&. u - the Society for Reproductive Biology

&. u

The Australian Society for Reproductive Biology
Twentieth
Newcastle University
29-31 August 1988
Programme and Abstracts of Papers
Copyright Australian Society for Reproductive Biology, 1988
ISSN 0705-6044
CONTENTS
Personnel
Acknowledgements.
Programme Guide
Programme
Author Index.
Abstracts.
iii
iv
v
vi-xx
xxi-xxvii
1-108

AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY
August, 1988
Chairman
Secretary
Treasurer
Committee Members
OFFICE BEARERS
Professor B. Setchell
Dr G. Evans
Dr L.A. Hinds
Dr B.M. Bindon
Dr I.J. Clarke
Dr L. Martin
Dr C.D. Nancarrow
Dr J.N. Shelton
Postgraduate Student
Representative
Mr H.J. Jabbour
PROGRAMME COMMITTEE
Chairman
Committee Members
Ass:x::. P.r:ofessar G.M. Stale
Dr D. Handelsman
Dr B. Miller
Dr R. Scaramuzzi
Dr C. Tsonis
LOCAL ORGANISING COMMITTEE
Dr J.R. Rodger
Mr J. Clulow
Dr R. Jones
Convenor
Dr R.Murdoch
Dr T. Roberts
iii

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AUSTRALIAN SOCIETY OF REPRODUCTIVE BIOLOGY
PROGRAMME 1988
Session 2 (Poster) : DEVELOPMENTAL BIOLOGY
chairman: Professor R. Wales
Time: 1100 - 1230 Venue: Function Room 3
REGISTRATION: The registration desk will be located at the top of
the Main Entrance Stairs (middle floor) of the Town Hall and will
be open from 1530-1800 on Sunday, 28 August and 0830-1200 on
Monday, 29 August.
The Goding Lecture will be presented in the Concert Hall and oral
sessi~ns will be in Function Room 2. Posters will be displayed in
Funct~on ~oom 3. All poster presentations will be in Function
Room 3 except for Session 12 where the presentation will be in
Function Room 2.
5
6
S.A. McKay and A. Lopata
The h.lock t:o po.lyspeI111.:ic fert:.:i.l.:izat:.:ion .:in mouse oocyt:es
P.L. Kaye
Ion.:ic requ.:ire.ment:s of g.lyc.:ine t:ransport: .:in mouse emh.zyos
7 E. De Luca and A. Lopata
Session 1 (Oral) FERTILITY
Chairman: Dr G. Evans
Time: 0930 - 1030
Monday 29 August
Venue: Function Room 2
1 P.A. Hamilton, 1.0. Killeen and J. Reeve
2
Effect:s of met:hod of freez.:ing ram semen and dose of PHSG on
fert:.:i.l.:it:y of ewes .:inse.m.:inat:ed .int:o t:he ut:erus
J.F. Smith, J.N. Clarke, D.L. Johnson and L.T. McGowan
D.:ifference het:ween t:wo genet:.:ic .l.:ines of Romney ewes,
se.lect:ed for .increased fecund.:it:y, .:in t:he.:ir ovu.lat:ory
response t:o PNSG
Effect: of an .:insu.l.:in/t:ransferr.:in/se.len.:it:e supp.le.ment: on DNA
synt:he~ ..is .:in mouse emh.zyos cu.lt:ured .:in v.:it:ro
8 I. Bruck and J.H. Hyland
Ana.lys.:is of t:he cont:r.:ihut:.:ions of t:he Emdben-./t:feyerhoff and
pent:ose-phosphat:e pat:hways t:o g.lucose met:aho.l.ism hy s.1:ng.le
equ.:ine e.m.b.zyos .:in cu.lt:ure
9 A. Szell and J.N. Shelton
10
Osmot:.:ic response of sheep and cat:t:.le emhiyos t:o peI111eat:.:ing
c.zyoprot:ect:ant:s
C. Herr, N. Holt, and K.C. Reed
Effect: of sucrose and ca.lc.:ium .:in t:he sp.l.it:t:.ing med.:ium on
surv.:iva.l of quart:ered ov.ine moru.lae
11 M.B. Harvey and P.L. Kaye
3
A.J. Ritar, P.O. Ball, P.J. O'May, T.M.
and N. Murray
Black, R.B. Jackson
AnahoLic effect:s of .I."nsu.l.:in
med.:iat:ed v.:ia .I."nsu.l.:in recept:ors
on mouse h.last:ocyst:s are
Superova.lat:.1:on response and e.m.b.zyo recove.zy from cashmere
and angora does aft:er t:reat:ment: w.:it:h FSH (fo.l.lt:roph.:inJ
12
J.L.Clarkson and C.D. Nancarrow
4
J.G.E. Thompson and G.W.
Asher
Superova.lat:.:ion and ova recovery .:in faI111ed fa.l.low deer (Dama
damaJ
13
Compar.:ison of t:rophob.last:.:ic ves.:ic.le and ov.:ine b.last:ocyst:
secret:ed prot:e.:ins and ana.lys.:is of ves.:ic.le met:abo.l.ic
act:.:iv.:it:y.:in v.:it:ro
A..Lopata, P.M.
Summers and J. P. Hearn
vi
Cont:ro.l of t:he ovar.:ian cyc.le .in maI111oset: monkeys for .:in
v.:i t:ro and .:in v.I.'vo fert:.:i.l.:izat:.I.·on and t:he t:ransfer of
vii

viii
ix
14
cu.ltured embryos to synchron.ized rec.ip.ients
M.J. Sinosich, S. Lanzendorf, M.D. Bonifacio, D.M. Saunders
and G.D. Hodgen
IJl1Illunof.luorescent stud.ies of pregnancy-assoc.iated e.lastase
.inh.ib.itor (PAEI) express.ion by act.i.rrated gametes and
pre.i.mp.lantat..ion hamster embryos
15 Y.C. Smart, L. Adamson, R.A. Wilcox and T.K. Roberts
Re.lease of qrowth factor(s) by embryo der..ived p.late.let
act.:ivat.:ing factor (EPAF)
III
16 L. Adamson, J.D. Stanger, Y.C. Smart and T.K. Roberts
PAF-Pretreat:ment ..impa.1.'rs embryon..ic deve.lopment
21
22
23
P.Pollanen and B.P. Setchell
The pe.I7lleab.i..l..ity to qamma-q.loba.l.i.n of the b.lood vesse.ls of
the rat test.i.s around the t..ime of puberty
A.J. Tilbrook, D.B. Galloway, A.H. Williams, R.C.
Oppenheim, W.J. Thiel and I.J. Clarke
Treatment w..ith a GnRH agon.ist de.lays reproduct..ive
deve.lopment .1.'n ram .lambs
C.J. Carati, K.E. Creed and E.J. Keogh
Hechan..isms of pen..i.le erect.1.'on .i.n
the doq
17 P.A. Batt, A.W.M. Cameron, D. Sakkas and A.O. Trounson
In v.:itro cu.lture of goat
embryos
SESSION 4
Chairman:
(Poster) PITUITARY HORMONES, INHIBIN AND OVARY
Dr J.K. Findlay
Time: 1530 - 1700 Venue : Function Room 3
Session 3 (Oral) : MALE REPRODUCTIVE FUNCTION
Chairman: Professor D. de Kretser
Time: 1330 - 1500 Venue Function Room 2
24 R. Bathgate, C. Sernia and R.T. Gemmell
Ident..if.i.cat.i.on by HP.LC and ..immunocytochem.i.stry of
hypotha.lam.:ic oxytoc.1.·n and mesotoc.1.'j] .:in the brashta.1.·.l possum
25 P.R. Lewis, M.B. Renfree and R.V. Short
18
Qihan Dong and D.J. Handelsman
Va.l.1.'dat.:ion of methodo.loqy for study of pu.lsat..i.le .LH
secret.:ion ..in the rat: cannu.lat.i.on route, samp.l.:inq
.:intens.1.'tyand durat.:ion
26
The re.lat.1.·onsh.1."p of breastfeed.ing patterns to the .lenqth of
.lactat..iona.l amenorrhoea and ovar.ian .i.nact..iv.1.·ty post partum
Qi Fa Wang, P.G. Farnworth, H.G. Burger and J.K. Findlay
19
A.E. Drummond, G.P. Risbridger and D.M. de Kretser
The ro.le of .Leyd..iq ce.l.ls ..in the requ.lat.i.on of .:inh.1.·b..in
product.1.'on
27
Inh.1.·b.i.tory effect of pure 31 kDa Bov.ine .1.·nh..ib..in on GnRH­
..induced up-requ.lat.1.·on of GnRH b.i.nd..inq s..ites ..in cv.ltured rat
anter..ior p..itu.1.'tary ce.l.ls
Zhang Zhiwen, J.K. Findlay, R.S. Carson, A.C. Herington and
H.G. Burger
20
J.A. Spaliviero, D.M. Robertson, E. Kidston, P.F. Hall and
D.J. Handelsman
H..igh.ly po.lar..ized secret.1.·on of .i.nh.:ib..in by ra t Serto.l.i. ce.l.ls
..in tw..in-chamber cu.lture system
28
Transform.1.·nq growth factor B enhances basa.l and FSH
st..imu.lated .inh..ib..in product..ion by rat: qranu.losa ce.l.ls ..in
v.i.tro
B.M. Bindon, T. 0' Shea, K. Miyamoto, M.A. Hillard, L.R..
Piper, R.D. Nethery and G. Uphill

viii
ix
cu.ltured embryos to synchron.ized rec.fp.fents
21
P.Pollanen and B.P. Setchell
14
M.J. Sinosich, S. Lanzendorf, M.D. Bonifacio, D.M. Saunders
and G.D. Hodgen
The permeab.f.l.ity to gamma-g.lobu.l.fn of the b.lood vesse.ls of
the rat test.fs around the t.fme of puberty
15
Immunof.luorescent stud.fes of pregnancy-assoc.fated e.lastase
.fnh.fb.ftor (PAEI) express.fon by act.fvated gametes and
pre.fmp.lantat.fon hamster embryos
Y.C. Smart, L. Adamson, R.A. Wilcox and T.K. Roberts
22
A.J. Tilbrook, D.B. Galloway, A.H. Williams, R.C.
Oppenheim, W.J. Thiel and I.J. Clarke
Treatment w.fth a GnRH agon.fst de.lays reproduct.ive
deve.lopment .in ram .lambs
Re.lease of growth factor(s) by embryo der.fved p.lat:e.let
act:ivat.fng factor (EPAF)
,~l
23
C.J. Carati, K.E. Creed and E.J. Keogh
Hechan.fsms of pen.f.le erect.fon .fn the dog
16 L. Adamson, J.D. Stanger, Y.C. Smart and T.K. Roberts
PAF-Pretreat:ment .fmpa.irs embryon.fc deve.lopment
17 P.A. Batt, A.W.M. Cameron, D. Sakkas and A.O. Trounson
In v.ftro cu.lture of goat emb.ryos
SESSION 4
Chairman:
(Poster) PITUITARY HORMONES, INHIBIN AND OVARY
Dr J.K. Findlay
Time: 1530 - 1700 Venue Function Room 3
Session 3 (Oral) : MALE REPRODUCTIVE FUNCTION
Chairman: Professor D. de Kretser
Time: 1330 - 1500 Venue Function Room 2
24 R. Bathgate, C. Sernia and R.T. Gemmell
Ident.if.fcat.ion by HP.LC and .fmmunocytochem.istry of
hypotha.lam.fc oxytoc.fn and mesotoc.in .in the brushta.i.l possum
25 P.R. Lewis, M.B. Renfree and R.V. Short
18 Qihan Dong and D.J. Handelsman
Va.l.fdat.fon of methodo.logy for study of pu.lsat.f.le .LH
secret.fon .fn the rat: cannu.lat.fon route/ samp.l.fng
.fntens.fty and durat.fon 26
The re.latJ:onsh.ip of breastfeed.ing patterns to the .length of
.lactat.iona.l amenorrhoea and ovar.ian .inact.iv.ity post partum
Qi Fa Wang, P.G. Farnworth, H.G. Burger and J.K. Findlay
19 A.E. Drummond, G.P. Risbridger and D.M. de Kretser
The ro.le of .Leyd.Ig ce.l.ls .fn the regu.lat.fonof .fnh.fb.fn
product.ion
Inh.fbJ.·tory effect of pure 31 kDa BovJ.·ne .inh.fb.fn on GnRH­
.fnduced up-regu.lat.ion of GnRB bJ.·nd.fng s.ftes .fn cu.ltured rat
anter.for p.ftu.ftary ce.l.ls
27 Zhang Zhiwen, J.K. Findlay, R.S. Carson, A.C. Herington and
H.G. Burger
20 J.A. Spaliviero, D.M. Robertson, E. Kidston, P.F. Hall and
D.J. Handelsman
H.fgh.ly po.lar.fzed secret.ion of .fnh.fb.fn by rat Serto.l.f ce.l.ls
.fn tw.fn-chamber cu.lture system
28
Transform.fng growth factor B enhances basa.l and FSH
stJ.·mu.lated J.·nh.fb.fn product.ion by rat: granu.losa ce.l.ls .in
v.itro
B.M. Bindon, T. O'Shea, K. Miyamoto, M.A. Hillard, L.R..
Piper, R.D. Nethery and G. Uphill

29
Superovu.lat:.ion .in pubert:a.l he.ifers .immun.ized aqa.inst: ov.ine
.inh.ib.in pur.if.ied by monoc.lona.l ant:.ibody aff.in.it:y
chromat:oqraphy
J.K. Finqlay, I.J. Clarke, H. Quigg, S. Katsahambas, P.
Juhola, M. de Blaslis and B. Doughton
Tuesday 30 August
Session 5 (Oral>: SP~RM AND THE MALE TRACT
Chairman: Professor R.C. Jones
Time: 0830 - 1000 Venue: Function Room 2
Inh.ib.in .in t:he sheep ovar.ian cyc.le
30 R.J. Rogers, S.J. Stuchbery and J.K. Findlay
Lack of express.ion of .inh.ib.in qenes .in ov.ine corpora .lut:ea
38
Yulu Tang, the late D.E. Brooks, A.M. Snoswell and B.P.
Setchell
Ga.lact:osy.lt:ransferase act:.iv.it:y on sperm surface and .in
ep.id.idyma.l p.lasma of severa.l ma.mma.lJ:an spec.ies
31
T. O'Shea, B.M. Bindon, J.K. Findlay, M.A. Hillard, L.R.
Piper and K. Miyamoto
Immun.iza t:.ion of ewes w.i t:h .inh.ib.in prepara t:.ions of
.increas.inq pur.it:y
32 R.A. Parr, I.F. Davis and M.L. Phillips
Inf.luence of shear.inq st:ress on oest:rus and ovu.lat:.ion
33 L.A. Fitzpatrick, T.J. Mullins and G. Fordyce
Pred.ict:.inq fo.l.l.ic.le popu.lat:J:ons .in Bos .ind.icus cows from
surface count:s of oVi1r.ies exam.ined aft:er ovar.iect:omy and by
endoscopy
34 J.D. O'Shea,. R.J. Rogers and M.J. D'Occhio
Ce.l.lu.lar compos.it:.ion of t:he cyc.l.ica.l corpus .lut:eUJ11 of t:he
cow
39 De Yi Liu and H.W. Gordon Baker
Corre.lat:.ion bet:ween spe.I7/1 charact:er.ist:.ics,
hUJ11an .in v.it:ro fert:.i.l.izat:.ion
40 De Yi Liu and H.W. Gordon Baker
zona b.ind.inq and
Corre.lat:.ion bet:ween human sperm morpho.loqy, acrosomes,
acros.in and fert:.i.l.izat:.ion .in v.it:ro
41 F.C. Molinia, P.A. Howe and M.A. Swan
A t:echn.ique of s.imu.lt:aneous.ly est:.imat:.inq forward ve.loc.it:y,
beat: frequency, ,. Live and the nat:ure of f.laqe.l.lar
wavefo.I7lls .in spe.I7/1at:ozoa
42 A.J. Conway, L.M. Boylan, M. Wood and D.J. Handelsman
Sem.inlJ.l p.lasma ret:.ino.l-b.ind.ing prot:ein as an .index of hUJ11an
Sert:o.l.i ce.l.l funct:.ion
35 E-M.A. Bugledich, L.A. Hinds and P.A. Janssens
Seasona.l fact:ors .inf.luence t:he t:.im.inq of f.irst: preqnancy .in
t:he t:ammar
43
\.
D.A.Taggart and P.D. Temple-Smith
Effect:s of mu.lt:.ip.le mat:J:ng on ep.id.idyma.l spe.I1ll d.ist:r.ibut:.ion
.in t:he brow.n marsup.ia.l mouse, A. st:uart:.i.i
36 M.B. Renfree, G. Shaw and T.P. Fletcher
Superovu.lat:.ion ..in a macropod marsup.ia.l, Hacropus euqen.i.i
37 M. Sathanandan, S.K. Walker, I.J. Clarke and C.D. Matthews
The effect: of qonadot:roph.in re.leas.inq hO.I7/1one aqon.ist: on
sheep p.it:u.it:ary and ovar.ian funct:.ion
xi

Session 6 (Oral):
Chairman: Dr C. O'Neill
DEVELOPMENTAL BIOLOGY
Session 8 (Poster):
Chairman:
Dr L.A. Hinds
UTERUS AND PREGNANCY
Time: 1030 - 1200 Venue: Function Room 2 Time: 1400 - 1530 Venue: Function Room 3
44 J.M. Shaw and A.O. Trounson 50 T.P. Fletcher and D.R. Blandon
U.ltrarap.:id freez.:ing of 2-ce.l.l mouse embryos
45 L.J. Wilton and L. Diotallevi
U.ltra-rap.:id cryopreservat.:ion of 8 ce.1.l mouse embryos w.:ith a
punctured zona pe.l.luc.:ida
46 G. ,Somers and L.J. wilton
A.l.locat.:ion of ce.l.ls to .:inner ce.l.l mass and trophectoderm .:in
mouse embryos b.:iops.:ied at the 4-ce.l.l stage
47 J.P. Ryan, N.R. Spinks, C. O'Neill and R. G. Wales
51
52
The ant.igest:agens RU486 and .3K2,g,g are not hound by the
uter.ine progesterone receptor of the tammar wa.l.laby"
Hacropus euqen.:i.i
R.A.
Cherny and J.K. Findlay
Prote.in secret.ion patterns of separated ov.ine endometr.ia.l
ce.l.ls cu.lt::ured .in dua.l env.ironment: chambers
N.R. Spinks and C. O'Neill
The effects of a spec.if.icp.late.let act.ivat.ing factor (PAF)
recept:or ant:agon.:ist (SRI 63441) on some aspects of materna.l
phys.io.logy
Illlp'.lantat.:ion potent.:ia.l and foet:a.l v.:iab.:i.l.:ity of embryos
cu.ltured .:in the presence of p.lat:e.1et actJ:vat.ing factor
53
R.J. Fairclough, T.M. Lau, L.G. Moore, A.J. Peterson and
W.B. Watkins
48
49
D. Sakkas, P. Batt, A. Cameron and A.O. Trounson
In v.itro deve.lopment of goat: pre.:imp.lantat.:ion embryos .:in
cocu.lture w.:ith ov.iduct ep.:ithe.l.ia.l ce.l.ls
S.K. Walker, R.F. Seamark, P. Quinn, G.M. Warnes, R.J.
Ashman, D.H. Smith and P. Ancell
Pregnancy .inh.ibits oestradJ.·o.l .:induced re.lease of uter.:ine
prostag.1and.in Fpand oxytoc.in-neurophys.in .:in the ewe
54 B. Williams and R.N. Murdoch
Resp.:irato.ry propert:J.·es of the mouse ut:erJ.·ne endomet:r.:ium
durJ.·ng ear.ly post:-J.mp.lant:at:.lon pregnancy
V.:iab.:i.l.ity of pronuc.lear embryos of sheep after cu.lture .in
v.itro for one" three or fJ:ve days 55 C.S. Pow and L. Martin
DJ.·str.ihutJ.·on of oestrogen receptors J.·n the fe.ma.le
reproduct.ive t:ract of the f.lyJ.·ng fox Pt:eropus scapu.latus
Session 7:
Chairman:
JAMES GODING MEMORIAL LECTURE
Time: 1200 - 1300 Venue: Concert Hall
Professor B. P. Setchell
56 P.A. Towers and L. Martin
Effect:s of gonadotrophJ.·ns on fo.l.l.ic.le deve.lopment dur.:ing
pregnancy J.·n t:he f.ly.ing fox Pteropus scapu.latus
Professor F.TY. Dazer: ESTABLISHNENT OF PREGNANCY IN SHEEP AND
PIGS
57 R.G. Alders and J.N. Shelton
Ov.ine uter.ine .lymphat.ics: J.·n v.ivo passage of IndJ.·a J.·nk
from ut:er.:ine subserosa" myomet:rJ.·u.m and .lumen
xii

58 F. Abdi and I. Pollard
The effect of pregnancy on the rate of the
b.iot:ransfo.r.mat.ion of caffe.ine
Wednesday 31 August
Session 11

69
R. Vishwanath, I.G. White,P.D. Brown-Woodman and J.R.
Mercer
Ant.ifert.i.l.ity act"iv1."ty of gossypo.l .in ma.le rats and
manganese status of d.iet
78
.M.J. D'Occhio and D.R. Gifford
P.itu.itary and ovar.ian responses of post-partum acyc.l.ic beef
cows to cont.inuous .lonq-t:entl treatment w.ith GnRH' and a GnRH
aqon.ist
70
J.L. Zupp and B.P. Setchell
79
W.M.C. Maxwell, S.K. Walker, D.H.
Smith and H.R. Wilson
Pro.lonqed effect of subfert.i.le ma.les .in reduc.inq numbers of
fetuses 1."n no.rma.l fema.le rats
Effect of GnRH' on fert.i.l.ityof Ker.ino ewes .insem.inated w.ith
frozen semen
71
B.C. Jefferies, B.P. Setchell and W.M.C. Maxwell
Fert.i.l.ity of ewes mated to Ner.ino rams from ·d.ifferent
sources
72 J.L. Reeve
Durat.ion of proqestaqen pr.im.inq on the response of Border
Le.icester X Jlfer.ino ewes to the 'Ram Effect /'
73 S.R.D. Sutherland and K.W. Entwistle
80
D.O.
D.H.
Kleemann, S.K. Walker, J.R.W. Walkley, R.W. Ponzoni,
Smith and R.F. Seamark
Factors .inf.luenc.ing .lamb surv1."va.l 1."n a h.iqh fecundJ."ty
Booroo.la X South Austra.l.ian· Ner.ino f.lock
81 A.W.N. Cameron and P.A. Batt
PNSG may d1."rect.ly st1.Jnu.late ova.lat.ion .in goats
82 B.M. Bindon, J.K. Findlay, L.R. Piper and M.A. Hillard
EffJ."cacy of me.laton.in .imp.lants (Regu.lJ."n)
cows
.in Bos .ind.icus
Ova.lato.ry potency' of ASRB-bFSH-1 .in sheep and catt.le
74 C.R. Earl, R.H. Male and E.A. Dunstan
Synerg.ist.ic effects of me.laton.in and .immun.isat.ion aga.inst
androstenedJ."one .in ma.iden BIt x JIf ewes
75 K.P. Croker, M.A. Johns and L.D. Staples
Session 13 (Poster): MALE 'R.EPRODUCTIVE FUNCTION
Chairman: Dr E.J. Keogh
Time: 1030 - 1230 Venue: Function Room 3
Use of Requ.l.in me.laton.in .imp.lant:s 1."n conjunct1."on w.ith
teas.inq of Jlfer.ino ewe f.locks jo.ined .in spr.ing
76 P.J. Wright, A.H. Williams and LJ. Clarke
Phe effects of nutr.ient restr.ict.ion and of .lamb remova.l on
ovar.ian cyc.l.ic.ity and on the .inh.ib1."tory effects of
oestrad.io.l on p.lasma concentrat.ions of LH and FSH 1."n postpartum
ewes
83
84
S. Wibullaksanakul and D.J. Handelsman
Gonadotroph.in-re.leas.ing hOntlone re.lease from med.io-basa.l
hypotha.lamus .in vIt:ro~ .va.lJ."dat:.ion of met:.'Jodo.loqy
D.J. Handelsman and R. Lazarus
Kat:hemat:.ica.l mode.l and camput:er s1.Jnu.lat:.ion of pu.lsat:.i.le
hOI11lonesecret:.ion w.it:h app.l.icat:.ion t:o LH
77 G. Fordyce, T.J. Mullins, C.A.J. Ridd and K.W. Entwistle
Phe ro.le of the p.itu.itary .in post-partum nutr1."t.iona.l
anoestrus .in cows
85 S.S. Raychoudhury, M.G.· Irving and A.W. Blackshaw
Phe effect:s of Serto.l.i ce.l.l matr1x/ fet:a.l ca.lf serum and
Serto.l1." ce.l.ls on JH-t:hym.id.ine 1."ncorporat:ed .int:o DNA of
cu.lt:ured myo.i-d ce.l.ls
xvi
xvii

86 G.F. Gonzales, J. Muir, G.P. Risbridger, and D.M. de'Xretser
Effects of seroton.in on .inh.ib,in and testosterone product.ion
by'adu1t rat' sem.in.iferous tubu1es and, Leyd.ig ce11s .in v.itro
94 L.K.-P. Leung and J.M. Cummins
Norpho1ogy of .immature speDTIatozoa of the Ch.inese pango.l.in
(Nan.i$ pentadacty.la : Phol.idota)
87 J.R. Ford, I.W. Purvis and G.B. Martin
88
.Effect of the Booroo1a F gene on p1asma FSH .in ent.ire,
c.ryptorch.id and castrate riflJl/1ambs
R.T. Gemmell and C. Sernia
Immunocytoche.m.ica1 1ocat.ion of oxytoc.in and mesotoc.in
W'.ith.in the hypotha1iH11us and reproduct.ive tract of the ma1e
marsup.ia1 badd.icoot
95.
96
M.. Lin, R.C.Jones and A.W. Blackshaw
phe cyc1e of the sem.in.iferous ep.ithel.ium of the Japanese
qua.i1, Coturn.ix coturn.ix
R. Vishwanath, I.G. White and S.A. Matlin
89 C.R. Earl, E.A. Dunstan and M. Schleuniger
Phe effect of day 1ength treatment on the reproduct.ive
perfoDTIance of Border .Le.:lcester rams 97
Effect of racem.ic gossypol and .its .isomers on surv.iva.l of
riH11 speDTI and on react.ivat.ion of speDTI models
5. Sujarit, G. Chaturapanich, M. Lin, R.C. Jones, B.P.
Setchell and G.M. Stone
Does rete test.isf.lu.id conta.in a
secretagogue
98 S. Sujarit, R.C. Jones, M. Lin, B.P. Setchell and G.M.
Stone
Session 14 (Poster): SPERM AND THE MALE TRACT
Chairman: Dr H.G. Baker
Time: 1300 - 1400 Venue: Function ROOm 3
90 D.P. Windsor and I.G. White
Regulat.J:on of the .J."n.it.ia.l segment of the ep.id.idym.is
99 S.J. Gatie, A.W. Blackshaw and T.D. Glover
Phe effects of b.ilatera1 castrat.ion and ethy.lene d.imethane
su1phonate (EDS) on ep.id.idyDlt!l1 funct.J.'on (speDTIatozoal
maturat.ion) .in gu.inea-p.ig
91
Effect of phosphat.idy1ser.ine on ca1c.ium uptake of co1d
shocked boar and riH11 speDTIatozoa
P.A. Howe and M.A.
Swan
100 J. Clulow and R.C. Jones
F1u.id and so.lute f1uxes .in the gen.ita.l ducts of the ma1e
Japanese qua.i1
92
Protect.ive effect of phosphat.idy1cho1.ine aga.inst co1d shock
.in goat sper:t1/iltozoa
H.N. Jabbour, G. Evans and N.W. Moore
101
G. Chaturapanich and R.C. Jones
Or.ig.in of 1um.ina.l prote.J.'ns .in the ep.id.idym.is of the tammar,
Nacropus euqen.i.i
Fert.i1.isat.ion .in superovu1ated ,ewesfo11oW'.ing .lnsem.inat.ion
W'.ith d.ifferent doses of fresh or frozen-thawed semen
93 G.M.. O'Bri~n, J. Clulow and R.C. Jones
Separat.ion of human speDTI for e1ementa1 ana1ys.is
102
P.O. Temple-Smith and G.J. Southwick
Problems .in screen.ing azoospeDTI.ic pat.J.'ents for correct.ive
m.icrosurge.z::y
xix

Session 15

Crane, L.R. . 63
Creed, K.E 23
Crocker, K. P. . .........•....•.....••...•..•..••..••.........•• 75
Cummins, J .M..............•..•....•••.•....•..•.•.....•...•••• 94
Cummins, J.T•...••..•...•.......•.•••.•.•...•..........••.••. 104
Czapala-Peeters, C. . ....••••..•.•.•..•.....'. . . . . . . . . • . • . . . • . .. 66
D'Occhio, M.J. . .•.......•...•.••.••••.•..........•..•.•.•.. 34,78
Davis, I.F•.•.............•..•....•...••........••............ 32
de Blasiis, M. ......•.....•..•••..•......•.••....••.•.....•.•• 29
de Kretser, D. M. ...•.••....•..•...••••...•..•...•••...•.••• 19, 86
de Luca, E.......•.....•..•.............•.•.•.••........ ,••..... 7
Diotallevi, L. . '................................... 45
Dong, Q••••••••••••••••••••••••••••••••••••••••••••••••••••••• 18
Doughton, B. . .....................•.................•.......•. 29
Drake, .B .L. ..•..•..•...••...........•..•..•..•.......•.....••. 64
Drummond, A. E. . ...........•........•.............•..........,.. 19
Dunstan, E.A•..........................•.•........•.••....• ,74.,89
Earl, C.R 74,89
Entwistle, K. W. • 73,77
Evans, G•........................•.•.........•...•.....•...... 92
Evans, J .R. . ............•..... '. . . . . . . . . • . • . . . . . . . . . . . . . . . . . . .. 67
Fairclough, R.J. ........................................•. 53,108
Farnworth, P .G. . ...................•...•.................. 26,103
Findlay, J.K......................•.......• 27,29,30,31,51,82,103
Fitzpatrick, L.A...................•....•.......•............. 33
Fletcher, T.P....................•••..••.•.............. 36,50,59
Ford, J .R. . ..........•.....•..•........•...................•.. 87
Fordyce, G. . ...•.........•........•.•.•..•..........•....'.. 33, 77
Fry, R.C•........'........•.........••..... ' 106
Galloway, D.B......................••..•.....•........•...•... 22
Garcia, J. . .....................•..••....•...••......•.•...•.• 62
Gatie" S. J • •..•••........••.••..•.••..••.•.•....•.•.•••••.•••• 99
Gemmell, R.T. •.............•.....•.•..•.................... 62,88
Gifford, D.R....•.....•'....•'..• '..•....•..•.•...•••........••.••. 78
Glover, T.D......•.........• '•......•.•.•.•..••...•..••..•.•... 99
Gogolin-Ewens, K...........•............•.•............•••.•.• 60
Gonzales, G.F. • ...•.....•...•.......••..•••..•.•.......•.....• 86
Hall, p.F.....•.........•.......•...•••.•.•....•....••.......• 20
Hamilton, P.A.............•.•.•..••..•....•........••.........• 1
Handelsman, D.J. . ......•........••..••••...••...•. 18,20,42,83,84
Harvey, M.B...........•.•.•...•....•••..............•..•....•• 11
Hearn, J.P. • •.•.....•..•.....••.•.•.•••..•......•..••........• 13
Henniawati .•....................•......•.........•........... 107
Herr, C. . .....••..........••.••.•...•••...•.•......••...•....• 10
Herrington, A. C • . .....•.•..•.....•..•••.•••.....•........•.,...' 27
Hillard, M.A. . .•...., ,. 28,31,82
Hinds, L.A. ••...........•.....•.•.•.•..•...•........••..... 35,59
Hodgen, G.D..•.....•......•.•.•.....••..•..•.........•.•.•. 14,65
Holt, N. • •••.•••••,••.••••••••••••••••••••••••••••••••.•••.••••• 10
Howe, P.A.•.........................•..••..•.........•..... 41,91
Howse, A.M. .. ........•.....•...•...•.................••....... 106
Hyland, J .R. . ...........•••.•.......•...•..•..............•.... 8
Irving, M.G...•........................•...................... 85
Jabour, R. N• ••.••..•....•...•••..••.••..•..•••...•....••..•••. 92
Jackson, R.B••...............................................•. 3
Janssens, P.A 35
Jefferies, B. C. . •..........•......•......•......•............. 71
Johns, M.A. . ........•...............................•.....•... 75
Johnson, D.L....................••.........'..•..•......•....•.. 2
Johnson, K.L.....•.....•......•.....•.•.•.......•...•.'•...•.• 106
Jones, R.C...•.........•.....•..•...•........... 93,95,97,100,101
Juhola, P. . ..••.•..•••...•......•.....•.•.......•.•...•...•.•• ' 29
Katsahambas, S. .....••............••.•.....•.•..•...........•. 29
Kaye, P.L.......•.•,.•.........••................•.....•..... 6,11
Keogh, E.J. . ••................•.....•....•...•........•... ~ . •. 23
Kidston, E•..........••...•..........•...•.................... 20
Killeen" I.D.••..•.........••......••....•............•..•..... 1
King" N.J. C. ....•...............•...••..•.........•...•....... 64
Kleeman, D.O....................•...•.......................•. 80
Lam, S-Y. • •••....•.•.•........•..••.•...••••.••.•••••.•.•.•... 67
Lanzendorf, S. . . • . . . • . • • . • • . • . • • . • . . • . • . . . • • . . . . . . . . . . . • . . . . ... 14
Lau, T.M.•.•........................•...•••...........•....... 53
xxii
xxiii

Lazarus, R. • ..•..•.....•..••••....•••.••••..•...•.•.•.•.•.•••'. 84
Lee, J. • ..••••..•....•......••...•.•••.••..••.•.••...•.••.••••• 65
Lee, C.S.. . . 60
Leung, L.K•- P • . .•.........•.....•••.•••..••..••......•...•...• 94
Lewis, P.R. • .•.•.••.•....•...••.••.•...•••.•••...•...•••••••.• 25
Lin, M. •...•..••....••.....•.••.....••••..••.•••.••..••• 95, 97 , 98
39,40
Liu, D.Y•••••••••••••••••••••••••••••••••••••••••••••••••••
Lopata, A. .••.•....•.•...••....•..•.•.....••••.•.•..••.... 5, 7 , 13
Male, R.H. . •....•....•.•...•••.••••..••••.•..•.••..•..•.•..... 74
Martin, G.B. . ...•......•...•..•.•.••••..••••••••...•••.•.•..•. 87
. L ••.••.•••..••••••••••••••••••••••••.••••••.••• 55,56,63
Mart~n, •
Matlin, S .A. . ••.•..•.........•....••...••........•..•.....•••• 96
Matthews, C. D. . •.............•..•.•••••.•..••....•..........•. 37
Maxwell, W.M.C. . .•.............••.••....•.•.....••.•.•.•.•• 71,79
Maxwell, L.E......................••..•.•.•.•.••••••........•• 64
McGowan, L.T•......•..............•.•.•.•...•...•...•.•...•.... 2
McKay, S.A....•..................•.•.••......•..••....•.•.••••. 5
Mercer, J.E..............•.....•...•..•.......•.•...•.•...••. 105
Mercer, J .R. . ...........•........•.•..•..................•..•. 69
Mercer, W.R. . ...........................• 60
~ .•............•.•...
Miyamoto, K. . ...........•.•... 28,31
~ ....•................•......
Molinia, F.C. . .............................•....•.....•....... 41
Moore, L.G. . ....•........•...•...•..•....•.•........•••..••.•. 53
92
Moore, N.W. . '.........•....••..••...••.....•••••
Muir, J .••••.......•..........•.....•...•......•....••..••.... 86
Mullins, T.J.................•...••...•.••..•.•....•.....•. 33,77
Murdoch, 'R.N...............•.......•....•••.•.•...•..•....•..• 54
Murray, N.•..••...........•...•..•.•..••.•...•......••...•.••.• 3
Nancarrow, C. D. . ...•......•.•....•..•..•.•..•......••.••.•...• 12
Nethery, R.D........•...•...•.......•....••.•.•.••.•.....••.•. 28
Nunan, K. • •.•.•.....•....•.••..•....•....•......••.••..••••'... 6 2
0, W-S ...•....•..•.•............•...•.......•........••.•••...
O'Brien, G.M.
. ••........•....•....•....••..•......•..•..•..•.•
O'May, P.J•...... ,; ..•.........•.••....•..•..•.....•.•........••
O'Neill, C....•...••.....•.......•••................•••••.• 47,52
O'Shea, J.D• ..•.............•...•.••••••.•..•..••...•..••.•..• 34
68
93
3
O'Shea, T. ...•........•......••••.•.•.....••.......•....•.. 28, 31
Oppenheim, R.C........•.••..••..•.....•.••........•........•.. 22
Parr, R.A. . .•....••.•••........••••••....•••...•..•........ 32,62
pepperell, R.J....•...•.•...........•......................... 67
Peterson, A.J. . ....••...•.•.•.....•.••....•••....•..•••.••..•. ·53
Phillips, D.J. ..•......••.....••....••.••.......•.•..•....... 104
Phillips, M.L•........•....••.•....••......•.•.•.•...•..•..... 32
Piper, L.R. . ..•••.•..•..••......•..••...•.••..•...•..••. 28,31,82
pollanen, P. •..••.•...•.•.••......•••...•••.....••.•..••...••• 21
pollard, r. . 58
Ponzoni, R.W•.........•..•..•.............••......••.......••• 80
Pow, C.S....•..•....•.•......•....•.••....•..•....•......••.•• 55
Purvis, r.w 87
Quigg, H. . •..........••.•..••......••.....•.......•...••...•.. 29
Quinn, P. . ..•......•....•....•.•....•...•.•.............••...• 49
Raychoudhury, S. S. ......•........•......•.••..........••...... 85
Raymond, S. . .........•.........•........•..................•.. 66
Reed, K. C • ......•............•••..........•..................• 10
Reeve, J. ...............•............•..•........•.....•.... 1 , 72
Renfree, M.B.....•.....................•••........... 25,36,59,68
Restall, B.J..................•.....•...•......•.........•.•. 107
Ridd, C.A.J.....•..•.........•................................ 77
Risbridger, G.P..•..•..........•..•........................ 19.,86
Ritar, A.J. ...........•........•....•..••.............•...•..•• 3
Roberts, T.K..•...•......•.•.......•.............•....•..•. 15,16
Robertson, D.M.•.. ~ ...•.....•..........•...••......•.....••... 20
Rodger, J. C. ............•...•.....••..........•.......•...•.•• 64
Rogers, R.J••.....·......•..........•.................•..... 30,34
Ryan, J. P • ..••....••....•.......•..•.............•...••....•.• 47
Sakkas, D. ............•............•.........•............. 17, 48
Sathanandan, M. .............•.....•....•.............•.....••• 37
Saunders, D.M..•...................•..............•........... 14
Scaramuzzi, R.J.......•.•...•••....•.........•.•.•....••..•.. 107
Schleuniger, M. . .....••..•.....•......................•.•.•... 89
Seamark, R.F..•......•.................••.....•.•.•..•...... 49,80
Sernia, C••.•..............•......••.•..•.........••.•..... 62,88
xxiv
xxv

Setchell, B.P.•..•........•.•••...••••••.....•. 21,38,70,71,97,98
Shaw, G.•.......•..•.•....•...•.•..•.••••.•..........••.. 36,59,68
Shaw, J .M. . ..••................•.•.••••••..........••••.•..•.• 44
Shelton, J.N. •...........•.•••..•..••..•....•......•...•••. .9,57
Short, R.V. •.....•...•.•....••...•..••••••.•.•....•••..•••• 25,68
Sinosich, M.J. . ......•..•...••••.•..•••••.•....•... ~ .•.•••. 14,65
Smart, Y.C..•..•..........••.....•.•.•.•••.••.....•••... 15,16,66
Smith, D. H. ....•..•.•....•..•••••.••••••••.••.....•.•... 490' 79 , 80
Smith, J .K. • •...•.•..•••..••.•.••....•.••.••..•.••.....•.•.••.• 1
Snoswell; A.M. . .•..•.......••••••.•••.••••.••.•......••••..••• 38
Somers, G.....................•............•.•••......•..••••. 46
Southwick, G.J. • ..............•........•.•...••..•......••.... 102
Spaliviero, J .A. . .........•.••...•.•.•...............•.....••• 20
Spinks, N. R. •...•..........•......•...••..........•.•..•... 47,52
Stanger, J. D. ..............•.....•...................•..•.•.•• 16
Staples, L.D. . ...•..................•.........•.....•••..•.••• 75
Stone, G.M...........•............•.•....•..........•...... 97,98
Stuchbery, S.J. . ..•........••.......•.........•.............•• 30
Sujarit, S........................•.•.••.......•..........• 97,98
Summers, P.M. . ............•...............•..........•.......• 13
Sutherland, S.R.D...........• ~ .......•....•................... 73
Swan, M.A. . .•....•..................•........•......••..... ·41,91
Szell, A.............•...............•.......•..............••. 9
Taggart, D.A. . ..•..................•...••......•..•...•••..••. 43
Tang, Y.•........................•...•.......••.....•......••. 38
Temple-Smith, P.D.•..••.•••..••.•.••••.•••.••.•.•••••.••.• 43,102
Thiel, W.J. . ............•.•.......••..•••.••..••.....•..•••••• 22
Thomas, W.G. . .•.........•..••......••..•.•..•..........•...•.• 62
Thompson, J .G.E...................•••....•...•..•...... ,; .•.•..• 4
Tilbrook, A.J...•......•...•.......••.•..•................•.•. 22
Towers, P.A......•...............•.......•...•....•....•••.•.• 56
Trounson, A.a••......•.....•.......•...•.••....•••.• 17,44,48,108
Tyndale-Biscoe, C.H......•..•.......•••.••.•.....•••..•....•.• 59
Uphill, G.....••..... ~ 28
Vishwanath, R. •......•.••........••.....•...........•.•...• 69,96
Wales, R.G...........•....•...•..•.......•.....•••.•....•. '•... 47
Walker, S .K. • .•.•.•..•.••.......••.....•.••.•.......•...• 37,79,80
Walkley, J .R.W. . ....•...•..........•••.••........•..•......... 80
Wang, Q•F • • .••...••.....••••,.............................. 26,103
Warnes, G.M•.••.•..•.......•...•......•...•.........•......... 49
Watkins, W.B.••...••....•••.•.....•.•.••.......•....•.•....... 53
White, I.G. •.••.....••...•..•.....•.•.•.........•...•... 69, 90 , 96
Wibullaksanakul, S. ..•........•.............•...•...•......•.. 83
Wilcox, R.A. . ••........•...••.•.•....•..•...•..•....•.......•. 15
Williams, R.F•.•....•..•............•..••.•.••.......•.•..••.. 65
Williams, A.H......•.•........•.•..•..•.•....•..••......... 22,76
Williams, B. •...•.......•.............•...•.....•...•.•....•.. 54
Wilson, H.R. • •.....•.......•.....••.....•...•.........••...... 79
Wilton, L.J·•...•••............•..••....•..............•.... 45,46
Windsor, D.P....•.•.......•..'..............•....••..........•. 90
Wolf, J.P•...•....•.....................•..••....•............. 65
Wood, M•.•.....•...•.•.•.•............•.•......•.............. 42
Wooding, P. . ••...•...•...........••.....•....•.. ,;............. 60
Wright, P.J.....•..•...•.•............•..•••................•. 76
Zhang, Z. . .•.•..................•........•..................•. 27
Zupp, J .L...•..........•.........•...•...............•.••..•.. 70
xxv;
xxvii

,;;:as
EFFECTS OF METHOD OF FREEZING RAM SEMEN AND DOSE OF PMSG
ON FERTILITY OF EWES INSEMINATED INTO THE UTERUS.
P.A. HAMILTON1, 1.0. Killeen2 and J. Reeve3
1. Elders Breeding Services, Tongala, Vic.
2. Heriot Agvet, 9 Edina Road, Ferntree Gully, Vic. 3156.
3. Agricultural Research Institute, Rutherglen,Vic.
The effect on fertility of inseminating ewes with semen frozen in straws or
pellets and of dose of PMSG was examined in 4 trials. The Border Leicester X
Merino (Trials I, II and IV and Merino Trial III) ewes were treated with
progestagen intravaginal sponges (Repromap, Upjohn), PMSG (Pregnecol, Heriot
Agvet or Folligon, Intervet) at sponge withdrawal, then 56-64 h later inseminated
intra-uterine, using glass pipettes and a laparoscope.
For each trial semen was collected from three rams and pooled before frezzing
in pellets or 0.25ml straws(l). A tris based diluent was used for the pellets(1)
which contained 200xl06 motile spermatozoa in a final dilution of 1 in 20. TThe
diluent for straw freezing contained 8.5gm trisodium citrate, 6gm fructose, 44g
skim .milk powder, 100mi egg yolk, 60ml glycerol made up to 1000ml with
distilled water. Each straw contained 60xl0b motile spermatozoa in a final
dilution of 1 in 20. Three ewes were inseminated with each pellet and one straw
was used per ewe. Pregnancy rates were determined by real time ultrasound and
presented in Table 1.
TABLE 1 Effect of method of freezing and dose of PMSG on fertility of ewes.
Main % pregnant (no. of ewes) Total
effect Trial I Trial II Trial III Trial IV
Freeze Straw 60(55) 47(53) 65(124) 60(106) 59(338)
Method Pellet 78(54) 49(59) 59( 119) 59(113) 60(345)
Dose 0 53(17) 53(19) 53(36) 30(37) 45(109)
PMSG 200 64(22) 39(18) 58(72) 53(15 ) 56(127)
i.u. 400 76(21) 39(23) 63(71 ) 61(69) 61 (127)
600 62(53) 62(53)
800 85(21) 76(21) 70(64) 73(15) 74(121)
1200 71(14) 40(15) 75(16) 62(45)
1600 57( 14) 38(16) 71(14) 57(44)
The method of freezing the semen had no effect on fertility. Increasing the dose
of PMSG, at least to 800 iou., increased fertility except in Trial II in which the
PMSG had been frozen and thawed after reconstitution, reducing potency.
(1) Evans,G. and Maxwell, W.M.C. Salamon's Artificial Insemination of Sheep
and Goats. Butterworth's, Sydney (1987).

2 3
DIFFERENCE BETWEEN TWO GENETIC LINES OF ROMNEY EWES, SELECTED FOR
INCREASED FECUNDITY, IN THEIR OVULATORY RESPONSE TO PMSG
J.F. Smith, J.N. Clarke, D.L.
Johnson and L.T. McGowan
MAFTech North, Ruakura Agricultural Centre, P.B., Hamilton, N.Z.
The ovarian response to exogenous gonodotrophin can provide a
useful indirect measure of the endocrine differences between genetic
groups (I, 2). This technique was used as part of a study into the
endocrinological and physiological basis for differences in fecundity
of two flocks of Romney sheep selected for high and low incidence of
multiple births (3,4). Ewes were allocated to 4 groups on the basis of
flock, ~election line, age and liveweight. All ewes were treated with
CIDRs for 14 d and joined with vasectomised rams. Pre-treatment
ovulation rate was recorded by laparoscopy 7 d later, 16 dafter CIDR
removal ewes received either 0, 375, 750, or 1500 iu PMSG (Folligon®
Intervet International B.V. Boxmeer-Holland; Batch 376551 and 7 d later
post-treatment ovulation rate was recorded. The pre-treatment
ovulation rates for the Ruakura Fertility flock were high = 1.47±0.06,
control = 1.02±0.04, and low = 1.03±0.04, while for the Hight flock
they were high = 2.04±0.07, and control = 1.64±0.05. The posttreatment
ovulation rates are presented in table 1.
Ovulation rate after PMSG
(mean±s. e .m.)
Flock Line n Dose of PMSG (iu)
0 375 750 1500
Ruakura High 22 1.79±0.12 2.00±0.17 2.09±0.14 3.l9±O.23
Control 19 1. 32±0.13 1. 21±0. 09 1. 7l±0 .18 2.20±0.35
Low 17 1.06±0.06 1. 33±0.11 1.63±0.23 1. 94±0 .46
Hight High 25 2.04±0.12 2.16±0. 11 3.28±0.37 5.48±0.87
Control 31 1. 48±0. 09 1.71±0.O9 2.47±0.22 2.97±O.33
The response of the Hight high line differed from that of the
other lines by having a greater slope which suggests a higher ovarian
sensitivity to gonodotrophin. This corroborates a similar suggestion
(5) based on data from prepuberal lambs from this flock.
(1) Bindon, B.M. , Chang, T.S. , Turner, H.N. (1971) . A.J.A.R. 11.:809.
(2) Smith, J.F. (1976). Proc.. NZ Soc. Anim. Prod. 1§.:247.
(3 ) Clarke, J.N. (1972). Proc. NZ Soc. Anim. Prod. 32:99.
(4) Hight, G.K. (1969). Ann. Rep. Res. Div. MAF 74.
(5) Bodin, L. et al (1986). Proc. Endoc. Soc. Aust. 29: Supl 2. E9.
SUPEROVULATION RESPONSE AND EMBRYO RECOVERY FROM CASHMERE
AND ANGORA DOES AFTER TREATMENT WITH FSH (FOLLTROPIN)
A.J. Ritar, P.D. Ball, P.J. O'May, T.M. Black,
R.B. Jackson and N. Murray
Department of Agriculture, Launceston South, Tasmania
superovulation in goats may be induced by numerous regimes of
gonadotro~hin adminis~ration. We examined the ovulation dose-response
of goats ~n the breed~ng season to a highly purified preparation of
FSH containing very low levels of LH contamination.
Mature Cashmere and Angora females (mean live weight + sem 37.3 + 0.72
and 33.4 ± 1.80kg) were continuously fed a high-energy diet (450g oats,
1100g hay; 13.1 MJ ME/day) and had CIDRs (AHI Plastic Moulding Co, NZ)
removed on the afternoon of the 18th day after insertion. FSH
(Folltropin, donated by Biocene Int Pty Ltd, Melb) was administered
7r~m 5h be 0re CIDR removal and continued twice-daily for seven
7
~nJect~ons ~n a decreasing regime (20, 20, 15, 15, 10, 10 and 10% of
total dose) to give total FSH doses of 3, 6, 9 and 12 mg. Testosteronetreated
wethers maintained a continuing male presence. Does were in
oestrus from 20-65h and were naturally mated at 39 and 47h after CIDR
removal. Embryos were surgically collected from females under halothane
anaesthesia 8 and 9 days after CIDR removal. Ovulation points (corpora
luteal were counted at this time.
Table 1.
Total FSH
Dose (mg)
3
6
9
12
Totals & Means
Superovulation rate of Cashmere and Angora does
treated with Folltropin
Ovulations/does treated (mean)
Cashmere Angora Total
25/4 ( 6.3)
56/3 '(18.7)
144/5 (28.8)
118/4 (29.5)
343/16(21.4)
21/4 5.3)
38/4 9.5)
17/3 5.7)
72/4 (18.0)
148/15 ( 9.9)
46/8 ( 5.8)
94/7 (13.4)
161/8 (20.1)
190/8 (23.8)
491/31(15.8)
Results are presented in Table 1. Folltropin appeared to be an
effective gonadotrophin for the induction of superovulation in goats.
There was an overall linear response in the ovulation rate to an
increase i~ the FSH dose (P

4
SUPEROVULATION AND OVA RECOVERY IN FARMED FALLOW DEER (Dama dama)
J.G.E. Thompson and G.W. Asher
MAFTech North, Ruakura Agricultural Centre, P.B., Hamilton, N.Z.
Fallow deer are highly seasonal breeders. Does are monovular and
can exhibit regular oestrous cycles of 21-23 days duration from April
to August on New Zealand farms (1).
Superovulation of 36 mature fallow does was attempted in May 1987
by gonadotrophin administration following intravaginal CIDR (12%
progesterone, Type-S, ARI) insertion for 14 days. Three treatments
were applied: (A) 1000 iu PMSG (Pregnecol, Heriot Agencies),
administered as a single i.m. dose 48 hr before CIDR withdrawal; (B)
20 mg FSH (Folltropin, Vetripharm) administered i.m. in a decreasing
dose regimen, twice daily for 4 days, the last dose coinciding with
CIDR withdrawal; (C) 750 iu PMSG + 14 mg FSH; PMSG administered as for
(A) and FSH administered as for (B). Immediately after CIDR withdrawal
does were joined with crayon harnessed fertile bucks. Onset of oestrus
was recorded by frequent observation over four days following CIDR
withdrawal. Ova recovery (OR) was performed after 6-8 days from CIDR
removal by uterine flush under surgical conditions. Numbers of corpora
1utea (CL) and total stimulation points (TS; including cystic and
1uteinised follicles) were also recorded.
Group n CL TS* O~ %REC %FEC
A 12 9.2±2.5 16.8±2.0 a 3.7±1.l a 40.0 70.0
B 12 6.3±2.9 7.0±3.1 b 1.1±0.5 b 17.7 84.6
C 12 11.2±3.3 20.4±3.0 a 1.9±O.5a ,b 17.2 52.2
* differing superscripts indicate significant differences (p

6
IONIC REQUIREMENTS OF GLYCINE TRANSPORT
IN MOUSE EMBRYOS
Peter L. Kaye,
Department of Physiology & Pharmacology, University of Queensland,
St.Lucia, QLD 4067
At the 2-cell stage mouse embryos possess a specific highly
concentrative glycine uptake system which is kinetically dependent on
2Na+ and resembles the gly-system of red cells. After development
to the blastocyst stage glycine transport is less specific, with
characteristics of other systems, however the kinetic dependence ,on
2Na+ remains. It has been proposed that the energy for concentrat7 ve
uptake ,was derived from the [Na+] gradien~, which presu~ably ~el~es
on Na+/K+-ATPase activity. However there ~s no such gra~~ent ~n 2­
cell embryos and significant ATPase does not appear unt~l the morula
stage. In order to further characterise the ionic requirements, ~he
role of ATPase and that of the Na+ ,H+-exchanger, we have stud~ed
glycine uptake in embryos of both stages.
Two-cell embryos and blastocysts were collected from
superovulated Quackenbush mice 48 or 9~h respectivelY,after hCG.
Uptake of 3H-glycine over 10 min was determ~ned. The requ~rement for
CI- was determined by replacement with S04= or acetate. Bo~h
substitutions inhibited uptake by 90% in 2-cell embryos and 80% ~n
blastocysts.The role of Na+ transport was invest~ga~e~ using ~he
Na+ ,K+exchange blocker, amiloride and the ATPase ~nh~b~tor ouaba~n.
Neither was effective on 2-cell embryos.
The ionic requirements of uptake by 2-cell embryos were
further investigated by removing K+ from the medium an~ measuri~g
uptake rate at increasing intervals. This was also done w~~h ouaba~n
in the absence of K+, since it has been reported that K+ b~nds close
to the ouabain binding site of Na+/K+-ATPase. Uptake was constant
when assayed at intervals up to 3h of incubation; omission of K+ had
little effect and ouabain reduced uptake by about 30%.
These results show that during development the major neutral
amino-acid transport system maintains its requirement for CI-. The
gly-system in 2-cell embryos is only marginally dependent on,the,K+
gradient, the Na+/K+-ATPase and the Na+ ,K+-e~changer to susta~n 7ts
concentrative activity. This suggests that ~n these embryos glyc~ne
uptake is independent of the Na+ gradient as might have been
predicted from the absence of such a gradient at this stage, and
possibly Na+ flux••
Funded by NHMRC.
EFFECT OF AN INSULIN/TRANSFERRIN/SELENITE SUPPLEMENT ON DNA
SYNTHESIS IN MOUSE EMBRYOS CULTURED IN VITRO.
E. De Luca, A. Lopata
University of Melbourne, Dept. Obstetrics and Gynaecology,
Women's Hospital, 132 Grattan st., Carlton, Vic., 3053
7
Royal
It is generally accepted that growth factors are important for
the development of mammalian cells in vitro. The aim of the present
study was to determine whether insulin/transferrin/selenite (ITS)
would influence cell proliferation of preimplantation mouse embryos
in culture. This was determined by assaying for the incorporation
of 3H-thymidine into DNA.
Preliminary experiments were performed to examine the optimal
conditions for measuring 3H-thymidine incorporation into DNA. Two
cell embryos were obtained from superovulated CBAxBalb/C (F1)
female mice, 6-8 weeks old. The embryos were cultured to the
hatching blastocyst stage in serum-free minimum essential medium
(~-MEM) and then examined for DNA synthesis. This was determined by
incubati,ng the embryos in 5 J,JCi/ml 3H-thymidine (specific
activity, 5 Ci/mmol) for 4 h under standard culture conditions.
Embryos were transferred to a multi-well "Bio-Dot" apparatus
(BIO-RAD) which contained a nitrocellulose sheet. The embryos were
treated with or without 2% 80S and/or 0.3M/3M NaOH. After washing
with 1M ammonium acetate the DNA bound to the nitrocellulose paper
was determined by liquid scintillation counting. These experiments
indicated that the optimum conditions for 3H-thymidine
incorporation into DNA involved treatment with 2% SDS followed by
a.3M NaOH. This method proved to be more sensitive than the TCA
precipitation method (1, 2) used by us previously.
Using this Bio-Dot method for the assay of 3H-thymidine
incorporation into DNA a dose response curve for ITS was
determined. Embryos were incubated with ~-MEM containing 0-10%
ITS. At the hatching blastocyst stage they were assayed for
3H-thymidine incorporation as described above. The results showed
that at a concentration of 5% (this corresponds to 25~g/ml insulin,
25~g/ml transferrin and 25ng/ml selenite), ITS stimulated
3H-thymidine incorporation to a maximum of 184% of control(ie.
291±46 cpm/embryo compared to 534±53 cpm!embryo, mean±S.E.M, n=4).
Following the observation that ITS increased DNA synthesis of
hatching blastocysts, preliminary studies have indicated that the
individual components of ITS were ineffective and that the growth
promoting effect required the presence of both insulin and
transferrin. Future studies will examine the stage of embryonic
development at which insulin and transferrin exert an influence on
DNA synthesis.
(1) Fi scher, B.
(2) De Luca, E.,
(1987) J. Reprod. Fert., 79, 115-123
Lopata, A. (1988) Proc. ANZSCB, I, 5

8
ANALYSIS OF THE CONTRIBUTIONS OF THE EMBDEN-MEYERHOFF AND
PENTOSE-PHOSPHATE PATHWAYS TO GLUCOSE METABOLISM BY SINGLE
EQUINE EMBRYOS IN CULTURE
I.Bruck and J.H.Hyland
Department of Veterinary Clinical Sciences,
University of Melbourne
Werribee, Vic.
The mare has the lowest reproductive efficiency among the
domestic animals. Seasonality, strict uniparity and a gestation
period of 11 months account for part of the problem.
Embryonic loss during the first 20 days of gestation (up to 45 %)
further contributes to poor reproductive performance.
To study the reasons for the high incidence of early embryonic
loss in the mare, our aim was to develop a culture system to
assess embryo viability, based on measurements of metabolic
activity. The contributions of the Embden-Meyerhoff (EMP) and the
Pentose-Phosphate Pathways (PPP) to the turnover of glucose
were measured separately.
Equine embryos were collected on Day 7 (n=6) or Day 9 (n=9) after
ovulation from 10 Standardbred mares using a non-surgical
transcervical flushing technique.
After determination of diameter and visual quality each embryo
underwent 2 x 3 h cultures in the inner well of a sealed two-well
system at 37 C. The culture medium (60 or 120 ul) contained
either (U-14C)- or (S-3H)-glucose. l4C-labelled carbon dioxide
(from activities of the PPP) or tritiated water (from activities
of the EMP) released by the embryo were trapped in 1.0 mol
sodium hydroxide in the outer well.
Metabolism of radioactively-labelled glucose was considered to
have occurred if the counts produced by the embryo were
significantly greater than the mean counts of the controls
(p

10
EFFECT OF SUCROSE AND CALCIlM IN THE SPLITTING Iv1EDIlM ON
SURVIVAL OF QUARTERED OVINE MJRULAE
C. Herr,l N. Holt,2 an d K.C. Reed l
lAdvanced Breeding Technology Pty. Limited
2Riverina Artificial Breeders Pty. Limited
Dissection of blastocyst stage embryos is more
successful than dissection of morulae. In morulae, cells
are tightly associated; when dissected (split) many cells
are damaged. In contrast, blastocysts have a fluid filled
coe I e so f ewe r c e I I s come inc0 n t act wit h the s p lit tin g
instrument. Commercial application of embryo splitting
technology requires that both morula and blastocyst stage
embryos yield acceptable results, i.e. there is a
significant increase in the number of offspring produced
from a given number of embryos. This study attempted to
improve the survival rate of quartered morula stage ovine
embryos.
The effect of the presence or absence of 500 roM
s u c r a s e (S) or 2. 2 1 mM c a I c i urn (Ca + + ) in the s p lit ting
me d i urn was i n v est i gat e d . Em b r y a s we r e su r g i c a I I Y
collected from the uterus of superovulated and
I a para s cop i c a I I y art i f i cia I Iyins eminated ewe s • Mo r u I a e
we reofun i form qua lit Y, and werea r bit r a r i Iy a I I 0 cat e d
the treatments. After microsurgical dissection, embryos
were cultured in bicarbonated medium at 39 0 C under a 5%
C02 atmosphere. Forty eight hours later, embryos were
evaluated for development to the expanded blastocyst
stage. Results are summarised in the Table 1.
In vitro development of quartered embryos dissected under
different media conditions.
Trea tmen t
Ca++ S
+
+ +
+
Total quarters (Unsplit) Percentage (Unsplit)
per treatment (control) Developing (control)
40
24
40
24
6
2
3
2
2.5
87.5
42.5
0.0
100
100
67
100
Mo r u I a e s p lit wit h out C a + + ten dedt a 10 s e
b I as tome res, wh i c h f a i led tor e ass a cia t e ins u b seq u e n t
cui t u r e . Na a t t emp twasmadeta i n d u cereass a cia t ion
because, in field use, embryos are transferred directly to
recipients. Splitting in standard conditions (without S,
wit h Ca + +) res u I ted in. the I y sis a f rna n y b I as tome res.
Mo r u 1a e s p lit inS and Ca + + can t a i n i n g me d i urn had no
o b s e r v a b Iely 5 i s 0 fbi as tome res, and par tit ion e din t a
quarters without loss of blastomeres.
11
ANABOLIC EFFECTS OF INSULIN ON MOUSE BLASTOCYSTS
ARE MEDIATED VIA INSULIN RECEPTORS
Mark B. Harvey and Peter L. Kaye
Department of Physiology and Pharmacology,
University of Queensland, St. Lucia, 4067.
The addition of hormones to the culture medium for preimplantation
embryos affects metabolism and improves developmental rate. In
particular, insulin doubles the protein synthetic rate in compacted
embryos (1) and stimulates cellular division. The specific mediation of
insulin's effect on embryos was investigated at varying concentrations
of insulin and utilising an anti-insulin receptor antiserum.
Two-cell embryos collected from superovulated Quackenbush mice were
cultured in BMOC2 medium containing varying concentrations of insulin
for 48h before blastocysts were transferred to fresh droplets of the
same medium plus 6p.M 3H-Ieucine (1Ci/L) and assay of protein synthesis
over 2h. Blastocysts from cultures with 0.17pM insulin were no more
active than those derived from culture in control medium, but culture
with 0.43-1~7pM insulin stimulated protein synthesis by 25-75% with an
EC~o=0.5pM (P

12
COMPARISON OF TROPHOBLASTIC VESICLE AND OVINE BLASTOCYST SECRETED
PROTEINS AND ANAYLSIS OF VESICLE METABOLIC ACTIVITY IN VITRO
Clarkson, J .L. and Nancarrow, C.D. CSIRO Division of Animal
production, P.O. Box 239, Blacktown, N.S.W. Australia, 2148.
The aim of this study was to compare the proteins secreted by
trophoblastic vesicles (TV's) with those secreted by day-17 ovine
blastocysts cultured in vitro, and to estimate the metabolic activity
of TV's during a 13 dayculture period.
TV's were produced by cutting the trophoblast from day-17 ovine
blastocysts into 2 rom sections. Each section was placed in 200 ~l B-2
medium and incubated at 37°C in a humidified, 5% 02, 5 C02, 90% N2
atmosphere. In Experiment I, the proteins secreted from TV'S and
intact blastocysts cultured in vitro were collected, washed and
concentrated using ultrafiltration:-Th"ey were then separated using a
Pharmacia FPLC with S-12 and S-6 gel filtration columns linked in
series and monitored at 280 nm. Experiment II was identical to
Experiment I except 1/10 the normal concentration of amino acids was
used and 16.5 ~Ci/ml [3 H]-amino acid mixture was added to the medium.
Additionally, the proteins were separated with a Pharmacia Mono-Q
anion-exchange column using a linear 0 to 0.25 M NaCl gradient in 0.01
M Tris-HCl (pH 8.2). One ml fractions were collected of which half was
added to scintillant and the presence of labelled proteins determined
on a Rack Beta counter. In Experiment III, 20 TV's were assigned to
one of four treatments (trt). 0.125 ~Ci [methyl-3 H]-thymidine was
added to each well in trt's I, II, III and IVan days 1, 4, 7 and 10
respectively. After 72 hr of culture with the label, the TV'S were
washed and placed in scintillation fluid and the thymidine
incorporation determined.
For statistical analysis, the morphological
state of the vesicles were given the following values: fragmented
tissue = 1, healthy, nonvesicle tissue = 2, TV-1 (multi-cell walled
small vesicles) = 3, and TV-2 (single-cell walled, large vesicles) = 4.
A vesicle which changed to a different stage during the label period
was given a positive or negative value equal to the number of stages it
progressed or regressed to. .
In Experiment I, the gel filtration profile of the blastocyst
secreted proteins consisted of nine peaks which corresponded to
molecular weights (MW) of > 120, 103, 82, 54, 32, 8, 3, 2 and < 1
kilodaltons (kD). The TV protein profile consisted of eleven peaks
which corresponded to MW's of > 120, 103, 39, 28, 18, 10, 6, 2 and
3 < 1 kD. In Experiment II, eight labelled protein peaks were recorded
from the blastocyst extract in contrast to five labelled protein peaks
recorded from the TV extract. In Experiment III, the mean % thymidine
incorporation was 4.0, 1.2, 2.8 and 2.9 in trt' s I, II, III and IV
respectively. The % incorporation was significantly less (p < .05) in
trt II than in the other treatments. There was also significantly less
(p < .05) progressive morphological change in trt II with mean changes
of 0.8, -0.2, 0.0 and 0.4 in the respective treatments.
We conclude that TV's produced several protein compounds similar to
those produced by intact blastocysts cultured in vitro. Additionally,
the TV's remain metaboli'cally active throughout a 13 day culture
period.
13
CONTROL OF THE OVARIAN CYCLE IN MARMOSET MONKEYS FOR IN VITRO AND
IN VIVO FERTI LIZATIoN AND THE TRANSFER OF CULTURED EMBRYOS TO
SYNCHRONIZED RECIPIENTS
A. Lopata,l P.M. Summers2 and J.P. Hearn2
Department of Obstetrics and Gynaecology, University of Melbourne
and Royal Women's Hospital, Melbourne, Victoria.
2 Institute of Zoology, London, United Kingdom.
It has recently been reported that an analogue of prostaglandin F2 alpha
(cloprostenol, Estrumate, ICI, U.K.) caused a prompt shut-down of luteal function in the
marmoset monkey (1) and that this agent could be used for initiating a new cycle and
subsequently inducing controlled ovulation in this primate species (2). We have applied
these procedures for timing the recovery of follicular and tubal oocytesfor in vitro
fertilization (IVF) and embryo culture. In addition we have evaluated the use of
cloprostenol for synchronizing the reproductive cycles of oocyte donors with that of embryo
reci pi ents.
The marmoset monkeys used in the present studies were proven breeders. In each
treatment protocol a new reproductive cycle was initiated by injecting 0.5 ug cloprostenol
between days 10 and 24 of pregnancy. In 40 animals the timing of ooycte collection was
based on the administration of 75 IU hCG at the following intervals after cloprostenol: at
9 am on the eighth day (protocol I, n=2o), at 9 am on the seventh day (protocol 2, n=6), at
5 pm on the seventh day (protocol 3, n=14). Animals that were to be used as embryo
recipients were treated simultaneously and in the same way as the oocyte donors. At 24
hours after hCG oocytes were obtained by aspirating ovarian follicles, or flushing the
Fallopian tubes, under general anaesthesia. Semen was obtained by electroejaculating
fertile males under general anaesthesia. The sperm used for in vitro insemination were
prepared by a swim up procedure. Insemination and embryo culture was performed in minimum
essentia1 medi um supplemented with 10% human cord serum. Embryos were transferred
surgically at the 4 to 6 cell stages to the uterine lumen of synchronized recipients.
In treatment protocol 1 9/20 (45%) marmosets had ovulated before oocyte collection,
none had ovulated in protocol 2, and 2/14 (14%) in protocol 3. There was no significant
difference in the mean !lumber of follicles aspirated and oocytes recovered between the
protocols. The fertilization rate of morphologically mature eggs was 64% (21/33) and 40%
(6/15) for eggs assessed as being immature. Of the 21 mature eggs that ferti lized 20
produced cleaving embryos. Five embryos were transferred to recipients at 4 to 6-cell
stages and of the 15 'that remained in culture six developed to the morula stage and three
progressed to advanced blastocyst stages. Two synchroni zed reci pi ents recei ved two embryos
each and one of the marmosets gave birth to twins. A third recipient -eceived a single
embryo and gave birth to a male infant. Two additional recipients received single embryos
that were cultured in vitro but were derived from oocytes fertilized in the oviducts. Both
recipients became pregnant but only one delivered a normal infant.
This is the first report of successful IVF and embryo transfer in the marmoset monkey.
Moreover we have shown that pharmaco1ogi ca1 agents can be used to accurately synchroni ze
the reproductive cycles of oocyte donors and embryo recipients following IVF in a nonhuman
primate.
(1) Summers, P.M., Wennik, C.J. and Hodges, J.K. J. Reprod. Fertil. (1985). U.:133-138.
(2) Hodges, J.K., Cottingham, P.G., Summers, P.M. and Yingnan, L. Fertil. Steril. (1987).
~:299-305.

14.
IMMrJIDFIOORFSCENr SlUDIES OF F'R.B3NANCY-ASOOCIATED ELASTASE INHIBI'IOR
(PAEI) EXPRESSION BY ACrIVATED GAMETES AND PREIMPLANI'ATION HAMSTER
EMBRYOS.
M.J. Sinosich, S. Ianzendorf l , M.D. Bonifacio, D.M. saunders, G.D.
Hoogen
Department of Obstetrics and Gynaecology, Royal North Shore
Hospital, st. Leonards NSW, Australia.
~e Jones Institute for Reproductive Medicine, Department of
Obstetrics and Gynaecology, Eastern Virginia Medical School,
Norfolk, Virginia 23507. (SFON: R. Stillman)
Human pregnancy-associated plasma protein-A (PAPP-A), a large (Mr
820kd) heparin-binding proteoglycan, specifically and potently
inhibited .granulocyte elastase activity. Wheras irranunological and
functional analogs of human PAPP-A were readily demonstrated in
pregnant high-order non-hLmlan primates, functional analogs have been
demonstrated in non-primate mammals with hemochorial placentation.
A large hep:lrin-binding elastase inhibitor was isolated fran term
guinea pig placentae and injected into N Z white rabbits to prepare
antisera for indirect irranunofluorescent studies on rna.ture oocytes
and embryos obtained fran superovulated golden hamsters.
Pronuclear, two-cell, four-cell arrl blastocyst embryos were
surgically removed at 1, 2, 3 and 4 days after rna.ting and fixed in
phosp:lte buffered forrna.lin for 60 minutes. No fluorescence was
Elanonstrated on intact oocytes, whereas in vivo derived
preimplantation embryos were strongly fluorescent. Furthermore, in
vitro microinjection (with or without sperm) irrluced PAEI expression
within 4 h. '!hese' findings suggest; 1) hLnnan PAPP-A is a more
recently evolved form of marmnalian PAEI. 2) sperm nuclei and gene
replication are not required for expression ,of PAEI, 3) oocyte
activation irrluces PAEI expression, 4) PAEI rna.y act as a barrier
against maternal phagocytic-proteolytic defenses, and 5) PAEI may
prove a useful target antigen for immunological contraception,
having high specificity for activated gametes or their early
embryonic derivatives.
15
RELEASE OF GROWTH FACTOR(S) BY EMBRYO DERIVED PLATELET
ACTIVATING FACTOR (EPAF)
Y.C. Smart, L.A. Adamson, R. A. Wilcox and T.K. Roberts
Faculty of Medicine and Dept. of Biological Sciences, University of Newcastle
Preimplantation pregnancy in the mouse is associated with a transient state
of thrombocytopenia induced by the release from the embryo of a platelet
activating factor (EPAF) (1,2). In vivo and in vitro studies have implicated EPAF
in a role which acts to initiate implantation and maintain foetal survival (3,4).
The mechanism by which EPAF triggers the events required for successful
pregnancy is not known.
This paper reports the results of an in vitro experiment which measures the
effect of a molecule(s), released by the action of mouse EPAF on human platelets,
on Balb/c 3T3 fibroblast cells. Day 5 embryo culture medium (ECM) with predetermined
thrombocytopenic activity in mice in vivo was incubated with washed
human platelet rich plasma (PRP) in microtitre wells at 37oC/5%C02 for 4
hours. Jhe wells were washed to remove ECM and platelets. Quiescent 3T3 cells
(2 x 10 ) were seeded into the EPAF/PRP treated wells. The cells were cu Itured
at 37 o C/5%C0 2 for 8 days. Each day the 3T3 cells were scored morphologically,
trypsinised and the cell concentration determined. Wells pre-treated with the
synthetic phospholipid platelet activating factor (PAF) and PRP served as the
positive control while wells containing ECM alone, control medium (CM) and PRP,
and PRP alone were the negative controls.
Results of replicate experiments showed that EPAF/PRP and PAF/PRP
treated wells supported continued proliferation of 313 cells with rapid growth
from Days 3-7, peaking on Days 6 and 7 of culture. 313 cells seeded into wells
pre-treated with ECM alone, PRP alone and CM/PRP proliferated poorly. Results
of a typical experiment are shown in Figure I. We conclude that EPAF and PAF
activates platelets to release growth factor(s) which binds to the tissue culture
wells and acts mitogenically on the 3T3 fibroblast cells. It is likely that in vivo,
EPAF activates platelets and/or oviduct cells to release growth factors important
in successful pregnancy.
Figure I. Effects of EPAF and ~
PAF on 3T3 cells
100
80
60
40
20
-0- EeM
... ,CM-PRP
.. CM-PRP
+ PAF-PRP
.. PRP
o .J--:¢;+::::=~~==8===&--.--,
o 4 6 10
DAYS IN CULTURE
(I) O'Neill, C. (1985) J. Reprod. Fert. 73:559.
(2) Roberts, T.K. et 01 (1987) Fert. Sterif. 47:848.
(3) Adamson, L.M. et al (1987) Am. J. Reprod. Immunol. Microbiol. 13: 117.
(4) O'Neill, C. (1985). J. Reprod. Fert. 73:567.

16 17
PAP-PRETREATMENT IMPAIRS EMBRYONIC DEVELOPMENT
ADAMSON LMi' STANGER JDj SMART YC
and ROBERTS TK.
Department of Biological Sciences, Department of Surgical
Sciences and Lingard Hospital, Newcastle, NSW.
The way in which the mammalian embryo signals its presence to the
maternal system remains an enigma to a large extent. Recently however,
work in this area has established the production of a factor by the
pre-implantation mouse and human embryo with platelet activating
activity(1,2). We call this factor, embryo-derived platelet activating
factor (EPAF), because of its biological resemblance to the
phospholipid platelet activating factor, PAF-acether (3,4). The result
of EPAF production during the first 5 days post-mating in mice is the
induction of a mild thrombocytopenia, early pregnancy associated
thrombocytopenia (EPAT), in the maternal system.
We have previously demons trated that, in the mouse, pre-mating
treatment with semi-synthetic PAF-acether has two obvious effects.
Firstly, the treatment inhibits the onset of early pregnancy associated
thrombocytopenia, presumably by inducing platelet desensitization to
PAF-acether and to EPAF. Secondly, examining these animals on day 10
post-mating reveals that PAF-pretreated animals have significantly
fewer implantation sites than the PBS-pretreated controls (4).
We have extended this study to determine the effects of
PAF-pretreatment on pre-implantation embryo development. QS female
mice were given single injections of sub-threshold levels of
PAF-acether or PBS on three consecutive days immediately prior to
mating. Oviducts and uteri were removed from these animals on either
day 1, 2, 3 or 4, and the embryos flushed out to record both embryo
number and developmental stage. Results illustrated that eventhough
PAF-pretreatment had no effect on embryo development during days 1, 2
and 3, by day 4 post-mating, PAF pre-treated animals had fewer embryos
and their development was impaired (Table 1). We suggest that
PAF-pretreatment affects embryonic development, perhaps by inducing
uterine asynchrony resulting in a utero-toxic effect on the implanting
embryos. If this be the case, EPAF may be an initial embryonic signal
to the maternal system for uterine receptivity.
TABLE 1: EMBRYONIC NUMBER AND DEVELOPMENT DURING
PREIMPLANTATION STAGE IN PAF AND PBS TREATED MICE.
DAY
MEAN EMBRYO NUMBER % EMBRYO DEVELOPMENT TO
(No. Animals)
EXPECTED STAGE
PAF
PBS
PAF
PBS
1 21+3(9) 29+3(9) 98.5
93.6
2 2ft"5(7) 30+6(8) 77.0
80.7
3 22+6(10) 18+2(7) 74.1
78.6
4 9±2(15) 19±2(13)* 30.6
81.1
* P

18 19
VAI.JDATICN OF MEIHXXJ[(X;Y FeR Sl'ODY OF PULSATILE IH SECRRl'ICN IN '.!HE
RAT: CANNUIATICN RaJrE, SAMPLIN::; INI'ENSITY AND DURATICN
Qillan Ibng, David J Handelsrran
Department of Medicine, University of Sydney
Pituitary-gonadal function is regulated by the hypothalamus through
episodic GnRH release into pituitary portal blcxxi which induces
pulsatile LH secretion. The experimental study of pulsatile LH
secretion as a non-invasive index of reproductive neuroendocrine
function of the hypothalamus has been validated in large a.niJnal
species however there has been little systematic study of the
requirem:mts for valid LH pulse studies in the rat which species has
econanic and ethical advantages as well as having the rrost well
studied reproductive neuroendocrine system. In particular there is no
co~sensus about which vessel to cannulate (carotid artery vs jugular
vell) or about standards of sampling intensity and duration re:;ruired
to obtain stable and valid estiJrates of pulsatile LH secretion.
Therefore we have examined (a) the short-tenn effect of unilateral
carotid artery ligation on pulsatile LH secretion and (b) compared
para:rreters of pulsatile LH secretion (mean, max:iJnurn, minimum, pulse
frequency and interpulse interval, pulse amplitude, pulse length)
estiJrated fran an intensive sampling regime (ql0 min, 6hr) with (i)
the first 1,2,3,4 and 5hr at q10 min and (ii) with less intensive
sampling (q20 min, 6hr). Castrate mature male rats (n=18) underwent
serial blcxxi sampling (0.25 rnl, q10 min, 6 hr) via right external
jugular vein cannula while freely rrobile and under minimal stress
conditions and with volumetric blcxxi replacerrent by thrice-washed
donor rat erythrocytes resuspended in a charcoal-extracted plasma
P70te~ substitut~. Half the rats ·also unde:rwent left carotid artery
1J.gatJ..on at the tJ.n'le of venous cannulation. Pulse study samples were
assayed together in duplicate and pulses analysed by an objective
method (PULSAR) optimized for pulsatile LH studies in the rat. .
(g10 min) 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr
Mean LH(ng/rnl) 4.0+0.6 3.7+0.5 3.5+0.4 3.5+0.4 3.5+0.4 3.4+0.5
Peaks (/6hr) *16.0+1.0 *12.7+0.8 11.8+0.8 11.2+0.7 10.9+0.6 10.4+0.5
Amplit (ng/rnl) 3.9+0.7 3.8+0.6 3.7+0.6 3.6+0.6 3.6+0.5 3.6+0.6
Mean LH (ng/rnl)
Peaks (/6hr )
Amplitude (ng/rnl)
(Mean+SEM,
Carotid ligation
q20 min/6 hr ql0 min/6 hr ql0 min/6 hr
3.5+0.3 3.4+0.5 *1.8+0.3
*4.9+0.3 10.4+0.5 *6.2+1.2
3.6+0.4 3.6+0.6 2.7+0.4
* P0.05 repeatedmeasures.ANOVA).
~r intensity sampling regime (q20 min) gave marked
underestJ..mates of para:rreters of. pulsatile LH secretion canpared with
qlO min sampling within a 6 hr pulse study. we conclude that for valid
estiJrates of pulsatile LH secretion in physiological studies of the
castrate mature male rat (i) carotid artery cannulation is undesirable
and (ii) sampling intensity of at least q10 min and durations of at
least 3 hours is required.
THE ROLE OF LEYDIG CELLS IN THE REGULATION OF INHIBIN PRODUCTION
A.E. Drummond, G.P. Risbridger, D.M. de Kretser
Department ofAnatomy, Monash University, Melbourne, Victoria.
It is established that the Sertoli cell is the source of inhibin in the testis and
the production of this hormone is controlled by FSH (1-2). The role of testosterone
in regulating inhibin synthesis is equivocal of Leydig cell function (3). The aim of
this study was to determine the effect of acute stimulation of Leydig cell function by
the administration of hCG/LH (human chorionic gonadotrophin/Luteinizing hormone),
or depletion of Leydig cells by implementing the Leydig cell specific cytotoxin ethane
dimethane sulphonate.
Adult male rats received a single injection of saline or 100 IU hCG (Pregnyl,
Organon). Serum was collected at time intervals up to 5 days after hCG
administration. A second group of animals received EDS, which destroys Leydig cells,
75mg/kg body weight, or the vehicle dimethyl sulphoxide (DMSO) in a single
intraperitoneal injection. Serum was collected 1, 2 and 4 weeks after injection. A
third group of animals received combined EDS/hCG treatment. Four days after EDS
injection a single subcutaneous injection of 100 IU hCG was administered. Serum was
collected 6 hours after hCG injection. Serum samples were assayed for inhibin using
a specific double antibody radioimmunoassay (4).
Serum inhibin levels were significantly elevated within 6 hours of hCG injection
peaking at 24 hours and did not return to control levels within the 5 day period of
the study suggesting a stimulatory effect of Leydig cell products on inhibin
production. Serum inhibin levels were significantly elevated 2 and 4 weeks after EDS
injection. The destruction of Leydig cells by EDS blocked the rise in serum inhibin
levels after hCG injection (Table 1). Paradoxically, after EDS injection alone inhibin
levels were unchanged until 2 and 4 weeks later when they were significantly elevated
suggesting that an inhibitory influence of the Leydigs on inhibin secretion had been
removed by EDS treatment.
These data suggest an effect of Leydig cell products in regulating inhibin
production. The role of testosterone and other factors in this phenomenon remains to
be elucidated.
Table 1.
Saline
hCG (6h)
DMSO
EDS (4 days)
EDS/hCG (6h)
Serum inhibin and testosterone concentrations after hCG, EDS or
combined treatment.
Inhibin (Ujml)
2.77 ± 0.43
4.84 ± 0.24*
2.64 ± 0.78
3.03 ± 0.55
2.89 ± 0.64
*p

HIGHLY roIARIZED SECREl'ICN OF INHIBIN BY RAT SERIOLI CEUS IN
'lWllH::IWffiER CULTURE SYSTEM:
Jermifer A Spaliviero~ *David M Robertson, Elsa Kidston,
Peter F Hall, David J Handelsman
Deparl:m9nt of Medicine, University of Sydney, Sydney and
*DeparbUent of Anatcmy, Monash University, Melbourne
20
Sertoli cells are the princir.al source of circulating inhibin in
the male however the ho:t:IOClnal regulation of inhibin secretion and its
route of entry into the blood-stream remain unclear. The central role
of Sertoli cells in spenratogenesis includes secretion of n'UllErotis
peptides and the maintenance of the blood-testis barrier which
secludes ,developing genninal cells within the diffusion-tight
adluminal canpartrnent away fran the extracellular fluid. The
distinctly polar aspects of sertoli cells in-situ pennits secretion
via the basal surface into the testicular interstitial and
extracellular fluid and/or via the apical surface into the adluminal
carnpartment and thereby into the rete testis fluids and seminal
plasrra. Therefore using a twin chamber culture system, which :mimics
the polarised state of sertoli cells in vivo (1), we have investigated
the vectorial secretion of inhibin and its ho:t:IOClnal regulation.
Bertoli cells isolated f:rom 2l-day old rats were cultured in a twin
c~r system using fully defined medium (supplerrented Eagles MEM) in
trlp1J.cate wells under basal conditions or stinmlated with oFSH
(250ng/ml), testosterone ([TESJ ,1000nM), insulin ([INSJ 5ug/ml)
retinoic acid ([RAJ ,500nM) or pai:rw:i.se cernbinations of FSH with INS'
TES or RA or all 4 stimuli (FIRT). Rat inhibin and transferrin ~
media (24 hr, days 5-7) were measured by specific RIA.
UPPER CHAMBER RAT INHIBIN Wlml)
BASAL FSH TES INS RA FSH/INS FSH/TES
1.4 5.3 1.0 3.1 1.1 5.9 7.9
(0.1) (0.1) (0) (0.1) (0.3) (0.5) (0.5)
Mean (SEM), * p

22
TREATMENT WITH A GnRH AGONIST DELAYS REPRODUCTIVE
DEVELOPMENT IN RAM LAMBS
A. J. Tilbrookl, D.E. Galloway2, A.H. Williams!, R.C. Oppenheim 3 ,
W.J. Thie1 3 and LJ. Clarke 4
1 Animal Research Institute, Dept. of Agric. & Rural Affairs, Werribee, 3030.
2 Veterinary Clinical Centre, Univ. Melb., Werribee, 3030,
3 Victorian College of Pharmacy, Parkville, 3052,
4 Medical Research Centre, Prince Henry's Hospital, St. Kilda, 3004.
Prolonged continuous infusion of adult Soay rams with an agonist of
gonadotrophin rp.leasing hormone (GnRH) initially stimulated the secretion of
luteinizing hormone (LH) and testosterone (T), followed by a suppression in the
secretion of LH, T and follicle stimulating hormone (FSH)(l). There was also a
small decrease in testicular diameter and disappearance of the sexual flush (1) but
sexual behaviour was not studied. The aim of our study was to determine if
development of sexual behaviour and testicular size could be delayed in
prepubertal rams by continuous administration of a GnRH agonist, and if this
treatment would influence their growth.
Preliminary work (Tilbrook et al., unpublished) demonstrated that a GnRH
agonist ({D_trpli_Pro 9 N-ethyl amide}GnRH) was effective in inhibiting LH and FSH
secretion in adult wethers when administered using s.c. mini-osmotic pumps
(Alzetminipumps) or prototype slow-release pellets (pellets).
In March, rams (aged 20-28 weeks) weighing 30.7±O.7 kg (mean±SD) were
either untreated (n=10; entire), treated with 50llg of the GnRH agonist/day using
minipumps for 16 weeks (n=lO), given a pellet containing 100 Ilg of the GnRH
agonist every 4 weeks for 16 weeks (n=lO) or surgically castrated (n=15; castrate).
Rams that mounted and/or ejaculated during .15 minutes of individual exposure to 4
oestrous ewes were defined as sexually active. Testicular volume (ml) was
estimated using calibrated beads, and liveweight (kg) was also measured.
Table 1. Effect of GnRH agonist on the testicular volume (TV) and liveweight
(LWT) oframs and the proportion oframs sexually active (SA)
Before treatment End of treatment 8 weeks after treatment
Group TV" LWT* SA TV" LWT* SA TV" LWT*
Entire 45±8 3l±1.0 9/lO x 118±12 ax 44±1.Ox lO/lO x 158±1 IX 51±1.0 xa
Castrate 32±O.5 0/15 Y 41±1.0a 0/15 Y 45±1.0 Yc
Minipump 45±7 29±OA 2/lO Y 68±lO b 40±1.0 zb 7/l0 x 91±1 JY 41±1.0 z
Pellet 39±6 31±0.5 3/10 Y 82±lO cy 42±1.oya 7/lO x 92±5 Y 49±2.0 b
"means±standard errors; x.y,Z differ at p

24
IDENTIFICATION BY HPLC AND IMMUNOCYTOCHEMISTRY OF
HYPOTHALAMIC OXYTOCIN AND MESOTOCIN IN THE
BRUSHTAIL POSSUM
R. Bathgate!, C. Sernia! and R.T. Gemmel1 2
tDepartment of Physiology and Pharmacology, 2Department of Anatomy,
University of Queensland, St. Lucia, 4067
All of the nine species of Australian marsupials investigated to
date appear to synthesise mesotocin, which is typical of non-mammalian
tetrapods, instead of oxytocin (1,2). Since much of the reported data
are based on bioassay of pituitary extracts and are qualitative only,
we decided to examine the hypothalamus of one of these species, the
brushtail possum, using high pressure liquid chromatography (HPLC) and
immunocytochemistry.
Fresh hypothalamic tissue was homogenized in 20 vols (w:v) of
1 molll HCI containing 15% TFA, 5% formic acid and 1% NaCI. The
homogenate was centrifuged and the peptides in the supernatant
extracted on C-18 cartridges and eluted with 0.1% C-18 HPLC column
(LKB UltroPac) using 0.1% TFA in a 10-35% ACN gradient and" assayed for
mesotocin and oxytocin by specific radioimmunoassays. The major
peptide was mesotocin, found at 9 to 12 ng per hypothalamus. An
additional peak with the relative mobility of oxytocin was also
detected but at the lower quantity of 1.5 to 2.5 ng per hypothalamus.
Serial sections of paraformaldehyde-fixed hypothalami were
i~munostained for either oxytocin or mesotocin using a biotinstreptavidin-peroxide
system (Amersham). Heavy mesotocin staining was
found in the Paraventricular and Supraoptic nuclei with only light
oxytocin staining in the same cells.
These results show that at least one Australian marsupial
expresses both mesotocin and oxytocin genes; a situation which has
also been reported for the opossum, D. virginiana (3).
(1) Chauvet, M.T., et. a1. (1981) FEBS Lett. 129: 120-122.
(2) Hurpet, D., et. a1. (1982) Int. J. Pept. Prot. Res. 19: 366-371.
(3) Chauvet, J., et. a1. (1984) BBRe 123: 306-311.
25
THE RELATIONSHIP OF BREASTFEEDING PATTERNS TO THE· LENGTH OF
LACTATIONAL AMENORRHOEA AND OVARIAN INACTIVITY POST PARTUM
P.R. Lewis, M.B. Renfree, R.V. Short
Depts. of Physiology and Anatomy, Monash University, Melbourne, 3168
The duration and frequency of breastfeeding are major factors
contributing to the length of lactational amenorrhoea. Starting in
1984, a study was conducted at Monash University to provide additional
information on the influence of these and other factors on the duration
of lactational amenorrhoea. A total of 146 breastfeeding women were
recruited in their first month post partum. They kept records of all
feeds given to the baby during two 24 hour periods each week and
collected saliva samples 2-3 times per week for progesterone assay to
monitor corpus luteum function following ovulation. A subset of women
also collected urine twice per week and this was analysed for total
oestrogens and pregnanediol.
Menstruati.on resumed within 1 year post partum in 70% of the women
(shortest amenorrhoea: 1 month); for the remaining 30%, amenorrhoea
lasted from 13 to 22 months. Three women became pregnant in their
second year of lactation without experiencing a menstrual bleed. First
post partum ovulations occurred from less than 1 month (1%) to 22
months (1%) after birth; median time to first ovulation was 10.5
months. Initial menstrual cycles were irregular in length and
frequently anovulatory; women who first ovulated early in lactation
often had luteal phases characterised by short durations or low
progesterone concentrations. Of women with amenorrhoea lasting less
than 3 months, 30% ovulated before their first menstruation. However,
70% of women experiencing their first menstruation after 12 months
ovulated before that menstruation.
Breastfeeding patterns varied greatly between women both with
respect to frequency and total time spent feeding during the 24 hour
recording period. During the first 1-2 months of lactation there were
no differences in these parameters between groups of women with
different lengths of amenorrhoea. The use of dummies and introduction
of supplements, however, were signiyicantly different between groups;
women who menstruated early used dummies more frequently ~nd introduced
supplements earli.er and more rapidly.
As yet, no specific formula can be given to predict the length of a
woman's lactational amenorrhoea from her breastfeeding pattern alone.
Total duration of feeding may prqve to be lI.lore important than
frequency, hut. an interacti.on between factors may explain the wide
variation in breastfeeding patterns seen between women with similar
periods of amenorrhoea.

2~
INHIBITORY EFFECT OF PURE 31 kDa BOVINE INHIBIN ON GnRH-INDUCED
UP-REGULATION OF GnRH BINDING SITES IN CULTURED RAT ANTERIOR
PITUITARY CELLS
Wang Qi Fa, P.G. Farnworth, H.G. Burger and J.K.
Findlay
Medical Research Centre, Prince Henry's Hospital Campus of
The Monash Medical Centre, St Kilda Road, Melbourne, Victoria, 3004
GnRH modifies gonadotroph functions, having both acute effects
(minutes) on hormone release and delayed effects (hours) on receptor
up-regulation and gonadotrophin synthesis. It is well documented that
inhibin, a gonadal glycoprotein hormone, suppresses the acute effects
of GnRH both in vivo and in vitro (1). However, whether inhibin also
modulate~ delayed effects of GnRH remains to be determined. The
present study was designed to investigate the effect of inhibin on
GnRH-induced up-regulation of GnRH binding sites in cultured rat
anterior pituitary cells.
Pituitary cells from adult male Sprague-Dawley rats were dispersed
and cultured for 2 days, after which the media were replaced and the
cells were then exposed to stimuli, with or without test substances,
for 10 h. Upon the completion of incubation, the cells were removed
and the binding of GnRH was determined using iodinated Buserelin as
tracer (2). Exposure to GnRH (10 nM) resulted in a 90% increase in
specific binding sites for GnRH. Inhibin (0.01 to 10 U/ml) suppressed
GnRH-induced up-regulation of GnRH binding sites in a dose-dependent
manner with an IC of 0.13 U/ml (5.5 pM). Unlike inhibin, neither
Transforming Grow~R Factor-~ (0.4-400 pM) nor Mullerian Inhibitory
Substance (0.1-100 nM), two inhibin-related peptides, had any
detectable effect on the GnRH-stimulated increase in GnRH binding
sites; suggesting that the effect was specific to inhibin. In
addition, inhibin (10 U/ml) significantly inhibited the up-r~gulation
of GnRH binding sites induced by the calcium ionophore A23187 (0.1
~M), indicating that this effect 2f inhibin can occur, at least in
part, at a stage subsequent to Ca + mobilization. Finally, inhibin at
a concentration up to 300 Ulml did not compete with iodinated GnRH-A
for GnRH binding sites.
We conclude from this study that inhibin at low concentrations
modulates delayed effects of GnRH on its own receptors and that
inhibin might therefore participate in the physiological regulation of
GnRH receptors.
(1) Findlay, J. Fertil. Steril. 46(5):770-783, 1986.
(2) Loumaye, E. &Catt, K.J. Science 215:983-985, 1983.
27
TRANSFORMING GROWTH FACTOR ~ ENHANCES BASAL AND FSH STIMULATED
INHIBIN PRODUCTION BY RAT GRANULOSA CELLS IN VITRO
Zhang Zhiwen, J.K. Findlay, R.S. Carson, A.C. Herington
and H.G. Burger
Medical Research Centre, Prince Henry's Hospital Campus,
Monash Medical Centre, St Kilda Road, Melbourne, Victoria, 3004
Transforming growth factor ~ (TGF-~), a member of the inhibin gene
family, is produced by ovarian thecal tissue (1), and has a paracrine
influence on granulosa cell steroidogenesis (2) and EGF receptor
number (3). We investigated the effects of TGF-~ on inhibin
production by granulosa cells from diethylstilbestrol-treated,
immature rats.
Granulosa cell primary cultures and inhibin radioimmunoassay of
conditioned media were performed as described previously (4). TGF~
(0.01-3 ng/ml) caused a dose-dependent increase in both basal and
FSH-stimulated inhibin production by rat granulosa cells in culture.
The TGF~ dose-response curve in the absence of FSH was approximately
parallel to that in the presence of either a minimally effective d~se
(1 ng/ml) or a maximally effective dose (30 ng/ml) of FSH, suggestlng
an additive effect of these two agents on inhibin production. There
was also a suggestion of an increased sensitivity of granulosa cell
inhibin production to FSH when the cells were coincubated with TGF~.
The time course study showed that similar to FSH, the stimulatory
effect of TGF~ on basal and FSH-stimulated inhibin production was
evident on day 1 and was maximal by day 4. In addition, EGF reduced
FSH-stimulated inhtbin production with an I~O value of 1.3 ng/ml.
Coincubation of cells with EGF and 1 ng TGF~ml enhanced greatly the
inhibitory action of EGF on FSH-induced inhibin production (ID 50
< 0.1
ng/ml).
It is concluded that: (a) TGF~ directly stimulates inhibin
production by rat granulosa cells and the combined effect with FSH was
largely additive, (b) the inhibitory effect of EGF on FSH-induced
inhibin production was enhanced by TGF~, (c) individual members of the
TGF~/inhibin gene family regulate ovarian function, not only by direct
action on follicle cells but also indirectly in influencing the
production rate of other members of that family.
(1) Skinner, M.K., Keski-Oja, J., Osteen, K.G. and Moses, H.L. (1987)
Endocrinology 121:786-792.
(2) Dodson, W.C. and Schomberg, D.W. (1987) Endocrinology 120:512-516.
(3) Knecht, M., Feng, P. and Catt, K. (1987) Endocrinology
120:1243-1249.
(4) Zhiwen, Z., Carson, R.S., Herington, A.C., Lee, V.W.K. and
Burger, H.G. (1987) Endocrinology 120:1633-1638.

28
SUPEROVULATION IN PUBERTAL HEIFERS IMMUNIZED AGAINST OVINE INHIBIN
PURIFIED BY MONOCLONAL ANTIBODY AFFINITY CHROMATOGRAPHY
1, 2 3 1
B.M. Bindon, T~OIShea, K. MiYimoto, M.A. Hillard,
L.R. Piper, R.D. Nethery and G. Uphill
2 CSIRO Division of Animal Production, Armidale
De~artment of Physiology, University of New England, Armidale
Gunma University School of Medicine, Gunma 371, Japan
Increased ovulation rate has been observed in cattle immunized
against ovine follicular fluid proteins (1). To explore this further,
crossbred beef heifers aged 10 months and weighing an average of 216 kg
were immunized against either ovine serum albumin (Group C; n=8) or
ovine .inhibin (Group I, n=8) purified from follicular fluid using a
monoclonal antibody against bovine inhibin (2). This procedure
resulted in an approximate 300-fold enrichment of the inhibin. The
animals were injected at subcutaneous and intramuscular sites with
approximately 350 JIg protein (for each immunogen) mixed with Freund's
complete adjuvant on Days 1, 25 and 65 of the experiment. Only one
heifer had shown oestrus when the experiment began. Laparoscopic
ovarian examinations were made on Days 57 and 87. On Day 57, three
heifers from Group C had ovulated, all with one ovulation while four
from Group I had ovulated, with ovulation rates of 3, 2, 1 and 16.
Ovarian data on Day 87, which is 22 days after the third
immunization are shown in Table 1. Inhibin-immunized heifers had
substantially higher ovulation rates and follicular development.
Table 1. Ovulation and follicle development of heifers immunized
against ovine serum albumin (Group C) or ovine inhibin (G~oup I)
Mean + S.E.
Live-
Group weight No. No. with Follicle % INH
(n=8) (kg) ovulated >1 ovulation Ov. rate score* binding
C 258+14 4 0 1.0+0 10.3+ 1.9 0
I 260+7 7 7 11.6+3.9 44.3~14.7 19.8
(range-2-32)
* Sum of follicle diameters (mm) (1 cm follicle = score 10)
Heifers from Group I showed measurable plasma binding of the
iodinated inhibin preparation from Day 38 and reached peak binding (13%
to 27% binding with 100 pI of 1/500 plasma dilution) by Day 87. Plasma
from Group C heifers did not show detectable binding of inhibin.
These results confirm the importance of inhibin as a feed-back
regulator of ovarian function in the bovine and promise useful
techniques for superovulation in this species.
(1) Cummins, L.3., O'Shea, T. and Bindon, B.M. (1986). Proc. Aust.
Soc. Reprod. BioI. 18: 39.
(2) Miyamoto, K., Hasegawa, Y., Fukuda, M. and Igarashi, M. (1986).
Biochem. Biophys. Res. Com. 136: 1103-1109.
29
INHIBIN IN THE
SHEEP OVARIAN CYCLE
J.K. Findlay, I.J. Clarke, H. Quigg, S. Katsahambas, P. Juhola,
M. de Blasiis and B. Doughton
Medical Research Centre, Prince Henry's Hospital Melbourne,
Victoria, 3004.
Inhibin is an ovarian glycoprotein hormone consisting of 2
dissimilar, disulphide-linked subunits, termed ~ and S, which inhibits
the production and/or secretion of pituitary gonadotrophins,
preferentially FSH. According to the inhibin hypothesis, there should
be (a) higher concentrations of inhibin in follicular fluid and
ovarian venous plasma than in peripheral plasma, and (b) a reciprocal
relationship between peripheral concentrations of inhibin and FSH
during the ovarian cycle. We examined these hypotheses in the ewe
using a radioimmunoassay developed recently for ovine inhibin (1).
Inhibin concentrations are expressed as nl equiv. ovine follicular
fluid standard/ml plasma or follicular fluid.
To examine inhibin release from the ovary, 6 Corriedale ewes on
days 12-13'of the cycle were subjected to midventral laparotomy under
general anaesthesia. Paired jugular and ovarian venous blood samples
were taken immediately before and 10 min after cauterizing the visible
follicles on one ovary. Where possible, follicular fluid was also
harvested before ablation of follicles. Before cautery, the mean +
s.d. (n) plasma inhibin levels were 130 + 72 (5) in the ovarian veIn
and 39 + 9 (6) in the jugular vein, a ratio of 3.37 + 1.90 (5). The
follicular fluid concentration was 936000 + 371000 (9 follicles),
approximately 7400-fold higher than ovarian venous plasma. After
cautery, inhibin in ovarian venous plasma fell to 65 + 20% (44-95%
range) of control, with little or no change in jugular concentrations
(97 ~ 10%; 83-114% range).
To examine the relationship between peripheral inhibin and FSH, 4
Corriedale ewes (days 8,-12) were treated with prostaglandin and bled
via the jugular vein twice daily across the ensuing 2 episodes of
estrus and daily during the luteal phase (days 3-15). Plasma samples
were assayed for LH, FSH and inhibin. During the luteal phase of the
cycle of all 4 ewes, 'inhibin concentrations varied in waves lasting
several days and there was an inverse relationship between inhibin and
FSH, with the onset of a rise in FSH preceding that of inhibi~ by 1-2
days. At the onset of luteolysis, FSH and inhibin levels decreased
together. During the follicular phase nadir in FSH, 3/4 ewe~ had
increasing inhibin concentrations which were terminated by tre LH
surge. The second FSH peak followed this decrease in inhibin. The
fourth ewe did not exhibit a rise in inhibin prior to the LH surge.
We conclude that (a) the ovarian vein is a major route of inhibin
secretion by large follicles, and (b) inhibin and FSH are inversely
related during the ovarian cycle except during the follicular phase
when we hypothesize that LH rather than FSH stimulates inhibin
production.
(1) Findlay, J.K., Quigg, H., Juhola, P., Katsahambas, S., Clarke,
I.J., Dough ton , B. and Robertson, D.M. (1988) Proc. 70th Ann.
Meet. Endocr. Soc. USA, Abstr.488.

30
LACK OF EXPRESSION OF INHIBIN GENES IN OVINE CORPORA LUTEA
R~J.
Rodgers, S.J. Stuchbery and J.K. Findlay
Medical Research Centre, Prince Henry's Hospital, St Kilda Road,
Melbourne, Victoria, 3004.
Pituitary FSH secretion is regulated by the gonadal hormone
inhibin, a glycoprotein composed of two subunits, «and ~) each the
product of a separate gene. It was originally postulated that in
females inhibin would be produced by ovarian follicles, however, the
recent detection of inhibin mRNA in human corpora lutea (1) and other
more circumstantial evidence in the sheep (2) suggest that ovine
corpora lutea (CL) may also produce inhibin.
To investigate this possibility Northern RNA blotting was performed
on RNA extracted from two pools of ovine follicles «4 mm and >6 mm in
diameter) and CL from both cyclic (n=7) and pregnant (n=6; foetal
crown-rump length 90 to 270 em) animals killed at an abattoir. Total
RNA (25 ~g) was subjected to electrophoresis through agarose/
formaldehyde gels and then electroblotted onto Biodyne A membrane.
Prehybridizations and hybridizations were carried out at 60 0 C in
4xSSC, 10 x Denhart's solution, 0.5% SDS, 1 mM EOTA and 100 ~g salmon
sperm DNA/ml for eDNA probes and at 60 0 C in 50% formamid, 5xSSPE, 5 x
Denhart's splution, 0.1% 50S and 100 ~g DNA/ml for RNA probes l (3). The
cDNA probes were bovine ~ inhibin (720 base SphI-SmaI fragment; 4),
and bovine cholesterol side chain cleavage (SCC) cytochrome P-4S0 ·(890
base EcoRI-Pvu II fragment; 5) as a positive control. An RNA probe
was made from pGEM containing an insert of bovine ~A inhibin (320 base
PstI - RsaI; 4). Stringency washes were in O.lxSSC at 4SoC for
cytochrome P-450 scc
' 50 C for ~ inhibin and 60 0 C for ~ inhibin.
Both ~ and ~ inhibin mRNA were readily detected in the pool of
follicles < 4 mm in diameter but not in the pool of larger follicles
and not in any of the CL. Cytochrome P-450 mRNA was readily
detected in the pool of large follicles andsrg all of the CL. It is
not known why inhibin mRNA was undetectable in the pool of larger
follicles but it is highly likely that these follicles were partially
atretic as cytochrome P-450 mRNA levels were high. However it is
clear that ovine CL do not ~~~ress inhibin genes, consistent with
bovine CL (6) but not with human CL (1). Interestingly ovarian
inhibin gene expression in CL of these three species appears to be
linked to expression of cytochrome P-450 aromatase activity.
(1) Davis, S.R. et al. (1987) J. Endocr. 115:R21-R23.
(2) Tsonis, e.G. et al. (1988) J. Endocr. 116:R3-R5.
(3) Manniatis, T. et al. "Molecular Cloning", Cold Spring Harbor
Laboratory. 1982.
(4) Forage, R.G. et al. (1986) Proc. Natl. Acad. Sci. USA
83:3091-3095.
(5) John, M.E. et al. (1984) Proc. Natl. Acad. Sci. 81:5628-5632.
(6) Rodgers, R.J. et al. (1987) Proc. Aust. Soc. Reprod. BioI. 19:117.
31
IMMUNIZATION OF EWES WITH INHIBIN PREPARATIONS OF INCREASING PURITY
T. O'Shea 1 , B.M. Bindon 2 , J.K. Findlay3, ~.A. Hillard 1 , L.R. Piper 2
and K. Miyamoto
~Department of Physiology, University of New E~gland, Armidale,
CSIRO, Division of Animal Production, Armi~ale, Medical Research
Centre, Prince Henry's Hospital, Melbourne, Department of Obstetrics
and Gynecology, Gunma University School of Medicine, Gunma 371, Japan.
Immunization of ewes with an inhibin-enriched fraction from
bovine follicular fluid (bFF) results in an increased ovulation rate
(OR). To confirm that the effects (1) were due to inhibin, ewes were
immunized with inhibin preparations (from bFF) of increasing purity.
bPPI (enriched 20-fold) was prepared as preViously described
(1). bMPI (enriched 200-fold) was prepared using a monoclonal
antibody agAinst bovine inhibin (2). bMPI 2
(enriched 1000-fold) was
prepared from bMPI 1
using antibodies raised in ewes immunized with
steer serum. Groups (n=20) of Merino ewes were immunized (using
Freund's adjuvant) with bovine serum albumin (bsa; 2.25 mg protein on
Days 0, 30 and 60), bPPI (2.25 mg on Days 0, 30 and 60), bMPI 1
(0.2
mg on Days 0 and 30), or bMPI 2
(0.1 mg Days 0 and 30). Laparoscopic
ovarian examinations were made on Days -14, 21, 51, 79 and 105.
Means ± sem of OR data are presented in Table 1. Immunization
with the purer preparations increased OR after two injections, and
resulted in a transient increase (P < 0.01) in plasma FSH at this
time (bsa, 1.4 ± 0.1 ng/ml plasma; bPPI, 1.7 ± 0.1; bMPI 1
, 2.0 ± 0.2;
bMPI 2
2.3 ± 0.3). Inhibin antibody titres were greater with the
purer preparations (Day 40; bPPI, 2 ± 0.5% specific binding at 1:500
dilution; bMPI 1
, 19 ± 3%; bMPI 2
, 20 ± 4%). Inhibin antibody titre
was correlated with OR (Day 40; N= 77, r = 0.44, P < 0.01).
Table 1
Ovulation rate following immunization (means ± s. e.m.)
Day
Group 21 51 79 105
bsa 1.7±0.1 1.6 ± 0.1 1.6 ± 0.2 1.1 ± 0.1
bPPI 1.7 ± 0.2 2.2 ± 0.6 2.0 ± 0.5 1.2 ± 0.1
bMPI 2.1 ± 0.3 3.8 ± 0.6 n 3.1 ± 0.9 2.3 ± 0.5*
bMPI 1
2
1.8 ± 0.1 5.2±1.0 n 2.5 ± 0.4· 2.2 ± 0.6
• P < 0.05, .* P < 0.01.
Significantly different from bsa group
These data are consistent with the postulate that the results
with crude preparations (1) were due to their inhibin content.
(1) Cummins, L.J. et al., J. Reprod. Fert. (1986),11: 365-372.
(2) Miyamoto, K. et al., Biochem. Biophys. Res. Commun. (1986),
.13Q.: 1103-1109.

32
INFLUENCE OF SHEARING STRESS ON OESTRUS AND OVULAnON
RA.Parr, LF.Davis, and M.L.Phillips*
Animal Research Institute, Werribee, 3030 and *Dookie Agricultural College,
Dookie,3647.
Shearing is associated with severe stress (1) and can delay the onset of
oestrus in ewes shorn just prior to mating (2). In the study reported here, we
investigated the effect of shearing on the incidence of behavioural oestrus and
ovulation rate in Merino ewes.
Mature Merino ewes, (n=212) were given intravaginal sponges (1ntervet)
for 12 days during September. Sponges were withdrawn on the day of shearing
and all ewes were injected (im) with 400 iu of PMSG (Heriot,Aust.). Ewes had
been randomly allocated to either a shorn or an unshorn group. Unshorn ewes
were crutched and were joined together with shorn ewes and 5% fertile rams.
During the expected period of mating, a group of 50 shorn ewes were isolated
with rams for a 6h period. Mated ewes and a sample of 30 unmated, shorn
ewes were endoscoped one week after shearing and, 2 days later, were given a
prostaglandin (1C1,Aust.) injection (im) and 400 iu PMSG. At this stage, shorn
and unshorn ewes were separately joined with 5% fertile rams. All ewes were
endoscoped one week later.
Table L Number and (percentage) of shorn and unshorn ewes detected in
oestrus and mean (+/- sem) ovulation rates (OR) of ewes at fIrst and second
oestrus.
GROUP 1st OESTRUS 2nd OESTRUS
n (%) OR n (%) OR
SHORN 5 a (4.7) . L9(0.18t 76 b (71.7) 2.3(0.21)
UNSHORN . 76b (71.7) 2.1(0.29) 71 b (67.0) 2.8(0.21)
~~ted ewes plus a sample of thirty unmated ewes.
a, ; differ at P2 mm recovered and classified as
above. Correlation coefficients between surface and dissected small
medium and large follicles were 0.77, 0.05 and 0.58 respectively. '
Data from another previously reported experiment (2) ill ustrates the
use of the three methods to assess ovarian follicle populations (Table
1) . Endoscopy was carri ed out when the group's average postpartum
interval was 81 days (s.d. = 14).
TABLE 1. Follicle populations at 80 days postpartum in ovaries from
cows fed three pr.epartu!J1 diets assessed by endoscopy (E) (n=19), or
surface (OS) and d1ssect10n (00) counts (n=33) following ovariectomy.
Prepartum Small Medium Large
Diet E OS 00 E OS 00 E OS 00
Control 6.3 8b 13.5 8 20.0 1.6 1.9 8.3 8 1.3 0.7 0.4
Supp. 1 9.4 b 19.5 8b 28.5 1.5 1.9 9.5 8 0.6 0.6 0.7
Supp. 2 5.4 8 28.8 b 38.5 1.2 1.2 16.2 b 0.8 0.4 0.4
Values within columns with different superscripts differ (P

34 35
CELLULAR COMPOSITION OF THE CYCLICAL CORPUS LUTEUM OF THE COW
J.D. O'Shea 1 , R.J. Rodgers 2 and M.J. D'Occhi0 3 .
2 1School of Veterinary Science, University of Melbourne, Victoria.
Medical Research Centre, Prince Henry's Hospital, Melbourne,
3 Victoria.
C.S.I.R.O., Division of Tropical Animal Production, Rockhampton,
Queensland.
It is important to know the numbers of large and small luteal
cells in the bovine corpus luteum (CL) to assess claims (1,2) that
granulosa-derived luteal cells disappear from the CL during pregnancy.
These ~laims, based on immunocytochemistry on enzymatically-dispersed
populations of luteal cells, depend in part on the assumption that
dispersed populations accurately represent the populations in intact
tissue. This study investigates this assumption.
Luteal tissue from 6 CL obtained on Day 12 of the oestrous cycle
was studied by ultrastructural morphometry (3). Data (mean ~ s.d.)
are shown below.
Volume density (%6
No. cells/CL x 10 10 3 )
Cell volume (~m x
Large luteal
cells
40.2 + 7.0
51.5 + 15.4
29.6 + 6.3
Small luteal
cells
27.7 +
392.4 +
2.7 +
6.3
135.1
0.4
The CL studied weighed 3.8 + 0.8 g. Large and small luteal cells g
combined occupied 67.9% of the luteal tissue. Of a total of 1.5 x 10
cells of all cell types/CL, large luteal cells comprised 3.5% and
small luteal cells 26.7%, a ratio of 1:7.6. Mean volumes of the large
and small luteal cells would convert, in spherical form, to diameters
of 34 ~m and 17 ~m respectively.
These estimates of cell diameter agree closely with measurements
from dispersed populations (4). However, numbers of luteal cells/g
luteal tissue as estimated here are 10-fold higher than numbers
counted after tissue dispersion (2) for large luteal cells, and 5-fold
higher for small luteal cells. We conclude that substantial and
selective losses of luteal cells may occur during enzymatic
dispersion.
(1) Alila, H.W. and Hansel, W. (1984) Biol. Reprod. 31:1015-1025.
(2) Hansel, \1. et al. (1987) Aust. J. Biol. Sci. 40:331-347.
(3) O'Shea, J.D. et al. (1986) J. Reprod. Fert. 76:685-691.
(4) Ursely, J. and Leymarie, P. (1979) J. Endocr. 83:303-310.
SEASONAL FACTORS INFLUENCE THE TIMING OF FIRST PREGNANCY IN
THE TAMMAR
E-M.A.
Bugledich, L.A.Hinds* and P.A.Janssens
Department of Zoology, Australian National University,
Canberra, ACT and * CSIRO Div. Wildlife and Ecology, PO Box
84 Lyneham ACT 2602.
Tammar wallabies emerge from the pouch in October at
approximately 10 months of age and females will enter
oestrus and conceive at this time. However the resulting
embryo enters diapause at the blastocyst stage and remains
in quiescence as is normal in adult females at this time of
year. Following the summer solstice reactivation occurs and
birth follows at the end of January (1). The aim of this
study was to determine the potential of females born late in
the reproductive season to produce young in the following
year.
Females from two different groups have been checked for
reproductive status at fortnightly intervals from early in
pouch life. Group 1 contained 6 females born in late
January-early February 1987. Permanent pouch exit occurred
when the young were 35-38 weeks old (1150-1600 g body
weight). They produced young in February or early March 1988
when they were 53-57 weeks of age (2800-3400 g body weight) .
Group 2 contained 3 females born June 2-4 1987. Permanent
pouch exit was at 34 weeks (600-900g body weight) and young
were produced in early May 1988 when they were 48 weeks of
age (2250-2380 g body weight) .
Thus the first blastocyst of female juveniles need not
enter diapause if conception occurs during a period of
decreasing daylength. This study further shows that female
tammars less than 2kg body weight can conceive and
subsequently undergo a successful pregnancy.
(1) Tyndale-Biscoe, C.H. and Hawkins J. (1977) In
"Reproduction and 'evolution" ed. J.H. Calaby and C.H.
Tyndale-Biscoe, pp. 245-252. Canberra: Australian Academy
of Science.

36
SUPEROVULATION IN A MACROPODID MARSUPIAL, MACROPUS EUGENII
M.B. Renfree, G.
Shaw and T.P. Fletcher
Department of Anatomy, Monash University, Victoria, 3168
Macropodid marsupials are monovular, so a technique for inducing
multiple ovulations to provide cleaving eggs and blastocysts for
experimental embryological techniques is increasingly needed. Whilst
it is easy to induce multiple follicular development using exogenous
gonadotrophin, ovulation usually fails and the ovary remains
hyperstimulated.
In a series of trials in 1979 and 1986-1988, we have tested a
,variety of stimulatory treatments during the follicular phase of
animals whose cycle had been initiated by removal of pouch young (RPY).
In 1979, 12 animals were treated with 5 to 125 III PMS (Folligon,
Intervet) on day 23 RPY. Those receiving 5 IU had no stimulation,
whilst 25 III animals were slightly stimulated, with one animal having 4
new CL's. The 6 animals receiving 50 IU PMS or more had grossly
enlarged ovaries with multiple >7 rom diameter follicles at laparotomy
on day 30, but no new CL's and all died within the next month. A
further 9 animals receiving either 50 IU or 75 IU PMS on day 23 and
100 III anti.-PMS (courtesy Dr B.M. Bindon, CSIRO Armidale) on d~y 26
and 5 J.1g LH-RH on day 27 had more controlled follicular stimulation.
One had 3 newCL's and 2 eggs were recovered, while 4 others had 2
CL's. In animals treated with 50 IU PMS on day 23, anti-PMS on day 26
and either 5 I1g LH-RH (HRF, Ayerst) or 50 IU HCG (Primogonyl,
Organon), 3/4 mated on day 27 and on day 30 at autopsy one RCG treated
animal had 4 uterine blastocysts of 40 cells.
In 1986-88, we measured plasma luteinising hormone (LH) ,
progesterone (P) and oestradiol 173 (E2B) to further monitor response.
Eleven animals treated with 50, 25 or 15 IU PMS at day 22, 100 1 anti­
PMS at day 25, and 5 I1g LHRH or 50 IU HCG at day 27 led us to adopt
either 15 or 26 IU as the optimum dose for follicle stimulation
although no ovulation resulted. All animals receiving 50 IU PMS had
grossly elevated concentrations of E2 3 (80-100 pg/ml) while P & LH
concentrations were normal for that stage of the cycle whereas those
receiving 15 or 25 IU had levels similar to those ~t oestrus (15
pg/ml). In 1988 8 animals were treated with 15 IU PMS on days 22 & 23
100 111 anti-PMS on day 25, and 50 l1g LH-RH at 09.00, 13.00 and 18.00 h
either on day 27 (n=4) or day 26 (n=4). Four and 3 blastocysts were
recovered from 2/4 animals in the last treatment group.
We conclude that it is possible to superovulate macropodid
marsupials, but that results may be highly variable, and that timing is
critical. Further refinements of the last treatment groups should
allow us to develop a more reliable method.
-
37
THE EFFECT OF GONADOTROPHIN RELEASING HORMONE AGONJST ON
SHEEP prrUrrARY AND OVARIAN FUNCTION
M. sathanandan, S.K. Walker*, LJ. Clarke**, C.D. Matthews
Def>C!l!ment of Obstetrics and Gynaeoclogy, University ~ Adelaide
and Department of Agri.cuJt.ure, Rcsedale, 5350 and MRC, Pr:ince Henry's
Hcspital, Melbourne, 3003.
Variation in response to gonadotrophin treatment is a limitation to embryo
collection in the sheep. GnRH agonists (GnRHa) have aided synchronous
follicular development in the human and this phenomenon may be applied to
domestic species. The aims of this study were (a) to determine the time
required for pituitary down regulation fallowing GnRHa and (b) to assess the
effect of PMSG on ovulatory response fallowing GnRHa. Four prC9'esterone
treated Merino ewes were administered Leuprolideacetate (GnRHa) (Abbott;
1mg/ewEV'day) for 14 days. GnRH Ohtervet; 50ug iv/ewe) challenge tests were
performed at day 0, day 7 and day 14.
Table:
Peak LH and FSH concentrations (ng/m}) fallowing GnRH challenge
prior to (day 0) and during GnRHa treatment (day 7 and 14). Mean
basalleve1s (n=3) in parenthesis.
Sheep Day 0 Day 7 Day 14
no. LH FSH LH FSH LH FSH
1 107.3
(1.0)
9.2
(9.0)
2.8
(1.3)
9.7
(2.5)
2.8
(1.7)
15.9
(9.3)
2 205.0
(2.0)
24.6
(13.4)
4.3
(3.3)
3.6
(3.0)
2.0
(1.4)
15.8
(13.9)
3 113.1 26.6 4.5 10.3 2.1 16.3
(1.4) (11.0) (1.6) (8.8) (1.6) (14.0)
4 45.2
(1.7)
22.4
(14.9)
2.4
(1.4)
10.0
(8.6)
2.1
(1.5)
18.7
(16.3)
Peak LH and FSH responses to GnRH (peak/base1ine ratio) were significantly
rerluced from day 7 compared with Day O. Baseline FSH was elevated at day 14
compared with day 7.
Ovarian response (either number of corpora lutea (CL) or CL + large unruptured
follicles) was assessed by Japarcscopy at two dcses of P MSG (1200ID and 24001U)
following 14 days GnRHa treatment (n=12/group). Contro1s received similar
PMSG dcses without GnRHa. GnRHa reduced the number of CL (P

38
GALACTOSYLTRANSFERASE ACTIVITY ON SPERM SURFACE AND
IN EPIDIDYMAL PLASMA OF SEVERAL MAMMALIAN SPECIES
Y. Tang, the late D. E..Brooks, A.M. Snoswell and B. P. Setchell
Department of Animal Sciences, Waite Agricultural Research Institute,
Glen Osmond, SA 5064
Galactosyltransferase (GalTase) activity is present on mouse sperm surface (1) and in
epididymal plas~a of mouse and rat (1, 2). It has been reported that in mouse the sperm
surfa~e GalTase IS the sperm receptor for the zona pellucida (ZP) in sperm-zona binding,
and hIgh sperm surface GalTase activity is related with preferential fertilization ability (1).
Sperm-zona binding is not strictly species specific, ego mouse sperm can bind with rat ZP
(3). Therefore, it is interesting to see if GalTase can be found on sperm surface of other
species. The GalTase activity in caudal epididymal plasma (CEP) has also been examined,
because of the absoption of soluble epididymal proteins by epididymal sperm.
C~P and sperm v:ere coll~c~ed from rats, rabbits, rams and boars either by
punctunng or by cannulatmg the epIdidymal duct. Mouse sperm were collected by mincing
the tissue (1). The enzyme assay procedures are essentially the same asreported (1, 2).
Sperm surface GalTase activity (pmole substrate transferred I 1.5 hr. ) in mouse rat
sheep and pig , ,
Sperm Number xlO-6 0.4 0.8 1.6 3.2
Mouse (1 Group of 24) 11.7 28.5 62.7
Rat (2 Groups of 2) 12.3±2.8 21.9±1.9 33.4±1.3
Ram n=3 10.9±3.5 18.9±10.5 48.2±20.9
Boar n=3 2.2±0.8 6.2±3.7 10.6±1.8
CEP. GalTase activity (pmole substrate transferred I ug protein· 20 min. ) in mouse, rat,
rabblt, sheep and pig
Mouse
n=3(groups)
12.3±4.1
Rat
n=8
5.5±1.7
Rabbit
n=4
9.9±3.9
Ram
n=8
7.1±2.3
Boar
n=8
9.4±2.1
The results show that GalTase is present on sperm surface and in caudal
epididymal plasma in several mammalian species. The enzyme activity was lower·in
plg sperm (p

4[1
41
CORRELATION BETWEEN HU}ffiN SPEID1 MORPHOLOGY, ACROSOMES,
ACROSIN AND FERTILIZATION IN VITRO
De Yi Liu and H W Gordon Baker
Department of Obstetrics & Gynecology, University of
Melbourne and Reproductive Biology Unit, Royal Women's
Hospital, Melbourne, VIC 3053
The human sperm acrosome reaction is important for zona
binding and penetration of human oocytes both in vivo and in
vitro. Acrosin, a neutral proteinase, is one of the most
important acrosomal enzymes. In order to determine whether the
proportion of sperm with normal intact acrosomes, and acrosin
levels are related to fertility, sperm samples remaining after
preparation for in vitro fertilization (IVF) in 119 treatments
were studied and related to the proportion of oocytes which
fertilized in IVF. Sperm normal intact acrosomes in semen and
in insemination medium were examined by staining with
fluorecein isothiocyanate conjugated pisum sativum agglutinin
(FITC-PSA) according to the method described previously. 1,2
Acrosin activity of sperm in semen was determined by a
spectrophotometric method. 3 Sperm concentration in semen and
in insemination medium, motility and motility index, viability
(% live) and normal morphology in semen were also examined by
standard methods.
The results showed that acrosin levels were only
correlated with motility index (p < 0.05) and proportion of
sperm with normal intact acrosomes (p < 0.05). The percentage
of sperm with normal intact acrosomes in semen was strongly
correlated with motility (p < 0.001), motility index (p
< 0.001), viability (p

SEMINAL
42
PlASMA RErINOLr-BINDING PROI'EIN AS AN INDEX OF
HUMAN SERIOLI CELL FUN:TIOO
Ann J Conway, Lyn 11 Boylan, *Margaret Wocx:i, David J Handelsrran
DE:;partment of l1edicine, University of Sydney and Andrclogy Unit &
*DE:;partment of Biochemistry, Royal Prince Alfred Hospital, Sydney
Sertoli cells synthesize vitamin A-binding proteins in-vivo and
these ITB.y be involved in the essential requirement for vitamin A by
spenre.togenesis. Therefore VJe sought to determine whether hUITB.n
seminal plaSITB. retinol-binding protein (REP) could represent a direct,
non-invasive ITB.rker of Sertoli cell secretory function in-vivo
comparable with seminal plasma transferrin (TRFN) which is prinlarily
of testicular origin and the levels of which are correlated with rate
of spenre.togenesis. Therefore VJe have studied the relationship betvveen
seminal and blocx:i plaSITB. levels of REP and TRFN in 61 men aged 25-49
yr undergoing semen analysis as spenn donors or during infertility
investigations covering a wide range of sperm output and quality from
nonre.l to various degrees of abnonre.l spenre.togenesis (range spenn
density 0-458 MlOO, ootility 0-70%). REP and TRFN were measured by an
imnunoturbidometric assay in spenn-free seminal plasma and concurrent
blocx:i samples.
Number
REP (ug/ml)
(ug/ejac)
TRFN (ug/ml
(ug/ejac)
Number
REP (ug/ml)
(ug/ejac)
TRFN (ug/OO
(ug/ejac)
SPERM DENSITY
10M/ml
20 41
21+6 21+5
84+28 80+25
34+5 * 54+6
118+18 * 178+20
MarILE SPERl'1
10M/ml
22 39
20+6 21+6
83+25 80+26
33+4 55+7
118+17 181+21
(Mean+SEM,
MOI'ILITY
50%
36 25
26+7 12+3
105+31 47+12
50+7 44+4
162+20 1 53+23
PLASMA FSH
nUll
17 20
24+10 14+4
81+32 62+23
58+13 38+5
191+35 129+18
*p 10IU/l
29 6
18+6 23+10
67+22 96+52
49+8 45+7
162+23 161+30
Similar lack of significant differences in REP VJere also observed
for total and total nntile sperm output and plaSITB. testosterone
levels. REP and TRFN were correlated in blocx:i (r=O. 285, p=0. 026 ) and
(weakly) in seminal (r=0.200, p=0.122) plaSITB. hOVJever there was no
correlation between blocx:i and seminal plaSITB. levels of TRFN (r=0.054)
or REP (r=O. 006). We conclude that seminal, but not blocx:i, plaSITB. TRFN
is correlated with spenre.togenic output but not sperm rrotility or
testicular endocrine function whereas neither seminal nor blood plaSITB.
REP are correlated with testicular exocrine, endocrine or sperm
function. This indicates that seminal plaSITB. REP is unlikely to be a
useful ITB.rker of human Sertoli cell function in-vivo.
43
EFFECTS OF MULTIPLE M..ATING ON EPIDIDYMAL SPERM DISTRIBUTION
IN THE BROWN MARSUPIAL MOUSE, ASTUARTII
D.A Taggart & P.D. Temple-Smith
Department of Anatomy, Monash University, Melbourne, Victoria.
The cauda epididymidis in Amechinus stuartii (brown mars~pial mouse) is
characterized by an extremely variable epithelial height ~sso~iat.ed.wIth. a n~rrow a~d
irregular lumen(l) suggesting that sperm stora~e capa~Ity IS limIted. m thIs sp~cles
(2,3). This study examined the effects of multIple matmg to test thIS hypothesIs by
assessing sperm distribution in the epididymis.
Males were hemicastrated in July before the mating season and sperm
concentrations in various segments of the epididymis were determined in groups of
animals given either no access (controls) or restricted access to females (caug~t J~ly,
mated three times in August), before complete castration in August and the estImatIOn
of sperm concentrations in the rema.ining ep~didymis. T~ese result~ were compared
with a group caught and mated three tImes durmg the breedmg season m August.
Epididymal sperm numbers reached a maximum in July (approx. 2 million), with
peak concentrations of s~e:m i~ the distal 7aput a~d distal corpus segments. Sperm
distribution along the epIdIdymIS changed m relatIOn to the date the sample~ were
collected. Very few sperm remained in the epididymis, even in cont.rol groups, ill late
August (approx. 200,000), indicating a significant loss of sp.erm vIa ~permatorr~ea.
The mean sperm content in the caput and proximal corpus regIOns comb~ed, the dI~tal
corpus region and the caudal region of each epididymidis in August pnor t.o matmg
totalled 494,000 ± 263,000, 717,000 ± 214,000 and 417,000 ± 123,000 respectIvely.
than 300,000 sperm were delivered (per epididymidis) with each matir:g (e~timated by
determining the total epididymal sperm number before and after matmgs m August),
suggesting that epididymal structure may be influencing sperm release.
This study provides important information on the effects of mating. on ep~didymal
sperm distribution in A. stuartii and suggests that spermatozoa from thIS specIes must
have a remarkabIv high success in the female in terms of the absolute number and the
proportion of spermatozoa inseminated.
(1)
(2)
(3)
Taggart, D.A & Temple-Smith, P.D. (1985) Proc. 17th Ann. Conf. Aust. Soc.
Reprod. BioI. p101, Adelaide, Sth. Australia.
Bedford et al (1984). Proc. R. Soc. Lond. B221,221-233. .
Taggart, D.A & Temple-Smith, p.o. (1988). Cell Tiss. Res. (submItted).
Less

44
ULTRARAPID FREEZING OF 2-CELL MOUSE EMBRYOS
Shaw 3.M.
and Trounson A.a.
Centre for Early Human Development, Monash Medical Centre, Monash
University, Clayton Vic. 3168.
The ultrarapid embryo freezing technique developed by Trounson et
at. (1,2) for early cleavage stages, gave promising results with
2-cell mouse embryos (up to 53% blastocysts and 26% fetuses), but
there was scope for improvement.
They used cryoprotectant solutions containing up to 4 M Dimethylsulfoxide
(DMSO), and 0.5 or 0.25 M sucrose in Dulbecco's phosphate
buffered saline with 4 mg/ml bovine serum albumin (BSA). Embryos were
exposed to this solution in a dish for 1 minute, then loaded into
plastic insemination straws containing 3 beads of cryoprotectant
solution. Two minutes later the straw was plunged directly into
liquid nitrogen.
We have modified this technique and are acheiving excellent
results in vitro and following transfer of intact embryos to
pseudopregnant recipients (Table 1). The development of the ultrarapidly
frozen and thawed embryos in vitro is not statistically less
than their non-frozen controls in the 4.5 M DMSO group.
TABLE 1.
Development in vivo and in vitro of slow cooled, and
ultrarapidly frozen 2-cell embryos.
BLASTOCYSTS/TOTAL N. FETUSES/N. TRANSFERRED
Frozen-thawed Solution contr. Frozen-thawed Control
N (%) N (%) N (%) N (%)
slow cool 43/60 (71 )** 46/50 (92) 92/130 (71) 62/84 (74)
3.0M UR. 242/280 (86)* 151/163 (92) 106/157 (68) 124/179 (69)
4.5M UR. 257/280 (92) 143/150 (95) 48/79 (61) 29/47 (62)
* Lower than own control P

2.4
46
ALLOCATION OF CELLS TO INNER CELL MASS AND TROPHECTODERM IN
MOUSE EMBRYOS BIOPSIED AT THE 4-CELL STAGE.
Gino Somers and Leeanda Wilton.
Centre for Early Human Development, Monash Medical Centre, Clayton.
It has been proposed that pre-natal diagnosis could be carried out
in the pre-implantation embryo by biopsy of a single cell. However, it
has recently been shown that such embryos have a significantly reduced
implantation rate (1). In the developing embryo, the first
differentiative step occurs at the transition from morula to blastocyst,
with the emergence of the inner cell mass (ICM) and trophectodermal (TE)
lineages. As the ICM gives rise to all tissues of the foetus proper, it
is critical that the number of cells in this lineage is sufficient for
successful implantation and development. Consequently, we have examined
the possibility of a disturbed ICM:TE ratio being responsible for the
decreased implantation rate of mouse embryos biopsied at the 4-cell
stage.
Four-cell embryos were flushed from F1 (CBA x C57) mice which had
been superovulated with sIU PMS followed 48 hours later by 5IU hCG. The
zona pellucida was removed by brief exposure to aCid-Tyrode's solution
(pH 2.5) and the embryos cultured in Ca-H-/~1g-H--free medium 2 U12) for
60-90 minutes. Gentle pipetting with a flame-polished micropipette
dislodged one blastomere and produced 3/4 embryos. Controls included
untreated embryos and embryos in which one cell was dislodged and
immediately re-aggregated. After 42 hours in culture (96 hours post-hCG)
in medium' 16 (M16) in 5% CO, embryos had reached the early blastocyst
stage and were fixed using -the fluorochromes propidium iodide and
bisbenzimide to stain the TE or IC~l respectively (2). The number of
cells allocated to each lineage was counted.
EMBRYO NUMBER CELL N~ffiER(~mAN ± S.D.)
TREATMENT OF EMBRYOS TOTAL ICH TE RATIO ICH:TE
UNTREATED 37 28.9+4.7 11.9+2.6 17.0+3.1 0.70
RE-AGGREGATED 37 26.9+4.0 1l.0+2.2 15.9+2.9 0.69
BIOPSIED (3/4) 35 20.3±4.3 7.3±2.0 13.0±3.3 0.56*
*Significantly different to re-aggregated embryos, P

48
IN VITRO DEVELOPMENT OF GOAT PREIMPLANTAnON EMBRVOS IN
COCULTURE WITH OVIDUCT EPITHELIAL CELLS
D. SAKI1:ment of o~etrics and Gynaecology, University of Adelaide, Box 498
GPO, Adelaide, and Turret&.-ld RESearch Centre, Department of AJTiculture,
R03eda1=, SA 5350.
Pronuc1-ar embrycs are required for studies on the microinjection of foreign
gens; and on embryogenesis. A previou:3limitation to such studiES in sheer; was
the availability of a culture system which \tHS able to support the development
of embryos of this age. In our laboratory, h.igh rate of development of
pronuclear embryos (50-80 % blastocysts) are now regularly obtained in
synthetic oviductuuid medium (SOFM) (1) without soma::ic cell support. This
atBtract prESents information on the viability of embryos after 1, 3 or 5 days of
culture.
Pronucleac embrycs ~',1ere cultursJ in SOul droplets of mediu m under
paraffin oil in an atmosphere of 5 % CO), 5 % 02 and 90 % N 2
. He::tt inactivated
hum3.n serum (20 % concentration) 1';3.3- the preferred protein source and the
NaHC 03 concentration of 25m M was par-ely rebJIace:1 \o,'ith Hepes at
concentrations of 12.5-17.5mM. 'T'empe..rature of the incub3.tor was 38°C,
humidity 93 % and pH of medium was calailatei.l to be 7.47±0.01l.
Viability after culture was as..sessed by transfer of embryos to synchron.i~2d
c.::ci;Jients which were slaught:~red on day 13 of pre;Jnancy and the nll11 :.:2c-:Jc
,;::1Dngated conC89b~ses deter mined.
C3.t>le 1.
Effect of cult:uce in SOFM on the viability of oronuclJ":;"I.r'".:,:'n;xy-:J;;.
;::; ay; ill culture
Control 1 3 5
,0. embryo transferred 36 42 51 36
Jo. recovered 35 40 51 35*
10. elon'jatej 34 39 48 19
(% ) (97.1) (97.5) (94.1) (54.3)
* ill elongated concer>tusES obtained from ern~ry:::B which had
com menced blastulation at the time of transfec (19/22 v. 0/13).
Th,:; results indi':::ate that SOF M can be used for the cllltJre of pr-onuclear
e :nrXy03 f:x 3t least 3 days without jeopanlizinj 'liability. Bmbryos which had
not com mencei blastul3.tion at the time of transfer (day-5 'jroup) f;ill.e::1 to
de-Jelop beyond the Jila.:,""tocy.-3t stage possibly due to an asynchrony between the
developmental age of the embryo and the stage of the recipient's repro::1uctive
cycle. Despite this, the culture .system de3cribec1 provides a simple and easy
means of asse'""~g emtlryo 'Viability.
(1) Tervit, H.R., Whittingham, D.G. and Rowson, L.E.A. (1972).
,J. Repro::1. FeLt. 30: 493-497.
Supported by A['1 LROC.

50
THE ANTIGESTAGENS RU486 AND ZK299 ARE NOT BOUND BY THE UTERINE
PROGESTERONE RECEPTOR OF THE TAMMAR WALLABY, MACROPUS EUGENII
51
PROTEIN SECRETION PATTERNS OF SEPARATED OVINE ENDOMETRIAL CELLS
CULTURED IN DUAL ENVIRONMENT CHAMBERS
~__Uetcher and D.R. Blandon R.A. Chernx and J.K. Findlay
Department of Anatomy, Monash University, Victoria, 3168 Hedical Research Centre, 110nash l'kdical Centre, Prillce !h=llry'
Hospital Campus, Melbourne, Victoria, ]004.
H[4813 (Mifr:pristone) and ZK299 are structurally rel.ated syntheLic
st.eroids which have anti-progest.erone and anti-glucocorticoid
properties. R[486 binds to the uterine progesterone receptor of rats,
rabbits, dogs, monkeys and humans with an affinit.y eCjual to, or greater
than, progesterone (1,2). We have devc-doped an a~,SHY to measure the
proge~d.erone receptOl" in cytosol preparations from the endometrium of
the talluuar, using [3H]-ORG 2058 as the specific competitor. Binding
was measun'd by incubat.ing different. concentrations of Lhe test
compounds with the cytosol preparation and comparing the effect on
tutul binding in the absence of competit.or. The assay has been
validated using rat and rabhit uterine cytosoL preparations which gave
values consistent. \oJith published recept.or concentrations at similar
stages of the reproductive cycle.
Table 1. The effect of competitor concentration on h:inding of (3H]-ORG
2058 to tammar endometrium progesterone ['("ceptors
Prog
I 3
(nM/I)
10 100
Hal n 37 22 10 25
ftahbi t 49 26 11 4 27
TalJUnar n 72 72 51 38
ORG 2058 (nM/l)
1 3 10 100
14 9 8
15 7 :3
30 L7 l5
RU 486 (oM/I) ZK 299 (nM/I)
10 lOO 1000 10 100 1000
40 1 B
68 2 t3
80 T~ 100
Both progesterone and ORG 205H displaced the [3H]-OHC; 2058 from
tammar cytosol preparations, but neither RU486 or ZK299 displaced label
at concenlr

52
THE EFFECTS OF A SPECIFIC PLATELET ACTIVATING FACTOR (PAP) RECEPTOR
ANTAGONIST (SRI 63441) ON SOME ASPECTS OF MATERNAL PHYSIOLOGY
N. R. Spinks and C. O'Neill
Human Reproduction Unit, Royal North Shore Hospital of Sydney, St.
Leonards. NSW, 2065
To test the influence of embryo-derived PAF on the earliest stages
of implantation, eight week old Quackenbush strain mice were induced to
ovulate with 3 i.u. pregnant mare serum gonadotrophin and human
chorionic gonadotrophin, given 48 h apart, and mated with fertile
males. Animals were injected. intraperitoneally, with 40ug SRI 63-441
(Sandoz. USA) in 200ul Dulbecco's phosphate buffered saline (PBS) or
200ul PBS alone.at 1600 h on day 1 (day of vaginal plug) and 0900 h on
days 2-4 of pregnancy. On day 5, animals were injected intravenously
with O.15ml of a 1% solution of pontamine sky blue. The uteri were
examined. 15 minutes later, for the presence of blue bands. indicating
the site of embryo implantation.
The mean number of implantation sites per uterine horn in the SRI
63-441 treated animals (2.17 ~0.69) was significantly (P

54
RESPIRATORY PROPERTIES OF THE MOUSE UTERINE ENDOMETRIUM
DURING EARLY POST-IMPLANTATION PREGNANCY
Barbara Williams and R.N. Murdoch
Department of Biological Sciences, University of Newcastle, NSW 2308
A transient decline in the respiratory activity of mouse uterine
endometrial tissue on day 6 of pregnancy was a finding stemming from
recent investigations (1). Although similar responses were earlier
described in the rat (2,3), very little work has since been undertaken
to explain the regulation of this phenomenon or its importance to the
maintenance of decidual tissue for the continued support of embryonic
development. In view of this, the present study was initiated to
further - investigate the respiratory properties of the early
post-implantation uterine endometrium.
The O 2
consumption of OS mouse endometrial tissue, measured by
Warburg manometric techniques and expressed in relation to its DNA
content on days 5, 6 and 7 of pregnancy and pseudopregnancy (day 1 :
day of vaginal plug), transiently decreased on day 6 only when the
uterus had been adequately stimulated to mount a decidual cell
reaction. This was achieved naturally by implanting bIastocysts
during pregnancy, and artificially by the introduction of 30 ul of
peanut oil into the uterine lumen on the afternoon of day 4 of
pseudopregnancy. Adenosine (50-500uM) had no significant effect on
the respiratory pattern suggesting that the response was not due to a
competition for a limited supply of cofactors common for both
glycolysis and oxidative phosphorylation. In addition, the response
was not effectively altered by supplementing incubation media with
either glucose (5mM) or glutamine (1 mM), indicating that endogenous
s~bstrates support respiration at these times. Ho~t.ver, the tissue
dlsplayed 14
some capacity to oxidise exogenous [U- C] glucose and
evolved CO 2
in a pattern consistent with that of 0 uptake.
Respiratory quotients suggested that utilizable &ndogenOUs
carbohydrates were rapidly depleted during incubation in vitro and
that, subsequently, lipids were probably utilized as respiratory
fuels. Experiments conducted with rotenone implicated mitochondrial
processes as being responsible for the 0 consumption of the tissue.
However, no significant changes were dete€ted in uterine levels of S ­
hydroxybutyrate to account for the lIballooninglt of mitochondria that
has been suggested to also occur transiently on day 6 of pregnancy
(4) •
In conclusion, the results indicate that the transient decline in
respiratory activity of the endometrium on day 6 of pregnancy is a
function of decidual cells and involves alterations which are
ultimately exerted at the mitochondrial level. Further work is
required to elucidate the regulatory mechanisms concerned and to
establish the extent to which the phenomenon is important for the
maintenance of decidualized tissue.
(1) Murdoch, R.N. (1987) Teratology 35, 169-176.
(2) Saldarini, R.J. &Yochim, J.M. (1967) Endocrinology 80, 453-466.
(3) Surani, M.A.H. &Heald, P.J. (1971) Acta Endocrinol.156, 16-24.
(4) Jollie, W.P. & Benscombe, S.A. (1965) Amer.J.Anat. 116, 217-236.
55
DISTRIBUTION OF OESTROGEN RECEPTORS IN THE FEMALE REPRODUCTIVE
TRACT OF THE FLYING FOX PTEROPUS SCAPULATUS
Craig S. Pow and Len Martin
Department of Physiology and Pharmacology, University of
Queensland, St Lucia, Qld 4067
Flying foxes are monovulatory seasonal breeders. Both ovaries
function and are thought to ovulate alternately in successive seasons.
Both horns of the uterus function, but preimplantation endometrial
development is limited to the cranial tip of the horn adjacent to the
single preovulatory follicle and subsequent corpus iuteum (1). We have
proposed that localised growth is due in part, to preferential
delivery of sex hormones from ovary to ipsilateral uterus via countercurrent
transfer between the ovarian vein and coiled ovarian artery,
the major blood supply to the cranial tip of the uterus (2). However,
endometrial growth may also be limited by a restricted uterine
distribution of hormone receptors. A peroxidase anti-peroxidase (PAP)
immunocytochemistry kit (Abbott) was used to explore this possibility.
Two bats· were used: one untreated, one given 5 pg of oestradiol
(Ez) subcutaneously 24 h before autopsy. Tissue was dissected out,
frozen in liquid Nz, and cryostat sections (10 pm) fixed in 3.7%
formaldehyde-PBS (15 min), cold methanol (5 min), and acetone (3 min).
After treatment with blocking reagent (goat serum, 15 min), they were
incubated (30 min) with rat monoclonal antibody H222, which was
raised against human oestrogen receptor (ER), but cross-reacts with
many mammalian ERs (3). ER positive MeF7 breast cancer cells were
treated similarly. As negative controls, MCF7 cells and bat sections
were incubated with normal rat antibody. Goat anti-rat antibody was
used to link primary antibody and PAP complex. Diaminobenzidine was
used as chromogen with haemotoxylin counterstain.
Negative control sections and cells did not stain. In
preparations incubated with H222, endometrial gland and luminal
epithelial nuclei stained intensely throughout each uterine horn.
Stromal and myometrial nuclei also stained, as did those of MCr7
cells. Intensity of staining of individual nuclei varied, but there
was no difference in the degree of nuclear staining between Ez primed
and unprimed horns, and no cytoplasmic staining. Overall the pattern
is similar to that obtained with H222 in primate uterus (4).
Significantly, cells of the vaginal epithelium, which is unresponsive
to oestrogens in Pteropus (5), did not stain.
Since staining occurred along the length of each horn, it appears
that limitation of endometrial development is not dictated by receptor
distribution.
(1) Marshall, A.J. (1949) Proc. Linn. Soc. Lond. 161: 26.
(2) Pow, C., &Martin, L. (1987) Proc. ASRB 19: 1~
(3) Greene, G.L., et ai. (1984) J. Ster. Biochem. 20: 51.
(4) McClelland, M.C., et ai. (1984) Endocrinol. 114: 20oi.
(5) Martin, L., et ai. (1987) Aust. Mammal. 10: 115.

56
EFFECTS OF GONADOTROPHINS ON FOLLICLE DEVELOPMENT DURING
PREGNANCY IN THE FLYING FOX PTEROPUS SCAPULATUS
Philip A.
Towers* and Len Martin
Department of Physiology and Pharmacology, University
of Queensland, St Lucia, Qld, 4067
Flying foxes are monotocous seasonal breeders. Both ovaries
function. Ovulation, alleged to alternate from side to side, is not a
simple reflex response to copulation. The 4-6 week breeding season
involves repetitive copulation which continues well into pregnancy.
Over this period preimplantation endometrial growth of the duplex
uterus is restricted to the tip ipsilateral to the single preovulatory
follicle or corpus luteum (CL). Consistent with this, peripheral
oestradiol and progesterone levels remain low until mid-pregnancy when
placental secretion predominates (1). Supplementary ovulations/CLs do
not occur. There are no authentic records of twin births. Nevertheless
our small series of specimens contains two cases of superfoetation in
which a foetus was found in each horn and appeared to have been
conceived -2 months apart. We therefore used gonadotrophins (GNs) to
determine if ovulation could be induced in pregnant Pteropus.
Six mid-pregnant (March) P. scapulatus were used: 4 received 4 iu
pFSH (Calbiochem, USA) s.c. daily for 4 consecutive days; 2 were then
killed on d4 while 2 received 250 iu hCG (Intervet, NSW) s.c. on d4
and were killed on d7. As controls, 2 received saline s.c. on d1, and
were killed on days 1 and 7. At autopsy ovaries were fixed, embedded,
sectioned and examined for follicular development.
In control animals, ovaries ipsilateral to the foetus contained 1
CL and small preantral follicles; contralateral ovaries contained
preantral/small atretic antral follicles. All ovaries contralateral to
the foetus, in GN treated bats contained large (>800 ~m) healthy
antral follicles but none were preovulatory, luteinized or had
ovulated. Each ovary ipsilateral to the foetus in these animals
contained 1 CL, but none showed any follicular growth.
The failure of GNs to induce ovulation in ovaries contralateral
to the foetus contrasts with non-pregnant non-breeding season
P. scapulatus in which they induce multiple ovulations by d4 (2).
This suggests that high circulating levels of placental hormones may
inhibit ovulation. The failure to induce any follicle development in
ovaries ipsilateral to the foetus could result from the same
inhibitory factors reaching the ovary at much higher concentrations
because of preferential transport from uterus to ipsilateral ovary by
counter current transfer. In Pteropus the uterine vein runs cranially
fusing with the ovarian vein to form a common sinus which completely
encloses the ovarian artery for much of its path (3). The preferential
inhibition of the ipsilateral ovary may reflect mechanisms involved in
the alleged alternation of ovulation from season to season.
(1) Towers, P.A., &Martin, L. (1984) Proc. ASRB 16: 9.
(2) Towers, P.A., & Martin, L. (1985) Proc. ASRB 17: 115.
(3) Pow, C., & Martin, L.(1987) Proc. ASRB 19: 100.
*Present address Riverina-Murray Inst. Higher Educat., Wagga, NSW 2650
57
OVINE UTERINE LYMPHATICS: IN VIVO PASSAGE OF INDIA INK FROM
UTERINE SUBSEROSA. MYOMETRIUM AND LUMEN.
R.G. Alders and J.N. Shelton
Department of Immunology, The John Curtin School of Medical Research,
Australian National University, Canberra, ACT.
The results of histological investigations into the location of
lymphatic vessels within the ovine uterus have b~en equivocal. T~e
work has been conducted in post mortem speclmens and has pald
particular attention to the distribution of vessels within the various
layers of the uterus. However, post mortem injection of dye provides
little indication of the ability of uterine lumen contents to enter the
peripheral lymphoid apparatus in vivo. Given that immunologi:al
responses within lymph nodes are usually dictated by the materlal
carried to the node by afferent lymphatic vessels and that several
sites which are recognised as immunologically privileged have been
found to have poor or absent lymphatic drainage, it was decided to
investigate the ability of a foreign substance placed within the
various layers of the uterus to be carried to the regional lymph nodes
in vivo.
The' uterus was exposed via a ventral midline laparotomy following
general anaesthesia in both pregnant and nonpregnant merino ewes.
Subserosal injections of 80 ~l of sterile india ink (II) were made
through a 30 gauge needle at 6 sites along both uterine horns.
Subserosal injections were made by inserting the needle just beneath
the outer serosal covering. Lumenal and myometrial injections were
made with the aid of a blunt 30 gauge needle inserted into either an
interplacentomal or placentomal region of the gravid horn. Lumboaortic
and medial iliac lymph nodes (DLN) were collected at necropsy
and fixed in 10 % neutral buffered formalin. Specimens were mounted in
paraffin blocks and 10 ~m sections stained with Giemsa.
In the majority of cases the II injections were made subsequent to
cannulation of a peripheral lymph vessel dr~ining the uterus. Cells in
the lymph collected post-operatively were examined under the light
microscope after cytocentrifuge smears were stained with a modified
Wright-Giemsa stain (Diff Quik; Lab-aids, Narrabeen, N.S.~I.).
Subserosal injection of II resulted in rapid transport (0: 10
mi nutes) to the, DLN in pregnant and nonpregnant ewes. Free and
phagocytosed II was evident within the subcapsular sinus and medulla of
DLN. The passage of II from deposits within the myometrium was less
rapid. In constrast, when II was injected into the uterine lumen of
pregnant and nonpregnant ewes no II was evident within DLN up to 2
weeks after deposition. II within the uterine lumen was found both
free and within mononuclear cells.
In ewes of various reproductive states the DLN displayed an
eosinophilic infiltrate and occasional mast cells within the medulla.
A dramatic eosinophilic infiltrate was observed at the site of the II
deposit in the myometrium.
These results indicate that there is effectively a barrier
preventing transport of II from the uterine lumen to the DLN. This
lack of lymphatic drainage may form part of the immunological
mechanisms involved in the maintenance of pregnancy.

58 59
The effect of pregnancy on the rate of the
biotransformation of caffeine
Fatmida Abdi and Irina Pollard
School of Biological Sciences, Macquarie University N.S.W. 2109
Caffeine, a methylxanthine, is consumed in coffee, tea, some cola
soft drinks or in analgesics and other over-the-counter drugs. Perhaps
most importantly, caffeine is consumed by children in chocolate and
cocoa drinks. Until recently caffeine was considered a mild stimulant
and harmless if taken in amounts not exceeding the equivalent of 6
cups of coffee per day. Recently, however, adverse effic~s of low
level caffeine intakes has been demonstrated on the fetus ' .
The concentration of caffeine and its major metabolites in the
blood 'and tissues of the pregnant rat was measured by HPLC and
compared to the non-pregnant rat. It was demonstrated that in the
pregnant rat the biotransformation and elimination of caffeine was
significantly (P

60
STUDIES OF BINUCLEATE CELLS IN THE PLACENTA OF GOAT, COW AND DEER USING
THE MONOCLONAL ANTIBODY SBU-3
C.S. Lee, K. Gogolin~Ewens, W.R. Mercer, #P. Wooding and M.R. Brandon.
Department of Veterinary Preclinical Sciences, University of Melbourne
and #Department of Cell Biology, Agricultural and Food Research Council,
Babraham, Cambridge, England.
The monoclonal antibody, SBU-3, raised against sheep trophoblast
microvilli, which recognises a population of binucleate cells and the
syncytium in the sheep placenta (1) also recognises the binucleate
cells in other ruminant placentae. The present studies investigate
the distribution of the binucleate cells and the subcellular localization
of the SBU-3-positive antigen in the placentae of the goat, cow
and deer.
Ti ssues of p1acentomes and interp1acentoma1 areas were obtained
from goat, cow and deer at different stages of pregnancy. Tissues for
immunohistochemical studies were fixed in 95% cold alcohol and the sections
prepared were stained with the indirect immunoperoxidase technique.
All tissues for electron microscope immunocytochemical studies
were fixed in 2% glutaraldehyde and ultrathin sections reacted sequentially
with SBU-3 and colloidal gold labelled goat anti-mouse antibody.
In all the specimens (goat, cow and deer) studied, the appearance
of SBU-3-positive binucleate cells was associated with the formation of
chorionic villi-The number of SBU-3-positive binucleate cells increased
dramatically with advancement of pregnancy and the most striking
feature was the high concentration of SBU-3-positive binucleate
cells at the tips of the chorionic villi. In the goat, the syncytium
covering the caruncular septa.was stained positively. However, in the
cow and the deer trinucleate cells were found at random in the epithelium
covering the caruncular septa. Electron microscopy revealed that
gold particles were local ized in the granules and the Golgi apparatus
of the binucleate cells and in the granules of the syncytia.
It is concluded that the syncytia in the goat placenta are derived
from the SBU-3-positive binucleate cells whereas in the cow and deer no
syncytium is formed and granule transfer seems to be the primary function
of binucleate cell migration. The histological classification of
the goat placenta is syndesmochorial while that of the cow and deer is
epitheliochorial.
(1) Lee, C.S., Gogolin-Ewens, K., White, T.R. and Brandon, M.R. (1985)
Journal of Anatomy ~, 565-576.
OCYTOCIN RECEPTORS
61
IN THE UTERUS OF THE BRUSHTAIL POSSUM
C. Sernia 1 , J. Garci~l, W.G. Thomas 1 and R.T. Gemmel1 2
lDepartment of Physiology and Pharmacology, 2Department of Anatomy,
University of Queensland, St. Lucia, 4067.
The role of pituitary oxytocin (OT) in marsupial parturition is
uncertain. However, evidence that the gravid uterus of the tammar
(H. eugenii) is sensitive to exogenous OT (1) suggests the presence
of uterine OT receptors in marsupials.
Receptors were prepared from uteri of ovariectomized, oestrogentreated
(30 ug/kg) possums using a published procedure. (2) For
comparison, rat and sheep uterine tissue was similarly prepared.
Receptors were measured by radioreceptor assay using a 3H-oyxtocin
tracer and PEG precipitation of receptor-bound OT. Receptor number
(Ro) and affinity (Kd) were estimated from Scatchard plots of the
data. Receptor specificity was characterized by inhibition
experiments with related peptides (dOT=des-amino-oxytocin, M=
mesotocin, AVP=Arg-vasopressin, LVP=Lys-vasopressin, IT=isotocin, VT=
vasotocin), an oxytocin-selective agonist (TGO= [Thr 4 , Gly7]­
oxytocin) an antidiuretic antagonist (PIA= [d(CH2)~, D-Phe 2 , Ile 4 ,
Ala 9 -NH]- AVP) and unrelated peptides (AII= angiotensin II, NT=
neurotensin). Table 1 shows that the Kd of the possum OT receptor is
similar to that of the rat and sheep. Under our experimental
conditions the Ro was lowest in the possum.
Table 1.
Affinity and Concentration of OT Receptors.
SHEEP RAT POSSUM
Kd (nmol/l) 2.5 ± 0.5 (n=5) 2.5 ± 0.5 (n=5) 3.8 ± 0.4 (n=8)
Ro (fmol/mg prot) 590 ± 40 250 ± 30 200 ± 60
Data shown as mean+/-s.e.m. n=number of estimates.
Table 2. Inhibition (%) of OT binding by analogues.
OT VT TGO AVP M dOT LVP IT PIA AII NT
SHEEP 100 120 96 96 94 85 64 55

A RELATIONSHIP BETWEEN NUTRITION AND PROGESTERONE AT PARTURITION AND
THE' IMPLICATIONS FOR LAMB SURVIVAL
62
63
IN VIVO MYOMETRIAL ACTIVITY IN PREGNANT RATS DURING THE
PERI-IMPLANTATION PERIOD
M.A. Brockhus, 1 K. Nunan 2 and Ron A. Parr 3 Linda R~ Crane and Len Martin
1 P~storal Research Ins titute, Hamilton, Victoria
3Dookie Agricultural College, Dookie, Victoria
Animal Research Institute, Werribee, Victoria
Two essential conditions for the survival of lambs are a sufficient
milk supply and a strong mother-offspring bond. Both conditions are
hormonally controlled and probably linked. Progesterone has been
shown to effect lactogenesis (1), as well as the ewes receptivity to
the lamb (2). Nutrition in late pregnancy can alter progesterone
patterns and delay lactogenesis (3). This study aimed to test the
effect of nutrition on progesterone concentrations, ewe behaviour and
lactation in Merinos at parturition.
Twenty-two Merino. ewes were divided into two groups and kept on
diets of either ad libitum hay (LOW) or 800g lupins/day plus ad lib
hay (HIGH) between days 120 of gestation and term. Blood samples
were collected regularly, stored, then analyzed for progesterone using
a radioimmunoassay. Continuous observations about parturition gave
records of times of lambing, grooming, lamb standing, ewe nuzzling,
lamb suckling and ewe feeding. At 48 hrs post-partum milk volume was
measured using a standard technique (4). See Table 1 for results.
Table 1 Plasma progesterone levels (ng/ml), milk vo1um~s at 48hrs (ml)
and behaviour of ewes at lambing on two planes of nutrition.
HIGH
LOW
Progesterone conc.(ng/ml) @ -48 hrs 5.12 ± 1.09 10.67~2.01 -24 2.96 ± 0.79 6.43 ± 1.68 *
o 0.84 ± 0.16 0.77 ± 0.21 NS
+2 0.23 ± 0.02 0.33 ± 0.09 NS
+5 0.23 ± 0.04 0.28 ± 0.09 NS
+10 0.03 ± 0.03 0.18 ± 0.01 NS
Milk volume (mls)
73.3 ± 5.4 34.7 ± 3.8 **
Time from lamb born to ewe
allowing to suckle (min)
32 ± 8 59 ± 12 NS
Time from lamb born to ewe
recommencing feeding (min) 124 ± 24 39 ± 10
Values are means + SEM: n=ll. * P < 0.05 ** P < 0.01 (ANOVA)
**
The slower decline of progesterone in the ~OW ewes supports the
view (3) that undernutrition affects progesterone secretion and/or
metabolism, and could contribute to the lower milk volume (p

64
INDUCTION OF MHC ANTIGEN EXPRESSION ON MHC-NEGATIVE PRIMARY
AND SECONDARY TROPHOBLAST GIANT CELLS
KinQ
N.J.C.*, Rodger, J.C.t, Maxwell, L.E.§ and Drake, B.L.·
*Dept. of Anatomy, University of Sydney N.S.W. 2006.
tDept. of Biological Sciences, University of Newcastle, N.S.W.
§John Curtin School, A.N.U., Canberra, A.C.T.
·Dept. of Cell Biology, University of Texas, Dallas, Texas.
Absence or low expression of surface major histocompatibility
complex (MHC) antigens on semi-allogeneic trophoblast cells during
implantation is a possible strategy for evading the maternal
cellular immune response, as MHC expression by target cells is
required for recognition by immune cytotoxic T (Tc) cells.
Cells of the preimplantation murine blastocyst and established
placenta express MHC antigens, but we have found, using immunogold
labelling and electron microscopy, neither primary (1°) nor
secondary (2°) (CBAxC57BLl6) F 1 trophoblast giant cells (TGC) outgrown
in vitro from 3.5 day post coital (pc) blastocysts, and 7.5 day
pc ectoplacental cones, respectively, express detectable quantities
of paternal class I (Kk) MHC antigens. Treatment of 2° TGC for 24­
40h with recombinant gamma-interferon (y-IFN) (Boehringer Ingelheim),
.produced significant paternal class I MHC antigen expression
but none was detectable on 1° TGC after ex, ~ or y-IFN treatment.
This suggests that during the early period of implantation; trophoblast
cell surface MHC is neither expressed nor inducible.
Concurrently, we have found infection of some embryonic cell
types with the flavivirus West Nile (WNV), causes an IFNindependent
increase in class I and sometimes class II MHC antigen
expression. Therefore, in an attempt to induce MHC antigens, we
infected 1 0 TGC monolayers with purified WNV. This induced de
novo paternal class I MHC antigen expression within 16h. Induction
is unlikely to be due to secreted WNV-induced IFN's or other
factors, as it occurred in the presence of high concentrations of
anti-exlB IFN antibodies, and was not induced by virus-inactivated
supernatants from MHC-induced 1° TGC cultures. Furthermore,
attempts to induce MHC expression with poly I:C or recombinant
tumor necrosis factor-ex in 1° TGC cultures failed.
Thus inhibition of constituent MHC expression on 1° TGC during
early implantation and the associated block to MHC induction is not
absolute. As such, manipulation of this phenomenon may
conclusively establish the significance of lack of MHC expression
in trophoblast cells of the implanting semi-allogeneic embryo.
65
DEVELOPMENr OF A CYNCM)LGUS mDEL 'IO SIUDY ffiffiNANCY-ASs::>CIATED
PLASMA PROl'EIN-A (PAPP-A)
;;1-._ Lee, J.P. WOlf l , R.F. Williams l , G.D. Hoogen l and M.J. Sinosich
Reproc1uctive Biochemistry and Immunology, Department of Obstetrics
and Gynaecology, Royal North Shore Hospital, St. Leonards NSW 2065
lpregnancy Research, The Jones Institute for Reproductive Medicine,
Eastern Virginia f.1edical School, Norfolk, Va. USA.
Pregnancy-associated plasma protein-A (PAPP-A) is a large
he:r;:arin-binding proteoglycan ",ith o(-electrophoretic mobility. In
the human, PAPP-A is uniquely distributed within the reproductive
tract, wherE' clinical and functicnal studies suggest a vital role
fOt this antigen in human reproduction. However to test this
hypothesis an animal mooel was required. Criteria for selection of
a model, included reversible binding to heparin, reversible
interaction with ilnrnobilized zinc ions, molecular size (Mr) of
820kd and secreted into maternal cirCUlation by a haemochorrially
implanted, placenta. The cynanolgus monkey proved a most suitable
model, and thus monkey (m) PAPP-A was purified fran cynanolgus
placental tissue and pregnancy serum. The antigen was injected
into NZ male rabbits and the resultant polyclonal artiserum used to
develop mPAPP-A radioimmunoasay. Distinct immunological
differences were apparent between cynanolgus and hmnan PAPP-A, with
the latter analog containing more recently evolved antigenic
determinants or epitoIA=s. P.s for t.he human anaJog, mPAPP-A was not
detected in blood of normal non-pregnant adults but was readily
measured in seminaJ plasma preovulatory follicular fluid and
ooternal blood. After initial detection at about 40 days gestation,
cirCUlating PAPP-A increased E'i

67
IMMUNOLOGICAL ASSESSMENT OF PATIENTS WITH RECURRENT PREGNANCY
LOSS AND UNEXPLAINED INFERTILITY.
S. Raymond, C Czapla-Peters and Y.C Smart.
Faculty of Medicine, University of Newcastle.
Recent studies have described immunological factors as important
in the aetiology of recurrent abortion and possibly unexplained
infertility.
To evaluate the importance of these factors 3 groups of patients:
were assessed:
a) 17 couples with proven fertility (two term deliveries
of normal hirthweight).
b) 30 couples with obstetrical histories of unexplained
recurrent pregnancy loss (URPL) divided into primary aborters (~3
miscarriages and no live children) and secondary aborters
(abortions after having children or stillbirths).
c) 17 Couples with unexplained infertility (UI).
Maternal sera were collected from all patients and screened for
the presence of antipaternal lymphocytotoxic activity, auto
antibodies, immune complexes (IC) and immunoglobulins (IG).
Results (table 1) showed that 4 of the 17 normal couples showed
antipaternal lymphocytotoxic activity. None of the 14 primary
aborters or the 17 patients with unexplained infertility showed
any antipaternal lymphocytotoxic ~ctivitx whereas 3 of the 19
secondary aborters did.
TABLE 1 Immunological Assessment
Antipaternal Auto
n Antibody Pos Antibodies
'1IC
IIG
FACTORS AFFECTING FETAL LOSS IN INDUCTION OF OVULATION WITH
GONADOTROPHINS:
INCREASED ABORTION RATES RELATED TO HORMONAL PROFILES IN CONCEPTUAL
CYCLES
suk-Yee Lam, H.W. Gordon Baker, James H. Evans, and Roger J. Pepperell
University of Melbourne, Department of Obstetrics & Gynaecology,
Royal Women's Hospital, 3053, Victoria, Australia
The aim of this study is to compare the fetal wastage in
gonadotrophin-induced pregnancies with that of spontaneous pre~nanc~es
before and after treatment and to analyse the risk factors pred1spos1ng
to such fetal loss. All the women treated with gonadotropin for
induction of ovulation at the Royal Women's Hospital between 1963 and
1985 were studied. Among 230 women, 36 first-trimester abortions
(9.7%), 16 second-trimester abortions (4.3%), 11 ectopic pregnancies
(2.9%) and 10 stillbirths (2.7%) occurred in 373 conceptual cycles
after gonadotropin induction of ovulation. Fetal loss (25 of 46
pregnancies, 54.3%) was higher in spontaneous pregnancies prior to
therapy (p

68
SEXUAL DIFFERENTIATION IN WALLABY POUCH YOUNG TREATED WITH
STEROIDS
G. Shawl, M. B. Renfreel , R. V. Shortl , 2 and Wai-Sum ()3
Departments of lAnatomy and 2physiology, Monash University, Clayton,
Vic., and 3Department of Anatomy, University of Hong Kong.
In marsupials much of gonadal differentiation occurs post natally,
so the young are readily accessible for experimental studies of the
hormonal control of sexual differentiation (1,2). We have investigated
how gonadal steroids can influence development of the reproductive
system in tammar wallabies by daily administration of estradiol
benzoate (E2) (n=4) to male neonates, and testosterone propionate (T)
(n=7) or oil (n=6) to female pouch young from the day of birth until
autopsy' on day 25. After dissection, the bodies were fixed and
serially sectioned.
In control females the ovaries were located in the abdomen, and
were well developed with the germ cells concentrated in the cortical
area. The Mullerian ducts (MD) were large, whilst the Wolffian ducts
(WD) were small and undifferentiated. A well developed pouch and four
mammary gland buds were present. The gubernaculum and processus
vaginalis terminated just inside the abdominal wall.
Testosterone treatment did not affect ovarian histology or
position, although it stimulated growth of both WD and MD. Pouch,
mammary glands and gubernaculum were all unaffected.
In control males the testes had well developed seminiferous cords,
and Leydig cells. The WD were well developed, but the MD were
regressing. The processus vaginalis and gubernaculum passed into the
centre of the well developed scrotum. The testes had completed their
trans-abdominal migration and were situated in the inguinal canal.
Testicular histology in the· E2 treated males was abnormal, with
reduced tubule diameter, necrotic germ cells, few, small Leydig cells,
and enlarged cortical areas containing germ cells. Although E2
treatment did not affect WD size, it prevented MD regression presumably
by inhibiting either Mullerian Inhibiting Substance (MIS) secretion by
the testis, or its action. The processus vaginalis terminated in the
inguinal region at the base of the scrotum, and the testes were located
high in the abdomen, at a similar level to that of females.
Thus, in the tammar wallaby, WD and MD development are affected by
exogenous steroids as they are in eutherian mammals, but scrotum, pouch
and mammary glands are unaffected by these hormones, supporting our
previous suggestion that differentiation of these structures is
independent of gonadal hormones (2). The prevention of both MD
regression and testicular descent further supports the hypothesis (3)
that MIS may mediate testicular trans-abdominal migration.
(1) Renfree, M.B., Shaw, G. & Short, R.V. (1987) in: Genetic Markers
of SeK Differentiation. pp 27-41. Eds F.P. Haseltine, M.E. McClure &
E.H. Goldberg (Plenum Press).
(2) 0, W.-S., Short, R.V., Renfree, M.B. & Shaw, G. (1988) Nature
(Lond.) 331:716-717.
(3) Hutson, J.M. (1985) The Lancet, Aug 24th, pp 419-421.
69
ANTIFERTILITY ACTIVITY OF GOSSYPOL IN MALE RATS AND
MANGANESE STATUS OF DIET
R. Vishwanath, I.G. White, P.O. Brown-Woodman,1 and J.R. Mercer 2
Department of Veterinary Physiology and 2Animal Husbandry, University
1 of Sydney, N.S.W.
Cumberland College of Health Sciences, Lidcombe, 2141, N.S.W.
There is good evidence that gossypol chelates with manganese (Mn)
and it has been suggested that the antifertil ity activity of gossypol
might be due to induction of Mn deficiency (1) which allegedly causes
degeneration of the testes of rats with loss of spermatids and
spermatozoa (2). This experiment was designed to investigate Mn
deficiency in male rats and to check if excess Mn in the diet would
mitigate the antifertility effects of gossypol.
Th i rty male rats were randomi zed into six groups and fed one of
three diets with or without gossypol in a factorial design. The diets
were Mn deficient, normal and Mn excess. Gossypol in 2% carboxymethylcellulose
vehicle was given orally (20 mg/kg body weight) each day.
On day 57 two female rats were caged with each male to test their
fertility and on day 64 the males were killed.
The mean body weight of control rats did not change significantly,
but rats given gossypol lost about 10% of their body weight. Neither Mn
nor gossypol significantly affected the weights of the testes,
epididymides, adrenals or seminal vesicles. However, there was a
significant decrease in weights of the coagulating glands of control rats
on the Mn deficient diet, and of all three groups receiving gossypol.
Fewer sperm coul d be recovered from the epi di dymi des of rats
receiving gossypol and most of these sperm were immotile and
morphologically abnormal with a large proportion of detached heads.
Gossypol greatly reduced the oxygen uptake of epididymal sperm from rats
on all three diets, and sperm from control rats on the Mn deficient diet
had a lower oxygen uptake than those on normal or Mn excess diets.
Gossypol rendered all male rats compeletly infertile, irrespective
of the level of Mn in the diet. The fertil ity of control rats on a Mn
defi ci ent di et was not affected although the Mn concentrati on of thei r
livers was reduced.
(1) Vishwanath, R. and White, LG. (1987) New Horizons in Sperm Cell
Research. Ed. H. Mohri. Japan Sci. Soc. Press. 431-437.
(2) Boyer, P.O., Shaw, J.H. and Phillips, p.H. (1942) J. Biol. Chern.,
143:417-425.

70 71
PROLONGED EFFECT OF SUBFERTlLE MALES IN REDUCING NUMBERS OF FETUSES IN
NORMAL FEMALE RATS.
J.L.Zupp and B.P.Setchell.
Department of Animal Sciences, Waite Agricultural Reasearch Institute,
University of Adelaide, South Australia.
When normal female rats were mated to males just before or after
a period of infertility produced by local heating of the testes, there
was an increase in embryonic mortality and partial fertilization
failure, even when the percentage of females becoming pregnant was no
different from control. This was reflected in a decrease in the
fetus/corpus luteum (F/CL) ratio between 7 and 14 days after the end
of a 7 'day mating period (1). In these earlier experiments, it
appeared that this effect continued for a considerable time after
fertility was regained, but because adequate controls were not
available for this aspect of the study, further experiments have been
undertaken to see how long the effect on F/CL ratio persisted.
Groups of 6 adult male rats (Porton strain in Exp.1, 3 and 4 and
Hooded Wistar in Exp 2) were anaesthetized with pentobarbitone sodium
(50mg/kg intraperitoneally) and their testes and scrotum immersed in a
water bath at either 43 or 33 0 C for 30 min. At various times after the
heating, each male was caged with 6 normal females for 7 days. The
females were then removed and autopsied after a further 7 days, when
the number of fetuses and corpora lutea were.90unted. There were·
significant differences in the F/CL ratio in 2 experiIilents for the
Il,ating period beginning 60 days after heating, associated with a
reduced percentage of feo.ales pregnant jthere was also a significant
difference in one experiment, but not in another in the F/CL ratio 90
days after heating, and no difference in 2 experiolents at 114 days.
Exp.No Days after %females F/CL ratio in
heating pregnant
pregnant females
Control Heated Control Heated males
90-97
50 42 0.80 0.58**
114-121
91 75 0.71 0.86
126-133
51 43 0.54 0.62
2 114-121 83 94 0.79 0.79
3 60-67
58 13* 0.64 0.34-
90-97
63 70 0.67 0.76
4 -7-0
53 81 0.68 0.69
60-67
54 29* 0.66 0.51.
.: P

72
DURATION OF PROOESTAGEN PRIMING ON THE RESPONSE OF
BORDER. LEICESTER. X MERINO EWES TO THE 'RAM EFFECT'
J.L. Reeve
Department of Agriculture & Rural Affairs
Rutherglen Research Institute, Rutherglen, Victoria.
Short tenn use of intravaginal progestagen sponges prior to the
introduction of rams to ewes in late Spring has been shown to
enhance the response to the 'rarn effect' (1),(2),(3). A high
proportion of primed ewes show oestrus at the 'rarn induced'
ovulation, when a majority of twins are conceived. 'Short' cycles
are eliminated and almost no responding ewes return innnediately to
anoestrus. In the initial experiment (1) progestagen sponges were
used for 4,6 or 10 days. The 6 day treatment was superior to the 4
,day in ewes conceiving at the ' rarn induced' cycle, and overall
fertility and fecundity. The 10 day treatment resulted in the
highest proportion of ewes lIIating at the 1st cycle but with fewer
ewes conceiving with lower fecundity than the 6 day. This study
examined the sensitivity of response to the duration of progestagen
priming between 5 and 9 days.
On 28 OCtober 1987 415 1.5 y.o. Border Leicester x Merino ewes were
treated with sponges (Repromap-60mg) and allocated at random to 5
equal groups. At intervals of 5,6,7,8,9 days the sponges were
withdrawn from a group of ewes which were then joined to 8% Poll
Dorset rams for 42 days. Mating was recorded daily and foetuses
were counted and aged by real time ultrasonography 13 weeks after
joining. In the table below, 1st CyCle refers to mating and
conceptions 2-3 days after joining.
Ewes
Foetuses
Conceiving Mean days Conceived Mean days Foetuses
Prog. cycle post cycle post per ewe
days n 1 2or3 joining 1 2or3 joining joined
-------------------------------------------------------------------
5 85 49 29 9.25 a 63 31 8.4 a 1.107 a
6 83 51 19 6.9 a 71 21 6.0 b 1.108 a
7 82 61 14 5.6 b 78 15 5.1 b 1.134 a
8 82 63 13 5.0 b 77 15 4.9 b 1.135 a
9 83 64 9 4.0 b 75 9 3.7 b 1.012 a
-------------------------------------------------------------------
Longer duration of progestagen priming increased the proportion of
ewes conceiving at the 1st cycle and concentrated oestrus behaviour
earlier in the post joining period. There was no overall effect on
foetuses produced per ewe joined.
The response to duration of progestagen priming has low, sensitivity
around an optimum of 7 or 8 days but it should be longer than 5
days.
(1) Reeve, J.L. and'Charnley, W.A. (1982). Proc. ASRB, 14:89.
(2) Reeve, J.L. and Charnley, W.A. (1983). Proc. ASRB, 15:71.
(3) Reeve, J.L. and Charnley, W.A. (1984). Proc. ASAP, ,15:161-164
Figure 1. Mean melatonin 1000
concentration in three pM
cows with four Regulin 500
implants. Shaded area is
endogenous night~time range.
(assay limit 112 pM)
73
EFFICACY OF MELATONIN IMPLANTS (REGULlN) IN BOS INDICUS COWS.
S R P
Sutherland and K.W. Entwistle
Graduate School of Tropical veterinary Science, James Cook University
Townsville, Queensland 4811.
Reproduction in Bos taurus cattle is affected by season and can be
modified by manipulation of the photoperiod environment (1). Therefore
it may be possible to use melatonin to modify reproduction in cattle.
The efficacy of Regulin implants (~egulin Ltd, Australia) to increase
circulating melatonin levels in Bos indiclls cattle was tested using 16
multiparous cows (mean liveweight 421± 7.7(SE) kg) kept on native
pasture. Each cow was treated with either 0, 1, 2 or 4 Regulin
implants (s . c.) at the base of one or both ears. All cows :were'bled
from the tail at 10pm and lOam at 0, 1, and 2 weeks, and at lOam, 3,
4, and 5 weeks after treatment. Night-time samples were taken by
torchlight. Before treatment melatonin was detectable (assay limit 40
pM) in the tail plasma of 12 cows at 10pm (95.2± 12.5 pM) and 4 cows
at lOam (50.2± 3.1 pM). One to tive weeks after treatment melatonin
was undetectable (assay limit 112 pM) in the tail plasma of most
animals in both day and night-time samples. At five weeks after
treatment jugular samples were taken from the cows without implants at
10pm, and from all the cows at lOam. In untreated cows the mean
jugular' concentration of melatonin at night-time was 426± 86 pM
(range 312 - 645). The night-time range reported for Jersey heifers is
190 - 1410 pM(2). Melatonin was undetectable «112 pM) in the day-time
jugular plasma of untreated cows and those with one implant. Melatonin
was detectable in the jugular plasma of three cows with two implants
(271± 97 pM) and all of the cows with four implants. One cow with four
implants had a very high concentration of melatonin (2645 pM)
suggesting abnormal liver function. The mean jugular concentration in
the other three cows with four implants was 227± 21 pM, about half the
mean endogenous night-time concentration. These three cows were
sampled for a further 13 weeks and during this period melatonin
concentrations were extremely variable, but were frequently within the
endogenous night-time range up to 16 weeks after treatment (Figure 1) .
6 8 10 12 14 16
weeks after 1reatment
conclude that' four subcutaneous Regulin implants elevate plasma
We
melatonin in cattle and may continue to deliver for up to 16 weeks. As
in sheep(3) melatonin released from the implants or from the pineal is
more readily detected in the jugular vein than in peripheral vessels.
(1) Hansen, P.J. and Hauser, E.R. (1984). Theriogenology 22:1-14
(2) Martin, T.C., Cunningham, N.F. and Saba, N. (1983). J. Endocr.
.9.6.:189-196
(3) Howse, A., Kennaway, D., Carbone, F., Staples, L. and Williams, A.
(1987). Proc. ASRB ~:20
The authors acknowledge Dr L.D. Staples (Regulin Ltd) for supply of
the implants and Dr D. Kennaway for melatonin assays.
18

74
SYNERGISTIC EFFECTS OF MELATONIN AND IMMUNISATION
AGAINST ANDROSTENEDIONE IN MAIDEN BL X MEWES
C.R. Earl, S: McPhee, R.H. Male and E.A. Dunstan
S.A. Dept. of Agriculture. Struan and Kybybolite Research Centres
Immunisation against androstenedione, Fecundin (Glaxo Aust.
Ltd.) and melatonin treatment, Regulin, (Genelink Aust. Ltd.) improve
the reproductive performance of crossbred ewes mated in summer. A
recent study by Dunstan et al (1) indicated that those two products
could act synergistically to improve lambing percentages but the
mechanism of action has not been identified.
In this study conducted at Struan, S.A. groups of maiden 1 1/2
year, old BL x M ewes received either no treatment (control n=63) a
Regulin implant at 4 weeks before mating (Regulin n=62) injections'of
Fecundin at 6 and 2 weeks (Fecundin n=64) or combined treatment with
both Regulin and Fecundin. All groups were grazed together and were
joined with 3% untreated Dorset rams on 4/1/88. Foetal percentages
were determined by mid pregnancy sonagraphy.
Table 1 - Fertility fecundity and distribution of litter size of
maiden ewes after no treatment, treatment with Regulin,
Fecundin or treatment with both.
Foetuses Per No. of ewes with
Group Ewe Ewe Preg. 0 1 2
Control n=63 1.11 a 1.37 a 12 32a 19a 0
Regulin 62 1.23 a 1.41 a 8 32a 22a 0
Fecundin 64 1.30 b 1.77 b 17 12b 34b 1
Combined 63 1. 56 b 1.81 b 9 15b 34b 5
a~b within
tables.
columns. P

76
THE EFFECTS OF NUTRIENT RESTRICTION AND OF LAMB REMOVAL ON OVARIAN
CYCLICITY AND ON THE INHIBITORY EFFECTS OF OESTRADIOL ON PlASMA
CONCENTRATIONS OF LH, AND FSH IN POST-PARTUM EWES
P.J
Wright, A.H. Williams and I.J. Clarke
Department of Veterinary Clinical Sciences, University of Melbourne,
Werribee 3030, Victoria, Australia
Corriedale ewes, 4-7 days post partum (PP) and non-post-partum
cyclic (C) ewes, were ovariectomized (OVX) during the ovulatory
season and received oestradiol (E) implants s.c. or remained
untreated. Ewes then received i) above-maintenance (M) nutrition,
ii) below maintenance (R) nutrition, or iii) R nutrition and lambs
rem9ved within 2 days of parturition (RW). Blood samples (3/ewe) for
the determination of plasma concentrations of LH and FSH were taken
over the period 18-24 days post partum or 12-18 days after
ovariectomy (C ewes). OVary-intact ewes also received treatments M
(n-20), R (n-26)" and RW (n-2l). Blood samples (2/week) were taken
from these ewes (from 7 to 60 days PP) for plasma progesterone
determination to assess the onset of ovarian cyclicity.
The plasma hormone concentrations (ng/ml) (mean (s.e.m.)) in
OVX ewes are shown below.
Treatment -==LH~ .-...F""SH ... _
group OVX OVX+E OVX OVX+E
n cone sig n cone sig conc sig cone sig
(a) values with no letters common are significantly different (P

78
P:t'rUITARY AND OVARIAN BESPOHSES OF POS':r-PAR'.rtJM ACYCL:tC :aBEl' COWS TO
CON'.rDlUOUS~ TREA'l'MENT WITH GnRH AND A GnRH AGONIST
M.J. D'Occhio and D.R. Gifford t
CSIRO, Division of Tropical Animal Production, Rockhampton, Qld
t SA Department of Agriculture, Turretfield Research Centre,
Rosedale, SA
Gonadotrophic hormone releasing hormone (GnRH) can be used to
promote ovarian activity and ovulation in acyclic post-partum (PP)
cows when given either by continuous infusion over 2-5 days or bolus
injection. Corpora lutea (CL) arising from GnRH-induced ovulations PP
tend to have a short life span, as do CL of the first spontaneous
ovulation PP. The present study examined whether sustained CL
function could be achieved with continuous long-term GnRH therapy.
Chronic GnRH could either ensure a normal life span of the first
induced CL by maintaining elevated LH or, alternatively, induce a
second ovulation with normal CL function. Multiparous PP (17.1 i: 0.6
days) acyclic beef cows received continuous GnRH or buserelin ([D-Ser
(tBu) 6, Pro 9 NEt] GnRH) from s.c. osmotic minipumps(2ML4) designed to
remain active for 28 days. Plasma LH, FSH and progesterone (P 4
) were
determined by RIA and P4 concentrations ) 1 ng/ml were taken as
indicative of active CL tissue. Results for P4 and CL are shown in
the Table.
P4
'Treatment n ) 1 ng/ml
corpus luteum
short-lived
extended
control 5 0 0 0
200 ng GnRH/kg BWjh 5 4 4 0
400 ng GnRH/kg BWjh 4 4 3 1
5.5 ng buserelin/kg BW/h 5 4 3 1
11.0 ng buserelin/kg BWjh 5 3 2 1
Plasma LH concentrations, but not FSH, were elevated at least to
day 10 in all cows receiving GnRH or buserelin. The outstanding
features of the P 4 profiles in 15/19 treated cows that responded were
the synchrony, both within and across groups, in P4 ) 1 ng/ml around
day 6, and the fact that most CL were short-lived (4-6 days). Only 3
cows showed evidence of extended CL function. The results suggest
that endogenous LH secretion in PP cows following GnRH-induced
ovulation is not limiting to CL function,and that other factors cause
early demise of the first CL. The most likely explanation is that
normal CL development appears to reqUire pre-exposure to P • In this
4
regard, it is not clear why cows failed to show a second ovulation
around day 10 that was followed by normal CL function; however, it is
considered that this was due to the doses of GnRH and buserelln used,
rather than physiological constraints within the cows.
Stu'ay supported in part by the Australian Meat and Livestock
Research and Development Corporation; GnRH was supplied by Peptide
Technology Limited, NSW; and buserelin by ~oechst AG, W Germany.
79
EFFECT OF GnRH ON FERTILITY OF MERINO EWES INSEMINATED WITH
FROZEN SEMEN
W.M.C. Maxwell l , S.K. Walker 1, D.H. Smith 1 and H.R. Wilson 2
Department of Agriculture, S.A., and
Collinsville Breeding Research Centre, S.A.
Tight synchrony of the time of ovulation may improve fertility
following AI of ewes. For this purpose, non-superovulated Merino ewes
have been treated with GnRH 36h after sponge removal (SR) (1). This
study examined the effect of GnRH treatment and time of insemination on
fertility of ewes inseminated with frozen semen.
Ewes treated with progestagen sponges (Intervet [Aust.] Pty. Ltd.) for
12 days and an injection of 400 Lu. PMSG (Intervet [Aust.] Pty. Ltd.)
at SR, received either no GnRH or 40ug synthetic GnRH (Intervet [Aust.]
Pty. Ltd.) 36h after SR. Semen was collected from three rams (A, B, C)
and frozen-stored in pellet form. The ewes were inseminated by
laparoscOpy (0.04ml semen containing 40 million motile spermatozol-)
either before (51-54h) or after (69-72h after SR [2]) the expected time
of ovulation. Pregnancy and number of foetuses were determined by
ultrasound 50 days after insemination.
Percentage ewes pregnant and foetuses present for rams A, B, C were
42.2 52.3,46.0 (p

Supported by the Australian Wool Research and Development Fund.
80
FACTORS INFLUENCING LAMB SURVIVAL IN A HIGH FECUNDITY
BOOROOLA X SOUTH AUSTRALIAN MERINO FLOCK
D.O. Kleemann,1 S.K. walker 1 , J.R.W~ wa~k~ey2, R.W. Ponzoni 2 ,
D.H. Smith 1 and R.F. Seamark 3
1Turretfie~d Research centre, Department of Agricu~ture, Roseda~e, SA
5350, 2Department of AgricUlture, Ade~aide, SA 5000 and 3Department of
Obstetrics and Gynaecology, University of Adelaide, Adelaide SA 5000
Reproductive potential of the South Australian (SA) Merino can be
improved substantially by incorporation of the Booroola Merino high
fecundity gene (F). However lamb survival is poor. Factors
associated with high lamb mortality need to be identified so that
appropriate strategies to reduce ~osses can be investigated.
Lamb surviva~ (birth to weaning) and birthweight data were
collected from a mixed-aged 1/2 Booroola x 1/2 SA Merino flock at Cape
Borda, Kangaroo Island, SA, in June-July of years 1981 to 1986. Ewes
were managed as in norma~ commercial practice. The effects of year,
ewe age (2-year-o~d vs older) and birthweight (0.5 kg categories
between 1.5 and 6.0 kg) on lamb surviva~ were arta~ysed within singleborn
and mUltiple-born categories, using the procedure GLM in SAS.
Least-squares means (+ s.e.) for birthweight and survival, of ~ambs
born in litter sizes ~f 1-4 from a 1/2 Booroola x 1/2 SA Merino flock
Litter size
Lambs born
Birthweight (kg)
Survival (%)
580
4.13 + 0.04
70.8 :; 1.6
Twin
784
3.28 + 0.03
53. 6 ~ 1.4
Triplet
321
2.80 + 0.05
35.9 "+ 2.2
Quadruplet
52
2.59.+ 0.15
15.9"+ 5.9
There was a decline in survival rate as litter size increased,
the reduction being paralleled by a reduction in birthweight (Table
1). In the analyses of both single and multiple-born categories
birthweight was the dominant factor inf~uencing survival (P

82
OVULATORY POTENCY OF ASRB-bFSH-l IN SHEEP AND
CATTLE
B.M. Bindon,l J.K. Findlay,2 L.R. Piper 1 and M.A. Hi11ard1
1 (On behalf of ASRB Pituitary Hormone Committee)
2 CSIRO Division of Animal Production, Armida1e, NSW
Medical Research Centre, Prince Henry's Hospital, Melbourne, VIC
Biological studies of pituitary hormones in vivo in domestic
livestock are restricted in Australia by the unavailability of suitable
reference preparations. Some years ago AMLRDC, AWC and NH&MRC funds
were provided to ASRB to begin the local purification of pituitary
hormones for use by the Society's members. The present
report deals with one of the preparations from this project.
ASRB-bFSH-1 was compared with commercial porcine FSH ("FSH-P"
Burns Biotec) in sheep and cattle. The latter has approximately fou;
times the FSH radioreceptor assay potency of ASRB-bFSH-1. Merino ewes
(8 per dose) were synchronized with progestagen sponges, then given FSH
as intramuscular injections twice daily for four days beginning on Day
11 after sponge insertion. Total FSH dose was given in decreasing
doses in the ratio 4:3:2:1 over four days and the sponges removed on
the morning of the third day. A similar protocol was followed in
cattle (three-year-old crossbred heifers, 300-350 kg !1veweight; 5-7
cows per dose), using two injections of prostaglandin analogue for
synchronization. In both species ovulation rates were assessed by
laparoscopy 5-7 days after oestrus. The results in Table 1 show that
in sheep ASRB-bFSH-1 has less than one-eighth the ovulatory potency of
FSH-P. In cattle, however, ASRB-bFSH-1 at a dose of 200 mg produced
useful superovulation with no evidence of ovarian over-stimulation,
suggesting a potency of about one-third that of FSH-P. ASRB-bFSH-1,
with

84 85
MN:IlIEWd'ICAL MDEL AND a:MIOl.'m SlMIJLATICfi OF PUI:SATILE JI:lMIilE
SOCRErr'ICfi wr:m APPLICATICfi 'It) III
David J Handelsnan., *Ross lazarus
Depart:.rrent of Medicine, University of Sydney and *Bundoora Extended
care Centre, Bundoora, Victoria
Episodic secretion of hypothalamic GnRH regulates pituital:y-gonadal
function via the pulsatile secretion of LH. Despite its importance,
mmy physiological aspects of pulsatile LH secretion remain
technically difficult to study arpirically. Various objective,
canputer-based pulse detection algorithmns have been applied to serial
LH :rreaSuretrents in blood as a non-invasive index of neuroendocrine
activity of the hypothalamus. Ccmparisons and validation of
algorithrnns is limited to a few intensive human LH pulse series and
ert1irical optimizing against false positive rates as detennined fran
signal-free noise series however the :i..nlp3.ct of these reccmrendations
on the false negative rates has not been studied SYSte:natically.
Therefore we have developed a mathamtical node! of pulsatile honrone
secretion with specific reference to LH and inplemented this in a
versatile canputer simulation package (SimPulse). The nodel is based
on pulses created f:ran a point source (pituitaJ::y) of LH secretion with
subsequent continuous decrementing by netabolic clearance in a
biexponential phanracokineticnodel using available ert1irical data.
Pulse Contour has adjustable shape and pulses are spaced at defined
ti.lTe intervals with net honrone level being the sum of all pulses
above a set threshold. All pararreters are user-definable and 4 key
variables - interpulse interval, secretory burst duration, secretory
rate, metabolic· clearance rate - may have user-defined gaussian
distribution of known nean and variance. Runs generate an output file
suitable for passing to pulse detection prog:r;ams and record files with
the actual pulse pararreters f:ran the simulation run to canpare with
output of pulse detection programs. A second nodule (SimError)
introduces stochastic variations due to sarrpling-time and assay
precision with definable statistical distributions and selectable
pararreters. This program will be useful for m:x:lelling of physiological
and pathological aspects of pulsatile LH secretion especially those at
present inaccessible to ert1irical estimation (burst durations and
pituitaJ::y secretory rates) as well as pulse detection strategies
including sarrpling schemata and canputer algorithrnns .
NO STOCHASllC VAIltATlON IN PNWolEreRS
1.0.,------ ---, 1.0,----_- -.
TlI.lE
0.8
STOCHAS!1C VNIlAnON ~ _ SAllE IGN·p~
TlI.lE
THE EFFECTS OF SERTOLI CELL lATRIX, FETAL CALF SERUI AND SERTOLI
CELLS ON 3H-THYMIDINE INCORPORATED INTO DNA OF CULTURED MYOID CELL8
cpmfug DNA
X on I XF on I 1 F on I S on I
2351
* 17. LSD values
8.8. Raychoudhuryl, I.G. Irving 1 and A.V. Blackshaw2
lSchool of Science, Griffith University, Nathan, Qld. 2De~artment of
Physiology k Pharmacology, University of Queensland, St. LUCIa, Qld.
Ve have reported radiolabel incorporation in~o myoid cell. P!ote~n
synthesis and secretion, GAG production, secretIon and deposlt.lOn In
the extracellular matrix (1). This study extends the work to Include
aH-thymidine incorporated into DNA.
8ertoli cells (S) and myoid cells (I) were prepared from 20-22 day
old rat testes and cultured (2). There were six treatment com~inations
of I S Sertoli cell matrix (X) and fetal calf serum (F), VIZ; I, S,
SI, XI, 'IF and IMF. All culture types were labelled with aH-thymidine
(1 JjCijml) for 96 h at 37° C and treated with (BU) 2 cAMP , (0.5 roM) or
FUT CFSH 25 ngjmlj insulin, 5 Jjgjml; retinol, 0.35 JjM; testo~terone,
0.7 Jjl). 'The extent of DNA synthesis in these culture combina~lons w~s
measured by both scintillation counting and autoradlographlc
examination.
The incorporation of 3H- thymidine into cultures was low in all
preparations except those containing feta~ calf serum. . In these
cultures the presence of Bertoli cell matnx had a relatlVely small
inhibitory effect on the incorporation of label.
The effect of culture conditions on the uptake of [3HJ thymidine by
myoid cells.
29555 497 34722
701
944*
7. labelled nuclei
X on 1 XF on M I F on I S on I
16.9
58.9 14.7 69.0 10.9
1.9*
The hormone mix (FUT) stimulated an~ cAMP inhib~ted ~ncorporation
under most culture conditions, although In the Sertoll-myold co-cu~ture
both FIRT and cAIP reduced the already low level of nuclear labelhng.
Ve conclude that DNA synthesis in myoid cells is slightly stimulated
by Sertoli cell extracellular matrix. The effect is minor compared to
the response to fetal calf serum, which clearly s~ppli~s necessary
growth factors. T~e inhib~to!yeffect of cAMP conhrms ItS.accepted
role of promoting dIfferentIatIon, whereas the hormonal cocktaIl (FIRT)
facilitates cell replication.
(1) Raychoudhury, S.S., Ir~ing, I.G. &Blackshaw, A.V. (1987)
Proc.Aust.Soc.Reprod.Blol. 19:84. .
(2) Skinner, I.K. &Fritz, I.B. (1985) Proc.Natl.Acad.Scl. 82:114-118.

86
EFFEcrs OF SEROTONIN ON INHIBIN AND TESTOSTERONE PRODUCTION BY
ADULT RAT SEMINIFEROUS TUBULES AND LEYDIG CELLS IN VITRO
G.F. Gonzales*, J. Muir, G.P. Risbridger and D.M. de Kretser
Department ofAnatomy, Monash University, Melbourne, 3168.
Hyperserotoninemia has been shown to be deleterious to spermatogenesis without
affecting serum testosterone levels in men (1) and male rats (2). Howeversome
authors have claimed that serotonin alters spermatogenesis, secondary to an inhibition
oftestosterone production (3,4). The aim of the present study was to determine the in
vitro effect of serotonin on both testosterone production by Percoll-purified Leydig
cells and inhibin production by isolated seminiferous tubules (ST) from normal adult
rats.
Leydig cells were isolated from the testes of adult rats and purified on discontinuous
gradients of Percoll (Pharmacia, Uppsala, Sweden). 50,000 cells were then cultured in
plastic multiwell dishes containing 250 J.lI Dulbeceo modified Eagle medium: F12 ± 2.5 IU
hCG at 32·C for 20 h. Serotonin was added in the dose range 10-1000 ng/well. 5 em
segments of ST were dissected free of interstitial tissue and cultured in 1 rnl DMEM
with the following additions: serotonin (Sigma) (1-1000 ng/rnl) ± either FSH (500
ng/rnl), dbcAMP (10 J.lg/ml) 0:' insulin (Sigma) (20 ng/rnl).
Serotonin.has no effect on basal testosterone production, but an increase in the hCGinduced
T production was observed with 10 ng serotonin (p

88
IMMUNOCYTOCHEMICAL LOCATION OF OXYTOCIN AND MESOTOCIN WITHIN THE
HYPOTHALAt4US AND REPRODUCTIVE TRACT OF THE MALE MARSUPIAL BANDICOOT
R.T. Gemmell l and C. Sernia 2
lDepartment fAt 2D t f
o na.omy,. epar ment 0 Physiology and Pharmacology,
Un1vers1ty of Queensland, Brisbane.
T~e structure o~ the ~eurohypophyseal peptide hormones isotocin,
mesotoc1 nand OXytOC1 n var1 es by either one or two ami no aci ds.
Isotocin hc:s been identified in all bony fishes, mesotocin in all the
non-mammal1an tetrapods, namely birds, reptiles amphibians and lung
fishes nd oxytocin in the placental mammals.O) In the marsupials
mesotoc;n, but no~ oxytocin, has been identified in nine species of
Austral;an marsup1al. In American marsupials, both oxytocin and
meso~oc1n have been reported in two species and oxytocin alone in one
spec; es. If one acce~ts the o~thodo~ evol uti onary pathway of these
pept1 de hormones as be1 ng from 1S0toc1 n through mesotoci n to oxytoci n
then t~e p~es~nce of mes~toci n wi thi.n the Austral i an marsupi a1s, and
oxytoc;n w1t~1n the A!"encan marsup1als, the more primitive form of
marsup1al, 1S puzz11ng.(2) In this study we examined the
neurohypophyseal hormones of the bandi coot (1. macrourus) an
Australian marsupial not examined previously.
'
Oxytocin, known to be produced by the hypothalamus has now been
obser~ed within the .ovary.. ~he testis and the adrenal gland.(3) The
loca~10n of oxytoc1n w1th1n the reproductive tract of the male
band;coot ~as also ~xamined. Tissue was fixed in paraformaldehyde and
se~t1on~ 1mmunos~a;ned b~ a biotin-streptavidin-peroxidase system,
us;ng h1 gh l~ spec1 f1 c l'abb1 t an.ti -oxytoci n and sheep anti -mesotoci n as
pnmary ant1 sera. Immunoreact1Ve oxytoci n cells were demonstrated in
the paraventri cul ar and supra opti c nucl ei of the bandi coot
hypoth~lamus. These cells did not stain with the mesotocin antibody.
OxytoC1n but not mesotocin was present in the Leydig cells and in the
spermatids of the bandicoo~. The bandicoot prostate has two segments,
~he dorsal and v~ntral reg10ns. Oxytocin and mesotocin were present in
l.he.ventral port1on of the prostate, but were not present in the dorsal
reg1on.
In conclusion, these results show that both oxytocin and mesotocin
are prese~t in Australian marsupials. Furthermore, these peptides are
not restncted to the hypothalamus but are also found in the testes
~ oxytoci n1 and prostate (.oxytoci nand mesotoci n) where they coul d be
1nvolved 1n spermatogenes1s, contractility of the seminiferous tubules
and in sperm transport in the female reproductive tract.
(1) Acher, R. (1980). Proc. Roy. Soc. Lond. Ser B. 210, 21-43.
(2) Chauvet, J., Roui11e, Y., Chauvet, M.T. and Acher, R. (1987). Gen.
Compo Endocr. 67, 399-408.
(3) Nicho~son,.H.D., Swann, R.W., Burford,.G.D., Wathes, D.C., Porter,
D.G. and P1ckenng·, B.T. (984), Reg. Pept1des 8, 141-146.
89.
THE EFFECT OF DAY LENGTH TREATMENT ON THE
REPRODUCTIVE PERFORMANCE OF BORDER LEICESTER RAMS
C.R. Earl, E.A. Dunstan and M.
Schleuniger
Dept. Ag. S.A. Struan « Kybybolite
Daylength is recognised as the principal environmental cue
determining the seasonal breeding activity of rams (1,2). In
Australia the majority of rams are used for mating during long days
at a time when testosterone levels and mating activity are low (3}.
Schanbacher (4) has reported that exposure of rams to short days
(L:D, 8:16) significantly improved the reproductive performance of
rams when singly mated.
In our experiment the reproductive performance of rams exposed to
long (L:D, 16:8) or short (L:D, 10:14) days for 12 weeks prior to
mating was compared.
Ten 1 1/2 year old Border Leicester rams were allocated to two
equal groups on the basis of liveweight on 16/9/85. Testicular
diameter and liveweight were recorded fortnightly prior to mating on
12/12/85. Each ram was individually mated to 40 merino ewes and
reproductive performance determined by the number of foetuses present
and mean lambing day.
Results
~eweight. testicular diameter. foetuses per ewe pregnant. and
meaning mating day for rams exposed to long and short day
len ths.
T est
1 Foetuses
c
u
per ewe
1
40 Wt.
a r 5.0 (kg)
60
Mean Lambing
day
0
1 Long day 1.18 143
a
II
e Short day 1.12 143
t 20
e N.S. N.S.
r
( 4.0,....---1._---1._......._ ...._.1-.......
em) 16i9 30/9 15/1027/10 11/11 28/11 12/12
Date
Although testicular diameter was significantly increased by
exposure to short day length no improvement in the reproduct.ive
performance of short day length treated rams was observed. The
proportion of rams to ewes (2 1/2%) is comparable with that used in
commercial practice with maiden rams. It is therefore unlikely that
treatment of rams with short day length or with products that mimic
short day length is likely to improve their reproductive performance.
(1) Yeates N.T.H. 1949. J. Ag. Sci. 39:1
(2) Ortavant R., Mauleon P., Thibault C. 1964.
Ann. N.Y. Acad Sci. 117:157
(3) D'Occhio H.J., Brooks D.E., 1983 Aust. J. Exp. Anim. Husb. 23:248
(4) Schanbacher B.D. 1979 J. Anim. Sci. 49:927

90 91
EFFECT OF PHOSPHATIDYLSERINE ON CALCIUM UPTAKE OF COLD SHOCKED
BOAR AND RAM SPERMATOZOA
D.P. Windsor and I.G. White
Department of Veterinary Physiology, Univer.sity of Sydney, NSW 2006
It has long been recognised that phospholipids play a role in
protecting mammalian sperm from cold shock. Phosphatidylcholine (PC) has
bee~ shown to protect ram (1) and boar (2) sperm, whil e phosphati dyl­
~er1ne (PS) has been shown to reduce acrosomal damage and motility loss
1n col d shocked boar sperm (3). The aim of this study was to further
assess the cryoprotective effect of PS on boar sperm by measuring uptake
of radioactive calcium and to test its efficacy in protecting ram sperm.
Boar sperm were flushed from cauda epididymides and ram sperm were
collected by ~lectroej8culation: PS (4 mg/ml) was added 25 minutes prior
to cO]d shock1ng at 0 C. Calc1um uptake was determined at 5, 10, 20 and
40 ffilnutes (2). Data were subjected to a split plot analysis of
variance.
TABLE 1:
Species
Boar
(n=8)
Ram
(n=7)
Ca uptake of sperm (nmoles/10 9 sperm, mean + S.E.)
Control 25+9ab 42+15ab 66+25b
Colq shock 117+8c 129+8c 135+6c
Control+PS 5+1 a 7+2 a 9+2 a
Cold shock+PS 62+21b 66+20b 57+18b
Control 21+7a 33+11a 46+13a
Col d shock 101+21c 125+17b 144+16b
Control+PS 35+16ab 49+19a 54+21a
Cold shock+PS 71+9bc 101+12b 133+25b
Different subscripts denote differences (p < 0.05) within the
and species.
40min
86+28b
128+7c
7+1 a
30+10a
42+13a
120+10b
52+18a
126+25b
same column
These data confi rm the observati on (3) that PS protects boar sperm
from col~ shock. Calcium uptake .(indicating plasma membrane disruption)
was cons1stently lower (p

92 93
FERTILISATION IN SUPEROVULATED EWES FOLLOWING INSEMINATION
WITH DIFFERENT DOSES OF FRESH OR FROZEN-THAWED SEMEN
H.N. Jabbour, G.
Evans and N.W. Moore
Department of Animal Husbandry, The University of Sydney, N.S.W. 2006
Intrauterine insemination is necessary to achieve satisfactory
fertilisation in superovulated ewes. The following study was
designed to examine fertilisation in superovulated ewes inseminated
with various doses of fresh or frozen-thawed spermatozoa.
TABLE 1. Proportion of ova fertilised (no. fertilised/no. recovered)
following insemination with different doses of fresh or frozen-thawed
semen.
100
50
25
12.5
6.2
3.1
SEPARATION OF HUMAN SPERM FOR ELEMENTAL ANALYSIS
G.H. O'Brien a ,
J. Clulow and R.C. Jones
Department of Biological Sciences, University of Newcastle, NSW 2308
O'Brien, Clulow and Jones (1) reported the elemental composition
of sperm that were pipetted onto electron microscopy grids and airdried,
as described by Chandler and Battersby (2). The concentrations
obtained in that study suggested serious inadequacies in the
technique, especially contamination of sperm by extracellular fluid.
We now describe a more appropriate technique developed in this
laboratory, and initial observations obtained with its application..
Semen collected from 3 Merino rams was pooled and then
inseminated either fresh after dilution with phosphate buffered
saline (PBS) or after frozen storage in glycerol-egg yolk diluent.
A formvar-coated nickel slot grid was placed in the bottom of a
Forty-nine mature Merino ewes were treated with intravaginal
0.5 mlmicrotestube and covered with a layer of di-n-butyl phthalate.
progestagen pessaries (Repromap, Upjohn) for 12 days and 1200 IU PMSG
An aliquot of semen was placed on the oil and centrifuged for 0.5­
(Folligon, Intervet) administered 48 h before pessary withdrawal
4 min in an Eppendorf centrifuge then the grid was rinsed in isopentane
(PW). Intrauterine inseminations were performed by laparoscopy
and freeze-dried at 0.5 x 10- 3 Pa overnight. The nuclei of 5
at 44 h after PW. A total dose of 100X106 , 50x106 , 25x106 , 12.5xl06,
sperm from each of 4 ejaculates (collected from fertile donors) were
6.2xl0 6 or 3.1x10 6 of either fresh or frozen-thawed motile
analysed by energy dispersive spectroscopy (X-ray probe microanalysis)
spermatozoa in a total volume of 0.1 ml PBS was deposited in the
in a scanning electron microscope.
uterine horns. Ova were recovered and ovaries inspected at
Mean concentrations of Na, Mg, P, S, CI, K and Ca are listed in
laparotomy under general anaesthesia 92 h after PW. Cleaved and
the Table below. Presented with them are results (for sperm in semen)
pronucleate ova were classified as fertilised.
from O'Brien et al. (1) converted to the same units (mmol.kgrl dry wt)
The mean ovulation rate was 11.9 ± 0.9 and the overall recovery
to allow direct comparison.
of ova (no. recovered/no. of ovulation points) was 56.5%: Multiple
X 2 analysis was performed and the data are presented in Table 1. Technique used Elemental concentrations in sperm nuclei
to prepare sperm
(mmol.kgrl drywt)
Dose of spermatozoa (X10 6 )
Fresh semen
Frozen-thawed semen
12/15
14/15
27/33
31/34
17/25
14/19
13/22
25/27
14/15
14/32
23/23
21/31
The overall fertilisation rate was 77.3%. Overall, there was no·
effect of the type of semen used on fertilisation, but there was an
interaction between the type of semen and the dose of spermatozoa
inseminated (P

94 95
MORPHOLOGY OF IMMATURE SPERMATOZOA OF THE CHINESE PANGOLIN
(MANIS P.ENTADACTYLA : PHOLIDOTA)
Luke K.-P. Leung and J.M.
Cummins
PIVET Medical Centre, 166 Cambridge Street, Leederville, WA 6007.
Pangolins are testicond eutherian mammals which for many years
were classified along with the Edentata because of superficial
resemblances to Armadillos and lack of teeth. This affinity is now
discounted, and their phylogenetic placement is uncertain (1).
Ballowitz (2) first described by light microscopy the spermatozoon of
a species of this Order (M. longicaudata - a frozen specimen from W.
Africa), and considered it to be similar to that of monotremes,
reptiles or birds. No subsequent work on the sperm morphology appears
to have been published. We were interested in further evaluating
Ballowitz's observations using electron microscopy. Material from a
small male M. pentadactyla Was fixed using 2.5% glutaraldehyde in
cacodylate buffer (pH 7.4). Representative areas of the reproductive
tract were excised, osmicated and embedded in Araldite follOWing
dehydration in an ethanol-acetone series. Ultrathin sections were
studied by TEM.
The animal was not fully reproductively active, and only a few
seminiferous tubules exhibited elongated spermatids. Immature
spermatozoa were found in the ductuli efferentes, but the rest of the
tract was devoid of gametes. Nevertheless, the spermatozoa showed
striking similarities with Ballowitz's descriptions for M.
longicaudata. The sperm head, unlike that of any other eutherian
mammal described, is not flattened, but has a pointed, elongated
nucleus (10-12 urn long) with a round cross-section (0.8-1.2 um
diameter). The chromatin in many of the spermatozoa of this specimen
was irregularly condensed, a feature also noted by Ballowitz. The
flagellum, midpiece and axoneme are round in cross-section, and there
is a spiral fibrous sheath underlying the principal piece
plasmalemma. The axoneme conforms to the common 9 outer doublet + 2
central microtubule pattern with 9 accessory coarse fibres extending
along the bulk of the flagellum. The acrosome (1.7-2.2 um long) has a
largely homogenous matriX, sits cap-like over the rostral fifth of
the nucleus, with no substantial subaerosomal material, and does not
appear to have a well-defined equatorial segment (ES). Lack of this
feature distinguishes M. pentadactyla from other eutheria with
flattened sperm heads, where the presence of an ES is possibly
related to common features of fertilization (3).
The sperm morphology of this group thus clearly differs from
the rest of eutherian mammals. Further work is needed to see whether
the ostensible resemblance to the sauropsid sperm of monotremes is o.f
taxonomic significance.
(1) Nowak, R.M., Paradiso, J.L. (1983). In, "Walker's Mammals of the
World" 4th Edn, pp 470-472. Johns Hopkins Press, Baltimore.
(2) Ballowitz, E.(1907). Zeits. f. Wiss.enschaft. Zool. 86:619-625.
(3) Bedford, J.M. (1982). In C.R. Austin and R.V. Short (eds)
"Reproduction in Mammals" 2nd Ed, Vol 1, pp 128-163. C.U.P.
THE CYCLE ot THE SEMINIFEROUS EPITHELIUM OF THE JAPANESE
QUAIL, COTURNIX COTURNIX
M.Lin"', R.C.Jones'" and A.W.Blaekshaw 2
'" Department of Biological Sciences. University of Newcastle. NSW
2 Department of Physiology & Pharmacology. University of
Queensland. Qld.
It is extremely difficult to identify the various cellular
associations which constitute the cycle of the seminiferous
epithelium of many birds with the routine histological methods
used for mammals and this problem has lead to the publication of
some unusual combinations of cellular associations (1). The
problem in the bird is that an individual cellular association
covers a very small area of the wall of a seminiferous tubule so
that several different adjacent associations overlap and it is
not possible to clearly distinguish the germ cells in one
association from the germ cells in an adjacent association.
This studY determined the cellular associations in the
Japanese quail using thin epoxy sections of single seminiferous
tubules. Tubules were isolated using a hypodermic syringe and
needle to flush them apart with 0.1 M phosphate buffer.
Subsequently they were fixed in glutaraldehyde and embedded in
Spurr's resin. The cytoplasm of individual Sertoli cells was
traced in 1-2 pm sections stained with toluidine blue. .
Germ cells were classified into 12 steps of spermatId
development. 3 types of spermatogonia, primary spermatocytes in
meiotic phases and secondary spermatocytes. Table 1 shows the 10
cellular associations of the cycle of the seminiferous
epithelium based on the morphological changes of the acrosome and
nuclei of the developing spermatids. There is some uncertainty
about the relationship between spermatogonia which can only be
resolved using a radiolabelled tracer.
.r---."..-.·--I-·-.--,r----~ u _.-~'-...------u
11' I 11 I 11 U 12 B 12 I U • H I B
1---1-·-·..··-,-1",·....,·,-···1·..····,,",·-1--.-....---'11---,.11--- 1 1---1
11 I 2 I 3 I 4 B 5 I 6 J 7 I B I 9 1 10 I
I'_'__'_I_'__-II,_"""·_,,~,,,,_~ '_""'·" I-"_""__·_I_"_·"'.._·........---·1----·1---·-1
BPI PIP 1 PIP I P I Dp I Dk I M-An I I I 1
1-"'-'1·-.."".-..-,,,0.-,,••·-.,,·--·1..,,,,-,,"'-'--1-,-----..1,,·,,-,----11·-,--·-·-.----11---1-----1
UB 1 BIB n LIZ 1 PIP 1 PIP I P I
'1----8----1-.-...-,"'1-·-'--1----......----11·---·1---1----1 1
UAp I Ap I Ap I Ap I Ap n Ap I BIB I BIB 1
DAd 1 Ad I Ad I Ad 1 Ad 1 Ad lAd Ap I Ad Api Ad Api Ad A~I
I I H II I I II I IV I V VI I VI I VI I IH IX I X
=-=~.::SYM:BOLEt;=cA'd;::'''·~d';;:)~==t~~~A·-~~7.~~~~~=~~;ti~~:::.A~.~ie ty~~ A
spermatogonia; B. type B spermatogonia; L. leptotene primary
spermatocytes; Z, zygotene primary spermatocytes; P. pachytene
primary spermatocytes; Dp. diplotene primary spermat0cytes; Ok.
diakinesis of primary spermatocyt~s; M. metaphase primary
spermatocytes; An. anaphase primary spermatocytes; 1-12. step 1­
step 12 spermatids; I-X, stages of spermatogenesis.
The
duration
birds.
results provide a
of spermatogenesis
(1) Yamamoto.S; Tamate.H &
Res.1B, 27-37.
basis for further studies of the
and renewal of spermatogonia in
Itikawa,O (1967)
Tohoku Agric.

96 97
EFFECT OF RACEMIC GOSSYPOL AND ITS ISOMERS ON SURVIVAL
OF RAM SPERM AND ON REACTIVATION OF SPERM MODELS
R. Vishwanath. I.G. White and S.A. Matlin 1
1 Department of Veterinary Physiology. University of Sydney. NSW. 2006
Chemistry Department. City University. Northampton Sq .• London. ECIV OHB
Gossypol is an optically active molecule and preliminary studies
indicate that the (-) isomer renders male rats infertile when given
orally. but the (+) isomer is ineffective (1). The aim of this study was
to investigate the stereospecificity of the action of gossypol on sperm
function in vitro.
Ram sperm were washed free of seminal pl asma by dil uting with
Krebs Ri nger phosphate gl ucose. centrifugi ng. removi ng the supernatant
and resuspendi ng the sperm pellet. Demembranated sperm models were
prepared by extracti ng the washed sperm in Triton-X medi urn and were
reactivated with ATP (2). Motil ity of sperm was scored on a scal e of 1
to 4, and reactivati on of the sperm model s as the percentage movi ng.
Oxygen uptake of sperm was determined with a Clark electrode and glucose
and lactic acid concentration of the medium by enzymatic assays.
Sperm motil ity decreased 1i nearl y with i ncreasi ng concentrations
of racemic (i.e. .:!:.) gossypol or its isomers; 50 IlM of the (+) or (-)
isomer or 100 IlM of the racemer depressed motility within 10 seconds.
Sperm'motility ceased after 30 minutes exposure to 50 IlM of each of the
compounds but weak motility persisted for 60 minutes in the presence of
25 IlM of the (+) or (-) isomers. A strong uncoupling (i.e. increase) of
sperm oxygen uptake was evident at low (25 and 50 IlM) concentrations of
racemic gossypol or its isomers, but higher concentrations ([50 and 200
IlM) of racemic gossypol reduced oxygen uptake. At 50 IlM the (+) or (-)
isomers decreased glucose utilization of sperm and 100 IlM of each of the
compounds completely inhibited glucose utilization and lactic acid
accumulation.
Demembranated sperm models were more resistant to gossypol or its
isomers; 25 or 50 IlM had no effect on reactivation of sperm models which
immediately became motile on addition of ATP. At 100 IlM. the %
reactivation (mean + S.E .• n=5) was 66.6 + 8.9 for the racemer. 53.6 +
7.1 for the (+) and-zero for the (-) isomer compared to 65.5 + 11.3 for
the control; 200 IlM of the compounds inhibited reactivation completely.
Overall, the in vitro spermicidal activities of racemic gossypol
and its isomers were not remarkably different. This suggests a stereospecific
in vivo interaction of gossypol at the testicular target site
leading to disruption of spermatogenesis.
(1) Wang. N.G., Zhou, L.F .• Guan, M.H. and Lei, H.P. (1987). J.
Ethno~harmacOl.• 20:21-24.
(2) -hite.I.G. and Voglmayr. J.K. (1986). J. Reprod. Fert.• 34:183-193.
Perfusion fluid
DRTF
sRTF
DOES .RETE TESTIS FLUID CONTAIN A SECRETAGOGUE 7
S. SUj~rit1, G. ChaturaP1nich1, H, Lin 1 , !.c. Jones 1 ,
B.P. Setchell ,.and G.M. Stone
1 Department of Biological Sciences, University of Newcastle, NSY 2308
2 Department of Animal Physiology, Yaite Agricultural Research
Institute, University of Adelaide, SA 5064
3 Department of Veterinary Physiology, University of Sydney, NSY2006
Last year we described a method of perfusing the initial segment of
the epididymis of the rat (1). It involved anaesthetising rats with
Inactin (Byk Gulden Konstanz, 'West Germany), cannulating region 1A (2)
with polyethylene tubing and perfusing at 0.5 ~l/min.
The present study examined the ef£ects of perfusing the initial
segment for 7.5 h with native rete testis fluid (nRTF) and modifications
of nRTF as weH as a synthetic medium (sRTF) based on the inorganic
compositions ofnRTF(3). None of the treatments affected the structure
of the duct epithelium. However., Table 1 shows that protein secretion
was higher when nRTF or a high molecular weight (M'IJ) fraction
(concentrated 3-fold) of nRTF was used to perfuse the duct than
modifications- of nRTF which damaged or removed the protein, or the sRTF
was used. The addition of bovine serum albumin (BSA) to sRTF enhanced
protein secretion into the lumen, but it was not as great as with nRTF
or the high MY fraction of nRTF.
PAGE of the perfusates indicated that the composition of the
perfusion fluid did not effect the type of protein secreted.
TABLE 1. Rate of protein secretion into the initial segment of the rat
epididymis during perfusion for 7.5 h wi th nRTF, sRTF or various
modi~ications of the fluids. (MEAN ± SE)
Unmodified
Steroid extracted
High MY fraction
Low MY fraction
Trypsinized
Protein-free
1.3 ~g/~l BSA
No. of
animals
It is concluded that nRTF may contain a secretagogue.
7
4
5
3
3
8
6
Rate of protein secretion
(ng/min)
93 ± 22
99 ± 45
145 ± 36
19 ± 3
41 ± 18
25 ± 4
45 ± 21
(1) Sujarit, S. et ~l (1987). Proc. 19 tho Ann. Meet. Aust. Soc. Reprod.
BioI., Sydney, August, p. 90 (Abstract).
(2) Reid, B.L. & Cleland, K.Y. (1957). Aust. J. Zool. ~, 223-246.
(3) Setchell, B.P. et al (1969). J. Physiol. 22, 73-85.

Table 1.
Untreated (n=7)
EDL (n=5)
98
REGULATION OF THE INITIAL SEGMENT OF THE EPIDIDYMIS
S. Sujarit 1 , R.C. Jones 1 , M. Lin 1 , B.P. Setchel1 2 and G.M. Stone 3
1 Department of Biological Sciences, University of Newcastle, NSY 2308
2 Department of Animal Physiology, Yaite Agricultural Research
Institute, University of Adelaide, SA 5064
3 Department of Veterinary Physiology, University of Sydney, NSY 2006
The initial segment (IS) of the mammalian epididymis has a
characteristic ultrastructure and the epithelium regresses following
efferent duct ligation (EDL). Nicander et al (1) showed that regression
occurs within 6 hours of efferent dUlc~ligation, we developed a
microperfusion technique to study the effects in vivo of modifying the
composi tion of the luminal fluids of the initialsegment (2).
Mature Yistar rats were anaesthetised with Inactin(Byk Gulden
Pharm., Y. Germany) and prepared for microperfusion. The duct was
flushed free of sperm wi th a synthetic medium (sRTF) with the same
inorganic composi tion as native ram rete testis fluid (nRTF) , and the
duct was perfused for at least 6 hours at 0.5 lli/min with either sRTF or
nRTF. The perfusate was analysed quantitively for protein and after
perfusion samples of the duct were fixed and epoxy sections were
prepared for light microscopy.
. It was found that the structure of the duct epithelium was much the
same after 7.5 h perfusion wi th nRTF and sRTF .as in unperfused duc ts
whilst 7.5 h after EDL some of the structural signs of regression
described by Nicander et al (1) were present. In order to distinguish
the effect of EDL from the effect of withdrawing the luminal fluids from
the lumen of the IS we examined the effect of EDL just prior to
perfusing the IS with nRTF (Table i). The reduction in protein
secretion following EDL indicates that the main effect of EDL on the
initial segment· may not be due to the withdrawal of the luminal fluids.
Protein secreted into the IS perfused with nRTF (ng/min).
Perfusion time (h)
1.5-3.5 3.5-5.5 5.5-7.5
76+1. 9
70+0.6
105+5.9
68+0.2
97+2.6
31+0.7
(1) Nicander, L. et al (1983) Int. J. Androl. 6, 91-103.
(2) Sujarit, S. et al (1987) Proc. 19th Ann. Meet. ASRB, Sydney,
August 24-26.
99
THE EFFECTS OF BILATERAL CASTRATION AND ETHYLENE DIMETHANE
SULPHONATE (EDS) ON EPIDIDYMAL FUNCTION (SPERMATOZOAL
MATURATION) IN GUINEA-PIG
S.J. Gatie 1 , A.W. Blackshaw 1 and T.D. Glover 2
lDepartment of Physiology and Pharmacology, University of
Queensland, St Lucia, Qld and 233 Lyddon Tee, Leeds,
LS2 9JT, West Yorkshire, England
Epididymal function is androgell dependent (1) and the secretory
functionof the rat epididymal epithelial cells is inhibited by
castration. After EDS treatment in the rat, testosterone is decreased
to the castrated level (2) and the present studies examine the effects
of androgen withdrawal and replacement on spermatozoal maturation
(rouleaux formation) in the guinea-pig.
Five groups were used in the bilateral castration study with
five guinea-pigs in each group: group 1 - castrated for 1 week + 10 mg
(SIC) of testosterone propionate (TP) daily: group 2 - castrated for 1
week without TP: group 3 castrated for 2 weeks + TP: group 4­
castrated for 2 weeks without TP and group 5 - sham operated control.
Three groups were used in the EDS study (five animals in each. group) ,
group 1 - received one dose of 75 mg EDS (liP): group 2 -received 2
doses of EDS two weeks apart and group 3 - received vehicle only. At
the end of each period of the two studies, epididymides and also
testes in the EDS treatment, were removed and processed for
histological study. Glycoproteins in groups 2 and 3 of the EDS study
for epididymal tissues and smears for epididymal spermatozoa were
examined using lectins. Protease activity of . acrosomes in smears of
spermatozoa was studied using gelatine coated slides.
In castrated animals: without testosterone (TP) replacement, the
epididymal epithelium was thin and atrophic with nuclear degeneration.
Stereocilia were absent from the luminal surface. Spermatozoa,
contained in the terminal segment, were totally separated from
rouleaux; some were decapitated and the concentration was reduced.
Epithelial and spermatozoal changes were prevented in animals
recovering TP. EDS treatment demonstrated almost the same histological
and spermatozoal pictures as in the castrated group. The testes
showed massive damage to the seminiferous tubuleS with only a few
degenerated germ cells present. Lectin binding to glycoproteins was
reduced markedly in the epididymal tissue and spermatozoa after EDS
treatment. Protease activity in the acrosomes was almost abolished in
the seminal smears collected from EDS treated guinea-pigs.
The present observations confirm the view (1) that androgens
control epididymal function in spermatozoarmaturation.
(1) Moore, H.D.M., & Bedford, J.M. (1979) Anat. Rec. 193: 293-312.
(2) Morris, I.D., Phillips, D.M., & Bardin, C.W. (1986) Endocrinology
118: 709-7.19.

--~-------------------------------------------------------------------
Fluid Na Cl K Protein
lJlIh nmoles/h nmoles/h nmoles/h lJg/h
Testicular output 221.9 40,000 38,000 2,800 355.2
Reabsorption in the:
Ductuli efferentes 205.7 30,OOOU 26,0000 2000 365.7
Ductus epididYJllidis 0.4 87U 54D 12D 0.6
Ductus deferens 0.4 63U 480 60 ..,.0.8
The data in Table 1 indicate that most of the fluid and solutes
produced by the testis are reabsorbed in the ductuli efferentes.
Subsequent rates of reabsorption in the more distal ducts are very
low. NaCl is the principal solute produced by the testis, and the
principal solute of the reabsorbate. from the ductuli efferentes.
Reabsorp~ion of Na is against the electrochemical gradient in all
regions of the ducts and may indicate that water and solute
reabsorption is secondary to active transport of sodium.
100
FLUID AND SOLlfl'E FLUXES IN THE GENITAL DUCTS OF THE
MALE JAPANESE QUAIL
J. Clulow and R.C. Jones
Dept. of Biological Sciences, University of Newcastle, N.S.W., 2308.
The extratesticular genital ducts of the Japanese quail are
involved in the transport, concentration, maturation and transient
storage of spermatozoa released from the testis (1). The
transepithelial transport of fluid and electrolytes is, presumably, of
fundamental importance in these processes through the reabsorption of
fluid and the modification of the luminal milieu of spermatozoa, but
has not been well characterized in any avian species. Consequently,
testicular output and transmural fluxes were studied in the quail.
Net fluid fluxes were determined by stereological procedures (2).
Solute concentrations were determined by X-ray microanalysis for
elements (3) and by the Goomassie procedure for protein (4).
Transmural solute fluxes were calculated from data for solute
concentrations and net fluid fluxes.
Table 1 Estimates of testicular output of fluid and solutes and rates
of reabsorption (positive values) and secretion (negative values) in
the extratesticular ducts. U indicates transport against, 0 transport
down the electrochemical gradient.
It is concluded that the active reabsorption of water and solutes
in the ductuli efferentes concentrates spermatozoa in the ductus
epididymidis and ductus deferens, which then require only low rates of'
absorption of inorganic electrolytes and secretion of protein to
complete sperm maturation and to store spermatozoa.
(1) Clulow, J. and Jones, R.C. (1982) J. Reprod. Fert. 64, 259-266.
(2) Clulow, J. and Jones, R.C. (1988) J. AnaL In press~
(3) Clulow, J. and Jones, R.C. (1986) Proc. 9th Aus. E.M. Conf.p24.
(4) Bradford, M.M. (1976) Analyt. Biochem. 72, 248-254.
101
ORIGIN OF LUMINAL PROTEINS IN THE EPIDIDYMIS OF·THE TAHHAR,
~
Q. Chaturapanich and R.C. Jones
MACROPUS
Department of Biological Sciences, University of Newcastle, NSW 2308
We have studied the origins of the proteins in the epididymis of
the tammar by comparing PAGE patterns of micropuncture samples of
luminal fluids (LF) from the epididymis and rete testis (1), perfusates
after microperfusion of the duct (2), blood plasma and spermatozoa, and
autoradiographs (AR) of ~ubation media following protein synthesis in
vitro in the presence of S-methionine.
--Table 1 shows the occurrence of proteins which were i!l the
epididymal fluids, but were not present in blood plasma or rete testis
fluid. The autoradiographs revealed that there were a·lso la,belled
protein bands, with apparent melecular weights of 43, 37, 34.5, 27 and
14.4 which migrated to thesame'position on the gels as blood proteins.
PAGE' of spermatozoa showed that .they lost 3 bands (corresponding to MW's
47, 29.4 and 28) and gainedonoe (corresponding to aMWof 30.4) during
passage through the epididymis.
Table 1. Protein secretion by the epididymis of the tammar determined by
different methods.
A\l1
Caput &corpus
LF
Perfusate
AR
90.675.76356.339.8 32.3 31.3 30 21.418.717.412.812.110.9
epididymis
Cauda epididymis
LF + +
Perfusate
AR + + +
+ + + + + + + + +
+ + + + $
* + + + + + + +
+ +
+
+ +
'l' secreted by caput only if secreted by corpus only
It is concluded from the in vitro incubation studifes hthad t tt he
hi' t"h same in all regions 0 t e uc us
pai~~~nm~:iSProt:~ns:::\~S t~e l.;rot:ins have the same MW as proteins in
~iood,yaddi~io~aldproJeinsr:~edth~;ethSeh~:nct~~ ~~~ t~~ii~:.li~:v~~t~:~;:s~
may be sYb nt es ze dand St~Cat some proteins which are synthesi~ed and
it cane cone l u e . C" f gels of
secreted in vitro are not secret~~ ~~divci:tO~s th~~pa::bsso;an~es in the
i~:i~:i i~f1sa:: t:ro~~~1;t~~~ec~~n1 the ~~:ep::t:irnosterie;e~~~~e~~~~~~:
the MW of some of the secrete pro e ns. be art of the
sperm do not appear in the ePhidiod:mt;se~l~Smath~od~~~ epiihelium (3).
cytoplasmic droplet which is p:g Yt . n of ~lJ 30 binds to spermatozoa.
It is suggested that the secrete pro e1.
Jones R.C. (1987) J. Reprod. Fert. 80: 193-199.
~~~ Chatu~apanich, G. and R.C. Jones (1987) Proc. 19th Ann. Conf.
ASRB Sydney, p.75. . (1984) C 11 Tiss
(3) Jones: R.C., Hinds, L.A. and Tyndale-Blscoe, C.H. e.·
Res. 237: 525-535.
+
+
+
+

102
PROBLEMS IN SCREENING AZOOSPERMIC PATIENTS FOR CORRECTIVE
MICROSURGERY
P.D. Temple-Smith & GJ. Southwick
Department ofAnatomy, Monash University, Clayton, Victoria 3168
Azoospermia associated with normal FSH levels and testicular volumes is
diagnosed as obstructive azoospermia and scrotal exploration with corrective surgery is
usually the only option available to reinstate fertility. This paper discusses
difficulties in preoperative diagnosis of efferent duct agenesis or germ cell arrest in .
azoospermic patients. Failure to identify these causes of azoospermia results in
unnecessary and unsuccessful surgery in a small number of azoospermic patients.
In each patient, azoospermia was confirmed by semen analysis. Semen fructose
and serum FSH, LH and testosterone levels were measured preoperatively. The
presence of vas and epididymis, and an estimation of testicular size, were determined
by preoperative clinical examination. Spermatogenic activity of the testis was
assessed from testicular biopsies and testicular volumes were confirmed from testicular
measurements taken during scrotal exploration.
Corrective surgery was not possible in 16 of 139 patients recommended for
scrotal exploration and epididymovasostomy during the period 1980 to 1987. In 12
patients microdissection showed that the epididymis was completely disconnected from
the testis and in the remaining group of four azoospermia resulted from spermatogenic
arrest at .the primary spermatocyte stage (2) and germ cell aplasia (2). In the twelve
patients with testiculo-epididymal discontinuity, five had normal testicular activity,
five had spermatogenic arrest and the remaining two had germ cell aplasia. In all
patients the epididymis showed no signs of obstruction, and hormone· levels and
testicular size were within the normal range, except for two patients with germ cell
aplasia who had raised FSH levels (>7IU/I).
As with germ cell aplasia (1) failure to identify patients with bilateral agenesis
of the efferent ducts of germ cell arrest using standard preoperative clinical and
laboratory assessment procedures results in unnecessary surgery. FSH levels and
testicular volumes are a guide for patients with germ cell aplasia but these parameters
were not able to distinguish patients with germ cell arrest or disconnection of the
epididymis from the testis. Further studies are needed to develop. simple diagnostic
tests which can be used to identify preoperatively these causes ofazoospermia.
(1) Jequier,A.M., Ansell, l.D. & Bullimore, N.J. (1984). Brit. J. Urol, 56: 537-539.
MECHANISM OF ACTION OF INH1BIN ON GnRH-INDUCED RELEASE OF FSH AND LH
103
Wang Qi Fa, P.G. Farnworth, H.G. Burger and J.K. Findlay
Medical Rese~rch Centre, Prince Henry's Hospital Campus of
The Monash Medical Centre, 5t Kilda Road, Melbourne, Victoria, 3004.
Inhibin, a gonadal glycoprotein hormone, suppresses GnRH-mediated
FSH and LH secretion by a mechanism that ma.y·be common to both
gonadotrophins (1), in addition to inhibiting FSH biosynthesis. We
previously observed that inhibin treatment decreased the number of
GnRH binding sites in cultured pituitary cells with no change in
receptor affinity (2). In the present study, we have used various
secretagogues, which stimulate gonadotrophin release by acting beyond
theGnRH receptor, to further characterize the anti-GnRH action of
inhibin.
Primary cultures of anterior pituitary cells were prepared using
tissue from adult male Sprague-Dawley rats. On day 2 of culture, t.h~'
cells were treated with pure 31 kDa bovine inhibin (0.1-3 U/ml) or the
protein synthesis inhibitor, cyc:J,oheximid:e (0.01-100 llM), for.

104
INDUCTION OF LH SURGES BY OESTROGEN IN OVARIECTOMIZED
(OVX) EWES REQUIRES A LARGE AMPLITUDE PULSE OF GnRH
OR A RAPID VOLLEY OF PULSES.
D.!. Phillips!, IT. Cummins 2 and 1.1 Clarke 3 .
lSchool of Agriculture & Forestry, University ofMelbourne, Parkville, 3052.
2St. Vincent's Hospital, Fitzroy, 3065.
3Medical Research Centre, Prince Henry's Hospital, Melbourne, 3004.
We have reported previously a modest 2-3 fold rise in plasma LH levels in
OVX-hypothalamo-pituitary disconnected (HPD) ewes receiving hourly GnRH
pulses and 17-19 hours after an oestradiol benzoate (OB) injection (1). Thus
!he 10-20 fold rise in pl~sma LH concentrations seen in hypothalamo-pituitary
mtact (HPI) OVX ewes gIVen OB reflects more than just a change in pituitary
responsiveness to GnRH. In this study we tested whether the LH surge may be
initiated by a large pulse of GnRH or a rapid volley ofpulses.
Six OYX-HPD ewes were given 250 ng pulses of GnRH (peninsula
Laboratories, U.S.A.) each hour and 50 ~g OB (Lm.) at t=O hours. Blood samples
were taken each 15 minutes for 15 hours beginning at t=16 hours. Two GnRH
pulse patterns were employed, with 3 ewes per treatment, after which 3 of the
6 ewes received the reverse treatment the following week. Treatment A
involved a bolus injection of 2.25 ~g GnRH at t=16 hours, then half-hourly 250
ng pulses from t=1630-3100 hours. Treatment B involved a volley of 500 ng
GnRH pulses each 10 minutes from t=1600-1630, a 500 ng pulse at 1645 hours
and then half-hourly 250 ng pulses from 1700-3100 hours. In HPI-OYX ewes
(n=lO) plasma LH concentrations were measured in half-hourly samples taken
across OB-induced LH surges. The area under LH vs time curves were
computed in all ewes across the LH surge (Table 1). There were no significant
differences in the responses of the 3 groups.
Table 1. Plasma LH responses (mean +/- s.e.m.) in OVX-HPI and GnRH-pulsed
OYX-HPD ewes given 50 ~g OB.
Group
OVX-HPD; Treatment A (bolus)
OVX-HPD; Treatment B (volley)
OVX-HPI
In HPX-OVX ewes, both treatments were able to release an eqUivalent
amount of LH to that obtained in OVX-HPI ewes given OB, with GnRH pulse
patterns similar to those measured during OB-induced LH surges in HPI ewes
(2). This suggests that initiation of the positive-feedback event requires a
large amplitude GnRH pulse or a rapid volley of pulses.
(1) Clarke, 1.1 & Cummins, IT. (1984) Neuroendocrinology 3..2.: 267-274.
(2) Clarke, 1.1 & Cummins, IT. (1985) Endocrinology ill: 2376-2383.
N
5
4
10
Response; Area under
curve (n&/ml h)
259 +/- 55
250 +/- 24
346 +/- 37
105
REGULATION OF PITUITARY LEVELS OF MESSENGER RNA (mRNA) FOR THE
SUBUNITS OF FOLLICLE STIMULATING HORMONE (FSH) AND LUTEINIZING
HORMONE (LH) BY GONADOTROPHIN-RELEASING HORMONE (GnRH) IN THE EWE
Julie E. Mercer and lain J. Clarke
Medical Research Centre, Prince Henry's Hospital,
Melbourne, Australia 3004
Secretion of LH and FSH often shows divergent patterns. In
hypothalamo~pituitary disconnected (HPD) ewes LH secretion falls
rapidly, while FSH secretion continues for a number of days following
GnRH withdrawal. Similarly, in HPD ewes treated wi th a constant
infusion or small pulses (25 ng) of GnRH, LH secretion diminishes much
more rapidly than does FSH (1,2). To determine the extent to which
these secretory patterns reflect ongoing gene transcription we have
moni tored mRNA levels for LHt3, FSH/3 and the cdmmon a. subunit in these
circumstances.
Pituitary glands were collected from OVD/HPD ewes which received a) no
further treatment,b) 250 Rg pulses of GnRH each 2h, c) 25 ng pulses
of GnRH, d) cons taRt infusion of 250 ng/h of GuRH, e) one week of 250
ngl2 h GnRH pulses and withdrawal of GnRH for 30 h. Total RNA was
prepared from individual pituitaries and analyzed on Northern blots.
Table 1. Relative pituitary mRNA levels for FSHI3, LH/3 and a.-subunit
GROUP
OVX/HPD 6
OVX/HPD+GnRH (250 ng/2 h) 8
OVX/HPD+GnRH (25 ng/h) 4
OVX/HPD+GnRH 3
(constant infusion, 250 ng/h)
OVX/HPD+GnRH (pump off 30 h) 4
FSHI3 LH(3 a.
0.30+0.07 0.19++0.0q 0.42+0.13
2.06+0.38 5.46+1.17 4.69+1.47
3.63+0.64 3.55+0.95 3.77+0.33
1. 36+0.16 1.43+0.21 2.15+0.45
2.90+0.46 3.89+0.54 2.50+0.26
GnRH (250 ng pulses) in the OVX/HPD ewe significantly elevated mRNA
levels for all gonadotrophin subunits. 25 ng pulses further increased
FSH/3 mRNA levels over those seen with 250 ng GnRH while levels of LH(3
and a..mRNA did not further change. Withdrawal of GnRH for 30 h does
not change FSHj3 or LH(3 mRNA levels. In contrast, administrati.on of a
constant infusion of GnRH (250 ng/h) lowers mRNA levels for both
subun~ts compared with pulsatile administration.
These data show that secretion of the gonadotrophins does not
necessarily reflect cellular mRNA levels, in that LH(3 mRNA levels
remain elevated in the face of treatments which reduce or abolish
secretion. High amplitude pulses of GnRH are required for maintained
secretion of LB, whereas lower doses are sufficient to maintain LH(3
mRNA levels. For FSB, secretion appears to be more closely coupled to
cellular FSH/3 mRNA levels, as evidenced by both the pump-off and
constant infusion studies.
1. Clarke lJ &Cummins JT. J. Endocrinol. 113:413-418, 1987.
2. Clarke et al. J. Endocrinol. 111:43-49, 1986.
n

PMSG (iu)
oFF (rnl) 0 7.5 10 15 20
0 0 8A a 18.2 b 39.6~ 64Ad
1.5 6A a 8.6 a 23.6 39.8 b
% inhibition 24 53 40 38
a,b,c,d, differ at P

108
INVOLVEMENT OF PROSTAGLANDINS IN PREMATURE LUTEOLYSIS
IN THE SUPEROVULATED GOAT
R.M.. Battye, A.W.N. Cameron,
Regression
Fig. 1
R.J.
Fairclough,l A.O Trounson
C 1
E.H.D. Monash Medical Centre, Clayton, Vic.,
Animal Research Institute, Werribee, Vic.
of corpora lutea within 6 days of superovulation in goats
results in low rates of embryo recovery (1). The cause of early luteal
regression in the goat has not been elucidated. However, luteal
regression in the naturally cycling goat is characterised by surges in the
concentration of 13, 14-d1hydro-15-keto-postaglandin F2~(PGFM) (2). The
administration of the PGF synthetase inhibitor. indomethacin, suppressed
PGFM peaks and prolonged the oestrous cycle. This study investigated
whether prostaglandins were the cause of prematurely regressing corpora
lutea in the superovulated doe.
Ten does were superovulated in June 1987, by treatment with
intravaginal progestagen impregnated sponges (Repromop, Upjohn, 60 mg
medroxy progesterone) for 16 days and 1200 iu PMSG (Pregnacol Heriot J,
Agencies). administered 1m, 2 days before sponge removal. Six does were ~
injected twice daily with the prostaglandin synthetase inhibitoX-f~nixlJl ~
meglumine (Finadyne, Schering) at 2.2 mg~·7rom·-·Day3-7 of the
synchronized" cycle. Four does served h "untreated controls. Jugular
blood samples were collected from 2 days before sponge removal to Day 7.
On Days 4 and 5 blood was sampled hourly between 0900 and 1700h.
Laparoscopy was conducted on Day 7 to determine the incidence of corpus
luteum regression.
Significant peaks of PGFM were detected in the 4 untreated does on
days 4 or 5 or both but only in 2 of the treated females on these days
(P