A Study of Embryonic Quiescene in Diprotodont Marsupials

A STIIDY Of' EMSRYONIC qUIESCENCE

TN DIPROTODONT MARSUPTAI,S

by

Meredith J. Smith, B. Sc. (Hons).

Departrnent of Zoologyr University of Ad'elaide, South Australía.

]?68

A thesis submitted for the clegree of, Doctor of Phllosopby in the Universúty of Adelaid-e. L¡

CONTENTS

SUMMARY vl_l_

DECTARATION x

AC K]\IOÏI'I,EDGEIIENT S xl-

1. TNTRODUCTTON

Defjnition quiescence 1.1 of 1

1.2 The d-iversity of Épecies that nay wrd.ergo 2 enbryonic quiesoenoe

1.3 Growth ancl development of the quiescent blastocyst 6

1.4 The corpora lutea and_ uteri during enbryonic guiescence I .{tr l.J the pituitary gIand. in enbryonic quiesoence 10

1.6 The effect of the ovaries and_ ovarian ho:rrones 13

1.7 Sunnar¡r 15

2. IIATERIAIS AND IVTETHODS 2.1 Classification of species 17

2.2 Mai-ntenanoe of experimental animals 17 213 Examination of aninals 18

2.4 Estimati¡g age of pouch young 1g

2.5 Hornone preparatíons 19 prolactín (i) 1g

(ii) tuteinizing hormone 20 l.L.

(iii) Progesterone 20

(iu) oestrogen 20

(") Th¡rrorine 20 j-a 2.6 Anaesthes and. gurgery 21

2.7 Collection and. transfer of blastocysts 22

(i) Transfer to eye 23 (t) Transfer to uterus t7

(ii-i) Transfer to nillipore fiLter ohamber 2)+

¿.ö Histology 24

219 Counting 25

2.10 Photonicrography ¿o

3. HVIBRTONIC QUIESCENCE IN TI{E RED I(ANGAROO

7A )¡ I Introduction 27

3.2 Development before quiescence 28

3.3 Quiescençt phase of lactation 28

3.4 Changes that oecur after 2@ d.ays of pouch sucklíng 32 3.5 Changes that occur after removaf of pouch- young 35

3.6 Dis cus sion 38

lr.. IMBRYONIC QUIESCÐ{CE IN OTTIER DÏPROTODOIi]| I\IAB.JUPIAI,S

4.1 Introd.uction 4o l-l-l- ¡

+,2 Speoies without post-partun xoating and in which the length of gestation is ind.epend-ent of lactation 40

Trichosums vul-peoula 40

Petaurus breviceps 42 l+.3 Species without post-partr:n roati-ng in lvhich gestation period. nay be lengthenetl during suckling 43

4.+ Llonotocous species with post-partr;m nating 45 (") Species capable of breeôing throughout the year 45

Thylosale billarclieri +5

l,iacropus robustus 46

L'laeropus rufogriseus +7

Macropus agil-is 52

Potorous tri-d.actvlus 52

(t) Species that breed. in an annual seasorl 53

IÍacropus eugenii 53

Setonix brach.yurus 55

4.5 Pol¡rbocous species with post-partr:rn mating 56

(u) Cercartetus concirurus 56

(r) Post-partr:m femal-e 58

(ii) Pregnant females 59

(iii) Anoestrous fenales 66 Ív.

(¡) Acrobates p-yHîaeus 66

+.6 Species that nate before parturition 66

ïfal-labia bie.olor 66

trÍacropus dorsalis 68

+.7 Dis oussion 69

5. fiIE PHYSIO],OGY OF M,4BRYONIC QUÏESCENCE

5.1 ïntroduction 76

5.2 Pituitary hormones 77

(") Prolaotin 77

(¡) L,uteinizing homone 78

(") Discussion 81

5.3 Developnent of blastocyst ind.uced_ by progesterone B3 (") Histological observations after injection of progesterone 84

(i) Blastocysts 84

(ii) Ovaries 86

(iii) uterí 87

(t) Sustainect- d.evelopment of the enbryo B9

(l) Red. kangaroos injectect with 10 ng progesterone; RPy d_ay 4 B9 (:-i) Red- kangaroos injected with ! or 20 mg progesterone; Rpy d.ay 4 9o (i:-:-) Progesterone treatroent continued. after RPY 9t Vr

(iu) Pouch-young removed_ later tha¡ daY 4 94

(") Discussion 96 5.+ Deve] opment of blastocys-b induced_ by oestrogen 100

(") Histological observatj-ons i_n red_ kangaroo 100 (¡) Sustained. d-evelopment of enbryo in red. kangaroo 101 (") Development of ernbryo in ovariectonized_ K.I. løaliaby 103

(i) Ovariectomized. control_ animals 103 (t) Ovariectoni-zed. alimals treated v,¡ith oestrogen 104 (iii) In,cact, oestrogen-treated_ control animals 107

(a) Discussion 110

5"5 Extra-ovaria¡ honnone - th¡rroxine 111 5"6 Maintenance of extra-uterine blastocyst in irivo 112

(u) Tntroduotion 112

(t) The ovi-duct as an ectopi-c site 11+ (") Transfer of blastocysts to an ectopic site 115

(i) Anterior chamber of eye 115

(ii) Millipore fÍlter chanber 117

(¿) Disoussion 119 l¡f_ o

6. DTSCUSSTON 6.1 The evolution of enbryonio quÍesoenoe 120 6.2 fubryonic quiescenoe and. the phylogeny of dÍprototlonts 121 6.3 The arrest of eurbryonic d.evelopnent 123 6.4 The stimulus for renewed. enbryonic clevelopnent 126 (") Direot effect of steroj-cl ho:mones 126 (t) Indíreot effect of steroid. hormones 128 6.0 Conol-usions 131

APPENDIX 7. B]B],TOERAPHY 133

8. RXFERENCES 131+ IriÍ.

SU1,0.,lARY

Embryonic quiescence has been investigated. in fifteen spocies

of ùiprotod.ont rnarsupials by observation of live aninals or by microscopic examination of fenar-e reproduotive organs.

fn all the macropoùids studied- (12 species) there is a

variable euiesss¡¿ period. in the d.evelopment of embryos formed.

lvhile a femal-e is suckl-ing a pouch-young that is not near independ.ence

of the pouch. The enbryo beoomes quiescent before the protod.e:m d-ifferentiates. The resting stage is sinil-ar Ín arl species, irrespecti'¡e of whether it is fo*ed. a felv d.ays before parturitíon, a d.ay or two after parturition or many weeks after parturÍtion. The spherícal, 'nilaninar protod.eru consists of an average of 69 ee1ls (i\4acropus robus tus and- Setonj-x brachJrurus) to an average of

92 cells (Maoropus Siganteus) utrd is surroundetL by a spherical shel1 joO about u i-n d-iameter. The embryo does not grow whir_e a pouoh- young suckles continuously.

corpora lutea aseociated. ürith quiescent enbryos are smarl, with snall luteal cells and. ínconspicuous blood- vesseLs. The uterÍ are non-lutea1. After renoval of the pouch_young of Meealei-a rufa the cozpus luteun grows by h¡rpertrophy and- h¡ryerplasia of ce11s, but growth near the termination of pouch life of the young ís by h¡ryertrophy only.

The absence of post-parturition nating has been confinoed. i¡ the phalangerid.s Trichosr.ur:s vulpecula a¡d. Petaun¡s brevÍcen S. vl-l_I .

Cercartetus concinnus m ates soon after parturition and. the embryos grow s1ow1y while the litter is suckled..

The effects of some pituitary and. ovarian hor:mones on the quiesoent reprod-uctive organs and. blastocyst rvere stud.ied. in

M. rufa and. ì,iaoropus e nl_l- . Systemic injection of nei_ther

prolactÍ-n nor luteinizing honnone caused_ d.evelopnent of the quiescent corpus luter:m.

The ovarÍarr hor:mones progesterone a¡d oestrogen both induoed. luteal changes in the uteri of L,l. rufa that continued. to suckle

pouch-young. The blastocysts resumed. grorvth and. in some aninaÌs

this growth continued. to term after the pouch-young was removed.,

The bírth rate v,¡as verxr 1or,,r in oestrogen treated. animals. Exogenous progesterone did not change the nicroscopic

appearance of quiescent oor?ora lutea, but oestrpgen causecl vascular d.evelopment. Holvever ttie effect of oestrogen ín 1\{. eusenii was independent of the ovaries, for blastocysts of bil-ateralIy ovariectonized anjmals grev,r in r€sponse to oestrogen.

This grorrth was not sustained- a¡rd- d.egeneration had. begun seven d.ays after the first of J injections of oestrogen. The th¡rroid. hormone, thyroxine, did. not stinulate enbryonic grolvth i-n l/1. euAenii..

The effect of the uterus on the embryo was stud.iecL Ín l,{. eusenii Thbal pregnancy was not ind_uced_ by ligation of the oviducts after mating, a¡rd- blastocysts d-egenerated. vyhen transferred. a]i. fron the uterus to ttre a¿torrior ohanber of the eye. Eight blastocysts were transferrecl to millipore filten oha¡nbers and. praoed in the peritoneal oavity of quiescent phase anrmars; five cLays later al-1 blastocysts were degenerate, but cel_l-s wore ðiuiùing in tu¡o of them.

The macropod.id. blastocyst nay resenbr-e blastooysts of the mouse in remainÍ-ng quiesoent only when oonfined. to the utèrus. rt fs possible that oestrogen and. pr"ogesterone do not aot clirectly on the maoropocritl blastocysto but permit tt to grow by ohangrng intra-uterine condj. tions . X¡

DECIARATTON

Ihis thesls oontains no nateriaL previously subnitteaL by ne for a d.ogree in any UnÍversity, ancL contains no matorial previously published. or r¡rritten by an¡r other porson, e:roopt when d.ue referenoe is nacle jn the text of the thesfs.

Meroùith J. Snith Xf- .

ACKNOITTLEDG¡TÍENTS

The worlc d-escri-bed- in this thesis ilras begun in the C.S"I.R.O.

Division of nïil-d-life Research, Canberra, vrhil-e I i,vas employed- by

C.S.I.R.0. It v¡as continued. briefly at the School- of Biological .lyas Sciences, University of New South T,/ales . The ',vork cornpleted in the Zoology Department of the University of Ad.elaide, and was supported. by a University Researsh èrant, for which I a"n 6ratefu1. Professor G.B. Sharroa¡ of the School of Biological Sciences, University of New South Ïrlales " adrised and- gu-ided. me during the initíaI phases of the study, and. I am grateful to him. I wj-sh to thank Dr. P.G. Martj¡r, Department of Botany, and Dr" S. Barker,

Department of Zoology, for their advi-oe and- supervision throughout the rest of the work. Professor H"G. Andrewartha r:nfailingly gave advice anil encouragement.

Several people provid-ed- me v,¡ith preservecl material . I wish to thank llessrs. J.H. Calaby, R.L. Hughes, J.C. Merchant, a¡d 1Àr"8. Poo]e for their gifts, &d Dr. TI"D.I. Ride and. I\ilr. P"F. Aitken for penrission to work on preserved. material in the

triiuser.¡rns Vfestern Austra]-ian and. South Austra"].i-an " f an grateful to the Corrncil of the Royal Zoologioal SocÍety of South Australia, and especially to llr. Y[.E. ],a¡rcaster, for their pennission to stud-y certain wallabies in the Zoologícal Gard.ens. 1.

rNîRODUCTION

1.1 Definition of quiescence The ma,mmalian ovum begins to d.ivid-e i^¡ithin hours of fertil-ization and in most species it maintains a rapid, rate of d.evelopment throughout gestation. But the ova of some species d-evelop rapid.ly into blastocysts which then enter a period of dormancy while lying free in the uterine lumen. This inacti.vity always occurs before the embryo attaches firmly to the uterine mucous membrane, that is, before implantation of the embryo, and. the phenomenon was termed. 'rd.e1ayed" implantationrr (Hamlett l-935). This term suggests that the blastocyst is held. in check beca.use it cannot achieve anatomical union with the uterrrs, and- ad-equately d-escribes the situation in rod-ents (Sctrlafke and End.ers l-963). However it is not appropriate for many other species. At the end- of quiescence roe d-eer bÌastocysts grow rapid.ry for about a month before implanting, and. a shorter period_ of rapid. growth before implantation occurs in the European bad.ger,

Mel-es me1es, and the weasel, Mustela frenata ( r,urignt t948.-

Canivenc and. Bonnin-Laffargue 1!61, Short and, Hay f966). The embryo of macropodid- marsuplals never implants in the strict sense of erod.ing the maternal end,ometrium (Sharman lp6ta). As inplantation does not always occur immediately, the enbryo is reactivated, a tern is need.ed- that implies that the limiting 2, mechanism is embryonic growth and. not uterine attachment. frEmbryonic d-iapausetr has been proposed- (Tyndale-Siscoe L963c,

Short and. Hay f966).

The name rrd.iapauserr rüas coined to describe a stationary phase in the development of a grasshopper embryo but it is no¡r used by insect physlologigts to describe a d.evelopmental arrest which is spontaneous and does not respond immediately to amelioration of the external environment. Inlhen development is temporarily inhibited. by an unfavourable environment and may be resumed as soon as the hindrance is removed, rtquiescencerr is a preferable term (And.rewartha I)J2). The dornant embryo of at

l-east some manmalian species can be reactivated. by changes in its hormonal- environment - by injecting the pregnant lactating mouse with oestrogen (wtri-tten f955), by removing the ovaries from the nÍne-band.ed armadillo t Das.ypus novemcinctus (Enders 1966) or by subjeoting the marten, Martes americana to increased photoperiod-s (Pearson and Enders 1944). ttQuiescencefr describes the oondition better than does triliapause'r. In this thesis the term ttembryonic quiescencerr will be used to desoribe the prolonged- inactivity that occurs in the blastocyst stage of many mammalian embryos.

1.2 The diversltv of snecies that may underso embr.yonic qulescence Embryonic quieseence occurs in some species of at least

seven mammallan orders (natte 1.1). Anong the rodents, shrews, 2

TABI.E 1.1 characteristícs of the gestation period. of some species i¡r which the embryo becomes quiescent. lhis table is not comprehensive; enbryonic quiescence is known to occur in other nustelid.s (wnigt'rp 1g$) and narsupials (Sharnan, Cataby and poole ßá6) ui¿-ír-r"rp"ãlea i" many further species.

0rder Species Quiescent Post-quiescent period. period. in d.ays in d.ays (approx. ) (approx. )

Marsupialia 150 26

20+ 31

338 27

Insectivora Sorex ara¡reus (shrew) Eãffir CÐ'

Rod.entia l,,ius musculus (rnouse ) o- 16 13 suclcling more than 2 yotmg (r ¡)

Rattus norvegieus (rat) o- 21 15 suchling more than 6 young (9, 16)

Chiroptera 150 9O

9o 120

Ed.entata noveno go- 135 150 -band.ed- azrn (+)

...... /4 4.

ÎABLE "l .1 (cont.)

0rd.er Species Quiescent Post-quiescent period- period. in days in days (approx. ) (approx. )

Carnivora Ursus americanus (Utact 150- 1Bo 50 bea¡) 117 ) rÐ-IViartes anericana- (narten) 21o - 255 28 (river 500 60

Ùlustela vison (nínt) (¡) 12-49 3o iÍustela frenata (1ong-tai1ed. 255 25 weasel) (18) Iieles meles (European 300 +5 badger) (3, 7) 60- 3oo +5 Ta^:ciclea taxus (Anerican 180 +5 ¡a¿eer (19) Phoca vitulina (con:non seal) ßo 180 Q) Halichoerrrs grypus (g""v 9o 180 sear) (Ð

Artiod.actyla C apreolus capreol-u.s (roe 150 Ito aeer) (14)

966, 5,

and most marsupíals ín which it occurs, a d.eLay in d.evelopment is facultative and. occurs only wheù the female 1s lactating. Tbe batsr armad-i11o, carnivores and- d.eer bave reprod.uctive cyclee that show a strict annual period.icity, and. in these speciee quiescence is obligate and- occurs in every pregnanoy (with rare exceptions ) . rnvestlgations into the physiology of embryonic quiescence have been hampered. by the nature of the species in which it oocurs, for very few of these are suitable for experimental manipulation. The armad-illo has not yet been bred. in the laboratory (End.ers f966)9 bad"gers mope and- behave abnormally in captivity (Harríson 1963); most of the mustelid.s are fierce and, intractable (tdrigÌìt L963). Of necessity, information in these specÍes ls malnly limited, to a-natomical and_ cytological observations on naterial from wlld.-caught ani-ma1s. 0n the other hand.r extensive studies have been mad-e of the hormone requirements for continuation or termination of qulescence in the rat and. the mouse r

1.3 Growth and. d.evelopment of the uiescent blastoc.yst

In all species in which quiescence occurs, rapid. development ceases soon after the formation of the blastocyst cavity. The eutherian embryo then consists of a hollow sphere of ce1ls, the trophoblast, and. an inner cell mass, which ad-heres to part of the 6

inner surface of the trophoblast. The marsupial embryo consists

only of a hol-1o'¡ sphere of appalently undifferentiated. ce11s, known as the protoderm (loyd- and- Ilamil+,on I)JZ)" In neither marsupial nor eutherian bl-as-bocys'u is there any d-ifferentiation

of ectoderm and enctoderm at the onset of o.uiescence. However

slow growth and- differentÍation me,l¡ occur d-uring quiescence.

The amount varies from species to species and-, as can be seen from the folloliing examples, the marsupials are at one extreme, roe deer at the other.

Blastocysts of the qr-ioi:ka and. recl kangaroo are of the same size irrespective of their age, ancl d-o not grow or differentiate d-uring quiescerice (Sharnan 1955s 1963; T;rndale-Biscoe f963c). llrmad.ill-o 'blastocysts rilnge in cliameter from 0.19 to 0. ! mm, but because of the impossibility of deterrnining the exact age of blastocysts, the rate of grol+th d-uring quiescence cannot be deterrnined. Some celi- d-ivision occurs. The looseì-y- associated- cel-ls of the inner cel-l- nass are d-evoid- of evid.ence of specializatíon, and give no inclication of the four centres of organization from which arise the identical quad-ruplets (End-ers 1962). Quiescent blastoc;'sts of the rat expand- slightly from

60- 85 u to as much as Ì10 r-L in d.iarnetei a,fter sixteen d-ays" Ulitotic activity is slight (ScHt-afte and. Enclers 1963) or absent 7

(Baevsky 1963) and. d.ifferentiation ís limited to attenua.tíon of the inner cell mass as the cel1s grad.ually elongate parallel to the long axis of the blastocyst. The zona pellucid.a is lost

frorn the blastocyst d,uring quiescence in both rat and. mouse

(Sctrtafte and Enders L9639 Mclaren 1967)" fn the carnivores, as in the rodents, changes during

quiescence are limited to expansion of the trophoblast accompanied. by a very loru rate of cell- d-ivision, and to rearrangement within the inner cell- mass. The overall d-iameter increases two- to four*fo1d, in the seven-month quiescent period of the American

badger and. in the ten-month period of the European bad.ger, and. in both the number of cells doubles (approxirnately) during quiescencc (Nea1 and Harrison 1!!8, llright f966). The expansion of mink and sable quiescent blastocysts is the prod-uct of celI expansion and. fluid- uptake rather than cel1 replication (Baevsky 1!63). The zona pelJ-ucida persists throughout quiescence in aII carnivores

( i,Iimsatt 19 63 ) . Greater d-ifferentia,tion occurs in the roe deer than in other species. Blastocysts increase in size frorn 100 u, d.iameter

in early Âugust to BOO r-r, in Octoberg by the end" of November they may measuîe 2,0 mm anrl proliferation of endoderm has changed. the trophoblast into the bilaminar omphalopleure. Quiescence end.s in mid.-December when the blastocyst measures a.pproxinately B.

3.3 x 1. / mni, and. there is a 4-week period. of rapid growth before the embryo implants (t

I.4 The corpora lutea and. uteri durins emb ryonic quiescence

The survival and- growtb of und.elayed" embryos depend.s on a

suitable environment, which is provid.ed- by the uterus in response to ovarian hormones; the release of ovarian hormones is in turn controlled by pitultary gonad.otrophins (Rowlands and parkes r!66). 1ln investigation of the structure of pituitary gland.s, corpora

lutea ancl uteri of animals uith quiescent embryos might give some ind-ication of a d-ifference in the hormonal environment of quiescent and- active embryos.

The gland.s of the marsupial uterus are larger and. more extensive d.uring quiescence than d.uring anoestrous period-s, but the gland- cells are low (in contrast to their talr columnar shape j.n the post-quiescent phase) and. there is no sign that they are secreting. The corpus luteum is srnarr, its d.iameter being less than half the maximum it attainsí it is poorly vascularized. and- the luteal- cells are srnall (Sharman 1955¡ I)6{a, Tynd.ale-Biscoe t963c)" The corpora lutea and uteri of carnivores are similarly i11-d.eveloped. during embryonic cluiescencei at the time of reactivation of the embryo, blood. vessels of the corpora lutea d-iIate, the luteal cells increase j-n size, the uterine grand.s become more ooiled- and- the uterine gland. cel-ls begin to secrete o

(Canivenc 1966, Enders and Enders 1963¡ Harrison 1963, Wimsatt f9æ)" The poor development of the uteri- in these species suggests that progesterone is in short supply, and assays of progesterone in corpora lutea of the bad-ger rcveal that their progesterone content is very much lower during quiescence than in later stages of pregnanoy. The rate of secretion of

progesterone has not yet been d-etermined (Canivence Short and. Bonnin-Laffargue 1966).

Corpora lutea of pregnancy in the rat are not larger than those of pseudopregnancy untir after implanta,tion. The ovaries of pregnant lactating rats contaÍn sma1l corpora lutea of pregnanoy and. slightly larger corpora lutea of lactation (Weichert and- í-jchurgast 1t{2). Progesterone Ís circulating in the rat d-uring quiescence, for a d.ecid.ual reaction occurs ín response to traumatization of the utems, even though the oontained embryos do not stimulate formation of d-ecid.ua (Krehbiel

1941b ) .

The uterus of the armad.illo is poorly-d-eveloped. and, non- secretory during d-elay but the corpora lutea are fu11y luteinized. by the time the blastocyst reaches the uterus, and the lutein cells are similar in appearance whether the ernbryo is quiescent or active (Enders, Suchanan and. Talma,ge l)Jï, Enders f966). The level of circulating progestins is five times higher d-uring d.elay 10.

than before ovulation, and- increases only a rittre over zo/o after implantation (Talmage et al. I9r4). Corpora lutea of the roe deer are mod.erately well vascularized and contain typical luteal cell-s throughout quiescence, renewed. development and. after implantation, they d-o not change in weight nor change in progesterone content (Short and Hay f966).

1.5 îhe pituitar.y 91and. in embr.yonic quiescence îhe inactive corpus luteum of the quokka resumes growth within a few d-ays of removar of the pouch young d.uring the broed.ing seasone and. embryonic quiescence 1s terminated. (Sharman I95j). lÍhen gonadotrophin secretion was blocked. by treating a quokka with reserpine after rer¡roval of pouch younge the blastocyst remained. tluiescent as long as the reserpine treatment continued (Tyndale-Biscoe L9ûc). fnjection of horse anterior pituitary extract ínto l-actating quokìcas caused h¡teinization of fol-licles in the ovaries, and., in two animals, growth of the inactive corpus Iuteum, but the quiescent blastocysts d.id- not grow (Tynda1e-Siscoe 1963c). In rats and. mice suckling large litters, ímplantation foLlows removal of the litter or temporary removal of the rat litter for 12 hours d.uring the fourth day (Zeitmaker 1964),

Implantation can be induced. at any tirne after the third d.ay (mouse) or fifth day (rat) by injection of serum gonadotrophín 11. or pituit¿ry extract (lütritten l-955t t¡[eichert 1940). The suckling stinulusr presumably acting via the pituitary, controls the length of embryonic quiescence in lactating marsupials, rats, and. mice; d.ay-length may simirarly controL quiescence in seasonarly-breed.ing species. Although the ruating season may extentl for weeks or even months in some carnivores and the arrnad.illor the time of parturition i-s rest¡icted. and. annually constant for each species. îhis is brought about by synchronization of the tine of irnplantation within the population, irrespective of the length of time the ernbryos bave been quiescent (pearson and. Enders 1944y Neal and Harríson 1p18, Ðnders L966).

Inplantation occuued. about four months earlier than normal when martens were exposed. to gratLuarly increasing photoperiod.s from early autumn, and. the gestation period of nink was about { d.ays shorter than the average when they ¡¡ere artificíalIy illurninated for 1S hours a d.ay after and,for before mating (pearson and Enders tg44).

The pitulta"ry t cells, which are presuned to secrete luteinÍzing hormonc, regress after or't.latÍon in the European bad'ger a,nd bent-winged. batr but enlarge prior to the reconmencement of ernbryonic activity (Canivenc 1966, peyre and Herlant 1p6l). Their regression suggests that the failure of the corpora Lutea and hence of the uteri, to d.evelop fully may be d-ue to a d,eficiency 12.

of gonad.otrophins. rnjection of forlicle-stimurating hormone (from mares) and luteinizing hormone (hunan) led- to the formation of new corpora Iutea, but these had. the same appearance of

functional inactivi-ty as the pre-existing corpora rutea, and. the blastocysts remaÍned- quiescent. prolactin was without effect on corpora lutea, or blastocysts (canívenc L966). Gonadotrophins

extracted fron bad.ger pituitaries apparently have not been injected.. rf there is a suppression of gonad.otrophin release d.uring the quiescent period-, it nust be selective rather than

complete, for ovulation d.uring quiescence is not uncommon (wea1 and" Harrison 1!!8, canivenc L966). Mink also may mato severar tirnes, at intervals of about six d-ays, in each breed-ing season, and. a ner¡r geïl.eration of ova are released. at each mating; blastocysts from each mating may contribute to the ritter born (Enders and Enders 1963).

îhe experimental shortening of the quiescent period_ ln the marten and. mink ind-icates that the ovaries are not refractory. Â gonad.otrophic substance must be lacking d.uring quiescence, but the substance remains unidentified- except in the rodents where fo1licle-stimulating hormone is probably the effective gonado- trophin (wiri.tten L955). The importance of gonadotrophins in the armad.lIlo and- roe d-eer is obscure, for in these species the corpus luteum changes little d.r;.ring quiescence and. active r3.

embryonic grovrth (End.ers 1966, Short and Hay 1966).

1.6 The effect of the ovaries and- ovarian hormones Despite the growth of the corpus luteum of carnivores coincident with renewed. enrbryonic activity, q,uiescence has not been artificially terminated with oestrogen or progesterone (Canivenc and Bonnin-Laffargue 1963)" Progesterone alone or with oestrogen d.oes not affect blastocysts of the bad-ger - they remain sma1l and. free within the uterus (Canivenc 1957)" In mink, inplantation is not hastened- by injection of progesterone and oestrogen, and- ernbryonic mortality occurs; oestrogen alone actually lengthens gestation (Cochrane and Shackleford 196?).

Nevertheless, oestrogen concentration must be high at the end. of quiescence, for the cpithelium of the vagina is highly cornified.

(Hansson 1947t Jlnders and Enders 1963). Oestrone caused a limited, unsustained expansion of mink blastocysts in cultureg growth was not achieved by any other treatment (Daniel l-967). Bilateral ovariectomy is detrimental to blastocysts of the weasel

(Wr:.ght L96J) but not to those of the bad-ger (Canivenc and. Laffargue 1958); the latter may even grow slightly after bilateral- ovariectomy (wcaI and- Harrison 1!l8).

ITo implantations ha,ve been achieved with injections of oestrogen anùfor progesterone into the intact armad.illo, and- whenever more than 0.3u8 oestrone was ad.ministered. the blastocyst I4

Ìüas lost from the ute¡us. rntrauterine Ínjection of hormones wa,s also ineffectual (End.ers and Buchanan I)J)). Bilatera1 ovariectomy causes implantation 18 - 20 d.ays later, and. at implantation the uterine end.ometrium of the castrate is

ind.istinguishabre from that of the intact animal (Enders 1966, End-ers, Buchanan and Talmage 1!JB)" uniLaterar ovariectomy

removing the corpus luteum does not hasten implantation and

total fertility is reduced (Enders 1966). To understand the maternal factors controlring embryonic quiescence, it rnay be necessary to extend investigations beyond. the sex hormones,

I seasonar return of the pituitary thyrotrophíc hormone rnay be involved (Canivenc t96O, cited. by DeanesJy l)66).

Bilateral ovariectomy of the rat or mouse prevents implantation not only in the lactating animal but also in the nonlactating animal if the ovaries are removed before d.ay 4 of pregnancy (rat) or on dry 3 (mouse) (wutt:.ng and. Meyer 1963¡

Yoshinaga and. .,\dams L966b). progesterone alone will not cause implantation but prolongs the 1if,e of quiescent embryos; in the presence of progesterone a sÍngle smal1 d-ose of oestrogen causes implantation 24 hours later (¡loct 1958, Nutting and Meyer 1963). Embryonic quiescence can be incluced. in the early pregnanct rat by transplanting the pituitary gland into the spleen; 1n this site FSH and LH are not released-, but the luteotrophic hormone, 15.

prolactinr is released- and. stÍmulates secretion of progesterone from the corpora lutea. The blastocysts remain quiescent until oestrogen is injected (Me¡rer, prasad_ and Cochrane 1!!g). similarly in the lactatíng rat or mouse, injected. oestrogen will ind.uce irnplantatipn (l¡Ieichert 1942, Ìthitten l?]rj) and. the effect of serum gonadotrophin in ind-ucing implantation is probably med-iated. by a release of oestrogen frorn the ovaries (wtr:_tten rg5il" Progesterone injected. systemically into the rat suckling ten young does not induce implantation (Zeirnaker l-964) arthough local application to the uterus of sma1l quantities of progesterone

ind.uces local implantation (canivenc, Drouvillô and_ Mayer lr953t Yoshinaga 1!61). Local application of oestrogen similarry induces implantation (yoshinaga r!6r.). Local_ apprication of progesterone is ineffective in the castrated pregnant rat (lsychoyos I)62). Â certain bal-ance between the concentrations of oestrogen and- progestcrone is necessary for the terrnination of quiescence in the rat and. mouse.

I.7 SJ.immary

Embryonic quiescence occurs in a d.iversity of mammalsg the hormonar cond-itions and- their morphorogical manifestations are equally d.iverse. /,mong the carnivores the poorly-d.eveloped- corpora lutea and- uteri suggest a d.eficiency of gonad.al hormones brought about by lack of a specific gonad.otrophin, but attempts 16,

to experimentally compensate for these d.eficiencies have been unsuccessful. The armad.i11o, rodents, and" probably the roe deer do not lack circulating progestins d_uring quiescence; a small dose of oestrogen terminates quiescence in the rat but causes loss of the arnadillo blastocyst; bilateral_ ovariectomy prevents implantation in the rat but precípitates it in the arnad.illo. Marsupials resemble the rodents in that suckling is the external stimtrlus for embryonic quiescence, but the stmcture of their corpora lutea and. uteri d.uring cluiescence is more like that of the carnivores than of the rod.ents. The response of the marsupial enbryo to exogenous gonadotrophins and gonadal hormones is uncertain. Different patterns of reproduction involving embryonic quiescence occur among the diprotodont marsupials (Sharman,

Calaby and" Poo1e 1966). Morphological stud.ies have been mad.e on corpora lutea of a few species (".g. Sharman 1955,1964a, Hughes L962, Tyndale-Biscoe I)63c, 1965), but structure of the blastocysts throughout the quiescent period. has not been examined.. The aims of the present stud.y were to gain more i-nformation on the reprod.uctive patterns of female d-iprotod-onts, to investigate the structure of the blastocyst in species with d.iffering patterns of reprod.uction, and- to gain some insight into the hormonal environment of the qulescent blastocyst. rT.

Lõ }IATERIALS AND 1VIETHODS

2.I Classification of species Taxonomists no longer divide narsuplals into two major groups accord-ing to the number of incisorsl but the term trdiprotodont mar,supialr' is conveniently retained. to group

together the familles Phalangerid.ae, Macropodid.ae and-

Shascolomidae (Sharman, Calaby and- poole, L966), These, together r,¡ith two extinct fanilíes, constitute the superfanil-y

Phalangeroidea ( Sirnpson L945). The generic names given by Simpson (L945) :.n his ctassification of marsupials are comrnonly used. Cal-aby (I966),

following Stirton flge¡),consid.ers that the generi-c name

Protemnodon shou]d- be restricted- to the fossil species, and.

prefers to place all the mod-ern wallabies wÍth 16 chromosomes

in the genus I'{acropus, together with the grey kangaroo and.

wallaroo group, The unique gait and- low chromosome number of

the sÌüamp wallaby are recognized- by placing it in a monotypic genuse ll¿;llabia. Calaby's mod-ification of Sinpson's classification has been used in this thesis.

2,2 lfaintenance of experimente"I animals

Red. kangaroos r¡rere penned. in open yards, approximately 100 sq. yard-s in area. The natural pasture ldas supplemented l¡ith lucerne hay and. sheep nuts. Some of the animals had. 18.

been taken in the wild- as juvenires, but nany had been bred- ín

captivity. Record-s were kept of the d-ates of parturitions and.

matings of each female" i1l-r the femalcs used. were suckling a

pouch-young less than 1!O d.ays o1d., and_ were known to have mated. at the post-partum oestrr¡s.

Kangaroo IsIand. wallabíes were naintained- in open,

concrete-floored. pens, 400 sq. feet in area. They were fed_ wheat, oats, bran, lucerne chaff or a conmercial compressed food., and green food was provid.ed. twice a week. The K. I. wallabies r^rere procured from a d.ealer, and. all had. beern t¿ken as ad.ults from Kangaroo fsland.. Some lost their pouch-young after capture, but the presenc€ of an elonga,ted teat and- enlarged mammary gland- ind-icatcd" that the female had recently suckled. a pouch-young and. should be carrying a quiescent blastocyst.

2.3 Exarrination of animals The aninral was grasped by the base of the tail and. placed- in a sack. One observer held the aninal on its b¡rck and- controlled the powerful hind. legs, while a second person examined the pouoh or urogenital opening. Oestrus was diagnosed from the composition of epithelial cells collected from the posterior vaginal sinus by the method, of Sharman and- Pilton (1964). A tapered glass tube was inserted into the urogenital sinus and. cells withd.rawn through this with t9"

either a polythene-tipped. syringe or a cotton T¿,¡ool s.wab. The cel-ls were smea¡ecl on a rnicroscope stid.e, covered. and obserwed. under phase contrast. sorne smears were fixed. in alcohol ether

(r:r) and. stained, with shorr's stain. At oestrus the smear was thick with cells, most of which were fully cornified. (shannan and Calabv t964)"

2.4 Estimating age of pouch _young

Pouch-young of animals shot in the wild. 'hrere measured. with vernier ca1lipers, the length of head-, 1eg and. foot being record.ed., together with the weight,. îhe erge was estilrated from growth curves compiled. from measurements of young of known age. The follol'ring growth curves r'¡erer used-; lvîqgareia rufa - sharman, Frith and Calaby I964a lVlacropus giganteus - poole unpublished.: lvlacropus robustus - Sad.leir 1963s l4acropus rufogriseus - Calaby and. llerchant unpublished.g potorous trid_act_yLus - Hughes I)622 hi¿rllabia bicolor - Sharman and Merchant unpublished.

2.5 Hormone preparations (i) Prolactin

Prolactin, NIII-P-S-I Ovine, was suppliccl and. stored. in crystalline forn. rt was weighed-, and- dissolved. in sterile d-istilled. water immed-iately before injection, the concentration þe1ng )-t- mglmlc 20.

(ir ) Luteinizing hormone ITIH-LH was d.issolved in sterílc distilled water imined-iately before injection to give a concentration of 3j rng/mI.

l{urnan chorionic gonadotrophin, trpregnyl'r, Organon,

rvas d-issoÌved. in water immed.iately before injection to give a concentration of 500 I.U.fnL.

\ 111,) ProEe sterone Progesterone, Fawns and, Mc1tllan, was supplied- in ampoures containing a sterile solution of 1o mg progestcrone in I ml synthetic oiI.

( i") 0estroqen

Oestradiol benzoate, rr0estrofornr aqueousil, BDti? was supplied, as a steril-e suspension in water (2 ngfnl-)" It

was diluted- r',rith sterilc d-istilled. water immed.iatery before use to give a concentration of IOO ugfml.

0estradiol bernzoate, Oe stroform, BDI{, was supplied.

in a sterile solution (2 l.gfnr) in a rnixture of ethyl oleate and

¿,,raohis oil-. It was d.il-uted- from one to three d.ays before use with sterile peanut oil (I'auld-ing), to give a concentration of

1OO u g/ml-. (") Thyroxine

L-thyroxine sodiuil, Glaxo-Állenburys, was ground. with a sterilizecl pestle and nortar, and. suspend-ed. in sterilized. 21.

peanut oil to give a concentration of either 1 or 2 ngfnr" rt was stored at 40c for not more than ! d-ays before injection.

2.6 Anaesthesia and. sur,qerr¡

Red- kangaroos and- K. f. wallabies hrere anaesthetized- with pentobarbitone sodium (Nembutal, Abbott, 6o ngfnt) injected into one of the two large lateral- tail- veins. An initial d.ose of

2 nt/5 Kg for kan6¡aroos anð.2 ntf3 xg for K.r. wall-abÍes was

supplemented- d.uring the operation with add-itional d-oses of about 1 ml injected- into an ear vein.

The fur was clipped, from around- the pouch, and from insid.e 'trrrinthrop. the pouch" The pouch was cleaned. with 'rphisohexr, A sterilized. tower was placed. over the incision site, and- the instruments were sterilized- before use. rn other respects,

surgical cond-itions r4rere clean but not steril_e. The pouch was

held- open with a retractor, and- the reprod.uctive system was

exposed by a mid-ventral- incision extending anteriorly from between the teats.

Ovaries that were to be removed- were supported. below the hilus with artery forceps and. were cut away without ligation, îhe cut surface was cauterized"

Before one utems was renoved., the uteri were separated b;- careful cutting of the membrane joining them" care was necessary to avoid. puncturing one of the two large veins that ¿¿.

l-Íe on the ned-iaf surface of ea,ch uterus and. almost meet in the nid.dle" The junction of the uterus with the vaginal conprex was ligated. with catgut (etain o), and- the uterotubal junction was Ìigated" Both ligatures inclucLed the large vein. The uterus was then cut away between the ligatures. The incision in the muscul_ature was cLosed ry!1¡ ¿¡ interrupted suture of c'tgut (rtain o), care being taken to includ"e the peritoneum in the suture. The skin íncision was closed. with a.n interrupted_ suture of braiC_ed_ sj.lk (rrlqersifk,r Oe Ethicon). The wound v,'as dusted- with rTerramyci'rr (rrizer). The animals rr¡ere returned to the yard, and reft undisturbed. while recovering frorn the anaesthetlc. Occasionally an antagoni-st of barbit'urates ('rl{egimid-e'r, Nicholas) r,ras ad-ministered. to rec kangaroos to hasterì recovery.

2.7 Collection and- t ransfer o f blastoc.yst s

Blastocysts of some anirnals shot in the wÍr. r¡ere fixed. in situ- together with the whole reprod.uctive system. Some d.ays later the ovaries ürere examined., and. the uterus ipse-laterar_ to the ovary bea.ring the quiescent cori-.us l-uteum was cut from the system. The uterus was carefulr¡rd-issected. und.er a dissecting microscope to reveal the blastocyst.

Other blastocysts r,¿ere isorated- frorn the fresh reproductive systenr by the neilrod of sharnan (t9i5)" The ovary bearing the c) L )¡ corpus luteum of lactation was cut off, together riith its x'a11opian tube and the tubaf tip of the uterrrs, A med.ian incision was mad.e in the vaginal cul-d-e-sac and_ a 1! gauge need.re fitted- to a syringe lüas inserted- into the os uteli_. The blastocyst w¿l,s flushed- from the uterus into a ruatchglass with a small stream of Ringer's solution ( Cosol, CSL).

Blastocysts of experimental- animals were flushed. from the excised uteri with Ringer's sol-ution at 3?oC, and. measured. with an ocurar micrometer in a d.issecting microscope before transfer.

Flushing, measuring and" transferhrere completed. within a few mínutes of excisíorr of the, uterus.

(i) Transfer to the e.ye

'rlhe eye of the wallaby was lightly compressed with a finger and. the cornea was punctured, with the tip of a No. 1I scalpel blad.e. some aqueous humor leaked. out th.rough the wound. The blastocyst was d-rawn into a fine-tipped. glass pipette, which was inserted- into the anterior chamber of the eye. The blastocyst, together with a small volume of saline, was expelled. into the anterior chamber. The wound closed itself and. the turgor of the eyeball was restored within minutes,

(ii) Transfer to uterus

The uterus l¡ras punctured. with a suture needle and. the blastocyst was inserted into the Lumen by means of a fine-típped- 24" glass pipette.

( ii-i ) Transfer to millipore fil-ter chamber

A millipole filter disc (pore-size O.22 ¡ or 0.45 u, ) was fixed wjth ethylene d.ichloride to a short perspen cylind.er,

13 mn wid-e and either ,2 mm or 1 mm hi,gh. The blastocyst in a sma1l volume of safine was pipetted" into the larger cylinder, which was then closed by fixing the smaller cylind-er over it. Care was taken to ensure that the ethylene d-i-chloride used- to seal the perspex d-id not leak into the saline, One blastocyst was introduced- through a hole in the sid-e of a Drepared. chambere the hole then being plugged r"rith paraffin wax.

2.8 Histology

Blastocysts, ovaries and uteri were fixed. for about {B hours in a solution of alcohol (90 nf 8O%), glacíal acetic acid- (5 rr) and formalin (! rnl), and were stored in alcohol (Bo%).

Portions of ovaries and uteri were embed.d.ed- in wax and. sectioned- at 6 r.r, on a rotary microtome. Sections were routinely stained vrith l-ihrlichrs haematoxylin and. counter-stained- with eosin. Some secti-ons of utori were stained- with Giemsa to demonstrate basophilia.

Blastocysts were prepared by a mod.ification of the method. used- by lrloog a,nd Lutwak-Mann (D>g) for relatively large rabbit blastocysts (6 aays post-coitums approx. 3 mn d.iameter). Each 25,

blastocyst v¡as trarisferred. with a pipette through successively

lower grades of alcohol to d.istilled. water. The shell membrane

and underrying protodern lüere punctured- with fine steel need_les

and. the blastocyst was stained-, either wi-th Mayer's haemalum and- d.ifferentiated. in tap water, or lvith l,{eigert t s haematoxylin

and d"ifferentia.ted. in saturated. picric acid- followed- by a d.istilled uater rinse. shelr niembrane and. protod.erm were torn by slits that extended- ahnost to the mid.d.le until the brastocyst

coul-d be frattened. on a glass slid.e. rt r,ras dehyd.rated" and

nountec] in a neutral ¡nedium.

.4, grad.uated. eyepiece lüas calibrated. against a micrometer slid.e and, used- for measurements from sections.

2.9 Counting

Nuclei of blastocysts h'ere counted from diagrams mad.e with a microscope d-rawing tube (tr.ii-fa), a I'eitz d.rawing attachment, or

iL projection mirror attached- to a monocul-ar 1¡lild. M20 microscope. For determination of the d.ensity of cell-s in corpora lutea,

the image of a section (6 u, thick) r^ras projected. onto paper with

the I'tri]d projection nirror. The outline was d-rawn of ea,ch nucleus in an area correspond-ing to 4.J6 x lr}-2 sq nm. As the

section was 6u thlck, :r volume of 2.135 x 1O-4 cubic mm uas sampled. Eight field.s were counted. for each corpus luteum. For both nuclei ancl corpora lutea, the number of cel1s 26.

T{as consiilered. to be equa} to the number of nuclei.

2.10 PhotomicroAraph.y

Photomicrographs were taken on Pan trr 35 mm film with

a Zeiss lkon camera rnounted on a Zeiss microscope, Films were developed in TD )6. 27.

3. EMBRYONIC QUTESOENCE rN THE RED KANGAROO

3.1 Introd-uction

The time betv¡een mating and. birth in non-lactating red.

kangaroos (Ir{egaleia rufa (Desmarest) ) averages 33.11 d.ays.

Post-partum oestrus and. mating occur usually within two d.ays of parturition, but this mating is not folrowed_ by bírth while

the young occupies the pouch. The embryo d-oes not d.everop beyond, the blastocyst stage. rf the young is removed- from the pouch lactation ceases and- birth occurs about 31 days after removal, without an intervening mating. If the,young is retained- until it emerges from the pouch permanently at about

216 d-ays of age, the mother prod-uces a second, young within a day or two of the firstrs emergence (Sharman 1963)" The corpus l-uteum of lactation, formed. at the post-partun ovul-ationr is uniformly small in size in females suckling young up to 190 days oldr whereas corpora rutea associated. with post- blastocyst stages are much larger. No ruteal changes occur in the uterus during embryonic quiescence (sharman 1963). To gain more information on the rorationship between corpus luteum, uteri anè. blastocystl the histology of these was Ínvestigated. d-uring early d-evelopment, quiescence and. rater d.evelopment, 28.

3.2 Development before quiescence

A four-ceIl embryo, sunound.ed. by sheIl membrane, was d-issected from the uterus of a female that had. recently nated. at the post-partum oestrus. The d,iameter of the shelI was 26Jt, the d-iameter of the protod-errn IlB u . The corpus luteurn was not fu1ly-formed-, but contained- a coagulum and. extra-vasated- blood- in the cerrtre. The granulosa cell-s contained little cytoplasm and. '¡rere separated by intercellular spaces. Strand.s of thecal cells extend.ed almost to the centre of the corpus luteurn.

Mitoses occuned but were not common in thecal and. granulosa ce1ls. The numerous uterine gland-s riere evenly scattcred throughout the endometrium and nany glands contained- clusters of sperm. The gland cel1s r,¡ere cofumnar, with slightly elongate nucleus maínly situated near the base of the cell. Many g1and. ce1ls were dividing (r'ie. 1B). A corpus luteum removed I d"ays a.fter post-partum mating nas fully-formed-, with sma1lr d-ensely-packed- luteal ceIlsr many of which were divid.ing (f'le. 1A).

3.3 e scent se of lactation The reproductive organs of females carryíng a pouch-young from I to 200 d.ays old- are in the quiescent phase of lactation (Sharman I)6!a).

The corpus luteum is a round. bod-y about I mm in d.iameter 29a

tr'igure 1

A. Smal1 d.ensely-paoked. cell-s of corpus luter:m of red. kangaroo three tLays after post- partun mating. Mitotic figure at upper

ríght. x 110O

B. Transverse section (T,S") of uterus of

red. kangaroo with newborn yowtg ín pouoh

and. four-ce11 enbryo in uterus. Mitotio figures oan be seen in gIand. ce11s (o.g,

ín lower nitl-ùle of fígure). Gland

lumina contain spentr. x 21O -t rt I a. J o. { ì¡ ttr{ D b ) I ü r¡ Þ. a Gþ 'ù t * a ) I Q a t t Ç #Éf t ,¡O a a I t ¡ a ü ¡ T t a l. a a ;, a o D $ L ¡ r I ,4 t .\rI T¡ b ¡a ) t j o çt i.) ,i \.o t- rÀ ^ 30.

(Sharman L963). The lutea1 cells are small and. densely-packed. (tatte 3.1); their nuclei are small and round, witb chromatin material condensed on the nuclear membrane, Clusters of luteaI cerls are sepalated- by tenuous strand.s of connective tissue. Blood- vessel-s are smalL anC few (f,ig, 4À). The histological appearance of the uterus d-Íffers from the post-oestrus stage (above) nainly in the absence of d.ivid.ing cells and sperm. The gland- cells are columnar, with slightly elongate nuclei occupying the basal third. of the cetl (fig. 5A). The cytoplasm is positively basophilic. The blastocyst consists of a unilaminar sphere of ceIIs,

the protoderm, surrounded- by a layer of so-caI1ed rraLbuminrt, and.

bound.ed. by a tougher layer, the shell membrane. Sperm are enbed-d.ed.

in the albumín layer and ad.here to the shelt membrane ( Ctara 1966). The external d.iameter of' 38 blastocysts recovered. from

females d-uring the -quiescent phase ranged, from 260 u to 3BO u, (mean 321 u,, standard deviation (s.d..) 29u); the diameter of the protoderm ranged from f iO ll to 310 u (nean 241u,, s.d. 4tp).

The age of the blastocyst was consid.ered- to be the same as the age of the pouch-young. Older blastocysts were not consistently larger than younger ones (fig. 2). The number of nuclei in the protod.erm of 2! blastocysts varied from 62 to L29 (mean BB, s.11. 13) and- d-id- not tend. to be greate¡ in old.er bl-astocysts 31.

r4c,

^ t20

2 o, U roo l^^ ¡ Àl I A o Ir I l¡ I A ¡l q, 80 ^t .o ¡ ^ e z2 l ¡^ óo

380 o o o oo o o o o \ 3¿3O oo oo oo oo o o o o ë o o o o oo oo o E 30 o oo o =\, E U' 2óo o

320 o o a o o \ O 280 \,a- o) o OO o o e o O o a €o 210 o o¡o oo e t- o oo O s, oo v 20 a oo o a a o À o o róo o óo 120 lso 210 Age ol blostocYst (Ooys ) Figure 2. Vorlotion with age in numbør of cells, diomøter of shell' ond diomctar of Protodørm of blostocyst of Megolølo rufo. 32.

(f'lg. 2). 1[o d.ivid.ing nucleus r¡ras seen in any blastocyst iuring the quiescent phase. All the cells were similar in appearance and, there 'hras no evidence of d-ifferentiation of end.od.erm (Fig. 3 A). Five unsegmented, d,egenerating eggs rrere recovered.

(Ci-art< f966) and also three d.egenerate 'blastocysts which were ind-istinguishable from eggs when observed, fresh. After staining the inner coagulum was seen to consist of from 9 to 28 nuclei, these nuclei being irregular in shape or pycnotic.

3.4 Chanees that occur after 200 d.avs of oouch sucklins Twelve femal-es that were shot in the r,¡ild- carried. pouch- young older than 2O0 d.ays. Two of the young escaped,, and. their age could- not be accurately determined.. ln aI1 these females the corpus luteum of post-partum ovulation d-iffered. from that associated" wi-th a pouch-young Iess than 200 d.ays old.. The diameter of the corpus luteum exceeded I mm and. lüas as great as

6 mm in a femaLe with an cnlarged blastocyst in the uterus (falte 3.I)" The luteal cells r¡rere larger than ín earlier stages, and- the d.ensity of cel1s d-ecreased. with increasing size of the gland (Table 3.1-, Fig. 4D)" fn the largest coïpora Lutea, connective tissue cells were al-so enlarged and formed- a network between the luteaf cel-ls (fie. 4 B). Dividing cells were not seenÒ Thus the increase in size of the corpus luteum was clue to hypertrophy of luteal cel-l-s rather than to hyperplasia" 33a

Figure J

A. Flattened. qui-escent blastocyst of red-

karrgaroo. Nuclei and- sperrn can be seen

overlying the sheII. x 21O

B. i,Íitosis in bl-as'Locyst of red- kangaroo approximately one week after loss of

pouch-young. x +5O ¿¿*

f t1l)

't i"li', rá' a;

\ ll rl -ll ;. Ì jÌ,.f I'j-' , ¡ I

al

a tt ;*/ a 'a 'tl I ! ;.¡l- a t a t "e{tr

A 34"

TABT,E J.1 tleasurenents of corpora lutea and- blastocysts of red. kangaroos with pouch-young older than 2OO days.

A¡inal Äge of Dia¡oeter of Denslty of Blastocyst pouch-yorxrg corpus lutel¡cr cel1s [no. (¿ays) (*') cel1s ner ("o. r*)-41 . òia.ur. No. cel1s (r-,)

K 1tg go 3.O 67,9 (3,o)o 263 Gr2 7+ 3.O 60.8 (3.4) 286 I 00

65/35 205 +.25 51 .g (t,o) 350 6s/+t 210 4.4 52.4 (2.1) 320 65 6s/t6t 21+ +.5 ,7.8 (1 .O) 357 65/J6 222 5.O 36.8 (2.7) 360 6S 66/+Sx 222 Not avaíIable 500 tp ',2g.6 51K 222 5.O (¡.t) 6rooo v2 29K 235 5.5 t 25.o (1.6) 2r5oo óa 5/31 236 Not available Ädvanoed ernbryo

7K 237 4.25 : 4o.9 (¡.0) 320 91 39K 2tù Not available 6r0oo (Ð 6/ tgtç Escaped. +.5 15.+ (6.2) 320 76 6/zltç Eseaped. 6.o 22,7 (2.2) 3to@ 6)

* Stand.ard. cLeviation in brackets 3r.

Although all the corpora lutea, were enlarged, síx of the twelve embryos recovered were bl-astocysts not larger than in the quiescent phase. they consisted. of between 6J anð. pl ce1ls, none of which was d-iviri-ing. Four of the unenlarged. blastocysts were recovered from uteri of which the histological appearance resembled- that seen during the quiescent phase. The other two uteri d.iffered. in that the nuclei were not regularly basal- in the gland" ceLls. Five enlarged- blastocysts and one ad-vanced- embryo were recovcred, all from uteri that were fully luteal. The very taI1 columnar gland- ceLls had. a smalI, round. nucl-eus at the base of the cell, and- the cytoplasm was not basophilÍc

( ¡'ig' 5 c) .

3"' ChanAes that occur after temoval of pouch-.young The corpus luteum and- uterus removed. at laparotomy from a female three d-ays after removal of pouch-young (nff) d-id, not d.iffer in appearance from those of the quiescent phase. A blastocyst was not recovered. At seven d.ays after RPY the colpus l-uteum was enlarged. (d,iameter 4.6 mm) but the d.ensity of luteal cells had. not decreased. (natte 3.2). Divisions were occurríng in both thecal and luteal cells. The uterine stroma had expand.ed- and. the nuclei of the gland- cell-s were not regularly basaI. There r'ras no evid.ence of hypertrophy of gland. cells. The blastocyst was not enlarged., and a single 36a

Figure /¡

Sections of oorpora lutea of red- kangaroo.

x IrTO

A. Assooiated. with quiesoent blastooyst.

B, Fu11y-d-eve1oped., associated. with 2.5 mm

embryo.

(¡¡ Approxinately one week after loss of pouoh-yotmg.

D Fron aninal with pouch-young oJ-d.er than

2OO d.ays; blastocyst quiesoent. v ruù' p. .t

I I a o (ì f'@

G oc'a a

.$ -a "â*

Ò'-17.

?

Oø t¿ o t.l ?, -o 37a

Figure !

Transverse sectj-ons of uteri of red. kangaroos.

x 160

A. Associated with quj-escent blastocyst.

B. Approximately one week after RPY.

C. Associated- with 2.J mm embryo. a¡ U i

[' t tl i

t p q

Ï .-- zr- t i -.:. ,.

'/Ç. 38.

nucleus was dividing. At B d-ays after RPY mítoses T/,rere common in tbe corpus luteum and. the cells were slightly enlarged

( Sharnan l-964io).

One female shot in the wild had. an elongated. teat from which watery milk coul-d. be expressed.; this milk looked- similar

to that of captive females approximately one week after RPYe and

ind-icated. that the pouch-young had. been lost about one week

before" The corpus luteum had- enlarged. but the cel1 d.ensity was only slightly less than in the quiescent phase (Table 3.2,

Fig. 4 C). The uterus was the same as at I d.ays after RPY

(above) (pie. 5 B). llhe blastocyst was slightly enlarged, it

consisted of 116 ce1ls, several of which were divid.ing (¡'lg. 3 B)"

3.6 Discussion

Gror,rth of the corpus l-uteum resumes d-uring the first 4

d.ays after removal of the pouch-young of tlie quokka. Both hypertrophy and. hyperplasia of luteal cells occur and. the corpus

luteum contains about twice as many celLs on ôay B as on d.ay 2 (Tyndale-Biscoe I963c), Development of the corpus luteum of

the red kangaroo after RPY resembles that of the quokka. I,trhile a large young occupies the pouch, growth of the corpus luteum is caused- largely or entirely by ce11 expansion, and. ce11 d.ivision has not been seen. TABIE,3.2 Measurements of corpora lutea ancl blastocysts of recL kangaroos after RPY

Aninal- Days since Dianeter of Density of cel1s Blastocyst per REY corpus luteum [no.- cells (rr) ("t. tt)-41 d.ian. No. cells (rr)

Èr2 0 3.o 60.8 (1.4) 286 100

K11 I 1 3"3 6g"4 (3.t+)

K132 7 l+.6 62.J (2"7) 286 86

65/e6 Approx. 7 4"3 49.1 (2.4) r+o5 116

\o\¡ a 4o

4. E]L{SIT.YONIC QUIESCENCE IN OTHER DIPROTODONT MARSUPIALS

4.I fntrod.uction Having exanined more than 1{ species of d-iprotod.ont marsupials, Sharman, Calaby and Poole (f966) ai-vi-aea them into four groups, accorcling to their pattern of reproduction. The first group contains species that d-o not have a variable quiescent stage in gestation. Enrbryonic quiescence occurs in all species of the other three groups but is associated, with d-ifferent patterns of rel-ated reproductive events in each groupc I have investigated- the pattern of reproduction ín five more species and. have made further observations on the breed,ing pattern or the quiescent embryo of 10 species mentioned. by

Sharman et al. (t966). I have divided- the species ínto five groups, characterized slightly differently fro¡n the groups of (ryee) sharman * g!. "

4.2 Snecies without post-'oartum matinE and. in which the length of gestation is independent of lactation.

Tricho surus vul-pecul-a The reproductive systems of 246 rnature female bzush possums Trichosurus vulnecufa (tcerr) ) were exarnined. In pregnant females, a corpus luteum about {. I mm in d.iameter protruded from the ovary ipse-lateral- to the gravid- utems. Seven females had. given birth ten d.ays or less before 4r. capture, and one ovary of each of these carried. a corpus luteum of slightly smaller d.iameter than in pregnant females. These seven animals were not in a pro-oestrous, oestrous, or post- oestrous cond-ition, for the d-iameters of the largest follicles were less than 2 mm (c.f. 4.5 rnn d-iameter of pre-ovulatory foIliole), there ïras no ner^r corpus luteum, and, the lateral vaginae were not enlarged. There was thus no evídence for a post-partum oestrus.

0f 160 femal-es with suckling young old.er than IO d.ays, 90 had" a single corpus luteum, l¡hich was smaLler in females with older young. Corpora lutea associated- with young old.er than

J0 days were less than 2 mn in d-iameter, and. a corpus luteum was not macroscopically visible in ovaries of 61 females with suckling young aged- from 43 to about 260 d-ays. This single corpus l-uteum was the degeneratinpç corpus luteum of pregnancy. The remaining nine females carried two corpora lutea, both of which were old and. non-secreting as ind.icated by their d-ark colour and- by the anoestrous condition of the uteri. Corpora lutea were not macroscopically visible in the ovaries of non-lactating femal-es. In the brush possums exanined- here, enbryonic quiescence occumed- neither in association with lactation nor with an annual season. Pilton and Sharman (t962) observed. that post-partum oestrus d.id- not normally occur in the brush possum. Pilton and.

Sharman (tgøZ) and- Tynd.ale-Biscoe (tgSS) each observed- one femal-e 42.

that mated- while carrying ¿ì,n early pouch-youngr but in neither animaL did- the embryo become quiescent. The l-actating possu.m mey nlate shortly before the young vacates the porrch, but there is no evidence that the ensuing gestation is lengihened by the concurrent suckling (Pilton and-

Sharman L962)"

?et¿¡,urus b:revíceps The sugar glid.er (fg!a.l-rr!¿1b-g"-uÆ. rr,laterhouse) is tentatively assiggned- to this group" Two pregnant females liele caged with a male untÍ1. they hac. given birthg they uere not examjned for post-partum mating. 'rrtrhen the pouch-young were a few d-ays o1d they v\ieïe removed, and- the fernales Tilere checked d.aily for parturition and- ma-bing" No young was born without an intervening mating (talte 4.1); this indicated. that a quiescent embr.vo lyas not present during l-actation. [[v¡o females became pregnant before weaning a litter, but the duration of preélrlancy was not deterrnined in lactating females. 43"

TABLE 4" 1 Time of mating and. birth after RPY in Petaurus breviceps

AnimaI RPY to birth RPY to mating Mating to ( aays ) ( aays ) birth (aays)

811 L2 r6

BII 13 L6 810 l1

4"3 S¡e cies without post-partum matinq in which gestation eriod. be1 hened. d suckl

The grey kangaroo ( Macropus giganteus Shaw ) d-oes not undergo a post-partum oestnrs (eitton 196L, Poole an¿ Pilton L964) but a small proportion of wild- females with young older than 110 tlays carry a quiescent blastocyst 1n one uterus (Kirkpatrick 1965, Cl-ark and, Poole 1967). A few captive animals came into oestrus ancl mated. while carrying young older than 112 d-ays, but d-id not give birth until the young were lost' or.vacated the pouch a,t about 3OO days (tCirkpatrick 1965, Clark and Poole 1967'), One female nated when the pouch-young was 1!l days otdg she was then isolated from males and. produced a second yountg 120 days later (Ctart< and- Poole f967). The 44,

gestation period- in nonlactating females d.oes not exceed_ lB d.ays (Poole and. Pitton f964)"

One 9v¿1.y of females carrying a blastocyst contained_ a corpus luteum similar in histological appearance to the quiescent corpus l-uteum of the red, kangaroo, The uterine end-ornetrium also resembled. that of the red- kangaroo in the quiescent phase (Ctart< ancl Poo1e 1967).

Sixteen blastocysts had- a mean external d.iameter of 262u, (s"d. 22.8u) and, nine of these consisted of an average of cells ( 15). Divid.ing cel-ls were not seen in 92 ".d. blastocysts associated. with cluiescent corpora lutea and uterí (except in one instance) Uut cel1 d.ivision occurreô in

blastocysts associated. with active corpora lutea (Clark and- Poole L967).

Three ad-d-itional females with pouch-young ha,ve been stud.ied. In one animal with pouch-young ageð. 22) d.ays, the

blastocyst was in the quiescent phase. The blastocyst was

255 p, in d-iameter and consisted of 82 cel-ls" The nuclei were

round or kidne¡p-shaped. with ¿çranular chromatint no nucleus was d.ividing. The second. animal carried. a young aged 2l{ d-ays and had a d.eveloping blastocyst (¡f+ r, d.iameter, 212 ce1Is)

ín the uterus. There had- been no recent rnting, for the

lateral vaginae d.id. not contain semen and. were not hypertrophied.. 45.

Numerous mitotic figures in the corpus luteum ind_icated. that hyperplasia contributes to the enlargement of the corpus luteum as the pouch-young approaches ind-ependence" Embryonic quiescence does not always follow a mating during pouch-suckling, for the

third animal, carrying a fu11y-furrec'l- young, had. recently rnated.,

but the bÌastocyst consisted, of several hundred- ce}Is, and. the

end.oderm had differentiated. The tr,ro serologically-different races of the grey kangaroo (tcirsch and. Poole 1967) have been consid-ered. together in the above studies"

4.4 Monotocous s¡ecies with È u- artum mat

(") Species capable of breeding throughout the .year The red. kangaroo belongs to this group (see Section l), which also includ-es Macro us rufo SEUS 14" robustus Potorous iridactylus and- other species.

ThyloEale billard.ieri-

Trr¡o female pademelons ( Th.yloEale billard ieri (lesmarest) ) with srnall-, naked pouch-young ÏIere caged with a male" Every second day, vaginal smears were taken and examined for evidence of rating. No mating r/üas d,etected. before the pouch-young emerged permanently after BJ and. 1{8 d-ays respectively. 'üüithin two days of the iarge young vacating the pouch the rnothers Save birth, and" both females mated- less than a day after parturition. 46,

One female hras isolated from the mal-e d,uring the subsequent lactation (except for a period. of 43 d-ays when regular vaginal smears showed that no mating occurred.). The pouch-young llras last seen in the pouch l¡hen 194 d"ays oId., ¿rncl on the following d-ay a neonatus was found" in the pouch. The male was introd.uced- into the yard and mated r,¡ithin 2{ hours with the post-parturient f emal-e.

Ì4a.cro'pus robustus

Blostocysts (diameter 31? u ) are founÔ in the uteri of lactatÍng euros ( l.[acro'ous robustus Gould ) (ryndare-Biscoe l-963c) and a young may be born from the stored. embryo (Sadleir and-

Shie1d" I)6Oa, b). Ten euros shot near Broken Hillr ld.S.Ïü., carried- pouch- young aged. between 1! anù 22O d.ays. A bl-astocyst was f-l-ushed. from one uterus of each femal,e" The blastocysts resenbled those of the rerl kangaroo. The mean external- diameter after fixation was 305 rr, ( s" d" 19 "6 u, ) " The protod.erm i-n seven blastocysts was adjacent to the she11 so that the al-bumin layer r^ras very thin if present at all. The protoderm of four blastocysts consistecl of lB, 59, 7l and. 88 cells respectively. The nuclei were round or very slight elongate, with granular chromatin material- and two d-arkly-stained nucleoli. No rlivid.ing nucleus was seen (nig" 7Â). 47.

The quiescent corpus luteum resembles that of the red-

kangaroo in size and. appearance of ce11s (fig" 6e). The

uterine grand-s are numerous and. scattered; the grand. celrs are cofumnar and the nuclei are not confined. to the basal third. of the ceIl (¡'le. 6B).

Macropus rufoAriseus Brastocysts were recovered. from six Bennettrs wallabies (Macropus rufogriseus frutica (Ogilby) ) with pouch-young of

undetermined. age. The mean d-iameter of the shell was 302 u (".d. 18.3u ) and. one protod.erm consisted of !B cells. Blastocysts were d-issected- from the fixed. reproductive

systems of three red.-necked- wallabies ( M. r. banksianus ( Q,.,oy and. Gaimard) ) shot near Goulbourn, N.S.ld. and. lp blastocysts

from red-necked wallabies taken at Bonalbo, N. S.lü. were al_so

examined." The ages of the pouch-young ranged_ from B to 23I d.ays. The young red-necked- wallaby reaves the pouch permanently

at about 2BO d.ays (Cataly and. Merchant, pers. corn. )..and_ so an age of 210 d.ays has been assigned. to five fuIly-furred pouch- young which escaped. and cou1d. not be measured..

The mean d.iameter of the shell of lT blastocysts was

3Ol-u (".d. 43.5), and the mean diameter of the protoderm of 13 of these was 2?-Bll (s.d.. 33.1). The protoderm consisted. of betr,¡een J2 and, I22 cel]-s (mean 83, sod. Zl-.6e n = lzf ). The LBa

Figure 6

-4. Seotion of corpus luteum of euro with

quiescent blastocyst. x 50O

B. T.S. of uterus of the sa¡ne euro. x 140

C. Seotion of corpus luteum of agile wallaby

with quiescent blastooYst. x 500

0 T.S. of uterus of the same agile walIaby.

x 140 -\!- rr-J -; . r_---

frs ¿ t ¿lr¡ \t' a l<. t ¿r Ð t{ Ê-_.ê ' f-r''-!r '

.. s_åt g :-i.; ,,'(3 , 49a

Figure /

A. Quiescent blastocyst of euro; protoderm has been fl-attened. without d-issection.

x 160

B. Quiescent blastocyst of agile wallaby;

both protod.erm and. shel1 have been torn

open before being flattened. x 160 t). r¡

6o { a q

A 50.

size of the blastocyst d.id not tend- to increase with the age of the pouch-young, and there 'hrere not consistently more cells in the protoderm of old.er blastocysts (nig. B).

One femaLe with an B-d-ay-o1d. pouch-young had- recently ovulated, and the corpus luteum contained a central- blood- spot. The fertilized. egg had. developed into a blastocyst consisting of 2I round nuclei" One nucleus was dividing" The nucLei of quiescent bla,stocysts were roundr or frequently elliptical- in shape, and- both round ancÌ elliptical nuclei were often seen in tire one protod.erm. The nuclei contained granular chromatin material and two d-ark1y-stained. nucleoLi. Except for slight variations 1n the shape of the nuclei, the cells were all- simil-ar in appearance and there r^ra.s no evid-ence of d-ifferenti¿rtion of protod.erm"

No female with a pouch-young yielded- an enlarged. blastocyst and. a quiescent bl-astocyst consisting of 69 cells rras recovered from a lactating female without a pouch-young. The interval between permanent emergenoe of one pouch-young and- p arturition is about 3 weeks in M. rufogriseuse whereas it is only a few hours in Ylegaleia rufa (Sharman L)6Ja)" The histological appearance of quiescent corpora lutea and. uteri of M, rufogriseus do not d.iffer appreciably fron those of the red. kangaroo. 51.

t20 ¡¡

A roo ø us, A A ¡ bco ¡ ¡ I ll \,L .o e3óO ^ ¡ z I

¡¡O

400 o

o 3óO o \ o o Ë 32o o o þ o o o eeo o o o Ë.c Ø o o o o o 210

280 a OO \ a a 210 o ê a a .o ! a E 200 a L 9U o o L óo o o- o 30 ôo 90 120 150 100 2lo 240 270 Age of blostocyst (doys)

Figure 0. Voriotlon with ogc in numb¿r of cells, diomctcr of shcll, ond diometer of protodørm of blostocyst of Mocropgg rufogriseus. trD

Macropus aEilis A blastocyst recovered from an agile wallaby (Macropus agilis (Gould.) ) with a 28) gm pouch-young had a shel1 25O v, in ùiameter and a protoderm consisting of '/l ce1ls. Slastocyst, corpus l-uteurn and uteri were histologically like those of the red kangaroo (¡'ie. 6C, De Fig. ? B)" A second femal-e that was shot carried a large fully-furred pouch-youngo The corpus l-uteum w¿rs enlarged- and the uterus luteal. The blastocyst r,ras lBOft in ùiameter and the protodcrm hacl d.ifferentiated. Until the occurïence of pos'i;-partum oestns is confirrnedt M. agilis can be only tentatively assigned to this group.

Po torous trid-actvlus

The potoroo ( Pc¡torous tridactylus (ferr) ) ovulates after givin.q birth and- blastocysts of 25O to 2BO ¡1 diameter are found- in fernales suckling young up to !! d.ays old (Hughes 1962). six blastocysts were stained and examineù" The number of cells varied between 83 and 143, less cells being found in ol_der blastocysts (tab1e 4"2)" The nuclei were round, with granular chromatin and a nucfeol-us. No d-ivid.ing cel-l was seeno

Cel1s may be lost fron the protod.erm with time, for a few cells of potoroo 20 contained- a smal1 pycnotic nucleus and- were apparently d.egenerate" The pouch-young of potoros l! was furred and approaching'indepenùence' The blastocyst from this 53. animal I4Ias enlargedr the end-od.erm had, d-ifferentiated- and mitotic figures were numerous in end-od.erm and. trophobLast"

TABLE 4.2 Characteristics of the blastocyst of of the lactating potoroo

Animal Age of Diam. No" cells pouch-young blastocyst ( aays ) (r)

5o 48 280 r43

20 72 ?7o r29

56 86 270 B3

51 9j 28O 9r

T6 9B 25o 9r

39 110 500 > 200

(r) Soecies that breed- in an annual- season

Macro'ous eugenir The breed.ing season of the Kangaroo Island- wallaby

( Macropus euEenii (Desmarest) ) extends from January to July. During the breed.ing season, birth is fo1lowed. by post-partum mating and. l-oss of pouch-young leads to development of the blastocyst. From August to Decemberr loss or weaning of the 54

pouch-young does not lead to developrnent of the blastocyst, which remains alive but quiescent until January, when it resumes growth

(Berger 1966)" Six blastocysts recovered from lactating femal-es d-uring

the breed-ing season ranged. in d,iameter from 260 to 3OO r-r " The protod-ern consisted. of '12 to 16 cells in the breed-ing season and. from 61 to 118 in the anoestrous seasonô t-tests ind.icated. that

the me an size and mean number of ce11s were not significantly

d-ifferent in breed-ing and. anoestrous seasons (natte 4"3). The nuclei of the protod-erm cell-s vüere round or slightly elongate, with granular chrornatin and a nucl-eolus, The nuclei in each

blastocyst varied considerably in size, the largest ha,ving a d-iameter more than twice as great as the smallest.

TABLE 4" 3 Size and number of cell-s in blastocyst s of M. eugenii in the breed.ing season and- in the anoestrous season

Diameter of shetl ( u) Number of ceIls

Breed-ing Anoestrous Breed.ing Anoestrous season season season season

n 6 T2 4 6

Mean 28O 294 83.5 88.6

Stand,ard. Devia,tion L7.9 20.6 11. B 20"I

t L.575 o.458 Significance 0.1

Setonix brachyurus Quokkas ( Setonix brachyurus (Quoy and Gaimard-) ) in the Rottnest Island. population are capable of breed.ing from late January to earì-y August, Parturition is foll-owed. by oestrus and- a blastocyst conslsting of about 70 cells can be flushed. from the uterus of lactating females (Sharman f9r5). The ability

to replace lost pouch-young from stored blastocysts d-ecreases

from 6Odþ in February to nil in August (Sfrlefa and. I¡loolley 1963). Blastocysts could not be d-emonstrated- in the uteri of anoestrous females and. they ar'e b,:iieved- to d.egenerate at the beginning of this phase (Sharman f955).

Six blastocysts recovered- on I'llay lst had- a mean shell

d.iameter of 27O Lr, (s.d." 26"8t-r") and. four of these consisted of 61, 68, 69 and- l1 ce1ls respectively. The nuclei rvere nainly round, with granular chromatin and- a nucl-eo1us, They varied little in size" One blastocyst taken in August measured 320rr, and. one in

September measurecl 25O l.', and. consisted. of l2 cells. The histological appearance of the protoderm of these two blastocysts was not appreciably d-ifferent from the May blastocystsand no degenerative changes ïüere noticed-. 56.

4.5 Pof.ytocous species with post-partum mating

(") Cercartetus concinnus

Bowley (tglg) reported. that a captive mundard.a (or pigmy possum, Cercartetus concinnus (Gould.) ) naa reared. and- weaned- one litter and- had- given birth to a second- litter about 11 d-ays after capture" No male other than her or,m immature young had access to the femal-e d.uring her captivity, and. Boi^¡l-ey believed- that d-elayed- fertil-ization had. occurred.

Casanova (fgæ) similarÌy observed- that a pigmy possum that had young in the pouch when captured produced a second litter in captivity without access to males. Both Sharman (l-9û) and. Hartman (t94O) consi

Sharman noted that Cercartetus is unique among marsupials with embryonic cr,uiescence, in that the female is polyovular, several quiescent blastocysts presumably being present during lactation. The reproductive systems of nine mundarda with pouch- young r¡rere examined. The cond.itíon of the ovaries and- uteri was not the sane in a,11 specimens and- each animal could- be classified from its reproductive system as (i) post-partum (ii.) pregnant or (iii) anoestrous (taute 4.4)" TABI,E 4-4 Reproductive cond.ition of C. conci¡mus stud.ied- anð observations on their pouch-young

lierial number Iocality Date Reproductive Pouch-young of aninal state Head- length Remarks (*')

Nr6537 'i[agil Nov.1964 Postlartun co No pouch or scrotr:m, ears covered- by epitrichium I'.,r106 iiiurat Bay 't9/8/12 Pregnant 4.O ItTo pouch or scrotum, ears covered by epitrichium, eye pignenteC. 1,í2768 Scad.d.en E4 ilg/ ua Pregnant ). I Pouch or scrotun risible; ears free at tips r¡,65tÐ l,iÍdgienooltha 25/1/65 Pregnant Âtr Pouch or scroturn visi-bIe M6616 I¿ke Grace 6/s/6s A¡roestrus 7.4 Ears pigmented, trace of fur on head. riLo67 ldo data Anoestrus 7.4 Trace of fur on head

M2952 Nyabing Nov.1 95J Pregnant 7.8 Ears pignented-, trace of fur on head- \n4983 ì,{erin4r Lpr.1)62 Anoestrus 8.4 Ears pigmented., traee of_fur on heacl .-l\¡ \n5872 Gidgieganup 22/9/63 Pregnant 9.1+ Ears pignented, trace of fur on heacl 58.

(i) Post-partum female The reproductive tract of the female with the smallest pouch-young (M65¡?) was much enlarged-; the anterior vaginal canals were distended, and- the uteri were {.2 mm long and had- a d.iameter of about 2.1 mm" Seven corpora lutea (Bf8u average d-iameter) associated- rslth the new-born young r^Iere present in the ovariesr four on the rigbt, three on the left. the l-utea1 cel1s were larget with large round. nuclei containing a nucleolus. mhe ríght ovary contained four and the left ovaly two older corpora lutea (??B rr average diameter, s.d. 5fu). The luteal cells were vacuolated- and- srlaller than those in the recent colpora lutea; they were isolated_ from each other by a connective tissue reticulum. Eleven tertiary fo11icles (right ovary 4e left ovary J) of d.iameter 280-42O u were present, each containing an ovum of about 130u diameter. The d-egree of maturation of the egg nuclei could not be determined. The left ovary also contained two lar¿çe, atretic follicles (2J2 and' ZSOr-¡ diameter), in v¡hich the membrana granulosa was attenuated- and degeneratingt and the ovï.m was vacuolated. No sperm llas seen in the ovid.uctS. The endometrium of the uterus was thrown into many d'eep, complex fold.s. The uterine glands (?O- 100¡.r, dianeter) haa wide lumina lined with low columnar epithelium, while the lining of ,9"

the uterine lumen was of cuboid.al ceJ-Is, with large, basally- situated. nuclei and- cytoprasm bulging into the lumen" cerl

d.ebris and. Leucocytes partially filIed. the uterine rumen and, many of the glands. The epithelium of the urogenital sinus and posterior

vaginar sinus consisted of about 12 layers of squamous cells,

many of which were being sloughed.. Some cells Ìùere partly- cornified-, but completely enucleatecells Írere rare (fig. 9A). The lateral vaginae posterior to the vestigial l¡lolffian ducts

had- a d-iameter of 1.1 mm (c.f. O.B mm in early pregnancy and.

0"3 mm in anoestrus) and. were similarly lined- with lO-12 layers

of cells, but none of these uere being shed_"

(li) Pregnant females The ovaries of all five pregnant females contained_ corpora lutea of two types (ra¡re 4.5) and- in one femare (uro6) contained three types. the corpora lutea of pregnancy T¡rere small in MlO6 (Table 4.6), and contained. much extra-vascular bl-ood.; the

luteal- celIs were smal1, with little cytoplasm (f'le. 9 B). Connective tissue was not prominent. fn females with older pouch-young the corpus luteum consisted- of densely-packed. luteal cells penetrated, by thin strands of connective tissue" The ce1ls were larger than in M106, but the nucLei remained. smal1

(¡'iS" 9 C). In ad.dition to five normal corpora lutea the left TABIE 4.5 Relation between corpora Iutea, enbryos and pouch-young in five specimens of C. conci¡rrus

1 number Total- nunbor of Ilumber of or?ou'a lutea Inbryos in Corpora lutea Blastocysts of ani-ma]- ol-d. cor¡cora lutea pouch-young of pregnancy l-eft uterus of pregnancy ríght uterus (tottr ovaries) ( rert ovary) (right ovary)

li106 6+B 6 2 2 6 6

\t2768 I /+ 5 4 3 2

\i65tÐ 6 3 4 4 z J

i¡12952 10 3 5 5 3 /+

It5782 I 6 4 4 1+ 3

oo\ 6r.

TABLE 4.6 Sizes of corpora lutea in ovaries of l-actating C. conci-nnus

Corpora lutea of pregnancy Corpora lutea of previ pregnancy Serial number of ani rnal Mean ùiam. Standard- dev. Ùiean d.iarn. Standard. d.ev. (u) (u)

M6537 818 53

¡,t106 380 U+ 7oo 25

\r2768 408 37 528 45

rir65¿,o 427 5o 473 45

tr6616 5+6 52

ri2g52 423 44 579 73

\/'4983 626 25

rr5872 +33 6B 784 7B

ovary of \[ 2]68 contained one corpus luteum atreticum which was distinguished from the corpora lutea of pregnancy only by the d-egenerating ovum contained in it" Corpora lutea. of the previous pregnency were larger than the corpora lutea of pregnancy (Table 4.6)" The l-uteaI cells 62"

r¡rere about twice as large as those in corpora lutea of pregnancy; some nuclei were large and- rouncl , but many !,¡ere irregular in shape and others l¡rere represented only by d-roplets of chromatin naterial. Individ-ua1 luteal cells r¡¡ere separated by a network of connective tissue cells (fig. 9D).

The ovaries of M1O6 contained. a set of eight olcler corpora lutea (5ZO,t, average diameter, s.d- {!rL ) in which the luteal cells l'üere vacuol-ated and few containcd a complete nucleuË"

From one to seven tertiary follicles (d-iam. 2OO - 3OOur,) hrere present in ea,ch ovary, and the right ovary of M6540 also conta,ined three large atretic folf icles (aian" ¿ìpproxo 3OOu).

The smal1 uterine glands of ItI106 and M2lóB (aiam. ¡O- TOu) were irregularly scattered. through the connective tissue and_ few r/ùfere open. The gland- cell-s were low columnar, with nuclei occurring at any l-eve.l in the ceIl. The epithelium of the lumen consisted- of very taI1u pseud-ostratified- columnar ceI]s, ad.jacent to a dense sheet of 3 - { layers of connective tissue celLs. The uterine glands of M654O anð, NI2)J2 were more clense, the cells were tall-er and- the rad.ially-elongated nuclei tend.ed- to occur in the lower half of the ceJ-1, although they were not regularly ba,salIy- situated.. -As in M2768, few glands !{ere open (Fig. 11 A). îhe inner gland.s of !'[5872 hacl a sma1l lumen, and- the cel1 nuclei were basal" 63u

L'ieurc.9

A T.S. thnough posterior vaginal sinus of post-partrnr munclard.a; the epitheliun

is thick a¡rd- cel-ls are being shed. into

the Iumen. x 2OO

B. Section through corpus luter:n of pregnancy

of nund.ard.a; associated. with 14-ce11

blastocysts. x 20O

C. Section through corpusr Luter.¡m of pregnancy

of rm.rnd.ard.a; assooiated. urith J00-oe11

embryos. x 20O

D. Section through oId.er corpus luter¡n of nund.ard.a; associated. with pouoh-young of

head.-fength 5.5 run. x 2OQ a a: ',-a

ì- l-(lrv -r¡ì e

{ s ¡t

-.r l-

íj ." '{:.,r

-¡.-. 64,

Embryos found. in the uteri varied- in size from e.n average d.iameter of 267 t¡ in Mt06 to 617 p, in Mj87Z (¡'iS. tO)" Thc uteri of M2l68 contained" six unsegmented. eggs¡ consisting of an inner coagulum (f3f ,, average d.iameterr s.d. 20) surrounded. by a shel-l membraneg no blastocysts were present. Female UI106 carried. one unsegmented. egg (shell ZJOrt,, coagulum 12Ou ) and- seven blastocysts. The blastocysts consisted- of a she11 (267 ,t, average diameter) insid.e which was the protod.erm (t74,-t, average diameter) mad-e up of l-2, 13 or 14 (3) cells surrounrLing the blastocyst cavity, fn blastocysts from the rema.ining pregnant females the protod.erm was ad-jacent to the she1l membrane. Tn six blastocysts of M654O the protoderm contained 261, 27O, 3OO, 3OIg

312 and- 318 cells respectiveÌy, and- mitotic figures Ì{eïe seeni one blastocyst of I\I29J2 contained- 340 ce11s. The protoderm r¡¡as unilaminar and- the enrlod.erm had not d-ifferentiated." The d-egree of d-ifferentiation of the blastocysts of M5872 couLd not be determined. sperm ad.hered to the protoderm of arl blastocysts.

The urogenital- sinus and- posterior vaginal sinus were lined. wj.th 4 - B layers of squamous epithelial ce1ls, few of which were hecorning cornified- or r¡rere being shed- (fig. 11 B)" The epithelium of the posterior portion of the lq,teral- vaginae was similar, but was slightly thinner (+-ø ce11s). 65..

o

T o { I

u, IJ o ,n âp, a-o

2Ê. o o T a- ù, ù, E o 6 Þ

t¡l 3ó749þ Heodlength ot pouch young (mm) Flgure lO. Increosø ln dlqmetcr of blostocyst of C.concinnus wlth oge ol pouch young os lndlcot€d by heod lcngth. Vcrtlcol llnes show gtondord d¿vlotion. I Ovum ln folllclc¡ O Blostocyst¡ O Egg. 66"

( i:.i ) /rnoestrous females The anoestrous reproductive tracts r^rere smaI1, the uteri being about 2.8 mm long and- I"O mm wid.e. The ovaries contained only old- corpora lutea and small folliclesg no recently-forned. corpora futea or tertiary follicles were seen. The uterine gland.s

ïi/ere sma1l (aiam. 30 rr, ), sparse and- almost straight (fig. 11 C). The gland.s and- the uterine lumen were l-ined- with a single-layered, low cuboid-al epithelium. Itro embryos hrere found. The epithelium of urogenital sinus, posterior vaginal sinus and. the lateral vagi-nae posterior to the vestigial I''Iolffian duct consisted of 3-5 layers of squamous celIs, few of which showed. signs of degenerating or of being shed (lig. IlD).

(¡) Âcrobates p.yqmaeus

.ê, single sp ecimon of Acrobates pygmaeus (snaw) was examined. Although no pouch-young were preserved with the female, cavities in the pouch and four enlarged mammary gland-s anrL teats ind-icated- that pouch-young had- been present when the animal was fixed-.

Four bl-astocysts (approx, d-iarneter 4OO wcre in the uteri. ':, ) Poor fixation of the reproductive system prevented- a d.etailetl examination bcing made of the corpora lutea and. blastocysts.

4.6 Species that mate before parturition

1¡,lallabia bicolor

The oestrous cycle of the sl,rlamp wallaby ( I¡fa11abia bicolor 67u

Fi-gure 11

A a T.S. through uterus of nund.ard.a witl¡

JOO-oelI embryos; glands weIL

tLevelopod.. x 19O

B. T.S. through posterior vag5naJ_ sjnus of pregnant mund.arda; the epithelir¡m is

low and- few ce1Is are being shed. into

the lunen. x 21O

c. T.S. of uterus of anoestrous mtmd.arrla;

glands snall. x 190

D. T.S. of postorior vaginal si_nus of the sa.me animal; epithelir:rn flattened.,

few cells being shed, into lunen. x 21¡O .w f

rI t; 7 I.t i \ \ .\ ¡ lr

a I

Ír-\ c ,lr,'

¡l 4 i ol \ 68.

(lesmarest) ) is shorter than the gestation period. Pregnancy does not suppress oestrus and a mating occurs from one to seven d.ays before parturition" Õyc1ic activity ceases d-uring lactation and. the embryo derived from the pre-parturition mating is not born untíl the prevíous young vacates the pouch (Sharman, Calaby and Poole 1966)"

One swamp wallaby shot in the wild- carried- a newborn pouch-young and- was feed.ing a young-at-foot from an elongated- teat. Â large d.egenerate corpus luteum 'hra,s trrresent on the right ovary and- the left ova,ry contained- a ful1y-fornred. corpus luteum (3.5 *r d.iarneter) r,rhich consisted of sma1l luteal cel1s with round nuclei, none of which r¡¡ere divid-ing. It resembled. the quiescent corpus luteum of the red. kangaroo. The blastocyst found- in the left uterus l^ras 300 p in d-ia.meter; the protoderm (f6Ou d.iameter) consisted. of 103 cells with smal1 round. nuclei. Dividing cells were not seen in the blastocyst, nor in the uterus. The uterine gland-s contained many sperm, Three blastocysts recovered. from females suckling older pouch-young consisted- of 62, 63 and Jl cells respectively, and the cells were similar in appearance to those of the post- parturicnt female.

Macropus dorsalis  blastocyst was recovered from a black-striped- wallaby 69

(Macropus d.orsalis (Cray) ) tfrat carrj-ed. a finely-furred- pouch- young of head. length 69.0 mm. The shell was 25Or_¿ in d.iameter, and. the protoderm fBOu, in cliameter" The latter consisted- of 12 cel1s, One black-striped. wallaby was examined rcgularly d.uring the final 66 days of pouch suckling and- no ma,ting occurred d.uring that time" I'tithin a day of the young emerging permanently a second. young was born and the fenale mated- a day Iater" The neonatus survived less than one day" The a.nimal mated- aga,in l{ d-ays later, gave birbh 2 days after mating, and, d-id. not ¡nate after parturition" This pouch young r¡ras removed- when 1! d.ays old, and 30 days after RP'Y the female had- both given birth and mated. This pouch-young I^ras r€jmoved. when {! d.ays old-, The femal-e mated. 28 d-ays after RPY and. gave birth 2 d.ays later. Until morc black-striped- wallabies have been examined, the significance of the two instances of pre-parturition rnating cannot be ascertained.. The potoroo may nate before and after

( parturition IIughe s I)62) " 4"7 Discussion Embryonic quiescence has been found. in all macropodids whose reprod-uction has been investigated. (fatte 4"1). These species are d.i-strj-buted- in two of the three sub-families, and constitute over 30Úþ of thc modern species, 70"

TÄBLE 4.7 Breeòlng d-ata of species of macropod.id_s for which infornation is available.

Subfarnily Species Breeùing pattern*

Þotoroinae Potorous trid-actylus Post-partum oestrus ; quiesoent blastoeyst carried. until young nea;rs i-nd.epend.ence of pouch (5)

BettonEia leseuri As above (5, 9)

Bettoneia cuniculus As above (8)

Lagos trophus fascíatus Post-partur oestrus; quiescent blastocyst d.evelops after RPy (t r )

aJ.e thetis ï,ac tation-controlle d- embryonic quiescence (no rl.etaits ) (B)

Thylosale billarclieri Post-partur oestrus; quiescent blastocyst camied_ until young nears of pouch (4. I ,itd"lutta"nce Setonix brach-yurus Post-partum oestrus; quiescent enbryo clevelops after RPY tluríne breed.ing season onfy (7)

ïtlal-labia bicolor Prelartrm oestrus; quiesoent embryo carried. r¡ntil young nears ind.epend.ence of pouofr (9)

ùlaoropus euEenii Postlartum oestrus ; blastocyst d-evelops after seasonal anoestrous perioct (t )

aaaaar/ 7r"

TABIE 4.7 (cont.)

Subfanily Species Breed.ing patterrrrr

Maoropodinae I,,{acronus ir"ma Qui-escent blastocvst in one lactating fenale \s)

Ìvlacropus agilis As above (lz¡

trlaoropus clorsalis Pre- or post-pgrtum urating; quiescent blastocyst d.evelops after nPV (tz)

i/iacropus robustus Post-partum nating; quiescent blastooyst carried. r¡ntil yor:rrg nears ind.epend.ence of pouch (6, 10)

Macropus rufoEriseus As above (9)

I/lacropus giganteus QuÍescent blastocyst forned. after orrrlation when pouch yor:ng > 1BO days (4, 2)

lleealeia rufa Post-partr.rm nating, quiescent blastooyst carried- until young nea.rs independence of poucfr (8)

{' Number in brackets indicates reference

References:: . (t) sers"r 1966, (z) cfari< and. Poole 1967, (3) ttughes 1.9ç2, (+) rcirþatrick i965, (¡) ¡:.+u ancl T¡rnd-are-gísoãe ßá2, (6) SaareÍr and. Shield- t)6oarb, (7) srrarman j955, (8) Sharnan 1963, (9) srt"tttan, Calaby and Þoole 1966, (ro) ryrraaíe-Biscoe 1963, (tt ) ry"dale-Biscoe 1965, (tz) rtr:-s thesis, 72"

Sharman (tgSZ) d.oscribed- the histological appearance of the quiescent corpus l-uteum and quiescent uterus of the quokka"

Later d.escriptíons for the red. kangaroo (Newsome 1964, Sharman t964a) g be.nd-ed hare wal.laby ( Lagostrophus fasciatus ) (ryndale- Biscoe L965): SreJ kangaroo (Clart< and, poole f967) and other species (ttris thesis) have shown a striking uniformity ín

appearance of the cluiescent corpora lutea and- uteri throughout

the illacropodinae. llhether the corpus luteum is formed. before parturition, a day after parturition, or many weeks after, its histological appearance during quiescence is simil_ar, Ilmbryonic d.evelopment ceases at a simiLar stage in all the Macropodinae stud-ied- (latte 4"8), irrespective of when the mating and. fertil-ization occur. Within a species, the cells of the blastocyst are similar, and. there is no evid-ence that the protoderm has d.ifferentiated., The size and shape of the resting

nuclei and- the number of rrucleoli- d.iffer slightly between species, The potoroo is the only species of Potoroinae for which the

blastocyst has been examined" The number of celÌs varies more wid-e1y than in any of the Macropod.inae, but the protoderm has not d-if ferentiated. The gestation period, of the red kangaroo is less than

2 d.ays longer than the intervaf betueen RPY and- birth, r¡here-as the d-ifference is four, d.ays in the red-necked waIlaby, This ÎABLE 4.8 Average si-ze and average number of cells of the qutescent blastocyst of some species of líacropod.inae

Dianeter of she1l Diarneter of protoclern Nunber of ce

Species I n Size (u) n Size (u) n Nurnber

Iliacropus giganteus* 16 262 16 246 9 g2

I¡1. robustus 10 305 4 69

L,i. rufogriseus banksianus 17 301 13 228 14 83

Irt. agilis 1 250 1 77

l'1. eugenü 18 289 10 87

Setonix brachyurus 8 274 5 69

tlfallabi¿ bi.color 1 300 1 1æ 4 75

illegaleia rrrfa 38 321 3B 2U 2g 88

___l (¡J

* Data fron Cl¿.rk and. Poole 1967 74.

greater d-ifference suggested. thet the quiescent blastocyst may have been at a more advanced, sta,6çt; in the rcd.-necked. wallaby

( sharman r965a) but the observations on the blastocyst show that it is not more ad_vanced than in the red kangaroo. rn the lack of differentiation of endoclerm, quiescent blastocysts of macropod.id-s resemble those of the rat (sctrtatxe

and Enders 1963), mustelids (e.g. Baevsky L963, Canivenc Lg66) and" armadillo (Enders 1962).

Reproductive patterns arc knorrn for fewer phalangerid_s than macropodid-s. pilton and sharman (t962) anrl H¿gþes, Thomson and- owen (ryel) found no evid-ence for a variable quiescent period in embryonic d.evelopment in the brush possum, nor in the ringtail possum Pseudocheirus re È ), The gestation period- is 1l d-ays in the brush possum (pirton and. sharman 1962) and 16 d.ays in the sugar glid.er, but in a lactating mund.ard,athe interval between mating and. parturition was at least l1 d-ays (Sowley 1939)' ,rnother lactating muncJ.arda gavo birbh after ll d.ays isol-ation from males (Casanova l95B).

The corpus luteum of the brush possum reaches its maximunr sjze I - 10 days after oestrus (litton and sharman r)62)" rn the mund.ard.a with the oldest pouch-young, which must have been about 20 d-ays old (from Bowl-ey 's (1939) d.ata)e the corpus luteum is about half the size it attains j-n lateï pregnancy and_ the lutea1 7r. cells are smalle as they are i-n the quiescent corpora lutea of the ma.cropod-id.s.

Embryoni-c development of the lactating mundard-a j-s much slower than in the brush possum, but is not completely arrested as it is in the red- kangaroo. In contrast to quiescent blastocysts of the red- kangaroo, which d,o not enlarge d-urÍng the first 200 d-ays, mundarda blastocysts grow slowly and. attain an average d.ianeter of 6ll¡¡, when the young are about 20 d.a.ys old.. Quiesoent blastocysts of the European bad.ger similarly grow slowly without differentiating (Canivenc 1966). Thc gestation period of the non-Ia.ctating mund"ard-a is not known.

The d.ifference between ernbryonic quiescence in Cercartetus and. in the Macropod-id.ae does not support Sharmant* (t963) view that the occurrence of embryonic quiescence in both families indicated- that it was of considerabl-e antiquity in the superfamily Phalangeroidea. 76"

, THE PHYSIOLOGY OF EMBRYON]C QU]ESCE}TCE

5" I Introd-uction The quiescent bLastocyst of the red kangaroo does not expand nor do the protod-errn cells d-ivid-e while a pouch-young is suckling continuously. Soon after the pouch-young ís 1ost, the blastocyst resumes growth, Suckling of one young-at-foot

d-id. not increase the time interval between Rpy and" birth.

r¡lhen thc duration of the tactile stimulus to the teat was

d-oub1ed. by fostering a second yor.rng-at-foot on the female, the time between RPY and brrth was lengthencd- in I of 6 aninals. This suggested that the blastocyst remaíned- quiescent while the d.uration of the tactile stimul-us remained. above a threshold.

( Sharman I965b). How is the tactile stimulus to the teat transmitted- to the blastocyst?

In jection of the mil-k-e jection Ìrormone, oxytocin, d-elayed-

the onset of d-evel-opment of thi: blastocyst after RPY in the red. kangaroo, and- the corpus luteum of lactation al-so remained_ quiescent (Sharman L964b)" Sharman Gge+A) suggested- that frequent or constant suckling causes a rclatively high level of circulating oxytocin, and- this inhibits the release of a pituitary luteotrophic facl,or necessary for growth of the corpus luteum of lactation and- hence of the blastocyst, 77"

To gain further information on the pituitaryy ovarian and

uterine factors responsible for the quiescence ancl resumed growth

of the embryo, attempts have been made to artificially terminate

the d.evelopmental arrest ín the red kangaroo and- K" f, wallabyg the results are reported- here.

5"2 Pituitary hormones The luteotrophic hormone d.iffers among species of eutherian mamnial-s" Prolactin is luteotrophic in the rat (Evans et al-,. f94l-) whereas in the rabbit, luteinizing hormone is sufficient for maintenance of the corpus Luteum (Kilpatri-ck,

Armstrong and- Greep L964). The l-uteotrophic hormone has not been identified. in any marsupial"

( ") Prolactiq Five red kangaroos l^ierê injected- daily for 3 d-a,ys with

prolactin (5 ngf aay or l-O ngf aay) and. the pouch-young ïüere removed

on the fourth d-ay. Four of the five animals subsequently gave birth, all at the expected- tine after BPY (Table 5.1)" In the d-oses tested, prolactin r¡¡as without effect on the quiescent blastocyst, 78.

TABTE 5.1 The effect of prolactin on the quiescent blastocyst of the red kangarooo Prolactin injected once d"aily for 3 d.ays and. pouch-young removed- on fourth day

Animal Dose proJ-actin Time from RPY Time from RPY (ng/aay) to birth (aays) to oestrus (aays)

Kg6 5 30 32

K103 5 32 3'

KI5 10 32 Not d-etected,

K66 10 33

Kl13 10 30 31

Average 31 32.8

Control injected. (B) 32" 4 33.1

(¡) Luteinizing hormone Luteinizing hormone (nCC or NfH-LH) was injected- into five red kangaroos and the pouch-young I^Ieïe removed. (Table 5"2). All showed behavioural oestrus and extensive desquamation of the urogenital sinus 3 to { d-ays after the first injection. At laparotomy on the sixth day the ovaries of one female were enlarged about four-fold, and consisted- mainly of 1arge, unruptured follicles" The corplrs luter-rm of lactatjon could not be seen. tÆIÃ 5.2 Effect of luteinizing hormone injecteô into the red kangaroo during enbryonic quiescence. Pouch-young vÍere removeð,3 d.ays after first injection.

A¡iroal Hormone Daily dose Ilunber of d.ays Oestrus (aays Result i¡jected- after first\ r_nJ ec tr-on.)

K2g HCG 500 ï.u" 5 3 Ovaries enlarged at laparotony. No birth. Oestrus 57 days after RPY.

K72 HCG 500 f.u. 3 4 Died- 1 2 days after first injection" Ovaries enlarged.. Blastocyst degenerate.

K82 NIH-1JI 33 ne 3 l+ Ì{o birth; oestrrrs J6 days after RPY"

K171 HCG 25O I.U. 1 3 No birth; oestrus J6 d.ays after RPY.

Kg2 HCG 25O I"TJ. 1 ¿+ No birth; oestrus J6 days after RPY. \o--.1 BO.

The ovaries were sinrilar i-n a female that d-ied. on the thirteenth day. A slightly-enlarged-, d-egenerate bl_astocyst was found in the uterus of this animal-, No LH-treated_ animal_ gave birth. T'he recurrence of oestrus at the expected, time or later after RPY showed- that l,H did- not stimul-ate development of the corpus luteum of lactation.

Two K.I" wal-labies camying pouch-young cluring the non- breeding season were given a single injection of HCG (I25 I"U" i/u).

One femal-e l-ost the pouch-young on the sixth d-ay and. was laparotomized five d-ays l:rter; the other femal-e d-ied, on the eighth clay. In both animals the ovaries r^rere enlarged-, with many large unruptured- follicl-es that rr¡ere becoming atretic without luteinizing. The corpus luteum of lactation had- not enlarged., ancl the cel1s appearecl quiescent (filg" 12 A, .B)" At autopsy on day 8, an enlarged-, d-egenerate blastocyst, in which cell d-ivision Ï/as occurring, lias recovered- from the uterine papilla that pro jects into the rned,ian vagina. The C-eveLopment of this blastocyst may have been stimul-ated" by oestrogen released. from the many foll-icles, rather than by a secretion frorn the corpus luteum (see 5.4). Its l-ocation in the uterine papilla suggests that it was about to be lost from the uterus, The ovaries of the K" f " walJ-aby contain l-uteinized- intcrstitial tissue at all times of the year" In quiescent 81.

phase animars, whether or not they are lactating, the luteinized_ interstitial cel-l-s are similar to, but slightry smaller than the

luteal- cells of the quiescent corpus l-uteum (¡'iS. 12A, C).

The luteinized. interstitial cell-s are larger in the luteal phase of the oestrous cycle than in the quiescent phase, but remain much smaller than the luteal- cells of the corpus luteum. At eight d-ays and eleven d.ays after injection of HCG, numerous blood. capi-1laries permeated the l-uteinized interstitial tissue, but the luteinized, cells r,,¡ere not enlarged. (fi_gure 12D). rnterstitial gland.ular tissue is characteristic of the ovaries of llustela frenata and. M. erminea when the co rpora Lutea are inactive and- the em'bryos are quiescent (Wright f9æ). l,uteinized- interstitial cells were not present in ovaries of the red. kangaroo. The functional signì-ficance of the interstitial gland- is obscure. Keycs and Nalbandov (fgeg) x-irrad-iated- one ovary of rabbits, mated the rabbits and removed the non-irrad-iated ovary.

They showed- that the irradiated- ovary, consisting entirely of interstitiaJ- gland-, secreted- 20- 1q-hyd.roxy- 4-pregnen- 3- one but not progesterone, and was unable to support pregnancy.

(") Di scussion

The anterior pituitary hormones of mammals are proteins which d-iffer among species of eutherians (Morris 1966) and- can Bza

f'ígure 12

Sections through ovaries of K.I. wa-llabies.

x 730

A. Quiescent corpus luteun.

B. Coryus luter¡m eleven d-ays after injection

of HCG.

C. Iatérstitial tissue cluring quiescent phase of prognancy.

D. Interstitial tissue eleven d.ays aftor

i¿jeotion of HCG. :

's) IJ .r;'?'or i a o qD\ t ;^tì ô a c i :tå I iF-* e i 'ì'o; 'o-,*t.*i,iôj 0r - $.!-,

I \ lL , a Ç 19 ì .,,.?.t a t o o a a T o b a o 3r T I lo a r I n ¡ ì : f I I ? ¡ a O¡ lç ! o Ð I s I ¡ o ¡ J t I o o o I O tia 3r d ì t -61 t, o a ¡ ! O ¡r ( I oo I O I o - a t q t ì qÊ t ìì t ?¿ t o ¡ ta _a (D ¡ ao ¡- I I D 83,

be expected- to d-iffer between narsupials and. eutherians.

However the failure of isorated- injections of prolactin, pure LH or HCG to stimulate corpora l-utea of red- kangaroo or K. r. wallaby d-oes not prove that eutherían LH is ineffective

in these marsupía1s" Although discrete injections, twice d.ai1y, of ovine I'H maintained the corpora lutea of pseud.opregnancy in

the hypophysectomized rabbit (tcilpatrick, Armstrong and. Greep

1964) interruptec. d,oses of LH were ineffective in the ewe.

Kal-tenbach elL al. (rye1) showc;d_ that crude ovine LH is

luteotrophic in the hypophysectomized- ewe, but the LH must be infused. continuousry into a vein; interruption of infusion for as littfe as one hour caused irreparabl-e d.ecay of the corpus

luteum, A l-ow dose of LH (Z"J ngfewe/Z+ n") was the most effective.

Cne red kangaroo and one K.I. wallaby d-ied a few d_ays after be ing injected" r,rith HCG" Kaltenbach ut (lq6g) "1. were unable to explain the d.eaths of 3 of ! hypophysectomized. ewes perfused. r,rith pure ovine LH.

5.3 Development of blastocyst ind-uced- b.y progesterone The corpus luteum of lactation in the red. kangaroo enlarges and. the cells begin to d.ivid-e slightì-y before cel_1 d_ivision begins in the blastocyst (Sectlon I above). It is possible that progesterone secreted. by the coïpus futeum may cause the 84" blastocyst to resume development.

(') Histolosical observations after iniection of progesterone

Five red kangaroos were injected- with progesterone (fO mg) on days 1 - 3. Two were laparotomized on day 4, three on d.ay B.

The pouch-young were not removed-. Pouch-young rvere removed. on d.ay 1 from two non-injected control animaLs which were laparotomized on d-ay { and- d-ay B respectively.

(r) Slastocysts The blastocysts recovered- from the two progesterone- treated" females on d.ay 4 had- shell d-íameters of 330u r which is simil-ar to the average shell d-iameter of the quiescent blastocyst. The d-iameters of the protoclerm were 260 and. 27O v, respectively, compared with the average of 240u d.uring d.iapause. However, these blastocysts consisted- of J{5 and 148 cel1s respectively, and several ce1ls of each blastocyst were d-ivid.ing.

Tlie average number of celfs in the quiescent blastocyst is BB, and the cell-s are not known to divid.e (Section l, above). The protoderm of the two developing blastoc¡'sts had- not d.ifferentiated." Blastocysts were recovered from only two of the three females laparotomized. on d.ay B" These blastocysts had. shell d.iameters of 420 and- 690 tr respectively, and. both consisted. of several hundred. cel-ls. Mitotic figures lrere numerous and the endoderm had. rLifferentiated. from the protoderm (fUg. 13). 85.

.'¡$r'

Figure 1J

Flattened- blastocyst of j/r711. This blastocyst was reeovered. five d.ays after the l_ast of three d.aily

i-:cjections of progesterone. The protodern has

d-ifferentiated-. x 150 86.

No blaetocyst was recovered from the d^y 4 control. The blastocyst frorn the d-ay 8 control had. a shell d-iameter of 2)Ou., protod-erm diameter of 24O t and. consisted- of 86 ceIls. A single nucleus was d-ivid-ing.

(ri) 0varies Corpora l-utea from animals on ùay 4 or d-ay B after progesterone treatment could- not be d-istinguished from typical quiescent corporr; lutea (r'ig" 14 A, c.f . Fig" 4 A Section 3). on d.ay {. after RPY, the corpus luteum of the control animal was simil_ar to the quiescent corpus luteum, but on day B ttre corpus luteum of the control r¡as enl-arged- (4.6 mrn diameter) ancl a few luteal and connective tissue cell-s were divid-ing

( r'is. t4 B) . The corpus Luteum of progesterone-treated animals is able to devel-op when the suckling stimulus ceases. Two animals were injected- with. progesterone on d.ays 1 - l, the pouch-young

1¡re1e removed on day 4, and the corpora lutea removed at laparotomy on d_ay 11 and d.ay tl respectively. 0n day 11, seven d-ays after RPY, the luteal cells were slightly larger than in the quiescent corpora futea, and- a few 1utea1 and. connective tissue celfs were divid-ing. 0n day 1!, eleven d.ays after RP! the luteal celfs were large, with large round nuclei; no d-ivid.ing cell hlas seen. 87,

( iii ) uteri Thc microscopic appearance of the uterus on day { after

RPY was similar to the uterus in the quiescent phase, but on day 4 after progesterone treatment, changes had. occurred. The gland.s were not larger or more numerous, but the cell_ nuclei r¡rere no longer regularly basa1. rn one animal the stromal cells of the endometrium had expanded. consid-erably so that the gland.s were widely-spaced- (¡'ig" f4C). On d-ay B after progesterone injection, the uteri were fully Iuteal. Blood vessels were prominent in the stroma, and in transverse sections, gland-s were moTe numerous than during the quiescent phase. The gland- celIs were tal-l, with vacuol-ated- cytoplasm and. smal1, round nuclei situated- almost at the bases of the cell-s and forming a regular layer around the gland.. The epithelium of the uterine lumen had become flattened., and the cell-s were almost cuboi-dal , in contrast to their columnar shape during the quiescent phase. îhe cytoplasm of the outer glands gave a negative reaction for basophilia, as in the luteal- phase of the brush possum (litton and Sharman L)62).

At d.ay B after RPY a full luteal phase had- not d.eveloped-, although the stroma had. expanded- and- the nuclei of the gland. ce1ls were not regularly basal (f'lg, 14D). The cytoplasm of the outer gland.s was basophilic" The âppearance vüas similar to that of uteri on day 4 after progesterone treatment. 88a

Figure 14

A. Section through oorpus luteum of retl. kangaroo four tLays after the first of threo daí1y inJeotions of progesteronor

x 410

Bâ Seotion through oorpus luterm of red.

kangaroo 6even days after RPY. A nitotic figuro can be seen at upper

rtght, x 5@

0. T.S, through uterus of sa¡ne a¡rinaI as

for A, x 170

D. ToS, through uterus of sane animal as

for B. x 195 ;r:îù¡t/ 'lþ

G riì \.t

t, è a I a, i:tJ ,l: I /,' t r), t .¡

ir ,tt ta+, ^ lr' r- -t

lr' - ¿l) lta t. I Þ\ '(_\ d

.it : 89

(¡) Sustained development of the embryo

Microscopic examination of blastocysts recovered. by heni-hysterectomy of progesterone-treated animals ind.icated, that injection of progesterone perrnitted- the blastocyst to resurne d.evelopment d.espite the presence of the suckling young. As herni-hysterectomized animals r¡rere unsuitable for further experi-ments, most animaLs were not laparotomized. and- the pregnancies proceed-ed- to term. A significant shortening of the time between RPY and- parturition indicated- that the blastocyst had resumed development before RPY.

\1) Red kangaroos in,iectecl with 10 mg 'progesteronec RPY d-ay 4 Ten red kangaroos t,üere injected- on days 1- 3 with progesterone (fO mg) or with peanut oi1 (1 ml). Pouch-young were removed on d.ay 4" The resul-ts are shown in Tables !. 1 and. ," 4.

A paireô t-test on the d-ecrease in time from RPY to birth after progesterone treatment (tne time from RPY to birth for each animal after peanut-oi1 injection being used as the stand.ard.) ind-icated- that the probability of the observed decrease being d.ue to chance was Less !,lnan 2dfo" The tine between RPY and- birth in progesterone-injected animals ranged. from 2l to 33 d.ays, and the variance was slgnificantly d.ifferent at the Lóy' l-evel- fron the variance of control-injected- animal-s, in which the range hras from 3l to 34 days. 9o.

TARLE 5" 3 Time from RPY to birth and to oestrus i_n animals treated- with progesterone before Rpy. Lengths of corresponcling periods in non-injected_ and" peanut-oi-l-injected- animal_s given for comparison

Mean intcrval- (aays) and- stand-ard- erïor

Treatment N RPY to birth RPY to oestrus

Not injectedx B 31.34 1 0.35 34"00 I 0.89

f Control-inj ected. B 32" 4 t 0" 33 33.1 : o.4o

I ôQ r aot.) tl Proge ste rone-inj ccted- l_o - 80 33.6 t o" Bo

x Da,ta fronr Sharman ( 1963)

A paíred. t-test on the interval between RPY and- oestrus in control-injected- and. progesterone-injected- animals faited- to indicate significant difference at the Jf" level. The variances of the mean times in the tlro classes r¡rere significantly d-ifferent at the 5/" l-evel-, but not at tine 16þ Level.

( ii ) Red kanAaroos iniected with 5 or 20 mg'orogesterones RPY d.ay 4 I^Then pïogesterone was given at the rate of 10 mg/d,ay for

I d.ays, the time between RPY and- birth varied- wideì-y among animals, and- this variation suggested that l-O mg/day may not have been the optimum dose, Eight animal-s were injected- daj-ly with I mg and. 9r.

nine with 20 mg progesteronei as in the previous experiment, the animals were injected- on days I - 3 and. the pouch-young were removed on d-ay {" The results are shown in Ta.b1e !. {. Seven progesterone-treated- animals gave birth 2'l d.ays or

less after RPY at one or more d.ose Ievel. One gave birth only 2l d-ays after RPY, i.e, 2'l d-ays after the first progesterone injection. Three animals failed- to give birth sooner than 30 days

after RPY at any d-ose levef given, and. two of the three gave birth ll days after RPY rrhen treated with the highest d.ose

(2o ngf aay) " The average time from RPY to oestrus in progesterone- treated animaf s ïüas 34.2 ! O. ! d.ays. Twefve times in 33 trials 3et¡7 progesterone'treated animal-s failed- to give birth, whereas only once in nine trials (fVl") did- a control-injected. animal fail to give birth" In non-injected. animals, about 8J/" of post-partum rnatings give rise to a neonatus after RPY (Sharman f964)"

( ii-i ) ProEesterone treatment continued after RPY Three rcd kangaroos liiere injected. d-aily with l, l0r or

20 mg progesterone. Iniections were given for 8 d-ays, the pouch-young being removed- on the 6tn aay. The results are shown in Table l. f" o)

TXSLÐ 5.¿+ Sffect of progesterone on the interval between Rpy and birth in the red. kangaroo. Progesterone injected for 3 days before RPY.

Aninal ÎÍme from RPY to birth (4"y")

Peanut oil Progesterone Progesterone Progesterone t_ t nl/day ,- ng/day 1o ne/day ß{ü.y

14 Kl3 t /t 3o 3t 35 + K2B not tested- D Ê'j:' 23

K32 33 26 27 ,r2 K36 not tested- not tested. -, 33 27

K58 )¿ 32 27r 27 33

K60 33 25 not tested-

K62 34 26 t

K85 32 12 30 35

Kg6 32 30 32

K171 32 not tested 27

L{ean 32"4 29.o 28"3 30.8 Stand.ard. o 1 0.80 Error "33 .48 1 "97

No parturition 'r Neonatus survived less than 2 ðays + Blooô released through urogenital opening J2 days after IPT 93,

TABLE 5.5 Effect of progesterone given six d.ays before and. two d-ays after Rpy

Animal Dosc of progesterone RPY tc¡ birth RPY to oestrus (mglaay) ( aays ) ( aays )

K3B 5 58

K62 10 3B 42

r{133 20 60

The single pouch-young born must have d.eveloped_ from a quiescent blastocyst, as a male hras present onJ-y d-uring the final zz ð.ays of pregnancy.

Progesterone injected- after Îpy d-id not always cause the long interval between Rpy and- oestrus seen in the three females above" Pouch-young r^rere removed- from four animalsg progesterone (ro mg) was injected. at the time of Rpy and, on the two following d-ays, The results are shown in Table 1.6. 94"

TABLE| 5.6 Effect of progesterone (fO mg/aay) given at the time of RPy and. on the two following d.ays

Animal- RPY to birth RPY to oestrus ( aays ) ( aays )

K4 35

KI5 34

K60 3B

Kr3z 30 35

The 1ow birthrate in these two experiments suggested- that

progesterone given after RPY nay have been harmful to the blastocyst,

{. iv) Pouch-.young removed- later than d.ay 4

Four red- kangaroos that had- given birth less than 30 d.ays

after RPY when injected. r,rith progesterone (t-O mg/aay) were again treated- for 3 d-ays with progesterone (fO mg/aay), but the young were not removed. tilI d-ay 11 (2 animals) or d,ay 18 (2 animals)" At least 1{ d"ays elapsed- between birth resulting from progesterone treatment and. first injection of the foltowing treatment" The results are shown in Table 5"1. 95"

îABLE 5.7 Tj-ne to birth and to oestrus after Rpy in red. kangaroos treated- with progesterone (fO mg/aay) on d-ays 1-l

Animal Day of RPY RPY to birth APY to oe strus ( aays ) (days )

K60 11 19* 35

K171 1I 3B

K32 1B I2x 34

K'B 1B 33 33

* Neonatus survived- less than 2 d-ays

At the tíme of d.eath of the new-born young of K60 and- K32t miLk could not be manually expressed- from the teat.

A neonatus transferred- to the pouch of K32 3I d-ays after Rpy d.ied- on the teat a day later. Apparently the mammary gland-s of K32 fail-ed- to produce milk in response to the stinulus of a young on the teat both at the time of parturition and also at the time after RPY when parturition would have been expected- in an untreated- animal.

Two recl kangaroos (i<32 and. K!8) were injected_ with progesterone (fO ng/aay) for 20 d.ays. The pouch-young vrere 96"

not removed. I'rom the Z)th ùay, the pouches T^rere examined- d,aily, but no neonatus was found" The poucb-young i¡rere f'emoved. on the

3?th day. One animal (K32) p;ave birth ll o.ays iater and. mated on the folfornring day" The other lff8) d.id- not give birth, and mated- ll d-ays after RPY"

(") Di scussion

Three or seven clays after 'uhe first i-njection of progesterone into red kangarocs, afl- blasiocysts recoverecl irere d-eveloping, d-espite the presence of a" pouch-your:g and- consequent inhibition of the corpus l-utc,:r "," Systenic injection of progesterone d"oes not stimulate growth of the quiescent enbryo of the rat (lUutti-ng and. l{eyer l-963, Zerlrraker f964), trìuropean badger (Canivenc L957), mink (Cochrane ancl Shackfeford f962) or the armadil-lo (End-ers and- Buchanan Lg59). Yoshinaga Gget) indrrced. local ii,rplantation of embryos of the lacta;ing rts.t by applying progesterone to the ad-ipose tj.ssue a:ounrl ì;he uterus.

Tyndale-Biscoe (fg6:e) shor¡ed that in the c¿uokka the corpus luteum is necessary .îor d-e..'el-opment of the blastocyst after RPY.

Ovariectomy earlj-er than I d-ays after RPY prevented- the appearance of a luteal phase in the uteri and the blastocyst clid- not d.evelop"

Blastocysts d.eve.:'-c¡peC. in animals ovarj-ectonized at RPY and. injected with progesterone (fO mg/a"y for J d-ays) (fyndale-Biscoe 1963a)" ïühile the stimul-us of a sucklirr.g young heid- the corpus luteum o.7

cluiescent in the red kangaroo, injection of progesterone

permitted the blastocyst to resume clevelopnent. Uterine and

embryonÍc development índ-uced- by progesterone injectíon could- continue r'¡ithout further exogenous hormone, d"espite inhibition

of the corpus l-uteum for as long as 1{ d-ays"

The response to progesterone was variabfe in the red- kangaroo both between animals and. in the same animal at d-ifferent times. Five of nine animals gave birth earlier than expected after RPY when treated- with 30 mg progesterone, while four gave birth at the expected. time. At a higher dose, two gave birth early, two at the expected- tíme and- two later than expected.. One aninaf (fl8) aia not respond. to 15 mg nor 60 mg progesterone nor d-id she respond- to a 20-day treatment with 10 mg/d-ay. Yet this animal ttrice ga,ve birth earlier than expected- when treated. for 3 d.ays with 10 mg/d-ay.

Depend-ing on the d-ose, progesterone i/üas capable of stimulating or inhibiting the growth of the red. kangaroo blastocyst, or of not affecting it at all. Ad"minístratíon of different d-oses of the thyroid" hormone can cause the same change to occur in d-ifferent directions (lata 1964)" I,rlhereas smalL amounts of thyroid hormone promote bod.y growth, larger amounts will- amest it or even cause a substantiaì- weight loss. gimilarly the anabolic effects of thyroid. hormones on protein gB.

and, lipid- metabolism or on glycolysis at near physiological amounts can be reversed- with larger or pharmacological doses (rata 1964)" In the red kangaroos that gave birth earlier than expected-, the interval between the first progesterone injection and. birth (ZB - 30 days) was shorter than the normaf interval between RPY and- birth (¡r.¡+ i o"89 days, sharman 1963)"

Sirnilarly the uterus an

compared with control-injected or untreated. animals suggests that some embryos d.ied- in the uterus. This may ha.ve resulted- from the asynchrony of corpus luteun and. uterus. Tynd.ale-Biscoe (l-9fi) found. that of five blastocysts ùeveloping in progesterone- treated ovariectomized- quokkas, three were degenerate or severely retarded"

Berger and Sharman (personal communication) injected. K.I. wallabies with progesterone (f-+ ngfaay) for three days and. found mitotic figures in all- but one of the blastocysts recovered at autopsy 1 to I d-ays later. Blastocysts responded to progesterone during the breed-ing season and d-uring the non- breeding season. Thirteen ad-d.itional vral-labics uere injccted- with progesterone (Z or ! ngfd'ay) for 3 days and laparotomized 1! or 20 days later. 0n1y four were scored as pregnant at laparotomy and. none subsequently gave birth (Berger and. Sharrnan, personal communication)" As in the red kangaroo, d-eath of the foetus may have been caused- by a d-irect detrimental effect of progesterone, or a.s a result of asynchrony of uterus and corpus luteum, but in thewallaby the laparotomy in the latter stages of pregnancy may have caused abortion. Berger and Sharman (personal communication) found that in the K. I. wallaby, as in the red kangaroo, progesterone d-id- not cause hyperplasia nor hypertrophy of luteal cel-1s in the corpus 100.

luteum. Blastocysts devefoped- in response to progesterone in

K.I. wall-abies bÍl-ateralJ-y ovariectomized in the breed.ing season and in the non-breeding season. However, in many blastocysts d-egenerative changes l¡rere evidenced. by irregular-shaped- or pycnotic nucl-ei, by abnormally large nucl-ei, and- by chromosome aberrations in d-ivid-ing cel-l-s. Quiescent blastocysts that were morphologically normal r¡rere recovered from non-injected" K, I. wa]l-abies that had- been bifaterally ovariectomized- up to 30 days /^ prevrousry \rerger and Sharman, p€rs. com.)" Four red kangaroo neonatuses d-ied. soon after birth.

They attached- to the teat in the normal way but died there a d-ay or two later, and- at that tirne neither milk nor the clear f.l-uid- of the first d-ay or tt¡o of l-actation could be squeezed from the teat. These young were born between 12 and. 2J ð.ays after removaÌ of a prevíous young. The hormone balance in the mothers at the time at parturition was apparently unsuitable for

marunary gland- development "

,.4 Development of blastocyst induced- by oestrogen

(") Histoloeical observations in red kangaroo Oestradiol benzoate (foo p,g/day for 3 d.ays) was injected into 3 red- kangaroos" Pouch-young îrerc not removed'. Two animals were laparotomized on day 4 anð- the third- on d-ay 8' The luteal cel-ls of corpora lutea on d-ay 4 resenbled- those of quiescent corpora Iutea, but the corpora lutea of the oestrogen=treated animals were d.istinguished- by an abundance of

Iarge blood- vessels. The uterine stroma had. expand"ed- so that the gland.s T¡rere widely scattered-" The gland-s were sirnilar in size and- appearance to those of the quiescent uterus and the cytoplasm was positively basophilic in afl gIand.s" The only blastocyst recovered. was not enlarged, the outer d-ianeter being 27O v" It consisted. of 186 cells, approximately ten of which were d-ivid.ing" As on ùay 4t the d-ay B corpus luteum was hyperaemi-c, but the cells remained- smaLl- and- no cell was d-ivid.ing. The uterine glands uere enlarged. The gland- ce11s were talI, with basal nuclei, and in some outer glands the cytoplasm geve a negative reaction for basophilia. A bl-astocyst was not found-.

(r) Sustained d.evel-o'oment of eimbryo ín red kangaroo Oestra,diol benzoate !üas injected, into 11 animals for three d-ays and- the pouch-young werc removed on the fourth day. Four animals were with a male ancl q.ll- mated- within I days of the first injection. Vaginal smears showed that in all animals, layers of epithelial cells in the urogenital- sinus were becoming cornified- and, were being sl-oughed- into the l-umen" Five animals subsequently gave birth and in three the time from RPY to birth was less than the expected. 31 d,ays (Table 5.8). LOz"

TABI,E 5" I Time of birth and of post-partum oestrus in red kangaroos injected- with oestraòiol- benzoate for 3 d-ays. Pouch-young lremoved on fourth day"

,Animal Dose oes'uïogen Tine from RPY Time from RPY ( r-r,g/aay) to birth to oestrus ( aays ) ( aays )

Klt3 20 33 33

Kl03 60 25x 34

K344 60 ¿c) 33

K86 tcc L.r " 34

Kg6 100 30 1D

* Neonatus survived- less than 2 da;rs

Five anima:Ì.s vere g:-ven a single injection of oestradiol benzoate (1OO u S) ancl the pouch-young vuere removed I d.ays l-ater. At the time of RPY one animal had" niateo" and- in all five, masses of cornified- cells fj-lled the l-umen of the urogenital- sinus. Only one femal-e gave birl;h, thc time from RPY to birth being 2l d-ays. In this animal-, a single sr,:afl d.ose of oestrogen was sufficient to stimul ate rener'Ied embr¡ronic growth and to maintain growth, at least uniil bhe corpus luteum was re.l-eased from inhibition" 103.

(") Development of embr.yo in ovariectomized K. f . wall-ab.y The hyperaemia of the corpus luteum of lactation after oestrogen treatment suggested that the effect of oestrogen may have been to leach progesterone from the corpus luteum, the released progesterone then acting on the blastocysts. As red kangaroos were not available for bilateral ovariectomy, the effect of oestrogen in the absence of the ovaries was investigated in the K, I. wallaby.

Hormone injections were begun seven d.ays after ovariectomy. Oestradiol benzoate in oil (fOO f,¡ g/m1) was injected intramuscularly at approximately 9c00 a.m. for I d.ays gf ð-ay (9 animals). at the rate of !O ,t, gf aay (6 animals) or loO '¡ Ovariectomlzed controf animals (J) were not injected. and- intact controls (7) were injected. with 50 ûg/day oestradíol benzoate. Seven d"ays after the first injection, each aninal was kj-11ed- with chloroform and the urogenital tract was Temovecl. Blastocysts were flushed from the uteri with Ringer's solutiont and. were measured before being fixed-.

(i) Ovariectomized controL animals The uteri of animals ovariectomized 14 d.ays before autopey rernained in the quiescent concìition typical of the period. of embryonic quiescence (fig" 154). The uterine glands were small enrl few wexe opün. The opithelial cells of the gland.o werc 1ow ro4" columnar, with oval-shaped nuclei and. basophilic cytoplasm. In the lateral vaginae the superficial ce11 layers of the epithelium Íiere not cornified and. few cells were being sloughed. into the lumen"

Five blastocysts and- one d-egenerate egg Írere recovered

(tatte 5.9)" Shell d"iameter ranged from 2lO¡r, to 32Ou, (mean 294w standard. error B.7rr,) and d-iameter of protoderm ranged. from lEO to 230 p, (2O2 I t3.61.r,)" The protod.erm consisted of from 60 to 97 ceLls Qg ! l.g) the nuclei of which were round or slightly elongate, with granul-ar chromatin.. No nucleus was d.ivid.ing. These blastocysts were similar in appeara.nce to those of intact aninral-s d-uring the quiescent phase (n:-g. 16 A).

Berger and Sharman (personalcommunication) showed that complete absence of the ovaries for as long as 30 d.ays had. no effect on the retention of blastocysts and. that for at least 21 d.ays afte¡ ovariectomy blastocysts lùere capable of resuming d.evelopment when treated with progesterone.

( rr ) Ovariectomized animals treated with oestrogen Seven d-ays after the first oestrogen injection the uteri r^rere slightly enlarged- and turgid. In transverse sections, the numerous uterine glands r¡rere seen to be irregularly scattered and many had- a wid-e lumen" The epithelial cell-s were tall columnar, and- the small, round nucLei Ïrere regularly basal ÎABI,I 5.9 Effect of oestrogen on the blastocyst of intact and ovariectonized. K.I" wallabies. Blastocysts were recovered- seven days after the first of three injections.

Ovaries Daily dose No" animals No. blastocysts Ave, no cel-ls oestrad-iol- recol-ered fron in blastocyst benzoate (ug) uterus

Renoved. 7 5* 79.2 Present -7 n I 1{' + 113

Removed. 5o 6 5 164.8

Removed 100 /+ 9 130 "3

'3 One egg also recovereil + One blastocyst fl¡as recovered- fron med-j_an vagina; it consisfed- of over 2OO cells, most of then d,egenerate P \¡o 106.

(¡'lS. fl¡). The cytoplasm of the outer gland.s was negative for basophilia in some animals, but positive in others" The anterior lateral vaginae were enlarged and thickened"

The stratified- squamous epithelj-um of the posterior lateral vaginae was hypertrophled- and several layers of cells were cornified. The lumen was partiall-y occlud-ed. with sloughed, cornified ceIls" Blastocysts were recoveled from I of 6 animals receiving 150 ug oestrogen and from 4 of 9 animals receiving 300u9 oestrogen, Shell d.iarneter ranged from ZJO to 4OOu (3ZB I f6.4r-r,) and protoderm díameter from 2OO to 360u, QIS ! r7"2 p). The protoderm of one blastocyst was so degenerate that it was seen as an irregular white mass insid-e the shell. The shell of two others was dented- although the protoderm was spherÍcal-. These

3 blastocysts were from animal-s that received 3OO U,g oestrogen; al-l- other blastocysts recovered were spherical. The number of cell-s in each blastocyst ranged from 82 to .+ 255 (150 I31.6). ivlitotic flgures r¡rere seen in nucl-ei of 4 blastocysts (ni-gure f 5 C) and, in general, d-ivid-ing cefls were seen in the blastocysts with fewer cefl-s. Some of the nucl-eí in each blastocyst were round or slightly elongater with granular chromatin, and resembled nucLei of blastocysts from ovariectonized controls, but nany other nucl-ei were d.egenerating. Degeneration 107.

in some nuclei was seen as an invol-ution of the nucl-ear membrane

in several places, so thai the outline of the nucLeusl,üas r'ravy (figure 15D)g other nuclei were shrunken and, pycnotic. Together with d.egenerate nucl-ei T^rere many d-arkly-stained d-roplets whose basophilic nature suggested- that they may have been chromatin material released from broken down nuclei. t-tests ind.icated- that the shell was not significantly enlarged in blastocysts from oestrogen-treated females, but the d-iameter of the protoderm and" the number of cells hrere significantly greater ( lalte 5. 10).

( r_r_1 ) Intact. oestrosen-treated. control- animals The microscopic appearance of uteri and- lateral vaginae were sinilar to those of ovariectomized animals treated with oestrogen, and, the corpora lutea did. not d,iffer apprecia,bly from quiescent corpora Lutea removed at ovariectomy. Five of the seven animals failed to yield. a blastocyst or egg fro¡n the uterus, and. from one a degenerate egg I^ras recovered. The singl-e blastocyst found- in a uterus had. a shel1 diameter of 2)Ou, , protodoerrn diarneter of 260 u and consisted of 113 cel-l-s. None of the cells were d.ivid.ing and- many nuclei were degenerate. The higher number of cells ín this blastocyst than in those of uninjected animals suggests that some cell d-ivision may have occurred" A degenerating TASIE 5.10 Comparison of measurements of blastocysts from oestrogen- injected and non-injected. ovariectonized. K.I" wallabies. Significance of ðifferences indi-cated. by t-test; variances assumed equal for mean protoderm dianeter but d,ifferent for mean shell ðiameter and number of cells.

n lvleen Variance t Significance

Shel1 diameter 0estrogen 9 327 "7 242O (u) .o977 N"S. Control 5 29+ 3æ

Protodern 0estrogen Õ 276.2 2369 dianeter (u) 3.O34 P<"02 Control 5 202 920

Nr¡mber of ce1Is 0estrogen 7 150 3260 J.08 P<.O2 Control 5 79.2 303.5 P oG 109a

Figure 1l

A T.S. of uterus of K.I. wallaby ovarieotonized.

'f4 days previously. x 10O

B. T.S" of uterus of ovariectomized_ K.I, vra11a.by after oestrogen treatnent.

x 100

C. Ce1ls of blastoofst,: of oestrogen-treated. K.I. wallaby; one nucl-sus is ùividing.

x 540

D. Ðegenerate ool1s of blastooyst of oestrogen-treated- K.I. wallaby. xfuo Ic 1r0.

blastocyst was located- in the median vagina of one animal. llhe poor yield. of bf astocysts and, the presence of one in the med.i-an vagina suggests that in the presence of the ovaries,

injected. oestrogen may cause the blastocyst to be expeIled. fronr the uterus"

(¿) Di scussion

lühen ovariectomized- K. L wall-abies were injected- with oestrogen, the mean shell d-iameter of blastocysts d.id- not d-iffer significantly froni non-injected- controls. But both

mean protoderm diarneter and- rnean number of ce1ls liere si6çnificerntly

increased-(f< O"OZ). The singJ-e uterine blastocyst recovered.

from an oestrogen-injected. intact animal had a greater protod.erm

d.iameter and- more ce1ls than had- any blastocyst from non-injected.

animals" The increase in protoderm diameter and. number of cells,

coupled. with the observation of mitotic activity in some

blastocysts showed- that oestrogen caused initial d-evelopment of the dormant blastocyst. This effect of oestrogen was not med.iated. by the ovaries.

In the intact red- kangaroo, oestrogen-stimula.ted- d-evelop-

ment could. conti-nue, and. four red kangaroos gave birth from

five to seven days earl-ier than expected. after RPY. Injection of oestrogen was frequently d-etrimental to blastocysts. The birthrate in oestrogen-treated. red- kangaroos 111. was loff ße.,¡¡ and. as early as d.ay 8, all blastocyst recovered. from K. I. wallabies ïüere partJ.y d.egenerate" The poor recovery of blastocysts from oestrogen-treated- K" L wallabies, and- the find.ing of one blastocyst in the med-ian vagina, suggested that oestrogen also caused- blastocysts to be expelled from the uterus.

Oestrogen is necessary for the implantation of embryos of the ovariectomized. rat treated- with progesterone. Doses of between 0.3 and- 3 rr,g oestrone per d.ay a.re effective. I¡ower doses do not prevent embryonic quiescence, but higher d.oses

(fO ug) cause al-most complcte loss of blastocysts (wutting and. Meyer f96]). Early pregnancy in the rabbit is read.ily terminated. by oestrogens (Greenwalù f9r7)"

Tynd-ale-Biscoe (f9û) found that the blastocyst resumed development in one of J pre:gnant cluokkas from which the ovary bearing the corpus Luteum l,ias removed. on d.ays 0 to 2 after RPY. This development may ha.ve been initiated- by a release of oestrogen associated with the ovulation that occurs after removal of the coïpus luteum. Two injections of oestrogen (10 ug each) failed- to cause blastocyst growth in a bifaterally ovariectomized- quokka (tyncla.le-Biscoe L963) "

Êtr Extra-ovarian hormone - th-yroxine Canivenc (t96O, cited. by Dcanesfy 1963) suggested that the pituitary thyrotrophic hormone might be involved in the Tl-2"

termination of ernbryonic quiescence in wil_d. animaÌs, but

Canivenc (f9æ) was unable to stimul-ate blastocyst growth by

injection of thyroxine in the bad.ger. Holl-and_ et al. (ryel) found. that hyperthyroid.ism ind-uced- by injection of L-thyroxine (+Bfl,g) aaj-ty for 2\ days was beneficial to blastocyst survival

in ovariectomized rats treated- with a sub-optj,mum d_ose of progesterone. They suggested- that thyroxine may alter progesterone metabolisn or arter thc sensitivity to progesterone of the uterus or blastocyst.

Six K. I. wallabies carrying pouch-young in April were

injected intra-muscularly with L-thyroxine in oil- for ! d.ays and. killed on the sixth day. The four blastocysts recovereil were morphologically ind.istinguishable from untreated quiescent brastocysts (lable 5"11).

L-thyroxine at the d,oses tested. Ïras apparently ilithout effect on the quiescent embryo of the K, I. waI1aby.

5"6 Maintenance of extra.-uterine blasto cyst 1n v]-vo

(") Introd-uction

Blastocysts in the uterus began to d.evelop when either progesterone or oestrogen was injected- systemically into the pregnant female. Both hormones caused luteal_ changes in the uteri and- it is possibl-e that one or both of them acted inclirectly on the blastocyst by changing the uterine environment. IABLE 5.11 Characteristics of blastocysts from K.f. rrallabies treated- nith l-thyroxine for 5 days before autopsy

Ani-ßa1 Dose Thyroxine Initi-al vr¡t" Blastocyst ('ela'Ð (ke)

Diaraeter of Dianeter of Nr:mber of she1l (u) protod.erm (u) ce1ls ìl

68/1+ P 1 3.15 IrTot Recovered

68/jD P I 4.Og 260 180 BO

68/8 P 2 r+.49 2f4D 140

6B/j3 P 3 4.1o 3æ 220 111

68/lo P 3 5.20 Idot Recovereð

6B/7t+ P 3 5.20 3oo 2l,p 111

ts (,H II4"

If the blastocyst could be maintained. in a site other than the uterus, i.e. an ectopic site, d.irect and" ind.irect effects of the hormones could be distinguished-"

(¡) The oviduct as an ectopic site Blastocysts could be confined. to an ectopic site by preventi-ng them from reaching the uterrrs, Sperm reach the site of fertilization within an hour of mating in many animals (l:-snop L96f) and over BJ/, of rabbit eggs aïe fertilized- if the lower ovid-uct is ligated 3- I hours after mating (Aaams 1956)" The fertilized- egg of the quokka takes about one day to pass into the uterus ( Sharman f955). If the ovid.uct of the

K" f. wallaby were to be tied- between four and- 24 hours of mating, the fertilized- egg should. be confined. to the ovid.uct.

Six K" I. wall-abies tr\rere anaesthetized- between four and-

24 hours of the post-partum mating. The reproductive system was exposed, To minimise d.amage to the ovid-ucts, the ovaries r^rere not examined-, and both oviducts were tied- wi-th fine mersilk at the uterotubal- junction, Pouch-young T^rere removed- from 3 femal-es at operationg the other I continued. to suckle their youngc Ten days l-ater the animafs were killed- and the oviducts and uteri r,rrere searched with a d-issecting mi-croscope. Swollen portions of the oviducts were embed.d.ed- in wax and. serially sectioned for microscopic examinatlon. No blastocyst or egg 115.

was found in any of the six animaLs. rt is possible that the nembutal anaesthesia interfered

with orrulation and- hence r¡ith fertifisation, although Adams

Gg>e) found- no evid.ence that it d.íd- so in the rabbit" Adams

Og>g) ligated one ovid-uct of recently-mated. rabbits and. ten d.ays later recovered. uterine embryos from the non-ligated side,

but d-egenerate eggs from the ligated tube. Eggs d-eveloped. normalJ.y in the ligated- tube for about 84 hours but then collapsed and. d.egenerated, (Ad.ams rgl,9). Bland. and. Donovan (t)6J) ai_a not find- blastocysts 1n the ligated- ovid-uct of the guinea pig after

uni]ateral ligation" Tr¡¡o scverely retarded- embryos were found-

in the oviduct sixteen d-ays after transection of the ovid-uct (llana and. Donovan r)6J). However tubal prcgnancy is not rare in women (rrry r9û).

(") Transfer of uterine blastoc.ysts to an ectopic site

Blastocysts were transferued. to ectopic sites d_uring

the non-breeding season, when intra-uterine blastocysts remained. quie scent.

(i) Anterior cha.mber of the e.ye

A blastocyst was inserted- into the interior chamber of the left eye of each of nine K. r. walrabies, seven of then receiving their own bLastocyst, the other two receiving a blastocyst from a d-onor. After five da.ys the animals were killerl anrl the 116,

anterior chamber of the eye r4ias dissected. in o.)fo sarj-ne and,

sea.rched for a blastocyst" six blastocysts hrere recovered.. None had enla,rged. and. five showed extensive degeneration, with loss of nuclei and breakd.own of nucreÍ to droplets of chromatin (r'ls. 16 c)" The protod,erm of the other blastocyst was collapsed- and. crumpled., but 86 nuclei coul-d be distinguished.. B1i;stocysts from five K. r" wal-Labies were transferred to the contra-lateral uterus and. four of these were recovered- at autopsy five d.ays later. The appearance of three of these was typical of the quiescent blastocyst (pig. 164, B)" The fourth blastocyst had, enlarged. and. consisted- of over 2OO ceIIs" The corpus luteum of this animal was al-so enlarged-, with hypertrophied. luteal cell-s. (Two instances of unseasonal growth of the corpus luteum and- embryo vÍere observed- in untreated animals d.uring this study. ) The recovery of morphologically-normal bJastocysts after transfer to the uterus suggested. that manipulation of blastocysts d-ld- not cause their d-eath, but that the anterior chamber of the eye lüas d"etrinental to them. Ahlgren and- Bengtsson (f962) recovered. JB/" of 128 rabbit eggs transplanted. into the anterior chambers of the eyes of twenty-nine rabbits. The eggs were transplanted- when at the 2- to {-ce11 stage. For {B hours their d-cvelopment was simi]ar to that of tubal ova, but between 117.

'12 and' 96 hours of transfer, j-ntra-ocular eggs showed retard.ation of growth and d-egenerative changes.

( ri ) l4illipore filter chamber

Eight blastocysts were sealed- into d_iffusion chambers and- inserted. into the peritoneal cavity, five being returned to the d-onor K" I. wallaby, three to a host K. I" wallaby" Five days later the animals were kill-ed- and. the d.iffusion chambers located., either free in the peritoneal cavity (3) or embedded- in fat masses surrounding the viscera (!). AII the blastocysts were recovereil. None had- expand-ed. and- in a]I of them many cells were degenerate. However, in two blastocysts, ceIls were d-ivid-ing (f'le. 16 D). Both dividing blastocysts were recovereil from diffusion chambers lying free in the peritoneal cavity of the d.onor. The corpora futea of these animals did. not d.iffer from the typicaJ- corpora l-utea of lactation. Cell d.ivision d.oes not occur in the intra-uterine quiescent blastocyst (serger 1966)" Ahlgren (tg66) transfened J0 hours-old rabbit ova (late morulae or early blastocysts) from the oviduct to the uterus, or to a millipore filter chamber wíthin the uterus. lühen the blastocysts were recovered 50 hours Iater, those within the chamber were retarded in comparison with the controls. fmpaired- contact with the endometrium or a toxic effect of the filter rnay 118a

Figure 16

A Flattened. quiescent blastocyst of K.I. wallaby. x j35

B. Ce1ls of protocl-enn of blastocyst recovered. five iLays after transfer to contra-lateral uterus; no evid.ence of d.egeneration of

cef1s. x 81O

c. Degenerate blastocyst recovered. five d.ays

after transfer to eye. x 21O

D. I/litotic figure in blastocyst fron rnillipore

filter chamber. x 8J0 { 18"

a

.t

'l A

Õ ù'tr

la .J { ¿ .r\'' -t. 'd I

a J

.a

D 119. have retarded- them (.Ahtgren 1966)"

(d) Di scussion

Mouse blastocysts grew and. implanted. in the anterior chamber of the eye and continued their development through mesoderm formation before d-egenerating (Runner \947). Kirby (f960, 1963a, b, 196T) transfered- blastocysts to the spleen, kid.ney or testis of mice and. found- that the blastocysts grew irrespective of the age, sex or hormone status of the host,

Mouse ova from the 2- to 16-ce11 stage and" early embryos grehr and- impla.nted. in millipore filter chambers in the peritoneal cavity (Bryson 1964). Embryonic d-evelopment of the nouse is therefore inhibited- only in the uterusg in ectopic sites the blastocysts proliferate, regard.less of thc horrnone status of the host.

The cell d.ivision of two K. I" wallaby blastocysts transferred. to filter chambers may indicate that in the macropodids, too, embryonic growth is inhibited. only in the uterus, 120.

6 DISCUSSION

6.I rl'he evolution of embr.yonic c{uiescence

Sharman (tg6Sa) suggested- that there has been a trend in marsupial evolution toward,s the production of a larger neonatus, with greater internal- and external organization. Greater development necessitates a l-onger gestation period-"

The young of the brush possum is born at the end. of the luteal- phase of the utcrine cycle, when the cel-ls of the corpus luteum show the first morphological signs of degeneration. This occurs about 1l d.ays after mating; oestrus occurs about 2{ ð.ays after an infertile mating but not after parturition

(litton and Sharman 7)62). The suckling pouch-young provides an inhibitory stimufus that prevents pro-oestrous changes in the ovaries and uteri. Lactation suppresses oestrus in the sugar glid-er also" In the macropodids, the gestation period- has been extended and the young is retained. in the uterus beyond the luteal phase. At parturition the ovaries anfl uterl have undergone pro-.oestrous changes and oestrus is imminent (Sharman

I955t Sharman and Calaby L964)" The mundard-a apparently resembles the macropod-id.s in that pro-oestrous changes precede parturition. A red kangaroo mated- at the post-partum oestrus is able to give birth to a, second- young 31 days after loss of the first. I2I"

Drought-stressed wild- females that had. not ovulated' after parturition took about eleven d-ays to return to oestrus when the d-rought broke; experimental- femal-es returnecl to oestrus 11.6 days (average) after surgical ablation of the quiescent corpus luteum when the pouch-young vuas r.emoved simUltaneously (Sharman and C]ark 1967). Females without a quiescent corpus luteum therefore require about {{ d.ays to produce a second young after foss of the first. Tn the evolution of macropod-id-s a saving of perhaps eleven d-ays in 44 nay have provided. sufficient advantage to animals ovulating post-partum and maintaining a quiescent embryo for the incorporation of the character into the species by natural sefection. A higher rate of loss of pouch-young would provide a higher sel-ection pressure in favour of post-partum ovulation and embryonic quiescence" The female macropodid may lose her pouch-young when fleeing from a predator, and Marshal1 Ggq) fras suggested that in prehistoric an¿ early historic times the herbivoïous marsupials were strongly preyed- on by such carnivores as the thylacine"

6.2 Ernb rvon]-c 0 uiescence and. the nh-vl-oEenv of d-iProtodonts It was considered- possibte that simil-arity in breed-ing pattern of different species of Macropod-id'ae night ind-icate evolutionary relationship. The shramp wallaby may stand alone r22.

in having a pre-parturi-tion mating, as it d_oes on chromosome number (Sharman L96Ib) and. morphological- features (Sharman, Calaby ancl Pooie f966), Among the remaining species, wi-thin one genus species rnay d-iffer in reprod-uctive pattern (e.g, Macropus giganteus and- Macropus rufogriseus ) rruhereas species so clistantly relaied as to be classified in d"ifferent subfamilies have a similar pattern of post-partum mating and- embryonic quie scence " The basic d.ifferences in reproductive physiology that

lead to rnating a'; cliffe¡enb times of pregnancy ori lactation may be quite small-" At least 'bwo gonadotrophins are probably

need-ed- for the formation and- fulf functioning of the corpus l-uteum of macropod-id-s. Release of the gonad,otrophin required for ovul-ation in the red- kangaroo is not inhibited- by lactation, for surgical removal- of the quiescent corpus l-uteum ís followed

in about 18 d-ays by ovuJ-ation and- formation of a new quiescent corpus luteum (Shar:man and Clark 1967). However the ovulation hormone is suppressed when drought cond-itíons severel-y stress the animal (Newsome 1964). fn the grey kangarooe both gonad.otrophins are initielly suppressed. by the suckling stimulust but from about 100 days onwards in lactatlon, suckling ceases to suppress the ovufation hormone in some animals. Reproduction

ín the red and" grey kangaroo nay d.iffer essentially only in the 12 3.

factors controlling the release of the pituitary ovul_ation hormone. Sharman et al" (1966) suggested. that the swamp wallaby pattern d-iffered. from that of the red. kaogaroo mainly in an incrcased- tol-erance of the uterus for the foetus, so that gestation lasted. longer than the oestrus cycle.

Hypsiprymnodon moschatus has sevcral mo rphological features that are consid-ered- primitive in the Macropod.id.ae

(Troughton I)62)" A knor,,rled-ge of its reprod.uction might help to d-istinguish between the possibilities that (a) post-partum mating and- embryonic quiescence was a feature of the ancestral macropod.id- and- that the sr¡ramp wallaby and grey kangaroo patterns have evol-ved. from this, or (t) embryoníc quiescence has evolved several- times ín the Macropod.id-ae.

6"3 The arrest of embryonic development The fcrtilized. egg of the post-parturient red kangaroo d.ivid-es and. d-evelops into a bJ-astocyst, but the protod-erm of the bl-astocyst d-oes not d-ifferentiate. Similarly in other macropod.ids and. in eutherian mammals the arrest in embryonic development always occurs before the d.ifferentiation of ectoderm and endoderm (references cited- in Sectiot 1.3). In non-Iactating macropodid-s there may be a short quiescent period-, for the protoderm of a quokka blastocyst had, not d-ifferentiated as long as five d-ays after mating ( Sharman f96I), The evidence L24,

suggests that fertilization stimul-ates development of the egg to the blastocyst stage, but the cells cannot d-ifferentiate until

they receive a further stirnulus or are released from an inhibition" Differentiation of the embryonic cell-s is a critical

period. in the development of the sea-urchin and- frog egg (Brown I964t Gross f964). During oögenesis the oöcyte accumulates nutrients. Rad.io-autography and- density-grad-ient studies have

shown that the nucl-eus is an active site of RNA synthesis and that the lliA is stored in the cytoplasn. Littler if any,

ribosomal- RNA is synthesized- d-uring cleavage, although this is a time of intense cell d-ivision when new proteins (e'g. for the mitotic spindle) and- new cefl surfaces must be synthesized.

Ribosomal RN.û synthesis begins agaín at gastrul-ation (Brown 1964, Gross 1964). There is evidence that before gastrulation the embryonic cells of the sea-urchin are not regulated. by new information from their nucl-ej-. At low concentration, actinomycin D inhibits the formation of RNA or DNA without preventing d.uplication of the

Dh'A. trnlhen sea-urchin eggs r/,Iere treated, with actinomycin D continuously before a.nd- after fertiLization ce11 d-ivision occurred- at a rate only slightly lower than in controls for the first {B hours, but there bras no morphogenetic pIlogress beyond- the earl-y blastula (Gross 1964). r25"

/\ block in RNÂ synthesis occurs in a nutant strain of the

clawed- toad-, Xenopus laevis" The heterozygous rnutant strain has only one nucleolus in each d.íploid- cell, whereas the wiId. type has two nucleoli. The homozygous mutant embryo j-s anucreolate,

and rad-io-autographic and. density-grad.ient stud.ies show that it is incapable of synthesizing ribosomal- RNA. rt d.evelops normally past hatching to an early swinmi-ng stage, but then growth ceases and the embryo d.ies (Brown L964). Ova of the toad-, like those of the sea-urchin carry in a stable, inactive form all the ribosomal RNÂ necessary for early development" Gastrul-ation or organization of the blastula into the three-layered- gastrula requires further informetion from the nuclear DNA. The stinrulus for the provision of this information must come from within the embryo of the sea-urchin and toad, for they d.evelop in a constant environment"

Perhaps the marnmalian egg also carries sufficient RNA for early d-evelopment but requires information from nuclear DNA for differentiation of the gern layers. The stimulus for renewed. synthesis of RNA cou.l-d be a maternal factor which is provid_ed_ soon after ovulati-on in animal-s which d.evelop continuously, but is Ìrithheldin those with embryonic qulescence. Míntz's (1964) investigations of mouse eggs developing

ín vitro show that considerable RNA is synthesized. Incorporation 126"

of labelled- RN.A was Low in the und.ivid-ed. and 2-ce11 egg but accelerated- after the,: {-ce11 stage" Rad,io-autographic stud.ies alone could" not ind.icatc the mol-ecular wcight of the RNA formed., but the presence of nucleoli after the 2-cel_J- stage suggested that some of the newly-formed RN.A woul_d_ be ribosomal_, Doses of actinomycin D sufficient to severely d.epress RN.A synthesis prevented d-evelopment beyond- the morula stage or even sooner (wtintz 1964)" Early d-evelopment of the mouse egg therefore requires new gene products. Simila"rly in the chick embryo ribosomal RNli is formed from as early as seven hours of incubation (Lerner, BeJ-l and. Darnell 1963). 1tl-though the genes of the early mammalian embryo actively transcribe information for translation in the cytoplasm it is possible that only portions of the genes are active. Nuclear involvement in early d-evelopment woul-d then d.iffer only in d-egree between amphibia and mamrnals.

6"4 The stirnulus for renewecl enibryonic developnent

(") Direct effect of steroid- hormones

Both oestrogen and- progesterone initiate d-evelopment of the quiescent embryo when injected- systemically into the red. kangaroo. Is it possible that these hormones activate a part of the gene system to transcribe the information necessary for further development and d-ifferentiation? r27.

Srnal-l mol-ecules absorbed. into the cytoplasm of simple

organisms may ind.uce the synthesis of specific enzymes. The

smarl mol-ecul-es are believed. to combine with the product of a

regulator gene; this prod-uct then acts on the operator of a group of structural genes and- so causes the information from

these genes to be released- (or suppressed if they r¡rere previously operating) into the cyto¡¡lasm, where it is translated- in the formation of new protein" It is conceivable that oestrogen and- progesterone are so sir,lilar structurally that either can combine with a regul-ator and- act on an operator controlling the genes needed- for cell differentíation.

Metazoan cel-l-s differ from microorganisms in having more stable messenger RIITA, shorter-Iived- proteins and. more nearly continuous generatj.on of ribosomes; regulation of translation of messenger RNA may be a more irnportant controL mechanism in higher organisms than rate of genetic transcription (,Iata I)66).

There is some evid-ence that steroid- hormones can induce enzyme synthesis in mammalian cel-ls. Tomkins et a1. Qgee) nave shown that the synthetic glucocorticoid, d-examethasone phosphate (1") ind,uced specifically the synthesis of the enzyme tyrosine- c<- ketoglutarate transarnini,se by cel1s of a hepatoma tissue culture.

Dx acted. at the level- of translation of the transamínase messenger

RNA by antagonízing a repïessor of messenger function (Tomkins T2B"

et al-" L966)" Other l-evel-s in translation that could be reguì-ated by hormones are transport of nRNA from nucleus to cytoplasm, stability of cytoplasmic nRNA and- attachnent of mRi\A to ribosomes (fata L966).

F\.rture experiments on the growth of macropod_id_ embryos i-n vitro may d-etermine the requirements of the growing blastocyst for steroid. hormones and, may ind.icate whether the hormones act by activating a repressed- portion of the genetic complement.

(¡) Ind-irect effect of steroid hormones The theoretical possibilities for the intervention of progesterone or oestrogen in protein synthesis are interesting, but there is as yet no evid.ence that renewed blastocyst growth requires the direct action of either progesterone or oestrogen. A specific ovarian hormone is not essential for growth of the blastocyst in an ectopic site. Kirby (t962, I)6Ja, b) transferred- mouse blastocysts to the kid-ney, spleen or testis of recipients of the same species and, found- that they grew regardless of sex or reprod-uctive state of the host. Blastocysts proliferated- in intraperitoneal filter chambers in hosts of either sex (Bryson f964)" Sirnílar1ye mouse blastocysts developed in chemically d-efined culture medium supplemented- only with calf or human seïum (Cote and Paul- f965). Siggers (in lüolstenhoJ.ne L)6J, p" 411) found that development of mouse r29.

blastocysts formed in culture i¡Ias arrested but that the blastocysts Iived. for at least five d-ays. Unfortunately,

Biggers d.id. not give d.etails. of hls culture methods. MiDk blastocysts d-id. not grow in culture med.ium supplemented. with oestrone or various nutrients, although oestrone d"id. ind-uce a transient expansion in two of three blastocysts (DanieI L967). Blastocysts may grow in an ectopic site while uterine embryos remain quiescent. In the K, I" wallaby two bl-astocysts underwent ce11 d-ivision in intraperitoneal filter chambers at a time of the year r,¡hen uterine bLastocysts remained quiescent. Kirby Qgq ) transfemed blastocysts from one uterine horn of ovariectomized mice to the kid.ney of the same mouse. The blastocysts in the kid-ney proliferated while those in the uterus remained ctruiescent.

The unprepared uterus inhibits growth of many tissues.

Kirby (in 'otolstenholme 1965, p. 392) commented that grafting of tissues into the uterus is very d-ifficult. A malignant tumourt the llalker carcinosaïcomar grew well outside the uterus of ratst but its growth within the uterus I¡Ias strictly hormone-dependent, and- it would only deveJ-op in those ,animals that were in an end.ocrine state which would have supported the growth of an embryo (Short 1968). Although ectopi-c blastocysts grow imespective of the 130.

hormone status of the host, ovarian hormones permit the growth

of uterine bl-astocysts. Injection of a single smal1 d_ose of oestrone (0.3-1 pg) into the ovariectomized rat lead.s to growth

of the blastocysts and- implantation within 36 hours (lUutting and" Meyer I963t Yasukawa a.nd }Ieyer 1966). In the red kangaroo, a single injection of oestrad-iol benzoate (fOO uS) or three injections of oestrad-iol benzoate or progesterone caused the blastocyst to grow (Section !).

The essential time of action of end.ogenous hormones may be short, for Tynd-ale-Biscoe (t963a, c) found that bilateral ovariectomy of the quokka as early as four days after RPY permitted. growth of the blastocyst, although the blastocyst does not increase 1n síze until 6 d-ays after RPY. The function of the ovarian hormone(s) may be to trigger a susta.ined. change in the uterine endometrium and so to provide conditions favourable to bl-astocyst growth. fn the pregnant nonlactating rat, oestrogen is released on day 4 (Shelesnyak and- Kraicor 1963). Blastocysts removed from such rats on day 5 would have been exposed- to oestrogen-ind.uced changed" in the uterus, but they are nevertheless unable to grow in the uteri of spayed rats treated- with progesterone (Yoshinaga and. Adams $66a)" Tynd.ale-Biscoe (fq6¡¡) found that blastocysts from lactating quokkas grew when transfemed. to animal-s from which young had been removed 6 d.ays r31.

previously" These blastocysts therefore greïú r'¡ithout being

subjected. to the hormonal- cond.itions of the first four d.ays after RPY, when growth normally resumes. Both oestrogen and, progesterone cause d,iverse changes in the uterus. 0estrogen causes increased protein synthesis,

vasodilation, hyperaemia, oedema, and" other effects on the

uterus (Spaziani and Sud.dicir- 1967). Effects of progesterone are more d-ifficult to demonstrate as they often occur only in a uterus previously exposed to oestrogen" Progesterone probably causes an increase j-n anino acid content of uterine fluid.,

secretion of glycogen and- movement of water from the extra- celluIar to the intracellular phase (Fotherbr 1964). After the

injection of either oestrogen or progesterone into the red. kangaroo, the uterine stroma expand-ed, ind-ícating a change in perneability of either (or both) tne blood capillaries or the stromal ce11s. Perrneability changes coul-d affect the concentration of oxygen and- metaboli-tes in the uterine fluid.. It is not known which, if any, of these effects of the ovarian hormones permit the blastocyst to grow,

6.5 Conclusions

Differentíation of the protoderm of the macropodid. embryo is a critical stage in development and can occur only in favourable cond.itions. These cond.itions are met (u) in the r32.

nonlactating aninal, (b) when the suckling stimulus is terminated. or its frecluency red.uced-, or (") after the sytemic injection of progesterone or oestrogen. The evid-ence of cell- d.ivision in

blastocysts transferred to the peritoneal cavity, consid-ered. together with published- observations on other .species ind.icates that the effect of the ovarian hormones on the blastocyst is indirect and. is med.iated. by the uterus. To gain further knowledge of the physiology of embryonic quiescence it r,¡i1l be necessary to make d.etailed. stud.ies of

changes in oxygen tension, pHr errzyme concentrations and distribution of metabolic substrates within the uterus at the tine the blastocyst resumes growth, either norma11y, or in response to hormones. It will also be refevant to determine the requirements for blastocysts to remain quiescent or grornr in cul-ture. Elucid-ation of the factors that terminate quiescence in the rat is complicated. by the proximity of implantation to the renewed- growth. The macropodid- blastocyst never implants in the strict sense of eroding the end.ometrium, and. in the macropod.id- uterus, biìochemical changes associated. with blastocyst growth will be more easily studied. 133.

7. APPTNDTX BÏBIIOGRAPTIY

The foll-olving papers have been publishecL under ny nraiden name.

Clark, XIereùith J. and. Sharman, G.B. (lg6E) Failure of hysterectony to affeot the ovarian cycle of the narsupial Trichosurus vulpeoula. J. Reprod.. ry. g +59-461 . Clark, L{eredith J. ?See¡ The blastooyst of the r.e d kangaroo, Megaleia rufa (Desm.) during èiapause. Aust J, ZooI . 14. 19- 25.

Clark, i\[eredíth J and- Poole, Tú.8. (lg6l) The reproductive systen and- enbryonic diapause in the fomale grey kangaroo, gallteus. .A,ust. J. ZooI. 1Dz h41 - 4i9. Clark, Mered.ith J. jgel) Pregnaney in the lactating pigmy possrntr, Cercartetus conct_nnus. Aust. J. Zoo!. 15: 673- 683. Shar."nan, G.B. and C1ark, l,[ered.ith J. (1167) Inhibition of ovulation by the cor?us luteum in the red. kaLgaroo, Ivïegaleia rufa. J. Reprod, Fert. 14: 129 - 137. C1ark, Mered.ith J. (tgøg) Grou'th of pouch-young of the red. kangaroo, }{9æ}Iþ. rufa, in the pouches of foster nothers of the same species. Tnternational Zoo Yearbook B: Íl press. Ci-ark, IÍered.ith J. (tfee¡ Ternination of embryonic diapause in the red. kangaroo, Megaleia nrfa. by injeotion of progesterone or oestrogen . J . Reprod. Fert. 15: h7 - 356.

Three of these papers have been referued. to in the text of the thesis, arrô oonstitute the remainder of this appen&ix. Clark, M. J. (1966). The bastocyst of the red kangaroo, Megaleia Rufa (Desm.), during diapause. Australian Journal of Zoology, 14(1), 19-25.

NOTE:

This publication is included in the print copy

of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

https://doi.org/10.1071/ZO9660019

Clark, M. J., & Poole, W. E. (1967). The reproductive system and embryonic diapause in the female kangaroo, Marcopodus giganteus. Australian Journal of Zoology, 15(3), 441-459.

NOTE:

This publication is included in the print copy

of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

https://doi.org/10.1071/ZO9670441

Sharman, G. B., & Clark, M. J. (1967). Inhibition of ovulation by the corpus luteum in the red kangaroo, Megaleia rufa. Journal of Reproduction and Fertility, 14(1), 129- 138.

NOTE:

This publication is included in the print copy

of the thesis held in the University of Adelaide Library.

13+.

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