n~po~ twenty-s i hth rnnurl conference - the Society for Reproductive ...

~ fa [Bu@4M ~ IN ~ POS BAS ~ [8G9~~M~!NJ ~ IPJ(Q)S[P>OST[E~SASfR{lBo9~N~POSTERSASlRi[83°~~M~[NHPOSf[E 0 AS~183°~4M~[N]~POSrE~SA\S~[8°~4M~M~[NJ~rPOSf[E~SASASlRiIBO~4M~lN~PODOSf[ErRUSAS~f8°~0~4M~[N~POS1[E~[E[RSAS~[Bi°~4M~[NHPOSu[E~SASfR{rs09 ~[NJ~[POSuE~SAS 0rs°9)4M~NHPOSuIE 0ASfR{l83°~4M~[N]~POSr[E[RSAS~[8°~4M~ . OSf[El~SASf1l[6°~M1~NHPOSflE~SAS0~4M~!Nl~POS1'E~ r~ SrRIB 0 94M ~lN~ POS1~fR{SASfRH8°Sl4M~lN~ POSllElFRSAS[R1[8Q~4M~fNJ[J°OSulE~SASRrs094M~fNl~POSll~fR{SAS[R{rsO~4M~N~POS"=~"ERSASrRi[80 ~41M] ~ [NJ ~pas1 IE fRiSIAS[Rl [8)°94 ~~ ~ IN ~ PCOSlE I~~§ J~\S!fit fB u@4M~ [NJ ~ Posr IE IR{SAS ~ [8) 094M ~ rNl ~ POS1 rE [RlSIASfRi [8) 094M ~ [1~ II Pcansrrr IE RSAS [R{ 18u~MJ ~ [N] ~ POS1IE RSAS~H8°94M ~ IN ~ rPOSllEfR{SIAS [riH8 U S,4M I] [i\~ ~ POS1E [R1AS[R{f8Q9)4M~lN~ POSulEfRiSAS[Rlf8Q~~rM~ U\JH POSu[E~\SASR[8°~4M~ fNH~(OSu[E ~SAS fR1rs o 94M ~ fNl ~ POSflEfR{SIAS fR1(83°~4M ~ fNj ~ [POS1IE RSAS~[8°~4M~~ PROrEED I ~I~S OF THE "'~~rN~POSf[EIM\SIA~ L I~ u ~SRlBO~~~~N~PO~ TWENTY-S I HTH RNNURLASfR1l8°~CONFERENCErlE lRSASR[8°9)4M ~ IN ~ pCQ)SlrlElKlSAS R[8U~4~tTI ~ [NJ ~ lPo)srr lF~USASt~~ ~~ ~ fpJOS1"IE [R{SIASlRi[8U9d{~M ~ IN~ POSllElKlSIAS~lB5USJc(!lJ,M~ fNl II~\S ·[R1[8°s)4M ~ IN ~ ~051llE RSAS [Rlf8°G)~M~ N ~ ~OSl'lE 11~SASiPOST[E R1S lAS fRU8°9~M ~ N ~poslE~SASRlB O~~M ~ [~~ [POST IE[8 u9J1~rM ~ ~\lH [pOSIT fE RSlAS ~ [8 o~~ 1M ~ [NJ ~ POS1 fE RSAS ~ [8U~~,M~ IN ~ [Posr rE rR1SAS [R{ [8°~4M ~ IN ~ P(() 511 rE fR{SlAS IRi [8 0 ~~, [MH [NJ ~ ~osrr [E lR{§ 10\S L~{ ~ uS)~ ~M~[NHP)OSl~ HILTON HOTEL HR I SHRNE ~~[POSlr[ERSA~fRl~o@4M~ 26-28 SEPTEMBER 1994 :IRl~o~4M~!N]~~h©lrE~~~ J ~~~D~~~~Ie ''J) \\d) lJ::;:;;, ln1~ In ... ..olI ~ U b Ln\ ~ 11\\ ~,2)[8U~~,M~ [N ~ [poSLrlE[R{SASfRl[8U9)~M~[NJ~ rOSlfElRiSASlR\[8)°~&1j}M~[N~ [POSf =[E!RlSAS[R{[B)°9~,M~~~~rPO lJ~rERSASR18U ~rrtA]~N 0(0

The Australian Society for Reproductive Biology Inc.Twenty Sixth Annual ConferenceThe Hilton Hotel, BrisbaneSeptember 26 - 28 1994PROGRAMME AND MINIPOSTERS OF PAPERSCopyright Australian Society for Reproductive Biology, 1994ISSN 0818-4712PersonnelAcknowledgementsHotel mapsProgramme guidesProgrammePlenary LecturersJunior Scientist FinalistsMinipostersAuthor indexCONTENTS11111IVVIIXXIXXX1-1171

THE AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY INC.September 1994THE AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY INc.wishes to thank the following for their supportof the 1994 conferenceChairmanSecretaryTreasurerCommittee MembersOFFICE BEARERSClinical Interests RepresentativePostgraduate RepresentativePROGRAMME COMMITTEEChairmanCommittee MembersProf Alan TrounsonDr Lois SalamonsenDr Leeanda WiltonDr Jim CumminsDr Graham JenkinDr Simon MaddocksDr Kris BattyeDr Conrad SemiaDr Rob NormanMr David MillerDr Simon MaddocksDr Bill BreedDr Rob NormanDr Mark NottleDr Bob SeamarkProfessor Brian SetchellSPONSORSMeat Research Corporation of AustraliaOrganon (Australia) Pty. Ltd.Serono Australia Pty. Ltd.William A. Cook Australia Pty. Ltd.TRADE EXHffiITORSAlphapharm Pty. Ltd.Astra Pharmaceuticals Pty. Ltd.Bayer Australia Ltd.Baxter Diagnostics Pty. Ltd.Bio-mediq DPC Pty. Ltd.Boehringer Mannheim Australia Pty. Ltd.Bristol-Myers Squibb Pharmaceuticals Pty. Ltd.Children's Growth FoundationCiba-Geigy Australia LimitedCryologic Pty. Ltd. - Freeze ControlCSL PharmaceuticalsEli Lilly Australia Pty. Ltd.Fisons Pty. Ltd.General Diabetes ServicesMedtel AustraliaNovo Nordisk Pharmaceuticals Pty. Ltd. (Australia)Organon (Australia) Pty. Ltd. Division of AKZOParke Davis Pty LtdPharmacia (Australia) Pty. Ltd.Roche Products Pty. Ltd.Sandoz Australia Pty. Ltd.Upjohn Pty. Ltd.LOCAL ORGANISING COMMITTEEChairmanCommittee MembersDr Peter KayeDr Len MartinDr Michael Demden (ESA)Mrs Ruth Lillian (ICMS)The contents of these Proceedings have not been edited by the Society and are reproduced assubmitted. Responsibility for the accuracy of the communications and for carrying outexperimental work on animals or humans in accordance with the approval ofthe appropriate ethicscommittee, or with the appropriate legislation, rests with the authors.Material in these Proceedings may not be reproduced without permission of the Society.Price of the Proceedings to Non-members: $25 plus postage.11iii

FLOOR PLAN FOR MEETING VENUE(JTY Jv{APFire Exit Stair 6FACILITIESBarbeques, Picnic Tables @]~Post Offices [§]Taxi Ranks •lllJToiletsTourist Information [)Car Park LobbyoMargauxsNight CluboNORTHMoreton65sq.my -.-­Xes;;:)TLockyer112 sq.mRedlands105 sq.mYX:50 ::~~.~ :r. >-"'::;~'Ej ~Fire Exil Stair iFire Exil Stair 5Fire Exit Stair ~Ballroom LobbyService Elevators~sunmapSCALEC==lOcO=2:IOO==3IOO==4:I00=::55?~etresivh~e ...nc{f.=i·:;;~r;..Fire Exil Stair 3gillCarparkAccessx X• xo"'0xBallroom C295 SQ.mA. X XIX .0Ir•IJL,Anterooms,~I.-II•Grand Ballroom I' .... .1II-,IIIIIIIII~~JIBallroom B200 sq.mIIIIIIII --,I_IX---~Ballroom A295 sq.mxO I xo XO XOY

Programme GuidesProgramme GuidesMonday September 26 Tuesday September 27ASRB ESA ASRB ESA0800 Snack breakfast in the Trade Exhibition area 0800 Snack breakfast in the Trade Exhibition area08300930100010301130Concurrent Miniposter sessions:-Hypothalamo-Pituitary-Gondal axis-In-vitro maturation & In-vitrofertilizationTeaConcurrent Miniposter sessions:-Cryopreservation & Transgenesis-Uterine structure & PregnancyPhannacia Lecture: David Hill"Peptide growthfactor interaction duringdevelopment "TeaConcurrent Oral sessions:-Sandoz Junior Scientist Award-Thyroid-clinical-Fetal development-Steroid honnone action-Servier Award0830090009301000Miniposter session:-Growth Factors and the UternsTeaMerck Sharp & Dohme Lecture: Ralph Buttyan"Death genes, anti-death genes andprostate cancer "TeaConcurrent Oral sessions:-Endocrine cancers-Effects ofIGFs-Bone/Calcium12001300140015301600Goding Lecture - llpo Huhtaniemi "Molecular aspects ofthe ontogeny ofthe mammalianpituitary-gonadal axis"LunchJunior Scientist OralsTeaThe Great Debate"lcsi or !cnon"LunchConcurrent Poster sessions:-HPA Axis-Stress, metabolism and peptides-Reproduction 1TeaConcurrent Miniposter sessions:-Thyroid-Insulin & IGF's103012001300140014301530Concurrent Miniposter sessions:-Somatic cells and gonadal regulation-Regulation of fertilityHarrison Lecture - Allen Spiegel "Defects in G protein-coupled signal transductionpathways in human disease "LuchConcurrent Miniposter sessions:-Genn cells, spennatogenesis &spenn function-Embryonic DevelopmentTeaLunchConcurrent Poster sessions-Growth Honnone-Clinical endocrinology-Reproduction 2Tea17151815FinishAnnual General MeetingFinish1600Miniposter session:-Growth factors in early pregnancyConcurrent Miniposter sessions-Testis-HPA Axis & 2nd messengers1700 Annual General Meeting Henning-Berlin Lecture: Marc Pannentier"Molecular genetics and transgenic models (~fthyroid proliferative diseases "1800Finish1830 Finishvivii

0800083009001000ASRBProgramme GuidesWednesday September 28ESASnack breakfast in the Trade Exhibition areaMRC Lecture: Bill Thatcher"Control and management ofovarianfollicles in cattle to optimize fertility "TeaPincus Taft Lecture: Ron Kahn"Insulin resistance, diabetogenes and the causeoftype II diabetes "TeaAUSTRALIAN SOCIETY FOR REPRODUCITVE BIOLOOY INC.PROGRAMME 1994Registration: The registration desk will be located on Level 4 of the Hilton Hotel. On ~unday, September25th, it will be open from O9OOh, on Monday - Wednesday between 0830 and 1700h.The Goding and Harrison Lectures, and the Junior Scientist finalis,t orals will be presented in Ballroom A,the MRC Lecture and the Annual General Meeting of the Society will be held in the Redlands Room, withother session venues as indicated in the detailed programme. All venues are on Level 4 ofthe Hilton Hotel.Trade displays will also be found on this level in Ballroom C where morning and afternoon teas will also beserved. Each morning between 0800-0830 a snack breakfast will also be available in the Trade Exhibitorarea.Following the ASRB Silver Jubilee Symposium on Sunday, September 25th, there will be an informalsocial function at the Queensland Art Gallery starting at 1830h.1030Miniposter session:-Artificial insemination & Embryo transferCombined symposium with ADS:Gene TherapyMONDAY SEPTEMBER 261130FinishSession 1 (Concurrent)HYPOTHALAMO·PITUITARY·GONADAL AXIS123013001500LunchLunchCombined symposium with ANZBMS:Corticosteroids and BoneFinishChairman: Dr. lain ClarkeTime: 0830 - 10001Venue: Ballroom BBarker-Gibb,M.L., Boublik,J.H. and Clarke,I.J.NPYeffects on LH secretion in the ovariectomised ewe: Y2 receptor involvement, seasonal variationand site ofaction within the hypothalamus2 Sawangjaroen,K. and Curlewis,J.D.In vitro effects ofPACAP and VIP on sheep pituitary cells3 O'Brien,G.M., Curlewis,J.D. and Martin, L.Physiological and pharmacological challenges to prolactin secretion in Pteropus species (jlying foxes)4 Drummond,A., Dyson,M., Mercer,J. and Findlay,J.Localisation ofinhibin and relatedpeptides in the neonatal rat ovary: potential mediators ofgonadotrophin sensitivity5 O'Shea,T.,Anderson, S.T. and Hillard,M.A.Immunization ofmerino lambs. Effects on inhibin binding and gonadotrophin response toovariectomy6 Klein,R., Clarke,I.J. and Robertson,D.M.Studies in sheep exploring plasma follistatin elevations due to frequent blood sampling or surgery7 Tilbrook,A.J., Clarke,!.J. and de Kretser,D.M.Follistatin suppresses FSH in castrated rams8 Garcia,S., Findlay, J.K. and Mercer,J.E.Regulation ofovarian activin receptor type II expression across the rat oestrous cycle9 Rabiee, A.R., Lean, I.J., Gooden, J.M. and Miller, B.G.Glucose and cholesterol uptake by the ovary ofthe sheep10 Bergfeld,E., D'Occhio,M.J., Whyte, T.R., Aspden,W.J. and Kinder,J.E.Recovery ofpituitary and testis function in young bulls cifter treatment with the LHRH agonistdeslorelinviiiix

MONDAY SEPTEMBER 26 (cont)11 Turner,A., Hemsworth,P.H., Lohuis,H. and Tilbrook,A.J.The role ofstress in boar induced puberty in gilts12 Stobart,S.J. and Fitzpatrick,L.A.Seasonal changes in ovarian activity ofbos indicus crossbred heifers in a tropical environment13 Wright,P.J., Philip,C. and Hariadi,M.Progesterone treatment ofewes reduced corpus luteum weight and oestradiol treatment reducedovulation rate14 Hamemick, D.L., Clay, C.M., VanKirk, E.L. and Moss, G.E.Oestradiol increases amounts ofmRNAfor gonadotrophin-releasing hormone receptors in sheepSession 2 (Concurrent) : IN-VITRO MATURATION & IN-VITRO FERTILIZATIONChairman: Dr.l..eeanda WiltonTime: 0830 - 1000Venue: Samford Room15 EarLC.R., Irvine, B.J., Rowe,J.P., Kotaras, P. and Armstrong,D.T.Production ofin vivo matured oocytes from 6 week old calves16 Smith, J.F., Tervit,H.R., McGowan,L.T. and Pugh,P.A.Effect ofaspiration system on the recovery and development ofsheepfollicular oocytes17 Udy,G.B., McGowan,L.T., Nemaia,F.A., Ankersmit,A.E.L. and Tervit,H.R.Cattle oocyte recovery is affected more by aspiration pressure than needle size18 Grupen,C.G. Nagashima,H. and Nottle,M.B.Clsteamine enhances synchronous pronuclearformation in porcine oocytes matured andfertilized inVItro19 McMillan,W.H., Pugh,P.A. and Peterson,AJ.Early .ernkryo survival is higherfollowing twin rather than single transfer offrozen-thawed, but notfresh In VItro produced bovine embryos20 Richings,N.M., Boume,H., Harari,O., Jones,G.M., Mathews,P.M., McDonald,M.,Baker,H.W.G. and Johnston,WH.Intra-cytoplasmic sperm injection (ICSI) as a treatmentfor severe male infertility in humans and itspotential as a tool in endangered species programmes21 Lacham-Kaplan,O. and Trounson, A.Effect ofsperm motility on the fertilization rate by intracytoplasmic sperm microinjection22 Ca~t,J., Morton,M. and Saunders,D.Should all oocytes fenilise after intracytoplasmic sperm injection?23 Smitz.J., J~nssensw~llen,C., Lui,J., Joris,H., Camus,M., Devroey,P. and Van Steirteghem,A.Retrospective analySIS ofsuperovulated cycles for ICSI yielding an increased number ofimmatureoocytes24 Fry,. R.C., Simpson,T.L., Squires,T.J. and Findlay,J.K.Follicular growth and oocyte collection in heifers immunised against inhibinMONDAY SEPTEMBER 26 (coot)25 Xia,P., Rutledge,J., Watson,A.J. and Armstrong,D.T.Characterization ofin vitro synthesized proteins secreted by porcine non-attached oviductal epithelialcell spheres treated with estrogenSession 3 (Concurrent)Chairman: Dr. Mark NottleTime: 1030 - 1130CRYOPRESERVATION & TRANSGENESISVenue: Ballroom B26. Brennan,A.P. and Holden,C.A.Cryopreservation ofhuman sperm with pentoxifylline27 Sanchez-Partida, L.G., Zupp,J.L., Maxwell,W.M.C. and Setchell,B.P.Effect oflevels ofglycerol and compatible solutes on the post-thaw motility ofram spermatozoa28 Molinia,F.C. and Rodger,J.C.Pellet-jreezing ofmarsupial spennatozoa29 Withdrawn30 Pugh,P.A., Ankersmit,A.E.L., McGowan, L.T. and Tervit,H.R.Effect ofprotein type and concentration in the freezing medium on the survival ofIVP bovineembryos31 Nagashima,H., Kashiwazaki,N., Ashman, R.J. and Nottle, N.B.In vitro and in vivo survival ofcryopreserved hatched blastocysts in pigs32 Kashiwazaki,N., Nagashima,H., Ashman,R.J. and Nottle,M.B.Cryopreservation ofporcine in vivo and in vitro derived blastocysts with glycerol and ethylene clycol33 Nottle,M.B., Nagashima,H., Kashiwazaki,N., Ashman,R.J., Du,Z., Grupen,C.G.,McIlfatrick,S.M., Harding,M., Cheah,C., Crawford,R.J. and Robins,A.J.Production ofpigs containing a metallothionein porcine growth hormone gene construct34 Sutton,R., Fawcett,A.A., O'Grady,J., Ward,W.G. and Brown,B.W.An evaluation ofa tyrosinase minigene as a markerfor transgenesis in Quakenbush mice35 Tatham,B.,Giliam,K., Dowsing,A., Mahaworasilpa,T. and Trounson,A.Effects ofthe zona pellucida on electrofusion and development oftwo-cell mouse embryos36 Du,Z.T., Dai,Y.F., Owens,P.C., Armstrong,D.T. and Seamark,R.F.Fertility oftransgenic female rats expressing pig growth hormoneSession 4 (Concurrent)Chainnan: Dr. Len MartinTime: 1030 - 1200UTERINE STRUCTURE & PREGNANCYVenue: Samford Room37 Riley,S.C., Wong,E., Findlay,J.K. and Salamonsen,L.A.The localization ofneutral endopeptidase in the ovine uterus during the oestrous cycle and earlypregnancyxxi

MONDAY SEPTEMBER 26 (cont)38 Meier,S., Jenkin,G. Trewhella,M.A. and Fairclough,R.J.Changes in endometrial phospholipids andfatty acids during the oestrous cycle and early pregnancyin the ewe39 Massa,H. and Martin,L.Porcine-relaxin differentially inhibits uterine longitudinal & circular muscles in vivo in oestrous &early pregnant rats40 Sakoff.J.A. and Murdoch, R.N.The period ofuterine receptivity in the pseudopregnanct quackenbush special mouse as defined by thedeciduogenic lectin, concanavalin A41 Shaw,T.J. and Murphy,C.R.Early deciduogenic response ofthe uterine mucosa to the lectine concanavalin A and sesame oil42 Hosie,M.J. and Murphy,C.R.Effects ofthe interaction between clomiphene citrate and ovarian hormones on the surfaceultrastructure ofthe uterine luminal epitherlial cells43 Terry,V., Murphy,C.R. and Shorey,C.D.Vaginal epithelium ofovariectomised rats treated with combined clomphene citrate and exogenousoestrogen or progesterone44 Kleemann,D.O., Walker,S.K. and Seamark,R.F.Origin ofenhancedfetal growth in sheep administered progesterone during early pregnancy45 Shaw,G., Gehring,H. and Bell,E.C.Prostaglandin production by placental tissues in late pregnant tammar wallabiesMONDAY SEPTEMBER 26 (cont)49 McDougalLS. and Macmillan,K.L.Anovulatory dairy cows have lower LH pulsefrequency and intrafollicular concentrations ofoestradiol (£2) and testosterone (T) than cyclic cows50 Russell,D.L., Robertson, D.M. and Findlay, J.K.Pla.filma FSH elevation in ewes immunised with inhibin aN subunit is attributed to neutralisation (~fcirculating high molecular weight inhibin51 Robinson,S.J., Squires,E.L., Graham,J.K. and Maxwell,W.M.C.Effects ofcalcium ionophore A23I87 and heparin on stallion sperm52 O'Brien,J.K., Ryan,J.P., Waite,K.M., Maxwell,W.M.C. and Evans,G.Energy metabolism ofin vivo and in vitro matured oocytes from pre-pubertal and adult sheep53 Lane,M. and Gardner,D.K.Eagle's essential amino acids stimulate development ofinner cell mass cells ofcultured mouseembryosSession 7 :Chainnan: Assoc. Professor Jock FindlayTime: 1600 - 1715Si:THE GREAT DEBATE: ICSI OR ICNONRob McLachlanChris O'NeillLeeanda WiltonVenue: Ballroom ANon:Robert SeamarkAlan TrounsonJim Cummins46 Shaw,G., Gehring,H. and Bell,E.C.Prostaglandin production by endomentrium in late pregnant tammar wallabies47 Cox,n.B., Kent,J.C., Owens,R.A. and Hartmann,P.E.Mammary morphological andfunctional changes during pregnancy in womenSession 5Chairman: Prof. Alan TrounsonGODING LECTUREProfessor I. HuhtaniemiMolecular aspects ofthe ontogeny ofthe mammalian pituitary-gonadal axisSession 8 :Chairman: Assoc. Professor Bob SeamarkTime: 0900 - 1000TUESDAY SEPTEMBER 27GROWTH FACTORS & THE UTERUSVenue: Ballroom B54 Wathes,D.C., Perks,C.M. and Stevenson,K.R.Insulin-like growthfactor-l (IGF-l) Synthesis in the ovine reproductive tract: possible interactionsbetween uterus and ovaryTime: 1200 - 1300 Venue: Ballroom A 55 Cann,C.H., Fairclough,R.J., Sutton,R. and Gow,C.B.Expression ofinsulin-like growthfactor-l mRNA in endometrium during early pregnancy in thesheepSession 6 :Chairman: Assoc. Professor Bob SeamarkTime: 1400 - 1530SERONO-ASRB JUNIOR SCIENTIST AWARDVenue: Ballroom A48 HotzeLM.J., Martin,G.B., Walkden-Brown,S.W. and Fisher,J.S.Nutritional effects on testicular growth and LH and FSH secretion in Suffolk and Merino rams in thebreeding and non-breeding seasons56 Peterson,A.J., Ledgard,A.M., Hodgkinson,S.C. and Tervit,H.R.IGFBP proteolytic activity in the uterineflushings ofpregnant and nonpregnant sheep57 Clements,J., Ehrlich,A., Marsh,M. and Salamonsen,L.Prostate-specific antigen and insulin-like growthfactor binding proteins-3 are co-localized in thehuman endomentrium: patterns ofexpression across the menstrual cycle58 Robertson,S.A., Seamark,A.C. and Seamark,R.F.GM-CSF mediates the post-mating inflammatory reaction in the murine uterusxiixiii

TUESDAY SEPTEMBER 27 (cont)81 Jiang,F.-X. and Short,R.V.Characterisation ofthe primordial germ cells in ratfetuses82 Simorangkir,D.R., de Kretser,D.M. and Wreford,N.G.M.Synergistic effect oftransient neonatal hypothyroidism and neonatal hemicastration on testis size,Sertoli cell and spermatid numbers in adult rats83 Harris, M. and Rodger,J.C.The generaion and partial characterisation ofantibodies against marsupial gametes84 Kim,Y.H., Almahbobi,G., Temple Smith,P.D., de Kretser, D.M. and McFarlane, J.R.Characterisation ofmammalian sperm tail proteins using ratfibrous sheath antibody85 Kim, Y.H., Temple Smith,P.D., de Kretser,D.M. and McFarlane, J.R:Isolation and characterisation ofrat sperm tail outer densefibers proteins86 Loveland,K., Zlatic,K., Hayes,T., de Kretser,D.M. and McFarlane,J.MAP2 expression in the adult rat testis87 Setchell.B.P., Zupp, J.L., Ekpe,G., Maddocks,S. and Grigg,G.The effect ofenvironmental temperature on scrotal temperature measured by telemetry in rams atpastureTUESDAY SEPTEMBER 27 (cont)95 Waite,K.M. and Ryan,J.P.Metabolism ofenergy substrates by polyspermic mouse embryos96 Pantaleon,M. and Kaye,P.L.Insulin and IGF-I stimulate glucose transport in mouse blastocysts via the IGF-I receptor97 Dunglison,G. and Kaye,P.Endocytosis offluid by mouse blastocysts98 Roberts,C. and Breed,B.Marsupial viviparity - shell membrane, implantation and placentation99 Mate,K.E.,Robinson,E.S. and Pedersen,R.A.A·timetable ofin vivo embryonic development in the grey short-tailed opossum (Monodelphisdomestical)100 Whitworth,D.J. and Renfree, M.B.Gonadal sex reversal in female tammar wallabies is induced by MIS101 Buaboocha,W. and Gemmell, R.T.Thyroid gland development in the brushtail possum, Trichosurus vulpecula88 Young,S., Bradley, M.P., Gidley-Baird, A. and Hinds,L.A.The effects ofmelatonin implantaton ofspermatogenesis in the European redfox (Vulpes vulpes)Session 14 :GROWTH FACTORS IN EARLY PREGNANCY89 Murdoch,R.N. and Jones,R.C.Chairman: Professor Alan TrounsonGlucose and acetate utilization by marsupial epididymal spermatozoa is inhibited by a prostaticsecretion Time: 1600 - 1700 Venue: Ballroom B90 Bailey,J.L. and Storey, B.T.Calcium influx into mouse sperm activated by solubilised mouse zonae pellucidae is inhibited by threeinhibitors ofthe zona-induced acrosome reaction: 3-quinuclidinyl benzilate, tyrphostin A48, andpertussis toxin91 Setiadi,D., Sistina,Y. and Rodger,J.C.Acrosomal integrity oftammar wallaby (Macropus eugenii) spermatozoa after treatment withpenetrating (TCEP) and non-penetrating (aSH) reducing agents92 SetiadLD. and Rodger,lC.The effect of2-mercapto-ethanol (2ME) on the motility and acrosome oftammar wallaby spermatozoaSession 13 : (Concurrent) :Chairman: Professor Marilyn RenfreeTime: 1400 - 1530EMBRYONIC DEVELOPMENTVenue: Ballroom B93 Mate,K.E., Villar, A.J. and Pedersen,R.A.Relaxation ofRI9 imprinting in mouse androgenetic embryonic stem cells94 Ryan,J.P. and Waite,K.M.Changes in the metabolism ofenergy substrates within the first cell-cycle ofmouse embryos102 Cavanagh,A.C., Kaye,P.L. and Morton,H.Early pregnancyfactor (EPF): purification to homogeneity from human platelets and identification aschaperonin 10103 O'Neill,C.Platelet activating factor stimulates cell-cycle progression in 2-cell murine embryos104 Marshall.J.T.A., Brownlee,A.G. and Nancarrow,C.D.In vitro amplification ofan ovine oestrus-associated oviductal glycoprotein cDNA105 Boatman,D.E. and Magnoni,G.E.The role ofan estrogen dependent glycoprotein, oviductin, infertilization in the golden hamster106 Harvey,M.B.,Arcellana-Panlilio,M., Zhang,X.Q., Edwards,D.R. and Schultz,G.A.Proteinases important in implantation are down-regulated in parthenogenetic mouse blastocysts107 Athanasis-Platsis,S., Kaye,P.L., Cavanagh,A.C. and Morton,H.Early pregnancyfactor is required at implantation to optimize embryonic development108 Somodevilla Torres,M., Murphy,R.M., Hillyard,N.C, Cavanagh,A.C. and Morton,H.Passive immunization ofpregnant mice against chaperonin 10 causes loss ofembryonic viability109 Lash,G. and Legge,M.The identification ofearly pregnancy factor in red deer (Cervus elaphus)xvixvii

TUESDAY SEPTEMBER 27 (cont) PLENARY LECTURERS FOR 1994Session 15 :Chainnan: Professor Alan TrounsenTime: 1700 - 1830Session 16WEDNESDAY SEPTEMBER 30MRC LECTUREProf. Bill ThatcherControl and management ofovarian follicles in cattle to optimize fertilityChainnan: Assoc. Professor Jock FindlayTime: 0900 - 1000ANNUAL GENERAL MEETINGVenue: Redlands RoomVenue: Redlands RoomThe Goding Lecturer: Professor IIpo Huhtaniemillpo Huhtaniemi is Professor and Chainnan ofthe Department ofPhysiology, Medical Faculty atthe University ofTurku, Finland. He completed an M.D. and then a PhD at the University ofHelsinki by1974 and then trained as a specialist in chemical pathology in 1979. He was appointed as a Senior Fellowof the Academy ofFinland from 1981-83, and was Associate Professor of Clinical Chemistry at theUniversity of Helsinki from 1984-85. He was appointed to the Chair of Physiology at the University ofTurku in 1986. He has spent time in other laboratories as a postdoctoral fellow in the Department ofObstetrics & GYnaecology at the University of California, San Francisco in 1975-76, as a visiting associateat the NICHD with the National Institutes ofHealth, USA in 1979-80, and was visiting professor at theCenter of Molecular Biology, University ofHeidelberg, Germany in 1990-91.Professor Huhtaniemi's research interests include the ontogeny and regulation of the hypothalamicpituitary-gonadalaxis, testicular endocrinology, the molecular biology of gonadotrophins andgonadotrophin receptors, and the use of transgenic animals.The Goding Lecture for 1994 is entitled "Molecular aspects ofthe ontogeny ofthe mammalianpituitary-gonadal axis".Session 17 :Chainnan: Dr. Chis MaxwellTime: 1030 - 1130ARTIFICIAL INSEMINATION & EMBRYO TRANSFERVenue: Redlands Room110 Smith,J.F., Parr,J., Beaumont,S., Oliver,J. and Upreti,G.C.Comparison ofcervical, transcervical and laparoscopic insemination ofewes with chilled stored ramsemen111 Eppleston, J., Pomares,C.C., Stojanov,T. and Maxwell,W.M.C.In-vitro and in-vivo fertility ofliquid-stored goat spermatozoa112 Vishwanath,R., Piu,C. and Shannon,P.A unique sperm agglutinating factor from egg yolk113 Prendergast,E., Vishwanath,R., Shannon,P. and Molan,P.Charged lipoprotein fractions ofegg yolk in bovine semen diluents114 Chairussyuhur, A., Sanchez-Partida, L.G., Zupp, J.L., Maddocks, S. and Setchell B.P.Motility characteristics ofsemen from rams subjected to scrotal insulation, qfter storage at chilledtemperatures for one weekThe MRC Lecturer: Professor William W. ThatcherBill Thatcher is Graduate Research Professor in the Department ofDairy and Poultry Sciences at theUniversity ofFlorida, Gainesville, USA. He completed a B.Sc. and an M.Sc at the University ofMaryland, before undertaking a PhD at Michigan State University (awarded 1968). He was appointedAssistant Professor in Animal Physiology at the University ofFlorida in 1969, and then AssociateProfessor before becoming Professor ofPhysiology in 1978. He was appointed Graduate ResearchProfessor in 1988. He has been the recipient of numerous awards including an NIH predoctoralfellowship, a Fulbright-Hayes award and awards from the American Society ofAnimal Science and theAmerican Dairy Science Association. In 1994 he was awarded the Society for the Study of ReproductionResearch Award.Professor Thatcher's research interests are focused on elucidating the mechanisms by which theoestrous cycle is controlled to optimize reproductive efficiency in cattle. Interactions between the embryoand the uterine endometrium, the hormonal regulation of gene expression ofkey enzymes, honnonereceptors, hormones and paracrine regulators in ovarian follicles are being investigated, as is the paracrinedialogue between the trophectodenn and uterine endometrial epithelium.The MRC Lecture for 1994 is entitled "Control and management of ovarian follicles in cattle tooptimize fertility".115 UI?r~ti,G.C., Jensen,K., Munday,R., Vishwanath,R. and Smith,J.F.Studles on ram spermatozoal aromatic amino acid oxidase116 Stojanov,T., Rhodes,S.L., Maxwell,W.M.C. and Evans,G.The effect ofantioxidants on the pregnancy rate after insemination with liquid stored ram spermatozoa117 Lehrer,A.R. and McMillan, W.H.Is peri-oestrous intravaginal impedance an aid in screening for prospective pregnant recipient cows?xviiixix

lISIrI!IIIrti ar i alistsMaria HotzelFaculty of Agriculture,UWA, Nedlands, WASimon RobinsonDepartment of AnimalScience, Sydney, NSWIIEScott McDougallDairy Research Corporation,Hamilton, New ZealandJustine O'BrienDepartment of Animal Science,Uni. Sydney, NSWDarryl RussellPrince Henry's Institute ofMedical Research, Melbourne, VICMichelle LaneInstitute of Reproduction andDevelopment, Monash Uni., Clayton, VICxx

NPY EFFECTS ON LH SECRETION IN THE OVARIECTOMISED EWE:Y2 RECEPTOR. INVOLVEMENT, SEASONAL VARIATION AND SITE OF ACTION WITHIN THEHYPOTHALAMUS.Mandi L. Barker-Gibb 1, Jaroslav H. Boublik 2 and lain J. Clarke 11Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168; 2Baker Medical ResearchInstitute, P.O. Box 348 Prahran, Victoria 3181.INTRODUCTIONNeuropeptide Y (NPY) action is mediated throughY1(post-synaptic) and Y2 (pre-synaptic) receptor subtypes.NPY plays a key role in modulating LHsecretion in many species, although the extent towhich this involves action through the differentreceptor sub-types is not clear. In rats, LH secretionappears to be controlled through activation of the Y1receptor, and NPY acts at both the hypothalamic andpituitary level (1). In contrast, NPY does not act onthe pituitary gland in sheep (2). We haveinvestigated possible hypothalamic mechanisms bywhich NPY modulates GnRH/LH release in ewes.Intracerebroventricular (i.c.v.) injections of NPY1-36which acts at the Y1 and Y2 receptors and a specificY2 receptor agonist NPY18-36 were given to identifyreceptor specific actions on LH pulse secretion.Microinjections (1 Ill) of NPY were also given into thepreoptic area (POA) to examine effects at the level ofthe GnRH cell bodies. The ewe is also a highlyseasonal species and the activity of the GnRH 'pulsegenerator' is directly influenced by changes inphotoperiod which occur with season. The effect ofphotoperiod on the GnRH system is mediated via anunidentified neuronal circuit. Thus, we alsoinvestigated seasonal variations in the effects of NPYon LH secretion by giving NPY1-36 into the thirdventricle (3V) during both the breeding (BS) and nonbreedingseason (NBS).METHODSOvariectomised (OVX) ewes were fitted with guidetubes into a lateral ventricle (LV), 3V or POA.Jugular venous blood samples were taken every 10min for 3h. During the SS, sheep were given 10lJgNPY1-36, 100lJg NPY18-36 or saline into theLV;101l9,100lJg NPY1-36, and 100119 NPY 18-36 orsaline into the 3V, and 1.5lJg NPY into the POA.Injections of NPY were also given into the 3V (100lJg)and POA (1,5, 10lJg) during the NBS to examineseasonal effects Sampling continued for 3h postinjection,and samples were assayed for LH. Pre- andpost- injection differences were analysed byStudent's paired t-test.RESULTSInjections of 10 (LV) or 100lJg NPY1-36 (3V) had littleeffect on LH secretion in OVX ewes during the B.S.except to extend (P5 400::r:C!J 200;--0- PACAP 5 min-+- PACAP 30 min-0- VIP5min-tI- VIP 30 min--0- Control____ PACAP 16 7 M__ VIP16 7 MPACAP 16 6 MVIP 16 6 Mo+---r---,--_r_----.---...,.--r-----.a 234 5 6 7Incubation time (h)80 -0--+-~ --­~ 60§8 40~.s::i 20o0-t---,---r---,----.----,------.----,o ~ ~ 00 ~ 100 1~ 1~Incubation time (min)Fig.1 Time-course relationship for PACAP and VIP on intracellular cAMPDose (M)Fig. 2Dose-response curve for PACAP and VIPFig. 3Time-course relationship for the effects of PACAP and VIP on GHsecretion from sheep pituitary cellso+--,---,----,r-~-r-_r_---r-..--~__r_~---.a 2 4 6 8 10 12 14 16 18 20 22 24Incubation time (h)Fig. 4Time-course relationship for the effects of PACAP and VIPon LHsecretion from sheep pituitary cells1 2

Physiological and pharmacological challenges to prolactin secretion inPteropus species (flying foxes)G.M. O'Brien, J.D. Curlewis & L. MartinDept Physiology & Pharmacology, University of Queensland, Q4072.INTRODUCTIONPlasma prolactin in female flying foxes(Megachiroptera: Pteropus po!iocepha!us & P.scapu!atus) was examined after removal ofsuckling young, and after a dopamine agonist,bromocriptine, or an antagonist, domperidone.METHODS50pL aliquots of plasma were assayed intriplicate, using a heterologous double antibodyRIA (1, 2).RESULTS - SUCKLING".........52-J 4E0)3c............2---l~0-I00-2 -1RESULTS - BROMOCRIPTINE2.5--..~ 2.0E0) 1.5c............-lCt:0..I01.00.51-0.0-1 0• T A Individuals012Time (h)Fig. 1. In 3 lactating P. scapu!atus, little redflying foxes, plasma prolactin was3.01 ±O.74ng mL- 1 (mean±s.e.). Young wereremoved, and after 2h prolactin wassuppressed (1.24± 0.02ng mL- 1 p < 0.05). 5­30min after resumption of suckling, prolactinwas 1.25 ± 0.18ng mL- 1 (p > 0.05) and had notrecovered after 4h.2Time (h)Fig. 2. Early-mid pregnant P. poliocephalus,34354grey-headed flying foxes, had similar initialprolactin levels in test & control groups(1.40 ± 0.33ng mL-' and 1.31 ± O.19ng mL- 1p > 0.05). In vehicle controls prolactin wasunchanged after 2h (p > 0.05) and suppressedin the group treated with bromocriptine(0.72 ± 0.19ng mL-' p < 0.05); response wasmaintained at 4h (p > 0.05).RESULTS - DOMPERIDONE: 10I---lE 80)--S 6---l 4~a...b 212 ,..-----r----r--~---r-----r--...,A·•• A • T Domperidoneo ~ 0 \7 Vehicle0'-----'---""-----1.---'----1.__-'-1 o2 3 4 5Time (h)Fig. 3. Prolactin was 2.03 ± 0.22ng mL- 1 &1.35 ± 0.20ng mL-' (p > 0.05) in 2 groups ofpregnant P. poliocepha!us. After Domperidone,prolactin increased to 7.56 ± 1.63ng mL-' by20min (p < 0.05, n = 3: no response in 4thanimal) with no further change by 60min(p > 0.05); vehicle controls remained constant(p > 0.05).CONCLUSIONSSuppression of prolactin by removal of thesuckling stimulus or by bromocriptine, and itsstimulation by domperidone confirm that thesensitive heterologous assay is measuringPteropus prolactin. The pharmacologicalresponses indicate that, as in other species,prolactin secretion is under dopaminergicregulation in flying foxes.REFERENCES1. O'Brien, G.M., Curlewis, J.D. & Martin, L.(1990) Proc Aust. Physio!. Pharmacol. Soc.2..1:65P.2. McNeilly, A.S. & Friesen, H.G. (1978)Endocrinology 102:1539-1547.LOCALISATION OF INHIBIN AND RELATED PEPTIDES IN THE NEONATAL RAT OVARY:POTENTIAL MEDIATORS OF GONADOTROPHIN SENSITIVITY. Ann Drummond, Mitzi Dyson, Julie Mercer and Jock FindlayPrince Henris Institute of Medical Research, Clayton, Victoria 3168, AustraliaIntroductionThe rat ovary at birth is insensitive to gonadotrophins andremains so until approximately day 7 (1), suggesting thatspecific functional receptors are not present during thistime. The acquisition of FSH receptors by preantralfollicles is independent of gonadotrophin stimulation andmay involve the inhibin-related peptides. We postulatethat activin is responsible for stimulating FSH receptorsin vivo, given that this phenomenon occurs in vitro (2).To investigate this hypothesis, the presence of mRNAarid protein for the a and ~A inhibin/activin subunits inthe neonatal rat ovary, at times pertinent togonadotrophin sensitivity, were examined. The suitabilityof an in vitro neonatal ovarian cell bioassay for testingthe effects of these peptides on steroidogenesis wasalso assessed.MethodsFrozen sections of ovary from 21 day old PMSG-treatedand neonatal rats were used for immunohistochemistryand in situ hybridisation. Digoxygenin-Iabelled riboprobesfor inhibin-a and inhibin-~A were used to localise mRNAin ovarian tissue. Immunohistochemical localisation ofinhibin/activin subunits, was performed using threepolyclonal antisera raised against synthetic peptidefragments of the inhibin/activin a, ~A and ~B subunits(3). For cell bioassays, neonatal rat ovaries werecollagenase/Dnase dispersed and the cells cultured inthe presence or absence of FSH and MIX for 48 hours.Conditioned media was assayed for androstenedione,progesterone and inhibin by RIA.ResultsThe a, ~A and ~B proteins were present in neonatalovaries (Table 1), but only a could be consistentlydetected in PMSG ovaries, by immunohistochemistry.Granulosa cells, or in less differentiated ovaries, follicularcells, expressed these proteins, whereas ~A was theonly subunit present in oocytes. Both inhibin-a andinhibin-~A mRNAs were expressed by granulosa cells ofPMSG and neonatal ovaries, as early as day 4. Notevery follicle expressed the mRNAs at any time. FSH inthe presence of MIX, stimulated androstenedione andprogesterone production by neonatal ovarian cells inculture (Fig 1). By day 8, androstenedione production ismore prominent than progesterone. Inhibin productionwas stimulated by FSH (Fig 2), in the absence of MIX,but only at day 8.References1. Funkenstein, B., Nimrod, A. and Lindner, H.R. (19aO) Endocrinology106: 9a-106.2. Xiao, S., Robertson, D.M. and Findlay, J.K. (1993) Endocrinology131: 1009-1016.3. Gurusinghe, C.J., Healy, D.L., Jobling, T., Mamers, P. and Burger,H.G. (1994) Gynaecologic Oncology (SUbmitted)Supported by the NHMRC of AustraliaFigure 1: Dispersed Neonatal Ovary Culture1816 • progesterone14 & androstenedione12- 10.§g> 8642O~-ccC 10 20 C 1 10 20Day4DaySAll treatments with MIX ± FSH (1-20 ng/ml)a p,0.01 b p

IMMUNIZATION OF MERINO LAMBS. EFFECTS ON INHIBIN BINDING ANDGONADOTROPHIN RESPONSE TO OVARIECTOMYT. O'Shea], S. T. Anderson] and M. A. Hillard 2IDepartment ofPhysiology, University ofNew England, Annidale, NSW 2351, and 2CSIRO, Division ofAnimal Production, Armidale, NSW 2350STUDIES IN SHEEP EXPLORING PLASMA FOLLISTATIN ELEVATIONS DUE TO FREQUENTBLOOD SAMPLING ORSURGERYB Klein 1 , I.J. Clarke and D.M. Robertson1Department of Anatomy and Physiology, RMIT, Bundoora, 3083 and Prince Henry's Institute of Medical Research, P.O. Box 152, Clayton, 3168, AustraliaINTRODUCTIONImmunization of lambs with inhibin preparationsresults in pubertal advancement (1,2), withincreased ovulation rate and testicular growth ratebut no changes in gonadotrophin concentrations(1,3). Further studies on Merino sheep immunizedat 3, 7 and 15 weeks of age against a syntheticpeptide fragment (porcine alpha inhibin 1-32,Peninsular Labs., Belmont, California) conjugatedto albumin (1,3) are reported.MATERIALS AND METHODSThe ewes were ovariectomized at 32 months, onemonth after an additional immunization. Bindingof iodinated native bovine inhibin (supplied by Dr.D. M. Robertson, Monash University, Melbourne)was determined on plasma diluted 1:500. Plasmagonadotrophin concentrations after ovariectomywere analysed by AOV with repeated measures.RESULTSNo binding was seen in the control animals, exceptat age 3 weeks by passive transfer from someimmunized mothers. In one ram passive transferappeared to interfere with active immunization anddata from this ram have been excluded. In peptideimmunized animals plasma binding was seen inboth sexes after the first booster immunization,reached a maximum at 24 weeks, and haddecreased at 28 months (Table 1).In ewes itrapidly increased after reimmunization at 31months but then decreased again (29.6 ± 1.11% at32 months; 29.2 ± 0.89% at 33 months; 17.9 ±1.84% at 50 months).Binding of inhibin was less in ram plasma than inewe plasma at all ages (P < 0.01). The rate ofdecline in plasma binding was the same inovariectomized ewes (0.65% per month) as inentire ewes (0.62% per month), showing that thepresence of endogenous ovarian inhibin did notcause a persistent titre.One ewe was undergoing a gonadotropin surge atovariectomy and data from this ewe have beenexcluded. During 8 days after ovariectomy plasmaFSH concentrations were greater (P < 0.006) inewes ovariectomized in the luteal phase (4.02 ±0.18 ng Iml) than in the follicular phase (3.50 ±0.17), and peptide immunized ewes showed aslower response (P < 0.002) to ovariectomy thancontrol ewes (Fig. 1).Plasma LH concentrations.showed a significant interaction (p < 0.04), suchthat LH concentrations in ewes ovariectomized inluteal phase were greater than those of ewesovariectomized in follicular phase in controlanimals (2.20 ± 0.24 v. 1.66 ± 0.16) but not inpeptide immunized ewes (1.07 ± 0.12 v. 1.16 ±0.13).At 32 days after castration neither FSH (8.75 ±DAD ng/ml) nor LH (4.51 ± 0.34 ng/ml) showedany residual effect from immunization or phase ofcycle at ovariectomy.DISCUSSION/SUMMARY1) The presence of endogenous ovarian inhibindoes not affect persistence of antibody titres toa synthetic alpha - inhibin peptide.2) The phase of the oestrous cycle at ovariectomyaffects the subsequent gonadotropin response.3) The delay in the post-castration rise in plasmaFSH in peptide immunized ewes is possiblydue to a longer retention of biologically activeinhibin in plasma by antibodies.Table 1: Binding of native bovine inhibin byplasma (diluted 1:500) of Merino sheepimmunized with porcine alpha 1-32 peptide.Results are expressed as percentage cpm bound tototal cpm added. Values are means ± SE for 12females and 11 males.AnimalsAge (weeks)8.4 15 16.4 24 32 121Female 6.6% 14.8% 24.0% 24.5% 21.4% 9.2%(0.95) (1.31) (1.57) (1.78) (1.78) (1.17)Males 2.8% 5.5% 10.1% 10.7% 8.2% 2.2%87-;;-E 6rI.l.$ I=l-.5~ 4 -5-:I:f£ 3210(1.06) (1.09) (1.08) (2.32) (1.93) (0.39)**n = 6........•......... ..LT .........••••••••• ..10.•...................48144Fig. 1: FSH after ovariectomy of controls (D) andewes immunized with peptide (. ).96HOURS AFTER OVARIECI'OMYT192REFERENCES1. O'Shea, T. et al. (1989) J. Reprod. Ferti!. Abstr.Series 1-.: Abstr. 6.2. O'Shea, T. et al. (1993) Reprod. Fertil. Dev. ~:173 - 180.3. Anderson, S. T. et aL (1990) Proc. Aust. Soc.Reprod. BioL 22: 133.INTRODUCTIONAprotein hormone termed FSH-suppressing protein (FSP) (Robertson ataI., 1987) or follistatin (Ueno et aJ., 1987) has been isolated from ovarianfollicular fluid which is functionally similar to inhibin in suppressing FSH.In a previous study (Klein et a/., 1993) aimed at exploring thephysiological role of FSP in plasma, FSP levels were observed to riseacutely during periods of frequent blood collection. This rise in plasmaFSP coincided with the time during which animals underwent surgery andperiods in which an increase in bleeding frequency had occurred.The aims of this study were to investigate the cause of the rapid risein plasma FSP levels by (1) examining the effects of the anticoagulantheparin used during blood sampling; (2) determining whether frequentbleeding per se acts as a stimulus for FSP elevations and (3) and todetermine whether a rise in plasma FSP seen 10-12 hours after surgerywas due to a concomitant rise in the acute phase protein a-2macroglobulin (a-2M) shown to-bind FSP (Vaughan et al., 1993).MATERIALS AND METHODSIn the first study eight Corriedale ewes were bled via indwelling venouscatheters at hourly intervals for 3h, then at ten minute intervals for 2h,followed by two samples taken at hourly intervals. The catheters wereflushed with heparinised (50IU/ml) saline and the collection vials wereheparinised. At the 4 h point (ha~ way through the phase of rapidsampling), 4 sheep received a large intravenous injection of heparin(3200IU/1.5ml) and control sheep received an equal volume of saNne. Inthe second study, 4 ewes were sampled (via jugular catheters) at hourlyintervals for 4h, then at ten minute intervals for 2h followed by the takingof 3 samples at hourly intervals using the anticoagulant citrate glucosedextrose instead of heparin. A further 4 ewes acting as controls weresampled at hourly intervals. In the third study, samples were taken athourly intervals from 8 ewes for 27h, and the sheep were thenlaparotomised and hOUrly samples were taken 10-12h later. Plasmasamples were collected for subsequent determination of a-2M byimmunoprecipitation and FSP by a radioimmunoassay method developedfor sheep plasma (Klein et aI., 1993).RESULTSPlasma FSP levels were observed to rise acutely (1)

Follistatin Suppresses FSH In Castrated RamsA.J. Ti/brook 1 , !.J. C/arke 2 and D.M. de Kretse,31Department of Physiology and 31nstitute of Reproduction and Development, M.ona~h2prince Henry's Institute of Medical Research, Clayton, 3168, VictoriaUniversity andREGULATION OF OVARIAN ACTIVIN RECEPTOR TYPE II EXPRESSION ACROSS THERAT OESTROUS CYCLESonia Garcia, Jock K. Findlay and Julie E. MercerPrince Henry1s, Institute of Medical Research, PO Box 152, Clayton, Vic. 3168.INTRODUCTIONFollistatin is structurally distinct from inhibin and has beenshown to suppress the release of follicle stimulatinghormone (FSH) by pituitary cells in vitro (1). We found thattreatment of castrated rams with a 50pg Lv. injection and800j./g infusion of human recombinant inhibin A (hr-inhibin)over 12h suppressed plasma concentrations of FSH to thelevels found in intact rams (2). There have been no studiesin sheep to determine if follistatin will also suppressplasma FSH in vivo. Therefore, we tested the hypothesisthat follistatin suppresses the plasma concentrations ofFSH in castrated rams. The effect on plasma luteinizinghormone (LH) was also investigated.MATERIALS AND METHODSEight rams, castrated within the first 3 weeks of birth,were allocated to 2 groups of 4. One group was given a50j./g Lv. injection followed by an 800pg Lv. infusion ofhuman recombinant follistatin (hr-F) over 12h. The controlgroup was given an Lv. injection and 12h Lv. infusion ofvehicle. Jugular venous samples were collected for 3hbefore and for up to 132h after treatment. FSH and LHwere measured by radioimmunoassay.RESULTSInfusion of castrated rams with hr-F significantly (P.. I CToSample A2 3 4 5CJ)Sample 82 3 4 5_o:::::l!!!!ilI!I•••S. AetRII eDNAr:::::.::::::::::::::::::::::::::::::::::::::::::J' CTCJ) CJ) (J)::J ::J ::::>::Ja: a: a: c::~~~CJ) CJ) I-CJ) (J)wWW ill0C50a: a:00- 0->- W...Ia:~« ...Iw~AetRII eDNA..(458 bp CT..( 408 bp AetRIIeDNAFig 1 (A) Schematic representation of amplified 408 bp region ofActR11 cDNA and of 458 bp competitive template (CT). Solidregion in CT represents a 50 bp oligonucleotide subcloned into aNarl site. (B) Titration curve of PCR products containing a fixedamount of ActRII cDNA and increasing amounts of CT DNA.Fig.2 Relative levels of ActR11 mRNA in rat ovaries across theoestrous cycle. * p< 0.05, different from oestrus.Summary and ConclusionThis increase in ActR11 mRNA levels occurs acrossthe period when the dominant follicle is developingand persists into, and indeed is maximal at oestrus,when ovulation occurs, consistent with a role foractivin in development of the follicle. These dataprovide further evidence for the important role ofactivin as a local regulator of reproductive function inthe ovary.7 8

-GLUCOSE AND CHOLESTEROL UPTAKE BY THE OVARY OF THE SHEEPA.R.Rabiee, I.J. Lean, J.M. Gooden and B.G. MillerDepartment of Animal Science, University of Sydney, Camden NSW 2570INTRODUCTIONThe dramatic cyclic changes in growth and steroid and protein output of the ovary suggest that theovary is a highly metabolically active organ. Knowledge of ovarian affinity and demand for energysubstrates used for steroid and protein hormone production should lead to improved understandingof ovarian nutrient requirements to ensure successful reproductive performance. Also important is thestudy of relationships between uptake of different variables because factors influencing ovarianuptake of nutrients may be identified. We have previously found that the small ovarian mass of sheepmay limit our ability to detect small, but significant uptake of metabolites, glucose uptake wassignificant for all sheep pooled (P

THE ROLE OF STRESS IN BOAR-INDUCED PUBERTY IN GILTSA.I. Turner 1 • 2 , P.H. Hemsworth 1 , H. Lohuis 1 and A.J. TilbraalCIVIAS, Dept Agriculture and 2Dept Physiology, Monash University, Victoria.IntroductionIt is well accepted that exposure of pre-pubertal gilts toboars stimulates puberty in gilts. This stimulation ofpuberty may be due to stimuli from the boar, primarilypheromones (1), and/or an acute stress response (2). Therelative importance of these factors is unknown. In thisexperiment we tested the hypothesis that stress plays amajor role in boar-induced puberty in gilts.Materials and MethodsWe used a behavioural approach to reduce the stressresponse when gilts are exposed to boars. Frequentexposure of pre-pubertal gilts to boars in an arena mayallow habituation to boars and to the arena in which thisexposure occurs. Thus, the degree of novelty of thisexposure for these gilts should decrease over time andconsequently the gilts should experience a lesser stressresponse than gilts inexperienced with this exposure.Evidence of habituation to this exposure should bereflected in a reduced level of exploration (activity) bythe gilts. We assessed the role of an acute stressresponse in boar-induced puberty by comparing theeffects of boar exposure at 170 days of age on gilts thathad previously been exposed frequently to boars in anarena with gilts which had not received this previousexperience.Twenty four gilts were allocated to the following 4treatments (N::;::6). Controls: gilts were introduced from130 days of age for 1h daily to an empty pen adjacent to2 pens containing ovariectomised females; DMA 13o : giltswere introduced from 130 days of age for 1h daily to adetection mating arena (DMA) that allowed auditory,olfactory and only limited physical contact with boarsthrough the arena walls; DMA 17O : gilts were introducedto the DMA for 1h daily from 170 days of age; INTRO:gilts were introduced to a boar in his own pen for 15mindaily from 170 days of age. From 170 days of age thestanding response to pressure on the back of the gilt wasused daily to determine the occurrence of puberty.Ovaries were examined at slaughter to confirmattainment of puberty. With the exception of the INTROgroup, the level of activity (locomotion) of the gilts wasmeasured during treatment by recording movementacross a grid marked on the floor of the arena.ResultsThe mean (±SEM) age of puberty (Fig 1) did not differbetween the control and DMA I30 treatments (P>0.05). Incontrast, the DMA 170 (P

PROGESTERONE TREATMENT OF EWES REDUCED CORPUS LUTEUM WEIGHTAND OFSfRADIOL TREATMENT REDUCED OVUI.ATION RAlEPatrick J. Wright. C. Philip and M. HariadiDepartment of Veterinary Sciences, University of Melbourne, Werribee 3030OESTRADIOL INCREASES AMOUNTS OF mRNA FORGONADOTROPHIN-RELEASING HORMONE RECEPTORS IN SHEEPD.L. HamernilC, C.M. Clay2, E.L. VanKirk!, G.E. MostIDept. Veterinary & Biomedical Science, University of Nebraska, Lincoln, NE; USA.2Dept. Physiology, Colorado State University, Ft. Collins, CO; USA.3Dept. Animal Science, University of Wyoming, Laramie, WY; USA.INIRODUCTIONWe have assessed i) the effect of exogenousprogesterone on corpus luteum (CL) weight (todetermine whether the reduction of CL weightsin ewes of reduced nutrient stat~ (1) could bedue to increasing plasma' progesteroneconcentration reflecting reduced metabolicclearance rate (2», and ii) the effect ofexogenous oestradiol on ovulation rate (todetermine whether ovulation rate could bemodulated by an inhibitoryeffect of oestradiol).MATERIAL AND METIiODSIn Study 1 mature Merino (M) ewes (n=20,body condition score 1.5-2) receivedprostaglandin (PG; Cloprostenol, 125 ug im)each 14 days on 4 occasions to synchroniseoestrous cycles. Ten of these ewes were treatedwith progesterone for. 4 cycles from Day 6-14(Day 0 = day of PG treatment) (group Mp).Pessaries (CIDR-G) were inserted on Day .6,replaced on Day 10 and removed on Day 14/0.The remaining 10 PG-treated ewes wereuntreated controls (Group Me). On .Day 14 ofthe 4th progesterone-treatment cycle the eweswere ovariectomized and CLs counted andweighed.In Study 2 mature Border Leicester (BL) ewes(n=22, body condition score 3-4) were treatedwith an oestradiol implant (3.5 em sc; groupBLo, n=11), or were untreated controls (groupBLc, n=11). The occurrence of oestrus (crayonmarks from vasectomised rams) was observedover 5 weeks. The ewes were then treated twicewith PG 14 days apart to synchronise oestrouscycles, ovariectomized 14 days later and thecorpora lutea counted and weighed.RESULTSIn Study 1, in the single ovulators (9 ewes ineach treatment group), progesterone-treatedewes had lighter CL's (367 (38), mean (sem»than did control ewes (489.3 (24.3» (P=0.02,Fig. 1).In StUdy 2, oestrogen treatment did not affectoestrous cyclicity, but did result in a lowerovulation rate (1.8 (0.2» than in control ewes(2.45 (0.16» (P=O.04, Fig. 2).DISCUSSION/SUMMARY/CONCLUSIONSThe results indicate that plasma concentrationsof progesterone and oestradiol may be factorsinfluencing corpus luteum weight and ovulationrate in ewes undergoing normal oestrous cycles.Ṣ5500:i:'ij '" ...5031E:JII-4OD2" :Je- 3500u.s~c:~ c""5>o550 P = 0.023002.700ControlContlolTreatmentlrealll1entProgesteroneFig. 1: Corpus luteum weights (mean, sem) inprogesterone-treated (Mp, n=9) and in control(Me, n=9) ewes.OestradiolP = 0.041.700L__~lIgll_~mm~ -Fig. 2: Ovulation rate in oestradiol-treated(BLo, n=11) and in control (BLc, n=ll) ewes.REFERENCES1. Wright, PJ, Pantke, P and Clarke IJ (1991)Proc. AS.R.B. abstr 82.2 Parr, RA (1992) Reprod. Fertil. Dev. 4, 297­300.INTRODUCTIONGonadotrophin-releasing hormone (GnRH)stimulates synthesis and secretion ofgonadotrophins in mammals by binding tomembrane-bound receptors on gonadotrophs.Recently, cDNA encoding the GnRH receptor(GnRH-R) were cloned from several species,including sheep (1). In sheep, amounts of mRNAfor GnRH-R increased prior to the preovulatorysurge of LH (1). Oestradiol (E), alone or incombination with inhibin also increased amountsof GnRH-R mRNA in sheep pituitaries in vitro(2). This study was conducted to test thehypothesis that E increases amounts of mRNA forGnRH-R during the periovulatory period in ewes.MATERIALS AND METHODSPituitarieswere collected from five ewes on d14 ofthe oestrous cycle (dO=oestrus). Another 20 eweswere ovariectomised (OVX) on d14 of theoestrous cycle, and E implants were inserted in 5OVX ewes (OVXE) to mimic low concentrationsof E during the early follicular phase of theoestrous cycle. Sixteenhours after OVX, pituitarieswere collected from 5 OVX and 5 OVXE ewes,and the remaining 10 OVX ewes received anintramuscular injection of E (25JLg) to induce apreovulatory-like surge of LH (OVX+E).Pituitaries were collected from OVX+ E ewes 18or 54 hr after injection of E (n=5/group). Bloodsamples were collected at hourly intervals followinginjection of E for measurement of LH. Number ofGnRH-R were quantitated in half of eachpituitary. Polyadenylated RNA was isolated fromhalf of each pituitary, applied to Northern and slotblots, and hybridised with a radioactive cDNAprobe encoding the ovine GnRH-R. Hybridisationsignals were quantitated in arbitrary densitometricunits.RESULTSInjection of E induced a preovulatory-like surge ofLH within approximately 18 hr in OVX ewes.Northern blot analysis revealed GnRH-Rtranscripts of - 5.6, 3.8, 2.1, 1.3, and 0.8 kbp. Eimplants increased amounts of mRNA for GnRH-R at 16 hr in OVXE ewes compared to theamount of GnRH-R mRNA at d14 of the oestrouscycle or at'16 hr in OVX ewes (Table 1). Amountsof GnRH-R mRNA in OVX ewes were alsoelevated (p < .05) 18 hr after E injection comparedto 0 or 54 hr after E (Table 1). Number of GnRH­R were elevated (p

PRODUCTION OF IN VNO MATURED OOCYTES FROM 6 WEEK OID CALVESC.R. Earll, B.J. Irvinel, J.P. Rowel, P.Kotaras 2 , and D.T. Armstrong 21 Strnan Research Centre, S.A Research & Development Institute, Naracoorte, Australia 2Department of Obstetrics and Gynaecology, University of Adelaide, Australia1N1RODUCTIONIt has recently been demonstrated that viableembryos can be produced from young lambsand calves using in vitro techniques onoocytes collected from follicles stimulated bya combination of FSH and PMSG followedby an injection of GnRH (1, 2). Preliminarydata obtained from 6 week old ewe lambs (1)suggests that the development rate offoetuses from oocytes is greater from in vivothan in vitro matured oocytes (3). Oocytes atcollection were classified into two groups:mature, those with expanded cumulus cells;immature, those with tight, intact cumulusinvestments. For these experiments with theyoung lambs, mature oocytes at collectionwere submitted directly to IVF whileimmature oocytes underwent IVM cultureprior to IVF. The young lamb differs fromthe calf in that it appears much easier toobtain in vivo matured oocytes from the lambthan the calf.The current study therefore investigatedthree different in vivo maturation inducingtreatments in an attempt to increase thepercentage of matllre oocytes obtained fromcalves at aspiration.MATERIAlS AND METHODSThe stimulation treatment employed was acombination of 200mg of FSH s.c.(Folltropin, Vetrepharm, Canada) and 400J.U. PMSG Lm. (Folligon, Intervet, Holland).Progesterone sponges were inserted 5 daysprior to the stimulatory injections andremoved 48 hours later. The 3 maturationinducing treatments were; Treatment 1, 500J.Lg GnRH Lm.; Treatment 2, 40 mg FSH Lv.with 10 mg LH Lv. (Lutropin, Vetrepharm,.Canada); Treatment 3, 1500 J.U. HCG i.v.and 1500 J.U. Lm. (Chorulon, Intervet,Holland). The maturation treatments weregiven 24 hours post sponge pull. Oocyteswere aspirated 24 hours post maturationtreatment and scored for maturation status.RESULTSTreatmentFolliclesAspiratedOocytes 6%Recovered Mature1 (n=8) 35.9 ± 23.1 17.1 ± 10.9 43%2 (n=7) 56.5 ± 34.8 31.9 ± 23.5 69%3 (n=8) 23.9 ± 21 10.9 ± 8.9 82%The between calf variation in follicle numbersand oocytes collected was large so that withthe small number of calves in each treatment(n=8) the differences in follicle numbersbetween treatments were not significant.Treatment 3 produced a greater percentageof oocytes that were mature but a lowernumber of matured oocytes than treatment 2(p < 0.05). It is of interest that such a largedifference in the number of oocytes collectedcan be caused by the different maturationinducing treatments.DISCUSSIONExperiments to determine the relativedevelopment rates of in vivo and in vitro calfoocytes are in progress. If in vivo maturedcalf oocytes develop to embryos at ratesbetter than in vitro matured oocytes thentreatment 2 would be a better maturationtreatment than those previously employed.REFERENCES(1) Armstrong, D.T., Irvine, B.J., Earl, C.R.(1994) S.S.R in press.(2) Earl, C.R., Irvine, B.J., Armstrong D.T.(1994) Proc. Aust. Soc. Animal Prod. (inpress).(3) Armstrong, D.T., Holm, P., Irvine, B.,Petersen, B.A., Stubbings, R.B.,McLean, D., Stevens, G., Seamark R.F.(1992) Therio 38:667-678.Supported by Meat Research Corporation.EFFECT OF ASPIRATION SYSTEl\1 ON THE RECOVERY AND DEVELOPMENT OF SHEEPFOLLICULAR OOCYTES.IF. Smith, H.R. Tervit, L.T. McGowan and P.A. PughAgResearch, Ruakura Agricultural Research Centre, PB 3123, Hamilton, N.Z.INTRODVCTIONRepeated collection of ooctyes from living animals followedby IVMlIVF/IVC has the potential to produce moreoffspring than traditional MOET procedures. Varioussystems for follicular aspiration in the sheep have beenreported (1, 2!) and we compare here two systems involvingcombinations of needle types and vacuums.l\IETHODSSixty four. Coopworth ewes were synchronised withintravaginal sponges (Repromap Upjohn! NZ) for 14 daysand given 0.96 units FSH (Ovagen; Iep! NZ) in 6 equaldoses on days 11! 12 and 13 of sponge treatment. Follicleaspiration was performed via laparoscopy on day 14. AStortz (STZ; 20 g double lumen; 45 cm long) needle at 3vacuums (15, 25! 50 mm Hg) was compared with a systemsimilar to that reported by others (2, A). It involved needlesof two gauges (20 v 18)! lengths (Long, 45cm; Short, 22cm)at 15 mm Hg. Two operators performed the aspirations over4 days and the STZ 50mm Hg treatment was used as thecontrol treatment on each day. Recovered oocytes wereIVM, IVF and rvc in our standard system (3) anddevelopment to blastocysts recorded. Oocytes recoveredfrom slaughtered untreated ewes were run as controls.RESULTSThe mean number of follicles aspirated in the treated eweswas 14.7 and mean number of oocytes recovered was 8.0(54.1 %). Recovery rate was influenced by operator (62.9 v44.7%; P

CATTLE OOCYTE RECOVERY IS AFFECTED MORE BY ASPIRATION PRESSURE THANNEEDLE SIZEG. B. Udy, L T. McGowan, F. A. Nemaia, A. E. L. Ankersmit and H. R. Teryit.AgResearch, Ruakura Agricultural Research Centre, PB 3123 Hamilton, New Zealand.CYSTEAMINE ENHANCES SYNCHRONOUS PRONUCLEAR FORMATION INPORCINE OOCYTES MATURED AND FERTILIZED IN VITROC. G. Grupen,:\H. Nagashima and M. B. Nottie ,Bresatec Limited, Department of Obstetrics and Oynaecology, University of Adelaide, Adelaide, SA,5005, AustraliaINTRODUCTIONA range of needles (16G-20G; single and doublelumen) and vacuum pressures (50-200mmHg)have been used for oocyte recovery in humansand domestic animals (1). Oocyte recovery andquality vary with some combinations used (2).To enable the appropriate needle and vacuumchoice to be made for cattle transvaginal oocyterecovery (TVR), we investigated the effects ofneedle size and vacuum pressure.MATERIALS AND METHODSFollicles (2-7mm) were aspirated from ovariescollected from slaughtered cattle using a 50cmlong, single lumen needle. Oocytes were gradedforthe presence of intact cumulus (G1), partiallydamaged cumulus (G2) and partially orcompletely denuded (G3). Between 210 and 220follicles were aspirated for each of three needlesizes (16, 17 or 18G) at four vacuum pressures(25, 50, 75 & 100mmHg).In addition to the in vitro ovarian aspirations,TVR was performed on 25 fifteen-month oldHereford nulliparus cattle using a 50cm, 17Gneedle with 75mmHg vacuum pressure. AnAloka (SSD-500, Japan) 7.5MHz linearultrasound probe was used to visualise theovaries.RESULTSThe mean number offollicles aspirated per ovarywas 13.1 (2575/197) and overall oocyte recoveryrate was 53.2%. Recovery rate was affected byrecovery day (P

EARLY EMBRYO SURVIVAL IS HIGHER FOLLOWING TWIN RATHER THAN SINGLETRANSFER OF FROZEN-THAWED, BUT NOT FRESHIN VITRO PRODUCED BOVINE EMBRYOSW.ij. McMillan, P.A. Pugh and AJ. PetersonAgResearch Rualrura, PB 3123, Hamilton, New ZealandINTRODUCTIONThere appears to be little published data comparingsingle and twin post-transfer embryo survival· (ES)of in vitro-produced (IVP) bovine embryos indesigned experiments. In one report where eithersingle or twin IVP embryos were transferred freshthere was a tendency for higher survival in singleembryos (1). The survival of each of twintransferredIVP embryos appears to be a dependentevent when transferred fresh, but an independentevent when transferred frozen-thawed (2). Thepotential therefore exists for an interaction betweenthe number of embryos transferred and freezethawing.The aim of the current study was to testthe hypothesis that the difference in ES betweensingle and twin transferred embryos was independentoftransfer as fresh or frozen-thawed embryos.MATERIALS AND METHODSNinety (Trial 1) and 109 (Trial 2) I8-month oldHereford x Friesian heifer recipients were randomlyallocated to one of 4 treatment groups in a 2 x 2factorial design (single or twin transfer; fresh orfrozen-thawed transfer). ES (n=300 embryos) wasassessed by slaughtering a random sample of heifersat d 18 (n=85 embryos assessed); slaughtering nonreturningheifers at d 25 (n=49 embryos); recordingreturn oestrus to d 50 (n=118 embryos) andslaughtering the remaining non-returning heifers at d50 (n=48 embryos). ES was defmed as the number ofembryos surviving to the time of assessment/totalembryos transferred, summed over all assessmenttimes.RESULTSAn interaction was apparent in Trial 1. There wasno difference in ES between embryos transferredfresh as singles (n=23 embryos) or as twins (n=46)(39 vs. 28%, Table 1). However, ES was higher fortwin (n=46) compared with single (n=21) transfers ifembryos had been frozen-thawed (26 vs. 0%, P =0.025, Table I). Single fresh embryos were morelikely to survive than single frozen-thawed embryos(39 vs. 0%, P

EFFECT OF SPERM MOTILITY ON THE FERTILISATION ~TE BYINTRACYTOPLASMIC SPERM MICROINJECTIONOrly Lacham-Kaplan and Alan trounsonInstitute of Reproduction, Centre for Early Human Development,Monash Mniversity, 246 Clayton Rd, Clayton, Melbourne, Ausralia.INTRODUCTIONThe use of intracytoplasmic spermmicroinjection(lCS1) as a treatment for severe male infertilityhas enabled most of these pateints to fertilizetheir wives oocytes(l). The insertion of asingle sperm into the ooplasm results in anincreased fertilization rate when compared to invitro insemination or sub zonal sperm injection.High recovery rates of motile sperm can beobtained from most samples of ejaculated andepididymal sperm (70%-100%). However, in somemale factor patients and frozen thawedepididymal sperm the recovery of motile sperm isbelow 10%. The present study examines the effectof sperm motility on the fertilization rateusing intracytoplasmic sperm microinjection ofejaculated and epididymal sperm.MATERIALS AND METHODSEjaculated sperm were washed by high speedcentrifugation(1800g)through a Percoll gradient(90%,45%) for 20 min. The 90% percoll layer wasretained and washed with HTF+30mg/ml BSA (1800gx 5min), followed by swim up of the pelletedsperm into fresh HTF+30mg/ml BSA until they wereneeded for microinjection. The top flraction ofthe sperm solution which contained the motilesperm was separated, and washed (1800g x 5min)in PBS+3mg/ml BSA. The pelleted sperm werediluted to lx10" sperm/ml in PBS+3mg/ml BSA.Fresh epididymal sperm which were obtainedsurgically from the epididymis (2) and frozen/thawed epididymal sperm were treated the same asejaculated sperm. A 100pl drop of the spermsolution was placed on the microinjectionchamber (3). Cumulus oocyte complexes obtainedafter follicular aspiration were transferredinto HTF+10% pateint serum. The cumulus cellswere removed. by a brief exposure to 0.1%hyaluronidase. Up to 5 mature oocytes wereplaced in the sperm drop located on themicroinjecton chamber. A single sperm wasselected, immobilized and ins~rted into theoocytes as described previously (3). Inaddition, fully motile and mechanicallyimmobilized sperm from a motile popUlation wereinjected into oocytes. The ocytes were examinedfor fertilization 17-19h after microinjection.RESULTSThere was a significant reduction, in thefertilization rate by non-motile ejaculatedsperm and epididymal sperm when compared tomotile sperm (Table 1 and Table 2) .Only 38% ofthe oocytes were fertilized by immotileejaculated sperm compared to 59% of oocytesinj ected with sperm from a motile population(Table 1) (p

RETROSPECTIVE ANALYSIS OF SUPEROVULATED CYCLES FOR ICSI YIELDINGAN INCREASED NUMBER OF IMMATURE OOCYTESJ.Smitz C. Janssenswillen J. Liu, H. Joris, M. Camus, P. Devrgey and A. Van Steirtegh~mCentre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University, BelgiumINTRODUCTIONWomen undergoing in-vitro fertilization by intracytoplasmicsperm injection are stimulated routinely by a combinedgonadotropin releasing-hormone agonist (GnRHa) andhuman menopausal gonadotropin (HMG) desensitisationprotocol. The final oocyte maturation is induced by humanchorionic gonadotropin (HCG). The nuclear maturationcan be precisely evaluated by removal of the surroundingcumulus-corona granulosa cell layer (CCG) and observationfor the presence of the first polar body (PB). The cohort offollicles stimulated by HMG is usually asynchronous andfollicles of different diameter are punctured 36 hours afterHCG injection.In a first series of of 227 consecutive GnRHa and HMGstimulations 80% of the oocytes were at the metaphase IIstage (MIl) at the moment of retrieval and 20% of theoocytes were either at the metaphase I stage (MI) orgerminal vesicle (GV) stage.In the present study we wanted to evaluate the stimulationprofiles and the outcome of cycles which yielded more than40% immature oocytes. We questioned whether thepresence of an elevated oocyte immaturity rate wouldinfluence fertilisability, developmental and implantationpotential of the mature oocytes from the same cycles.PATIENTS AND METHODSThe origin of infertility in these couples was severeandrological impairment. The majority of women in thisgroup were regularly menstruating. Out of 1471consecutive IC51-treatments (over a 2 year-period) thecycles with ~ 40% immature oocytes were listed. For thisanalysis we considered exclusively the cycles with a normalovarian response i.e. when ~ 5 oocytes were recovered.These cycles were compared to matched control cyclesperformed in the same period but with a percentage ofimmature oocytes < than 40%.Ovarian stimulation treatmentAll cycles were stimulated with a combined Buserelin andHMG long protocol as described previously (1). Thedecision for HCG injection was decided on the presence offollicular diameters of the largest group of folliclesexceeding 17 mm and/or concentrations of serum17 ~-estradiol > 300 ng/L per large follicle. Lutealsupplementation with 600 mg micronised naturalprogesterone intravaginally was systematically given (2).Oocyte retrieval fertilization and embryo cultureOocyte retrieval was done 36 hours after the ovulatorystimulus. The CCG were kept for less than 1 minute inmedium with the hyaluronidase and the removal of theCGG was enhanced by aspirating the oocytes in and outhand-drawn pipettes. The oocytes were put under aninverted microscope to assess the maturation stage by theobservation of a germinal vesicle (GV) and the presence(Mil) or absence (MI) of the first polar body (PB). Alloocytes that had extruded the first polar body were injectedwith a single sperm (3). Fertilization was observed byvisualisation of 2 pronuclei 16 hours after single sperminjection and embryo quality was based on the equality ofsize of the blastomeres and the proportion of anucleatedfragments (4). Transfer of embryos was limited to 3embryos. Supernumerary embryos with < 20% fragmentswere cryopreserved (5). .RESULTSOut of 1471 stimulation cycles the absence of a secondpolar body was revealed in more than 40% of the retrievedoocytes in 124 cycles (110 women). In 13 women theappearance of a high proportion of GV oocytes happenedrepeatedly. We compaired the frequency distribution of thenumber ofoocytes per cycle in the GV ~ 40 % group withthe matched control group. The two distributions had asimilar profile. We analysed the outcome of these cycles inaccordance with the number of oocytes per retrieval. Theresults are summarised in table I.We aimed to find differences in the ovarian stimulationprofiles that might explain the abnormally high rate ofimmature oocytes per retrieval. These figures are listed inTable III.OocyteslcyclePreovulatory E2 conc (mglL)(P fJglL)N° of days of exponentialE2 riseTotal number of HMGamps usedN° of follicles with meandiameter ~ 18 mmon day of HCG injectionN° offollicles ~ 18 mm perN° of retrieved oocytes (%)Study group Control groupGV ~ 40% GV < 40%Oocytes/cycle < 15 ~ 15 < 15 ~15N° cycles 75 49 84 40Fertilization (%) of Mil oocytes 65±28 56±23 64±20 59±28Cleavage rate (%) to embryosof good and/or fair quality 74±27 69±24 67±28 66±29N° of embryos transferred 1.93 2.51) 2.50 2.63per cycle (± 1.03) (±0.65) (±0.68) (±0.54)Implantation rate (%)per embryo 12.5 20.2 22.5 25.0N° of embryos frozen forN° of patients 16 for 8 69 for 23 96 for 32 146 for 23The detailed results of the embryo transfers shown intable II.Study group (GV >40%)Study group< 15 ~ 151831 2527(±646) (±530)0.84 0.86(± 0.58) (± 0.60)7.6 6.2(±1.93)a (±0.93)a39 25.5(±19)b (±9.1)b4.1 5.1(±1.95)c (±2.52)da, b, c, e, f: P < 0.001; d : P < 0.01; g, h : P < 0.05Control group< 15 ~ 151899 2621(±537) (±649)0.83 0.87(±0.92) (±0.71)7.0 6.7(±1.43) (±1.1)35 27.2(±15) (±10.5)5.4 6.6(±1.84)c (±2.69)d48.6 26.2 56.9 33.7(±26.1)e,g (± 15.5)e,h (±25.7) f,g (± 16.1) f,hCONCLUSIONSIn 8.4% of consecutive GnRHa/HMG cycles an oocyteimmaturity rate of more than 40% was observed. Themetaphase II oocytes from these cycles had a similarfertilization and cleavage potential than those from thematched control group. The lower implantation and takehome-babyrates in cycles with a high proportion ofimmature oocytes are due to the lower availability of goodquality embryos for transfer.A lower proportion of preovulatory follicles with a meanfollicular diameter of ~18 mm in cycles was found incycles with a high oocyte immaturity rate. In the cyclesproducing more than 15 oocytes the ovulatory stimulus wasgiven when only 25 to 35% of the follicles were larger than18 mm diameter.REFERENCES1) Smitz et aL, Hum. Reprod. 3, 585-590,19882) Smitz et aL, Hum. Reprod. 7, 168-175, 19923) Van Steirteghem et aL, Hum. Reprod. 8, 1055-1060, 19934) Staessen et aL, J. Vitro Fert. Embryo Transf. 6, 22-29, 19895) Van Steirteghem et aL, Hum. Reprod. 2, 593-598, 1987ControlsN° of cycles without a transfer 11 9Embryo transfer (% of total)with 1 embryo 21 12 embryos 35 383 embryos 44 61Transfer of 3 embryosNumber 51 65Embryo quality distribution% of E's with exellent 13 16good quality 67 74fair 20 10FOLLICULAR GROWTH AND OOCYTE COLLECTION IN HEIFERS IMMUNISEDAGAINST INHIBIN.R. C. Fry, T.L. Simpson, T.J. Squires and 1.K Findlay *.VIAS, Werribee, Vic 3030 and *PHIMR, Clayton, Vic 3168IntroductionThe repeated aspiration of follicles from elite heifers canproduce numerous oocytes for embryo production (1).We can aspirate all follicles >2 mm diameter on theovaries using a 17 gauge needle attached to a 6.5 MHzultrasound probe (Toshiba Capasee, 2). This studyinvestigated the influence of immunisation againstinhibin' or repeated FSH on follicular growth, oocyterecovery and oocyte quality in heifers.Materials and MethodsTwenty five 15 month old Friesian heifers were allocatedto 5 groups (n=5). Four groups (PLC, INHI5, INH30,INH250) were immunised against inhibin (Biotech Aust)at dose rates of 0, 15, 30 and 250 Ilg given 4 weeks priorto and at the start of the treatment period. The 5th group(FSH) received 16mg FSH (Follitropin) + 200 ill PMSGat each aspiration. All heifers received progestagen earimplants (Crestar) throughout the experiment to preventovulation and serum samples were taken for antibodyresponse.All heifers were aspirated twice weekly for 1 week priorto and during the 4 week treatment period. At eachaspiration, follicle number and size were recorded, allcandidate follicles aspirated, and the oocytes collectedand graded according to the cumulus vestment. Resultsfor the 4 week treatment period were analysed byANOVA (Genstat).ResultsThere was a mean of 16 follicles 2mm in diameter orgreater observed on the ovaries at each aspiration (9x 2­4mm, 4 x5-9mm, 2 xl0+mm). A mean of 9.0 follicleswas aspirated and 4.6 oocytes recovered (51 %). Of these,3.6 (78%) were classified as viable for IVMIIVF(1.5A,O.8B,1.4C) while 0.5 were Denuded and 0.6Expanded cumulus). Antibody levels against inhibin weredetected at the time of booster and reached a plateau 2weeks later (Fig. 1). Immunisation against inhibin ortreatment with FSHlPMSG did not alter the total numberof follicles on the ovary, but did change the follicle sizedistribution, with less 2-4 mm follicles and more 10+mm follicles (P.".I:>0:;; 20c:«

CHARACTERIZATION OF IN VITRO SYNTHESIZED PROTEINS SECRETED BYPORCINE NON-ATTACHED OVIDUCTAL EPITHELIAL CELLSPHERES TREATED WITH ESTROGEN.Ping Xia, Jean Rutledge, Andrew J. Watson and David T. AnnstrongDept. of Obstetrics and GynaecoIogy, The University of Western Ontario, London, ON,Canada N6A 5A5INTRODUCTIONRecent studies by Buhi et a1. (1992) have demonstrated thatestrogen is responsible for induction ofde novo synthesis andsecretion of certain oviductal secretory proteins (OSP) andinhibition of other OSP in porcine oviductal explant cultures. Inthe baboon (Boice 1990), sheep (Gandolfi 1991), hamster (Kan1988) and mouse (Kapur 1988), E:2-dependent oviductalglycoproteins contribute to the extracellular matrix ofthe embryoby associating with the zona pellucida and to the exclusivemicroenvironment of the embryo by entering the perivitellinespace. Studies in our laboratory have shown that E:2-treatedoviductal epithelial cells facilitate early porcine embryonicdevelopment in vitro (Xia and Armstrong, 1994). The presentwork was undertaken to detect in vitro synthesis of secretoryproteins by ~-treated porcine oviductal epithelial cells used forcoculture with early embryos.MATERIALS AND METHODSEpithelial cell sheets were squeezed from ampullae of oviductsand cultured in TCM 199 with 10% FCS with or without E 2 (1~g/ml) for 48 hr, after which the oviductal cell aggregatesformed epithelial cell spheres (ECS). The ECSs (40 ECSs) werecultured in 200 ~I MEM without methionine containing 5 mg/mlglucose, 500 ~Ci/ml L)SS-Methionine and with or without E:2 (1~g/ml) under paraffin oil for 6 hr. The culture medium wasdialysed against 10 mM Tris-HCI buffer, pH 8.2, and deionizedwater. Dialysed culture medium (10,000 or 50,000 cpm) wasanalyzed by both ID- and 2D-SDS-PAGE.RESULTSThe results from ID-SDS-PAGE showed that the in vitrosynthetic pattern of secretory proteins by ECS was altered by E 2treatment. These alterations included the appearance of onepolypeptide (33,000 Mr) and the disappearance of a secondpolypeptide (82,000 Mr) in the E:2-treated group, compared to the200-200-116-97-116-97- 66-82~tV 45-ctVC ~~45-31-33~31- 21-I I I2 3 4 7.6 7.0 6.0Figure 1. ID-SDS-PAGE.patterns observed for the controls. Additional proteins ofapproximate Mr 45,000 and 97,000 were increased in abundanceby E:2 treatment (Figure 1). Upon analyses by 2D-SDS-PAGE ofECS culture medium, the Mr 97,000 protein band was resolvedinto two acidic proteins of PI 4.5 and PI 5.1. The PI 5.1 proteinwas clearly suppressed in the ~-treated group (arrow 7) whereasthe PI 4.5 protein was increased by E:2 treatment (arrow 8). TheMr 45,000 protein complex in the ~-treated group was resolvedinto three acidic proteins, Mr 45,000 and 43,000, both with PI 5.5(arrow 4,5) and a Mr 36,000-45,000 protein complex with PI 4.8(arrow 6). Two basic proteins with PI 8.0 (Mr 36,000) (arrow 1)and PI 6.8 (Mr 25,000) (arrow 3) were both inhibited by E:2treatment. Another protein (Mr 25,000, PI 7.6) was increased inthe E:2-treated group (arrow 2).CONCLUSIONIn vitro synthetic patterns of secretory proteins by porcineoviductal epithelial cells used for embryonic coculture areinfluenced by E:2 treatment.REFERENCESBuhi WC et aI., 1992 J Exp ZooI262:426-435.Boice ML et aI., 1990 Bioi Reprod 43:340-346.Gandolfi C et aI., 1991 Eur J Basic Appl Histochem 35:383-392.Kan FWK, (1988) J Histochem Cytochem 36:1441-1447.Kapur RP and Johnson LV (1988) Anat Rec 221:720-729.Xia P and Armstrong DT (1994) Therigenology 41 :340.Figure 1: 1. ECS conditioned medium, control.2. E:2-treated ECS conditioned medium.3. ECS, control.4. E:2-treated ECS.Figure 2: A: ECS conditioned medium, control.B: E:2-treated ECS conditioned medium.Figure 2. 2D-SDS-PAGE11'1 A2 3I I I L5.1 4.5 7.6 7.0pHI6.0IBI5.1 4.5-CRYOPRESERVATION OF HUMAN SPERM WITH PENTOXIFYLLINEMonash IVF, 185 RoddIe Street, Richmond, Victoria 3121INTRODUCTIONAddition of 3mM pentoxifylline (PF), a cyclicadenosine 3'-5'-monophosphate (cAl\.1P) phosphodiesteraseinhibitor, to cryoprotectant has been shownto increase recovery of motile sperm post-thaw (1).This improvement may be due to (i) increasedintracellular cAl\.1P levels and direct stimulation ofmotility or (ii) PF acting as an antioxidant therebyreducing membrane damage associated with thefr~eze-thaw process. We have previously shown thatmotility and normal acrosome morphology (AM) aresignificantly reduced by cryopreservation and thatdecreases in normal AM may be due to cell death or tosubletllal damage including membrane disruption (2).The aim of this study was to investigate the effect ofPF on sperm during cryopreservation and to assess itspotential usefulness in improving the recovery offunctional sperm post-thaw.MATERIALS AND METHODSSemen samples from donors (n=15) with normalsemen characteristics (WHO, 1992) were assessed forprogressive and total motility and AM. Motility wasassessed on a warm-stage-equipped phase contrastmicroscope. AM was assessed by labelling ethanolfixed sperm with fluorescein-conjugated concanavalinA lectin (2). Samples were diluted 1: 1 with HumanSperm Preservation Medium (3) and divided into 4aliquots to which PF was added to the following finalconcentrations; 0 (control), 1, 3 and 10mM. Thesealiquots were mixed, aspirated into O.5m1 straws,frozen in liquid N 2 vapour and stored in liquid N 2 •Samples were thawed at 25°C for 10 minutes for reassessmentofmotility and AM which were reported asa percentage oftheir initial values (mean±sem).RESULTSPost-thaw progressive motility was found to beincreased in the presence of all concentrations of PFcompared to control (Fig. I). However, this increasewas only significant (p

-EFFECT OF LEVELS OF GLYCEROL AND COMPATIBLE SOLUTES ON THEPOST-THAW MOTILITY OF RAM SPERMATOZOAL.G. sanchez-Partida l , J.L. Zuppl, W.M.C. Maxwelz2 and B.P. Setchelzi.lDepartment ofAnimal Sciences at 1Waite Agricultural Research Institute, University ofAdelaide, Glen Osmond,SA 5064 and 2University ofSydney, NSW 2006.INTRODUCTIONSuccessful cryopreservation ofram spermatozoadepends on the presence of glycerol in thefreezing medium. However it has been suggestedthat glycerol may have a negative effect onfertility and lambing rates (1). The addition ofcompatible solutes (CS) has been reported toenhance post-thaw motility (PTM) of ramspermatozoa in the presence of 5% glycerol (2).In order to investigate the effect of differentlevels of glycerol and CS the following studieswere conducted.MATERIAL AND METHODSTwo factorial experiments were conducted,using tris-citrate diluents containing six levelsof glycerol (0,1,2,3,4,5 % v/v) and three levelsof CS (0, 54 and 81 mM). The CS were prolinein experiment 1 and glycine betaine i"nexperiment 2. All the diluents contained 15%egg yolk (v/v) at pH 7.0. Semen collection,dilution and freezing was done as previouslydescribed (2). Three pellets per diluent per ramwere individually thawed and assessed using aHamilton-Thorn motility analyser afterincubation in a water bath (37°C) for 0,4 or 8 h.REFERENCESRESULTSResults are presented in Figures 1 and 2. Theaddition of54 mM ofCS to the diluents improvedPTM in the presence of glycerol (P

EFFECT OF PROTEIN TYPE AND CONCENTRATION IN THE FREEZING MEDIUMON THE SURVIVAL OF IVP BOVINE EMBRYOS.P.A. Pugh. A.E.L., Ankersmit, L.T. McGowan and H.R. TervitAgResearch, Ruakura Agricultural Research Centre, PB 3123, Hamilton, N.Z.·- Paper withdrawn -INTRODUCTIONThe survival of in vitro produced (IVP) bovineembryos following freezing can be low (1) and isinfluenced by the type of culture system used (2).There. are reports of successful freezing of NPembryos in media with raised protein levels (3). Sinceproteins can have cryoprotective effects (4,5), thisstudy examined the effect of freezing medium BSAor FCS concentrations on the survival of IVP bovineembryos.MATERIALS AND l\1ETHODSEmbryos were produced by IVMlIVF and then IVC inSOF/amino acidslBSA as described previously (1).Good quality late morulae/early blastocysts; (LMlEB;n=187); expanded blastocysts (ExB, n=332) andhatching/hatched blastocysts (HB, n=142) wereselected for freezing. The basal freezing medium(FBI, 4 mg/ml BSA) was supplemented with 10%,20% or 50% v/v FeS or 5, 10 or 25 mg/ml BSA and1.5M ethylene glycol. Embryos were equilibrated atroom temperature in the freeze solution, loaded intostraws and frozen at -0.6 0 C/min to -35 0 C beforeplunging into liquid nitrogen. Straws were thawed(30 0 C) and recovered embryos held in PB1 + 0.5Msucrose at room temperature for 5 min. Embryoswere then cultured in SOF + 5% FCS for 48 h. Thosethat had re-expanded or hatched and maintainedintegrity were judged to have survived freezing.RESULTSSurvival rates are presented in Figure 1. There was asignificant negative effect of FCS concentration onsurvival (control, 64%; BSA, 64%; FCS, 53%;P

IN VITRO AND IN VIVO SURVIVAL OF CRYOPRESERVEDHATCHED BLASTOCYSTS IN PIGSH. Nagashima, N. Kashiwazaki, R.I. Ashman, M.B. NattIeBresatec Limited, Department of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, S.A. 5005CRYOPRESERVATION OF PORCINE IN VIVO AND IN VITRO DERIVED BLASTOCYSTSWITH GLYCEROL AND ETHYLENE GLYCOLN. Kashiwazaki, H. Nagashima, R.I. Ashman, M.B. NattIeBresatec Limitid, Department of Obstetrics & Oynaecology, University of Adelaide, SA 5005INTRODUCTIONThe production of live piglets from cryopreserved embryos isstill in its infancy and is restricted to embryos from the perihatchingstage (1). Survival of cryopreserved embryosfollowing transfer is extremely limited (1) and may bebecause the cryoprotective effect of 1.5M glycerol used inprevious studies is insufficient. The addition of 0.25Msucrose to freezing media containing glycerol has also beenreported to be beneficial for post-thaw survival of cattleembryos (2). The present study was undertaken to determinewhether increasing the concentration ofglycerol or addition ofsucrose to the freezing media could improve the efficiencywith which pig embryos can be successfully frozen.MA1ERIALS AND METHODSEarly hatched blastocysts were collected from superovulatedcrossbred (Large White/ Landrace) gilts six days after matingwith Large White boars. Embryos were cryopreserved in thepresence of 1.5M glycerol + 0.25 M sucrose (GS), 1.5Mglycerol (1.5G) or 1.8M glycerol (1.8G) using a conventionalslow cooling method. Embryos were cooled directly fromroom temperature to -6.5°C, seeded, and then cooled at a rateof 0.3°C/min to -30 (GS) or -35°C using a programnlablefreezer (Freeze Control, CryoLogic) before being plunged intoliquid nitrogen. Mter thawing in a 30°C water bath followedby stepwise dilution of the cryoprotectant, the post-thawsurvival of embryos was assessed by in vitro culture for 24 hin Whitten's medium supplemented with 15mg/ml BSA andtransfer. Frozen-thawed embryos were classified according totheir morphology into 3 categories (A: prefreeze morphology,B: damaged, C: extensively damaged) after culture.RESULTSAs shown in Table 1, post-thaw survival rates of embryoswere significantly improved by either increasing glycerolconcentration (1.8G) or adding sucrose to the freezingmedium (GS). More A-grade embryos were obtained in GSgroup compared with 1.5G and 1.8G groups. Transfer offrozen-thawed embryos from the GS, 1.5G and 1.8G groupswhich had been graded after culture for 24 h to 4 recipients didnot result in pregnancy (Table 2). Transfer of embryos fromGS group to a recipient 3 h after thawing without beinggraded resulted in the birth of4 piglets (Table 2; Fig.!).DISCUSSION/SUMMARY/CONCLUSIONSThe results demonstrate that addition of 0.25M sucrose to thefreezing media improves post-thaw survival compared withfreezing with glycerol alone. A marked feature of thisimprovement was the increase in the percentage ofblastocysts with little or no visible sign of damage (A-grade).Frozen-thawed embryos were evaluated morphologically afterin vitro culture for 24 h in an attempt to ensure that onlyviable embryos were transferred to recipient animals. Neithertransfer, however, resulted in the establishment ofpregnancy.In contrast, a litter of 4 live piglets was obtained followingthe transfer of 66 embryos, 3 h after thawing to one recipient.Although this result needs to be confirmed using furthernumbers of transfers, it is possible that in vitro culture afterthawing may have had a detrimental effect on embryoviability. In conclusion, the present study has demonstratedthat the in vitro survival of hatched blastocyst followingfreeze-thawing using 1.5M glycerol can be increased by theaddition of 0.25M sucrose and that this treatment can be usedto produce live piglets from frozen embryos.Table 1. Survival of cryopreserved porcine hatchedblastocyst in vitro.Cryopro- Embryo DeyelQpment after culture fQr 24 htectants survival A-grade B-grade C-grade(%) (%) (%)1.5G 40148 4 8 28(85.1)a (10.0)C (20.0) (70.0)1.8G 55/61 16 26 13(91.6)b (29.1)d (47.3) (23.6)GS 136 63 36 20(95.2)b (52.9)e (30.1) (16.8)a vs b, c vs d P

PRODUCTION OF PIGS CONTAINING A METALLOTHIONEIN PORCINE GROWTHHORMONE GENE CONSTRUCTM.B. Nottle, H. Nagashima, N. Kashiwazaki, R.I. Ashman, Z. Du, C.G. Grupen, S.M. McIlfatrick,M. Harding, C. Cheah, R.I. Crawford, A.I. RobinsBresatec Limited. PO Box 11, Rundle Mall, Adelaide, South Australia, 5005, Australia-An evaluation of a tyrosinase minigene as a marker for transgenesis inQuackenbush mice.R. Sutton, ~.A. Fawcett, J. '0 Grady, W.G. Ward, and B.W. BrownCSIRO Division of Animal Production, Locked Bag 1, Delivery Centre, Blacktown, NSW,2148INTRODUCTIONIntegration rates for pigs containing variousgrowth hormone gene constructs vary from 0.04to 1.73 % of embryos injected and from 5.6 to35.3 % of piglets born (1). In this abstract wereport integration rates for pigs containing ametallothionein porcine growth hormone geneconstruct. Data are from two trials conducted inApril (Trial 1) and November (Trial 2) 1993.MATERIALS AND METHODSLarge White x Landrace gilts were mated at theirsecond oestrus and then aborted by intramuscularinjection (Lm.) of 1 mg of the prostaglandin F2uanalogue, cloprostenol (Estrumate; Pitman-MoorePty. Ltd., New South Wales, Aust.) between 25 and40 days after mating, followed by a secondinjection of 0.5 mg cloprostenol 24 h later.Animals were superovulated with 1000 Lu. eCGLm. (Pregnecol, Heriot AgVet, Vic., Aust.) given atthe same time as the second cloprostenol injection.Ovulation was induced by Lm. injection of 500Lu. hCG (Chorulon, Intervet Pty. Ltd., New SouthWales, Aust.) 72 hours later. Animals werenaturally mated 28-30 hours after hCG injectionand in Trial 2 were also inseminated with freshsemen (lxl0 10 sperm) 2 hours after naturalmating. Ova were recovered by retrogradeflushing of the oviducts with PBS plus 1% FCS,50-56 hours after hCG injection. Zygotes werecentrifuged at 12,000 x g for 8 minutes to enablethe pronuclei to be visualised. Zygotes were heldin Dulbecco's Minimal Essential Media with 25mM Hepes and 5mg/ml BSA. One pronucleus wasinjected with 4~10 picolitres of 10 ng/Ill DNA inPBS. 25-35 microinjected embryos weretransferred to each recipient. Integration of thetransgene was determined by PCR using DNAfrom a sample of tail tissue taken at birth.RESULTSEmbryo and animal data are shown in Tables 1and 2.Table 1. Mean (± sem) ovulation, recovery andfertilisation rates and the numbers of zygotes injected forthe two trials. Values are expressed on a per donor sowbasis.Ovulation No. ova No. ova No.rate recovered fertilised zygotesinjectedTrial 1(n=229) 16.6± 0.3 13.3±0.3 6.8 ±0.4 5.7±0.4Trial 2(n=151) 18.9+0.3 14.9+0.4 10.8+ 0.5 10.1+0.5Table 2. Number of recipients, farrowing rate, number ofpiglets born live and the number of these that weretransgenic for the two trials.Trial 1(n=229)Trial 2(n=151)No. of No. No. No.recipients recipients piglets transgenicfarrowing born live48472524DISCUSSIONACKNOWLEDGMENTSThe authors gratefully acknowledge the assistanceof Bunge Meat Industries Ltd., Corowa, New SouthWales, Australia.REFERENCES148141Data from the two trials indicate that between 5-10zygotes suitable for microinjection can beobtained per donor using the current protocol andthat 3-6 donors are required to provide 25-35embryos for one transfer. Overall integration ratesfor the transgene were 2.9 % of embryos injectedor 30.4% of piglets born live.(1) Brem, G. (1992). In 'Embryonic Developmentand Manipulation in Animal Production' EdA. Laurie and F. Gandolfi . p 147. PortlandPress. London.(2) French, AJ. et al., (1991) Theriogenology 35:202.4345INTRODUCTIONThe aim of the present study was to test the utility ofa tyrosinase minigene (ptrTYR5) as a marker fortransgenesis as described by Beermann et al.(1). Thisgene can genetically rescue albino mice, restoringpigment to hair and eyes.These authors suggested that ptrTYR5 could be coinjectedas a marker with other transgenes (3 out of 5of their transgenics carried both genes). The coinheritanceof pigmented hair with a transgene ofinterest would greatly reduce the amount of screeningrequired to maintain the transgenic line.MATERIALS AND METHODSWe generated transgenic mice by microinjection ofptrTYR5 into pronuclei of single cell newly fertilisedeggs. This 11.1 kb construct includes a short 270bppromoter, the first exon and first intron from themouse gene; and remaining 4 exons from mouseeDNA; and 850bp SV40 polyadenylation signal. Wetested tested for transgenesis by phenotype, PCR andSouthern blotting. We compared the microinjection ofptrTYR5 into Quackenbush mice at 2 concentrations,with that of 3 other genes (all 5ng/ul) injected into F1mice. Gene 1 was injected before the 5ng/ul series ofptrTYR5; Gene 3 and then Gene 5 were microinjectedin the period as the 2.5ng/ul ptrTYR5 often on analternate days in the same week.TABLE 1. TRANSGENESIS SUCCESS RATESRESULTSWe had a lower success rate with this transgene inQuackenbush mice than other genes microinjectedinto the CBAxC57BU6(F1) mice normally used in thislaboratory. In Quackenbush mice, the pronuclei werevisible in fewer eggs (57% cf 72%) so that fewerembryos could be injected, embryo survival of themicroinjection was similar, so that the numbers ofembryos transferred are comparable; and the level oftransgenesis is lower than usual. The 5ng/ulconcentration appeared to be toxic.DISCUSSIONWe have successfully restored the pigmentation toalbino Quackenbush mice by transgenesis with atyrosinase mini-gene, ptrTYR5.However, we would not at this stage recommend theco-microinjection of ptrTYR5 as a marker with gene ofinterest using albino Quackenbush mice as analternative to use of C57Black/F1 mice. Other optionswhich could be explored would be to use an F1 crossof 2 albino lines, to engineer a shorter transgene andto further reduce the concentration of the tyrosinaseminigene used.REFERENCEBeermann, F., Ruppert, S., Hummler, E. andSchutz,G.(1991) Nucl. Acids Res. 19:958Total Embryos Embryos Pups TransgenicEmbryos Injected Transferred Born MiceptrTYR5 314 183 140 9 0(5ng/ul) 58% 77% 6% 0%ptrTYR5 462 260 194 10 1(2.5ng/ul) 56% 75% 5% 10%Gene 1 748 68% 69% 11% 5% (n=2)Gene 3 505 76% 78% 13% 13% (n=5)Gene 5 748 73% 75% 13% 28% (n=15)33 34

Effects of zona pellucida on electrofusion and development of two .cell mouse embl)'osBrendan Tatham, Kim Giliam, Aneta Dowsing, Tohsak Mahaworasilpa 1 , and Alan TrounsonCEHD Monash University Melbourne and IDepartment Physi~s University New South Wales SydneyAustralia.INTRODUCTIONElectrofusion is a process where electric fields induce cell to cellfusion and is a valuable technique used to fuse embryonic cells fornuclear transfer. The modification of the nuclear transfer procedurewhich alleviated the requirement for micromanipulation so that oocyteswere enucleated by centrifugation. The new technique involved theremoval of the zona pellucida, the effect of this on the electrofusionproperties ofcells is unknown. This study in vestigated the electrofusionofbiastomeres of 2 cell mouse embryos with the zona intact or absent'and the development of these tetraploid embryos when cultured invitro.MATERIALS AND :METHODSFemale C57/BIJxCBAlWehi mice were superovulated then matedbefore embryos at the 2 cell stage were collected. The zona pellucidawas removed with Pronase. Embryos were equilibrated for 10 minutesin fusion media (0.25 M sucrose, pH 7.4) \vith 4 washes. Blastomereswere then aligned for 5 to 10 seconds with an AC pulse of 8 voltsamplitude in an electrofusion chamber which consisted ofparallel wireswith a diameter of 0.5 mm and a separation of 0.5 mm. Electrofusionwas then achieved with a single DC pulse of 10, 50 or 100 ~s duration,with the amplitudes from 0.4 to 4.8 kV/cm . Electrofusion was scored1 hour after electrical treatment when groups of control and fusedembryos were cultured in M16 until the blastocyst stage.RESULTSThe results of approximately 1500 electrofusion attempts showthat when DC pulses of less than 2.0 kVIcm amplitude were used theelectrofusion was not significantly different for embryos with the zonaintact (52.0%) or absent (61.5%). At amplitudes greater than 2.0 kV/cmDC the electrofusion of embryos with the zona intact (61.3%) wassignificantly greater than when absent (33.8%; P

· THE LOCALIZATION OF NEUTRAL ENDOPEPTIDASE IN THE OVINE UTERUS DURINGTHE OESTROUS CYCLE AND EARLY PREGNANCYSimon C Riley, Evelyn Wong, Jock K Findlay &Lois A SalamoQsenPrince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria, 3168Changes in Uterine Endometrial Phospholipids and Fatty Acids during theOestrous Cycle and Early Pregnancy in the Ewe,8 Meier, MA Trewhella, RJ Fairclough, G JenkinDepartment of Physiology Monash University Clayton Vic. 3168 AustraliaINTRODUCTIONNeutral endopeptidase (NEP; EC 3.4.24.11;'enkephalinase') is a membrane-bound cell surfaceectoenzyme with an extracellular active site thatcleaves and inactivates several bioaetive peptides. Itis expressed in the uterus in women and rats (1,2)and its substrates, which include oxytocin andendothelins are important in the control of uterinefunction. In women, NEP has been implicated in thecontrol of menstration, because NEP activitydecreases during the pre-menstrual phase allowing anincrease in the availability of endothelin to inducevasoconstriction of the spiral arterioles. The role ofNEP in the ovine uterus during the oestrous cycle andat the time of implantation, in which peptidesubstrates of NEP have been implicated (3) has notbeen determined. This study examines for thepresence and localization of NEP in the sheep uterusduring this period and investigates its regulation. byovarian steroids in vivo.MATERIALS AND METHODSThe uteri of parous Corriedale ewes (n=64; 4/group)throughout the oestrous cycle and the first 20 days ofpregnancy, and of ovariectomised ewes receiving notreatment (ovx), or progesterone (P), or oestradiol (E),or E+P, were flushed and collected by hysterectomy.Pregnancy was confirmed by identification of anembryo. Tissues were fixed in formalin forimmunohistochemistry. Specific NEP immunostainingwas identified using a monoclonal antibody tocommon acute lymphoblastic leukemia antigen (CloneJ5; Coulter Corp.), and visualised using the VectorABC kit. Sections were scored on a scale of 0-3 (0 =no staining; 3 = strong staining throughout a cell type)in comparison with both positive and negative controlsections.ReSULTSSpecific_ NEP immunostaining was present in theuterus throught the oestrous cycle and early pregnancy.In the endometrium,' NEP was' localised instromal cells except those immediately underlying theepithelia, . in increasing intensity to I day 14 of theoestrous cycle, which was maintained from Days 15-.20 of pregnancy (Fig.1). Weak staining was presentin luminal and glandular epithelium. Strong stainingwas observed in myometrium, in particular in a bandadjacent to the endometrium, and in endothelial andsmooth muscle cells of the vasculature (Fig.1). In theDay 20 conceptus, staining was localized inuninucleate, but not in binucleate cells, and in heartand brain of the embryo. In ovx ewes untreated, ortreated with E or P, immunostaining in all tissuecompartments was strong, whereas E+P treatmentresulted in a weaker pattern of staining (Fig.1).DISCUSSIONNEP immunoreactivity is present in the ovine uterusand is regulated at least in part by ovarian steroids.NEP is distributed in specific regions that may conveya partitioning effect within the tissue on its bioactivepeptide substrates. The band of NEP in themyometrium may prevent the passage of oxytocin andendothelin, potent myometrial constrictors frompassing into the myometrium. The presence of NEPwithin the vasculature may regulate the paracrineeffect of endothelin vascular tone, and the passage ofpeptides between the uterus and systemic circulation.The absence of NEP in luminal and glandular epitheliawould maintain the high concentrations of suchpeptides that are required for preparation of the uterusand the implantation processes in the uterine lumen.The presence of NEP in the trophoblast and embryoindicates an ability to regulate bioaetive peptides atthis early stage of development.FIGURE 1: Specific NEP immunostaining in caruncular andintercaruncular stroma (a,c) and myometrium, vascularsmooth muscle and endothelium (b,d) during earlypregnancy (a,b) and in steroid-treated ovariectomised ewes(c,d).3 (a)2.521.5E' 1'c 'ro§ 0.52_ 0..ut+-'-..lCf-I--t4-l-l"l""'-"4-JL....t'4-L.t.::j--1-l:'t-'-E.4 8 10 12 14 16 17 20'0,?;~ (e)~3j 2.521.510.5O~~t:a4-~l-l-Ovx P E E+PEarty pregnancy3 (b)2.521.510.5oOvariectorrised steroid-trsated4 8 10 12 14 16 17 20Ovx P E E+PREFERENCESCasey, et al. (1991) J BioI. Chem 266, 23041-7.Ottlecz, et al. (1991) J Cell Biochem 45,401-11.Riley, et al. (1994) J Reprod Fert 100, 4551-9.Supported by the NH&MRC and a PHIMR VacationScholarship to EW.IntroductionThis study was conducted to determinechanges in endometrial phospholipid andfatty acid content in association withprostaglandin synthesis and release duringluteolysis and early pregnancy.MethodsUterine endometrium was collected ondays 3, 12 and 15 of the oestrous cycleand on corresponding days of pregnancy.Lipids were extracted using chloroform andmethanol (2: 1 v/v), containing the antioxidant, BHT. Phospholipids wereseparated from total lipids using thin layerchromatography (TLC), with a solventsystem of chloroform:mathanol:aceticacid:water (65:60:6:4 v/v). Areascorresponding to standard phospholipids(PC, PS, PI, PE) were removed separately.The fatty acids associated with eachphospholipid were measured as the fattyacid methyl esters (FAME), using gaschromatography, and a BPX-70 columnpacked with 70% cynopropyl siloxane,(0.25 ~m; Ld. 0.22 mm;25 m length). EachFAME was quantitated against an internalstandard C17:0. Different stages of thecycle and pregnancy were comparedusing ANOVA and expressed as the mean± standard error of the mean (sem).ResultsTotal lipids extracted from the uterineendometrium during the oestrous cycle andearly pregnancy are summarised inTable 1.TABLE 1. Total Lipids (mg/g tissue)Stage * Mean semDay 3 c. 17.93a,o 3.79Day 12 c. 20.53°'c 1.72Day 15 c. 27.62 c 3.64Day 3 p. 15.77 a 1.01Day 12 p. 25.72°'c 1.68Day 15 p. 17.03 a 1.15* c. oestrous cycle, p. pregnancya,b,c statistical significance (p

PORCINE-RElAXIN DIFFERENTIALLY INHIBITS UTERINE LONGITUDINAL & CIRCULAR MUSCLESIN VIVO IN OESTROUS & EARLY PREGNANT RATS.Helen Massa and Len MartinDepartment of Physiology and Pharmacology, University of Queensland, Q4072, AustraliaThe period of uterine receptivity in the pseudopregnant Quackenbush Special mouse asdefined by the deciduogenic lectin, Concanavalin AJennette A. Sakofjand R. N MurdochDepartment ofBiological Sciences, The University ofNewcastle, N.S.W., 2308INTRODUCTION Myometrial contractions increase incomplexity and frequency in early pregnant rats (1,2). Thecontractions are essential for normal embryo spacing andorientation since these are disturbed when myometrialactivity is inhibited with porcine relaxin (p-RXN; 3,4).MATERIALS AND METHODS Oestrous (Oe) & day 5pregnant rats (d5P; d1=day of sperm) underwent videolaparoscopy(VL; 1,2). VL started at 09:00h, 1h afteranaesthesia (Ketamine:Xylazine;60:8 mg kg- 1 ) and insertionof a jugular cannula, and lasted for 3.5h. In each rat, vehicle(V, 0.9% NaCI), oxytocin (Ot, 20mU) & p-RXN (0.2, 1, 5~g,one dose/rat, 5 rats/dose) were given 20 min apart asboluses (0.5mL IV) in the sequence V, Ot, p-RXN, Ot, Ot(Fig 1). Myometrial activity, analysed from video-tape replay(1,2) was recorded as the number 10min- 1 of longitudinal &circular contractions propagating towards the cervix or ovary(respectively Lc, Cc La, Co) and examined by ANOVA.RESULTS At Oe, most contractions were Lc: on d5P, theirfrequency fell whilst that of Lo, Cc & Co rose, as before(1,2). A bolus of Ot increased Lo & Co in both Oe & d5Prats [P

EARLY DECIDUOGENIC RESPONSE OF THE UTERINE MUCOSA TO THELECTIN CONCANAVALIN A AND SESAME OILINTRODUCTIONThe Decidual Cell Reaction (DCR) is vital for theestablishment of pregnancy and involves thespecialised differentiation of the uterine stromasurrounding an implanting blastocyst. It has longbeen recognised that agents other than a blastocystare capable of initiating a Decidual like response.Few studies have investigated the interactionbetween deciduogenic agents and the uterineepithelium -the first point of contact between theblastocyst and maternal tissue. Given the widelydiffering composition of deciduogenic agentsavailable, it would be of interest to compare themanner in which these agents interact with theuterine epithelium. This investigation compares theinitial stages of deciduomata fonnation using oil, acommonly used deciduogenic agent and the lectinConcanavalin A (ConA).MATERIALS AND METHODSPseudopregnancy (PSP) was induced on themorning of oestrus in adult virgin rats. On themorning of day 5 PSP, Animals were anaesthetisedand given an intraluminal injection of either 1 mgof ConA in 50 pl of saline, 50 JIl of sesame oil or50 JII of saline alone. Animals were sacrificed 6, 12and 24 hours after injection. Fifteen minutes prior tosacrifice animals were injected with 0.3 ml of 0.1 %Pontamine Sky Blue to visualise stromal oedema.Tissue was processed for routine light microscopyand Electron Microscopy. Observations of tissuemorphology were made from wax serial sectionsand 1 JIm thick semi-thin sections.RESULTSTwelve and 24 hours following injection of eitherConA or oil, blue bands were clearly visible withinthe uteri. Tissue sections revealed marked stromaloedema. Other indicators of a decidual-like reaction,including differentiation of stromal cells and thefonnation of the Primary Decidual Zone (PDZ)were visible in the uterine hom of both ConA andoil treated groups at these times. The reaction of theepithelium of the two groups however variedsignificantly. The f epithelium of animals injectedwith oil was greatly attenuated within the bluebands (fig 1) and in places was entirely missing,often appearing to be contained within luminal oildroplets. The epithelium of ConA injected rats didnot display 'such damage. However, within blueTJ. Shaw and C.R. MurphyDept. Anatomy and Histology, University of Sydney.bands from ConA injected animals, white bloodcells were often aggregated under the epithelium,and preliminary results suggest that within the blueband the white cells appeared to breach the uterinelumen (fig 2).DISCUSSIONWe conclude that both ConA and Oil Inducedeciduomata formation on Day 5 of PSP. Themanner in which the.se two agents interact with theuterine mucosa, however, differs significantly. Oilappears to cause marked uterine trauma supportingthe theory of Lundkvist 1982, that oil is in fact atraumatic deciduogenic agent. The exact manner inwhich ConA induces deciduomata is unclear. It ishowever apparent th&t ConA alone does not causethe degree of trauma associated with oil, and it ispossible the white blood cells present followinginjection are themselves involved in the initiation ofdeciduomata.Figure 1. Uterine mucosa following injection of oil,attenuated epithelial cells (arrow) are visible. x830.Oil droplet is out of field of view.Figure 2. Uterine mucosa following injection ofConA, White blood cells are visible in lumen (A),and arrow indicates point of epithelial crossing.x830.Lundkvist, O. & Nilsson, B.O. 1982, Cell Tiss Res.225,355-364.EFFECTS OF THE INTERACTION BETWEEN CLOMIPHENE CITRATE AND THE OVARIAN HORMONESON THE SURFACE ULTRASTRUCTURE OF THE UTERINE LUMINAL EPITHELIAL CELLSMJ.Hosie and C.R.Murphy, Dept Anatomy and Histology, The University of Sydney, NSW, 2006.INTRODUCTION: The widely used super-ovulatory drug,Clomiphene citrate (CC) is a synthetic non-steroidal impededoestrogenlantioestrogen thought to act via the hypothalamicpituitaryaxis resulting in the maturation of multiple follicles (1).Although CC is a valuable and efficient super-ovulator (70-80%of women) the ensuing pregnancy rates are relatively low (2). Ithas. been suggested that this may be due to altered endometrialdevelopment (3), which is thought to be critical for implantation(4). Currently there is little research on the ultrastructural effectsof CC on the uterus. This study therefore focuses on the surfaceultrastructural effects of CC at the time of blastocyst implantation(around day 6 of pregnancy in the rat). Scanning electronmicroscopy (SEM) was used to evaluate uterine surfaceultrastructure from ovariectomized rats treated with CC andoestradiol-17p (E2) or progesterone (P 4 ) or these two hormonesgiven in combination, as well as giving CC prior to the P4-P4.P4E2sequence of hormones, given over 3 days, which has been shownto induce both implantation and an ultrastructural picture similarto that seen at implantation in ovariectomized rats.MATERIALS AND METHODS: This study used 60, 12-14week old sexually mature rats, bilaterally ovariectomized andallowed to recover for 4 weeks before treatment. The animalswere treated with: O.lml injections of: 0.51lg E 2 ; 5.0mg P 4 ; and/or0.25mg CC or 1.25mgCC. The experimental groups consisted of:1) P4+E2; 2)E2+O.25mgCC; 3)P4+O.25mgCC; in a single dose; 4)P­P-PE; 5)0.25mgCC-P-P-PE; 6)1.25mgCC-P-P-PE; 7)P-P-P; thesewere given over 4 days. Group 8 consisted of day 6 pregnantcontrols, and group 9 consisted of the appropriate placebo groupswhere the results were combined as the tissue appeared similar.The rats were killed 24 hours after the last treatment and thetissue routinely processed for SEM.RESULTS: It was found that when CClE2 (Fig 3) wereadministered together, the surface ultrastructure was similar inmany respects to that seen in the control (Fig 1) or E/P4 (Fig 2)or P4 alone were administered, suggesting that in this situation CCis behaving as an anti-oestrogen or' a progesterone. Typicalfeatures include short relatively sparse microvilli and raised cellborders. At low power these groups all resembled each other inthat the tissue had an overall smooth appearance with few smallgland openings. When CCIP 4(Fig 4) was given the result wasoestrogenic with oval to round domed cells with recessed borderscovered with dense blunt sometimes long MV. At low power theepithelium had a roughened appearance with some foldingevident, suggesting that CC is acting as an oestrogen in thissituation and/or in some way preventing progesterone action in theexpression of the progestogenic features usually qbserved. WhenCC is given prior to the PPPE sequence (Fig 5), the surfaceultrastructure of the tissue most closely resembled that observedwith CC alone showing very long microvilli, many droplets andareas of doughnut shaped cells (Fig 6). CC appears to override theusual surface ultrastructure seen with PPPE (Fig 7). Features seenwith both PPPE and day 6 pregnant tissue (Fig 8) include a lossof mv, and the appearance of many irregular protrusions anddroplets.CONCLUSION: These results indicate that when CC is given inconjunction with E2 or P4 it is capable of either behaving in asimilar fashion to P4 or E2. Clomiphene administration prior to thePPPE sequence of hormones appears to change the tissuetopography to a surface similar to that seen when CC is used onits own (5). Thus CC changes the ultrastructural appearance of theuterine epithelium and so may interfere with implantation by thismechanism.l.Clark lA, B.M. Markaverich (1982) Pharmac.Ther. 15,467-519.2.Navot D., N. Lufer (1989) IReproduct.Med. 34,3-9.3.Cummings A.M. et al (1991) Fundam.Appl.Toxicol. 16,506-16.4.Martel D.C. et al (1981) (Ide Brux, R.Martel, IP.Gautry,eds). Plenum Publishing Corporation.5.Hosie MJ., C.R.Murphy (1992) Acta Anat. 145,175-178.All Micrographs x6000.Fig 1, Placebo control.Fig 3, CCIE 2 .Fig 5, CCIPPPE Droplet (D).Fig 7, PPPE Protrusion (P).Fig 2, E21P4.Fig 4, CCIP 40Fig 6, CCIPPPE (X) Pit.Fig 8, Day 6 pregnancy_41 42

-VAGINAL EPITHELIUM OF OVARIECTOMISED RATS TREATED WITH COMBINEDCLOMIPHENE CITRATE AND EXOGENOUS OESTROGEN OR PROGESTERONEV. Terry, C.R. Murphy, C.D. ShoreyDept. Anatomy and Histology, University of Sydney, NSW, 2006INTRODUCTIONClomiphene citrate (CC), a non-ste.roidaloestrogen derivative, has been used In theinduction of ovulation in anovulatory women formany years (1) .Emphasis has been placed onresearching the effect of this compou~d on. thereproductive tract of neonates WIth httleattention to the adult reproductive tract alreadyexposed to steroidal hormones. Our previousstudies with CC on the adult rat vagIna haverevealed an oestrogenic component of activityand an ability to modify cell appearance (2,3).Since CC rarely acts in isolation, or in a systemdevoid of hormonal priming, we decided tostudy the interactive effects of CC and oestrogenor progesterone on the vagina of ovariectomisedadult rats.MATERIALS AND METHODSThirty-five Female virgin Wistar rats (12-14weeks of age) were bilaterally ovariectomisedunder halothane anaesthesia and maintail1ed for4 weeks prior to experimentation. Groups 1,2,3and 4 were designated control groups and givenno treatment or a single injection of carrier A orB or A+B only. Other groups were treated asfollows: Group 5 - 0.25mg CC + 5mg P4 (PC);Group 6 - 0.25mg CC + O.51lg E2 (EC); Group 7- 5mg P4 + O.5Jlg E2(PE). Animals were sacrificed 24 hrs afterinjection and tissue processed for light andscanning electron microscopy.RESULTSResults indicate that a 24 hr exposure to thecombined treatments can stimulate a dormantvaginal epithelium by initiating cell division,cause cell proliferation and modification.Analysis showed that there was no statistical orstructural differences between CC incombination with exogenous oestrogen orprogesterone (Fig 2). Both treatments resulted inincreased epithelial area, height, number oflayers and mitotic activity compared to controltissues (Fig 1). Statistical comparison with ourprevious study indicated that the two combinedtreatments using CC were similar to E2 alone.Scanning Electr~n microscopy revealed achanged surface morphology in all threetreatment groups. Control groups exhibitedlongitudinal vaginal wall folding, the covering ofwhich comprised polygonal cells clearlydemarcated by ridges (Fig 3a + 3b). Treatedgroups exhibited increased folding; surface cellswere irregularly arranged with slightly raised43apicies and separated by recessed borders.Numerous cells exhibited individually elongatedmicrovilli and/or rosette formations (Fig 4a +4b).CONCLUSIONIn conclusion it can be suggested that when CCis given in conjunction with P4 there is anenhancement of CC's oestrogenicity plus anincreased ability. to modify cell surfaceappearance. When CC is administered with E2there is no apparent cumulative effect from thatof E2 alone, but there is a modification of thesurface appearance.Fig 1. Control (x 500)Fig 3a. Control (x 920)Fig 2. PC, EC, PE (x 500)Fig 3b. Control (x 4600)Fig 4a. PC, EC, PE (x 920) . Fig 4b. PC, EC, PE (x 4600)1. Greenblatt R.B.,Barfield W.E., Junck E.C., Ray A.W.(1961) 1. Am. Med. Assoc. 178,101-1042. Terry V., Murphy C.R., Shorey C.D. (1992) Acta Anat.145,212-2153. Terry V., Murphy C.R., Shorey C.D. (1993) Acta Anat.148, 14-21..vOrigin of enhanced fetal growth in sheep administeredprogesterone during early pregnancyD. O. Kleemann J , S.K. Walker and R.F. Seamark1South Australian Research and Development Institute, TurretfieldResearch Centre, Rosedale, SA 5350 and 2 Department of Obstetrics andGynaecology, University of Adelaide, SA 5000INTRODUCTIONModification of the embryo's early environment can actto influence subsequent development. For example, insheep the transient exposure of day 3 embryos for threedays to an advanced uterine environment resulted inlarger fetuses compared with control fetuses whenmeasured on day 37 of pregnancy (1). Similarly,subjecting sheep zygotes to in vitro culture for 3-5 days(2) or transferring bovine nuclei to host oocytes (3) canresult in progeny of excessive birthweight. In the cow(4) and sheep (5) progesterone administered early inpregnancy enhanced fetal development compared withthat observed in control animals . In all these studies,changes to the embryo or to the embryo's environmentoccurred within the first few days of development thushighlighting the significance of this period on subsequentfetal performance. In the present experiment weexamined if the effect of progesterone on fetal growth ismediated during the days of treatment or is longer termin nature and whether any effect of progesterone isdependent on the exposure of embryos to the uterineenvironment during supplementation.MATERIALS AND METHODSZygotes (n = 949) were collected and transferred to theoviducts of initial recipient Merino ewes (n = 36; 6ewes per replicate) that were randomly allocated toeither a control group or a group treated withprogesterone pessaries (CIDR G) for three dayscommencing 13-18 h after the expected time ofovulation. In the latter group embryos were ligated inthe oviducts of 12 of 24 ewes. Embryos from eachtreatment group were recovered on day 3 andtransferred to uterine horns of final recipient ewes. Halfof these ewes were similarly treated with progesteronepessaries, giving a 3 x 2 factorial design. Bloodsamples were taken from initial recipients on day 3 andassayed for plasma progesterone concentrations. Eweswere slaughtered on day 76 and the number, weight, sexand crown-rump length of fetuses was recorded.RESULTSPeripheral progesterone concentrations were significantly(P

Prostaglandin production by placental tissues in late pregnant tammarwallabiesG. Shaw, H. Gehring and E.C. Bell.Department of Zoology, The University of Melbourne, Victoria Australia 3052.Prostaglandin production by endometrium in late pregnant tammarwallabiesG. Shaw, H. Gehring, and E.C. Bell.Department of Zoology, The University of Melbourne, Victoria Australia 3052.INTRODUCTIONProstaglandins (PGs) are released at parturitionin tammar wallabies, leading to a brief peak ofPGF2 metabolite (PGFM) in the peripheralplasma (1). There is increased PO production bythe uterine endometrium at term (2). This studyinvestigates the contribution of the placenta to PGprcxluction in late pregnancy.MATERIALS AND METHODSTammars have a thin, membranous,choriovitelline, or yolk sac, placenta. It ispossible to separate the placenta andendometrium cleanly, by gently pulling themembranes free, even at'full term. Females atday 18 of pregnancy (primitive streak: stage) tod26 of pregnancy (full term) were killed byanaesthetic overdose, the gravid uterus wasopened, and the fetus and placenta were gentlyremoved. Placenta was chopped into pieces 0.5 ­1 mm across. Portions of chopped tissue (10-20mg) were cultured on polycarbonate membranesat the liquid-air interface in 5 ml Ram l s-F10medium with antibiotics. Two replicates fromeach animal were set up. After a 4 hour preincubationthe medium was replaced. At 24h themedium was removed and frozen at -80°C inindomethacin coated tubes until assayed byspecific RIAs for PGF2a, PGE2 and PGFM.Tissue from each well was collected and assayedfor protein content against BSA standards.Results are expressed as mean ± s.e.m. of PGproduced (ng per day per ~g protein). Data aregrouped by stage: d18-22 (early organogenesis);d23-24; and d25-26 (term: viable neonates can beobtained at day 25, though they are usually bornon day 26).RESULTSPlacenta produced both PGF2a and PGE2, butlittle or no PGFM Figure 1. Production ofPGF2a was about 10 times that of PGE2.Production rates of by placenta increasedsignificantly during late pregnancy200180160e: 140ot5::J ..-.. 120-0 .f:o Q)0.. e 100e: Q.::g 0) 80~.€0)0)jg ..s 60enea.. 4020oI ~ .PGE2PGF2a~PGFMd18-22d23-24Days of gestationd25-26Figure 1. Prostaglandin production by explal1tsof yolk sac membrane from late pregnant tammarwallabies.DISCUSSIONPG production by the placenta increasesdramatically at term. What regulates this increaseis unknown. This increased placental PGproduction may contribute to the fetal (or fetoplacental)signal that triggers parturition.REFERENCES(1) Lewis PR, Fletcher TP & Renfree MB(1986) J. Endocrinol. 111:103-109.(2) Shaw G, Gehring, H & Bell, EC (1994)These Proceedings.INTRODUCTIONProstaglandins (ros) playa key role at parturitionin mammals, inducing uterine contractions andsoftening the cervix. In tammars there is a briefpulse ofPO F2a metabolite (PGFM) in peripheralplasma in the minutes around birth (1) probablyassociated with the uterine contractions that expelthe fetus. PGs may also synchronise thestereotyped parturient behaviour with birth (2).The source of the PGs has been assumed to be theuterus. This study examined the profile of POproduction by uterine tissue through latepregnancy.MATERIALS AND METHODSFemales at day 18 of pregnancy (primitive streak:stage) to d26 of pregnancy (full term) were killedby anaesthetic overdose and the uteri dissected out.Tammars have two separate uteri with separatecervices, and only carry a single fetus, so it waspossible to compare gravid and non-gravid uteri ateach stage. The uteri were opened, the fetus andplacenta were gently removed, and theendometrium removed form the myometrium usingforceps. Endometrial tissues were chopped intopieces 0.5 - 1 mm across. Portions of choppedtissue (10-20 mg) were cultured on polycarbonatemembranes at the liquid-air interface in 5 mlRam l s-FI0 medium with antibiotics. Tworeplicates for each tissue type from each animalwere set up. After a 4 hour pre-incubation freshmedium was supplied. At 24h the medium wasremoved and frozen at -80°C in indomethacincoated tubes until assayed by specific RIAs forPGF2cx., PGE2 and PGFM. The tissue from eachwell was collected and assayed for protein contentagainst BSA standards. Results are expressed asmean ± s.e.m. of PG produced (ng per day per Jlgprotein). Data are grouped by stage: d18-22 (earlyorganogenesis); d23-24; and d25-26 (term: viableneonates can be obtained at day 25, though theyare usually born on day 26).RESULTSProduction rates of PGF2a and PGE2 in thegravid endometria increased dramatically towardsterm, and substantially more PGF2a wasproduced than either PGE2 or PGFM. Nongravidendometrium produced significantly lessPGs than gravid endometrium, and production didnot peak: at term.DISCUSSIONThus, the uteri may be the source of the peak: ofPO seen in peripheral plasma at parturition. Thea) Gravid Endometrium1001 o IIlII PGF2aPGE280 0 PGFMe:ot5::J..-.. 60-oSo Q)0.0l-e: Q.:0 0) 40~.€0l0)cu e:w-e 20a..e:ot5od18-22 d23-24 d25-26Days of gestationb) Non-gravid Endometrium::J C 60 -0 ._o Q)0..0l-e: Q.:0 0) 40.ffi.€0)0)cu e:w-o 200::100rb PGF2a1_ PGE280 Iq PGFMd18-22 d23-24 d25-26Days of gestationgravid uterus is more likely than the non-graviduterus to be the major source because thiscontains 2-3 times more tissue (3), and is moreactive in PG synthesis at term. The mechanismsregulating the increasing PG synthetic activitytowards term are still unknown.REFERENCES(1) Lewis PR, Fletcher TP & Renfree MB(1986) J. Endocrinol. 111:103-109.(2) Shaw G (1990) J. Reprod. Fert. 88: 335­342.(3) Renfree MB & Tyndale-Biscoe CH (1973)Dev. BioI. 32: 28-40.45 46

MAMMARY MORPHOLOGICAL AND FUNCTIONAL CHANGES DURING PREGNANCY IN WOMEN:METHODSIn this ongoing study of five women, we have measuredbreast volume during pregnancy, using the ComputerizedBreast Measurement (CBM) System (1) and the 24-houtput of lactose in urine, using a spectrophotometricassay (2).RESULTS AND DISCUSSIONThe timing and magnitude of the increase in the output oflactose in urine during gestation was similar involunteers 1-3, with volunteers 4 and 5 showing lowlactose excretion during the early stages of pregnancy(Figure 1).Changes in breast volume are shown in Figure 2 with acomputer generated breast reconstruction for volunteer 3shown in Figure 3. Although there was close agreementin the changes in breast volume between right and leftbreasts of the individual volunteers. considerablevariation was observed between the volunteersthroughout pregnancy (Figure 2). In addition, therelative volumes of the right and left breasts of volunteerI were considerably different. The preconception breastvolume of volunteer 5 (indicated by asterisk), is likely tobe elevated as a consequence of her breastfeeding aprevious infant at the time of measurement, who wasweaned 4 weeks prior to conception (Figure 2).Assuming that the 24-h urinary lactose is a qualitativemeasure of the amount of lactose synthesized, theseobservations suggest that lactogenesis 1 begins duringmid-gestation in women. However, the growth of thebreast during pregnancy, measured by the increase inbreast volume, showed considerable differences betweenthe women. These differences may be explained byvariation in the extent to which breast adipose tissue wasreplaced with lobular-alveolar tissue. We conclude fromthese preliminary studies that the measurement ofchanges in the output of lactose in urine and in thevolume of the breasts during pregnancy may offer a noninvasivemeans of relating the endocrinology ofpregnancy to mammogenesis and lactogenesis 1 inwomen.DB. Co."), i.e. KetU l , R.A. Owens 2 and PE. Hartmantz llThe Department of Biochemistry, 2The Department of Computer ScienceThe University of Western Australia, Nedlands 6009INTRODUCTIONDuring pregnancy the mammary gland undergoesextensive lobular-alveolar growth (mammogenesis). Atsome time during this growth, the epithelial cellsdifferentiate and initiate the secretion of milkconstituents albeit at a low rate (lactogenesis I). Lactosesynthesized by these newly-differentiated alveolarepithelial cells is reabsorbed into the blood and excretedin the urine. We have measured changes in breastvolume and urinary lactose during pregnancy, todetermine the relationship between mammogenesis andlactogenesis LREFERENCES1) Daly, S.EJ. et al. (1992) Exp. Physiol. 77:79-872) Arthur, P.G. et al. (1989) Anal. Biochem. 176:449-456ACKNOWLEDGEMENTS -- NH&MRC432o1000900- 5-Q)800E::::l0 700>.....C/)~~ 600.0Q).2:~Q)a:500400300o 10 20 30 40Time post-conception (weeks)Figure 1. Excretion of lactose in the urine duringpregnancy. Volunteer 10 ,2" ,3. ,4+,5....200*~~~-10 0 10 20 30 40Time post-conception (weeks)Figure 2. Breast volume changes during pregnancy, Opensymbols, Right breast, closed symbols, Left breast.Volunteer l' ,2" ,3. ,4+,5....Figure 3. CBM generated reconstructions of left breastshape during trimesters 1,2 and 3, for volunteer 3...pNutritional effects on testicular growth and LH and FSH secretion in Suffolkand Merino rams during the breeding and the non-breeding seasonsMaterials and MethodsTwo experiments were carried out, one in the non-breeding season (July/August; n=6/group) and one in the breeding season(FebruarylMarch; n=5/group). Merino and Suffolk rams were fed to maintain liveweight (0.9 kg chaff + lOOg lupins;"Maintenance") or the Maintenance diet plus 1.5 kg lupins ("Lupins"). On Days -1 and 12 after the change in diet, blood wassampled every 20 minutes for 24 hours and assayed for LH and FSH. Liveweight and scrotal circumference were measuredweekly. Data were analysed by repeated measures analysis of variance. In the case of LH and FSH, where an interaction wasfound to be significant, analysis of the change between Days -1 and 12 was carried out.BREEDING 30% change in LW % change in SC FSH (ng/ml) LH pulses/24h2015 3 1210102 8SEASON(February~arch) 10 5 6NON-BREEDING 30M.J. HotzeIl, G.B. Martin 1 ,2, S.W. Walkden-Brown1 and J.S. Fisher11Faculty of Agriculture, The University ofWestern Australia, Nedlands, WA 6009, Australia2CSIRO Division of Animal Production, Private Bag, PO Wembley, WA 6014, AustraliaIntroductionTesticular growth parallels changes in live weight in Merino rams, but in Suffolk rams testicular growth appears to be driven byphotoperiod, independently of changes in live weight (1). We tested the hypothesis that the reproductive responses to changes indiet differs during the breeding and the non-breeding seasons in Suffolk, but not Merino rams.ResultsLupin supplementation increased live weight in both breeds in both seasons (p

ANOVULATORY DAIRY COWS HAVE LOWER LH PULSE FREQUENCY ANDINTRAFOLLICULAR CONCENTRATIONS OF OESTRADIOL (E 2 ) AND TESTOSTERONE (T)THAN CYCLIC COWS.S. McDougall and K.L. MacmillanDairying Research Corporation, Private Bag 3123, Hamilton, NZINTRODUCTIONSome pasture-fed dairy cows in New Zealandhave extended (>50 day) periods of postpartumanovulation (ppa) despite the presence of largefollicles (>10 mm) undergoing regular turnover.Low LH pulse frequency (1) and lowintrafollicular E 2 concentration (2) are reported insuckled beef cows during ppa. Failure ofovulation may occur due to a low LH pulsefrequency producing insufficient T and hence E 2to induce the pre-ovulatory gonadotrophin surge.As intrafollicular E 2 concentrations vary with thestage of follicle development (3), this hypothesiswas tested at two stages of follicle developmentin the ovaries of ppa dairy cows.MATERIALS AND METHODSUsing a 2 x 2 design, 28 anovulatory (Anov) orcycling (Cyc) cows at either growing (Grow) orplateau (Plat) stage of development of thelargest follicle (as determined by daily transrectalultrasound) were ovariectomised 24 h afterinduction of luteolysis (25 mg of Dinoprost). LHconcentration was determined in jugular samplescollected at 15 minute intervals for the 8 h beforeovariectomy and the number of pulses definedusing an alogorithm (4). The largest follicle wasdissected out, its diameter (Diam) measured andthe E 2 , T and P4 concentrations of its follicularfluid determined. Data were analysed by GLMwith cow status, stage of follicular developmentand the interaction as the main effects. Thesteroid concentrations were log transformedbefore analysis. The relationships among LogE 2 , Log T and LH pulse frequency wereexamined by regression analysis.RESULTSAnov cows had a lower LH pulse frequencyGrowPlatAnoveycAnovCyeLH tpUlses/8h3.1 bc5.4 ab2.4 c6.0 aDiam tmm10.0 c11.4 bc16.2 a15.0 ab58.6 a274.2 b37.6 a504.9 band lower E 2 , T and P4 concentrations than eyccows. Plat follicles were larger, had lower Tconcentrations and had higher E 2 :T ratios and P4concentrations than Grow follicles (Table 1). Thestatus by stage interactions were not significant.The Log E 2 and Log T concentrations werepositively related to the number of LH pUlses. 2(Log E 2 = 0.12xLH freq.+1.56, R =26.3, p

EFFECTS OF CALCIUM IONOPHORE A23187 AND HEPARIN ON STAWON SPERM1l0r--r------------,-------------rllO"'0 70 . ~..~.~~ ,. 1.?9..~1.l}................................. 70Q)'0::J"if..fDJUalS.J. Robinson\ E.L. Squires 2 , J.K. Graham 2 and W.M.C. Maxwell 110epartment of Animal Science, University of Sydney, NSW 20062Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523 USAIntroductionIn vitro capacitation of sperm is an essential pre-requisite for in vitrofertilisation (IVF). Alterations to the sperm head membranes duringcapacitation enable the sperm to undergo the acrosome reaction(1), and thus the proportion of sperm which have reacted may beused to assess sperm capacitation. Incubation with calciumionophore A23187 (2) and heparin (3) increase the occurrence ofthe acrosome reaction in stallion sperm. Experiments wereconducted to study the effects of A23187 and heparin on themotility and acrosome reaction of stallion sperm in vitro.Materials and MethodsSemen was collected from four stallions on separate days.Immediately after collection, the gel-free fraction was extended to50 x 10 6 sperm/ml with a modified, glucose-free TALP medium(mTALP-g; 2mM Ca 2 +) at 3rC. The extended semen was thenwashed < once by centrifugation (500g, 15 min) to remove theseminal plasma. The sperm were then resuspended to 50 x 10 6sperm/ml in mTALP-g and the sperm suspension divided into 6parts (A,B,C,D,E,F), equilibrated at 37.5°C. A stock solution ofA23187 in DMSO was added to each part so that the finalconcentration was 0 (A,B), 2.5~M (C,D) or 5.0~M (E,F). The spermsuspensions were then incubated at 37.5°C for 5 min beforewashing by doubling the volume with mTALP-g and centrifugation(300g, S min). The sperm were resuspended to 25 x 10 6 sperm/mlin mTALP·g, and a stock solution of heparin (in mTALP·g) wasadded to parts B,D & F so that the final concentration was1OO~g/ml. The sperm suspensions were then incubated for 2 hoursat 37.5°C in 5% CO 2 in air. Immediately (0) and at 30, SO and 120min, aliquots were removed for assessment of % motile sperm(objective assessment; Hamilton-Thorn motility analyser), %acrosome reacted sperm (spontaneous), and the % of sperm whichwere able to undergo induced acrosome reactions. The % of liveacrosome-intact and acrosome-reacted sperm were determined byviewing a drop of the sperm suspension under fluorescentmicroscopy after staining with propidium iodide and fluoresceinconjugated~sativum agglutinin«4). To assess the ability ofsperm to undergo an induced acrosome reaction, the sperm wereincubated in mTALP~g containing 1OO~g/ml Iysophosphatidylcholine (LPG) for a further 15 minutes (5) and the acrosomereaction assessed as above. Mean % of motile and acrosomeFigure 1: Effects of A23187 and heparin on the motility andacrosome reaction of stallion sperm. Means with different lettersdiffer (P.EJ A-IVVu 00 800[g A-IVT;:e;(l)~.~ 600~~CI:l 400,.0:::lCI:l"0~ 200 03 H-Glu 14 C-Glu 14 C-Glut 14 C-PyrEnergy substrateFigure 1. Metabolism of energy substrates by in vivo(IVV) and in vitro (IVT) matured pre-pubertal (P) and adult(A) sheep oocytes.* P-IVV oocytes not allocated to these treatments.# 14C_Pyr (fmol x 10-1).1000~ >..u 8000.Qn"'dQ) S.~ c3 600=3 eno +1Q)I::::Cd C'::l.b (l) 400CI:l S,.0 "-"0CI)"0 200~oPre-pubertalAdultOocyte classificationFigure 2. Metabolism of glutamine by mature pre-pubertaland adult sheep oocytes (in vivo and in vitro data pooled).REFERENCES1. Palma, G. A., Clement-Sengewald, A. & Krefft, H. (1993).Therio. 39, 278 abstr.2. Pinkert, C. A., Kooyman, D. L., Baumgartner, A. &Keisler, D. H. (1989). .T. Reprod. Fert. 87, 63-66.3. Ryan, 1. P., Hunton, 1. R. & Maxwell, W. M. C. (1992).Reprod. Fert. Dev. 4, 91-97.4. O'Fallon, .T. V. & Wright, R. W., .Tf. (1986). BioI. Reprod.34, 58-64.51 52

Eagle's Essential Amino Acids Stimulate Development of Inner Cell Mass Cellsof Cultured Mouse EmbryosMichelle Lane and David K. GardnerEmbryo Physiology Laboratory, Institute of Reproduction and Development, Monash University, Clayton, Victoria.IntroductionDevelopment and cleavage of mouse zygotes to the blastocyststage in culture are significantly increased by Eagle's non-essentialamino acids and glutamine (NEGLN), whilst essential amino acidswithout glutamine (ESS) have no apparent beneficial effects ondevelopment in vitro (1) However subsequent studies have shownthat NEGLN do not effect fetal development after blastocysttransfer. In contrast, fetal development is significantly increasedwhen blastocysts are developed in the presence of ESS (2). It ispossible that the two groups of amino acids have different effectson the cell lineages in the blastocyst. The aim of this study wastherefore to assess the role of amino acids on the development ofinner cell mass (lCM) and trophectoderm (IE) cells in culture andsubsequent blastocyst viability.Materials and MethodsZygotes were collected from Fl hybrid (C57BU6 x CBA/Ca)female mice and cultured in amino acid free medium mMTF (1) ormMfF supplemented with NEGLN, ESS or Eagle's 20 aminoacids (20AA). Embryos were cultured in 20111 drops ofmedium ingroups of lOin 5% CO 2in air at 37'C for an initial 48h in one ofthe above groups and then allocated for a second 48h ofculture inone of the three amino acid treatments. Control embryos werecultured in mMfF throughout giving a total of 10 cultureconditions. In vivo derived blastocysts were recovered 88hpost-hCG. lCM and IE cells in blastocysts were assessed usingimmunosurgery and differential nuclear staining (3). Viability ofblastocysts was detennined by transfer to pseudopregnant femalesand fetal development assessed on day 15 ofpregnancy.ResultsTotal blastocyst cell munber and number of IE cells weresignificantly increased by culture with NEGLN for the first 48h,irrespective of the medium in the second 48h (Fig. 1). Culturewith either ESS or 20M for the second 48h period significantlyincreased the number of rCM cells compared to NEGLN or noamino acids and were equivalent to in vivo derived controls.Similarly, fetal development was significantly increased by ESS or20AA for the second 48h culture period compared to NEGLN(Fig.2). Fetal development in all treatments was significantlyreduced compared to in vivo derived embryos. There was apositive correlation between the mean number of inner cell masscells in blastocysts in each treatment and subsequent fetaldevelopment (Fig.3).DiscussionThis study demonstrates that the preimplantation mouse embryoundergoes a switch in nitrogen requirements as developmentproceeds from the zygote to the blastocyst stage. NEGLN stimulatecleavage when present during culture of. the pre-compactedembryo whilst ESS stimulate the development of the rCM ofcultured embryos post-compaction. This has significantimplications for the formulation of media for the culture of thepreimplantation embryo as currently only one medium is used forall stages of development. This study demonstrates a positivecorrelation between allocation of cells to rCM and subsequentfetal development. Detennination of rCM number in blastocysts istherefore a useful in vitro viability index.80"070~c:'*60~ 5002:-.cE40l.1JQ) 30j807060a; 50 .cE:J40zQj()3020100c: 20~OJ0..102520a;.cE:J15c:::EQ1050010aNo M N/N EIN 2O/N NIE EIE 2O{E N/20 E/20 20/20 In V'NoFig.! Effect of amino acids on the development of rCM (solidbars) and IE (open bars) in cultured mouse blastocysts. a-f: likepairs significantly different for rCM; P

EXPRESSION OF INSULIN-UKE GROWTH FACTOR-! mRNA IN ENDOMETRIUM DURING EARLYPREGNANCY IN THE SHEEPC.R. Cann, RJ. Fairclough 1 ,R. Sutton 2 and C.B. GowSchool of Agriculture~La Trobe University~ Bun.doora, Vic.~ 3083.1 Dept Food Technology~ Victoria University ofTechnology~ Werribee, Vic.• 3030.2 Division of Animal Production~ CSIRO~ Blacktown~ NSW, 2148.IGFBP PROTEOLYTIC ACTIVITY IN THE UTERINE FLUSHINGS OF PREGNANTAND NONPREGNANT SHEEPA.J. Peterson, A.M. Ledgard, S.C. Hodgkinson and H.R. TervitAgResearch, Ruakura Agricultural Research Centre, Hamilton, New ZealandINTRODUCTIONInsulin-like growth factor-I (IGF-l) of maternal origin hasbeen implicated in the cellular proliferation anddifferentiation of the preimplantation mammalian embryo.Although mouse embryos do not produce mRNA encodingIGF-I until after implantation (1), mRNA encoding the IGF­I receptor is present in mouse embryos from the 8-cell stage(2). Studies of uterine flushings from ewes at days 10-16 ofpregnancy have shown the presence of immunoreactive IGF­I (3). indicating that the ovine embryo is in the presence ofIGF-I prior to implantation. This study aimed toinvestigate the expression patterns of mRNA encoding IGF­I in the endometrium of early pregnant ewes.MATERIALS AND METHODSEndometrium and uterine flushings were collected fromMerino ewes at oestrus (day 0) and on days 3, 6~ 8, 15 and20 of pregnancy. There were 5 ewes per group except onday 20 where n=4. Uterine flushings were examined for thepresence of an embryo to confrrm pregnancy. Ewes wereincluded in this study if an embryo was found in the uterineflushings.Endometrium was immediately snap-frozen and stored at-70°C. Northern blotting was performed to screen formRNA transcripts for IGF-I using sheep liver as a positivecontrol for IGF-I mRNA expression. Total RNA wasextracted using the Guanidine lsothiocyanatelCsCI cushionmethod. Total RNA (15 mg) was electrophoresed on 1%agarose/formaldehyde gels before transfer to nylonmembranes (Hybond-N+; Amersham. Australia).Membranes were hybridised with a cDNA probe encodingpart of the protein coding region for ovine IGF-I. The probewas labelled with 32p-dCTP using random primingprocedures. Prehybridisation was for 30 min at 65°C inRapid Hybridisation Buffer (Amersham, Australia) andhybridisation was for 3 hours 65°C in Rapid HybridisationBuffer (Amersham, Australia) with the addition of the 32p_labelled probe. Blots were washed to a stringency of 0.1 xSSC/0.1 % SDS at 65°C prior to autoradiography for -6days.Fold differences in IGF-I mRNA signals were quantitatedusing scanning densitometry. Signals for IGF-I mRNAwere adjusted for RNA sample loading differences determinedfrom densitometric analysis of photographs of the 28Sribosomal RNA bands on gels stained with Ethidiumbromide prior to transfer. Signals for IGF-I mRNA werealso adjusted for differences between autoradiographs. Datafrom one ewe on both days 3 and 6 of pregnancy wereexcluded from analysis due to poor preparation of samples.Data were analysed by Analysis of Variance to evaluate theeffect of day ofpregnancy on expression of IGF-I mRNA.RESULTSA major transcript of -8.3 kb in size encoding IGF-I wasdetected in the endometrium of early pregnant ewes. Therewas an effect (p-0.002) of day of pregnancy on theexpression of IGF-I mRNA. The highest level of expressionof IGF-I mRNA occurred on the day of oestrus whereas thelowest level occurred on day 15 of pregnancy. The temporalvariation in expression of IGF-I mRNA~ relative to themean value recorded on day 15, is shown in Figure 1.Relatively high IGF-I mRNA expression at oestruscompared to various stages of the cycle and pregnancy havebeen previously reported in endometrium of the cow (4).Our study extends these findings to early pregnancy in thesheep~ suggesting a role ofovarian steroids in the regulationof IGF-I mRNA. Previous studies have also shown thatuterine flushings from ewes on day 16 of pregnancy havelower concentrations of immunoreactive IGF-I compared toewes on day 10 ofpregnancy (3). We have also shown verylow expression ofIGF-I mRNA in the endometrium of eweson day 15 of pregnancy. Further studies are underway todetermine if the present observations are pregnancy specific~but it appears that the synthetic capacity of the uterus toproduce IGF-I may playa role in the development of theovine embryo.REFERENCESo 3 6 8 15 20Day of PregnancyFigure 1: Changes in endometria! IGF-I mRNAe~ cillirge8rty pregl8I1Cy In the ewe. Values are expr8ssed re&aliw 10 themean varue for-IGF-I mRNA expression on day 15 of pregl8I1Cy.Values presented are means; wrticaJ lines are $EM.DISCUSSION(1) Heyner. S., Rao~ V., Jarrett~ L.• & Smith, R.M. (1989)Developmental Biology 134:48-58.(2) Heyner, S., Smith~ R.M.~ & Schultz, G.A. (1989)Bioessays 11:171-176.(3) Ko~ Y.~ Lee, C.Y.~ Ott, T.L., Davis, M.A.• Simmen.R.C.M., Bazer, F.W., and Simmen, F.A. (1991) Biology ofReproduction 45: 135-142.(4) Geisert. R.D., Lee. C-Y., Simmen. F.A.~ zavy. M.T.,Fliss, A.E.~ Bazer, F.W. & Simmen. R.C.M. (1991)Biology ofReproduction 45:975-983.INTRODUCTIONInsulin-like growth factors (IGFs) -I and -II belong toa family of peptide hormones that includes relaxinand insulin and share a high degree of structuralsimilarity with proinsulin(1). They are importantmitogenic peptides and are significant autocrineparacrinefactors in cell proliferation(2). They arecomplexed to a family of at least six binding proteins(IGFBPs) which modulate their mitogenic activity invivd 3 ).Recently it has become apparent that thebioavailability of IGF is influenced not only by theIGFBPs themselves but also by specific IGFBPproteases(4). IGF-I and -II are present in the uterinelumenal fluid of sheep and IGFBPs of 16-18 kDa and22-24 kDa, specific for IGF-II, are seen in bothpregnant and non-pregnant sheep only on days 10­15 after oestrus. The aim of the present study wasto determine whether these low molecular weightIGFBPs were proteolytic fragments of an unidentifiedparent IGFBP.MATERIALS AND METHODSTimed uterine flushings were obtained from pregnantand non-pregnant sheep. After centrifugation, theywere lyophilized, reconstituted in sterile water andprotein concentration determined by the Bradfordmethod. Recombinant human (rh) non-glycosolatedIGFBP3 and rh IGFBP2 were iodinated usingiodogen. Labelled rh IGFBP3 and IGFBP2 wereincubated overnight at 37°C with uterine flushingsequivalent to 30 ~g protein followed by SDS-PAGEunder non-reducing conditions. The gels were driedand autoradiographed. In a separate experiment theproteinase inhibitors EDTA (10 mM) and PMSF(10 mM) were added.The flushings were also subjected to Weaternimmunoblotting using anti-b IGFBP2 and anti-hIGFBP4.RESULTSDay 15 uterine flushings from both pregnant and nonpregnantsheep reduced both rh IGFBP3 and IGBP2to lower molecular weight fragments; the former wascleaved to four fragments of 5.5, 14, 16 and 20­22 kDa, the latter to two fragments of 14 kDa and18 kDa. This proteolytic activity was inhibited byabout 70% by EDTA but was unaffected by PMSF.The activity was absent on days 3 to 10 and presenton days 12 to 15 after oestrus in both pregnant andnon-pregnant sheep. It disappeared in both casesfrom days 16 to 17 after oestrus.Although similar Western immunoblotting revealedIGFBP2 and IGFBP4 in ovine and bovine follicularfluid(s) negative results were obtained from the ovineuterine flushings.DISCUSSIONThe profile of proteolytic activity suggests that in bothpregnant and non-pregnant sheep it is underprogesterone control. Thus in the latter it appearsduring the luteal phase and disappears duringluteolysis. In the former it also disappears despiteelevated plasma progesterone levels, suggesting thatthe conceptus is secreting a factor that inhibits IGFBPproteolysis. The physiological significance of theIGFBP2 and 3 proteases in uterine fluid is unknowna~hough they appear to be divalent cation dependent.The parent binding proteins themselves have notbeen detected in sheep and the source and regulationof the IGFBP proteases must await purification andsubsequent identification.REFERENCES1. Daughadey, W., Rotwein, P. (1989) Endocr. Rev.10: 68-91.2. Rechler, M.M., Nissley, S.P., Roth, J. (1987) N.Engl. J. Med. 316: 941-3.3. Rechler, M.M., Brown, A.L. (1992) Growth Reg.2: 55-68.4. Binoux, M. (1994) Growth Reg. 4 (SuppI.1): 53.5. Peterson, A.J., Ledgard, A.M., Hodgkinson, S.C.(1994) Growth Reg. 4 (SuppI.1): 113.55 56

PROSTATE-SPECIFIC ANTIGEN Ar;.JD INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 ARECO-LOCALIZED IN THE HUMAN ENDOMETRIUM: PATTERNS OF EXPRESSION ACROSS THEMENSTRUAL CYCLE.Judith Clements. Allison Ehrlich, Marion Marsh and Lois Salamonsen.Prince Henry's Institute of Medical Research, PO Box 152, Clayton, Vic. 3168.IntroductionProstate-specific antigen (PSA) is a member of thehuman kallikrein (KLK) gene family of serine proteasesand is encoded by the gene KLK3. We have recentlydemonstrated that KLK3 and two other KLK genes(KLK1 and 2) are expressed in the human endometrium(1). Seminal PSA has been shown to degrade theinsulin-like growth factor (IGF) binding protein, IGFBP-3,so that it is no longer capable of binding to IGF-I or IGF­II (2). These growth factors have been implicated ineither the proliferative response mid-cycle (IGF-I) ordecidual cell differentiation (IGF-II) in the humanendometrium (3). PSA may thus act to regulate thebioavailability of the IGFs in the uterus as has beensuggested for the prostate. In this study, we wished todefine to which cell-type(s) PSA localized in the humanendometrium, its pattern of expression across themenstrual cycle, and whether it was co-localized withIGFBP-3.Materials and methodsEndometrial tissue (n=31) was collected from essentiallynormal cycling women (cycle days 2-28) undergoingdilatation and curettage during routine investigation ofinfertility and was histologically dated. Tissue blocksused for PSA immunocytochemistry were divided into 5arbitrary phases of the menstrual cycle: menstrual (day2-4, n=4), proliferative (day 6-12, n=11), and early (day17, n=4), mid (day 21-24, n=8) and late (day 25-28, n=4)secretory phases. Ten of the 31 tissue blocks werechosen for subsequent immunocytochemistry forIGFBP-3. Immunocyto-chemistry was performed withcommercially available antibodies to PSA (Dako) andIGFBP-3 (UBI) using the avidin-biotinylated horseradishperoxidase complex method (Vectastain ABC kit).Intensity of staining was graded, by 3 independentobservers, using a scale of 0-4 to indicate absence ofstaining (0) to intense staining (4).ResultsPSA was localized primarily to the glandular and luminalepithelium and was also present in secretions fromindividual glands. There was less staining in the stroma.There was no apparent difference in staining intensity orpattern of localization across the menstrual cycle (Fig.1). The endothelium and/or the smooth muscle cells ofendometrial blood vessels were also positive for PSA tovarying degrees, but with no consistent pattern,throughout the cycle. IGFBP-3 was localized to theglandular and luminal epithelial cells, stromal cells andendometrial blood vessels in a similar fashion to that ofPSA (Fig. 2).References1. Clements J & Mukhtar A. 1994. J Clin Endocrinol Metab 78:1536-1539.2. Cohen P, Graves HCB, Peehl DM, Kamarei M, Guidice LC,Rosenfeld RG. 1992. J Clin Endocrinol Metab 75: 1046-1053.3. Seppala M 1994. In Molecular biology of the femalereproductive system. Academic Press p379Fig. 132.5o~ 2en£1.5encQ)E 10.5Fig. 2PSA3-2.5-~ 2-;:;:Mens.(4)Prolif.(11 )o glandularepithelium~ ::::>'1 5 .....~ . -:::: ITC .;.; II~ 0.: ~.::!·.!:i=.:::~.i,.:.:. •.:::.::.• ::.:i••iUla-'-'-",""",,"L-.,&;:;';": :.c.::::;w:.I}:--.I.':"':'=Mens. Prolif. Sec.(1) (5) (4)Early(4)Mid.(8)IGFBP-3Late(4)SecretoryI28l luninal ED stromaepitheliumII!IMens. Prolif. Sec.(1) (5) (4)Staining intensity scores for PSA alone (Fig. 1)and PSA/IGFBP-3 co-localisation (Fig. 2) in themenstrual', proliferative and secretory phases ofthe menstrual cycle. n/group is indicated inparentheses below each figure.Summary and ConclusionPSA and IGFBP-3 are co-localized to the same celltypesin the human endometrium. These data suggestthat PSA, as in the prostate, may function as aprocessing enzyme for IGFBP-3 in the uterus to regulatethe bioavailability of IGF-I and IGF-II. The lack of anyapparent change in intensity of staining for PSA or itssubstrate, IGFBP-3, across the menstrual cyclepresumably reflects the need for bioactive IGF-I and/orIGF-II in the human endometrium at all phases of themenstrual cycle.GM-CSF mediates the post-mating inflammatory reaction in the murine uterusSarah A Robertson, Anna C Seamark and Robert F SeamarkDepartment of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, SA 5005INTRODUCTIONGranulocyte-macrophage colony-stimulating factor (GM­CSF) is a lymphohaemopoietic cytokine synthesised byoestrogen-primed epithelial cells in the murine uterus. Adramatic but transient peak in its release is evoked by seminalplasma-mediated stimulation of epithelial cells at mating1.In recent studies we have investigated the role of GM­CSF in the pre-implantation uterus. Transcripts for both the ex.and ~ components of the GM-CSF receptor have beendetected by RT-PCR in uterine tissue at oestrus and duringthe pre-implantation period, with expression being severalfold higher following mating than at oestrus (S. Robertson,unpublished). In inflammatory sites, GM-CSF regulates therecruitment and activation of myeloid leukocytes, and cellsexpressing GM-CSF receptor in the day 1 pregnant uterushave been identified as leukocytes on the basis of theirpredominantly sub-epithelial distribution in the endometrium,using autoradiography following incubation of ethanol-fixedfrozen tissue sections in 125I-GM-CSF2. The aim of thecurrent study was to determine whether epithelial GM-CSFhas a role in the post-mating inflammatory response which ischaracterised by infiltration of large populations ofmacrophages, eosinophils and neutrophils into thesubepithelial endometrial stroma and luminal cavity.EXPERIMENTALTo investigate the effect of GM-CSF on the number anddistribution of uterine leukocytes, groups of fourovariectomised mice were anaesthetised and given anunilateral intraluminal injection of either 40, 200, or 1000 Uof recombinant GM-CSF in 50 jll of 1% BSA, or carrieralone, through a dorsal incision. Sixteen hours later, the micewere sacrificed and the numbers and distribution ofleukocytes in right and left uterine horns were assessedimmunohistochemically using lineage-specific mAbs.RESULTSIn each of two experiments, the numbers of leukocytes inendometrial tissues were altered dramatically by intraluminalGM-CSF (Fig. 1). The most notable of the cellular changeswas a dose dependant effect of GM-CSF on the numbers ofeosinophils (endogenous peroxidase+) within the endometrialstroma and the myometrium (p = 0.005), with the numbers ofmacrophages and neutrophils (F4/80+ and Mac-l+ cells), andto a lesser degree activated macrophages/dendritic cells (Ia+)also increased in response to GM-CSF. F4/80+ and Ia+ cellswere distributed throughout the stroma but were mostconcentrated in areas underlying the epithelium. Neutrophils(Mac-l+ F4/80- polymorphonuclear cells) were absent incarrier-treated mice but present in large numbers in GM-CSFtreated mice where they were located predominantly betweenepithelial cells in a pattern suggestive of trafficking into theluminal cavity, similar to that observed during the postmatinginflammatory response.Interestingly, GM-CSF also had a dose-dependant effecton both the number of endometrial glands and the diameter ofthe uterus, with a four-fold increase in the mean number ofglands (p = 0.0001), and a 70% increase in the mean uterinediameter (p =0.0001) after intraluminal injection of 200 U ofGM-CSF. Both the number of glands (r = 0.845, p = 0.0001)and uterine diameter (r = 0.859, p = 0.0001) correlated withthe number of endometrial eosinophils.No differences in leukocyte numbers or endometrialglands were found between injected and ipsilateral horns,most probably reflecting the contiguity of the lumen ratherthan any systemic actions of th~ injected cytokine.Figure 1: Immunohistochemical localisation of leukocytes(LCA+ cells: A,B), eosinophils (endogenous peroxidase+cells: C,D) and macrophages / neutrophils (Mac-l+ cells: E,F)in the endometrium of ovariectomised mice treated eitherwith 200U of rGM-CSF (B,D and F) or carrier (A,C and E)SUMMARYThese data identify specific populations of GM-CSFresponsivemyeloid leukocytes within the uterus, and supportthe view that GM-CSF produced by uterine epithelial cellshas a physiological role in regulating the trafficking andbehaviour of these cells to participate in the dramaticremodelling processes characteristic of this tissue during theoestrous cycle and during pregnancy.1. Robertson SA, Mayrhofer G and Seamark RF (1992),BioI. Reprod. 46, 1069-1079.2. Robertson SA, Mayrhofer G and Seamark RF (1993), inSerono Symposia 97 'Reproductive Immunology' (Dondero Fand Johnson P, eds), pp 117-122, Raven Press, New York.57 58

ENDOTHEllN CONTENT AND CHARACTERIZATION IN THE SHEEP UTERUS DURINGTHE OESTROUS CYCLE AND AT IMPLANTATIONSimon C Riley, Lois A Salamonsen & Jock K FindlavPrince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria, 3168--Endothelin in the Endometrium of Women with MenorrhagiaM.M. Marsh1, D.L. Healy2, J.K. FindlaY' & L.A. Salamonsen 1 • .1Prince Henry's Institute of Medical Research and 2Monash University Department ofObstetrics and Gynaecology Clayton Vic 3168.INTRODUCTIONEndothelins (ET) are a family of three 21 amino acidpeptides with potent vasoconstrictors and mitogeniceffects. ETs have been demonstrated in the ovineuterus in luminal and glandular epithelial cells usingimmunohistochemical techniques (1) with the greateststaining intensity at the time of implantation.However, their role in the control of ovine uterinefunction has not been defined. These studies havecharacterised and measured the content of ET withinthe ovine uterus during the oestrous cycle and earlypregnancy around the time of implantation to try andelucidate the role of ET in these processes.MATERIALS AND METHODSUteri of Corriedale ewes (n=75) were flushed withsaline and removed by hysterectomy. Pregnancy wasconfirmed by the presence of an embryo.Endometrium and myometrium were dissected andfrozen. Tissues were homogenised in 1M acetic acidand the homogenates were boiled, centrifuged andthe supernatant collected and Iyophylized. ET contentof the extracts and flushings were measured by aradioimmunoassay that recognises ET-1, -2 and -3equally, but not the precursor big ET. The isoform(s)of ET present in the uterus were characterised byreverse-phase HPLC.RESULTSET-1 was the principal ET isoform present in bothtissue extracts and uterine flushings. In tissueextracts there was no significant change in ET contentduring the oestrous cycle, but during pregnancy, ETcontent significantly increased on and after Day 10-12(Table 1).TABLE 1 Amount of IR-ET (±SEM; pg g-1n=4/group) in endometrial extracts.Period Day IR-ETOestrous 4 327 ±9S acycle12 476±97 a16 629 + 129 aPregnancy 4 279 +40 a10-12 453 +39 015-17 477+25°20 616 +43 cwt wt;Within anyone group, values denoted with the samesuperscript are not significantly (P>0.05) different.In uterine flushings, the amount of ET increased significantlyon Day 14 of the oestrous cycle thendecreased by Day 16 (Fig. 1). During early pregnancy,ET content in flushings increased on Day 14and increased further on Days 15 and 16 (Fig. 1).CONCLUSIONSET-1 was present in the ovine uterus in tissue extractsand in uterine flushings in increasing amounts aroundthe time of implantation (Day 16).FIGURE 1. Immunoreactive ET content (±SEM) inuterine flushings of sheep during the oestrous cycleand early pregnancy.300022500~.... 2000gg. 1500~ 1000~a,b4 10 12 14 16 4 7 8 9 10 12 14 15 16oestrous cycleearlyDaypregnancyWithin anyone group, values denoted with the samesuperscript are not significantly (P>0.05) different.REFERENCES(1) Riley et aJ (1994). J. Reprod. Fert. 100: 451­459.(2) Marsh et al (1994). J. Clin. Endo. Metab. (inpress).Supported by the NH&MRC.This is the sameisoform as produced by the human endometrium invitro (2), and is present in the uterus in similarconcentrations to that found in other tissues where afunction for ET has been demonstrated. ET mayhave a role in the control of uterine blood flow duringimplantation. ET may elicit a mitogenic effect on theblastocyst during the phenomenal expansion thatoccurs between Days 13-16, and on the endometriumin association with the remodelling and angiogenicresponses related to implantation and placentaldevelopment.x,IntroductionEndothelin (ET) a growth factor and potentvasoconstrictor (1) is present in human endometriumthroughout the normal menstrual cycle. Expression ofET protein and mRNA shows cyclical variation,maximum levels being observed in the premenstrualphase (2). The aim of this .study was t? ex~mi~e thecellular localization of ET In endometnal bIopSies ofwomen with documented menorrhagia and to comparethis to that observed in the normal menstrual cycle.Materials and MethodsEndometrial biopsies (n=17) were obtained withinformed consent from women who presented withmenorrhagia confirmed by a monthly menstrual bloodloss greater than 80 mls. These samples were classifiedhistologically according to Noyes criteria (3) intoproliferative (n=6), early secretory (n=3), mid-secretory(n=4) and late secretory phase (n=4). Normalendometrium (n=55; proliferative n=21 , early secretoryn=11, mid secretory n=10, late secretory n=5 andmenstrual n=8) was obtained from women of provenfertility undergoing laparoscopic sterilisation or womenundergoing assessment of tubal patency. Tissue wasfixed in 10% buffered formalin, paraffin embedded,sectioned and subjected to immunohistochemistry usinga polyclonal antibody to ET (cross-reactive with ET-2 &ET-3) and the components of the StrepAviGensupersensitive kit (2) and counterstained with Mayer'shaematoxylin (Sigma). ET immunoreactivity was scoredby two independent observers in relation to knownpositive and negative controls included in each staining.Individual cellular compartments within each tissuesection were scored from 0 (negative) to 4 (maximalstaining intensity) and transformed to a percentageimmunostaining intensity where negative = 0 and 4 =100%. Mean immunostaining intensity ± SEM wascalculated for each cellular compartment within eachphase of the menstrual cycle.ResultsET immunoreactivity in the endometrium from thosewomen with menorrhagia (Fig 1) is substantially differentfrom that of the normal menstrual cycle (Fig 2).Compared to normal endometrium in menorrhagia thereis little variation in the immunostaining pattern acrossthe menstrual cycle. ET immunoreactivity is less intensein glandular and luminal epithelium than that observedat the same stage of the normal menstrual cycle, andalthough the stromal ET immunoreactivity is of similarintensity to the normal menstrual cycle, at all stages itremains more intense than that of luminal and glandularepithelium.Acknowledgments: Supported by the NH & MRCFigure 1:e 'E 100o()~0) 75c::'c 'cu(j) 50o c:::::JE.§ 25I­ w§O-+J~"""""(l)~Figure 2:--- e 'E 100o()~(6, 2, 4, 4) (6, 3, 4, 4)Luminal Epithelium Glandse..... 750)c::'c 'cu(j) 50o c:::::JE.§ 25Iii~(l)0 -+-J:......&.t-(21,11,10,6,8) (9, 5, 4, 5, 8) (20,10,10,5,8)~ Stroma Luminal Epithelium GlandsFigure LegendsCellular localization of ET immunoreactivity (Mean±SEM)across the menstrual cycle - D Proliferative, [21 EarlySecretory, II Mid Secretory, E8I Late Secretory, and •Menstrual phase. Numbers below each bar denote the numberof samples in each cellular compartment. Endometrial biopsiesfrom subjects with menorrhagia (fig1 ) and normal endometrium(fig 2).Summary & ConclusionsThe immunostaining pattern of ET in the endometriumin menorrhagia exhibits a different pattern to thatobserved during the normal menstrual cycle. Inmenorrhagia lower levels of immunoreactivity areobserved at all stages of the menstrual cycle in luminaland glandUlar epithelium compared to the normalmenstrual cycle where maximal staining intensity isobserved in the premenstrual phase. This difference isconsistent with a role for ET in the control of menstrualbleeding. Further studies are being performed toelucidate the function of ET in normal and abnormalmenstrual bleeding.References1. Masaki, T. (1993) Endocrine Reviews 14:256~268.2. Salamonsen L.A., et aI., (1992) Am J Obstet Gynecol.,167: 163-167.3. Noyes, R.W., et aI., (1950) Fertility & Sterility 1:3~25.59 60

· LEUKAEMIA INHIBITORY FACTOR (LIF) IN OVINE ENDOMETRIUM DURING THEOESTROUS CYCLE AND EARLY PREGNANCYDaphne Vogiagis1,2, Richard C. Fry2 & Lois A. Salamonsen1.1 Prince Henry's Institute of Medical Research, PO Box 152, Clayton, Vic, 3168.2 Victorian Institute of Animal Science, Werribee, Vic, 3030.THE USE OF THE POINT SM1PLED INTERCEPT F.M1ILY OF N1ETIIODS FOR TIIEESTIMATION OF SERTOLI CELL NUCLEAR VOLUME.Nigel G Wreford l • 2 andAnne O'Connor l ,Institute ofReproduction and Development! and Dept. ofAnatomy 2, Monash University,Wellington Rd. Gayton, Victoria 3168 , AustraliaINTRODUCTIONLeukaemia inhibitory factor (L1F) is a cytokine with abroad range of actions on various in vitro culturesystems. In the mouse its uterine expressioncoincides with the onset of blastocyst implantationand females lacking a L1F gene are fertile but theirblastocysts fail to implant (1,2). This study wasdesigned to investigate the expression and cellularlocalisation of L1F in the ovine endometrium, duringthe oestrus cycle and early pregnancy. Specifically itwas of interest to establish whether L1F is temporallyassociated with either embryo hatching, developmentor blastocyst implantation. Previously we havelocalised L1F immunohistochemically in humanendometrium throughout the menstrual cycle wheremaximal staining was detected in the mid-latesecretory phase particularly in the glands prior tomenstruation (3).MATERIALS AND METHODSParous Corriedale ewes (n=56) were mated witheither castrate or fertile rams (day 0 = oestrous) andhysterectomised on given days of the oestrous cycle(days 4-16) or early pregnancy (days 4-20) (n=3-4per day). Endometrial tissues were snap frozen forRNA extraction and fixed in Carnoy's fixative forimmunohistochemistry. Ovine endometria (total RNA,20l-lg per lane) were tested for L1F expression byNorthern blot analysis using an ovine L1F eDNA probe(0.6-kilobase) a generous gift from WEHI. Thecorresponding protein was localised in these tissuesby immunohistochemistry with a commerciallyavailable rabbit anti-human L1F antibody whichrecognises both native and recombinant human L1F(AMRAD, Pharmacia Biotech, Victoria). TheVectastain ABC immunostaining kit was used fordetection. Intensity of immunostaining was graded bytwo observers, to indicate absence of staining (0) tointense staining (4).RESULTSImmunoreactive L1F was present in all endometrialsamples and was markedly diminished followingpreadsorption of the antibody with humanglycosylated L1F. Positive immunoreactivity waspresent in all cellular compartments of theendometrium (intercaruncular luminal and glandularepithelium and stroma, caruncularluminal epitheliumand stroma, blood vessels and myometrium).However, staining intensity was low in the bloodvessels and myometrium. In the majority of thecellular compartr'f1ents there was a maximal increaseof L1F immunoreactivity at day 12 of the oestrouscycle and early pregnancy (Fig. 1 & 2).Northern analysis also demonstrated the expressionof the 4.2-kilobase L1F messenger RNA transcript inall samples of ovine endometrium, confirming theimmunohIStochemical data.Figuresl.A 2.A4 43 3>.~c2 2~ 1 1go 0'c'(ij1.8 2.8"@ 4 4c~ 3 3§221 10 0I I I4 10 12 14 16 4 7 8 9 10121415161720DayDayDistribution and relative intensity of positiveimmunoreactivity for L1F in ovine endometrium during theoestrous cycle (Fig. 1) and early pregnancy (Fig. 2). A;caruncular, luminal epithelium, B; intercaruncular stroma.CONCLUSIONSIn the ovine endometrium, expression of L1F is notconfined to the time of blastocyst implantation as it isin the mouse. We postulate that in the sheep, L1Fmay have a paracrine role in endometrial function andblastocyst development since it is expressed andlocalised throughout early pregnancy and theoestrous cycle. Whether L1F is essential forimplantation in the sheep, as has been demonstratedin the mouse remains to be established.REFERENCES1. Bhatt, H. et al. (1991) Proc. Natl. Acad, Sci. USA.88:11408-12.2. Stewart, C.L. et al. (1992) Nature. 359:76-79.3. Vogiagis, D. et al (1994) Proc. Endo. Soc. Aust.(abst).DV is supported by the Victorian EducationFoundation.LAS is supported by NH&MRC of Australia.INTRODUCTIONThe volume of a nucleus or cell is animportant but little used parameter indicatingits functional activity. In combination withvohune density data, it can also be used toobtain numerical density. Traditionally,nuclear volume has been detennined using ageometric model based approach. This workswell in the case ofspherical nuclei but is notsuited to irregular or oval nuclei. Thealternative method of serial reconstructionwith sunnnation of areas has been usedextensively in the testis but is extremely timeconsuming and dependent on an accuratedetennination of section thickness.The recent development ofthe point sampledintercept (psi) family ofmethods (1,2&3) hasprovided the basis for the rapid and unbiasedestimation of nuclear and cell vohune. Themethods can be applied in 2 contexts. In the:first, nuclei are assumed to be isotropicallyoriented (ie. as a group have similar geometricproperties in all directions) and in the second(vertical section method), no assumptions aremade with respect to spatial orientation andnuclei may be anisotropic (ie have differentgeometric properties in different directions).With both approaches, nuclei can be sampledin either the vohune (psi) or nmnberdistributions (nucleator and rotator) to giveestimates of volume (vv) or nmnber (v N )weighted mean volume.In this report, these methods are appliedindependently to the estimation ofSertoli cellnuclei.MATERIALS AND 11ETHODSSections (1~) were obtained from Epon­Araldite embedded glutaraldehyde fixed rat(120 days) testis (n=5). Two sectionorientations were used. In the first, the usualtransverse sections were prepared whilst in thesecond sections were cut parallel to the longaxis (vertical sections). Vertical sections weresampled to allow different rotations in thehorizontal plane (2). Sections were sampledusing a motorised stage to give systematicuniform random fields. Nuclei were sampledwith a point grid (i.e. with a probabilityproportional to their volmne) for thedetennination of Vv and unifonnly using thenucleolus for determination of v N .Measurements were performed on a videoimage at a magnification of 2708x using aninteractive software package (GRID,Medicosoft, Denmark). This package willgenerate a variety of grids as well assystematic sets of lines of appropriate (sineweighted) directions for application to verticalsections.RESULTSl\1ean ± sd Setfuli cell nuclear vol~ (,mf)~~uredltiing different ~dtods.Assumption Isotropy NoneSectionOrientationVv (Psi)V N (nucleator)V N (rotator)TransverseVertical666 ± 76 m,± 105553 ± 42 612± 55672±48DISCUSSIONThe assumption of isotropy carries aconsistent negative bias in both the psi andnucleator estimates. There is reasonableagreement between the nucleator and rotatorestimates applied to vertical sections. Thesquare root of the ratio (Vv/v N ) minus 1 (i.e.0.4-0.5) is equal to the coefficient ofvariationofnuclear volmne in the number distributionsuggesting a substantial intra animal variationin Sertoli cell volume at this stage. Thequestion of whether this variation is stagedependent remains to be investigated.(1) Gundersen, R1.G. (1993) J. Microsc. 170: 3-45(2) Baddeley, AJ. (1986) 1. Microsc. 142: 259-276.(3) Vedel Jensen, E.B. & Gundersen RJ.G (1993) 1.Mcrosc.170: 35-44.61 62

SITES OF SYNlHFSIS AND ACIION" OF PDGF IN 1HE RAT TESTISKate Loveland, Kristina Zlatic, Gail Risbridger, Alicia Stein-Oakley* and David deKretserInstitute of Reproduction and Development and *Dept ofMedicineMonash University, Clayton, VictoriaIntroduction: The importance of platelet-derived growth Figure: Northern blots hybridized with cDNAsfactor (pOOF) in growth and differentiation has been encoding POOF ligand and receptor subunits. Eachdocumented in a wide variety of normal and pathological lane contains 10J.lg of total RNA from (a) Leydig cells,systems, though only recently has expression of receptors (b) Sertoli cells, (c) cytoplasts, (d) primary,for this growth factor been described in the adult testis. spennatocytes, (e) round spermatids. Positions ofBinding sites for POOF have been described on rRNA bands are indicated.preparations ofrat Leydig (I) and peritubular myoid cells(2), and mRNA encoding the a-subunit of the POOF abc d ereceptor (3) has been detected in isolated rat Leydig andSertoli cells. POOF has been implicated in regulation oftestosterone production by Leydig cells (3,4) and in PDGF-Ramodulation of peritubular myoid cell function in vitro(2,5). To more fully explore the role of POOF in thetestis, we sought to determine the sites of its synthesisand action. ....288EXTRACELLULAR MATRIX PRODUCTION BY BOVINE GRANULOSA CELLSH.F. Rodgers*, T.C. Lavranos, C.A. Vella, K. Scott and R.I. RodgersDepartments ofMedicine, and *Anatomy and Histology,Flinders University ofSouth Australia, Bedford Park, S.A., 5042, Australia.Bovine granulosa cells were cultured underanchorage-independent conditions (1), and theECM produced by these cells examined bymicroscopy and Western immunoblotting.The colonies of cells (Fig 1, x190) producedan ECM (*) identified as basal lamina collagentype IV (~) by immunohistochemistry (Fig 2,x190).Materials and Met/rods: Northern blot analysis wasperformed using 32p random· primed cDNA probesspecific for each of the POOF ligand (A and B) andreceptor (pOOF-Ra and POOF-Rf3) subunits. Leydigcells, pachytene spermatocytes, round spermatids, andcytoplasts were isolated from adult rats as previouslydescribed (3); Sertoli cells from 20 day old rats (3) werecultured in serum-free medium for 24 hours prior tocollection. RNA was prepared as described (3), and 10~g of total RNA was loaded in each lane of afonnaldehyde agarose gel. RNA was transferred toHybond N membrane (Amersham) and hybridizationperformed using Rapid Hyb (Amersham). Membraneswere washed to O.2X SSC, 0.1% SDS at 65°C.PDGF-~PDGFB---288Fibronectin was detected by immunostaining andWestern immunoblotting (Fig 5).Results: The POOF-Ra cDNA hybridized to mRNAs inLeydig and Sertoli cells,confrrming our previous work(3). No signal was evident in the germ cell preparationswith this probe. In addition, the Leydig cells containmRNA encoding both POOF-Rf3 and POOF B, althoughreactivity with the POOF A cDNA was barely detected.The Sertoli cell preparations contained mRNA whichhybridized with cDNAs encoding the POOF-Rf3, POOFB and POOF A cDNAs. No cross-reactivity of any ofthese probes with germ cell mRNAs was detected. Allbands observed were of expected sizes.Discussion: Our results demonstrate that bothSertoli cells and Leydig cells have the capacity tosynthesize POOF and to respond to this growth factor.Thus the role of POOF in the testis is likely to be muchmore extensive than has previously been appreciated, asboth paracrine and autocrine interactions are likely to beinvolved. POOF thus has the potential to mediatecommunication between the interstitial and seminiferousepitheilium compartments.PDGFAReferences:(1) Gnessi L, Emidi A, Farini D, Scarpa S, Modesti A,Ciampani T, Silvestroni L, Spera G (1992)Endocrinology 130:2219-24.2) Gnessi L, Emidi A, Scarpa S, Palleschi S, Ragano­Caracciolo M, Silvestroni L, Modesti A, Spera G(1993) Endocrinology 133: 1880-90.(3) Loveland KL, Hedger MP, Risbridger GP, HerszfeldD, deKretser DM (1993) Mol Reprod Dvlt 36: 440-7.(4) Risbridger GP (1993) Mol Cell Endo 97:125-8.(5) Tung PS, Fritz IB (1991) J Cell Physiol 146:386-3.Ruthenium red stalrung for proteoglycansrevealed spaces between cells (Fig 4a, x27500,b, x65000) that were rich in fibers decoratedwith ruthenium red (--»). Follicular fluid alsohas ruthenium red postive proteoglycangranules.ABC D EF. Lanes: A - preincubation, B - control, C - bFGF,D dibutyryl cArv1P, E - Matrigel, F - plasmafibronectinIn these studies control colonies produced ECMand components, bFGF (50ng/ml) promotedtheir production and the cells remainedundifferentiated. Dibutyryl cAMP (1mM)inhibited ECM production and the cellsdifferentiated as determined by theirultrastructure, progesterone production andlevels ofsteroidogenic enzymes.Granulosa cells produce basal lamina whoseproduction can be regulated.1 Lavranos TC, Rodgers HF, Bertoncello I,Rodgers RJ (1994) Expt. Cell Res. 211, 245­251.63 64

Steroidogenesis Is Mediated By Heparan Sulphate ProteoglycansIn The Adult Rat Leydig CelLJim Mc Farlane, Andrew Laslett, Milton Hearn*, David de Kretser, & Gail Risbridger.Institute ofReproduction and Development and *Department ofBiochemistry, Monash University,Clayton, Victoria 3168.Introduction: LH stimulated androgen production by adult and immatureLeydig cells has been shown to be modulated by paracrine stimulatory andinhibitory factors. The aim of this study was to investigate the role cellsurface heparan sulphate proteoglycans might play in the regulation ofsteroidogenesis in these cells.Materials & Methods: The Leydig cell bioassay used in this study has beenpreviously described l , briefly; cells are purified by centrifugal elutriation andPercoll density gradients, yielding Leydig cells at greater than 95% purity.Endogenous and exogenous heparin binding proteins were removed by theaddition of heparin (275 J.lg/ml) to the media during purification The cellswere plated at 5 x 10 4 cells/well (48 well plates), preincubated for 1h in0.25ml ofDMEMJF12 containing 0.1 % BSA at 32°C, 5% CO 2 . This mediawas then removed and replaced with fresh media containing 0.1mg/ml bovine.lipoproteins and test substances in the presence or absence of LH (16 ng/ml).The cells were incubated for a further 20h and assayed for testosterone. Usingthis experimental protocol varying doses of heparin (10, 100, 200 ug/m1),protamine sulphate (0.1,1,10 ug/ml) an inhibitor of heparin binding growthfactor activity, and sodium chlorate (3.13, 6.25, 12.5, 25mM) which blocksthe sulphation ofthe heparin sulphate proteog1ycans were examined for theireffect on testosterone production..Results: Heparin increased the testosterone production in unstimulated cellsin a dose dependant manner, in some experilnents the stimulation was to alevel achieved by a maximum dose LH (Figure 1). The effect ofheparin onLH stimulated cells was variable, but was predominantly inhibitory at dosesof 100-200 ug/ml (50%). Protamine sulphate had no effect on unstimulatedLeydig cell testosterone at the highest dose used (10ug/ml), however in LHstimulated cells the testosterone production was inhibited by 55% at the samedose. The addition of sodium chlorate had no effect on testosteroneproduction by unstimulated cells but was able to inhibit testosteroneproduction in a dose dependant manner up to 87% at the maximum dose usedas shown in Figure 2.Discussion: These results demonstrate that cell surface heparan sulphateproteog1ycans together with an autocrine factor or factors are an obligatorypart of the LH stimulated testosterone production pathway in the adult ratLeydig cell. Furthennore these data reinforce the emerging view that cellsurface and extracellular matrix heparin sulphate proteoglycans together withautocrine and paracrine factors play an important, if not fundamental role inthe regulation ofcell function.1. Sun, X-R. et al (1993) Endocrinology, 132: 186-192.50o 10 100Heparin (ugml)Figure 1: The effect of heparin ontestosterone production byunstimulated adult rat Leydig cells.~ a; 75(0ooo 3 6 13 25&xflll1 Qiorate (ntv'!)Figure 2: Effect of sodium chlorateon testosterone production by basaland LH stimulated Leydig cells.REDUCTION OF OVULATION RATE IN THE RAT BY ADMINISTRATION OF ANEUTROPHIL.DEPLETING MONOCLONAL ANTIBODYMats Brannstrom, Nigel Bonello, Robert J. Norman, Sarah Robertson.Department of Obstetrics and Gynaecology, The University of Adelaide, Queen Elizabeth Hospital andMedical School Campuses, Adelaide, South Australia.INTRODUCTIONNeutrophilic granulocytes have been implicated as importantcells in ovarian physiology, particularly in the processes ofovulation and luteolysis. There is an 8-fold increase in thedensity of neutrophils in the theca layer during the ovulatoryprocess in the rat (1) and peripheral leukocytes amplify the LHinducedovulation response in vitro (2). In the present study wehave examined the importance of infiltrating neutrophils inovul·ation by investigating whether selective depletion ofneutrophils from the peripheral blood prior to ovulation affectsthe ovulation rate.METHODSImmature 25 day-old rats were primed with 10IU eCG, prior tobeing injected intraperitoneally with 2.5m1 ascites fluidcontaining RP-3, a monoclonal antibody (mAb) known toselectively deplete rat neutrophils (3), on the day before and onthe day of an ovulation-inducing lOIU hCG injection. At thesame times, rats in the control group were administered withascites fluid containing an irrelevant mouse anti-rat mAb.Neutrophil counts in the blood were detennined at Oh and 20hrelative to hCG administration and ovulation was examined bycounting the released ova found in the ampullary region of theoviduct 20h after hCG injection. The number of neutrophils inthe walls of ruptured follicles were measuredimmunohistochemically using MCAI49, a mAb directedagainst a cytoplasmic detenninant in neutrophils. Circulatingprogesterone levels at 20h post-hCG were assayed byradioimmunoassay. All data was statistically analysed usingtwo-tailed unpaired student-t tests, except where otherwiseindicated.RESULTSThe total white blood cell count in peripheral blood was notaffected by administration of RP-3 (RP-3-treated group:5.8±O.4x10 6 cells/ml, Control group: 6.6±Q.3x10 6 cells/ml).The proportion of neutrophils in differential counts ofperipheral blood samples froJ!1 ~-3~freated rats wassignificantly decreased ~pared to ·>proportions at hCGinjection and 20h later- in the control group (Fig 1). Theovulation rate in the RP-3-treated group (32.4±3.6 ovulationsper treated animal) was significantly lower than in the controlgroup (44.1±3.1) (Fig 2), indicating that depleting peripheralblood neutrophils has partial inhibitory effects on ovulation ratein the rat. Immunohistochemical staining with MCA149revealed that at 20h post-hCG the density of neutrophils in theruptured follicles of RP-3-treated animals was significantly(p ='3530~ 25:a§"20tla3 15Z~ 105.~~ 200 10**ControlTreatmentD Control ~ RP30l..L-_--.J~~~.L-______I.____l_J~~~Oh20hBlood Sample Time Post-hCGFigure 1. Depletion of peripheral neutrophils as % of totalwbc (mean ± SEM) measured Oh and 20h post-hCG injectionfollowing the administration of the RP-3 antibody. **significantly different (p

-IntroductionThe penneability of testicular blood vessels is known to increase after administration of human ChorionicGonado~phin(hCG) ~d is associated with the adherence ofpolymorphonuclear leukocytes to the vascularendothelIum (1). T~stIcular .macrophages are found in the testicular interstitium in close proximity to both the~stoster?neprodUCIng LeydIg cells and ~e blood. v~ssels ?f the testi~ ~d v:hile the hCG response does not appear tod~ectly Involve androgens or prostaglandins (2), It IS abolIshed by elImInatIon of the Leydig cells with ethanedImethane s~lphonate (EDS) (1). The removal of the TMs does not affect this response (3). Nitric oxide (NO) is amolecule whIch has a broad s~trum ofpotent effects including the regulation of vascular penneability, immuneresponses and leukocyte adhesIOn (4).Release ?~ N? by the ~ and/or Leydig cell may contribute to the regulation of blood flow and the vascularpermeabIlIty In the test!s. !n th~ present study, we have assessed production ofNO by isolated testicularmacrophages cultured In vltro In the presence of the non specific stimulant, lipopolysaccharide (LPS), indomethacin(IDM) an~ hCG. In order to ~elate these observations to an in vivo situation, we have also observed NO productionby the testIcular macrophage In the presence of Leydig cells (LC),MethodTMs and LC. w~re isolated b~ a three step procedure involving mechanical dispersion of the seminiferous tubules,cellular elutnatIon and a denSIty-dependant separation with Percoll which allowed highly purified cultures of both celltypes to be obtained (5). The TM were then cultured alone (5xl05 cells/ml, lml) or in co-cultures consisting ofequal !1umbers ofLC and TM (5xloS cells/ml total) in RPMI1640/Hams F12 with 10%FCS and 2mM glutamine at34°C In 5% C0 2 in air. Combinations of LPS (10Jlg/ml), IDM (10Jlg/ml) and hCG (lOng/ml) were then added toTM, LC and T.M/LC co.-e~lt~es. After 24 hours the TM conditioned medium (TMCM) was collected, centrifuged:m d frozen untIl.use. NItrIte ~n culture supernatants was me~~red by a microplate assay method described in Ding etal, .1988 (4). Bnefly, 50JlI ~IqU?ts were removed from condItIOned medium and incubated in equal volume withGness reagent (1 % s~phanI1amIde, 0.1 % napthylethylene diamine dihydrochloride and 2.5%H3P04) at roomtemperature ~or 10 mI~utes. 1J1e ab~~bance at 550nm was detennined by a microplate reader and NO concentrationswere dete~med by ~smg sodium rntnte as a standard. The cell free medium alone was measured for NOconcentratIon and thIS value was subtracted from the value obtained with the cells.40[NO] 30JlM 2010oResultsNITRIC OXIDE SECRETION BY MACROPHAGES AND LEYDIG CELLS ISOLATEDFROM THE RAT TESTISS. Kern and S. MaddocksDepartment of Animal Science, The University of Adelaide Waite Campus, Glen Osmond 5064.* *li4 ~ mIDItlPS ~G i~ h~~ LCffM L~&MValues are the mean±SEM for n=6 experiments; *=P

Immunosuppressive activity in the rat testis that is not temperature or pH labileINTRODUCTIONThe mammalian testis is regarded as animmunologically privileged site, and testisextract is capable of suppressing lymphocyteproliferation in-vitro (1). In the present study wehave investigated some characteristics of theimmunosuppressive components of a rat testisextract (TE).METHODSTesticular extract (TE) was prepared byhomogenising decapsulated testes in phosphatebufferedsaline (PBS) and centrifuging this at250g for 15 min and then 10,OOOg for 30 mins.Aliquots of this extract were heated for 10 minsat 25 0 , 56 0 , 80 0 or 100 0 C, then cooled at 40 Cand recentrifuged. Additional aliquots had theirpH lowered to 1.9 using 1M HCl, raised to 11.2using IN NaOH, or kept at pH 7.2 withequivalent amounts of water added for controls.These aliquots were kept at room temperature for60 mins, then neutralised to pH 7.2, cooled to4 0 C and centrifuged. Further aliquots of theinitial extract were separated into fractions ofMW >5000, 1000-5000 and 5000 but no activity in the fractions ofMW 3500 Da)and was inhibited by a human recombinant IL-I receptorantagonist (IL-lRA) (Fig. 1A). The inhibitory activity wasretained following dialysis or ultrafiltration (100 kDa cutoff),and was not affected by the amine oxidase inhibitor,hydroxylamine (0.1 mM). Lymphocyte viability, asindicated by exclusion of propidium iodide, was notaffected by culture with IF. Purified testicularmacrophages secreted a similar non-dialysable (> 3500 Da)lympho-inhibitory activity in vitro (Fig. IB). Samples ofmedium collected from purified Leydig cell andseminiferous tubule cultures were inactive or stimulatoryin the assay, respectively. Inhibitory activity in testicularIF was reduced to 38 % of control by treatment with EDS(2 weeks) and to 18% of control by cryptorchidism (4weeks) (Fig. IC).DiscussionThe data indicate that IL-I, presumably from the Sertolicells, is secreted into the interstitial tissue, but its activityis prevented by a potent large molecular weight inhibitorof lymphocyte activation. This inhibitory activity is notattributable to testosterone, prostaglandins or oxidizedpolyamine products. The inhibitory factor may beproduced by the macrophages under Leydig cell control.References(1) Wang J, et al. (1994) BioI Reprod 51: in press.(2) Gustaffson K, et aI. (1988) J Reprod Irnmunol 14: 139-150.(3) P611linen P, et aI. (1992) J Reprod Irnmunol. 21:257-274.Figure 1: Mouse lymphocyte bioassay.A: Activity ofhuman IL-l (IV) and testicular IF (1.0 p.L), with andwithout addition of IL-IRA (10 p.g/mL). B: Activity oftesticular macrophage conditioned medium (MCM),with and without LPS-stimulation. Values are mean ±SEM (n = 4); * p < 0.05. C: Relative bioactivity (±95% CL) of the IF inhibitor from untreated control(CONT), DMSO-treated control (DMSO), EDS-treated(EDS) and 4-week cryptorchid (Co) rats, assayed atmultiple dilutions.C?0 no IL-1RAT""x:::JE0a.50 ~. +IL-1RA I -LPS rf..£.L()Q?.z010~~+~~0 0Q.10:~0 ffi0 w~ >wza* *~w0:~>-:cl- a a aI h1L-1 (1 U) IF (1.0 uL) a 50 100 CONT EDS Co~MCM (uL)DMSO60JA DB 2 C69 70

DEPENDENCE OF FLUID REABSORPTION IN THE EFFERENT DUCTS(ED) OF THE RAT TESTIS ON LUMINAL NA AND CLLA. Hansen, J. Clulow & R.C. JonesDepartment of Biological Sciences, University of Newcastle, NSW, 2308INTRODUCTIONPrevious studies from our laboratory (1,2) havedemonstrated that, in the rat, the efferent ducts,rather than the ductus epididymidis, are theprincipal site of net reabsorption of testicular fluid.By the time sperm have traversed the efferentducts, around 95% (2) of the rete testis fluid (RTF)accompanying the sperm has been reabsorbed.This fluid reabsorption occurs with no significantchange in the luminal osmotic pressure, and Na(RTF=136.9±2.7 mM, distal ED=136.8±2.4 roM) isreabsorbed at the same rate as water (isotonicreabsorption). These data ,suggest that fluidreabsorption in the efferent ducts is related to, andpossibly dependent upon, the transport ofNa acrossthe epithelium, as has been demonstrated for thehomologous proximal kidney tubule (3). Thecurrent study was undertaken to test thishypothesis.MATERIALS AND METHODSIndividual efferent ducts of anaesthetized Wistarrats were perfused as reported previously (4) withthe following isotonic solutions: (a) control with161 mM NaCl (b) varying concentrations of NaClmade isotonic with mannitol (c) Na only solutionsmade with sodium isethionate and mannitol and (d)Cl only solutions made with choline cWoride andmannitol.RESULTS AND DISCUSSIONThe results of perfusions at 0.1 pI min-I are shownin Fig. 1. The results show that perfusion with 161mM NaCl results in a high rate of fluidreabsorption similar to rates reported for KrebsBicarbonate Ringer or native RTF (4). As NaCl isreplaced with mannitol, reabsorption is abolished at[NaCl] = 81 roM. Above this concentration, fluidreabsorption occurs, below this concentration,secretion occurs. Removal of either Na or CI fromthe perfusion fluid is sufficient to abolishreabsorption. We conclude from these results thatfluid reabsorption in the efferent ducts of the rat isdependent on the presence of both Na and Cl in the71luminal fluid at the high concentrationsdemonstrated to occur physiologically (2).z0j::::c.4030a:0enaJ~w 20a:- cE 10- EE0,...- c- 0z0i=w-10a::0w en-20-0- ChCI--.- Nal___ NaCILUMINAL CONCENTRATION (mMJI)200Fig.1 Fluid transport in the BD after perfusion withvarying concentrations of NaCI, choline chloride (ChCl)and sodium isethionate (NaI).REFERENCES(1) Jones, R.C. & Jurd, K.M. (1987) Aust. J. BioI. Sci.40: 79-90.(2) Clulow, J., Jones, R.C. & Hansen, L.A. Bxperim.Physiol. (in press).(3) Giebisch, G., Klose, R.M., Malnic, G., Sullivan,W.J. & Windhager, E.B. (1964) J. Gen. PhysioI. 47:1175-1194.(4) Clulow, J., Hansen, L.A. & Jones, R.C. (1992) Proc.Aust. Soc. Reprod. BioI. 24: 10.IMPROVEMENT IN THE DEGREE OF SYNCHRONY OF THE PROSTAGLANDIN-INDUCEDOESTRUS IN COWSMas'ud Hariadi, D.Broomfield and P.J.WrightDepartment of Veterinary Clinical Sciences, University of MelbourneWerribee 3030, VictoriaINTRODUCTIONTreatment of cows with prostaglandin (PG) on day 12(D12) of the cycle (oestrus = DO) results in greaterspread in the occurrence of oestrus than treatment atother stages (1). This spread of oestrus reflects thestage on follicular maturity at luteolysis (2). We havestudied the effect of treatments on D5 of the cycle,aimed at synchronising follicular waves, on the degreeof synchrony of prostaglandin-induced oestrus in cows.MATERIALS AND METHODSThe study, comprising two experiments, used nonlactatingdairy cows of different breeds (Friesian, Jerseyand Crossbred) which exhibited normal oestrous cycles.In Experiment 1 cows (n = 56) were devided into threegroups. Cows were treated on D4 and D5 of the cyclewith saline (control), or charcoal extracted bovinefollicular fluid (CFF, 16 ml, iv, 12h apart), or on D5treated with oestradiol benzoate (ODB, 5 mg', im) andprogesterone (pessary/CIDR-B inserted for 7 days). InExperiment 2 cows (n = 92) were divided into four equalgroups, on 05 of the cycle Group 1 received 1 ml saline(control), Group 2 had CIDR-B inserted for 7 days andODS (5 mg, im). Groups 3 and 4 had CIDR-B and ODBrespectively. All cows in both experiments received500 /1g c1oprostenol (CS) on D12. Blood samples forprogesterone determination were taken on D12 (Exp.1)and on D5, 09, D12, D14 and at oestrus (Exp.2). Cowswere fitted with heat mount detector and observed twicedaily for sign of oestrus.RESULTSTreatments with CIDR-B/ODB tightened (P=0.002) andwith CFF worsened (P=O.01) the synchrony of PGinducedoestrus in cows (Fig.1, Exp.1). In Experiment 2,CIDR-B/ODB had better synchrony than saline treatedcows, and ODB treated group was the tightest(P

OESTROUS BEHAVIOl)R IN THE ABSENCE OF A DOMINANT FOLLICLEOR OVULATION AFTER NORGESTOMET TREATMENT IN HEIFERSA. Niasari-Naslaji"S, M.J. D'Occhio', T. Whyte' and D. Jillella!fCSIRO Division of Tropical Animal Production, Tropical Beef· Centre, Rockhampton, Queensland;!Department of Farm Animal Medicine and Production, University of Queensland, Brisbane, QueenslandIntroductionCrestar® is an oestrous synchronisation treatmentthat is used extensively in artificial inseminationand embryo transfer programs in beef and dairycattle. It involves the i.m. injection of oestradiol(OE 2 ) valerate (5 mg) and the syntheticprogestagen norgestomet (17a-acetoxy-11 p­methyl-19-norpreg-4-en3,20-dione, 3 mg),together with a s.c. implant of norgestomet (3 mg)that is removed after 8 to 10 days. Oestroususually occurs within 48 hours of implant removal.Crestar® has been used to induce oestrus inprepubertal and peripubertal heifers, butconception rates to artificial insemination (A.I.)have generally been relatively low (1, 2). In astudy in ovariectomised cows and heifers, astandard Crestar® treatment induced oestrousbehaviour in 55% and 57% of females,respectively (3). This finding suggested that inentire females Crestar® could induce oestrus in theabsence of ovulation, which has importantpractical implications. Therefore, in the presentstudy, we examined whether Crestar® could induceoestrous without ovulation in peripubertal heifers.Materials and MethodsOvarian status (follicle diameter, presence of acorpus luteum (Cl)) was determined in 23, 18­month-old Brahman (80S indicus) heifers (341 ± 6kg) by (1) trans-rectal ultrasonography using anAloka 210 real-time linear scanner and 7.5 MHztransducer and (2) assay of plasma progesterone(P4) concentrations by RIA. Heifers then received astandard Crestar® treatment described above (Day0). On Day 7, heifers were injected withcloprostenol (Estrumate®) and ovarian statusrecorded. Norgestomet implants were removed onDay 8 and oestrous behaviour was monitored overthe next 3 days with teaser bulls and twice dailyobservation of group behaviour. Furtherassessment of ovarian status was carried out 6days after oestrus. Follicles were classified asmorphologically 'dominant' if they were ~ 10 mmin diameter. Plasma P4 concentrations ~ 2 ng/mlwere taken as indicative of a functional CL.Results are presented as means ± SEM.ResultsOn Day 0, 3 heifers had a functional Cl suggestingthat the majority were acyclic. At the same time,13 heifers had a dominant follicle of diameter 10.9± 0.4 mm. On Day 7, only one heifer had adominant follicle and the diameter of the largestfollicle in other heifers was 5.7 ± 0.9 mm (n =22). After norgestomet implant removal on Day 8,16 (70%) heifers showed oestrous behaviour withthe distribution shown in Table 1. On Day 6 afteroestrus no heifer had a functional CL.Table 1 Distribution of oestrous behaviour afterremoval of norgestomet implants.Within 24 h9/16Within 48 h4/16Within 72 h3/16DiscussionThe relatively small follicles on Day 7 of treatmentwould not have been expected to secretesignificant OE 2 over the next three days. This,together with the absence of elevated P4 on Day 6after oestrus, strongly suggested that oestrousbehaviour occurred in the absence of folliclegrowth or ovulation. Escobedo et 8/ (4) reportedthat in 80S indicus cross heifers ranging from 8 to16 months of age norgestomet treatment for 9days induced oestrus in 60% of heifers, but noanimals showed a typical pre-ovulatory LH surge.A possible explanation for the above findings isthat circulating OE 2 concentrations were reportedto remain elevated at least to Day 8 after injectionof 5 mg oestradiol valerate (5). The elevated OE 2could presumably induce oestrous behaviour uponwithdrawal of norgestomet.The present data, together with previousobservations (4), have important implications. -Forexample, the results may explain low conceptionrates to A.1. after synchronisation of peripubertalheifers. Also, in superovulation and embryotransfer programs, heifers should be assessed forovarian status (ovarian palpation, ultrasound, P4assay) to confirm the presence of a CL aftersynchronisation, and before FSH therapy is started.References(1) Short RE et 8/ (1976) J Anim Sci 43: 1254(2) Spitzer JC (1982) Theriogenology 17: 373(3) McGuire WJ et 8/ (1990) Theriogenology 34:33(4) Escobedo F et 8/ (1989) J Anim Sci 67 (Suppl1): 410(5) Kojima N et 8/ (1992) Bioi Reprod 47: 1009-Ovulation and Oestrus among Dairy Cows with Anovulatory AnoestrusFollowing Progesterone Pre-TreatmentK.L. Macmillan, S. McDougall. V.K Taufa andA.M DayDairying Research Corporation, Private Bag 3123, Hamilton, New Zealand.INTRODUCTIONAnovulatory anoestrus (A-A) is the mostcommon form ofinfertility among New Zealand'spasture-fed dairy cows. Even when they dorecommence ovulating, it will frequently occurwithout oestrus. Studies in lactating dairy cowson responses to injected oestradiol benzoate(ODB) during the postpartum period showed that5 days of pre-treatment with progesterone (P4)increased the incidence of oestrus from less than40% without P4 priming to over 70% (1, 2).Ovulation usually accompanied the oestrusfollowing P4 priming. These interactions werestudied for their possible relevance as treatmentregimens for A-A.MATERIALS AND METHODSTwo trials were conducted with cows in 8 dairyherds in 1992 or 1993. Animals diagnosed withanovulatory anoestrus (A-A) were randomisedwithin herd taking account of age, breed andcalving date into 3 (Trial 1) or 5 sub-groups(Trial 2). A CIDR device (InterAg, Hamilton)was inserted into the vagina of each animal toprovide a source ofP4. This device was removed5 or 7 days later at which time some animals wereinjected with 400 ill eCG (NZ Pastoral REFERENCESainseminated through to 14 days after deviceremoval when the uninseminated animals werepresented for a second veterinary examinationand classified as unresponsive and still A-A, orovulating without being detected in oestrus (Ov­Oe). In Trial 1, a milk sample was obtained fromevery cow 14 days after device removal. Thissample was assayed for P4, with concentrationsof>1 ng/ml being accepted as indicative of lutealactivity.RESULTSThe ODB injection increased the percentage ofanimals inseminated within 2 to 4 days of deviceremoval (p

-PREPARTUM SlJPPLEMENTATION OF BOS INDICUS COWSENHANCES POSTPARTUM OVARIAN FUNCTIONLee A. Fitzoatrick!, Geoff Fordyce and Keith Entwistle.31 Department of Biomedical & Tropical Veterinary Sciences, James Cook University, Townsville, 48112 Queensland Department of Primary Industries, Swan's Lagoon Beef Cattle Research Station, Millaroo, 48073 Present address: Faculty of the Sciences, University of New England, ArmidaJe, 2351Immunisation Against Growth Hormone-Releasing Factor (GRF) Does Not affectThe Testicular Response To Nutrition In RamsS,W Walkden-Brown 1 , M,J. H6tzel 1 , R,D.G. RigbyZ and G.B. Martin 1 ,31Faculty of Agriculture, University of Western Australia, Nedlands, WA 6009. 2CSIRO Division of Animal Production,Blacktown, NSW 2148. 3CSIRO Division of Animal Production, Wembley, WA 6014.INTRODUCTION Introduction ResultsThe growth and function of ovarian follicles in the postpartumcow are considered to reflect the net effect of endocrineactivity at any particular time postpartum. However, previousstudies have shown that prepartum supplementation canenhance postpartum ovarian follicle populations, in theabsence of bodyweight (BW)/body condition (BC) effects (1).The aim of this study was therefore to test the hypothesis thatshort-term prepartum supplementation of Bos indicus crosscows can improve postpartum ovarian function by enhancingfolliculogenesis.MATERIAL AND METHODSFifty pregnant grade Brahman cows (~-% Bos indicus) wereallocated to either receive 1 kg of cotton seed meal/head/day(44% crude protein) fed twice weekly for six weeks prior tothe expected date of commencement of calving (CM), orremain unsupplemented controls (CaNT). Cows wereallocated to be either ovariectomised at 80 days postpartum orto remain intact. Blood samples were taken every 10 days forplasmaprogesterone (P,) estimation. Postpartum ovulation wasregarded as having occurred when plasma P, was >1 ng/m!.Ovarian follicles (~mm) were dissected, measured andcounted, and follicular fluid collected for oestradiol (Ez) andprogesterone (P,) estimation. Aromatase activity studies werecarried out on granulosa cells from the ovaries of 12 cowsfrom each treatment group by measuring E, production by4xlO' viable granulosa cells in tissue culture medium withadded testosterone (T,), luteinising hormone (LH) and(or)follicle stimulating hormone (FSH), after 3 hours incubation(2). To simplify statistical analyses, follicle data were pooledinto five diameter classes (2-3,4-5, 6-7, 8-10 & ~ll mm).RESULTSThere were no differences in BW or BC between the groupsat the end of supplementation or during the postpartumperiod. Supplemented cows ovulated 30 days earlier followingcalving (101 compared to 131 days for CaNT cows; P=0.08).At any given time 80 to 180 days after calving, the proportionof cows with cyclic ovarian activity tended to be higher forCM cows, although this was only significant at 100 dayspostpartum (64% and 10%; P5 mm) from CM cows produced more E, thansimilar follicles from CaNT cows (p=O.053), while smallfollicles (2-5 mm) from CM cows produced less P, thansimilar follicles from CaNT cows (p=0.003) (Figure 1).Supplementation had no effect on numbers of granulosa cellsin diameter-classes. However, follicles from CM cowscontained more granulosa cells overall than follicles fromCONT cows (80.3±12.6 x 10' vs 31.5±7.3 x 10'; P=0.OO3).Furthermore, granulosa cells from CM cows had greateraromatase activity compared to CONT cows (po etSmall Large Small LargeFollicles Follicles Follicles FolliclesCO NTCMFigure 1 Least squares means (±SEM) for a and P, in follicular fluidof small (2-5 rnrn) and large (>5 rom) follicles from CONT and CMcows0.40~E 0.35~ 0.30--S 0.25o 0.20""'C 0.15aJ:: 0.10(/)(J) 0.05o0.00CONCLUSIONS. T40 T4+FSH T4+LH T4o+FSH+LHCulture Medium CompositionFigure 2 Least squares means (±SEM) for aromatase a of granulosacells from CM and CONT cows, in the presence ofT., T,+FSH, T.+LHor T,+FSH+LHWe conclude that prepartum supplementation can enhancepostpartum ovarian function ofBos indicus cows. The effectsof this nutritional input on reproductive function may simplybe due to changes in gonadotrophin secretion, or local effectson the ovary mediated by metabolic hormones and (or)growth factors. However, the exact mechanisms involvedremain to be determined.1. Fitzpatrick LA (1994) PhD Thesis, James Cook University. Townsville2. McNatty et aI. (1984) Journal ofEndocrinology 102: 189-198This work was partially supported by the Meat Research CorporationNutrition-induced changes in sperm production inrams are only partially explained by changes ingonadotrophin secretion (Martin et al., 1994). Wetherefore investigated the role of the hormones ofthe somatotrophic axis by immunising rams againstGRF and observing their nutritional responses.Immunisation • 2 year old rams were immunisedagainst hGRF [1-29] conjugated to ovalbumin (GRFi),or ovalbumin alone (Control), on day -56, Boosterswere given on days 0 and 28 (n=10/group).Diet • Days -56 to -18. At pasture• Days -17to -1. Maintenance (M) in animal house• Days 0 to 35. 2.5 x maintenance (2.5M)• Days 36 to 77, 5 rams in each group returned to MMeasurements • Weekly Liveweight, scrotalcircumference and jugular plasma sample.• Days -1, 34 and 69 Plasma sampled at 20 minintervals for 12h for pulsatile-hormone assay.• Day 77 Slaughter and carcass analysis. Spermproduction estimated from testicular homogenates.~ 40ca) 70.5±1.

Use of a GnRH vaccine for immunocastration of coltsK.F. Dowsett*, U. Tshewang*, L.M. Knott*, A.E. Jackson*, DAV Bodero* and T.E. Trigg#*Departments of Farm Animal Medicine and Production, The University of Queensland, st. Lucia, Queensland;Sufiivan &Nicolaides Pathologists, PO Box 344, Indooroopi/ly, Queensland; #peptide Technology Ltd, PO Box 444, Dee Why, NSW.-Timing of progesterone supplement and embryonic survival inoverfed giltsIntroduction: Following an experiment with an oil based ovalbumenconjugated GnRH vaccine (Dowsett et al., 1991), a water solubleformulation was developed for immunocastration of horses. Whilethe oil based vaccine produced effective antibodies to GnRH andsuppressed testicular function it caused severe tissue reactions. Tominimise this effect but maintain antigenicity and efficacy of thevaccine, a watersoluble ovalbumen conjugate was tested in colts.Methods: Once testosterone concentrations and semen quality hadattained pUbertal levels, ten 2 year old colts were allocated to twogroups: 4 x 200 mg and 6 x 400 mg. One 2 year old monorchid wasalso treated with 200 mg. They were vaccinated on 6/3/92 and3/4/92. Additional vaccinations were given individually, as determinedby antibody titres and testosterone concentrations, to maintainimmunosuppression during the 1992/1993 breeding season.Colts were weighed and scrotal diameters measured weekly. Bloodsamples were collected twice weekly for testosterone assays,androstenedione assays and GnRH antibody titres until theconclusion of the experiment. Two semen samples were examinedprior to vaccination and after testosterone concentrations andantibody titres returned to normal.Following castration, the left testis of each colt was perfused fixed forhistology and electron microscopy. The right testis was used toestimate daily sperm production. The volumes of the seminiferoustubules, intertubular areas and Leydig cells were estimated by thepoint counting method. The testis of the monorchid was used for allexaminations.Results: Nine of the colts required third vaccinations. These wereadministered to two colts (200 and 400 mg) and the monorchid on26/5/92,three colts on 9/7/92 (2 x 200 mg and 1 x 400 mg) and theremainder (1 x200 mg and 2 x 400 mg) on 6/8/92. Third vaccinationswere not reqUired by the other two (400 mg). GnRH titres rangedfrom 0 to 1:4200 some 4 weeks after primary vaccination. Peak titresin the 200 mg and 400 mg groups ranged from 1:3300 to 1:9000and 1:2500 to 1:42000 respectively 2 to 4 weeks after the booster(Fig 1). Effective titres (1:1000) were maintained for 26 and 27 weeksin two 400 mg colts. The other 400mg colts had effective titres for 12to 20 weeks. Third vaccinations maintained effective titres for afurther 8 to 24 weeks in these colts. The 200 mg colts andmonorchid developed effective titres for 12 to 20 weeks. Thirdvaccinations maintained effective titres for a further 12 to 22 weeks.Prior to vaccination, testosterone concentrations varied between 0.31and 1.33 ng/ml (mean 0.65 ng/ml). They decreased to 0.08 ng/ml 6weeks after the booster and returned to normal concentrations forcolts (>0.30 ng/ml) 31 to 43 weeks after primary vaccination. (Fig 2.)Mean androstenedione concentrations followed a similar pattern totestosterone. They decreased from 0.51 ng/ml to 0.08 ng/ml 4 weeksafter the primary vaccination. Normal concentrations (>0.25 ng/ml)were reached 31 to 43 weeks after primary vaccination (Fig 3.)Mean scrotal width and length before vaccination was 9.1 em and 9.7em respectively. These decreased to 6.5 em and 7.6 em followingvaccination. Mean scrotal width and length at castration was 9.4 and11.0 em.Mean semen characteristics before vaccination were gel free volume16.5 ml, sperm concentration 295.5 x 10 6 /ml, total sperm perejaCUlate 4041 x 10 6 and 32% live normal spermatozoa. Beforecastration these parameters were gel free volume 13.0 ml, spermconcentration 319.4 x 10 6 /ml, total sperm per ejaCUlate 4729 x 10 6and 38.7% live normal spermatozoa for the 200 mg dose group.Before castration the 400 mg live normal spermatozoa.The testes of all animals showed active spermatogenesis with Leydigcells present. The mean testicular parenchyma weights for the 200and 400 mg group were 129.0 g and 109.8 g. Daily sperm productionper testis and per gram of testis for the 200 mg group was 3.7 x 10 6and 2.8 x 10 6 , while those of the 400mg group were 2.3 x 10 8 and2.0 x 10 6 respectively.180001600014000.. 12000,Ii!~ 10000.Jl 8000~ 60004000200010·10 10 20 30 40 50 60Weeks from primary vaccination1.20.80.60.40.2Fig.1. Antibody titres follOWing vaccinationPrima!y vaccinationPrimary vaccination10Dose ofvaccination-0-- 200mg-400mg10 20 30 40Weeks from primary vaccinationFig 2. Androstenedione levels following vaccinationDose ofvaccination2030Weeks from primary vaccinationFig 3. Testosterone levels following vaccination4050Surgical castrationo +-~--;"'-~~~-,---~--r-~-~~--,--..-'--,·101.2i 0.8..~ 0.6~.!!j 0.40.25060Surgical castranoDiscussion: The results indicate that the water soluble vaccinesuppressed testicular function,and both testosterone andandrostenedione production. Androstenedione appears to provide amore accurate measure of the effect of the vaccine on testicularfunction than testosterone does. Semen data, testicular histology anddaily sperm production data suggest that the effects of the vaccineare reversible. Further work is required to determine the reason forindividual variation in antibody production.References:Dowsett, K.F., Pattie, W.A., Knott, L.M., Jackson, A.E., Hoskinson,R.M., Rigby, R.P.G. and Moss, B.A. (1991) A preliminary stUdy ofimmunological castration in colts. J. Reprod. Fert., Suppl.44, 183­190.60. R.A.Par;, M.A.Miles 1 , M.P. Cash 2 , and J.M. Waters 1IVictorian Institute of Animal Science, Werribee, Vic.3030, 2University of Sydney, NSW2006,INTRODUCTIONGilts which are overfed during early pregnancy may havean increased incidence of embryonic mortaltiy. This hasbeen related to reduced levels of peripheral progesterone(prog) and may be corrected with exogenous progtreatment (1). We have previously shown that in sheep,there is a discrete period (days 11-12 after mating) ofenhanced sensitivity to prog reductions (2).Our aim in this study was to determine if a similarlydiscrete period of response to prog supplement could bedemonstrated in overfed gilts.MATERIALS AND METHODSLarge White x Landrace gilts (n=61) were housed ingroup pens until first pubertal oestrus when they weretransferred to individual pens and fed 2 kg/day offinisher diet. Gilts were double mated at their nextoestrus (Day 0) and from day 2, they were fed 3.5 kgdaily, split into two feeds. Treatments commenced onday 4 according to the following groups. Group 1­Control, arachis oil days 4 to 22; Group 2-Prog days 4 to22; Group 3-Prog days 4 to 10; Group 4-Prog days 10 to16; Group 5- Prog days 16 to 22. Prog (50mg/ml) wasdissolved in arachis oil and Iml was injected (im) eachday into alternate sides of the shoulder during themorning feed. One ml of oil was injected daily whenprog was not given. Gilts were slaughtered on day 23 ofpregnancy and reproductive tracts were collected.RESULTS and DISCUSSIONThere was a main effect of prog treatment in whichembryo survival was increased (Fig 1). For individualgroups, this difference was only significant when progwas given between days 16-22 but did not occur in giltstreated between days 4-22. In groups 2 and 5, CLweight was reduced, indicating a feedback effect whengiven prog between days 16-22 (Fig 2). The greaterembryo weight from gilts treated with prog from days 4­10 was not confirmed when gilts were treated with progfrom days 4-22 (Fig.3). The precise role of progesteronein the overfed pig requires further study.This work was funded by thePig Research andDevelopment Corporation.....u~ 0.4­:c.~ 0.3~0.0 L.--l-co-n-tr.J.ol-llpllrJ..JogLlJ.--I._ pr- oḡ• .I..-I'::pCL.roL£g."'--n;p~ro~g""'.'------0.35~ 0.20~·ir 0.10""10090BO70--0 60ḳ E:50.,40 ;;s~ 30lJIt200.70.6100.50.20.100.300.250.100.05Control Prog.(011) DaY'"Day. 4-224-22Fig.1 Percentage of viable embryos at Day 23 aftermating(Oil) Days Days Doys DoysDays 4-22 4-10 10-16 16-224-22Fig.2 Mean (±se) weight of corpora lutea from gilts onday 23 after mating0.00 L....l__-I--.LlUl-l..LU.JIL--=:==---'""",Control Pra9. Pro;(all) Oay. Dal'"Coy. 04--22 4-10+-22ProgCo)'.16-22Fig. 3 Mean (±s.e) embryo weights for gilts on Day 23after mating(1) Parr,R.A., Hughes,P.E., Davis,LF., Miles,M.A. andFerlazzo,lC. (1993) Proc. 4th Int. Conf. on Pig Reprod.93.(2) Parr,R.A. (1992) Reprod. Fertil. Dev., 4, 297-300.77 78

PROGESTERONE WITHDRAWAL, PLASMA LACTOSECONCENTRATIONS AND PIGLET PERFORMANCE: IS THERE ARELATIONSHIP?Brett D. Glencross, Janine K. Toussaint, Robert C. Tuckey and Peter E. HartmannDepmtment of Biochemistry, The University of Western Australia, Nedlands W.A. 6009IntroductionThe initiation of copious milk production(lactogenesis II) in the sow is triggered by thewithdrawal ofprogesterone from the circulatorysystem (1). Poor piglet perfonnance has beenassociated with high post partum plasmaconcentrations of progesterone (2). It washypothesised that low milk production was theprimary cause of this poor perfoffilance. Theconcentration of lactose in sow plasma aroundparturition provides a temporal measure oflactogenesis II (3). We have examined therelationship between progesterone withdrawaland the increase. in plasma lactose concentrationsin 5 sows and compared these changes to thegrowth oftheir litters.MethodsBlood samples were collected from sows from70 hours pre- until 44 hours post-partum at 4­hourly intervals (samples were only collectedfrom sow 2 up to 24 hours post-partum). Thesamples were analysed for progesterone andplasma lactose by radio-immunoassay andluminometric techniques respectively (Fig 1aand b). In addition the birth weights andgrowth ofthe piglets were recorded at 12-hourlyintervals for the first 3 days after birth and theweight gain was standardized by expressing thepiglet's growth as a ratio of its birth weight.Litter perfoffilance was detennined by the meangrowth rate of all the piglets within the litter.Results and ConclusionsThis preliminary study supports the observationmade by de Passille et at. (2), that incompletepr?gesterone withdrawal from sow plasma(~IgS la, lb and 2) is associated with poorpIglet perfoffilance. In addition, the positiverelationship between lactose in sow plasma (Fig3) and litter growth rate supports the hypothesisthat poor postnatal growth is caused by asuppression oflactogenesis II (2).References(1) Martin, C.E., Hartmann, P.E. & Gooneratne, A.(1978) Aust. J. BioI. Sci., 11: 517-525.(2) de Passille, A.M.B., Rushen, J.• Foxcroft,G.R., Aherne, EX. & Schaefer, A. (1993) J.Anim.Sci.1l.: 179-184.(3) Hartmann, P.E., Whitely, J.L.& Willcox, D.L.(1984) J. PhysioI. 347: 453-463.AcknowledgementsPig Research and Development Corporation255? 205Q)15t::0I-

»THE GENERATION AND PARTIAL CHARACTERISATION OF ANTIBODIES AGAINST MARSUPIAL GAMETESM.S.Harris and I.C.RodgerDepartment of Biological Sciences, University of Newcastle, NSW 2308INTRODUCTIONThe elucidation of the mechanisms involved in gametematuration and their interactions requires an understanding ofthe molecular nature of these cells. Monoclonal antibodieshave proven powerful tools in dissecting the molecularcharacter of eutherian gametes, their maturation andsubsequently the fertilisation process. Such characterisationof marsupial spermatozoa has been initiated with theproduction of monoclonal antibodies against tammar wallaby(Macropus eugenii) spermatozoa.CHARACTERISATION OF MAMMALIAN SPERM TAIL PROTEINS USING RAT FIBROUS SHEATHANTIBODYY.H. Kim~ G. Almahbobi, P.D. Temple Smith*, D.M. de Kretser & J.R. McFarlane, Institute of Reproduction andDevelopment, Department ofAnatomy*, Monash University, Clayton, Victoria 3168, AustraliaINTRODUCTIONSperm movement in the mammalian species depends on their flagellum which consists of several cytoskeletalcomponents, including the axoneme, outer dense fibres and fibrous sheath (FS). FS is a unique principal piececomponent ofsperm tail, which is composed oflongitudinal columns and numerous transverse ribs (1). The aim ofthe study was to characterise and compare sperm tail fibrous sheath proteins in different species using a polyclonalantibody to rat fibrous sheath by Western blotting and immunofluorescence microscopy.MATERIALS AND METHODSMale Balb/c mice (6-8wks) received immunisations (i.p) ofeither ejaculated or cauda epididymal spermatozoa at 3-4 weekintervals in Freunds adjuvant prior to a final i.v boost. Themouse spleen cells were then fused with NS-1 myeloma cellsand resulting hybridomas screened for antibody production byELISA and indirect immunofluorescence (IIF) usingparaformaldehyde fixed methanol permeabilised spermatozoa.Positive clones were subsequently single cell cloned. Cellsurface distribution was assayed by IIF on live wallabyspermatozoa at 40C. Species cross-reactivity was evaluatedusing eutherian sperm fixed as above. Electrophoreticseparation of wallaby sperm proteins was performedfollowing a sequential protein extraction. Proteins were thentransferred to nitrocellulose and immunodetection ofmonoclonal antibody binding performed using an anti-IgGalkaline phosphatase conjugated secondary antibody.RESULTSTwo promising monoclonal cultures were produced, wallabysperm 1 and 2 (WS-1 & WS-2). In immunofluorescence studiesWS-1 strongly labelled the tail, midpiece and either the wholehead or the acrosomal region of fixed wallaby spermatozoa(fig 1). The antigen recognised by the WS-1 antibody hasbeen shown to arise in the testis and its cellular distributionremained unaltered up to and after ejaculation. WS-1 did notlabel live spermatozoa implying· an intracellular as opposedto cell surface distribution. The WS-1 antibody alsodemonstrated species cross-reactivity with the fixedspermatozoa of another marsupial, the common brushtailpossum (Trichosurus vupecula). The midpiece of the possumspermatozoa labelled most strongly however the head and tailalso labelled distinctly. The WS-1 determinant was alsopresent on mouse, rat and rabbit spermatozoa. Again themidpiece and tail were most strongly labelled, weakerreactivity being associated with the acrosome.Another antibody, WS-2, recognised an antigen localised tothe tail, midpiece and peri-acrosomal ring of fixed wallabyspermatozoa (fig 2). Only the midpiece of live spermatozoawas labelled by this antibody suggesting both a surface andintracellular distribution of this antigen. The WS-2 antigenwas shown to be epididymal in origin and was not present onpossum, rat, mouse or rabbit spermatozoa. Immunoblottingrevealed that the WS-2 antibody recognised epitopes on threeproteins of approximate relative molecular weights 30, 35and 60kDa.Fig.I. Immunofluorescent localisation of WS-1 to theacrosome (a) , midpiece (m) and tail (t) of a tammar wallabyspermatozoan.Fig.2. Immunofluorescent localisation of the WS-2 antibodyto the peri-acrosomal ring (pa), midpiece. (m) and tail (t) of thetammar wallaby spermatozoan.DISCUSSIONSix fusions following whole sperm immunisations haveproduced clones exhibiting similar immunolocalisationpatterns. Work is therefore continuing with immunisationsusing an acrosomal extract produced after the spermatozoahave been induced to undergo an in vitro acrosome reactionusing the diacylglycerol DiCg (1). Sperm were induced toundergo the acrosome reaction and then removed bycentrifugation. The supernatant containing the plasmalemmaoverlying the acrosome, the outer acrosomal membrane andthe acrosomal contents were then concentrated for use asantigen.REFERENCES(1) Sistina, Y, Lin,M, Mate, K.E and Rodger,J.C. (1993)J.Reprod. Fert. .22.:335-341.MATERIALS & METHODSThe rat spenn tail fibrous sheath was isolated as previouslydescribed (2), and was solubilised in 1% SDS, 2mM DIT, and 25mM Tris-HCl. The proteins were emulsified in a Freund's completeadjuvant and injected into New Zealand white rabbits (75 ug perrabbit). The rabbits were boosted with antigen at 2 week intervalsand bled 1-2 weeks after each boost. Rat cauda epididymal spermand ejaculated rabbit and human sperm were prepared for indirectimmunofluorescence as previously described (3) or solubilised inurea, SDS, and DIT by shaking at room temperature and thensperm tail proteins were isolated from the gel-like DNA materialby centrifugation.RESULTSSDS-PAGE of these sperm tail proteins showed CoomassieBrilliant Blue bands of similar intensity, corresponding to 116kDa, 80-89 kDa, 57 kDa, 31 kDa, 28 kDa, and 15.5 kDa, althoughthere were some species specific bands of different molecular sizespresent (Figure 1). Western blots of sperm tail proteins with anti­FS reacted strongly with 116 kDa and 80 kDa polypeptides oftherat, rabbit and human sperm tail proteins. Rat and rabbit sperm tailproteins have similar cross reactivities in 80-89 kDa, 57 kDa, 31kDa and 28.5 kDa, but there was no cross reaction or weak crossreaction with human sperm tail proteins (Figure 1). Usingimmunofluorescence, isolated intact sperm denatured by 1% TritonX-I00 in PBS, reacted with rat fibrous sheath antibody mainly inthe principal piece of the sperm tail, but in the rabbit also reactedwith the equatorial segment ofthe head (Figure 2).STO 1 2 3116 ....66 ....45 ....31 ....21.5 ....14.4'"4Figure 1: SDS-PAGE of rat (1), rabbit (2), andhuman (3) sperm tail proteins, and rat FS (4).Western blot using anti-rat FS antiserum ofhuman (A), rabbit (B) rat (C) and rat FS (D).Figure 2: Immunofluoresence micrograph ofrabbit sperm using anti-rat FS antiserum.Immmunoactivity was seen on the equatorialsegment (E) of the head, and principal piece(P) but not the middle piece (M) of the spermSUMMARYSperm tail proteins are immunologically similar in the 3 species tail.although there were some species differences. The cross reactivity ofthe antiserum with the equatorial segment inrabbit sperm heads suggest that this protein has antigenic similarity to that ofrat FS proteins, interestingly no crossreactivity was seen in human or rat sperm.REFERENCES(1) Fawcett, D.W. Dev. BioI 1975;44:394-436(2) Kim, Y.H. et al., Proceeding ofthe XIIth North American Testis Workshop. 1993; p 51(3) Almahbobi et al., Histochemical J 1993; 25:14-183 84

•· ISOLATION AND CHARACTERISATION OF RAT SPERM TAIL OUTER DENSE FIBRE PROTEINS MAP2 EXPRESSION IN THE ADULT RAT TESTISY.H. Kim, P.D. Temple Smith*, D.M. de Kretser & lR. McFarlane, Institute of Reproduction and Development,Department ofAnatomy*, Monash University, Clayton, Victoria 3168, AustraliaINTRODUCTIONThe outer dense fibres which surround themicrotubules of the axoneme are located along theentire length of the sperm tail, and they may beimportant in maintaining the passive elasticstructures and elastic recoil of the sperm tail (1).The assembly ofrat outer dense fibres is initiated atthe proximal end of the flagellum and progressesdistally during step 16 of spermiogenesis (2). Theisolation of rat sperm outer dense fibres has beenpreviously reported (3,4). The aim ofthis study wasto extend this work and further examine the proteincomposition of outer dense fibres, by isolating andcharacterising the individual protein components ofthe outer dense fibres.DISCUSSIONA1nino acid analysis ofthe structure shows some similarity to the fibrous sheath, both being rich in aspartic acid,glutamic acid and serine (5,6), however it contains a higher content of proline and arginine. Although the 28-34kDa protein ofthe rat outer dense fibres has been identified as being alternatively spliced forms ofthe rt7 (7) gene,the derivation ofthe other proteins has yet to be determined.REFERENCES(1) Phillips, D.M. (1972) J. Cell BioI. 53:561-573.(2) Irons, MJ, Clermont, Y. (1982) Am J Anat 165:121-130.(3) Vera et al., ( 1984) J BioI Chern 259:5970-5977.(4) Oko, R. (1988). BioI. Reprod., 39:169-182.(5) Brito et al (1989) Gamete Res. 22:205-217.(6) Kim Y.H. et al (1994) Proc 8th European Testis Workshop p126.(7) Van Der Hoorn et al (1990) Dev. BioI. 142: 147-154.14.4.......METHODSFigure 1: Electron micrograph and SDS-PAGE of isolated ratouter dense fibres.Sperm were collected from the cauda epididymidesof 12 adult Sprague-Dawley rats. The sperm were filtered through a Nitex screen, and decapitated by sonicationand layered through a sucrose step gradient. Isolated sperm tails were assessed by phase contrast microscopy andextracted in 1% SDS, 2 mM DTI and 25 mM Tris-HCI pH 8 for 90 min with shaking at room temperature.Finally, the solubilised sperm tail suspensions were centrifuged through a sucrose step gradient and the purificationofouter dense fibres was verified by electron microscopy. Electroelution following SDS-PAGE was used to isolateabundant proteins.RESULTSAfter exposure to SDS-DTI, most sperm tail components were solubilised including axoneme, fibrous sheath andmitochondrial sheath while the more resistant outer dense fibres remained (Figure 1). SDS-PAGE ·of the outerdense fibres revealed at least 13 bands of varying abundance (Figure 1). The range and molecular size pattern ofthese proteins was similar to that reported by Vera (3) and Oko (4). The most abundant proteins were a large bandbetween 28 kDa-34 kDa, a band at 82 kDa, followed by one 21.5 kDa and 15.5 kDa. We have isolated 8 of themost abundant proteins. A1nino acid analysis of whole ODF shows this structure is rich in aspartIc (11 %) andglutamic acid (9%), serine (13%), proline (10%), arginine (10%) and leucine (10%).STOKate Love1Cl;nd, Kristina Zlatic, Terri Hayes, David de Kretser, and Jim McFarlaneInstitute ofReproduction and Development, Monash University, Clayton, Victoria, Australia 3168IntroductionThe testis is an abundant source ofmicrotubule networks which include mitotic and meioticspindles, the spermatid manchette and axoneme, and Sertoli cell cytoskeleton. Microtubulesare comprised of CJ. and ~ tubulin subunits, which are polymerised and stabilised by a varietyof microtubule associated proteins (MAPs). Previous studies have shown that the testiscontains a variety ofMAPs, and a putative MAP2 protein has been reported inbovine spenn(1). M.AP2 has been shown to bind to and induce polymerisation of microtubules, and isinvolved in cytoskeletal cross-linking and binding to the regulatory subunit of cAMPdependentkinase IT (2,3). MAP2 mRNA is processed into at least three alternatively splicedvariants, designated MAP2~ MAP2b and MAP2c. Ofthe 5.7 kb ofcoding sequence presentin the 9 kb mRNA which encodes MAP2a and MAP2b, approximately 4 kb of internalsequence is deleted to produce mRNA encoding MAP2c, which consists of only the aminoand carboxy terminal regions ofMAP2 (3).o 519 1500 2000-4603t='D-----I~--------1JJ___J'-.-----MAP2R.(--MAP2R2--MAP2R1- -MAP2R3-Figure 1: MAP2 coding sequence and cDNAprobes used in this study.Methods and ResultsMicrotubule preparations were isolated from adult rat testis and brain using taxol-mediatedpolymerisation (4). The polymerised testis samples were analysed by ELISA and Westernblot using an anti-MAPs polyclonal antibody (M7273) which is reported to primarilyrecognise MAP2 and Tau, anti MAP2 (IIM:-2) monoclonal antibody which recognises allforms ofMAP2, and anti-MAP2a+b (AP-20) monoclonal antibody which recognises MAP2aand MAP2b only.Immunoactivity was detected by ELISA using all three antibodies. Western Blots using theanti-MAPs antibody (Figure 2, lane A) showed numerous bands ranging from greater than200 kDa to less than 14 kDa with a major band ofimmunoactivity at 70 kDa, rather than theexpected 280-300 kDa region for MAP2. The anti MAP2 antibody (Figure 2, lane C)revealed a major band at 70 kDa, while the MAP2a+b antibody failed to recognise anyproteins (Figure 2, lane B), indicating that MAP2c is present in the adult rat testis. Theseresults were confinned using Northern blot analysis of total RNA from adult rat brain andtestis, and cDNAprobes which distinguish between MAP2a and MAP2b and MAP2c (Figure3). Brain contains a predominant mRNA of9 kb corresponding to MAP2a and MAP2b and aminor species at 6 kb corresponding to MAP2c. In contrast, the testis contains only onepredominant mRNA of6 kb which corresponds to MAP2c.References1. Carr DW and Acott TS (1990): BioI Reprod 43:795-805.2. Garner et al (1988) JBC 106:779-783.3. Kindler et al (1990) JBC 265: 19679-84.4. Valle RB (1982): J Cell BioI 92:435-442.5700200-Discussion 28 S ...These data demonstrate by ELISA, Western and Northern Blot analysis that a MAP2c-likeprotein is present inthe testis. Northern blots also indicate that multiple MAP2 mRNA arepresent in the testis including species of appoximately 9 kb which could encode MAP2a orMAP2b proteins. These will most like be in association with discrete cell types at specificstages ofdifferentiation and it will be important to establish the nature and site ofproductionofthe various MAP2 mRNAs.66-116 ­97 -55-ARl R2T B T BBR3TBcFigure 2: Western blots ofpurifiedmicrotubule preparations from adultrat testis probed with (A) anti MAPs,(B) anti MAP2a+b, and (C) antiMAP2 antibodies.R4TBFigure 2: Northern blot ofadultrat testis (T) and brain (B) totalmRNA probed with (1) Rl, (2)R2, (3) R3, and (4) R4 cDNAprobes.85 86

TIlE EFFECf OF ENVIRONMENTAL TEMPERATURE ON SCROTAL TEMPERATUREMEASURED BY 1ELEMETRY IN RAMS AT PASTURE.Brian P.Setchell 1 , James L.Zupp l, Grace Ekpel, Simon Maddocks 1 and Gordon Grigg 2 .1: Department ofAnimal Science, University ofAdelaide (Waite), Glen Osmond, SA 5064.2:Departrnent ofZoology, University of Queensland, Brisbane, Q 4072.It has been known for many years (see (1)) that underlaboratory conditions, the temperature of the scrotum andhence the testes is maintained at about 33°C, considerablybelow body temperature. However, there is littleinformation available on the temperature of the scrotum ofanimals out in the field. It is important to know the rangeoftemperatures to which the testes oframs are exposedunder field conditions to put into context our studies (2, 3)on the effects on semen quality, fertility and embryonicmortality of intermittent scrotal insulation, which raisesscrotal temperature by between 1.5 and 2.5°C.Two adult Merino rams weighing about 55 kgwere anaesthetized with pentobarbitone sodium andfluothane, and a small incision made in the skin over thecentre of the posterior smface of the scrotum, level with themiddle of the testes. A pocket was made in thesubcutaneous tissue between the testes and a temperaturesensitive transmitter (Sirtrack NZ) inserted. The signalfrom this device was a pulse, the frequency ofwhich wasrelated linearly to temperature. The skin was sutured withsilk, and after recovery, the rams were released into apaddock, from which the signal from the transmitters couldbe recorded with a Telonics TR-2E 150/154 receiver in thenearby laboratory. The transmitters were implanted onJanuary 1 1994, and recordings continued several timesdaily until the end of February. At the time ofeachrecording, air temperature was measured with a wet and drybulb thermometer fixed about 1 m from the ground on a4038~ (1);:,iiiQj 36Q.EQI....ea 34e()tIJ• • y = 31.611 + 0.12361x R"2 = 0.51132 ~Y = 31.459 + 0.11357x R"2 = 0.618 Ram (1)I]f] Y = 31.767 + 0.13356x RI\2 = 0.562 Ram (2)3010 20 30 40 50Air Temperature (degrees Celsius)post on the edge of the paddock.The summer of 1994 proved to be a particularlymild one, with only one day on which the temperature inAdelaide exceeded 400c. Nevertheless, sufficient recordingswere obtained to show that scrotal temperature was linearlyrelated to both dry and wet bulb temperatures. The linearcorrelations derived from 256 observations with airtemperatures ranging from 14 to 42°C, were highlysignificant (P < 0.(01) for both rams and for the pooleddata. Similar relationships were obtained with wet bulbtemperatures. At any given ambient temperature, smalldifferences in scrotal temperatures between the two ramsprobably were related to the fact that the transmitter in ram2 remained between the two testes, whereas that in ram 1was located at the bottom of the scrotum, wheretemperatures are usually about 0.5 0 C cooler (4).It is apparent from this study that at dry bulb airtemperatures above 400C, scrotal temperatures of greaterthan 360C can be anticipated. Therefore our studies withintermittent scrotal insulation, which raise scrotaltemperature to between 34.5 and 35.5°C for as little as8h/day, and produce abnormalities in sperm morphology,fertilising ability and embryo survival (2,3) are entirelyrelevant to the field situation, and therefore heat stress tothe males must be considered as a possible factor in thehigh level ofembryonic mortality observed in theAustralian sheep population.(1). Setchell, B.P. (1978) The Mammalian Testis. Elek Books, London. (2). Mieusset, R., Quintana Casares, P.,Sanchez Partida, L.G., Sowerbutts, S.F., Zupp, J.L. & Setchell, B.P. (1992) J. Reprod.Fertil. 94, 337-343.(3). Ekpe, G., Zupp, J.L., Seamark, R.F. & Setehell, B.P. (1993) Pmc ASRB, 25, 88. (4). Fowler, D.G. (1968)Aust.J.exp.Agric'. 8, 125-132.THE EFFECTS OF MELATONIN IMPLANTATION ON SPERMATOGENESIS IN THEEUROPEAN RED FOX (VULPES VULPES)S. Young AB , M.P. BradleyA, A. Gidley-Baird B and L.A. Hinds A .CRC for Biological Control ofVertebrate Pest Populations, CSIRO Division of Wildlife and Ecology, PO Box 84, Lyneham,ACT 2602, Australia A . Department ofAnimal Science, University of Sydney, NSW 2001, Australia B .INTRODUCTIONThe European red fox (Vulpes vulpes) is aseasonal breeder. The mating season occurs during Julyand August in the Southern hemisphere, and the gonadsof each sex exhibit an annual cycle ofregression andredevelopment. Both artificial photoperiod and melatoninimplantation have been used to advance the breedingseason in the silver fox, a colour mutant ofthe red fox.The aim ofthis study was to advance spermatogenesis inthe male red fox by administration ofexogenousmelatonin commencing 5 weeks after the summer solstice.METHODSOn January 25, 1993, 6 male red foxes (GroupMLT) were implanted with melatonin (Regulin®, 18 mg),and 6 were used as controls (Group C). Fortnightly bloodsamples were collected for measurement ofplasmamelatonin, and testis volume was determined weekly. Toassess the sensitivity ofthe pituitary-gonadal axis,challenge with a standard dose ofgonadotrophin-releasinghormone (GnRH, 40 ~g) was performed at monthlyintervals (February - July). On March 31 and May 26,blood samples were collected throughout a 24-hour periodto determine the pattern ofendogenous melatoninsecretion in V. vulpes. Four foxes (2 from each group)were autopsied in May, June and July, for determinationof histological differences in the testes and epididymides,and expression ofa fox-specific testis gene (FSA-l).RESULTSGroup lYfLT showed advances in spermatogenicactivity, expression ofFSA-l, and epididymalspermatozoal content. Testis volume was significantlygreater in Group lYfLT from April 11 - June 12 (Fig. 1).No change in GnRH-induced luteinising hormone (LH)release occurred as a function oftreatment or time. Incontrast, Group lYfLT showed a significantly lower releaseof testosterone in response to GnRH challenge duringFebruary - April~ thereafter, testosterone release increasedto equal that of Group C in June. A diurnal pattern ofmelatonin secretion was absent in Group C foxes on bothMarch 31 and May 26, and in Group lYfLT foxes on May26. In contrast, all members of Group lYfLT showed aconsistent nocturnal increase in plasma melatonin onMarch 31 (Fig. 2).CONCLUSIONThis study indicates that the continuous presenceof elevated plasma melatonin commencing 5 weeks afterthe summer solstice is effective in advancing the onset ofspermatogenesis in the male red fox by approximately 4weeks. These results extend findings of previous studies(1,2) in the silver fox in which melatonin implants placed3 or 8 weeks earlier were also effective in advancingtesticular redevelopment by 4 weeks.'i~ 140 El'l E 120.£.:E:OJ100'Qj~ 80>-"0.0 60iiiE:J40(5> 20III~

»Glucose and acetate utilization. by marsupial epididymal spermatozoa is inhibited by aprostatic secretionR. N. Murdoch and R. C. JonesINTRODUCTIONA knowledge of the energy metabolism of epididymalsperm is basic for understanding sperm storage in theepididymis and the effects of their dilution in seminalplasma during ejaculation. However, there are noreports on the metabolism of epididymal sperm from amarsupial and only one report of a metabolic study ofmarsupial spenn (1). The study (1) used ejaculates fromthe brush tailed possum and concluded that theendogenous metabolism of the sperm was greater thanreported for eutherian sperm. Also, the rate was notaffected by addition of glucose, or N-acety 0­glucosamine (NAG) which is the naturally occurringsugar in accessory gland secretions of marsupials.However, the study confounded the metabolism ofsperm with that of prostatic bodies which were alsopresent in the semen (2).This report describes the metabolism of epididymalsperm from the mo~t studied marsupial (the tammarwallaby, Macropus eugenil).MATERIALS AND METHODSSamples of luminal fluid (1 00-200 ~I) were flushed withparaffin oil from the distal cauda epididymidis of thetammar, washed once in 0.5 ml Krebs-Ringerphosphate (pH 7.4) and incubated (1 ml; 0.7-1.6 x 10 8sperm! flask) at 37°C in Warburg flasks containing 0.1ml 20% (w/v) KOH in the centre well. Depending uponthe design of the experiment compounds were added tothe incubation media: D-[U- 14 C]glucose (5 ~moles; 500nCi), C 4 C]acetate (5 ~moles; 500 nCi) and NAG (5~moles). Oxygen consumption was measured by usingair as the gas phase. Glucose and acetate oxidationwere detennined by measuring 14C02 trapped in theKOH while glucose and lactate concentrations weremeasured by enzymatic methods. The data shown inTable 1 are for the first 2 h of incubation.RESULTSA preliminary study showed that in the absence ofexogenous substrate sperm can oxidise endogenoussubstrate for up to 8 hours. Table 1 shows that additionof exogenous substrate stimulated oxygenconsumption, with acetate having a greater stimulatoryeffect than glucose or NAG. Experiment 1 shows thatglycolysis produced more lactate from NAG thanglucose, and that glucose utilization was completelyinhibited by the presence of NAG. Experiment 2 showedthat NAG also reduced the utilization/oxidation ofacetate, but only by about 50%.Department of Biological Sciences,University ofNewcastle, NSW, Australia 2308DISCUSSIONIt is concluded that tammar epididymal sperm have ametabolic rate about the same as bull and ram sperm,but probably have a greater store of endogenoussubstrate for energy metabolism than sperm fromeutherian mammals. Oxygen consumption by tammarsperm can be stimulated by the addition of exogenoussubstrate with glucose and NAG having about the sameeffect, but a smaller effect than acetate. However,glycolysis is greater with NAG than glucose assubstrate, and the presence of NAG reduces theutilization of glucose and acetate by tammar epididymalsperm. Since glucose utilization is completely inhibitedby the presence of NAG it is concluded that NAG actsto inhibit the transport of glucose into the cell.Table 1. Effects of exogenous substrate on themetabolism ofsperm from the tammar wallaby. Means± sem from 5 tammars expressed as rate/ 1(f sperm!2hours.SubstrateOxygenuptake(~I)Glucoseutilized(~mole)Lactateproduced(~mole)Glucoseoracetateoxidised(~mole)Experiment 1:None 62 ± 4.8 0.0 ± 0.00Glucose 79 ± 7.3 1.8±0.47 2.2 ± 0.40 0.5 ± 0.1NAG 78 ± 8.2 4.2 ± 0.42Glucose+ NAG 79 ± 7.9 0.0 ± 0.00 3.7 ± 0.39 0.01 ± 0.0Experiment 2:None 47 ± 5.7 0.0 ± 0.00Acetate 75 ± 6.4 1.3 ± 0.00 1.3 ± 0.1NAG 54 ± 3.9 2.3±0.12Acetate+ NAG 58 ± 4.9 2.0 ± 0.23 0.7 ± 0.01REFERENCES1. Rodger, J.C. & Suter, D.A.1. (1978) GameteResearch 1, 111-116.2. Rodger, J.C. & White, I.G. (1976) J. Reprod. Fert.46, 467-469.CALCIUM INFLUX INTO MOUSE SPERM ACTIVATED BY SOLUBILIZED MOUSE ZONAEPELLUCIDAE IS INHIBITED BY THREE INHIBITORS OF THE ZONA-INDUCED ACROSOMEREACTION: 3-QUINUCLIDINYL BENZILATE, TYRPHOSTIN A-48, AND PERTUSSIS TOXIN.Janice L. Bailey and Bayard T. StoreyDepartment of ObstetriCs & Gynecology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USAINTRODUCTION. The acrosome reaction in mouse sperm bound to the zona pellucida of the mouse egg is induced by thezona glycoprotein component ZP3 [1]; this induction is blocked by the muscarinic antagonist, 3-quinuclidinyl. bcnzilate(QNB) [2]. Exogenous calcium has long been known to be obligate for occurrence of the acrosome reactIOn. Wehypothesised that a zona-induced rise in intracellular calcium [Ca2+]i would be an early precursor reaction to acrosomalexocytosis and that acrosome reaction inhibitors should block the zona-induced [Ca 2 +]i increase. We report here a test ofthis hypothesis, using the fluorescent Ca 2 + indicator fluo-3 to monitor mouse sperm [Ca 2 +li.MATERIALS AND METHODS. Male mice were CD1 retired breeders. Sperm were obtained from excised caudaepididymides by swim-out into bovine serum albumin (BSA)-free medium MJB [3] and incubated with 5 pM fluo-3 tetraacetoxymethyl(AM) ester for 20 min at 25° to load the cell-penneant ester, which then hydrolyses to g~ve the ce~limpenneanttetracarboxylate indicator. Exogenous dye was removed by centrifugation, ~d the cells w~re .the~ lllcu?a~ed IIIMJB containing 20 mg/mL BSA for capacitation. Microscope examination showed umform fIuo-3 dlstnbutlOn wlthlll thecells; chlortetracycline (Cfe) assay [4] showed 75-80% capacitated spenn. Spenn [Ca 2 +]i was monitored at 37° by fIuo-3fluorescence at 530 nm, using 498-436 nm dual wavelength excitation. Intracellular fluo-3 fluorescence was stable over theexperiments' duration in control tests, showing acceptable dye retention. Acrosome reactions were scored using the CfCassay [4]. Solubilised mouse zona pellucida protein (SMZP) was prepared as described by Ward et al. [5].RESULTS. The parallel effects of added SMZP in the absence and presence of 50 pM QNB; 12.5 pM tyrphostin A48, atyrosine kinase inhibitor; and 10 ng/mL pertuss"is toxin (PTX) , an inactivator of Gi proteins, on [Ca 2 +]i and inducedacrosome reactions(% AR) incapacitated mouse spenn are 150....,----------------------,-50shown in Figure 1.40Fig. 1 Increase in [Ca 2 +]i~ 100c:over baseline of 231 ±58 nM ,..:r(filled bars) and in % AR of ~intact cells (stippled bars» ~ 50over 20±5 % spontaneous ARinduced by SMZP (X ± SE, n:±ZP, 9; +QNB, 7; +A48,5; o+PTX,4).noZP+ZP_..0.&.-+-0+ ZP + QNB + ZP + A48 + ZP + PTXACKNOWLEDGMENT. This work was supported by U.S. National Institutes of Health grant HD-06274.REFERENCES.1. Bleil, J.D., and Wassarman, P.M. (1983) Dev. BioI. 95, 317-324.2. Horman, H.M., and Storey, B.T. (1982) Dev. BioI. 91, 121-130.3. Storey, B.T., Hourani, c.L., and Kim, J.B. (1992) Mol. Reprod. Dev. 32,41-50.4. Ward> C.R., and Storey, B.T. (1984) Dev. BioI. 104,287-296.5. Ward, C.R., Storey, B.T., and Kopf, G.S. (1992) 1. BioI. Chern. 269, 13254-13258.30 a:(20 #The increase in [Ca 2 +]i induced by addition of SMZP (+ ZP) was significant (P < 0.05) compared to control (no ZP);increases in [Ca 2 +]i induced by SMZP in the presence of inhibitors were not significant compared to control (P =0.65).Addition of the ionophore Br-A23187 after establishment of the SMZP-induced stea~y state [Ca2+li resulted in the followingfurther [Ca 2 +]i increases (nM; X ± SE, n as in Fig.I): no ZP, I04±29; + ZP, 110±17; + ZP + QNB, 157±42; + ZP +A48, 114±42; + ZP + PTX, 124±49. These further increases in [Ca2+:k were not significantly different (P = 0.65). Theincrease in percent acrosome reacted sperm was significant only for + ZP, P < 0.001; for the other conditions, P = 0.2.DISCUSSION. The increase in [Ca 2 +]i induced by SMZP is tightly correlated with induction of the acrosme reaction, inagreement with results reported earlier [2]. Of particular interest is the observation that the three inhibitors, QNB, A48, andPIX, each of which acts on a different intracellular signal transduction reaction, all block the SMZP-induced [Ca2+]iincrease and the SMZP-induced acrosome reaction. We conclude from this observation that the sperm [Ca 2 +Ji increaseinduced by the zona pellucida is an early reaction in the sequence leading to zona-induced exocytosis. Still to be resolved isthe question of whether the Br-A23187-induced increase in [Ca 2 +]i, which is not affected by the inhibitors and appears to beaddtive to the SMZP-induced increase, represents a pathway for Ca 2 + influx independent of that for the zona-induced ea 2 +influx, and whether Ca 2 + influx per se is sufficient to induce acrosomal exocytosis.1089 90

Acrosomal Integrity of Tammar Wallaby (Macropus eugenii)Spermatozoa After Treatment with Penetrating (TCEP) and Non-Penetrating (GSH)Reducing AgentsDadi Setiadi, Yulia Sistina and John C. RodgerDepartment of Biological Sciences, University of Newcastle, NSW 2308INTRODUCTIONStudies of the acrosome of marsupial spermatozoa have shownthat it is relatively stable compared with that of placentalmammals and this appears to be at least partially due to theproperties of the acrosomal membranes (1-2). The marsupialacrosome contains significant concentrations of disulphides andthiols, which are likely to contribute to its stability (3). This studyfurther examined the basis of marsupial acrosome stability byexamining the effects of S-S reducing agents on acrosomalintegrity and sperm motility. The two agents used were nonmembranepermeable agent glutathione (GSH) and permeable Tris(2-carboxyethyl)-phosphine Hydrochloride (TCEP).MATERIAL AND METHODSSpermatozoa were obtained by electroejaculation, washed by"swim up" in Phosphate Buffer Saline (PBS) and adjusted to afinal concentration of 60x10 6 sperm/ml. Glutathione (GSH,Sigma, St. Louis, USA) and Tris(2-carboxyethyl)-Phosphinehydrochloride (TCEP, Pierce, illinois, USA) were diluted in PBSand added to spermatozoa to achieve final concentrations of: 0.0,0.5, 1.0,2.0,4.0, 8.0, and 16.0 mM; then incubated for 180 minat RT. At the end of incubation. motility was assessed andspermatozoa fixed with 2% paraformaldehyde in PBS; stained byBryan's stain and acrosomes classified as normal, altered orabsent (2). In addition spermatozoa were processed for mBBrlabelling of SH groups as described previously (3). Two hundredspermatozoa per treatment were counted for each replicate.Statistical analysis was by Student t test.RESULTSBryan's staining revealed that the number of altered acrosomeswas maximum at 8.0 mM GSH (7.2%10.60) and 4.0 mM TCEP(15.9%±2.68). The percentage of spermatozoa without anacrosome was maximal with: 16.0 mM GSH (98.4%±0.44) and8.0 mM TCEP (99.5%±0.14). Moreover the percentage ofspermatozoa without an acrosome was significantly greater forTCEP treatment compared with GSH treatment at 2.0, 4.0 and 8.0mM (P81 % and 3.5) or 1.0 mM TCEP (>79% and 3.0).However, all spermatozoa were immotile with; 8.0 mM GSH at60 min and 4.0 mM TCEP at 30 min incubation. After 3 hoursincubation in 4.0 mM GSH or TCEP, 75% and 97% ofspermatozoa respectively had fluorescent acrosomes. In additionmBBr labelling also allowed visualisation of swollen alteredacrosomes (Fig. 1).DISCUSSIONThe motility data suggested that both TCEP and GSHcompromised the survival of tammar wallaby spermatozoa andTCEP was deleterious at a lower concentration. Similarlyacrosomalloss and alteration was seen at lower concentrations ofTCEP than GSH. The greater potency of TCEP was probablydue to the fact that it penetrated the plasma membrane. Theeffects of GSH, which does not penetrate the plasma membrane,Fig. 1. Acrosome (arrow) of tammar wallaby spermatozoa treatedwith the fluorescent SH label mBBr. (a) Untreated - Acrosome isnot visible (b) TCEP treated - A veil of moderately fluorescentacrosomal material lies above the brightly f1uorescent condensedacrosome seen in lateral view.must have been mediated through the surface of the spermatozoa.Wallaby spermatozoa appear very sensitive ~o GSH. Guinea pigspermatozoa retained 85% motility after incubation in 40 mM for70 min (4). GSH at concentrations >4 mM and TCEP > 2 mMlead to rapid loss of motility by wallaby spermatozoa.CONCLUSIONSTreatment with either reducing agent resulted in disruption of theacrosomal contents (altered) and acrosomal loss with thepenetrating agent being effective at lower concentrations.However, in both cases major acrosomalloss was not seen untildeleterious concentrations of reducing agent were present. SinceGSH treatment had little effect on the acrosomes of livingspermatozoa S-S stabilisation ofthe plasma membrane is unlikelyto playa part in the stability of the marsupial acrosome.REFERENCES1) Mate,KE. and Rodger,J.C.(l991) 1.Reprod.Ferti1.91,41-48.2) Sistina, Y., et al. (1993) Reprod. Ferti!. Dev. 5,1-14.3) Mate, KE., et al. (1994) Molec. Reprod. Dev. 37, 318-325.4) Fleming, A.D., et al.,(l986) Gamete Res. 13, 93-102.The Effect of 2-Mercapto-ethanol (2-ME) on the Motilityand Acrosome of Tammar Wallaby SpermatozoaDadi Setiadi and John C. RodgerDepartment of Biological Sciences, University of Newcastle, NSW 2308INTRODUCTIONFertilisation in vitro is now a routine procedure in many speciesof placental mammals, but has not been achieved for anyAustralian marsupial. The remarkable stability of the marsupialacrosome (1) may contribute to the problem of obtaining-spermeggbinding in vitro. The stability of the acrosomal membranesis due at least in part to disulphide (S-S) bonds (2) and reductionof S-S may have a role in capacitation. The present studyexamined the potential of the reducing agent 2-ME to convert S-Sbonds to SH (thiol) groups at concentrations which maintainedmotility.MATERIAL AND METHODSSpermatozoa were collected by electroejaculation, washed by"swim up" into Phosphate Buffer Saline (PBS) and theirconcentration adjusted to approximately 6OxlQ6 sperm/ml. One mlof spermatozoa was added to 1 ml 2-ME (Sigma, St. Louis, USA)for final concentrations of: 0.0, 0.05, 0.5, 5.0, 25.0, 50.0 and100.0 mM and then incubated for 3 hours at RT. The motility(%) and progressive motility (1-5) were assessed at 0,30,60, 120and 180 minutes. Fluorescent labelling of thiol groups withmonobromobimane (mBBr) (Calbiochem, La Jolla, CA) wascarried out as previously described (1). Acrosomes were classifiedas Non-Fluorescent, Fluorescent around the Perimeter or EntirelyFluorescent. Two hundred spermatozoa were assessed pertreatment and the data were analysed using Student's t test aftersquare root transformation.RESULTSPrior to culture in vitro less than 10% of untreated wallabyspermatozoa had an entirely fluorescent acrosome. The numberof spermatozoa with fluorescent acrosomes (Fig. 1) was higher inall treated groups than in the control. After treatment with 5, 25,50 and 100 mM 2-ME the percentage of spermatozoa withentirely fluorescent acrosomes was >95%, and with 0.05, 0.5 mM2-ME, it was between 85-90%. However after 180 min, 50% ofcontrol spermatozoa also had fluorescent acrosomes. Spermmotility remained high during the incubation in 0.05,0.5 and 5.0mM 2-ME, but motility dropped to: 35% after 120 min in 25 mM2-ME; 28% after 60 min in 50 mM 2-ME and 13% after 30 minin 100 mM 2-ME. Progressive motility remained high (>3.5)during 3 hours incubation with concentrations of 2-ME under 25roM, but it dropped to: 1.5 after 60 min with 25 mM 2-ME , 1.33after 30 min with 50 mM 2-ME and 1.00 after 30 min with 100mM 2-ME (Fig. 2).DISCUSSIONIncubation of wallaby spermatozoa with or without addedreducing agent resulted in reduction of acrosomal disulphidebonds as indicated by an increase in the number of spermatozoawith entirely fluorescent acrosomes. However, essentially allspermatozoa had fluorescent acrosomes in 2-ME treated groups.In addition spermatozoa treated with 2-ME, below 25 mM werehighly motile for up to 60 min. Spermatozoa retained highmotility (>80%) and a high percentage of entirely fluorescentacrosomes (>90%) after 180 min incubation in 5 mM 2-ME. Theresponse of spermatozoa to 2-ME was more encouraging thanwith other reducing agents (GSH and TCEP) which resulted inFig. 1 The entirely fluorescent acrosome (arrow) of tammarwallaby spermatozoa after treatment with 2-ME.-.LOI~'-"4>.....-+oJ0-1.r-!-+oJ~ 3CIJ~ 2MQCJ5__---I~~"I_--...f'AoM0-.1o 60 120 180Incubation Time (min)o.0mM\l .05 mM .5 rnM.6.=5mM• 25 mM.1 ". 50 mM• 100 mMFig. 2 The effect of 2-ME on progressive motility of tammarwallaby spermatozoa.loss of the acrosome and poor motility at low concentrations (3).We are now examining 2-ME treatment as a capacitating strategyon the assumption that after S-S cleavage the acrosomalmembrane may become less stable, able to bind the zonapellucida and undergo an acrosome reaction.REFERENCES1) Mate, KE. and Rodger, J.C. (1991) 1. Reprod. Fertil.91,41-48.2) Mate, KE., et al' (1994) Molec. Reprod. Dev. 37,318-325.3) Setiadi, D., et al. (1994) Proceedings ASRB.91 92

------------------------------------------------------1IIIIIIIIIIIIl.-----------------------------~RELAXATION OF H19 IMPRINTING IN MOUSE ANDROGENETIC EMBRYONIC STEM CELLSKaren E. Mate, Angela J. Villar & Roger A. PedersenLaboratory of Radiobiology & Environmental Health, University of California, San Francisco, USAINTRODUCTIONMammalian development is unique in itsrequirement for both the maternal and paternal genomes,owing at least in part to genomic imprinting, which resultsin inequality in the activity of paternally and maternallyderived genes (1). The abnormal development ofparthenogenetic and androgenetic embryos has thereforebeen attributed to inappropriate expression patterns ofthese imprinted genes. R19, one of the five imprintedgenes identified to date, is expressed exclusively from thematernal allele in the postimplantation mouse embryo, andis lethal when over expressed (2). We have thereforeexamined the expression ofR19 in wild type, androgeneticand parthenogenetic embryonic stem (ES) cells todetermine whether parental-specific gene expressioncontributes to the abnormal development of isoparentalembryos.MATERIALS AND METHODSES cell lines derived from wild type, androgeneticand parthenogenetic embryos were used to overcome someofthe constraints ofworking with isoparental embryos. EScells are the in vitro counterpart of blastocyst inner cellmass cells and their differentiation into embryoid bodiesmimics events that occur in the peri-implantation embryo.Androgenetic ES cell lines were derived from androgeneticembryos produced by pronuclear transfer using hybridzygotes from the mating of CD-l females (GPIa) andB6CBA Fl (GPIb) males. Parthenogenetic ES cells werederived from parthenogenetic embryos produced byactivation with 7% ethanol for 4 min at ambienttemperature. ES cells were plated initially with STO cellfeeders in medium containing cytokine leukemia inhibitoryfactor (LIF). Before cells were harvested for RNApreparation, undifferentiated ES cells were grown in feederfree gelatine coated dishes for at least three passages inmedium containing LIF. Differentiated embryoid bodieswere produced by culturing feeder free ES cells inuncoated bacteriological plates. Total RNA was preparedby the GuSCN-CsCI gradient ultracentrifuge technique and15 Ilg per lane was loaded onto a formaldehyde gel fornorthern blotting.RESULTS & DISCUSSIONThe morphology of wild type, androgenetic andparthenogenetic ES cells appeared similar during cultureand after differentiation into embryoid bodies, indicatingthat they followed a similar developmental timetable. Anysignificant contribution to the levels of Ri9 mRNAdetected in ES cells by contaminating STO feeder cells wasdiscounted, as less than 151lg of STO cell RNA did notcontain detectable levels of H19 RNA.RI9 RNA was detected at varying levels in threelines of undifferentiated androgenetic ES cells (Fig la).Overall the level of expression was similar to that detectedin wild typeES cells. The levels ofRi9 RNA increased inall three lines of androgenetic cells after differentiation intoembryoid bodies. Ri9 RNA was detected in parthenogeneticES cells at high levels (Fig 1b), consistent withover-expression due to the presence of two activelytranscribed alleles. The levels ofR19 RNA increased afterdifferentiation of parthenogenetic ES cells into embryoidbodies.Detection ofR19 expression in androgenetic EScells indicates an alteration of the imprint status normallyassociated with this gene. Alternatively these levels ofR19may reflect those present in normal early embryonic cells,implying the existence of a multistep imprinting process asrecently suggested (3). The over expression of R19 inparthenogenetic ES cells raises the possibility thatexcessive R19 mRNA may be a factor in the abnormaldevelopment of parthenogenetic embryos, which is furthersupported by the in utero lethality ofR19 transgenic mice(2). The occurrence of relaxed imprinting also providesopportunities for examining the concordance between lossor maintenance of parental allele-specific patterns oftranscription and methylation of specific sites in imprintedgenes.(a)Figure 1: Expression of H19 by (a) three lines ofandrogenetic ES cells (ES) and embryoid bodies (EB); (b)four lines ofparthenogenetic ES cells (BS) and two lines ofembryoid bodies (EB).REFERENCES(1) Surani, M.A.H., Barton, S.C. & Norris, M.L. (1984)Nature 308, 548-550.(2) Brunkow, ME. & Tighlman, S.M. (1991) Genes Dev.5, 1092-1101.(3) Latham, K.E., Doherty, A.S., Scott, C.D. & Schultz,R.M. (1994) Genes Dev. 8, 290-299.CHANGES IN THE METABOLISM OF ENERGY SUBSTRATES WITHIN THEFIRST CELL-CYCLE OF MOUSE EMBRYOSINTRODUCTIONMATERIALS AND METHODSRESULTSExperiment 1: Data for the metabolism ofenergy substrates byQs embryos is presented in Table 1. The metabolism of allsubstrates examined varied with time, though the pattern ofchange varied between substrates (Figure 1). Utilization ofglucose via glycoll.sis eH-Glu) and the amount of glucoseoxidized to CO eC-Glu) decreased with time, the greatestdecrease being observed between time periods commencing at 24and 28 hours post hCG. Pyr metabolism was characterised by anincrease between 24 and 28 hours with a significant decreaseobserved between 28 and 32 hours post hCG. Lact metabolismremained relatively constant for the first two time periods butdecreased significantly during the time period commencing 32hours post hCG. The metabolism of GIn for embryos developingin vivo was characterised by a substantial increase between 24 and28 hours post hCG followed by a decrease at 32 hours.Experiment 2: Whereas time-course changes in Glu, Pyr andLact metabolism for in vitro grown embryos did not differsignificantly from their in vitro cultured counterparts (Experiment1), the metabolism of GIn was markedly different (Figure 1). Thatis, in contrast to the increase observed at 28 hours post hCG forfreshly collected embryos, no significant change with time wasobserved for embryos cultured in vitro. Significant differencesdue to strain of male used for matings were also observed for 14C_Glu (P=0.025) and GIn (P=0.034) parameters (Figure 2). Forboth substrates, values observed for embryos derived from Qsmales were higher than for C57BL males at all observation times,the pattern of change with time also differing for GIn metabolism.DISCUSSIONChanges in metabolism between embryos of different cell-stageshave been well documented in the mouse (3,4,5). The presentstudy demonstrates significant variations in the metabolism ofenergy substrates within the first cell-cycle of the mouse embryoand for glutamine metabolism, a marked interaction with theextent of culture in vitro prior to the period of assay. Thesignificant effect of paternal genotype on metabolism wouldappear therefore to reflect embryos at varying stages ofthe firstJohn P. Ryan and Kathleen M. WaiteHuman Reproduction Unit, Royal North Shore Hospital, St.Leonards, NSW, 2065.A previous study (1) demonstrated differences in the metabolismof energy substrates of pronucleate and 2-cell mouse embryosderived from Quackenbush strain (Qs) females mated to males ofdifferent strains. In the mouse, paternal influences have also beenobserved for the duration of S phase of the first cell-cycle andtime to first cleavage (2). The present study investigated whetherthe metabolism of energy substrates differed within the first cellcycleof mouse embryos.Experiment 1: Pronucleate embryos were collected at 24,28 and32 h post hCG from superovulated Qs females that had beenmated to Qs malrs. The ab~lity of embryos to conv~rt 1.0 mMD[5- 3H]glucose ( H-Glu) to H 0 and 1.0 mM D[U- C]glucose,0.4 mM [2-14C]p:xruvate (Pyr); 4.0 mM DL[1- 14 C]lactate (Lact)and 0.5 mM L[U-14C]glutamine (GIn) to 14CO were immediatelyassessed during 2 hour incubation period~ using a methodpreviously described by O'Fallon and Wright (1986) (3).Experiment 2: Pronucleate embryos were collected at 24 posthCG from Qs females mated to Qs or C57BL males and eitherprocessed immediately to assess their abilities to utilize the abovementioned substrates or cultured in Human Tubal Fluid (HTF)media + 3 mg BSA!ml until 28 or 32 h post hCG at which timethey were assessed. Statistical analysis: Data were analysed byanalysis of variance with means compared by Newman-Keulstests.cell-cycle as a result of possible differences in the timing offertilisation, rate of early embryonic development or theirsusceptibility to in vitro culture conditions. Further studies appearwarranted to assess the association between metabolism andspecific phases ofthe cell-cycle and to investigate the relationshipbetween changes in metabolism and subsequent embryonicdevelopment.Table 1. Metabolism of energy substrates (fmoles!embryo.h- 1 ,mean ± s.e.m.) by Qs derived embryos 24, 28 and 32 hours posthCG.Expt Time 3 t~belledEnergy Substrate(h) H-Glu C-Glu Pyr Lact:( c:h

METABOLISM OF ENERGY SUBSTRATES BY POLYSPERMIC MOUSE EMBRYOS INSULIN AND IGF-I STIMULATE GLUCOSE TRANSPORT IN MOUSE BLASTOCYSTS VIA THEIGF-I RECEPTORKathleen M. Waite and John E RyanMarie Pantaleon and Peter KayeDepartment of Physiology and Pharmacology, The University ofHuman Reproduction Unit, Royal North Shore Hospital, St.Leonards, NSW, 2065.Queensland, Brisbane, 4072, AustraliaINTRODUCTIONIn the mouse, significant increases in the metabolism ofglucoseand pyruvate have been observed as a result offertilization (1).No such effect was observed for parthenogenetically activatedoocytes, suggesting that the fertilizing sperm or a sperm relatedfactor plays an important role in initiating and/or maintainingappropriate metabolic processes within the developing embryo.This study investigates the metabolism of energy substrates bypolyspermic mouse embryos.MATERIALS AND METHODSExperimental animals. Oocytes were collected fromsuperovulated Quackenbush strain (Qs) females at 8 weeks of age,15 hours post hCG. Cumulus cells were removed in Hepes­Human Tubal Fluid (HTF) media supplemented with 150i.u.hyaluronidase/ml. The zonae pellucidae were removed from themajority of oocytesby brief exposure to an acidified Tyrode'ssolution (pH 2.5). Zona-free oocytes \\jere rcubated in RTF + 3mg BSA/ml in the presence of 10 -10 motile sperm /ml,collected from Qs males. Experimental design. A randomselection of these oocytes were assessed individually during a 2hour incubation period ~t 6 h post igsemination for their abilitiesto convert 1.0 mM D[5- H]glucose ( H-Glu) to 3H 0 (an estimateoft~i total amount of ~lucose utilised via glycolysis) and 1.0 mM[U- C] glucose to 1 CO (an estimate of the total amount ofglucose oxidised), simultineously (2). Th amounts of 0.4 mM[2_r 14 C]pyruvate (Pyr) or 0.5 mM L[U- 4C]glutamine (GIn)oxidised to 14CO during 2 hour incubation periods were alsoassessed. Zona-intact (ZI) and zona-free (ZF) oocytes not exposedto sperm were used as controls. Following the assay period, alloocytes were fixed, stained with orcein and examined for thepresence of sperm tails and pronuclei (PN, includingdecondensing sperm heads). Statistical analysis. Data wereanalysed by analysis of variance with means compared byNewman-Keuls tests.RESULTSThere was no significant difference in metabolism of glucose,pyruvate or glutamine observed between zona intact and zonafree oocytes. Glucose metabolism, both the total amountutilised via glycolysis and the amount oxidised to COincreased significantly with fertilization as evidenced by th~presence of 2 PN (Figure 1). A linear increase in the metabolismof glucose was also observed as the number of PN identified inpolyspermic embryos increased. In contrast to glucose, nosignificant changes in the metabolism of pyruvate or glutaminewere observed irrespective of the number of PN (Figures 2 and3).DISCUSSIONThese data support a role for sperm or a sperm related factor inregulating the metabolic competency of the early mouse embryo.The significance ofthe increased metabolism ofglucose with anincrease in the number ofpronuclei is unknown. It may, however,represent a mechanism whereby development of polyspermicembryos is different to normally fertilised oocytes. The two mainhypotheses proposed as to how sperm cause oocyte activation andthe generation of calcium oscillations are by the binding of spermto cell-surface receptors and the introduction into the oocyte of acytosolic sperm fraction (3). The present study has not addressedwhether it is the fusion of multiple sperm to the plasma membraneor an increased dose of a sperm specific factor into the oocyte thatis responsible for the observed effect on glucose metabolism.How the metabolism of energy substrates might be associated tothe complex series of events associated with oocyte activationawaits further investigation.fmoles/embr."o/h (mean +/- s.e.m (n»2500,...------"------------------..,_ Total Utilisation l22:I Oxidisedd2000 .1500 ': .1000 .30500 .200ZI ooc."te ZF ooc."teZI ooc."te ZF ooc."te 2 PH 3 PHTrea1;Ment GroupZI ooc."te ZF ooc."te 2 PH 3 PHTrea1;Men1; Group4 PH4 PH4 PH5 PHFigure 1. Metabolism of 1.0 mM glucose eH-Glu, 14C_Glu) bymouse oocytes and embryos having 2, 3, 4 or 5 pronuclei. Valuesfor each metabolic parameter with different superscripts differsignificantly (P

•ENDOCYTOSIS OF FLUID BY MOUSE BLASTOCYSTSGreta Dunglison 112 and Peter Kaye 11Department of. Phtsiol~gy and Pharmacology, The University of Queensland, Brisbane,4072, Australla; Nuffleld Department of Obstetrics and GynCEcology, John RadcliffeHospital, Oxford, OX3 9DU, UK .INTRODUCTIONEndocyto~s and subsequent lysosomal hydrolysis provide b1astocysts with accessto considerable pools of external maternal protein in the extra cellular fluid (ECF).This protein may be transported into the cells in the bulk fluid phase as well asadsorbed to invaginated plasma membrane. Using iodinated bovine serum albumin(BSA), we estimated that the blastocyst internalises fluid at a rate about equivalentto the whole blastocyst volume per hour (1). However, as albumin may adhere tothe cell membrane (2), it may be endocytosed in the two phases mentioned aboveand so the measured rate of uptake may overestimate fluid transport. Toaccurately assess fluid phase endocytosis we used a non-adsorbing solute, 3Hdextran.MATERIALS AND METHODSBlastocysts were collected from superovulated mice by standard procedures andincubated under varying conditions with 3H-dextran (17.4 ~M, 18,5 GBq/LI 73 kD)in BMOC2 medium containing 1 g BSA/L. They were washed free of external 3Hdextranand radioactivity analysed.RESULTSAt 4°C in BMOC2 b1astocysts took up dextran at a rate equivalent to 0.9 ± 0,2pL/rnin/b1as~ when incubated at 3rC, the initial rate of uptake rose to 4.3 ±0,6pL/min/blast Insulin stimulated uptake at 3rC by 30% to 5.7 ±0.7 pL/min/b1ast lP

A TIMETABLE OF IN VIVO EMBRYO~ICDEVELOPMENT IN THE GREY SHORT-TAILED OPOSSUM(MONODELPHIS DOMESTICA)Karen E. Mate!, Edward, S.Robinson 2 & Roger A. Pedersen!•Gonadal sex reversal in female tammar wallabies is induced by MISD.l. Whitworth and M.B. RenfreeDepartment of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia!Laboratory of Radiobiology & Environmental Health,University of California San Francisco &2Department of Genetics, Southwest Foundation for Biomedical Research,San Antonio, TexasINTRODUCTIONThe grey short-tailed opossum (Monodelphisdomestica), has emerged in recent years as one of the majormarsupial models, and offers great potential for cellular andmolecular analyses ofmarsupial embryo development. Thetiming of early cleavage and some later stages ofMonodelphis domestica embryo development have beenexamined (1,2), however a comprehensive timetable ofembryonic development throughout gestation has not yetbeen established. The aim of this study was to establish thesebasic embryological parameters for Monodelphis.MATERIALS & METHODSThe timing of development was examined in a total of501 embryos from female Monodelphis domestica, collectedat known time intervals after video recorded matings. Whena pair mated more than once, the first mating was taken asthe initiating event. A total of 60 pregnant females weretransported by air from San Antonio to San Francisco,usuallythe day after mating. An additional 23 animals werekilled and examined in San Antonio to collect early stages ofgestation and to ensure that transportation had not caused adelay in development. Embryos were recovered andexamined fresh, or in the case of blastocysts also afterfixation and sectioning.RESULTS & DISCUSSIONA total of 403 embryos (mean 8.8/female, range 1-17)as well as 47 degenerate embryos (mean 1.0/female, range 1­12) were recovered from 46 ofthe animals shipped to UCSF.The remaining animals contained only unfertilized eggs ordegenerate embryos. Preimplantation development inMonodelphis occupied nine of the 13.5 days of gestation.Most of the time during the preimplantation period wasoccupied by cleavage and the bilaminar blastocyst stage(Table 1). The interval from copulation to birth inMonodelphis was approximately 14.5 days.The developmental sequence and timetable ofMonodelphis is similar to that of the other well characterizeddidelphid, Didelphis virginiana (3), but with several notabledifferences: (i) transit of fertilized eggs through the oviductwas rapid (as quick as 6-12 h), which may contributing to thevariable asymmetric mucoid covering of Monodelphisembryos; (ii) early cleavage stages were slower than inDidelphis, in particular the four-cell stage was variable andlasted up to 24 h; (iii) primary endoderm cells proliferatedfrom an already distinct embryonic area of the non-expandedunilaminar blastocyst.Table 1: A summary of development in the grey short-tailedopossum, Monodelphis domestica.Day AfterMatingDay 12Day 13Day 14No. oraEmbryosDay 1 96 (7)Day 3 46 (5)Day 4 32 (3)Day 5 30 (3)Day 6 30 (4)Day 7 9 (2)Day 8 14 (2)Day 9 36 (4)Day 10 40 (4)Day 11 34 (4)72 (7)14 (2)10 (2)a No of animals in parenthesesMcCrady Stage (m bold)1: Ovulation, fertilization, PN formation2-5: 2-6 cell (mostly 4-cell)4,6: 4-8 cell (mostly 4-cell)8-9: 16-32 cell9-10: Complete unilaminar blastocyst11-14: Primary endoderm formation;complete bilaminar blastocyst (335~)14: Bilaminar blastocyst (607llm)14: Bilaminar blastocyst (Imm) withobvious embryoblast18-22: Primitive streak & first somite14-26: Heart & blood vessel formation,fusion of neural folds, cephalic flexure,amnion formation, implantation30: Cervical flexure, paddle-likeforelimbs, lateral amniotic folds form32: Digital buds on forelimb; club-likehind limb, well developed allantois33-34: Claws on forelimb digits, digitalridges on hind limb, mouth/nostrils openDay 15 29 (3) 35: Elongated body, digital buds on hindlimb, birthREFERENCES(1) Baggott, L.M. & Moore, H.D.M. (1990)-J. Zool. Lond.222,623-639.(2) Harder, J.D., Stonerook, MJ. & Pondy, J. (1993) J. Exp.Zool. 266, 463-479.(3) McCrady, E. (1938) Am. Anat. Mem. 16, 14-226.IN1RODUCTIONJost's observations of the freemartin condition in cattleshowed that fetal ovaries could be transformed into testislikestructures by hormones derived from the male co-twin(1). MIS could be the factor responsible, since testicularstructures develop in fetal rat ovaries exposed to MIS invitro (2). Similarly, fetal mouse ovaries transplanted underthe skin of adult male hosts also develop seminiferoustubule-like structures (3)t and female mice transgenic forMIS show the transitory appearance of seminiferous tubules(4).This study was designed to determine if an XXundifferentiated gonad could be transfonned into a testis-likestructure in the tammar wallaby (Macropus eugenii) bytransplantation into older male pouch young. We alsocultured undifferentiated tammar ovaries with MIS todetennine ifthis could cause testicular development.MATERIALS AND :METHODSTransplantsOn the day of birth, gonads with or without mesonephrosattached were removed from male and female pouch young,and transplanted under the abdominal skin of 10 day oldmale and female recipients. Donor gonads from each sexwere transplanted into recipients of the opposite sex and thesame sex. After surgery, recipients were reattached to theirmothers teat and returned to the pouch. After 25 days,recipients were killed and the transplants removed.Transplants were fixed in Bouin's fixative, paraffinembedded,sectioned at (7Jlm)t and stained withhaematoxylin and eosin before analysis with lightmicroscopy.Organ cultureGonads with or without mesonephros attached wereremoved from female pouch young on the day of birth andplaced onto individual polycarbonate membranes.Membranes were floated onto D:MEM/Ham's FI2 culturemedium containing L-glutamine, to which had been addedpenicillin/streptomycin (1 OOU/mVIOOJlg/ml), transferrin(5llg/ml), insulin (20Jlg/ml) and retinoic acid (50ng/ml).Recombinant human MIS (9Jlg/ml)1 was added to onegonad from each pair. The corresponding gonad from eachpair was cultured without MIS as a control. Gonads werecultured for 4 or 8 days before processing for routinehistology.RESULTSThe majority (11/15) of male gonads transplanted into malerecipients were either resorbed by the hostt or uponhistological analysis were found to be dysgenic. However,of those which did survive, all continued to developmorphologically as normal testes, with Sertoli cells alignedinto seminiferous tubules surrounding the primordial germcells. A tunica albuginea and Leydig and peritubular myoidcells were also visible.Of the 2/8 female gonads transplanted into female recipientswhich survived, both developed as normal ovaries, with1We thank Dr. P. Donahoe and R. Ragin, PediatricSurgery, Massachusetts General Hospitalt for providing therhMIS.mesenchymal cells delineating nests of mitotically-activegerm cells.Of 18 female gonads transplanted into male recipients, 4survived and all developed seminiferous tubule-likestructures which were devoid ofgenn cells (Fig. la).Figure 1: Transdifferentiated ovaries after transplantationinto male recipients (a)t or after culture with added MIS (b)showing the formation of seminiferous tubule-likestructures. Note the lack of germ cells in (a).We did not find that the mesonephros had any effect ontransplant survival, or to the degree of development as atestis or an ovary.Of the 4/8 female gonads which developed seminiferoustubules in vitro, 3 were cultured with their mesonephrosattached. These gonads showed seminiferous tubulesforming around clusters of germ cells, in a patterncharacteristic of male control gonads (Fig. Ib). The singlegonad cultured without attached mesonephros was muchmore masculinised and the well-developed tubules weredevoid ofgerm cells. One ovary remained undifferentiatetd..All three gonads which were cultured for 8d becamedysgenic, suggesting that the medium cannot support thetissue for this length of time.CONCLUSIONSMIS can induce the masculinisation of neonatal tammarovaries in vitro, and could be the factor responsible for thetransdifferentiation of ovaries transplanted into male pouchyoung. Thus, MIS may playa role in normal testiculardifferentiation.REFERENCES(1) Jost, A., Vigier, B. and Prepin, J. (1972) J. Reprod.Fert. 29, 349-379.(2) Vigier, B., Watrin, F., Magre, S., Tran, D. and Josso,N. (1987) Development 100, 43-55.(3) Taketo, T., Merchant-Larios, H. and Koide, S.S.(1984) Proc. Soc. expo Biol. Med. 176, 148-153.(4) Behringer, R., Cate, R.L., Froelick, GJ., Palmiter,R.D. and Brinster, R.L. (1990) Nature 345,167-170.99 100

Thyroid gland development in the brushtail.possum,Trichosurus vulpecula.W. Buaboocha and R. T. GemmellDepartment of Anatomical Sciences, University of Queensland•EARLY PREGNANCY FACfOR (EPF): PURIFICATION TO HOMOGENEITY FROMHUMAN PLATELETS AND IDENTIFICATION AS CHAPERONIN 10.Alice C Cavanagh!, Peter L KayrJ and Halle Morton!.IDept of Surgery, 2Dept of Physiology and Pharmacology, University of Queensland, Brisbane.INTRODUCTIONThe initiation of thyroid gland function usually occursprenatally or at birth in eutherian mammals. Inmarsupials. this endocrine gland is nOt present at birth.The gland becomes functional during the latter half ofpouch life. In this smdy the ultrastrUcwre ·of the thyroidgland will be correlated with the plasma concenrrationsof both thyroxine (TJ and rri-iodo thyronine (T 3 ) in thedeveloping brushrail possum to determine whether theincrease in thyroid hormones in mid-lactation areproduced by me young or are passed across in the milkfrom me mother.~fATERIAL AND METHODSThyroid tissue from seven possums aged 40. 65. 96.113. 115. 129 days POSt partupl and an adult wereexamined \vith me light and the electron microscope.The body weights and the weights of excised thyroidglands from the seven animals were also recorded.Blood samples were obtained weekly via hean puncmrefrom pouch young and the plasma concenrrations ofboth T 4 and T:; were determined by radioimmunoassayusing ICN Diagnostics RIA kits (ICN Biomedicals. Inc..Diagnostics Division. Costa Mesa. USA).RESULTS:The weight of thyroid tissue in the possums increasedwith age and body weight (Table 1). Lysosomesrequired for the conversion of thyroglobulin to thyroxinewere first observed within the thyroid follicular cells ofthe 96 days old possum. The plasma thyroid hormonesconcentrations varied in the young possum during thefirSt 150 days after birth. Plasma concentration of T..rose from less than 10 ng/ml prior to day 60~ to 12ngiml on day 90 and [0 a peak of 45 ngiml on day 120post panum. Thereafter. the values tended to diminishco below 10 ng/ml by day 160 post parwm (Figs. 1 and2). Plasma T;; concenrrations were similar in profilewith a peak concentration of 0.25 ng/ml beingsdetected on day 120 post panum.CONCLUSION:The plasma concenrrations of T.. and T 3 in the youngpossum do not correlate with the morphologicalevidence for the initiation of activity of the thyroidgland of the young. The thyroid hormones in theplasma of the young, prior to day 96, are presumablyproduced in the mother and are transpOrted to the youngvia the milk (1). The activation of the thyroid glandcoincides with the reported increase in growth rate ofthe young possum on day 96 (2).Table 1. Weights of thyroid glands and body weightsof possums.Age Thyroiddays gm (paired)40 0.001465 0.003596 0.0086113 0.0184115 0.0314129 0.0400adult 0.3520EOic504030lDCX0 20~10EOic:00so r40I30 ~lD.S x I>.I-=10 L II0 20 r250 1 0 25Bodygm12.4124.0170.0087.0080.00275.002249.007575,e.,... ~,. .~.% thyroid weightto body weight0.01130.01460.01230.02110.03930.01450.0157" ..,...:.' .II ~I.... I. .... . ... I. e ........:- .I. I. I • .'I • • a ~ •••• I, •5050100 125 150 175 200days post partum100 125 150 175 200days post partum.....Fig. 1. The plasma concemration of thyroxine (T 4) inthe developing brushtail possum.Fig. 2. The weekly plasma concentration of thyroxine(T 4) in two developing possums.REFERENCES1. Gemmell. R.T.. and Sernia. C. (1992) CompoBiochem. Physiol. 103A:541-543.2. Gemmell. R.T.. and Hendrikz. J.K..(1993) Aust. J.Zoo1. 41: 141-149.101INTRODUCTIONEarly pregnancy factor (EPF), detectable in maternal serumwithin 24 h of fertilisation in all mammalian species tested. isindicative of and necessary for embryonic well-being. EPF hasimmunosuppressive properties and is important also as agrowth regulator of transfonned, neoplastic and nonnal cells;its presence in platelets implies a role in inflammation andwound healing. A molecule possessing this exceptionalcombination of properties has great biological significance but,until now, the strocture of the biologically active molecule hasremained elusive.MATERIALS AND METHODSAll major classes of source materials with activity in the EPFbioassay, the rosette inhibition test. including pregnancy serum(1.2), were fractionated by a 6-step scheme including reversedphaseHPLC(1). Each purified active protein was treated withan immobilised anti-EPF monoclonal antibody: activity ofbound and unbound fractions was tested (l). Platelet-derivedEPF was analysed by SDS-PAGE (15% Tris-Tricine gels),electrospray mass spectrophotometry and amino acidsequencing of both the whole molecule and proteolyticfragments (l).RESULTSFor all source materials. activity followed an identical patternthroughout fractionation and each purified active protein wasspecifically recognised by a monoclonal antibody to EPF (datanot shown)(l). These biochemical and immunologicalsimilarities indicate that we are dealing with a single substanceor closely related family of substances, acting in diversebiological situations.Only human platelets provided enough active protein forstroctural studies (45 pg/300 units - 150 litre blood). On SDS­PAGE (Fig Ia), EPF migrated as a single band coincident withbio-activity; thus the protein, not a small associate, is active.Even more compelling evidence of homogeneity was providedby mass spectrometry, which also detennined an accuratemolecular size of 10843 (Fig 1b). The amino acid sequence of-70% of the molecule was detennined. This was identical torat liver mitochondrial chaperonin 10 (cpnlO). except for asingle residue (Fig Ic). Further experiments using EPF. cpnlOand its functional associate cpn60 demonstrated that thisstroctural relationship extends to functional identity. Briefly,rat cpnlO was active in the EPF bioassay and this activitycould be neutralised by a monoclonal anti-EPFantibody. Inaddition, platelet-derived" EPF fonned an ATP dependent stablecomplex with cpn60 while imrnobilised cpn60. with ATP,could remove all EPF-activity from pregnancy serum; activitycould be recovered by removing ATP from the complex (datanot shown)(l). This requirement for ATP demonstrates thespecificity of the interaction between cpn60 and the activemoiety and illustrates that, in pregnancy serum as in platelets.cpnlO is the molecular entity initiating response in the EPFbioassay.CONCLUSIONOur studies identify EPF as an extracellular fonn of cpnlO, amember of the highly conserved heat-shock family ofmolecules, which functions as a molecular chaperone. This isthe first example of such a molecule perfonning anextracellular role. We are now investigating extracellular targetmolecules to determine whether EPF influences otherregulatory molecules (as might be expected for a molecularchaperone) or whether it exerts its effects more directly.C102BIHuman EPl' 1AIIo o \---14.,( IIIJto 15SlIc.c noBOO "00 ICOO 1100 1200 0(10 1400mIlIC.

CP·PLATELET ACTIVATING FACTOR STIMULATES CELL-CYCLE PROGRESSION IN 2-CELL MURINE EMBRYOSC.O'NeillHuman Reproduction Unit, Royal North Shore Hospital, St. Leonards, N.S.W. 2065•IN VITROAMPLIFICATION OF AN OVINE OESTRUS-ASSOCIATED OVIDUCTALGLYCOPROTEIN eDNAINTRODUCTIONWe have previously demonstrated that culture of embryos invitro compromised the release of embryo-derived plateletactivatingfactor (PAF) (1), while supplementation of mediawith synthetic PAF resulted in increased metabolism ofcarbohydrate substrates and increased implantation, pregnancyand birth rates in humans and mice following embryo transfer(2,3). It was also demonstrated that 8-cell embryos andblastocysts had a greater mitotic index when treated with PAFthan did controls (4). This latter observation could possibly beconfounded by the substantial asynchrony of the cell-cycle inthe later stage preimplantation embryos. In this study weexamined the effect of embryo density and PAFsupplementation ofmedia on the rate of cell-cycle progressionof2-cell mouse embryos which were synchronised by exposureto a reversible mitotic spindle poison at the I-cell stage.MATERIALS AND METIIODSQs female mice were superovulated and mated with fertilemales. 30h after hCG zygotes were collected from thereproductive tract. Zygotes were cultured in modHTFM(contains glutamine and EDTA)+3mg BSA/ml and observed attwo-hourly intervals to determine the kinetics of the first celldivision.Since prolonged culture in nocodazole is detrimentalto development, a 4 hour period was chosen to capture themaximum number of I-cells in the shortest period of time(Figure 1). In all experiments synchronisation was achieved byculturing zygotes for several hours, followed by transfer to 5~M nocodazole in modHTFM for 4 h. Upon removal fromnocodazole they were washed in 5 changes of HepesHTFM toremove all Nocodazole and then placed in modHTFM for 90min. At the end of this time all embryos which had divided to2-cell embryos were collected (synchronised 2-cells). Culturewas in 1O1l1 drops of modHTFMlBSA under paraffm oil andsupplemented with 1.86~M PAF/ml or vehicle (Control).Embryos were allocated at random at a density of 1 or 10/drop.After 20h culture embryos were observed at regular intervalsand the proportion progressing to the 4-cell stage recorded. Allexperiments were repeated at least 3 times.RESULTSFollowing synchronisation the first 4-cell embryos wererecorded at 24 hours. When embryos were cultured as groupsof 10 all embryos capable ofcleaving had done so by 38 hourswith 50% cleaving by 29h. When embryos were culturedindividually, cell-cycle progression was delayed, with the 50%progression occurring at 33h (4 hour delay, P < 0.001, logisticregression analysis). The addition of PAF to medium had nosignificant effect on the development of embryos in groups of10, but significantly shortened by 2.2h (p

... »THE ROLE OF AN ESTROGEN,DEPENDENT GLYCOPROTEIN, OVIDUCTIN, INFERTILIZATION IN THE GOLDEN HAMSTER D.E. Boatman and G.E. Magnoni,Dept. ofAnimal Health & Biomedical Sciences, University ofWisconsin, Madison, U.S.A.INTRODUCTION• Fertilizationin vivo is more efficient compared tothat in vitro: fewer sperm per egg are requiredand sperm interact for a shorter time with thezona pellucida prior to penetration.• Oviductal ampullary molecules may increase invivo fertilization efficiency by interacting withsperm or eggs or both to [1]:a. alter binding affinities ofcapacitated spermfor the zona pellucida.b. increase efficiency ofacrosome reactions(ARs) ofsperm on the zona pellucida.c. alter biophysical properties ofzonaepellucidae.• We now present evidence that oviductin (OVN), amammalian member ofthe chitinase family [2],is an oviductal fertilization factor that binds tosperm as well as to eggs and embryos [3].MATERIALS AND METHODS• Isolate OVN from oviducts ofPMSG primedhamsters using Helix pomatia lectin affinitychromatography (no. ofisolations=4) [3,4].• Bioassay OVN fractions for follicular eggpenetration enhancing activity (1 h pulse withpre-eapacitated sperm) [4].• Add ± OVN to sperm at two capacitation times.a. capacitate cauda epididymal sperm 0 and 5.5h in TALP (BSA= 3mg/rnl, 37 0 C, 5% CO 2 ),b. add ± OVN to sperm (1.6 X 106/rnl) 30 min.c. layer sperm suspensions onto 2-step Ficollgradients; centrifuge to wash and fix (1 %paraformaldehyde on bottom).• Assess ARs by phase and bright field microscopy.• Assess binding ofOVN to sperm (immunolocalizationusing anti-OVN MAb, I C4, andgoat anti-mouse IgG FITC conjugate).RESULTS• Follicular egg penetration: OVN fractions,increased penetration 2.9 fold (OVN= 49±2.7 %,vs. BSA alone= 17±4.0) [4].• ARs: ARs increased between 0.5 and 6 h, butOVN (effective in increasing egg penetration) didnot increase ARs at either time (Fig. I).• OVN binding to AR intact sperm: at 0.5 h OVNbound to the acrosomal crescent (82.5%labelled); at 6 h OVN bound over the equatorialand post-acrosomal regions ofthe heads (60.4%labelled) (Fig. 2).105CONCLUSIONS• We conclude that OVN increases penetration andfertilization by increasing sperm binding affinityfor eggs, because:a. OVN i penetration offollicular eggs [4].b. as a result ofcapacitation, binding ofOVN tosperm shifts regionally and temporally fromthe acrosomal crescent to the equatorialsegment.c. sperm binding affinity to oviductal comparedto follicular eggs is increased [1].d. ARs are not stimulated by OVN.Fig. 1 Effect of OVN Added at TwoCapacitation Times on Acrosome ReactionsUJa:

»THE ROLE OF AN ESTROGEN.DEPENDENT GLYCOPROTEIN, OVIDUCTIN, INFERTILIZATION IN THE GOLDEN HAMSTER D.E. Boatman and G.E. Magnoni,Dept. ofAnimal Health & Biomedical Sciences, University ofWisconsin, Madison, U.S.A.INTRODUCTION• Fertilization in vivo is more efficient compared tothat in vitro: fewer sperm per egg are requiredand sperm interact for a shorter time with thezona pellucida prior to penetration.• Oviductal ampullary molecules may increase invivo fertilization efficiency by interacting withsperm or eggs or both to [1]:a. alter binding affinities ofcapacitated spermfor the zona pellucida.b. increase efficiency ofacrosome reactions(ARs) ofsperm on the zona pellucida.c. alter biophysical properties ofzonaepellucidae.• We now present evidence that oviductin (OVN), amammalian member ofthe chitinase family [2],is an oviductal fertilization factor that binds tosperm as well as to eggs and embryos [3].MATERIALS AND METHODS• Isolate OVN from oviducts ofPMSG primedhamsters using Helix pomatia lectin affInitychromatography (no. ofisolations=4) [3,4].• Bioassay OVN fractions for follicular eggpenetration enhancing activity (1 h pulse withpre-eapacitated sperm) [4].• Add ± OVN to sperm at two capacitation times.a. capacitate cauda epididymal sperm 0 and 5.5hinTALP (BSA= 3mg/ml, 37 0 C, 5% CO 2 ),b. add ± OVN to sperm (1.6 X 106/ml) 30 min.c. layer sperm suspensions onto 2-step Ficollgradients; centrifuge to wash and fix (1 %paraformaldehyde on bottom).• Assess ARs by phase and bright field microscopy.• Assess binding ofOVN to sperm (immunolocalizationusing anti-OVN MAb, I C4, andgoat anti-mouse IgG FITC conjugate).RESULTS• Follicular egg penetration: OVN fractions,increased penetration 2.9 fold (OVN= 49±2.7 %,vs. BSA alone= 17±4.0) [4].• ARs: ARs increased between 0.5 and 6 h, butOVN (effective in increasing egg penetration) didnot increase ARs at either time (Fig. I).• OVN binding to AR intact sperm: at 0.5 h OVNbound to the acrosomal crescent (82.5%labelled); at 6 h OVN bound over the equatorialand post-acrosomal regions ofthe heads (60.4%labelled) (Fig. 2).105CONCLUSIONS• We conclude that OVN increases penetration andfertilization by increasing sperm binding affinityfor eggs, because:a. OVN i penetration offollicular eggs [4].b. as a result ofcapacitation, binding ofOVN tosperm shifts regionally and temporally fromthe acrosomal crescent to the equatorialsegment.c. sperm binding affinity to oviductal comparedto follicular eggs is increased [1].d. ARs are not stimulated by OVN.Fig. 1 Effect of OVN Added at TwoCapacitation Times on Acrosome ReactionsCI.Ia:«-c:a>o L-a>ll..50. r-------------,40302010oco 5.5Time of Oviductin Addition (hr)OVN:o minuso plusFig. 2 Temporal and Regional Shift inOVN Binding to Acrosome-Intact Sperm• OhD 5.5h!EmREFERENCES5.5 h1) Boatman DE et al. (1994) Molec Reprod Dev(in press).2) Hakala BE et al. (1993) J BioI Chern 264:25803.3) Kan FWK et al. (1990) Anat Rec 226:37-47.4) Boatman DE & Magnoni GE. (1994) BioI Reprod51(SuppI1): 129 (abs 297).Proteinases important in implantation are down-regulatedin parthenogenetic mouse blastocystsMark B. Haroe:i, Mayi Arcellana-Panlilio 2 , XingQi Zhang 3 , Dylan R. Edwards 4 , and Gilbert A. Schultz 2School of Life Science, Queensland University of Technology, Brisbane, Qld 1 , Deparbnents of Medical Biochemisn-y-2,Obstetrics & Gynaecologf and Pharmacology & Therapeutics 4 , University of Calgary, Calgary, Alberta, CanadaINTRODUCTIONMuch of the embryo's activity in the implantation process comesfrom the trophectodermal component of the blastocyst in whichthe uterine tissue is invaded by trophoblast cells. Human andmouse trophoblasts produce two types of proteinases, gelatinaseB (MMP-9) and urokinase-type plasminogen activator (uPA), thathave been implicated in the process of implantation becauseinvasion is blocked by neutralizing antibodies or inhibitors.Parthenogenetic mouse embryos cleave and develop to theblastocyst stage but show implantation defects. (Pedersen et al.,1993). Because of this/ we examined parthenogenetic blastocysts, and blastocyst outgrowths in comparison to normal fertilizedembryos in terms of their ability to express the genes and geneproductsfor uPA and MMP-9.MATERIALS AND METHODSParthenogenetic embryos obtained by ethanol activation of CD1mouse oocytes were cultured to the blastocyst stage andsubsequently transferred to 50pl culture droplets pre-treated withfibronectin (50-100 embryos/droplet). After 48 hr culture in TCM199 plus Img/ml, the medium was changed and embryos thathad not attached discarded. After a further 24 hr culture (day 8 ofdevelopment), the conditioned media droplets from normal andparthenogenetic embryos were collected and assayed. for (uPA)activity by a chromogenic assay and (MMP) activity byzymography. To investigate the expression of the genes encodingthese proteinases in embryos, RNA was extracted fromparthenogenetic and normal blastocyst outgrowths on day 8 ofdevelopment and subjected to RT-PCR analysis for detection ofuPA and MMP-9 transcripts.RESULTSAfter 24 hr culture of blastocysts (day 6 of development), 11±8%of parthenogentic blastocysts had attached compared to 18±3% fornormal counterparts (Fig 1). After another 24 hr culture (day 7)only half the number of parthenogenotes had attached comparedto normal blastocysts (37±14% vs 77±2%, P

wEarly pregnancy factor Is reqJ,Jired at implantation to optimize embryonic development.Athanasas-Platsis, S/ Kaye PL} Cavanagh, A.C./ and Morton H. iThe University of Queensland, 1Dept of Surgery, Clinical Sciences Building, RBH 4029; 2Dept ofPhysiology and Phannacology, Brisbane 4072, Qld.INTRODUCTIONEarly pregnancy factor (EPF) has a role in earlydevelopment of the embryo. Passive immunization ofmated mice with anti-EPF antibodies (Abs) on d 1 and 2post coitum (pc,d l=day of mating) demonstrated a failureto maintain pregnancy, when examined at d 7 pc (1);embryos flushed from treated mice on d 3 pc showed asignificant retardation at the 1-2 cell stage of development(2). This effect apparent in vivo was indirect, asdevelopment proceeded uninterrupted to the blastocyst stagewhen embryos were cultured in vitro in the presence of anti­EPF Abs (2). The aim of the study presented here was toextend these findings, to determine if, as well as beingrequired for preimplantation development, EPF is alsorequired by the embryo for the processes of trophoblastoutgrowth and implantation.MATERIALS AND METHODSConfirmed-mated Quackenbush mice were passivelyimmunized with rabbit anti-EPF Abs #816 (1,2) on d 3 and4 pc (500 JIg IgG x 2 daily); control groups received normalrabbit IgG or 0.9% NaCI (saline). On d 7, implantationsites and corpora lutea were counted. Oviducts and uterinehorns were flushed and development of unimplantedembryos recorded. In in vitro experiments, morulae werecollected from untreated mice on d 3 pc, washed andcultured under oil in Eagle's medium containing 10% foetalcalf serum and anti-EPF monoclonal antibodies (rnAbs;l) orcontrol rnAbs 1Dy (see Table 1) After 168 h., thedevelopment of trophoblast outgrowths was recorded. In thelowest concentration of anti-EPF mAbs tested (2.5 pg/ml),in which % blastocysts which outgrew was not significantlydecreased, the surface area of outgrowths was measured.RESULTSFollowing passive immunization on d 3 and 4 pc, only 30%embryos (ie embryos as a % of corpora lutea) had implantedby d 7 compared with 90% and 100% in the control Absand saline groups respectively (anti-EPF Abs v. control Abs,p < 0.001, t test, Fig 1). Unimplanted embryos in thecontrol Abs group (10%) were hatched whereas unimplantedembryos in the anti-EPF group (25%) were not; 45%embryos in the latter group were not accounted for.Examination of embryos after 24-48 hours in culture withanti-EPF mAbs 5/341 revealed a progression of the majorityof morulae to the blastocyst stage. However developmentwas interrupted after this time. Culture of embryos for 168h with 25 or 250 pg/ml anti-EPF rnAbs significantly~ffected the development of blastocysts to the outgrowthstage when compared to control mAbs IDy (Table 1). Ofthe affected embryos, 29 or 34% were dead after treatmentwith 25 or 250 pg/ml anti-EPF mAbs respectively comparedwith only 0 or 13% after treatment with 25 or 250 pglmlcontrol mAbs IDy respectively (p < 0.001, '1.. 2 test). Cultureof embryos with 2.5 pglml anti-EPF rnAbs did not affectdevelopment to the outgrowth stage (Table 1) but did inhibitthe surface area of these outgrowths. Average area ofoutgrowths (x 10- 3 mm 2 ± sem) with 2.5 pglml anti-EPFmAbs was 64 ± 11.8 (n = 13) compared with 110 ± 8.1 (n= 54) and 108 ± 16.7 (n = 15) in the control medium and2.5pglml control mAbs IDy respectively (p < 0.05,Student's t test).CONCLUSIONThe observation that development of morulae to theoutgrowth stage was inhibited when cultured with anti­EPF mAbs together with the fact that administration ofanti-EPF to pregnant mice on days 3 and 4 disruptedimplantation, suggests a role for EPF as a growthfactor. The inhibition of trophoblast outgrowth or, in ahigh proportion of cases, death of the blastocystcoincides with embryonic production of EPF,suggesting that it is autocrine growth factor at thisstage of development.10080~ - a-EPF IgG§33 - control rabbll IgGITIJ - 0.9% NaCI:58 CELLCOMPACTED BLASTOCYSTS IMPLANTEDMORULAEEMBRYOSFig 1: Stage of development of embryos on d 7 pcafter passive immunization of mated mice, at 56, 61,80 and 85 h pc with 500 pg rabbit anti-EPF Abs #816, control Abs or 0.9% NaCl/ injection. Anti-EPFAbs v control Abs, p < 0.001 '1.. 2 test).Table 1: Effect of anti-EPF mAbs 5/341 ontrophoblast outgrowth in vitro.rnAbs (pg/ml)" No of No ofembryos outgrowths %b p*Control medium 72 54 (75)Control rnAbs 1Dy2.5 17 15 (88)25 23 22 (96)250 30 23 (77)Anti-EPF rnAbs 5/3412.5 18 13 (72) NS25 28 15 (53)

.. •THE IDENTIFICATION OF EARLY PREGNANCY FACTOR IN RED DEER (CERVUS ELAPHUS)Gendie Lash and Mike LeggeDepartment of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand.INTRODUCTIONEarly pregnancy factor (EPF) is one of the firstbiochemical indicators of pregnancy. It is producedby the mother in response to a signal from thezygote on fertilization. The aim of this study was todetermine whether farmed red deer (Ce rvuselaphus) synthesised EPF.MATERIALS AND :METHODSFarmed red deer hinds (n=28) from an ovulationstudy (1) were bled 13 times at 3-4 day intervalsfrom 11 to 15 days after cloprostenol administration(ie 24 to 26 days after the initial oestrussynchronization) via jugular venepuncture. Theblood was allowed to clot, serum removed andfrozen at -20°C until required for analysis.Pregnancy was diagnosed by rectal ultrasonography25 to 31 and 45 to 51 days after cloprostenoltreatment. The date of calving was determined bydaily observation, and the date of conceptioncalculated on a gestation period of 233 days (2).EPF was assayed for using the rosette inhibition testaccording to Rolfe et al. (1984)(3).RESULTSFour major results came out of this study.1. All hinds which calved (n=20) gave an EPFresponse (Figure 1), dilution range of 10- 3 to 10- 5 .2. One hind of the twenty which calved had twins,and had a corresponding change in the EPF doseresponse curve, dilution range of 10- 5 to 10- 7 (Figure2). Two other hinds who had singleton births werealso identified as twin pregnancies at around day 25pc but gave normal EPF activity after day 30 pc.r.:Ia:Eo-E:z0 20E:9:c~ 10r.:II: r.:I~0 a:10-1 10-2 10-3 ID-4 10-5 10-6SAMPLE DILUTION• day 9pcFigure 1. Changes in dose response curve duringblastocyst formation in red deer at approximatelyday 8pc.3. Of the eight hinds which did not calve three wereconsidered to have had a fertilization event asobserved by a positive EPF result in the first serumsample. No subsequent samples were positive.4. As is seen in Figure 1 the EPF dose responsecurve changed around day 9 pc. This correspondswith blastocyst formation (4). No further changeswere observed over a time course up to day 45 to 46pc. The twin pregnancies did not change until day15 pc (10- 3 to 10- 4 day 15pc).3010-1 10-2 10-3 ID-4 10-5 10-6 10-7 10-8SAMPLE DILUTION• Non-pregnant hindsII Single pregnancyra Multiple pregnancyFigure 2. Changes in the dose response curvebetween nonpregnant hinds and those with single ormultiple pregnancies.DISCUSSIONThis study has shown that EPF is detectable in reddeer and that EPF can be used to distinguishbetween a twin and a singleton pregnancy. It hasalso been shown that a change in the EPF doseresponse curve corresponds with blastocystformation, where there is either a down regulationof the inhibitor or the embryo itself starts to produceEPF, and not with implantation at day 40 pc (5). Itwas surprising to find the change in the twinpregnancies was much later than the singletons.This result can not be commented on at present asso little is currently known ab.out deer earlyembryology.REFERENCES(1) Fisher M W, Asher G W, Meikle LM andNewman R E (1994) Animal Reproduction ScienceIn Press(2) Guinness F, Lincoln G A and Short R V(1971) Journal of Reproduction and Fertility 27427-438(3) Rolfe B, Cavanagh A, Forde C, Bastin F, ChenC and Morton H (1984) Journal ofImmunologicalMethods 70 1-11(4) Berg D (1993) Proceedings ofthe sixth annualgeneral meeting ofthe Australasian Association ofAnimal Arificial Breeders (Abstract) 22-25(5) McMahon C D (1989) BSc (Hons) Thesis,University of Otago, Dunedin, New Zealand.COMPARISON OF CERVICAL, TRANSCERVICAL AND LAPAROSCOPIC INSEMINATION OFEWES WITH CHILLED STORED RAM SEMEN.IF. Smith, J. Parr, S. Beaumont, 1. Oliver and G.C. UpretiAgResearch, Ruakura Agricultural Research Centre, P B 3123, Hamilton, N. Z.INTRODUCTIONA technique for transcervical artificial insemination (TAI) ofthe ewe has been reported as an alternative to laparoscopicintrauterine insemination (LAl) (1). This study evaluated thetechniques under New Zealand conditions.METHODRomney ewes (365) were treated in February for 14 dayswith CIDR® devices and inseminated (50-56 h) after deviceremoval. Semen was diluted in a milk based diluent toconcentrations of 800xl0 6 /ml for cervical insemination (CAl)(0.2 ml) and 200xl0 6 /ml for TAl and LAl (0.25 ml straws)and cooled to 15°C until insemination either 12 - 18 hr CD0) or 36 - 42 hr CD 1) after collection. Frozen semen(100x10 6 sperm; 50% motile, in 0.25 ml straws) from thesame rams was used via the TAl and LAl methods. Allinseminations were on a time basis about 52h after CIDRremoval. Oestrous status of ewes at Al and the depth ofTA! were recorded. Pregnancy was determined byultrasonic scanning 50 days post insemination. Twoadditional trials were conducted in Nov-Dec (214 ewes) andMarch (115 ewes) in which TAl (D 0 semen) and LAl werecompared.RESULTSThe pregnancy rates for the main trial are shown in Figure1. LA! was superior to TAl with all semen types (P

•IN VITRO AND IN VIVO FERTILITY OF LIQUID-STORED GOAT SPERMATOZOAJ Epplestorfl, C. C. Pomares'J, T. Stojanovb and W.M C. MaxwellbaDep~rtment ofWool and Animal Science, Uruversity ofNSW, Sydney, 2052.bDepartment ofAnimal Science, University ofSydney, NSW, 2006.INTRODUCTIONThere are several reports on the liquid storage of ramspermatozoa and its fertility after AI (1) but there islittle information available on the fertility of liquidstoredgoat spermatozoa. The use of in-vitrofertilisation (IVF), if reliable, would simplify fertilityassessment and facilitate improvements in semenstorage procedures. This study reports both the in vitroand in vivo fertility of goat spermatozoa liquid storedin a Tris-based diluent at 5 0 C.MATERIALS AND :METHODSSemen collected by artificial vagina from 4 bucks waspooled and diluted 5;.fold with a Tris diluent (300 mMTris, 27.75 mM fructose, 94.7 mM citric acid, 2% eggyolk)previously bubbled with nitrogen (N). Thediluted semen was stored at 5 0 c under N in cappedtest tubes.The fertility of goat spermatozoa stored for 0, 2, 4 or 8days was determined by both IVF and after AI. Thesemen used for the in vitro and in vivo fertilityassessments were from different batches but werecollected from the same bucks and were treatedidentically. The fresh semen (Day 0) was treatedidentically to the stored semen except that it was notcooled.For IVF, sheep oocytes collected from an abattoir andmatured in TCM-I99 for 22-27 hr, were cultured infertilisation media (bicarbonate-buffered SOF + 20%sheep serum) with Percoll treated buck spermatozoa ata concentration of Ix10 6 /ml for 18 hr. Ova wereclassified as fertilised if, after fixing and staining (1 %aceto-orcein), they contained 2 pronuclei and a spermtail.For AI, adult Cashmere does were treated in Marchwith progestogen sponges (Chronogest 45 mg,Intervet) for 19 days and with 200 iu PMSG(pregnecol, Horizon) given 24 hours before spongeremoval. The does were inseminated 50-58 hr aftersponge removal by either the cervical or laparoscopicmethod using 150 or 45 x 10 6 sperm, respectively. Theproportion of does pregnant was determined byultrasound 50 days later.RESULTSFertility was maintained when semen stored for up to4 days was deposited into the uterus, but declined after·only 2 days storage when cervical insemination wasused (Table 1). The proportion of ova fertilised invitroalso declined after 2 days storage.The fertility of fresh semen was lower than semenstored for 2 days when inseminated into the cervix(P

CHARGED LIPOPROTEIN FRACTIONS OF EGG YOLK IN BOVINE SEMEN DILUENTSE. Prendergast*, R. Vishwanath, P. Shannon and P. Molan*Livestock Improvement Corporation Limited, New Zealand Dairy Board, Hamilton, and*Department ofBiological Sciences, University ofWaikato, Hamilton, NewZealand.•MOTILITY CHARACTERISTICS OF SEMEN FROM RAMS SUBJECTED TO SCROTALINSULATION, AFTER STORAGE AT CHILLED TEMPERATURES FOR ONE WEEKChairussyuhur, A., Sanchez-Partida, L.G., Zupp, J.L., Maddocks, S. and Setchell, B.P.Department ofAnimal Sciences, Waite Agricultural Research Institute,University of Adelaide, Glen Osmond, S.A., 5064INTRODUCTIONA bovine semen diluent containing positively chargedlipoprotein material (WTM) has previously been shown tobe superior to a 5% egg yolk control diluent in survivaland fertility studies. This superiority has been attributedto both the charged and lipoprotein nature of the WTMmolecule (1). The charged protein moiety of the lipoproteinis believed to bind to the sperm membrane whilstthe lipid portion blankets the membrane providingthermal protection. Fractionation ofWTM by chloroform:water (2: 1 v/v) produced three extracts. Extract 1 was anaqueous extract and Extracts 2 and 3 organic extracts.Both Extract 1 and Extract 2 showed multiple anioniccomponents. The extracts were incorporated into diluentsand tested for their ability to protect bovine sperm againstcold shock damage.METHODS100 ml WTM was mixed with 400 ml chloroform:water(2:1 v/v) for 4 hr, on standing at 4°C for 24 hours threedistinct layers were visible, the middle layer underwenttwo further extractions. The corresponding layers fromeach extraction were pooled and concentrated by rotaryevaporation. Protein concentration was determined byeither the Biuret method or a modified Biuret method forlipid-rich samples (2). The amount of lipid present wasdetermined by the method of Foulkes (3) and theproportion of neutral and phospholipid by silicic acidchromatography. Isoelectric points were determined byeither IEF using the Phastsystem or cellulose acetateelectrophoresis. Semen collected from 3 bulls was dilutedto 100 million sperm/ml in 14GC buffer containing 5%egg yolk or one of the EX1Iacts. Cold shocking of spermoccurred at 1°C for 20 minutes.RESULTSThe results from protein and lipid determinations areshown in Table 1. Extract 2 contained the bulk ofthe lipidand protein that was extracted, Extract 3 contained justlipid and Extract 1 protein. The lipid composition isshown in Table 2. The relative abilities of the extracts toprotect sperm against cold shock are shown in Table 3.The diluents containing the Extract 1 fraction were foundto afford slight protective action to the sperm against coldshock, the Extract 2 fraction was as good as the 5% eggyolk control,but superior after 24 h incubation at 37°C.Extract 3 was found to have no protective ability. Furtherexperimental work with the Extract 2 diluent showed it tomaintain sperm survival over time at both ambienttemperature and 37°C as well as the 5% egg yolk diluent,but not for as long as sperm in the original WTM.CONCLUSIONThe results from this study have shown that organicextraction of the cationic lipoprotein W1M producesanionic lipoprotein material that can provide bovinesperm with protection against cold shock and canmaintain sperm survival over time as well as a 5% eggyolk diluent. The changes in the lipid and proteinproportions and overall charge have not affected theability to protect against cold shock, but may have affected·the ability to sustain survival at 37°C over time comparedwith WTM. This may be due to the loss ofhomogeneity ofthe lipoprotein molecule due to the disruption of its nativestate as a result ofthe extraction procedure.Table 1. Protein and lipid determinations ofthe extractsofWTMPROTEIN (g) LIPID (g)WTM 3.80 15.2Extract 1 0.41 0Extract 2 3.36 4.48Extract 3 0 8.51Table 2. L' 101'dcompos1uon 0fExtracts 2 and3 0fW1M0/0 0/0 0/0TOTAL PHOSPHO NEUTRALLIPID LIPID LIPIDWTM 79.8 16.7 83.3Extract 2 57.1 83.7 16.3Extract 3 100 13.8 86.2Table 3. Ability of the Extracts of WTM to protect spermagainst cold shock. Values are means ± se (n=3). Meanswithin columns with the same letter are not significantlydifferentBEFORE AFTER SURVIVALCOLD COLD (after CS)SHOCK SHOCK 24 H/37°CExtract 1 80.0 ± 2.9 a 25.0 ± 2.9 b 8.3 + 1.7 cExtract 2 80.0 ± 2.9 a 68.3 ± 1.7 a 51.7 ± 6.0 aExtract 3 65.0 ± 2.9 b 16.7 ± 4.4 c 5.7 + 4.7 c50/0 EY 78.3 ± 4.4 a 61.7 + 6.0 a 30.0 + 7.6 b14GC 73.3 ± 6.7 a 10.0 ± 2.9 c 5.0 ± O.OcREFERENCES(1) Vishwanath R, Shannon P, Curson B (1992). Anim.Reprod. Sci. 29: 185-194.(2) Upreti GC, Ratcliffe RA, Riches PC (1988). Anal.Biochem. 168: 421-427.(3) Foulkes JA (1977). J. Reprod. Fert. 49: 277-284.INTRODUCTIONWhen normal ram spermatozoa are stored at 5°Cfor up to 10 days, their motilty is better when anovel CEQY diluent is used than when aconventional HYCG diluent is used (1). However,summer temperature extremes in the field maycompromise normal sperm quality. Increase oftesticular temperatures during summer or byscrotal insulation decreases sperm quality (2,3).The present study was therefore undertaken toinvestigate the effect of CEQY and HYCGdiluents on the motility characteristics of semencollected from rams subjected to scrotalinsulation, after storage at cool (5°C), rather thanfreezing temperatures, for one week.MATERIALS AND METHODSThe techniques of scrotal insulation, semencollection and processing were as previouslydescribed (1,2,4). Semen was collected weekly for2 weeks before, for 4 weeks during. and for 5weeks after scrotal insulation. Sperm motility(MOT) and progressive motility (pROG) wereassessed using a Hamilton Thorn MotilityAnalyser. After angular transformation, pooleddata for each treatment period were subjected toanalysis ofvariance and differences between meanswere determined by Tukey's test.RESULTSAt all time periods, and for both treatmentgroups, the percent motility and progressivemotility was significantly higher (p

••. STUDIES ON RAM SPERMATOZOAL AROMATIC AMINO ACID OXIDASEG.C. Upreti, K. Jensen, R. Munday, R. Vishwanath* and IF. SmithAgResearch, Ruakura Agricultural Research Centre, PB 3123,-Hamilton, NZ*Livestock Improvement Corporation Ltd, PB 3016, Hamilton, NZTHE EFFECT OF ANTIOXIDANTS ON THE PREGNANCY RATE AFTERINSEMINATION WITH LIQUID STORED RAM SPERMATOZOAT. Stojanov, S.L. Rhodes, W.M.C. Maxwell and O. EvansDepartment ofAnimal Science, University ofSydney, NSW 2006INTRODUCTIONThe addition of desferal to bovine semen diluentimproved the shelf-life of diluted bovine semen (1).Desferal is an iron chelator and hence blocks the'lconversion of hydrogen peroxide (H 2 0 2 ), producedby semen aromatic aminoacid oxidase (AAO), to theOhydroxyl radical (OH ) in an iron catalysed nonenzymaticreaction. Surprisingly, desferal did notimprove the ram spermatozoal motility in a syntheticdiluent, RSD-1 (2). One possible reason for the lackof a beneficial effect could be due to low levels ofAAO in ram spermatozoa. We therefore investigatedthe AAO activity of ram spermatozoa.[K = constant 4.45 III O 2 Iml, V = Volume (5 ml)]RESULTS:AAO activity in ram (R) and bull (B) spermatozoa(37.8 for R; 37.0 for B; s.e.d. 4.96; n=3) was similar.The relationship between AAO activity and proportionof dead sperm is shown in Fig. 2 and the equationsfor various combinations of D, L, Ram & Bull spermin citrate and in RSD-l are:LB/DBLR/DRLB/DBLB/DRRSD-lRSD-lcitratecitratey = 7.62 + 42.4xY= 7.85 + 57.0xy = -3.33 + 38.9xy = -1.81 + 45.0xINTRODUCTIONSpontaneous lipid peroxidation of mammalianspermatozoa in vitro is associated with loss ofmotility and decreased fertility. In vitro studiesshowed that adding antioxidants, specificscavengers or metal chelators to semen extenderscan improve survival time and motility of bovinespermatozoa (1) and in vitro fertilising ability ofram spermatozoa (2) stored in liquid phase. In thisstudy, the antioxidants superoxide disniutase(SOD) and catalase (CAT) were included in thediluent in an attempt to maintain the fertilisingcapacity ofram spermatozoa during liquid storage.Fig. 2: Effect of storage time and diluent onpregnancy rate (%) after insemination with liquidstoredram semen.IITGY.TGY+SO().j.CATMATERIALS AND METHODS:AAO activity was measured in washed ram (R) andbull (B) spermatozoa. The spermatozoa were killedby freeze-thawing (3x) and O 2consumption wasmeasured in 5ml of 2.9% citrate (pH 6.9) containing30 mM phenylalanine and 250 million ofspermatozoa. The AAO activity of live (L) and dead(D) spermatozoa of both species (R & B) wasmonitored by assaying AAO activity in populationscontaining known proportions of D and L sperm inRSD-l and in 2.9% citrate.AAO activity was calculated from 02 consumptioncurves as shown in Figure 1 (see figure & legend).MO activity (uI02/hr/250 mil. sperm)SO -----------")5040302010oRSD-160 80 100Proportion of dead sperm (%)MATERIAL AND METHODSPooled semen collected by artificial vagina from 4rams was diluted 4-fold with Tris-glucose-yolk(TOY) diluent (Tris-3oo mM, citric acid 94.72roM, glucose 27.78 mM, kanamycin 0.07 mg and20 % egg-yolk) or TOY + SOD (800 i.u.lml) +CAT (200 i.u.lml). The diluted semen was storedat 5°C for 9 hr (Day 0, control), 7 and 14 days andassessed for progressive motility of spermatozoabefore insemination (Fig. 1).--+-TGY---TGY+SOD+CATo 7 14STORAGE TIME (DAYS)RESULTSChi-square analysis showed that pregnancy rateswere higher after insemination with control sementhan with stored semen (P

4IS PERI-OESTROUS INTRAVAGINAL IMPEDANCE AN AID IN SCREENING FORPROSPECTIVE PREGNANT RECIPIENT COWS?A.R. Lehrer 1 ,* and W.H. McMillan 2lInstitute of Animal Science, Agricultural Research Organization, P.G.B. 6, Bet Dagan, Israel;2AgResearch Ruakura, P.B. 3123, Hamilton, New ZealandINTRODUCTIONConception rate of inseminated cows (l) and embryo yieldand quality in donor cows (2), are inversely related withperi-oestrous intravaginal impedance (IVI). No suchinformation exists on recipient cows. The main objectiveof this study was to determine the pregnancy rate (PR) ofrecipient cows relative to their IVI during peri-oestrus andon the day of embryo transfer (ET).EXPERIMENTAL PROTOCOLOestrus was synchronised in 36 mixed-age, non-lactatingbeef cows using a CIDR® (3). CIDR® removal was on d 0and IVI was measured once daily on d 0, 1, 4 and 9, andtwice daily on d 2 and 3. A probe developed andmanufactured at the first author's Institute was used for IVImeasurements. On d 9, a fresh single in-vitro producedembryo was transferred to all oestrous cows (4). PR wasdetermined using return oestrus and ultrasonography ond 35.RESULTS AND DISCUSSIONTwenty eight cows (78%) were in oestrus on d 2-4, with21 (75%) detected on the morning of d 2. PR was 13/28(46%). No IV! differences between pregnant and nonpregnantrecipients were significant, other than IVI on theafternoon ofd 2, i.e. -55 h after CIDR® removal, which waslower in pregnant compared with non-pregnant cows(36.1±1.8 vs 41.7±1.4 ohms, P

.'1"4AUTHOR INDEXAlmahbobi,G 84Anderson,S.T. . 5Ankersmit,A.E.L. . 17, 30Arcellana-Panlilio,M 106Armstrong,D.T 15, 25, 36Ashman,R.J 31, 32, 33Aspden,W.J. . , 10Athanasis-Platsis,S 107Atkins,R.C 70Bailey,J.L 90Baker,H.W.G 20Barker-Gibb,M.L. . 1Beaumont,S 110Bell,E.C 45, 46Bergfeld,E. . · ··· 10Boatman,D.E 105Bonello,N. . 66Boublik,J.H. . 1Bourne,H 20Bradley, M.P ~ 88Brannstr5m, M. . 66Breed,B 98Brennan,A.P. . 26Broomfield,D. . 72Brown,B.W 34Brownlee,A.G 104Buab()()Cha,W. ..··.. ·· ·· 101Camus,M 23Cann,C.H 55Cash,M.P 78Catt,J.W 22, 80Cavanagh,A.C. . 102, 107, 108Chairussyuhur, A 114Cheah,C 33Clarke,I.J. . 1, 6, 7Clay, C.M 14Clements,J. . 57Clulow,J 7.1Cox,D.B 47Crawford,R.J 33Curlewis,J.D. . 2, 3D'Occhio,M.J 10, 73Dai,Y.F 36Day, A.M 74~e Kretser,D.M 7, 63, 65, 82, 84, 85, 86g~~:~i::''',~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ::J~~~~~~~:A:··:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::~~g~~~~~~~:~:.::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ~~::~!~:;;d~:D:R'" .:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::j b~Ehrljch,A 57Ekpe,G 87Entwistle,K. . 75AUTHOR INDEX (cont)Eppleston, J. . 111Evans,G 52, 116Fairclough,R.J 38, 55Fawcett,A.A. .. 34Findlay,J.K 4, 8,24,37,50,59,60Fisher,J.S 48Fitzpatrick,L.A. . 12, 75Fordyce,G 75Fry,R.C 24, 61Garcia,S 8Gardner,D.K. . 53Gehring,H 45, 46Gemmell, R.T 101Gidley-Baird, A 88Giliarn,K. . 35Glencross, B.D 79G()()(ien, J.M 9Gow,C.B 55Graharn,J.K. .. 51Grigg,G 87Grupen,C.G 18, 33Hamernick, D.L. . 14Hansen,L.A. . 71Harari,O. .. 20Harding,M 33Hariadi,M 13, 72Harris, M 83Harry,J.L. . 68Hartmann,P.E 47, 79Harvey,M.B 106Hayes,R 70Hayes,T 86Healy,D.L. . 60Hearn,M 65Hedger,M.P. . 70Hemsworth,P.H 11Hillard,M.A ' 5Hillyard,N.C. .. 108Hinds,L.A. . 88Hoogkinson,S.C. . 56Holden,C.A. . 26Hosie,M.J 42Hotzel,M.J 48, 76Irvine, B.J ~ 15Jackson,A.E. .. 77Janssenswillen,C. . 23Jenkin,G 38Jensen,K. . 115Jiang,F.-X 81Jillella,D 73Johnston,W.H. . 20Jones,G.M 20Jones,R.C 71, 89Joris,H 23Kashiwazaki,N 31, 32, 33Kaye,P.L 96, 97, 102, 107Kent,J.C 47Kern, S 67, 69

AUTHOR INDEX (coot)Kim,Y.H 84, 85Kinder,J.E 10King,J.C 80Kleemann,D.O 44Klein,R 6Knott,L.M. . 77Kotaras, P 15Lacham-Kaplan,O. . 21Lane,M 53Lash,G 109Laslett,A 65Lavranos,T.C 64-I..ean, I.J. . 9I..edgard,A.M 56ugge,M 109I..ehrer,A.R. . 117Lohuis,H. . 11Loveland,K 63, 86Lui,J 23Macmillan,K.L. . 49, 74Maddocks, S 67, 69, 87,114Magnoni,G.E 105Mahaworasilpa,T. . 35Marsh,M.M 57, 60Marshall,J.T.A. . 104Martin, G.B 48, 76Martin, L. . 3, 39Massa,H 39Mate,K.E 93, 99Mathews,P.M. . 20Maxwell,W.M.C 27,51,52,111,116McDonald,M. . 20McDougall,S 49, 74McFarlane, J.R 65, 84, 85, 86McGowan,L.T 16, 17,30Mcllfatrick,S.M. . 33McMillan,W.H. . 19, 117Meier,S : 38Mercer,J.E 4, 8ti~~f:;, ~~~~ ~~~~ ~~ :~:~~: ::~~ ~~ ~~ ::~::::~ :~~: ~~:~:::::::::::::::::::::::::::::::::::::::::::::::~ ~~~~:~:::~ ~~ ::~~:~~::~ ::.~Molan,P. . 113Molinia,F.C 28Morton,H. . 102, 107, 108Morton,M. . 22Moss, G.E 14Munday,R 115Murdoch, R.N 40, 89Murphy,C.R 41, 42, 43Murphy,R.M 108Nagashima,H 18, 31, 32, 33Nancarrow,C.D. . 104Nemaia,F.A 17Niasari-Naslaji,A. . 73Nikolics-Paterson,D.J. . 70Norman,R 66Nottle,M.B 18, 31, 32, 33O'Brien,G.M. . 3AUTHOR INDEX (coot)O'Brien,J.K. . 52O'Connor,A 62O'Grady,J 34O'Neill,C 80, 103O'Shea,T 5Oliver,J 110Owens,P.C 36Owens,R.A 47Pantaleon,M. . 96Parr,J 110Parr,R.A. . 78Pedersen,R.A. . 93, 99Perks,C.M 54Peterson,A.J. .. 19, 56Philip,C 13Pitt,C 112Pomares,C.C. . 111Prendergast,E. . 113Pugh,P.A. . 16, 19, 30Rabiee, A.R. . 9Renfree,M.B 68, 100Rhooes,S.L 116Richings,N.M. . 20Rigby,R.D.G 76Riley,S.C 37, 59Risbridger,G 63, 65Roberts,C. . 98Robertson, D.M 6, 50Robertson,S.A 58, 66Robins,A.J 33Robinson,E.S 99Robinson,S.J. . 51Rooger,J.C. . 28, 83, 91, 92Roogers,R.J 64-Roogers,H.F 64Rowe,J.P. . 15Russell,D.L. . 50Rutledge,J. . 25Ryan,J.P 52, 94, 95Sakoff,J.A. . 40Salamonsen,L.A 37, 57, 59, 60, 61Sanchez-Partida, L.G 27, 114Saunders,D 22SawangjarClen,K. . 2Schultz,G.A ,106Scott,K. . 64-Seamark,A.C 58Seamark,R.F 36, 44, 58Setchell,B.P 27, 87, 114Setiadi,D 91, 92Shannon,P 112, 113Shaw,G 45, 46, 68Shaw,T.J 41Shorey,C.D. . 43Short,R.V 81Simorangkir,D.R 82Simpson,T.L. . 24Sistina,Y 91

AUTHOR INDEX (cont)Smith,J.F ~ 16, 110, 115Smitz,J. . , 23Somooevilla Torres,M. . 108Squires,E.L. . 51Squires,T.J 24Stein-Oakley,A. . 63Stevenson,K.R. . 54Stobart,S.J. . 12Stojanov,T 111, 116Storey, B.T. . 90Sutton,R. . 34, 55Tatham,B. . 35Taufa, V.K 74Temple Smith,P.D 84, 85Terry,V 43Tervit,H.R. . 16, 17, 30, 56Tian,L 80Tilbrook,A.J. . 7, 11Toussaint,J.K 79Trewhella,M.A 38Trigg,T.E 77Trounson,A 21, 35Tshewang,U 77Tuckey,R.C. . 79Turner,A 11Udy,G.B 17Upreti,G.C 110, 115Van Steirteghem,A 23VanKirk, E.L. . 14Vella, C.A 64Villar, A.J 93Vishwanath,R 112, 113, 115Vogiagis,D. . 61Waite,K.M. . 52, 94, 95Walkden-Brown,S.W 48, 76Walker,S.K. . 44Ward,W.G 34Waters,J.M. . 78Wathes,D.C 54Watson,A.J :.25Whitworth,D.J 68, 100Whyte, T.R 10Wong,E 37Wreford,N.G.M 62, 82Wright,P.J 13, 72Xia,P 25Young,S 88Zhang,X.Q 106Zlatic,K 63, 86Zupp, J.L 27, 87, 114A