Thesewomen also have a diminished responsiveness to exogenous gonadotrophintherapy (1). In the present study we have compared the IVF results overthe same period of time for women of 40 years of age and older to thosefor women of 39 years of age and younger. The results obtained for thetwo age groups are summarized in Table 1.TABLE 1Age;::40

56STUDIES ON THE CRYOPRESERVATION OF 2-CELL MOUSE EMBRYOS 57L.R. Mohr and L. Freemann EVALUATION OF FROZEN-THAWED HUMAN EHBRYOSDepartment of Obstetrics and Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Australia, 3000.cryobiological studies carried out on morula to blastocyst stagemouse, cow and sheep embryos indicate a good correlation between the lowpowermorphological appearance of frozen-thawed embryos and their developmentalpotential (1). We have investigated the correlation between cellularsurvival of 2-cell mouse embryos and their developmental potentialafter a number of cryobiological procedures.Mouse embryos were collected and cultured as previously described (2).Embryos were frozen in 1.5 molar dimethylsulphoxide (DMSO) using methodspreviously described for human embryo freezing (3). Termination of slowcooling and transfer to liquid nitrogen occurred at -39 (fast freeze) or-80°C (slow freeze). Thawing was carried out at +8°C/min (slow thaw) orby direct immersion in a 30-35°C water bath (fast thaw).The proportion of 2-cell mouse embryos recovered with both blastomeresintact or 1 blastomere intact was investigated after two differentfreezing and thawing techniques (Table 1). The developmental potentialof these embryos was evaluated by the proportion developing to theexpanded blastocyst stage after 72 hours culture in vitro.TABLE 1 Survival and development of frozen-thawed 2-cell mouse embryoswith either both cells intact or only 1 blastomere intact after thawing(mean ± S.E.M.)FreezingProcedureslow freezefast freeze% Post-thaw recovery2-cells 1-cell69 ± 3.867 ± 5.522 ± 1.521 ± 5.4% Development to blastocyst2-cells 1-ce1168 ± 5.257 ± 2.365 ± 752 + 6Results indicate no difference in the proportion of 2-cell mouseembryos surviving the two freezing procedures. Control 2-cell mouseembryos exposed to DMSO but not frozen, show over 90% development toblastocyst in vitro. There was no difference in the ability of frozenthawedembryos to develop to the blastocyst stage between those withboth b1astomeres surviving and those with one blastomere lysed. Only60-70% of the morphologically normal frozen-thawed 2-cell embryos werecapable of development to blastocyst stage in vitro. In conclusion, theloss of developmental potential after freezing and thawing 2-cell mouseembryos may not be accompanied by readily detectable changes inmorphological appearance.(1) Ciba Foundation symposium 52, Excerpta Medica, Elsevier/NorthHolland, Amsterdam, (1977).(2) Mohr, L. and Trounson, A. J. Reprod. Fert. 58: 189-196 (1980).(3) Trounson, A. and Mohr, L. Nature, 305: 707-709 (1983).stage of embryo4-cell8-cellL.R. Mohr, L. Freemann and A.O. TrounsonDepartment of Obstetrics and Gynaeco1ogy, Monash U~iversity,Queen Victoria Medical Centre, Melbourne, Austral~a, 3000.In developing human embryo freezing procedures ethical Ob~igations tofreeze and replace all embryos with any developmental potent~al mayrestrict evaluation to non-invasive techniques. Subsequent to the f~rstpregnancy established after transfer of a frozen-thawed 8-cell humanembryo (1)" we have examined the response of 4-cell and 8-cell huma~mbryos to two different freezing techniques. Human embryos result~ng~rom in vitro fertilization (IVF) were frozen in 1.5 mo~ar dime~ylsulphoxide(DMSO) in phosphate buffer and cooled at 0.3 C per m~n~te.to-390C (fast freeze) or -aooe (slow freeze) prior to transfer to.l~qu~dnitrogen. Embryos were frozen and thawed using proceduresprev~ouslydescribed.Embryo quality and outcome of freezing were evaluated.in 86 e~ryos byclassifying them as "good" or "irregular" prior t~ freez~g. Th~s.classification was made non-invasively on the bas~s of bl~stomere s~ze,number, arrangement and presence of fragments.. Embryos w~~h half or moreof the original cell number intact after fre:z~~g.and th~w~ng wereconsidered to have survived. There was no s~gn~f~cant d~f~erence betweenpost-thaw survival of embryos classified as "irregular", 1~/26 (42%)survival, and those classified as "good", 26/60 (43%) surv~v~l.Fifteen embryos classified as severely damaged after thaw~ng.wereexposed to fluorescein diacetate, FDA (2) and :ubsequently :xam~ned bylight and electron microscopy. Blastomeres wh~ch appeared ~ntact at thedissecting microscope level fluoresced after exp~s~re to FDA bU~ someintact blastomeres showed intracellular abnormal~t~es when exam~nedultrastructurally.The survival of 4-cell and 8-cell embryos was higher when frozen bythe slow procedure than by the fast (Table 1). There was no significantdifference between survival of 4-cell and 8-cell embryos.TABLE 1 Proportion of 4-cell and 8-cell human embryos showing survivalof 50% or more cells after freezing using the fast and slow proceduresTotal1967Fast freeze1/63/20(16%)(15%)Slow freeze6/1327/47(46%)(57%)Results of freezing and thawing 119 4-cell and 8-cell human ~ryosusing the slow procedure has resulted in 59/119. (50%) embryo surv~~aland the transfer of 59 thawed embryos to 41 pat~ents has resulted ~n 7pregnancies.(1) Trounson, A. and Mohr, L. Nature 305: 707-709 (1983).(2) Mohr, L.R. and Trounson, A.O. J. Reprod. Fert. ~: 189-196 (1980).

58GLYCOSYLTRANSFERASE LEVELS IN HUMAN SEMINAL PLASMA 59M.T. de Witt, P.J. Lutjen, J. Hoy and A.O. TrounsonDepartment of Obstetrics and Gynaecology, Monash UniversityQueen Victoria Medical Centre, Melbourne, Australia, 3000.'The mechanism of terminal glycosylation involves the direct transfer fsugars from ~e nucleotide-sugar complex to the oligosaccharide chain o~a glycoprote~n by the action of glycosyltransferases. Sialic acid andfucose.ar~ two.suc~ terminal sugars and previous studies have suggestedthat s~al~c ac~d m~ght playa role in the fertilization process (1).The present study reports on the quantitation of sialyltransferase andfucos~ltransferaseactivity in human seminal plasma.Sem~nal Pla~ma was separated by centrifugation from semen obtained from64 norm~s~erm~c donors and stored at -80°C. Prior to assay for transfera~eact~v~ty, ~-~ercaptoethanolwas added to the seminal plasma to af~nal c~ncentrat~on of 10mM and samples ultrasonicatedat 4 0 C for 2 x 5sec•. s~alYltransf~rase and fucosyltransferase were assayed as previouslydescribed for cerv~calmucus(2). Oesialylated bovine fetuin ( 800Ug) wasused a~ acceptor molecule and transferase activity was expressed as pmolof radWlabelle~ sugar transferred per mg protein of seminal plasma perhour. Correlat~on of the level of activityo~ both enzymes with thevolume of· the e~aculate,spermconcentrations, percentage" motility andpercenta~e of l~ve.sperm was analysed using Spearmans non-parametriccorrelat~on analys~s.The result~ (Table 1) show that there is a predominance of fucosyltransfer~se~n human seminal plasma with levels approximately 50 timesthat o~s~a~yltIansferase. Fucosyltransferase levels in the seminal plasmawere s~gn~f~cantly correlated with sperm concentration (P=0.017) and thepercentage o~ live sperm (P=0.046) but not with the volume of ejaculate(0.4~6) or w~th the percentage of motile sperm (P=0.165). The activityof s~alyltransferase was not correlated with any of the semen parameters.TABLE 1Fucosyltransferase Sialyltransferasepmol/mg/hrpmol/mg/hrActivity (Mean ± SE) 777.65 ± 47.08Range (n)15.24 ± 3.9414.57 - 2145.96 (64) 1.14 - 50.4 (21)The levels of both enzymes are much greater than previously reported .seminal plasma (3). The correlation of fucosyltransferase activity With~~e concen~ration of live spermatozoa suggests that fucose may be~portant ~n regulatin~ sperm interactions in the fertilization process,e~ther dur~ng penetrat~on through the cervical mucus or association withthe cumulus or zona pellucida.(1)(2)(3)Glycosyltransferase activity in human seminal plasmaOurr, R., Shur, B. and Roth, S. Nature 265: 547 (1977).Lutjen, P.J., Trounson, A.O. and McBain,-Y:c. Ferti1. Steril.41(2): 385 (1984).Ronquist, G. and Stegmayr, B. Int. J. Fertil. 28(4): 239-242 (1983).NormalCervical-factorSIALIC ACID DEFICIENCY IN CERVICAL MUCUSP.J. Lutjen, M.Mean ± SD3.1 ± 0.71.3 ± 0.4HOSTILITYde Witt, J.C. McBain* and A.O. TrounsonDepartment of Obstetrics and Gynaeco1ogy, Monash University,Queen Victoria Medical Centre and *Reproductive Biology Unit,Royal Women's Hospital, Victoria, Australia, 3000.There is an increase in the uptake of N-acetylneuraminic acid (sialicacid) and attachment to the mucin oligosaccharide chain in infertilewomen with a diagnosed cervical factor (hostile mucus) not associatedwith sperm antibodies (1). Although the enzyme responsible for transferof sialic acid to mucin (glycosyltransferase) appeared to be present atnormal levels in mucus, it was not known whether the observed deficiencywas due to a reduced content of sialic acid in mucus or to a dysfunctionof glycosyltransferase regulation. In the present stUdy we report arapid assay for free and glycosidal bound sialic acid in cervical mucususing cation exchange high performance liquid chromatography. Using thismethod we have examined the sialic acid content of mucus in fertile womenand women with cervical-factor infertility.Mid-cycle cervical mucus samples which had been previously assessed forsperm receptivity and binding of sialic acid were diluted 1:1 with SOmM~-mercaptoethanol and dispersed by ultrasonication. Samples werehydrolysed in O.lN H2S0q 80 0 c for 1 hour containing SOOng/ml sor~itol asinternal standard. Separation of sialic acid was achieved on Am~nexHPX-87H cation exchange resin (300 x 7.8mm) using O.OlN H2S0q mobilephase, 600u1/min flow rate and 1 x 10 6 NM-2 at 40°C. The retention timesfor sialic acid and sorbitOl were 8.99 ± 0.20, and 11.60 ± 0.20respectively. The concentration of sialic acid and sorbitol wasdetermined by refractive index measurement at 950nm; with the correctedvalue of sialic acid concentration being expressed per mg of protein at254nm.Preliminary results show a significant decrease (P

6061EFFECTS OF SURGICAL LUBRICANTS ON SEMEN ANALYSISJ.P.P. Tyler and M.N. SchoemanDepartment of Obstetrics and GynaecologyWestrnead Centre, Westrnead N.S.W. 2145It is an implicit assumption in the and 1collected by masturbation fUlly ro ogy laboratory that a sample, represents that deposited ' ,-at co~tus. However, there is little data ' . ~n a vag~nathis hypothesis (1) and some anecdot 1 ,~n the l~terature to support(2). These studies examined the effa t ev~fence to suggest the oppositeavailable surgical lubricants on thee~o~'~'ta ~umber of commerciallywith the aim of recornrnendin . ~ ~ yo spermatozoa with time,was not visibly detrimental g : non-tox~c water soluble compound whichsemen for routine analysis. 0 spermatozoa following collection ofEach lubricant tested was dissolved in .medium (3) supplemented with 2Omrnol/l an appropr~ate amount of culturewhen equal Volumes of medium d Hepes and 3mg/ml HSA such thatan semen were combined th f' 1concentrations were 10% 5% d 2 5 e ~na testwas quantitated at 15m ~nd l;Qrnn t' %. Objective spermatozoal motilitya room temperature.Each lubricant (KY, Aci, POI and Balfor ' II'and saliva at 10% contamination s' . f' ]e ~es, Lubafax and Surgilube)spermatozoal motility from contro~~n~t~cantlY re~uced (P < 0.01)decreases 12.6% - 26.6% and 27 7% _ 31 15m and l~~m (range of meane~fect Would be apparent befor~ routin~O~a~spect~velY), .Thus thisc~dal cream (OrthO-Gynor) as t' ratory analys~s. A spermispermatozoawith;n 15 ' t nega ~ve control also killed all... m~nu es.Following exclusion of t 1 b .packaged as single sach:~:' n~tr~::~~~ for ~ractical reaSOns (eg notonly KY and POI lUbricatin' , II' Y ava~lable; components "suspect")photography (4) at 5% and ~ ~= ~e~ w~re ~xamined by mUltiple exposuresignificant differences coui con am~nat~on. At these levels noand their velocity profiles ~ be fo~nd between spermatozoal motilityor up 0 two hours at room temperature.Consequently, because it is well known KYd ose"sachet is the lubricant of choice.' jelly packaged as a ". 1s~n9 el.2.3.4.Freund, M. (1962) J. Reprod. Fert. 4; 157.Tyler, J.P.P., Driscoll G L -191. ' " and Crockett, N.G. (1983) Lancet i,Biggers, J 0 Wh'tt"Methods .' ., ~. en, W. K. and Whittingham, D. G. (1971) in~n Mammahan Embryology" 1st Edn Ed C J D '1Pub San Francisco p86. . " an~e.Makler, A. (1078) Fert. Steril. , 30, 192.ACUTE CELLULAR ANDHUMORAL RESPONSES TO EQUINE STREPTOCOCCAL ENDOMETRITISMUNYUA, S., WILLIAMSON, P., PENHALE, J., MURRAY, J.School of veterinary Studies, Murdoch University, Murdoch, W.A. 6150.Bacterial endometritis is a major cause of infertility in mares. 1However, the factor(s) preventing the establishment of bacterial infectionin the equine uterus are not well understood. The present study reportsuterine response i~nediately following intrauterine infusion of B-hemolyticStreptococci, in a group of mares known to be either susceptible orresistant to uterine infection.Susceptible f6} mares, with either history of recurrent endometritisand/or diffuse endometrial fibrosis, and resistant (20) mares with healthyuteri, were selected for challenge. All challenge mares were bacteriologicallyand cytologically negative on uterine swabbing at least 14 daysbefore challenge. The mares were challenged by uterine infusion of 10 9viable B-hemolytic Streptococci in 5ml of distilled water, while thecontrol (3) mares received distilled water. Guarded uterine swabs andwashings were obtained for bacteriological culture. Uterine washingswere also examined for protein content and neutrophil numbers and 0 and10 day plasma assayed for progesterone (sampling intervals -0, 2, 4, 6,12 hours, 1, 2, 4, 10 and 28 days post challenge).The dynamics of bacterial infection showed a similar pattern in all mares,with bacteria being recovered from uterine washings and swabs for thefirst 6 hours but not at 12 hours. Large numbers of bacteria weresubsequently recovered, for up to >28 days from susceptible mares,whereas resistant mres cleared the infection within 4-8 days. In allmares, there was an influx of neutrophils and serum proteins within2 hours of challenge, but there were wide variation in neutrophilnumbers and protein concentrations between mares. Quantitative andqualitative protein assays indicated that many serum proteins includingalbumin and IgA, IgG, IgG(T) but only traces of IgM, were present inuterine lumen at the peak of protein influx.Infections became established in all mares designated as susceptibleprior to the trial, and was cleared in all resistant mares, regardlessof whether the mare was in oestrus, dioestrus or anoestrus at the timeof infection. This data suggests that the failure by susceptible maresto eliminate bacteria does not appear to be related to an inability tomobilise local cellular and humoral responses. It is also unlikely thatthe stage of the oestrous cycle has much influence on the establishmentand the subsequent resolution of endometritis in the mare.(1) Bain, M.A. (1966) Vet. Rec. 78: 168-173.

62BIOCHEMISTRY AND PHYSIOWGY OF PREGN.1l.NCY ASSOCIATEDPLASMA PROTEIN-A (PAPP-A)IN HUMAN REPRODUcrIVE FLUIDSM J SinosichDepartrrent of Obstetrics and Gynaecology,Royal North Shore Hospital, St I.eonards NSW 2065Pregnancy associated plasma protein-A (PAPP-A), a large glycoprotein,was detected by radioirrm.moassay in the peripheral circulation of pregnantwomen. More recently PAPP-A was detected in humna seminal and ovarianfollicular fluid, but not in the circulation of nomal non-pregnant individuals.Physicochemical characterisation of non-pregnancy and pregnancyderived PAPP-A showed both species to have a unique rrolecular sizeof 820K. Furtherrrore, all PAPP-a species reversibly round to heparinand zinc chelate affinity matrices. Imnunological characterisation, asdemonstrated by logit transformation of the binding displacerrent curvesfollowing serial sample dilution, showed all species to be inmunologicallyidentical.Seminal PAPP-A concentrations varied from undetectable to less than1% whereas follicular concentrations were less than 6% of late pregnancyserum (designated 100IU!L). No significant correlation was deInonstratedbetween sperm rrotility, number, rrorphology and. seminal PAPP-A levels.Analysis of split ejaCUlates showed the major proportion of PAPP-A wasejaCUlated with the latter contractions, indicating a non-testicularorigin for the glycoprotein. By contrast, follicular PAPP-A concentrationsvaried markedly throughout the spontaneotls rrenstrual CYCle, withPAPP-A concentrations increasing during folliculogenesis to peak imrrediatelyprior to ovulation. PAPP-A concentrations were significantlygreater in preOVUlatory than in atretic follicular aspirates. In addition,follicular PAPP-A levels strongly correlated with follicular diameter,number of oocytes retrieved ard follicular progesterone but not withestradiol concentrations.Purified PAPP-A derronstrated no imnunosuppressive activity asdetected by the mitogenic effect of phytohaemagglutinin on peripherallymphocytes. HONever, PAPP-A specifically and non-competetively inhibitedgranUlocyte elastase but did not inhibit trypsin, chyrrotrypsin,plasmin or collagenase activity. This data suggests that PAPP-Acontributes to the protease inhibitor pool, which includes alpha-2­macroglobulin, alpha-I-antitrypsin and antithrombin III in the maintenanceof proteolytic homeostasis. The checking of proteolytic attackoriginating from maternal leucocytes and. directed against the depositedsperm and subsequently the embryo and placenta, is essential for humanreproduction.ENSATORY HYPERTROPHY AND OVULATION IN SYMPATHETICALLYeO~ENERVATED RAT OVARIES AFTER UNILATERAL OVARIECTOMY63S.N. wylie and W.R. Gibson. el t n Victoria, 3168.Department of Physiology, Monash Universkty, ay 0 ,henomena displayed by the ovary remaining afterThe compen~atory p OLO have classically been explained by alterunilateralovar:Lectomy ( ) 1 Recent work however, suggests thatations in hormonal feedback o~Ps'and hypothalamus may also be involved· ls between the ovarkes d 1neuraIskgna .' thought to influence compensatory a rena(1). A ~eural mechan::: r~:ilateral adrenalectomy (2). In the p~esentgrowth wh:Lch .o~curs a 1 t 1: sympathetically denervated Ovarkes to5 tudy the abl. h ty of comp e e'yft OLO was determJ..ned.compensate a er . ther unilaterally denervated or sham-Cycling, adult rats were ek Denervations were achieved byt d on the day of metoes t rus. . tdenervae. . vascular pedicle and suspensory ll.gamenbriefly freez :Lng the ovarl.an (3) 'Ulree days later the other ovary. t ly 1 cm from the ovary • dapproxl.ma e . d Additional rats were unoperate •was either sham-operated or rem~ve • vulation animals were killed and1\.fter the first or second ensul.~g 0 ovaries were weighed and examinedovaries and :,vi~UC~~v:~~:nr:=~:: 'fluorescence microscopy. Numbers offor adrenergl. c knn . b countin tubal oocytes.owlations were determJ..ned Y g d . exhibited increases (pBoth sham-denervated. and ~e~~r::~e o:v;::::n rate after removal of< .05, unpaired t-test) ~n W~g increases in denervated ovaries weretheir partners (Table. 1 )d' t t e t) to those in sham-denervated ovaries.similar (p > .05, unpal.re - es. ovulation rates (mean ± SEM) of aenervated orTABLE 1. Wekghts a~d sham-o erating or removing the othersham-denervated ovar~es after p. t are significantly differentovary. values with the same. superscrkpfrom each other (p < .05, unpal.red t-test) •UNOP.SHAM ULOULOUnop.Sham den. Den. Sham den. Den.8 8 1ONo. of rats 9 12b a ba48±245±2 53±3 51±1ovarian 46±2Weight (g)f eg fg ge9.8±1.2 10.5±1.8 4.5±0.6Ovulation 6.3±0.9 5.8±0.5Rateadrenergic innervation is not required forWe conclude that ovarian ULOhypertrophy and ovulation after •the development of compensatoryTrends in Neurosciences .§: 87-88(1) Gerendai, 1., and Halasz, B.(1975). M.F. Neuroendocrinol. 19: 352-362(2) Engeland, w.e., and Dallman,(1975) •R.J. and wylie, S.N. Proc.(3) Gibson, W.R., Roche, P.J., summers,Aust. Soc. Rep. Biol. 15: 23 (1983).

6465CHANGE OF OVULATION RATE WITH TIME IN EW·ES IMMUNIZEDAGAINST ANDROSTENEDIONER.I. Cox, P.A. Wilson and M.S.F. WongCSIRO, Division f An'o ~mal Production, PO Box 239, IB acktown, NSW, 2148.I~munization of ewes against androstenedi .in the~r ovulation rate and sUbsequentl . one results, ~n an increase(1). The aim of this study was t y ~ncreased lamb~ng performanceovul t' 0 measure how long th .a ~on rate was maintained followin' " e ~ncrease inMedium-wool M ' g LmmunLzatLon.erLno ewes (n = 168) aged 4 6allocated to treatment and tr 1 - years were randomly( con 0 groups Treat t 'androstenedione-HSA in DEAE dextran s ' • ~en w~th FecundinR1984 and 19 days later (April 3rd). vOlutLon,) was g~ven o~ March 15th,harnesses and crayons were . tr d asectomLzed rams wearLng Sire-sin..." , Ln 0 uced at the ti f ­LnJectLon, and remained with th me 0 the secondweekly. Laparoscopy was car ,e ewes throughout. Oes trus was r ecordeddd ,. , rLe out 15 36 50 d 65 dsecon LnJectLon. Androstenedio ." an ays after theintervals. ne antLbody titres were measured at. The data (Table 1) show that the' , .~n ovulation rate (O.R.) that was main~:~unLzatLon produced an increaseA~ ~h~ fourth laparoscopy the O.R. fO~ned ov~r at least seven weeks.sLgnLfLcantly above the control 1 b the Lmmune group was stillthat recorded previously. va ue, ut had declined (P < 0.01) fromTable 1. Ovulation rates f dr,or an ostenedione-immune andover a period of nLne weeks after immunization control ewesGroupControlAndrostenedione-immunen8484corpora lutea per ewes ovulatingOvulation Rate*Days after Second Injection15 36 501.42 1.22 1.161.67 1.56 1.62Cox, R.I., Wilson P A Ho k'J.M. and Bindon ' B ~ ·'(19:2)L.nson, R.M., ScaramuZZi, R.J., George511-514. ,. • Proc. Aust. Soc. Anim. Prod. 14;651.151.32Antibody titres (± s em) f I1:4023±708, 1:701±127 and ;:;6~±490(~0:ed18the expected course and werethe second injection F' ) at 1, 5 and 9 weeks after" • ewer Lmmune ewes sho dLmmedLately after the sec d '" we oestrus in the week, 'd on LnJectLon (p < 0 01) bLnCL ence of oestrus was si '1 ' • ut thereafter theTh mL ar Ln both groups. us, although antibody titres decl' '.remaLned markedly elevated in th' Lned rapLdly, ovulation ratesd . . e Lmmune group for~econ LnJ ection. Accordingly '" seven weeks after theLmmunization should be effect' '. Jo~nLng :-4 weeks after the secondLve Ln fLeld sLtuations.THE EFFECT OF MONENSIN SUPPLEMENT ON OVARIAN ACTIVITY AND RESPONSE TOFSH CHALLENGE IN EWE LAMBSSUMBUNG, F.P. AND WILLIAMSON, P.E.school of Veterinary Studies, Murdoch University, Murdoch, W.A. 6150.Monensin sodium(C36H6LOllNa - Elanco, Sydney) is a growth promotingsubstance which is produced by Streptomyces cinamonensis 1 • In heifersmonensin has been shown to decrease age and weight at puberty and toincrease ovarian function, peak LH levels and duration of the LH surgein response to exogenous gonadotrophin or E~ injection 2 ,3. In sheepmonensin supplementation has been observed to significantly increase inthe number of ewes mated in a limited breeding season {Schlink, pers. com,(1982)). The present study reports on LH release, rate of ovulation andpersistence of corpora lutea in prepubertal ewe lambs fed on a monensinsupplement and subsequently challenged with FSH.Thirty Corriedale ewe lambs (28 weeks) were divided into 2 groups, 15control (C) and 15 monensin fed (M). Ruminal fluid san~le~ were obtainedon days 0 (sampling commences) and 14. Five (C) and 5 (M) ewe lambs wereintensively bled for 6 hours at 20 minute intervals on day 15 of monensinfeeding and ovariectomised on day 16. The remaining ewe lambs wereinjected with FSH, given twice daily for 4 days (total 10mg/head), bledat 12 hour intervals and ovariectomised on the 5th day (SC & 5M) or 12thday (5C & 5M) after initiation of FSH treatment. Ovaries were weighedand follicles and corpora lutea counted and dissected. Follicles weredivided into small «2mm), medium (>2-4mm) and large (>4mm) size. groups.Corpora 1utea were defined as being functional through macroscopic andmicroscopic appearance.The M ewe lambs had better feed conversion efficiency and their ruminaIfluid contained significantly more (p

d6667Treatrrent Cycle 2 Cycle 3group--n----=:'O"'::;=-;CL;-::/7-e-~-----n--=:.c:::::;;::..,CL~/:-e~----C 16 1.94±O.ll 17 1. 94±0 .10 dL 19 1. 95±0.09 19 2.06±0.10aH 15 1.87±O.17 14 1.54±0.14 bValues with different superscripts differ significantly (p

68EFFECT OF FSH DOSE AND TREATMENT REGIMEON OVULATORY RESPONSE IN SHEEPJ. Eppleston*, R.J. Bilton** and N.W. Moore****School of Wool and Pastoral Sciences, University of New South Wales** P.O. Box 200, Orangeand'***Oepartment of Animal Husbandry,University of Sydney, Camden.Purified Follicle Stimulating Hormone (FSH) usually of porcineori~in is widely used in cattle to induce multiple ovulation (1,2).Opt~mal responses have occurred when the total FSH dose 'is given astwice daily injections for four consecutive days on a decreasingdose regime (2). In sheep, Pregnant Mare Serum Gonadotrophin (PMSG)has remained the major superovulatory agent with only few reportsof the use of gonadotrophins of pituitary origin (3, 4).The present study was designed to examine the effect of dose FSH(FSH-P, Burns.Biotec, Omaha, USA) and of dose regime on ovulatoryresponse. Mer~no ewes were treated with intravaginal sponges(Repromap, Upjohn) for 13 days. On Day 10 after sponge insertionewes were tr~ated with either 4, 6 or 9 mg FSH divided equallybe~ween morn~ng and night injections. They were then injected twiceda~ly for the following 3 days using a decreasing or a constant doseregime.TABLE 1.Ovulatory response of ewes to FSHSTARTING DOSE DOSE TOTAL NO. MEAN NUMBERFSH REGIME DOSE EWES(mg/day)(mg)C.L. EmbryosOverall there was no effect of starting dose FSH, total dose FSHor of dose regime on either ovulatory response or number of embryosrecovered but there were marked variations within groups on the numberof ~orpo:a lutea. Clearly, a decreasing dose regime, with consequentsav~ngs ~n FSH was not associated with any major reduction in OVUlatoryresponse.1.2.3.4.4 Constant 16 7 7.9 4.9Decreasing 10 7 6.7 4.3Total 14 7.3 4.66 Constant 24 7 6.9 3.9Decreasing 15 7 9.3 7.0Total 14 8.1 5.49 Constant 36 7 13.9 5.0Decreasing 22 7 6.7 3.2Total 14 10.3 4.1TOTAL 42 8.6 4.7Elsden, R.P., Nelson, L.D. and Seidel, G.E. Theriogenology ~:17-26 (1978).Chupin, O. and Procureur, R. Anim. Repord. Sc. ~: 11-23 (1983).Moore, N.W. and Shelton, J.N. J. Repred. Fert. 7: 79-87 (1964).Boland, M.P., Crosby, T.F. and Gordon, I. Anim.-Reprod. Sc. 6:119-127 (1983).INHIBITION OF MITOSIS BY OVINE FOLLICULAR FLUID IN VITRO69Ronald Carson and Marianne deGarisMedical Research Centre, Prince Henry's Hospital, Melbourne 3004.Differentiation and proliferation of granulosa cells is necessaryfor development of ovulatory follicles. Whereas pitUitary gonadotropinsare known to be responsible for differentiation, estrogens andpossibly serum growth factors present in follicular fluid are thoughtto stimulate mitosis of granulosa cells. Thus, fluid obtained fromestrogenic, preovulatory follicles would be expected to stimulatemitosis. On the contrary, charcoal-adsorbedovine follicular fluid(oFF) inhibited follicular development and mitosis of gran~losa cellsby a direct ovarian effect when administered to intact ewes (1).In an attempt to resolve this question, we have examined theeffect of oFF collected from large ovarian follicles on mitosis invitro. . ­-----Swiss mouse embryonic fibroblasts (3T3) were cultured inDulbecco's Minimum Essential Medium supplemented with 1% (v/v) foetalcalf serum (DMEM-FCS) and 3H-thymidine (0.1 ~Ci). After 48 h, platedcells were washed (x3) and 3H-thymidine activity in cell lysatesdetermined. All determinations were quadruplicate and cell growthover the cultured period, determined as 3H-thymidine incorporation,was linear. Pooled oFF was aspirated from large (>3 rom) folliclespresent on the ovaries of animals at random times of the breedingseason. 3T3 cells were cultured in the presence of a range of dosesof this material (1-100 ~l/wellj 0.2-10% v/v) or oFF treated withcharcoal-dextran (Table 1).TABLE 1: Dose-dependent inhibition of 3H- t hymidine incorporation(mitosis) of 3T3 cells by oFF in vitro. Values represent mean %control (±SE). Control: DMEM-FCS.DOSE (7. v/v)PREPARATION0.2 0.4 1 2 4 10oFF 75.1 83.9 75.5 40.8 15.3 0.8(6.6) (6.6) (2.9) (1.4) C1.0) (0.2)oFF (charcoal) 50.7 55.5 55.3 28.4 10.7 3.0(4.1) (3.5) (4.2) (2.9) (0.1) (0.1)When cultured initially in oFF (4% v/v, 48 h) and subsequentlyfor 48 h in the absence of oFF, 3H-thymidine incorporation (48-96 h)was not different from control. (Control 3822 ± 414 cpm, oFF:wash3776 ± 698, oFF: 1381 ± 218). Neither cell number nor morphology wasaltered.This study indicates that oFF obtained from large folliclesinhibits mitosis of cells in a dose-dependent and reversible manner.Reversible inhibition of granulosa cell mitosis by intra-ovarianfactors might therefore have a role in determining patterns offollicular growth and ovulation ~.(1) Cahill, L.P., Driancourt, M-A, and Findlay, J.K. Proc. 17thAnn. Meet. Soc. Study Reprod. (Laramie, Wyoming) July 1984.

70SUPEROVULATION AND EMBRYO RECO\~RY IN THE MERINOM.B. Nottle, D.T. Armstrong. M.K. Holland and P.R. SharpeDepartment of Animal Sciences, Waite Agricultural Research Institute,The University of Adelaide. Glen Osmond, South Australia 5064.Superovulation and embryo transfer (SOET) in sheep has beenlimited by a) variable responses to PMSG and b) fertilisation failurefollowing insemination by conventional means. Porcine pituitarypreparations (FSH-P; Burns-Biotec) are now available as an alt€rnativeto PMSG. Together with intrauterine insemination FSH-P has producedpromising results in comparison with PMSG and cervical deposition(1,2). As part of an ongoing SOET programme in sheep we have extendedthese findings to the Merino and compared FSH-P with PMSG treatment andintrauterine with cervical insemination.Thirty two 4-6 year old Merino ewes were randomly assigned to oneof two groups. One group was injected with PMSG ( 1200 i~Folligo~Intervet) 48 h before sponge removal. The other group received FSH-P(22.5 mg; Burns-Biotec) in a series of 8 injections beginning with 5 mgand continuing with 2.5 mg every 12 h. 60 h before sponge removal.Inseminations were carried out 48±0.5 h after sponge removal.using techniques described previously (1). Semen was collected byelectroejaculation, diluted with Delbeco's PBS and maintained at 30 c C.A total of 80xl0 6 motile sperm were used for intrauterine inseminationand 400xl0 6 sperm for cervical deposition.Oviducts and uteri were flushed on days 2 and 6 (day 0 = day ofinsemination) via mid-ventral laparotomy using techniques describedpreviously (2). Ovulation rate, ova recovered and stage of cleavagewere recorded for each ewe. Results are summarised in Table 1.Table 1 Ovulation rates, egg recovery and embryo recovery in PMSFSH-P treated ewes inseminated at cervical or intrauterine sitesSOV Site of Meants.e.m. per ewetreat. insem. Ovulations Eggs recovered Fert. eggsPMS cervix B.OtO.S duterus 7.UO.S aFSH-P cervix l4.9±1.1~uterus 13.4±0.9Within each column, means withdifferent (P

Experiment 1 examined the response of three genotypes of Merinoewes (T, 0 {l} and normal fine wool) during the breeding season. Onday 7 of the cycle ewes received either one or three subcutaneousimplants each containing 375 mg of progesterone (Sil~Estrus, AbbottLaboratories) and 100 ~g Cloprostenol (ICI). The implants were leftin place for 9 days.TABLE 1. The effect of number of progesterone implants on ovulationrate (OR±SE) per ewe ovulating in Merino ewes72THE EFFECT OF PLASMA PROGESTERONE CONCENTRATION DURINGTHE LUTEAL PHASE O~~ SUBSEQUENT OWLATION RATE IN SHEEPL.J. Cummins and T. O'SheaPastoral Research Institute, P.O. Box 180, Hamilton 3300 andPhysiology Department, University. of New England, Armidale 2351High fecundity genotypes of sheep have higher plasma progesteronelevels during the luteal phase of their oestrous cycles than the lowfecundity genotypes with which they have been compared. Two experimentswere carried out to determine if the plasma progesterone concentrationduring the luteal phase influenced the ovulation rate (OR) atthe subsequent oestrus.Experiment 2 was a 2 x 2 factorial design to examine the effect ofthe number of Sil-Estrus implants and the dose of PMSG on OR in finewool Merinos during the seasonal anoestrous period. The implants wereleft in place for 14 days and PMSG (Folligon Intervet) was given on theday of implant removal.In both experiments blood samples taken during the period of implantationconfirmed the expected differences in plasma progesterone concentration(i.e. 1 implant maintained a concentration of ~0.7 ng/ml and 3implants ~2. 2 ng/ml). The results are shown in Table l.EXPERIMENT 1 EXPERIMENT 2No. of implants n OR No. of implants n OR1 28 l.2±0.1}ns 1 21 l.3±0.1}3 30 1.2±O.1 3 22 1. 7±0.4 nsGenotypePMSG0 21 l.O±O.O}p< 225 iu 22 l.l±O.l}P165min on d2, compared with saline: 79±8 min dl, 109±26 m~n d2. Thepreovulatory surge of LH which occurred in the saline infused ewes(peak 68±14 ng/ml) was supressed in the EGF infused ewes (peak 7±2ng/ml). FSH concentrations remained low after the LH peak in the .saline infused ewes, but rose to high values (10-20 ng/ml) on d3-S ~nthe EGF infused ewes. All of the saline infused ewes were marked ond3 but none of the EGF infused ewes mated. Despite these changes, EGFdid not interrupt the regular course of P cycles, as was observed inthe luteal phase infusions. . .Although EGF apparently does not affect luteal secret~on ~nthe ewe, it prevents the p:eovulatory E 2 se retion by the follicle.7It is not clear whether th~s results from d~sturbances togonadotrophin secretion, or from a direct effect on the ovary, withthe disturbance of the gonadotrophin patterns resulting from thealtered feedback.(1) Waters, M.J., Whip, T.A., McGrath, P., Manley, S.W. and Bourke,J.R. (1983) proc. Endocrine Soc. Aust. 26, 39.(2) Singh-Asa, P. and waters, M.J. (1983) MoL cell Endocrinol. 1.'189-99.

74ANTIBODY LEVELS IN THE EWE AND ITS PROGENY FOLLOWINGSTEROID IMMUNIZATIONP.A. Wilson, R.I. Cox and C.J. WastieCSIRO, Division of Animal Production, PO Box 239, Blacktown, NSW, 2148.In ewes immunized against steroids to increase ovulation andsubsequent lambing rates, the initial antibody level gradually declinesuntil it is relatively low at parturition. Hence the amount of antibodyavailable for transfer from ewe to lamb via colostrum is low and doesnot affect the SUbsequent performance of the female lamb (I). Howeverthe quantitative relationships between the maternal and lamb antibodylevels in steroid immunized ewes needed to be further evaluated.Border Leicester Merino ewes previously immunized in 1982 againstandrostenedione-7a-carboxyethylthioether:human serum albumin in DEAEdextranadjuvant (2) were given a booster injection in 1983 and 7 dayslater, the treated ewes together with control ewes were joined withDorset Horn rams for 5 weeks. Blood plasma samples were taken at thebeginning and end of joining and 11 days prior to the commencement oflambing. The ewes were confined to small lambing paddocks and kept underregular observation. Blood samples were taken from the lambs prior tosuckling (in those instances where the birth was observed), 2 days and14 days after birth. Antibody titre was determined by measuring thedilution of plasma which bound 50\ of the maximal specific binding of3H-labelled steroid at 4°C under standardized conditions (Table 1).Table 1. Antibody levels in immunized ewes (n ~ 28) and their progenyReciprocal antibody titres (± s.e.m.)EwesLambstBeginning End of Near Pre- 2 days 14 daysof joining joining Lambing suckling*4234 751 237 0 604 301±910 ±193 ±57 ±150 ±69* n ~ 5For multiple births, the mean titre of the lambs was taken, i.e. one"lamb titre" for each ewe; twin lambs had very similar antibody titre.Non immunized ewes and their progeny (N = 8) had no detectablestero~d antibodies. At two days, the immunity of the ewe and its progenywas hl.ghly correlated (r =:; 0.95). The antibody level of the progenyseveral days after birth is however significantly greater than that ofthe immune ewe (p < 0.05) suggesting that there is an enriched transferof antibodies via the colostrum.75EFFECT OF HIGH LEVELS OF ANDROSTENEDIONE IMMUNITY IN PREGNANT EWES ONTHE SEXUAL ACTIVITY DISPLAYED BY THEIR MALE PROGENYP.E. Mattner, B.W. Brown, M.S.F. Wong, P.A. Wilson and R.I. CoxCSIRO,Division of Animal Production, Prospect, N.S.W.In males, androgen influence at about the time of differentiationof the gonads is necessary for functional organization of the centralnervous system (CNS) centres which later control sexual behaviour(1). The present studies examined the effect of high levels ofandrostenedione immunity during early pregnancy in ewes on the levelof sexual activity in their male progeny.Fifteen Merino rams born to ewes actively immunized againstandrostenedione-7-HSA (antibody titres > 1:8,000 at mating) and 15Merino rams born to non-immunized ewes were given 3 libido tests (2)when 18 months old. The mean numbers (± S.E.M.) of services performedby rams from the 2 groups were 4.1±0.9 and 8.3±0.8 respectively (p

76NATURE OF "DEFECTS" IN CORPORA LUTEA FOLLOWING INDUCTION OF OVULATIONDURING SEASONAL ANOESTRUS IN THE EWEJ. D. O'Shea, R. J. Rodgers and P. J. Wright*Departments of Veterinary Preclinical and Clinical* Sciences,University of Melbourne, Victoria 3052.Ewes can be induced to ovulate during seasonal anoestrus by bolusadministration of LH-RH, but resultant corpora lutea (CL) are oftenassociated with subnormal plasma progesterone concentrations. Wehave investigated the nature of the IIdefects" leading to low plasmaprogesterone under these conditions.All of 31 seasonally anoestrous Corriedale ewes given 3 x 30 PgLH-RH i.v. at 90 min intervals showed a plasma LH surge, but only 16of these ewes ovulated. Resultant CL were smaller than those of cycliccontrol ewes during the ovulatory season (310 ± sem 45 vs 520 ±34 mm3, P

d78PROGESTERONE PRIMING OF POSTPARTUM OVARIAN FUNCTION IN BEEF COWSP.H. Sharpe*, T.D. Carruthers, P.E. McKibbon and J.G. MannsDept of Veterinary Physiological Science. University of SaskatchewanSaskatoon. Canada and *Dept of Animal Sciences, Waite Agricultural'Research Institute, Glen Osmond, South Australia 5064.Small, short-lived peaks of progesterone (P4) have frequently beenobserved in jugular serum of postpartum cows prior to the normal lutealphase rise (1). This transient P4 release may have a role instimulation of the hypothalamic-pituitary-ovarian axis and onset ofnormal oestrous cycles (2). Exogenous P4 treatments are sometimeseffective in stimulating the onset of postpartum ovarian activity (3).The objective of this study was to determine whether a p4 treatment(designed to mimic these short-lived P4 peaks) would influenceconcentrations of gonadotrophin receptors or steroids in the largestovarian follicles of beef cows on days 0 or 7 of their first postpartumoestrous cycle.Ovaries were collected within 6 h of onset of oestrus or 7 dayslater from 21 Hereford cows which had received daily i.m. injections of100 mg P4 or vehicle on days 32-34 postpartum. Steroids were measuredin follicular fluid (ff) by r.i.a. Gonadotrophin receptors werequantitated by measuring binding of radiolabelled hCG and FSH toovarian tissues.Concentrations of P4, testosterone (T) and oestradiol (OE) in ffand concentrations of receptors for luteinizing hormone (LH) andfollicle stimulating hormone (FSH) in thecal and granulosa tissue ofthe largest follicle from each ovary are given in Table 1.Table 1:Mean (±SE) concentrations of steroids in follicular fluid (ng/ml) and of receptors for LH and FSH in theca and granulosa (fmol/mg)Day Theca Granulosa GranulosaoP4T ...QL LH-R LH-R FSH-R271±59* 43± 17* 279±83* 0.16±0.03 3.2±1.5 0.43±0.097 130±47 l4±6 l18±43 0.15±0.03 3.l±0.7 1.5 ±0.7**p Once Once > Once10/20 (50)13/15 (82)9/11 (82)8/10 (80)79Ewes inseminated at their first oestrus after the withdrawalof progesterone impregnated sponges ar~ considered to require greaternumbers of spermatozoa than ewes insem~nated at a subse~u~nt oestrus,if optimum fertility is to be achieved (1). Under cond~t~ons ofnatural mating additional sperm could be acquired through more~r~quentservice. We have tested the hypothesis that the effect on fert~l~ty ofmultiple services of ewes will be greater when the ewes are mated at thefirst than when mated at the second oestrus after sponge removal.Merino rams had continual access to oestrouS ewes for sevendays. For the first 2 days mating was with spayed ewes in which oestrushad been induced using exogenous progesterone and oestrogen.This depleted epididymal reserves of spermatozoa, so tha~ ejaculatescontained relatively constant numbers of spermatozoa dur~~g the s~sequentfive days of mating with entire ewes. Oestrus was ~nduced ~nentire Merino ewes using intravaginal progesterone sponges (60 mg MAP:Repromap) inserted for 12-17 days, followed by 400 I.U. PMSG (Intervet)on the day of sp0!1ge withdrawal. Sponge withdrawal was staggere~ sothat 6-9 ewes were in oestrus, per ram per day. Half were exp~r~encingtheir first oestrus after sponge withdraw~l, and half the~rsecond. Mating was carried out on two farms, ~n late January andearly February. Six rams were used on each farm. Pregnancy was determinedby laparoscopy, 33-43 days after service.TABLE Ewes pregnant at 33-45 days after service (%)21/39 (54)21/27 (78)16/20 (80)23/27 (85)Fertility was not influenced by the oestrus at which eweswere mated. Multiple versus single insermination had a beneficialeffect on fertility. This was significant for Farm A (P < 0.01) butnot for Farm B. .The numbers of spermatozoa present in single ejaculates fromthe rams were derived by flushing spermatozoa from the vagina. Thenumbers of spermatozoa per ejaculate were similar for the two farms(460±50 million, n=22, Farm A, 450±90 million, n=13. Fa~ ~). ~ese.numbers are higher than are considered necessary for a:t~f~cal.~nsem~nation,suggesting that the beneficial effects.o~ mult~ple mating arenot mediated through the mere provision of add~t~onal spermatozoa.(1) Allison, A.J. and Robinson T.J. (1971) Aust.J.Biol.Sci. 24;1001-1008.

80SIZE DISTRIBUTION AND OESTRADIOL PRODUCTION OF RAM INDUCED OVARIAN FOLLICLES5. Atkinson and P. WilliamsonSchool of Veterinary Studies, Murdoch University, Murdoch W.A. 6150.The introduction of a ram to a flock of anoestrous ewes has beenshown to temporarily increase the circulating levels of LH but significantlydecrease the circulating levels of FsH (1). This study was undertaken toassess the effect of the presence of the ram on the size distribution ofram-induced ovarian follicles and their ability to produce oestradiol (E ).southdown ewes in deep seasonal anoestrous were in contact with rams for 2 thefollowing periods of time: Oh (control), 40h, 3 days, 10 days and 20 days.The ewes in contact with the rams for 3 days were sampled 17 days later;during the intervening time they remained isolated from rams. This was tocompare the effect of brief contact with continuous contact at 20 days.At the end of the period of ram contact, all ewes were bled hourly forgonadotropin determination and then ovariectomised. Follicles >lmm diameterwere dissected from the ovaries. The follicles were measured and openedand drained of follicular fluid, then incubated at 37°C for 24 hours in 1mlof modified minimum essential medium.Ewes in contact with the ram for 40h were the only group which hadcorpora 1utea (CL) but all ewes exposed to the ram had large follicles(>4mm in diameter). By contrast, the control ewes had no large follicles(>4mm) , but significantly more medium follicles (2-4mm) and less smallfollicles than any other group. Ewes ovariectomised at 40h and 10 daysafter ram introduction, had the greatest number of small follicles andtotal ovarian structures. Ewes in contact with the ram for 3 days andovariectomised at 20 days, had the least number of ovarian structures.Table 1. Ovarian structures of ewes in contact with rams. Samesuperscripts are not different (p4mm 2-4mm 4mm) washighest after 40h ram contact and decreased significantly between 10 and 20days of ram contact.Small follicle formation was increased following the introduction ofthe ram and the number of follicles remained high for at least 10 days ofram contact. Follicle growth beyond 4mm was also stimulated by ramintroduction. These processes took place in a period of depressed LH andFSH activity. Ovulation occurred in 4 of 6 ewes within 40 hours of ramintroduction, but functional CLs were not present at 10 days. Oestradiolproduction occurred in all follicles >lmm taken from anoestrous ewes but wasenhanced by the presence of the ram, independent of gonadotropin levels.(1) Atkinson,s. and Williamson, P. (1984). J. Reprod. Fert. (In Press).M.PPhysiolOgy*, Monash Universi ty, Clayton,Departments ot Anatomy andVictoriaA number of studies have shown that LHRH or LHRH ~gon~sts will' ect l' nhibitory effect on testicular functlon 1,n v-z,VO and .aexert a d l.r . . 't 0 It lS'ulato effect on Leydig cell steroidogenesl.s -z,n v-z, 7' • ,stl.rn d ~ha~ LHRH action is through LHRH receptors found on Leydlgpresume wever as yet LHRH has not been identifie~ ,in :esticularc~lls. ~ an attempt to measure testicular LHRH a pur~flca~l.on schemetl.ss~e., ed consisting of an acid extraction, bulk fractl.onat~on on ODSw~~iC:~:lfiltration on Sephadex G15 and reversed phase HPLC. LHRH ~ss~ ~ b an in vit7'o bioassay using mono layers of an~er~ordeterIDl.ned i in culture (1) and by LHRH RIA using an antlserump~tuita~y ce~ Stg~:~ the Nand C-terminal regions of.LHRH. Lyophilisedd~recte agal. ns 'ately 600 mature Sprague-Dawley rats were dividedtestes from approxl.m. . f' d LHRH was added to two groups tointo four groups, to whl.ch purl l.e ou s fractionated as describedassess procedural losses, and all four gr . p. ' . the LHRHLHRH waS detectable by the -z,n 1J1..t7'O bloassay Inabo:e. f ~he HPLC chromatogram for testis extracts without added LHRHregl.o~ 0 1 . 1 ctivity was found in these fractions. Based onbut l.ffillUno oglea a f 14 6% as determined from the extracted samplesaverage recoverl.es 0 • / m dry wt., dd d LHRH the levels of LHRH were 8.5 and 2.4 pg g .conta~nl.ng a e , '1 f the two pools, or equivalent tooriginal testis powder respectl.ve y or0.8 pg/testes.fILHRH found in the adult rat testis suggestThe very low leveso, ' effect on thethat a) LHRH is unlikely to exert a significant bl~loglcal ~ . be ad b) the resence of LHRH ~n the testls maytestis in the adult, an . ? ~ Th role of the LHRH receptors onresult of plasma LHRH contaml.natlon. eLeydig cells still remains enigmatic.1.Scott, R.S., Burger H.G. and Q.1igg, H. (1980)1536-1541.81THE MEASUREMENT OF LHRH IN RAT TESTICULAR EXTRACTSHedger,no.bertson, C A.D.:.:M.:...::-:",:.==-~:::.=-;:...;..._'This study was funded by the N.H.Browne* and D.M. de Kretser& M.R.C. of Australia.Endocrinology 2Ql:

82INTRA-TESTICULAR CONTROL OF LEYDIG CELL FUNCTION •H. Ishida, G.P. Risbridger, D.M. de Kretser.Department of Anatomy, Monash University, Clayton, Vic. 3168.We have recently demonstrated that rat testicular interstitialfluid contains a factor which is not LH, capable of stimulating Leydigcell testosterone (T) production and may thus play a role in the localcontrol of testicular function ( 1). Exposure of the testis to a singleepisode of heat treatment results in a temporary disruption ofspermatogenesis and an alteration in capacity of the Leydig cells toproduce T, indicating that the function of the Leydig cells isinfluenced by the state of the seminiferous tubules (2). The aim ofthe present study was to determine whether or not there were changes inthe stimulatory activity of interstitial fluid which were correlatedwith the disruption of spermatogenesis and alteration in Leydig cellfunction after exposure of the testis to heat.The scrota of adult rats were immersed in a 43°C waterbath for 15mins, and studied 1-8 weeks after treatment. Isolated Leydig cellswere prepared by collagenase digestion and the capacity to produce basalor gonadotrophin-stimulated T was examined by incubation in vit~o for 3hat 34°C. Two or 8 weeks after heat treatment interstitial fluid wascollected and the stimulatory activity determined as describedpreviously. Briefly, we have demonstrated that charcoal treatedinterstitial fluid (CIF) stimulates the production of T in a dosedependentmanner using an in vit1"o rat Leydig cell bioassay. In thepresence of maximally stimulating doses of gonadotrophin the addition ofCIF will further increase T production. This stimulatory activity ofCIF was not inhibited by pre-incubation with antiserum to rLH.Two weeks after heat exposure the capacity of Leydig cells toproduce T in vit7'o was significantly (p

....84AMINO ACIDS IN THE GENITAL DUCTS OF THE MALE JAPANESE QUAILJ. Clulow and R.C. JonesUniversity of Newcastle, Newcastle, N.S.W.S~u~ies o~ the domestic turkey and fowl (1) indicate that thecompos~tlOn av~an semen differs considerably from mammalian semen inthe maln organlc constituent is gluatamic acid (concentration =65-80mM) Howe~er, due to difficulties in obtaining uncontaminatedsamples of fluld from the male genital ducts, it was not possible toc~nclude whether the glutamate originated from the testis or mored~stal parts of the genital ducts. Consequently, we have developedmlcropuncture procedures to resolve this problem and to investigatethe occurrence of other amino acids in the male genital ducts of theJapanese quail.Sexually mature male Japanese quail were anaesthetized (InactinB~k Gulden, Konstanz) and maintained on a heated operating table whilemlcropuncture samples were collected from the testis epididymal regionand vasdeferens (3-4 replicates). Samples (0.5-1.0~1) were centrifugedto remove sperm and amino acids were separated by thin layerchromatography (TLC), using single and double dimension solventsystems (2,3) and stained with ninhydrin. Quantitative estimationswere made by spectrophotometer after recovering ninhydrin stainedsamples and standards from TLC plates.Glutamate (Gt) and Glutamine (Gn) were identified in fluids from~ach ~f the genital ducts sampled. Rf values for Gt and Gn wereldentlcal for sta~d~rds and duct samples with all solvent systemsemployed. Gn stalnlng was generally more intense in seminiferoustubule and effer~nt duct fluids and Gt in ductus epididymidis andvas ~eferens flulds~ Quantitative estimations for the efferent ducts(Gt x = 1.2 mM, Gn x = 4.9 mM) and distal vas deferens (Gt = 8.5 mM,~n = 1.3 ~M) also suggested a decrease in Gn concentration and anlncrease ln Gt along the efferent ducts..This study has shown that Gt and Gn are present in bothtestlcular and excurrent duct fluids in appreciable amounts. Gtappears to be presen~ in lower concentrations than reported for thefowl ~n~ tU~key. Whlle Gn has been reported in the fowl (4), itsquantl~lcatlon ~a~ not been reported. On the basis of these resultsa testlcular orlgln for Gt and Gn in avian genital duct fluids seemspro~able. Chan~ing concentrations along the tract may be due tofluld reabsorptlon, utilisation by sperm or enzymic conversionbetween Gn and Gt.(1)(2)Lake, P.E. (1966) Adv. Reprod. Physiol. 1, 93-123.Horton, D. et. al. (1966) J. Chromatog. 23,309-312.((~)) Jones, K. and Heathcote, J.G. (1966) J. Chromatog. 24, 106-111.Chubb, L.G. and Cooper, D.M. (1962) J. Reprod. Fert~±, 7-12 .85R.C. Jones and K.M.TABLE. Reabsorption of fluid by the ductuli efferentes.Echidna Elephant TarnrnarSample Sperm - % Sperm - % Sperm - %Site atocrit Reabsorbed atocr i t Reabsorbed atocrit ReabsorbedRete testis 1.5 1.2 1.5Prox. initial segment 5.8 74 26.3 96 11.3 87In studies on the echidna, elephant and tammar, quantitative proteindeterminations showed that the ductuli reabsorb about 50% of the protein leavingthe testis and PAGE of the micropuncture samples showed that this involves theremoval of a couple of specific proteins from the luminal fluid .(1 )(2)(3)(4)THE MAMMALIAN EPIDIDYMIS: STRUCTURAL DIFFERENTIATIONAND FUNCTIONS OF THE DUCTULI EFFERENTESIt is suggested that the ductuli efferentes play an important role in thematuration and storage of spermatozoa in the mammalian epididymis.JurdDepartment of Biological Sciences, University of Newcastle, N.S.W.We have studied the ductuli efferentes in the echidna (I), Africanelephant (2), rat and tammar (Macropus eugenii). Although there is somevariation between species the ductuli are usually anatomically differentiatedinto an initial zone where the ductuli course roughly parallel with one anotherand coni vasculosi where the ductuli are pigmented, follow a very sinuous pathand anastomose with one another. An ultrastructural study on the rat showedthat the epithelial cells in the initial zone contain more vesicles andvacuoles than in the coni where the cells contain numerous dense bodies.Micropuncture studies on the echidna, elephant and tammar have shown thatthe ductuli reabsorb most of the fluid leaving the testis (Table). Stereologicalstudies on the rat confirm these findings and provided an estimate of the rate offluid transport across the epithelium which shows that the transport is greaterin the ductuli than in the proximal convoluted tubules (12.7 vs 2.2 ~l .cm-Z.hr- 1 ,4). Further, estimates of the amount of fluid transported across the epitheliumin vesicles and vacuoles did not support the proposal (3) that these structuresare the vehicles for most of the fluid transport.Djakiew, D. and Jones, R.C. (1983) J. Reprod. Fertil. 68, 445-456.Jones, R.C. (1980) J. Reprod. Fertil. 60,87-92.Flickinger, C.J., Howards, S.S. and English, H.F. (1978) Am. J. Anat. ~,537-585.House, C.R. (1974) Water transport in cells and tissues. Ed. Arnold. P.429.

86TESTOSTERONE AND THE IMMATURE BOVINE EPIDIDYMISMichael K. Holland and David E. BrooksDepartment of Animal Sciences, University of Adelaide,Waite Agricultural Research Institute, Glen Osmond, SA 5064.Testosterone (T) and its metabolite dihydrotestosterone (DHT)influence both the overall rate and the regional pattern of proteinsynthesis and secretion by the epididymis of mature animals (1).Castration results in decreased protein synthesis and in selectivechanges in the regional pattern of proteins secreted. Immatureanimals, in which the testes are not producing T, have a higherrate of protein synthesis than mature animals but do not exhibitregionality of protein secretion (2). This study reports an invitro and an in vivo attempt to assess the relationship betwe;;peripheral T and the onset of regional protein synthesis by theimmature epididymis.Experiment 1: Epididymal tissue minces (25 mg) from bullcalves (10 weeks old) were incubated in 1 ml of Krebs Ringerphosphate buffer, pH 7.4, at 34°c for 5 h in the presence ofvarying concentraions of T (1-10,000 ng/ml). The medium wassupplemented with glucose and amino acids including 10-15 uCi of35 S methionine. Radioactive proteins were harvested and a~alysedby electrophoresis as previously described (2).Experiment 2 : Bull calves (10 weeks old) were implantedsubcutaneously with silastic T releasing implants. At varyingtimes thereafter the animals were castrated and the pattern ofprotein synthesis determined using essentially the method describedabove.In vitro incubation with T at up to 10 ~g/ml did not alter therate or pattern of proteins secreted by the testis or caput, corpusor cauda epididymis. Administration of T via silastic implantselevated peripheral blood T levels to 2-5 ng/ml which is comparableto adult values. As a consequence the epididymis showed precociousgrowth which was especially marked in the caudal region. "Thiseffect is rapid, being readily detected after 1 week of treatment.However, no concomitant change in the profile of proteins secretedwas detected.It is concluded that neither acute nor chronic exposure of theimmature bovine epididymis to T results in any change in theregional profile of proteins secreted. Chronic exposure to T doeshave an anabolic effect on the immature epididymis, especially thecaudal region. The relevance of this for the development of thenormal adult regional pattern of protein synthesis is uncertain butit would seem that a factor(s) other than T must be involved.(1) Brooks, D.E. Aust. J. BioI. Sci. Ji: 205-221 (1983).(2) Holland, M.K. and Brooks, D.E. Proc. Aust. Soc. Reprod. BioI.(Abstract 86 ) (1983).87PATTERNS OF PROTEIN SECRETION ALONG THE LENGTH OF THE MATURE OVlNEEPIDIDYMIS IN VITROMary J. J. Carabott and D. E. BrooksDepartment of Animal Sciences, The University of Adelaide,Waite Agricultural Research Institute, Glen Osmond, SA 5064A number of proteins which are synthesized in the epididymis andsecreted into the luminal fluid become associated with spermatozoa(1,2,3). The binding of these secreted proteins to the surface ofspermatozoa is believed to alter their motility and fertilizingcapacity.The epididymis was divided into nine consecutive segments and atissue sample was also taken from the testis, in the rete testisregion. Tissue (25 mg) was removed from the centre portion of eachsegment, minced, washed free of spermatozoa and incubated at 34°C, 5 hin Krebs Ringer Phosphate pH 7.4 containing 5 roM glucose and 20 ~Ci35 S-methionine (4). The incorporation of radioactivity into secretedand total homogenate macromolecules was analysed by polyacrylamide gelelectrophoresis (PAGE) as described (1).Secreted proteins exhibited differences in the level of incorporatedlabel in different regions and ranged from 0.45-0.13% of theinitial added label. The protein profile as revealed by autoradiographyshowed regionality of protein secretion with unique proteinsappearing in the caput and proximal corpus regions. In contrast theprofile of proteins synthesized in the total homogenate showed novariation in different regions of the epididymis. A report (5) that novariation exists in different regions of ram epididymis was the resultof analysing the total homogenate only.We are presently extending our observations to two-dimensionalPAGE of regions that exhibit unique proteins as this provides greaterresolution of the protein mixture. In conjunction we are employingpolyspecific antibodies to spermatozoa membranes to elucidate whichsecreted protein(s) is absorbed onto the spermatozoa surface and fromwhich region(s) of the epididymis the protein(s) originate.(1) Brooks, D.E. and Higgins, S.J. J. Reprod. Fert ~: 363-375(2)(1980).Lea, O.A., Petrusz, P., French, F.S. Int. J. Androl. Suppl. 1:(3)592-607 (1978).Courtens, J.L., Rozinek, J., Fournier-Delpech, S. Andrologia 1!!,:509-514 (1982).(4) Brooks, D.E. Biol. Reprod. ~: 1099-1117 (1981).(5) Jones, R., Fournier-Delpech, S., Willadsen, S.A. Reprod. Nutr.Develop. ~: 495-504 (1982).

88PRODUCTION AND OUTPUT OF SPERMATOZOA AND EPIDIDYMAL SPERMRESERVES OF SEXUALLY RESTED OR FREQUENTLY EJACULATED DOGSP.R. Davies and I.C.A. MartinDepartment of Veterinary Physiology, University of Sydney, NSW, 2006. Daily sperm o~tput (DSO) from 7 dogs (Group A) was measured fromda~ly s~men collect~ons for 14-21 days after an initial 7-10 days ofcollect~o~ t.o stabilise extragonadal sperm numbers. These dogs werek~~l~d ~~th~n 30 min of the final semen collection, testes andep~d~dym~des were dissected and homogenised portions of these tissueswere sampled for sperm counts. A similar procedure was followed for41 sexually r~sted dogs (group B). Estimates were made of the dailysperm product~on (DSP) and of the extragonadal sperm reserves (1 2)The ,dogs ranged in body weight from 7.5 to 37.7 kg with t~ta~test~cul~r ~a:enchym~l weights ly~ng between 4.49 and 39.37 g.No s~gn~f~cant d~fference was detected in DSP between groups A andB. :rhe mean (:t SE) 0.!1 sper!!'l production was 14.69 (:t 0 33) _x 10 6 (gtest~cular Pgrenchyma) day and the mean was 343 x lOb day 1 (ran127-637 x 10 ). geThe mean numbers of sperm contained in the epididymis and meantransit time of sperm through it were:-A. Daily semen collection B. Sexually rested(7 dogs) (20 dogs)spe6'm Count Transit Time* spe/5m Count Transit Time(10 ) :tSE (days) :t SE (10 ) :tSE (days) +SECaput 67 15 0.36 0.05 68 7 0.37 0.03Corpus 687 113 3.75 0.02 657 42 3.69 0.13Cauda 388 66 2.20 0.02 766 42 4.48 0.23* Count in region .divided by DSP of the corresponding testis andassuming sperm resorption in the epididymis to be negligable.The cauda epididymis appears to be the principal site ofstorage of sperm, but this "reserve" is only of the order of twicethe DSP.The mean ratio, DSO:DSP (+ SE), of Group A was 70.6 (+6.0) %. The mean sperm content gf th; vasa deferentia of 5 ~ogs fro;;;group B (mean DSP = 432 x 10 ) was found to be 57 x 10 per vas(transit time = 0.28 days) indicating that sperm do not accumulate inthe vas deferens of the dog. Thus much of the difference between DSPand DSO is likely to be in losses of sperm into urine.A BICARBONATE AND CALCIUM DEPENDENT INDUCTION OF RAPID GUINEA­PIG SPERM ACROSOME REACTIONS BY MONOVALENT IONOPHORES.Ross V. Hyne89Department of Obstetrics & Gynaecology, University of Melbourne,parkville, Vic. 3052Monensin (Monensin A, Eli Lilly) and niger~c~n (Sigma) stimulatedrapid guinea-pig sperm acrosome reactions in the presence of extracellularNa+, Ca2+ and bicarbonate (HC03-jC02)' Extracellular K+,in contrast, was not required for the stimulatory effect of the ionophores.The effect of HC03-jC02 is concentration, pH and temperaturedependent, with maximal responses obtained with 50pM monensin or 25pMnigericin at a concentration of 30mM HC03-' 2.5% C02 and pH 7.8 at 25°C.A constant HC03- concentration (30mM) , monensin stimulated acrosomereactions within the pH range 7.5-7.8, whereas a higher or lower pH didnot support acrosome reactions at 25°C. A constant extracellular pH(7.8), monensin stimulated acrosome reactions in the presence of 30rnMHC03-' whereas higher and lower concentrations did not support acrosomereactions at 250C. The perrneant anions pyruvate and lactate wereessential to maintain sperm motility when treated with monensin underthese conditions. NH4Cl, sodium acetate and DIDS (25pM), an aniontransport inhibitor, blocked the ability of monensin to stimulateacrosome reactions. Veraparnil (lOOpM), a putative ca 2 + transportantagonist, in contrast, did not prevent the monensin induced acrosomereactions. Physiological concentrations of Na+ were needed formonensin to stimulate acrosome reactions, but high concentrations ofMg 2 + prevented the monensin stimulation. The Ca 2 + ionophore A23l87(75nM), also required physiological concentrations of Na+ for therapid induction of maximal acrosome reactions at an elevated_pH(8.3) but did not require the presence of extracellular HC03 •These studies suggest that a monovalent ionophore induced rise insperm intracellular Na+ concentrations is a pre-ca 2 + entry event,that stimulates an endogenous Ca2+jNa+ exchange that allows a Ca 2 +influx which in turn induces the acrosome reaction. The requirementof HC03- for the ionophore stimulated acrosome reactions, and itsinhibition by intracellular modulators, suggests there may be acritical intracellular pH for the acrosome reaction to occur.(1) Amann, R.P. and Lambiase, J.T. J. Anim. ScL, 28:369-3741969.(2) Davies, P.R. and Martin} I.C.A. Proc. Ann. Conf. Aust. Soc.Reprod. Biol. ~:51

90ATP INDUCED ACTIVATION OF DEMEMBRANATED RAM SPERMI.G. White* and J.K. Voglmayr**worcester Foundation for Experimental Biology, Shrewsbury,Massachusetts, 01545, U.S.A.*p~esent address: Department of Veterinary Physiology, University ofSyaney, N.S.W., 2006.** Present address: Medical Research Institute, Florida Institute ofTechnology, 150 West University Boulevard, Melbourne, Florida, 32901,U.S.A.This communication reports the development of a system for thedemembranation . of ejaculated, cauda and testicular ram sperm in anextract~on med~um containing the detergent Triton X-l00. The cond­It~ons for activation of the sperm by ATP have also been examined inrelat~on to the mechanism of maturation.The demenbranation procedure was based on that developed forhamster sperm (1). Activation after adding ATP was scored as thepercentage of sperm moving in a carbon-coated chamber of a microscopeshde.It was pos~ible to d~m~mbranate and reactivate not only freshlycollected test~cular, epId~dymal and ejaculated sperm but also spermthat had. been stored for several days at DoC and for several months at~196°C In egg yolk citrate media. Bovine serum albumin (1.0%)Increased the survival of sperm durng the washing process employed toprepare sperm samples but more than 0.25% in the extraction mediumdecreased react~vation. Triton concentrations between 0.01 and 1.00%In the extraction medium were satisfactory for demembranating thesperm.EJaculated and cauda sperm were reactivated immediately on additionof ATP and activity was evident for up to 10 min. Testicular sperm,on the, other hand, required a latent period of about 4 min beforeact~vat~on by ATP. The optimal ATP concentration for activation ofsperm was 0.1-1.0 mM.Magnesium ions (D.1-1.DmM) were important for activation andtest~cular sperm required a higher magnesium concentration than caudaor eJacula;ed sperm. , Manganese ions were almost as effective asmagnes~um .. or act~vatIng cauda and ejaculated sperm; cobalt andcalc~um ~ons were much less active. None of these ions were effectivefor,test~cular sperm. Fluoride (25-50mM), which binds magnesium ions,Inh~bIted the activation process.,The presence of 50 ~M C-AMP in the extraction medium or pre-incubat~onof, testIcular sperm with the phosphodiesterase inhibitors,theophyll~ne or caffeine, Increased activation, but this was notev~dent w~th ejaculated or cauda sperm.It is concluded that activation conditions for ejaculated and caudaram sperm are s~m~lar, but testicular sperm differed in some respectspo~nt~ng to changes associated with the sperm motility mechanismdur~ng maturat~on.(1) Mohri, H. and Yanagimachi, R. Exp. Cell. Res. i27:191-196 (1980)CHANGES IN GUINEA-PIG SPERM INTRACELLULAR SODIUM AND ?OTASSIUMCONTENT DURING CAPACITATION.Kim P. Edwards and Ross V.Hyne91Depa~tment of Obstetrics & Gynaecology, University of Melbourne,Parkville, Vic. 3052Recent studies (1) have indicated that the ability of variousmedia having an elevated pH to support guinea-pig sperm acrosomereactions, is related to culture conditions that increase the spermintracellular Na+ concentrations. In this investigation, we determinedwhether there is a net change in intracellular Na and Kcontent in treatments associated with the induction of the gUineapigsperm acrosome reaction.Guinea-pig spermatozoa were collected from the caudae epididymidesin various isotonic solutions and the intracellular sodiumand potassium content was determined by atomic absorption spectroscopyafter washing the spermatozoa twice with a Na+- and K+-free isotonicsucrose solution. The sperm intracellular sodium and potassiumcontent was found to be influenced by large variations in the extracellularconcentrations of these ions. Treatment of spermatozoasuspended in a saline-based solution with the monovalent ionophoresmonensin (Eli Lilly) or nigericin (Sigma) caused an approximate 2­fold increase in the intracellular Na+ concentration and a 2-6 folddecrease in the intracellular K+ concentration. Incubation of thespermatozoa in a K+-free minimal culture medium (MCM-PL) at a pH of7.6 or 8.3 for 2h caused an approximate 2-fold increase in the spermintracellular Na+ concentrations and a 5-fold decrease in the intracellularK+ concentrations. The motile spermatozoa incubated for 2hat pH 7.6 showed less than 5% acrosome reactions, compared with 30­40% acrosome reactions after incubation at pH 8.3, in response tothe addition of 5mM-Ca 2 +.These studies show that extracellular bicarbonate is not essentialfor monensin or nigericin to modulate the sperm intracellular Na+jK+quotient, however it is a requirement for the stimulation of a rapidacrosome reaction by these ionophores (2). The data thus demonstratesthat there is no need for an elevated pH for a rise in intracellularNa concentrations to occur. However, the bicarbonate-dependentcontrol of intracellular pH appears to have an important regulatoryfunction in determining whether guinea-pig spermatozoa undergo anacrosome reaction.(1) Hyne, R.V., Higginson, R.E., Kohlman, D. and Lopata, A.J. Reprod. Fert. 70: 83-94(2) Hyne, R.V. and Edwards, K.P. J. Reprod. Fert. (In press)(1984)

92EFFECTS OF MALONIC, MALEIC, CITRICHUMAN SPERM ANDPENETRA~_ION AND CAFFEIC ACIDS ON THE MOTILITY OFOF CERVICAL MUCUSP.D.c.Brown-woo~man, ,I.G.White+ P.y.W.Chow~ and E.J.PostDepartment of Blologlcal Sciences Cumberla dP.O. Box 170, Lidcombe, Australia: 2141 a d n + College of Health ~ciences,Physiology, University of Sydney,Sydney,nAustralla,Depa~tment of Veterlnary2006.This study was concerned with evaluatin f "for possible inclusion in vaginal g ,our organlC aC1ds as spermicidesorganic acids when introduced intoO~nc~~~~~al c~ntracep~ive ~evices. Suchcontraceptive effect by (a) low ' th cervlcal devlce mlght have a dualsperm motility and (b) alteringe~~:g h e,pH of the m:dium and hence reducingto make it an effective barr' t p ySlcal propert1es of the cervicaller 0 sperm entry.The spermicidal activity of malonic, maleic, citric andassessed when human sperm were su d' , caffeic acids was(O~ 0.0001, 0.001, 0.01, 0.1 and ~:~~ ~d ~~ solu~lons containin~ these acidsaClds completely immobilized the )" 3 trla~s 0.1% maloOlc maleic andthe medium from 7.5 to 4 5-5 5 ~perm,wlth~n 30 mln. by reducing the pH ofsoluble and 0.01% only r;duc;d'th affe~~,acld was less spermicidal and lessafter 30 min., although more mark:dm~~~ ltt y score fr~m a mean of 3.0 to 2.7ec s were ObV10US after 60 min.The effect of the four 'dcervical mucus was asse:~~dsb~nt~perm survival an~ migration in humanoration of 0.01% maleic maloni e sdPer~ p:netr~tlO? test (1). The incorp-'th' ' c an cltr1c aC1ds 1nto capillarie f'll dWl cervlcal mucus was sufficient to reduc d s ,1 epenetration density, although the motil't e epth of sperm penetratlon andgreatly reduced Thus in 5 test th l Y score of the spermatozoa was notwere control 2 1'+0 3 l' s e mean ±SE penetration (cms) after 10• _ • , rna onlC 0 7+0 4 l' 0 6In the presence of 0.1% malei . -1' ~ rna elc, . ,±O.O~ and citric 0.7±0.4.th ' c, rna onlC and cltr1c aC1ds no sperme mucus; a h1gher concentration (1.0%) of caffeic a 'd' ,prevent sperm penetration. Cl was requ1red toThe ultrastructure of the acrosome of human'microscopy following penetration of hu sp:rm was examl?ed by electronacrosomal proteinases may assist th man cervlcal mucus, Slnce release ofhuman sperm do not undergo th e process., Howe~er the data indicate thatmucus in vitro.e acrosome react10n whlle penetrating cervicalThe reduced penetration of mucus bof 0.01% could be due to alterati y s~e~: exposed to an organic acid concentrationa decrease ' , , on 0 e structure of the mucus, rather thanunaffected ~~ :~:rm mot1l1ty ~ince sperm motility within the mucus appearedmotilit is 1S concen~ratlon: Furthermore the pH was 6.5 to 7 and sperm, y only reduced 1n cerv1cal mucus at a pH below 6.5 (2).Irt lS ~onclluded that ~alonic, maleic and citric acids have proml'seO vaglna contraceptlves. as cervical(1)(2)MKremer, h' , J. and Kroeks, M.V.A.M. Acta Eur. Fertil. 6: 377-380 (1975)og 15Sl, . K.S., Dabreich, D., Levl'ne, J . andHeuhaus, O.W. Fertil.. Stenl. 15: 15-23 (1964)93THE EFFECT OF (S)-a-CHLOROHYDRIN AND 6-CHLORO-6-DEOXYGLUCOSEON THE METABOLISM OF GUINEA PIG SPERMATOZOA IN VITROA.R.Jones and S.A. FordDepartment of Biochemistry, The University of Sydney,Sydney, N.S.W. 2006(S)-a-Chlorohydrin exerts its antifertility actionwithin the mature spermatozoa of susceptible species by atwo stage process. The first stage is its conversion byoxidative metabolism to (S)-3-chlorolactaldehyde and thesecond is the inhibition by this metabolite of the enzymeglyceraldehyde 3-phosphate dehydrogenase. Such an inhibitiondecreases the ability of the spermatozoa to metabolisecarbohydrate for the production of ATP which is essentialfor their motility when they are ejaculated. A speciesthat is susceptible to the antifertility action of administereda-chlorohydrin is the guinea pig and aspects ofthis action have been investigated on the spermatozoa ofthe guinea pig in vitro.A concentration of 2.5mM (S)-a-chlorohydrin inhibitedoxidation of fructose (lmM) to lactate by cauda epididymalspermatozoa over a period of lh at 34°C. There was anaccumulation of the glycolytic intermediates fructose-l,6­bisphosphate, dihydroxyacetone phosphate and glyceraldehyde3-phosphate which is characteristic of the inhibition ofglyceraldehyde 3-phosphate dehydrogenase. Furthermore,there was a decrease in the concentration of endogenouslactate which is compatible with a block in the glycolyticpathway before the production of lactate and confirms that(S)-a-chlorohydrin has no inhibitory effect on processesinvolving the oxidation of lactate. When spermatozoa wereincubated with fructose (lmM) and (R,S)-3-chlorolactaldehyde(2rnM), a similar effect was observed which probablYindicates an identical mechanism of action. Incubation ofspermatozoa with fructose (lmM) and (R,S)-{3- 36 Cl}-achlorohydrin(5mM) for 2h at 34°C enabled a radioactivealdehyde to be isolated. This was identified as 3-chlorolactaldehydeof unknown configuration.Another compound which has an antifertility action invivo paralleling that of (S)-a-chlorohydrin is 6-chloro--­6-deoxyglucose (6-CDG). When spermatozoa were incubatedwith fructose (lmM) and 6-CDG (lOmM) for lh at 34°C, asimilar inhibitory effect was observed. Chemical analysisof the spermatozoa enabled the presence of 3-chlorolactaldehyde(of unknown configuration) to be detected.We postulate that (S)-a-chlorohydrin is metabolisedwithin guinea pig spermatozoa to (S)-3-chlorolactaldehydewhich is the active antifertility metabolite. In addition,the demonstration that 6-CDG is converted to (s)-(presumably)3-chlorolactaldehyde is the first evidence so far reportedthat links the similar antifertility activities of (S)-~chlorohydrinand 6-CDG to a common metabolite.

95EFFECTS OF NON-DIALYSABLE SUBSTANCES IN RAM SEMINAL PLASMAON RAM SPERMATOZOA DURING DILUTION, COOLING AND DEEP-FREEZINGP. Situmoranq and I.C.A. MartinDepartment of Veterinary Physiology, University of Sydney,TABLE 1: Percentage of I. intact apical ridgesof II. broken plasma membrane and loss of acrosomeDiluent a EY 15% EY 0 EYComposition 0 NOS 0 NOS 1.3% NOSSemen treatment I II I II I IIChilled 49 10 79 6 63 8Deep-frozen 6 69 43 50 21 59NSW,2006Removal of seminal plasma from the medium in which mamma11an Spermwere suspended and replacement with simple, defined solutions free ofmacromolecules caused a loss in motility of sperm and a tendency forthem to agglutinate (11. These effects could be partially countered bythe inclusion of a protein such as serum albumin. The motility of bullsperm inhibited by washing or extensive dilution, was restored by theaddition of fractions of seminal plasma (2). However, neither of thesereports contain information on the morphology of sperm treated withmacromolecules.The effects of non-dialysable substances (NOS) obtained fromseminal plasma on the motility and morphology of ram spermatozoa arereported in this communication.NOS were prepared by dialysing seminal plasma obtained from a poolof 200 ml of ram semen and then freeze-drying it. The dry matter yieldwas 2.6% w/v. The solution used for 20-fold dilution of the 3eJaculates studied was 200 roM tris (hydroxymethyl) aminomethane, 55 ~~fructose, 64 roM citric acid and 6.4% vlv glycerol to which was added15% vIv egg yolk (E. Y.) and/or 1.3% w/v NOS as required by theexperimental design. Both morphology and motility were assessed withthe light microscope (phase contrast) and for ultrastructural studies,the methods outlined previously (3) were used.EY significantly improved the percentage of motile spermatozoa inchilled or deep-frozen samples. Both EY and DNS had significanteffects on the state of the acrosome as shown in Table 1.(phase contrast)content (e .m. ).15% EY1. 3% NOSI II83 365 41The proportion of sections of mid-piece showing condensation ofmitochondria was significantly higher when the diluents did not containEY or NOS but motility and the morphology of the midpiece were notmarkedly affected by the presence of NOS.It appears that the proteins of seminal plasma can function tostabilise the membranes of the sperm head during reduction oftemperature and the effect is additive to that of the non-specificmacromolecules of EY.(1) Harrison, R.A.P., Dott, H.M. and Foster, G.C. J. Reprod. Fert.52:65-73 (1978).(2) Baas, J.W., Malan, P.C. and Shannon, P. J. Reprod. Fert.68:275-280 (1983).(3) Situmorang, P., Martin, I.C.;,. Proc. Aust. Soc. Reprod. BioI.]2:37 (1983).andREDUCTION OF LH PULSE FREQUENCY BY STEROIDS:DIFFERENCES BETWEEN MERINO AND SUFFOLK EWESG.B. Thomas, D.T. Pearce, C.M.Oldham and G.B. MartinSchoel of Agriculture (Animal Science), University of WA,Nedlands, Western Australia 6009Photoperiod controls the breeding activity in the ewe bychanging the responsiveness of the hypothalamo-hypophyseal axis tonegative feedback by oestrogen (1). The 'depth' of anoestrus variesbetween breeds and could be related to differences between the breedsin their sensitivity to oestrogen. This experiment investigated theseasonal changes in negative feedback by oestrogen and progesteroneon luteinizing hormone (LH) in Suffolk and Merino ewes which havemarkedly different breeding seasons.Sixteen cyclic Merino and 16 cyclic Suffolk ewes wereovariectomized in March and given either no implant (0) or a 3, 6, or12 mm implant containing oestradiol (n=4/group). Blood was collectedat 10 minute intervals for 8 hours during the breeding season. Anintravaginal progesterone (p) dispenser was then inserted and bloodwas sampled 10 days later. This sampling procedure was repeatedagain during anoestrus. Plasma was assayed for LH. P treatment+increased the concentration (ng/ml) of P from 0.42 ± 0.10 to 2.29-0.12.TABLE 1. Effect of oestradiol and progesterone on LH pulse frequency(mean/8 h) in Merino and Suffolk ewes.MERINOSUFFOLKoestradiol (mm)oestradiol (mm)Season P 0 3 6 12 0 3 6 12Breeding 9.0 7.8 6.0 7.8 10.8 12.3 12.8 13.5season + 4.5 2.5 2.8 3.8 6.8 4.8 4.8 7.3Anoestrous 10.2 7.5 2.0 4.0 8.2 1.7 1.0 0.0+ 7.8 3.2 2.2 3.0 6.2 2.3 0.6 0.7seasonDuring anoestrus oestradiol reduced the frequency of LH pulses(P(O.OOl) in both breeds (Table 1). This response was dependent onimplant size and Suffolks showed greater sensitivity than Merinos(P(O.Ol). Oestradiol had no effect during the breeding season,although 4/12 Merino ewes appeared to show a negative feedbackresponse. Progesterone suppressed LH during both seasons (P(0.05)and there was no synergistic effect with oestradiol in either season.These results show Merino ewes are less sensitive to oestradiolthan Suffolk ewes during anoestrus and this may explain thedifferential seasonality of the breeds.(1) Legan, S.J., Karsch, F.J. and Foster, D.L. (1977).Endocrinology lQl: 818-824.

9697GnRH IN CONTINUOUS LOW DOSE INDUCES OVULATION IN BOTH POST-PARTUM ANDSEASONALLY ANOESTROUS EWESA.H. Williams, P.J. Wright and I.J. ClarkeDepartment of Agriculture, Animal Research Institute; Departmentof Veterinary Clinical Sciences, University of Melbourne,Werribee 3030; Medical Research Centre, Prince Henry's Hospital,Melbourne 3003.We have assessed the value of continuous low dose GnRH treatmentfor the induction of plasma LH surges, ovulation and normal lutealfunction in ewes post partum during the anovulatory season. In otherstudies we have shown that this treatment induces ovulation inseasonally anoestrous ewes (1).Corriedale ewes (n=28) that were 22-33 days post partum on Day 1(D1=5th Dec. 1983) of the stUdy and seasonally anoest~wes (n=28)were allocated to 3 treatment groups (1) C-untreated controls, (2)P-progesterone from D-12 to 01 (Silestrus implants, Ceva Aust., 2 s.c.),(3) PG-progesterone, then GnRH (250ng/h s.c. from a mini-osmotic pump(Alza Corp., U.S.A., Model 2001)} from 01 to 09. Blood for LHdetermination was taken via jugular venous cannulae each 3h for 96hfrom the start of GnRH treatment, and for progesterone determinationtwice weekly for 4 weeks by jugular venepuncture. Ovulation wasdetected by 1aparoscopy on 012.THE USE OF RAMS, GnRH, AND IMMUNIZATION AGAINST ANDROSTENEDIONEAND OESTRONE FOR THE INDUCTION OF FERTILE OESTRUS IN ACYCLICPOST-PARTUM EWESP.J. Wright, P.E. Geytenbeek, I.J. Clarke and R.M. HoskinsonDepartment of veterinary Clinical Sciences, University ofMelbourne, Werribee, Victoria 3030; Waite Agricultural ResearchInstitute, Adelaide, South Australia 5064; Prince Henry'sHospital, Melbourne, Victoria 3003; C.S.I.R.O., Divisionof Animal Production, Blacktown, N.S.W. 2148.The endocrine basis of post-partum anoestrus in Merino ewes is thelack of appropriate secretion of luteinizing hormone (LH) (1). We haveassessed the value of 4 methods of increasing plasma LH concentrationsfor the induction of oestrus. The methods were (i) the introductionof rams to ewes that had been isolated from rams for 3 months, (ii)the s.c. administration of GnRH (125ng/h for 7 days) by mini-osmoticpumps (Alza Corp., U.S.A.), (iii) immunization against oestrone (E 1),and (iv) immunization against androstenedione (A4)' On day 1 (D1),when the ewes were 20-37 days post partum, fertile rams were introducedto the flocks. Progestagen (P) pretreatment was by pessary (Repromap,Upjohn, Australia) from 0-7 to 01. Immunogen was administered around80 and 110 days of pregnancy, and on 0-14.TABLE 1.Incidence of oestrus within 7 days of ram introductionRAMS WITH FLOCKTABLE 1. Plasma LH surges (>10ng/ml for >6h), ovulation, and normalTREATMENTCONTINUOUS (a) FROM OAY 1Pluteal function (plasma progesterone >lng/ml for >10 days) induced inUntreatedO/lO(b} 0/10NSanoestrous ewes.P4/20 16/19

98EFFECTS OF AN ANALOGUE OF GnRH ON PREGNANCY RATES IN DAIRY CATTLEK.L. Macmillan, V.K. Taufa and A.M.DayRuakura Animal Research Station, Private BagHamilton, New Zealand.A single intramuscular injection of 5 mcg of the GnRH analogueHoe766 (Receptal, Hoechst AG) administered during dioestrus can extendthe interval to oestrus in dairy cows (1) and temporarilY protect thecorpus luteum (CL) from some of the luteolytic effects of exogenousprostaglandin F dl (2). These responses are not associated withinduced ovulations and can be prevented by CL-removal by unilateralovariectomy (2).Four trials were completed to determine whether the observed GnRHrelated effects could alter pregnancy rates (PR) in dairy cowsinjected with either 5 or 10 mcg dosages of Hoe766 at from 1 to 13days after first ~nsemination. The trials were conducted duringOctobe~fNovemher ~n 1981, 1982 and 1983 in commercial dairy herds int~e.wa~kato. There were from 7 to 12 herds in each trial, and as~m~lar number of cows in each herd was injected in each daypos~-inseminationwith either or both dosages of Hoe766 and isotonicsal~ne as a placebo. Herd owners maintained complete breeding recordsand cows were pregnancy tested in three of the trials to confirmconception date.TABLE 1. Effects of a single post-insemination injection of Hoe766 onpregnancy rates (%) in dairy cattle.DOSAGE OF Hoe766POST-INSEM INTERVAL5mcg10 mcg(days)Treated Control Treated Control1-360.2* 71.9( 123)t (121 )4-662.5 71.8( 136) ( 142)7-1065.2 65.2 64.9 65.4( 466) ( 474)( 299) (335)11-1356.3 60.9 72.4** 60.9( 263) (273 ) ( 225) ( 276)t no. of cows * P

100 101REDUCEDOESTROGENIC EFFECT OF YARLOOP PASTURE SPRAYED HITH BENOliYLN.R. Adams and K.P.CrokerCSIRO Division of Animal Production, Wembley, W.A. andW.A. Department of Agriculture, South PerthWe have taken advantage of the availability of ewes wh'chiiI ... had grazedan agronom c tr a comparing cultivars of subterranean clover to tr trelate infertility in the ewe to the proportion of oestrogenic clove~ i~the pasture. We measured the cervical sperm reservoir to indicate tdegree of permanent clover infertility (1), using the number of sperm ~ethe cranial cervix as the indicator of this relationship (2).nTwo replicates of 7 mixtures of the highly oestrogenic Yarlooand. the non-oestrogenic Trikkala cv. were sown in a randomized ~l~:kd~s~gn. Seven of the 14 blocks were sprayed in the spring of each yearw~th benomyl (Benlate, DuPont Aust.) to protect against the fungald~sease clover scorch. Groups of 4 ewes were run on each block fo 3years, after which all ewes were run together on non-oestrogenic 'past~refor. 4 ~onths. At the second oestru_s after synchronization ewes wereart~fic~al1y inseminated with 450x10 tl sperm and killed 24 hi·pentobarbitone injection. The reproductive tract was lignt:~er a~~remo~ed. Sperm wer~ washed from the cranial third of the cervix incalc~um-free Krebs-R~nger solution and counted.Block 2 3 4 5Unsprayed% Yarloop 98 68 47 44 36 27Sheep examined 1 3 3 4 3 4Hean Log sperm 0 0 0 1.07 l.89 2.41 2.00Sprayed% Yarloop 97 80 69 53 30 26 1Sheep examined 3 4 3 4 3 4 4Mean Log sperm 1. 07 1.37 3.28 2.14 3.75 3.95 1.39The most obvious effect was unexpected: sheep which had gra'7ed~astures sprayed with benomyl had more sperm in the cranial cervix (m;an_ s.e.m. log10 2.39±0.37 vs 1.24±0.36, P

102103DEVELOPMENTAL CHANGES IN THE METABOLISM OF PROGESTERONE ANDDIHYDROTESTOSTERONE BY THE BLOOD OF NEWBORN LAMBSC.O. Nancarrow and P.J. ConnellCSIRO, Division of Animal Production, Prospect, NSW, Australia, 2148.Progesterone (P) metabolism in the blood of newborn lambs declineswith age (1) whereas reduction of 5a-dihydrotestosterone (DHT) remainshigh. This occurs despite a switch in the main product from 5a-dihydroandrostane-3~,17~-diolto5a-dihydroandrostane-3a,17~-diol(2). Howeverabout .5% of adult sheep have a significantly lower rate (L) of OHTreduct~on (3) and we now have determined the developmental changes in Pand DHT metabolism in L lambs following birth.Nine lambs were produced by mating a L ram to 7 L ewes, and werebled weekly from birth. The 'equilibrium' position for P and OHTreduction and the rates (v) of P and OHT reduction were estimated bincubating 0.02 to 1.0 ml of whole blood with labelled steroid for 5-6~min (2,3). Radioactivity associated with each steroid was determinedafter they were separated by thin-layer and paper partition chromatog':'raphy. The true equilibrium position for two groups of 4-6 month-oldlambs with either L or high (H) rates of DHT reduction were alsocompared by constructing a time-course of metabolism by '.0 ml blood.Also, some parameters of DHT metabolism were estimated for a separategroup of H lambs and adults for comparative purposes.The value of OHT reduction in the 'equilibrium' reaction declinedfrom 92.0 ± 2.6 (S.D)% at birth to a minimum of 21.2 ± 6.5% at 6 weeks.Similarly V for DHT reduction fell from 1.32 ± 0.38 ).lmol/ml erythrocytesper h to a minimum of 0.38 ± 0.09 ).lmol/ml per h at 7 weeks. Thiscompared with V = 0.98 ± 0.46 ).lmo Ilml per h in another group of '8~ week-old H lambs. The diminishing proportion of foetal erythrocytesJ.n blood was also responsible for a decline in V for P reduction from0.49 ± 0.10 to 0.02 ± 0.01 lJ.mol/ml per h at 8 weeks. However the Pequilibrium conversion began to fall from 86.6 ± 6.4% after 2 weeks andrea~h~d its lowest value of 22.0 ± 9.3% after 9 weeks. True equilibriumposJ.tJ.on for DHT reduction in H sheep (92.4 ± 4.7%, n=6) was reachedwithin 1 h and for L lambs (78.0 ± 5.9%, n=7) within 6 h. Other datahave shown this equilbrium in 18 H lambs at 8 weeks of age to be93.8 ± 4.0%.The blood of lambs retains a greater capacity to metabolize DHTthan P even in the genetically determined group which have a lower v.The evidence suggests that the blood of developing lambs and adults isfunctional in modifying the circulating levels of androgenic steroidsand thus may be considered as a secondary endocrine organ.(1) Nancarrow, C.D. Aust. J. BioI. Sci. 36: 183-190 (1983).(2) Nancarrow, C.D. and Connell, P.J. Proc. Endocr. Soc. Aust. 25: 16(1982) •(3) Nancarrow, C.D. and Connell, P.J. Proc. Aust. Soc. Reprod. B~ol..:!..!: 26 (1982).INSULIN IN OVINE MILK: PRE-DELIVERY ANDPOST-WEANING.J. Falconer, R. F. Sheldrake and J. S. Robinson.Faculty of Medicine, University of Newcastle,Newcastle, NSW 2308.Milk has been shown to contain a large number of hormones,including peptide, steroid and thyroid hormones (1). Th~thyroid hormones in milk have been proposed as a potent~alsource of these hormones for the hypothyroid neonate. Milkis also a source of immunoglobulins which can be absorbed bythe infant. We have recently shown that ovine milk co~tainsrelatively high concentrations of insulin (2) which m~ght bea potential source of this hormone for.the newbor~ lamb. Wehave extended this study to look at th~s hormone ~ncolostrum and milk obtained prepartum, postpartum and afterweaning. .samples were obtained from 16 Merino ewes ~n the week beforedelivery, 8 ewes during the first 10 days postpartum and 17ewes 5 days post-weaning. Separate samples were taken from.both left and right glands. samp~es w~re st~re~ frozen unt~lassayed for insulin using ovine ~nsul~n (El~ L~~ly,Indianapolis) as tracer and standard and an ant~serumobtained from Dr. A. L. C. Wallace. Milk samples w:re .parallel to the standard in this ~ss~y, exo~enou~ ~nsul~nwas recovered from milk samples, ~od~nated ~nsul~n.w~s n~tsignificantly degraded in milk and the ~mm~noreac~~v~t~ ~nmilk was found in the same fraction as ~od~nated ~nsul~nafter chromatography on Sephadex G100. Significance wasassessed using the Mann-Whitney U-test.No significant differences were found between the ~eft andright mammary gland at either time. The concentrat~on ofinsulin in milk was lower pre-delivery than on the day ofdelivery ( 33 + 7 ng/ml (mean ~ SEM, n= 31) and 1~4 ~ 30ng/ml (n=4) respectively (P

104MATERNAL RECOGNITION OF PREGNANCY PRIOR TO IMPLANTATIONC. O'NeillDepartment of Veterinary Physiology, Un~versity of Sydney, NSW 2006It has not yet been clearly defined whether maternal responses topregnancy occur prior to implantation in mammals. Although there havebeen reports of alterations to lymphocytes during the preimplantationperiod (1), the physiological significance of these changes remainsunclear (2). A study was designed to determine whether meaningfulphysiological responses occurred as a sole consequence of pregnancy inmice.virgin, Quackenbush strain mice were mated with either fertile orvasectomized males to produce pregnancy and pseudopregnancy, respectively.Animals had full haematological evaluation and wereautopsied on each day for a week after mating and examined for grossmorphological changes associated with pregnancy.The most significant alteration observed was a retardation for 48h of a hormonally induced increase in spleen weight in pregnantcompared with pseudopregnant mice, this was due to splenic contractionand expulsion of the platelet content (Table lA). A reduction in theperipheral blood platelet count also occurred shortly after fertilization,but did not occur in pseudopregnant mice and was not,therefore, a consequence of mating (Table lA). Females with bilateraloviduct ligations mated with fertile males did not display a reductionin platelet count (Table lB), therefore, the response was not due tomating or the presence of spermatozoa. There was a significant (p

107106EFFECT OF PROTEIN-FREE CULTURE MEDIA ON MOUSE & HUMAN EMBRYODEVELOPMENT IN-VITROC. M. Caro, A. O. TrounsonDept. of 0.& G. Monash University, Q.V.M.C. Melbourne AustraliaMouse embryos will develop in-vitro from two-cell to blastocysts inmedia containing no fixed nitrogen source. (1) (2)We investigated further the effect of omitting protein from culturemedia by transferring blastocysts grown in protein-free medium to recipientmice to assess their potential for normal fetal development. Wethen studied the omission of protein in the human in-vitro fertilization(IVF) system.Embryos were transferred to superovulated, pseudopregnant Fl hybrid recipientmice. Two groups were transferred:- the first consisted ofblastocycsts grown in-vitro in protein-free medium and the second groupwere grown in medium containing 4mg/ml Fetal Calf Serum (FCS), to act asa control. In the protein-free group 7/9 (78%) of the recipients became~regnant with a total of 39/124(31.5%)of blastocysts transferred implant­~ng to become fetuses, compared with a pregnancy rate of 8/9 (89%) and42/144(29.2%)fetuses from blastocysts transferred in the control group.Although there was no significant difference in the number of recipientsbecoming pregnant or the number of fetuses achieved in both groups, therewere a number of resorbing fetuses in the control group. Therefore,protein is not essential for mouse embryo development in-vitro or normaldevelopment to advanced fetal stages in-vivo.The second part of the study was to look at the effects of protein-freemedia on human TVF. A protein source, usually the patient's own serumhas always been used for human embryo development in-vitro. A randomisedtrial was set up in which every second patient's oocytes were cultured inprotein-free media from the time of oocyte recovery to embryo transfer.A control group of embryos cultured in medium containing 10% patients'own serum was used. In the protein-free group, 236/345 (68.4%) ofoocytes fertilized and 12/81 (14.8%) of the patients became pregnant. Inthe control group the fertilization rate was 221/296 (74.6%) and the pregnancyrate 14/94 (14.9%).Although there is no significant difference in fertilization or pregnancyrates between the two groups, we have shown that omitting protein from ourculture media has had no deleterious effects on embryonic or fetal development.Eliminating the variable of serum may enable studies on embryo culture toprogress further as the chemical and physical culture conditions can beexactly defined.(1) Cholewa J.A. & Whitten W.~. (1971) J.Reprod.Fert. 22:553-555(2) Caro C.M. & Trounson A.O.T.(In Press)(1984) J. IVF & Emb.Transfer Voll.EFFECTS OF INSULIN ON PREIMPLANTATION MOUSE EMBRYOSHeather G: Gardner and peter L. KayeDepartment of Physiology and pharmacology, University ofQUeensland, St Lucia 4067, Qld.Studies of hormonal effects on preimplantation embryos have beenconfined primarily to the steroid hormones in relation to the maternalsystem and implantation. Recently, evidence for exogenous peptidehormone effects on mouse embryos in vitro was presented (1). Wereport here studies of the effects of insulin on metabolism and growthof mouse embryos in culture.Insulin effects on embryonic glucose transport were assessed byincubating freshly collected embryos for 5 min at 37° in M2 mediumcontaining 3-0-Methyl-D-(1- 3 H] 'glucose (5.5 mM, 1 mCi ml- 1 ) with andwi thout insulin. After extensive washing on ice, uptake was measured(Table 1).TABLE 1. 3-0-Methyl glucose uptake (fmol min-'embryo-l,±sem,n=6-10) in"fresh" embryos and blastocysts cultured from 2 cell stage ± insulin.stageMorulaeBlastocystsControl Insulin 170 nMFresh CUltured Fresh CUltured24±2 t44±5240±17 250±15 tt392±8 tt342±20(t P < 0.01, tt P < 0.001)Insulin at 170 nM but not 1 nM stimulated methyl glucosetransport in freshly collected blastocysts and morulae. There was noeffect on 2 cell embryos. Long term effects of insulin on growth andglucose transport were studied by examining the growth of 2 cellembryos in BMOC medium (2) ± insulin at 24, 48 and 72 h. Growthresults presented in Table 2, as percentage of 50 embryos developingto indicated stage are the means of two experiments which agreed towithin < 4%.TABLE 2. Development of embryos in culture.CUl ture time/Embryo Stage Control Insulin 1 nM Insulin 170 nM24 hrs t > 8 cell 15% 31'5 17%48 hrs % to early blastocysts 23% 31% 14%72 hrs % to late blastocysts 73% 85% 90%plasma levels of insulin enhanced developmental rate during thefirst 24 h in culture, suggesting that insulin at physiological levelswas affecting the pre-compaction embryo. This effect continued untilthe blastocyst stage. However, high levels of insulin seemed toretard the early developmental rate. Glucose uptake in theseblastocysts was determined as above (Table 1). The presence ofinsulin (1 nM) during 72 h culture stimulated methyl-glucose transportapprox 2 fold. A smaller increase in transport rate was observed forembryos cultured in high insulin concentration. This supports thefinding presented in Table 2.Thus insulin has acute metabolic and long-term growth effects onearly embryos. The relevance of insulin and other hormones in thedevelopment of the preimplantation embryo, and particularly in theimproved in vitro culture of embryos,(1) Kaye, P.L. (1983) Proc. Aust. Biochem. Soc. E' 36.(2) Brinster, R.L. (1965) J. Reprod. Fert• .2£, 227.Insulin 1 nMFresh CUltured30±4247±16 tt477±18pose interesting questions.

AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY108Minutes of 15thAnnual General MeetingMICROINJECTION OF A CHICKEN HISTONE GENE INTO MOUSE ZYGOTESA. Michalska, A.J. Robins, P. Quinn, J.R.E. Wells and R.F. SeamarkDepartments of Obstetrics & Gynaecology and Biochemistry,The University of Adelaide, S.A.To allow investigation of factors which regulate specific geneexpression in mammalian cells, techniques for transferring genes intoone-cell mouse zygotes have been developed and evaluated using genesfor B-galactosidase and chicken histone.One-cell in-vivo fertilised mouse zygotes from superovulated(C57BL x CBA) Fl mice were collected 19-20 hours after heG injectionand transferred to HEPES-buffered culture medium at room temperaturefor 1-3 hours.Micromanipulation was carried out under l25x magnification usinga Leitz microscope and micromanipulators. Injection pipettes (externaldiameter approximately 2 ~m) were pulled from Leitz capillary tubing(1 mm overall diameter) in a vertical pipette puller and opened byimmersing the tip in 30% hydrofluoric acid. Holding pipettes (80-100 umexternal diameter) were pulled by hand on a microburner and polished ona microforge. The holding and injection pipettes were connected byteflon tubing to an 0.5 ml threaded-plunger Hamilton syringe and acylinder of compressed nitrogen respectively. The holding system wasfilled with filtered and de-aerated paraffin oil. The tip of theinjection pipette was filled with DNA suspension by capillary action.Zygotes were placed in HEPES-HTF medium in a depression slide andcovered with paraffin oil. The eggs were held individually by suctionon the tip of the holding pipette. The injection pipette was insertedinto the male pronucleus and injection of 2 pl of DNA solution wasaccomplished by a short (about 1 sec.) pulse of pressure (10 psi)supplied through a 3-way valva connected to the compressed N2 cylinder.In preliminary experiments 2000 copies of pMT B-gal plasmidcontaining the structural gene of B-galactosidase were injected into themale pronuclei of the zygotes. After 22 hours of incubation, theactivity of B-galactosidase (using 4 methylumbelliferyl B-D galactoseas an enzyme substrate) was detected in cell-free extract obtained from29 injected zygotes.In a further experiment, approximately 600 copies of chickenhistone gene were injected into zygotes. After injection the zygotes(which appeared normal) after a further incubation for 1-3 hours inHEPES medium, were transferred to the oviducts of pseudopregnant(C57BL x CBA) Fl females. Of 272 injected eggs, 189 (69%) survivedinjection and 136 were transferred to 10 recipients. Eighteen offspringhave been born and seventeen have survived to weaning. Nucleicacid for DNA analysis will be isolated from tails samples of these miceto see if they have incorporated the injected DNA into their genome.Held at 4.00 p.m. on Tuesday, 6th September, 1983 in Haydon Allen TheatreA.N.U.Canberra.15.015.115.215.315.415.515.615.715.815.915.1015.1115.1215.13AttendanceApologiesChairman and 97 membersJ. ElkingtonMinutes of 14th Annual General Meeting: With one correction ofitem 14.9(1983 instead of 1984) the minutes were received.Matters arising from 15.2:NilTreasurer's Report (attached) The report was received. TheTreasurer reported that the increased liquidity was due to revenueand bounty from the Embryo Transfer book; the small amount allocatedto student travel in '82 and the profit from the '82 meeting.Membership was 360 persons. The Lorne workshop had profitted$200. In 1983, 21 student travel grants were issued.Matters arising from Treasurer's Report: It was resolved thatthe annual subscription remain as for 1983.Programme Committee Report (attached)The report was received.Matters arising from Programme Committee Report. Much discussionrelated to the use of posters and to the format of the abstractbook.Chairman's Report: The Chaimlan reported that: the post-graduatestudent member had attended both 1983 Committee meetings; the Societywas financially strong; the 185 meeting would be held in Adelaidewith R. Seamark as Local Organiser; the Committee had been set alimit of $1500 for reimbursement of travel costs; the Newsletterurgently needed the support of all the membership; there existed theopportunity for ASRB to run a Boden Conference; the POC for 184 isto be the same as for '83.The report was received.The post-graduate representative (P. Lutjen) was called on to commenton the post-graduate student meeting.Matters arising from Chairman's Report:1988 World Congress on Animal Reproduction: T. Robinson reported onthe progress of the steering committee.New Members:See 15.569 new members were electedElections: J. Findlay was elected Chairman, John RodgerFalconer were elected as committee men.Nil&John. .. /2

Page 2REPORT OF THE A.S.R.B. PROGRAMME ORGANIZING COMMITTEE 1983.15.14General Business: It was br?ught to th~ atten~ion of the meet' thatthe Aust. J. Biol. Sci. was lnterested In publlshing articles ~~fatedto reproductive biology.Signed as a true recordDateOne hundred and eleven abstracts were submitted for thisyear's Meeting and all papers were examined by at least tworeviewers. Papers of doubtful scientific merit were then reviewedby the entire Programme Organizing Committee and two papers wererejecterl. The papers were rejected because they did not containadequate scientific data to back up the conclusions reached by theinvestigators. Severa1 other papers also contained only limiteddata to back up the conclusions reached and the Committee wouldurge all authors to include sufficient data to back up theirconclusions. Of the 111 papers submitted a further paper waswithdrawn and one was rejected since the author had too manypresentations for the Programme. Thirty-eight papers wereselected for oral presentation and the remaining 69 were allocatedto poster presentations. The decision on whether a paper wasincluded as a poster or an oral presentation was' based purely onthe consideration of grouping presentations adequately to create acohesive scientific programme. '!he papers which were viewed asgood papers and bad papers by the Committee were distributed inthe oral and poster sessions. Of those members requesting oralpresentations 36 were accommodated and 23 were allocated postersession whereas those requesting poster sessions 46 were allocatedto poster sessions and two to oral presentations.The Committee took the decision to request the LocalOrganizing Committee for space to enable posters to remain up forthe entire Programme and organized the posters into groups naminga Chairman to carryon a poster discussion which was to take placearound the poster.The Committee made use of the fact that a number of speakershad been brought to Australia by the International Union ofPhysiologist for their Congress in Sydney and arranged for aSymposium on Embryo-maternal Relationships to be part of theProgramme with Drs. Bullock and Findlay being appointed Chairmanof this Symposium. Excellent rapport was established with theProgramme Organizing Committee for the Endocrine Society to obtainamutually acceptable Programme.The Committee will be introducing several changes for the1984 Meeting but would already indicate that a self addressed cardshould be included with the abstract for acknowledgement ofreceipt and a photograph of the chief investigator should beincluded on poster boards to enable members to identify thisperson for the purposes of discussion.Finally the Programme Organizing Committee was disturbed atthe late notification to members of the deadline for receipt ofabstract but would point out that the information for thesubmi~sion of abstracts had been passed onto .the Secretariatearly in March.23

· As Chairman of the Committee .I '...ould Like to thank allCornm~ttee members (Drs. Risbridger, Charnley and Staples) for theirg~eat. help in this year's activities and particularly Dr.R~sbr~dgerfor the help in organizing the format of the Programmefor inclusion in the abstract booklet.AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGYChalnnan: Dr. J. K. Findlay,Medical Research Centre.Prince Henry's HospitalMelbourne, Vic. 3004.(03) 62 0621.FORM OF APPLICATION FOR MEMBERSHIPAdd,." all correspondence to:-The Secretary rDr. T. K. Roberts,Department of Biological SCiences,University of NewcastleN.S.W. 2308. Australia.(049) 68 5511Note1. In the year of election as a member, the subscription rate is $10.00per year (students $1.50), these rates being one half of therespective sUbscription rates for a full year. In addition, it ispossible to bUy variOUS journals at reduced rates for personal use.The journals available are: .Australian Journal of Biological SciencesBibliography of ReproductionJournal of Reproduction and FertilityJournal of Andrology2. The proposer must be a member of the A.S.R.B.3. There is no entrance fee.4. Pay~ent ~ be enclosed with this form.( PLEASE PRINT OR TYPE l (Title..•..........•.... )1, ...................•.•....•.......•...•.•...•...•. (Degrees-if any )(Diplomas.....•••....... lof.................•................•......•.•.......•...•............... . .. ..... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ••",. .,; ..Post Code: ....•.....•••....•..•.........state: ...•..... ····•····· ....Signature: ••..•.•.•......•....•.•.......Date: .•....••...•......•...•.hereby apply for membership in the Australian Society of ReproductiveBiology.POsi tion held: .•••....•.•.• •·····•·······••···•····•···•••·•···••·····•Area of interest in Reproductive Biology: .......•• ···•·•••··•··••···•••Nominated by: •.........•.•.•.... · •.. ••· .Signature: ...•.••...•..• ···•·•·( PLEASE PRINT l Date: ...••...••..••.....•.·····I enclose a cheque/ money order, drawn in favour of the Australian Societyfor Reproductive Biology, for $10.00 (or $7.50, student*l.* I certify that .....•.••.•.....•...••.•.•.•.. is enrolled as apost-graduate student.Signed:•......................•••........•.... (Supervisor)4

Chairmen1969·19731973·19771977·19811981·19831983·1985T. J. RobinsonC. W. EmmensD. M. de KretserN. W. MooreJ. K. FindlayLife MembersC. W. EmmensT. J. RobinsonJunior Scientist Award19811982P. J. LuljenR. J. Rodger & C. B. Gow1983 S. P. Flaherty