Diapause, pregnancy, and parturition in Australian marsupials

transcript

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 266450-462 (1993)

Diapause, Pregnancy, and Parturition in Australian Marsupials

MARILYN B. RENFREE Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia

ABSTRACT Marsupial pregnancy is characterised by a long lactation and a relatively short gestation. Marsupials have, in effect, exchanged the umbilical cord for the teat. However, gestation can be extended for up to 11 months by the imposition of a period of developmental arrest known as embryonic diapause. Diapause may be under either lactational or seasonal control, and in the kangaroos and wallabies these effects are mediated by prolactin and melatonin, respectively. At the other end of gestation, namely parturition, it appears that marsupials are fairly typical mammals and require all the same physiological and behavioural cues essential for the delivery of a viable young. Parturition depends on a synchronised cascade of hormonal events triggered by the fetus itself. Pros- taglandin and prolactin concentrations pulse around the time of birth and progesterone falls. Successful parturition also depends on the adoption of the appropriate behaviour and birth posture by the mother. Despite the fact that the entire period of gestation is accomplished in such a short time, the neonate has perfectly adapted its growth and development to influence its mother's physiology to induce the change from nurturing the young in its uterus via a placenta, to a precise synchronisation of the birth process resulting in completion of its growth within the pouch sustained by a milk tailor-made for each developmental stage. o 1993 Wiley-Liss, Inc.

Marsupial pregnancy is characterised by a long lactation and a relatively short gestation. Marsu- pials have, in effect, exchanged the umbilical cord for the teat (Renfree, '83). However, gestation can be extended for up to 11 months by the imposition of a period of developmental arrest known as embryonic diapause. There are two groups of marsupials that use this strategy. In one, the gestation period is almost the same length as the cycle and extends into the follicular phase so that postpartum estrus and ovulation occur. If fertilization occurs the embryo develops to a unilaminar blastocyst of about 100 cells, and during lactation further development is arrested. Most of the kangaroo and wallaby family Macropodidae, which are monovu- lar, polyestrous species share this pattern (Tyndale- Biscoe and Renfree, '87). The other group are those polyestrous, polyovular, marsupials with a very pro- longed preluteal phase and gestation which includes a long period of embryonic diapause after a postpartum estrus. It is unclear as to whether diapause is controlled by lactation or is obligatory, controlled by as yet unknown seasonal cues. It appears that all members of the small possum families Bur- ramyidae (except Burramys), Acrobatidae, and Tarsipedidae have this pattern. A great deal of information now exists on the control of diapause in the Macropodidae, but almost nothing is known of

the control in the small possum families. Diapause in marsupials has been extensively reviewed (see Flint et al., '81; Tyndale-Biscoe and Renfree, '871, but recent data have shed additional light on the process in macropodids.

At the other end of gestation, namely parturition, it appears that marsupials are fairly typical mammals and require all the same physiological and behavioural cues essential for the delivery of a viable young. Much of the work on parturition is very recent, and almost all on just one species, the tammar wallaby Macropus eugenii. Much more needs to be done, especially on polyovular species, before a truly comparative study of gestation can be made within marsupials, and between marsupials and eutherians. This review will therefore con- centrate on the newer information on the beginning and the end of gestation: the control of embryonic diapause and the control of parturition in Austra- lian marsupials.

EMBRYONIC DIAPAUSE Dormancy is widespread among mammals and

usually occurs when the embryo is 200-400 cells (Fig. 11, with the exception of macropodid marsupials and rodents with 80-100 cells, the badger with 900-2,000 cells, and in tarsipedid, burramyid, and acrobatid marsupials in which blastocysts have up

0 1993 WILEY-LISS, INC.

DIAPAUSE, PREGNANCY, AND BIRTH IN MARSUPXALS 451

Fig. 1. A diapausing blastocyst of the marsupial Mucropus eugenii, the tammar wallaby. The tammar blastocyst has about 80-100 cells and is 0.25 mm in diameter. s , shell; sp, sperm trapped in mucin layer; t, trophoblast.

to 2,000 cells (Renfree, '81; Ward, '88; Ward and Renfree, '88). All species of the two subfamilies of the Macropodidae except M. fuliginosus that have been investigated exhibit the phenomenon of diapause (Tyndale-Biscoe et al., '74; Renfree, '81). Dur- ing diapause the blastocyst shell diameter varies from 0.25 to 0.33 mm and the diameter of the trophoblast from 0.20 to 0.25 mm (Tyndale-Biscoe, '63a; Renfree and Tyndale-Biscoe, '73; Smith, '81; Tyndale-Biscoe and Renfree, '87). The macropodid blastocyst is invariably unilaminar (Figs. 1 and 2) and mitoses are never seen in the 70 to 100 uni- form cells. Blastocysts in diapause are always associated with a quiescent corpus luteum.

In addition to the lactational inhibition of the blastocyst and corpus luteum, in at least two species, the tammar Mucropus eugenii and the Bennett's wallaby M . r. rufogriseus, there is a seasonal control of diapause, and the quiescent blastocyst and corpus luteum persist for several months after weaning, until they resume development after the summer solstice (Berger, '66; Sharman and Berger, '69; Renfree and Tyndale-Biscoe, '73; Tyn-

Fig. 2. Transmission electron micrograph of a single cell of a 100-cell quiescent tammar blastocyst taken from the uterus of a lactating female. The shell membrane(s) has the characteristic 3 layers, with the mucin coat separating it from the zona pellucida(z1. The individual trophoblast cells are flattened along the inner surface of the shell, with elongate nucleii. Con- voluted tight junctions join adjacent trophoblast cells. No sperm fragments are seen in this micrograph. Scale: x 5500: the width of the shell membrane is 0.0085 mm. m, mitochondrion; n, nucleus.

dale-Biscoe et al., '74; Merchant and Calaby, '81; Tyndale-Biscoe and Renfree, '87).

As mentioned above, diapause is not restricted to the macropodids, and occurs in the Tarsipedidae, a monospecific family consisting only of Tursipes rostrutus (the honey possum) (Renfree, '80, '81; Renfree and Calaby, '81; Woller et al., ,811, the Burramyidae (the pygmy possums) Cercurtetus

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concinnus (Clark, '67), C. nanus and C. lepidus (Ward, '88>, and the Acrobatidae, Acrobates pyg- maeus (the feather tailed glider) andDistoeuchurus pennutus (the feathertailed possum) (Ward, '88; Ward and Renfree, '88). In these small possums the diapause blastocyst is very large (1-2 mm) and con- sists of about 2,000 cells in Acrobates and Tarsipes. In contrast to the macropodids, there is a period of slow growth (for about 30 days) of both the blastocyst and corpus luteum early in the diapause, after which growth remains static until reactivation occurs (Renfree, '81; Ward and Renfree, '88). The endocrine control of diapause in these small possums is unknown but it does not appear to be under lactational control (Renfree et al., '84; Ward and Renfree, '88). Nutrition and availability of nectar and pollen bearing flowers may be important controlling factors (Ward, '88).

Initiation and maintenance of diapause The corpus luteum is not required to maintain

diapause because blastocysts can remain apparently viable for at least 4 months after ovariectomy or lutectomy (Tyndale-Biscoe and Hearn, '81). The inhibitory influence of the sucking stimulus on the corpus luteum begins on day 4 or 5 postestrus. In the absence of any sucking stimulus, there is a transient pulse of progesterone on days 6 or 7 postestrus (Hinds and Tyndale-Biscoe, '82a), preceding an increase in the weight of the endometrium and composition of the uterine secretions, and expansion of the blastocyst by about 2 days. The interval from the progesterone pulse to birth is relatively constant at 21-22 days, regardless of whether the female is lactating or not (Tyndale-Biscoe and Renfree, '87). Thus, progesterone appears to be the primary stimulus for reactivation.

Reactivation after diapause The role of the sucking stimulus

It is clear that the influence of the sucking stimulus is all-important in the maintenance of lactational quiescence, at least among macropodids (Tyndale-Biscoe and Renfree, '87). It is the neural stimuli from the teat that carry the inhibitory stimuli, since denervation of the mammary gland in the tammar causes immediate reactivation of the diapause blastocyst, even though lactation continues (Renfree, '79). However, development does not resume immediately on removal of the sucking stimulus, in that no size increases can be measured in the blastocyst before day 8, and no change in the corpus luteum before day 4.

Ovariectomy or excision of the corpus luteum between days 2 and 4 after removal of the sucking young is followed by resumption of blastocyst growth, but subsequent collapse, and by a failure of the endometrium to become secretory (Berger and Shar- man, '69). Ovariectomy or lutectomy before this stage blocks reactivation, and after this stage al- lows fetal development to full term (Tyndale-Biscoe, '70; Young and Renfree, '79; Bryant and Rose, '86). Similarly lutectomy at day 3 after removal of the pouch young (RPY) results in a failure to produce a neonate, but anew ovulation occurs instead 18-23 days after RPY (Fletcher and Renfree, '88). When reactivation is stimulated by exogenous progesterone which by-passes the corpus luteum, the fetus takes 3 days less to reach full term than after RPY suggesting that the corpus luteum takes 3 days to reactivate after sucking stimulus withdrawal (Ren- free and Tyndale-Biscoe, '73; Tyndale-Biscoe, '79). The sucking stimulus must be withdrawn for at least 72 hr to allow the corpus luteum to escape inhibition (Gordon et al., '88). If the young is re- turned to the teat and reattaches after 72 hr, reactivation is not inhibited and pregnancy proceeds concurrent with lactation. Thus the corpus luteum must be present for at least 3 days if reactivation is to be initiated, presumably to mediate the effects of the loss of the sucking stimulus, and the pituitary- hypothalamic axis must be sensitive to the lack of the sucking inhibition on 3 successive days.

The marsupial corpus luteum does not depend on a luteotrophic stimulus for reactivation and subsequent progesterone secretion, but rather needs the withdrawal of an inhibitory effect. In the tammar, the quiescent corpus luteum undergoes its normal growth and secretory functions after total hypophysectomy (Hearn, '74). After hypophysectomy only exogenous prolactin can inhibit reactivation and it is now generally accepted that during diapause prolactin is the inhibitory agent. The administration of prolactin after removal of the pouch young prevents corpus luteum growth (Tyndale- Biscoe and Hawkins, '771, and the corpus luteum contains prolactin (Sernia and Tyndale-Biscoe, '791, but not LH receptors (Stewart and Tyndale- Biscoe, '82).

Removal or replacement of pouch young in early lactation does not cause a marked change in prolactin concentrations (Hinds and Tyndale-Biscoe, '82b; Gordon et al., 'SS), and although a single injection of the dopamine agonist bromocriptine ini- tiates blastocyst reactivation, there is likewise no change in prolactin concentration measured in once daily samples (Tyndale-Biscoe and Hinds, '84). An-

DIAPAUSE, PREGNANCY, AND BIRTH IN MARSUPIALS 453

imals induced to reactivate by experimental ma- nipulation of photoperiod lose their characteristic early morning (dawn) pulse of prolactin (McConnell et al., '861, and there must be a loss of at least three consecutive dawn pulses of prolactin for the blastocyst to be irreversibly committed to reactivation.

Progesterone and reactivation There is evidence to suggest that both uterine

and blastocyst reactivation have already begun before the day 5 or 6 transient progesterone pulse. As noted above, between a critical period after day 2 and before day 6 after RPY in the quokka Setonix bruchyurus (Tyndale-Biscoe, '63b) and in the tammar (Sharman and Berger, '69; Tyndale-Biscoe, '70; Young and Renfree, '79) ovariectomy is followed by resumption of blastocyst growth and subsequent collapse and by failure of the luteal phase to develop in the endometrium. Ovariectomy after day 6 does not prevent the appearance of a luteal uterus and fetal development to full term. Thus, although initiation of reactivation precedes the time when an early pulse of progesterone occurs (Hinds and Tyndale-Biscoe, '82a), the corpus luteum is necessary to influence blastocyst reactivation, either directly or indirectly through increased or specific secretion from the uterus.

No gross changes can be observed in the blastocyst before the significant increase in blastocyst diameter that occurs by day 8 (Renfree and Tyndale- Biscoe, '731, but a few mitoses can be observed in the blastocyst on day 4 (Berger, '70). There is a three- fold increase in diameter by day 10 (Renfree and Tyndale-Biscoe, '73). The volume has increased 45-fold between day 5 and 10, and by day 15 by about 10,000-fold (Tyndale-Biscoe and Renfree, '87). Endometrial protein synthesis increases as early as day 4, with a significant increase in leucine incorporation by the endometrium (Shaw and Renfree, '86) and some prealbumins are present in uterine exudates at day 4 which are not present at day 0 (Renfree, '73a). RNA-polymerase activity and uri- dine incorporation are significantly higher in day 5 blastocysts than at day 4 and earlier stages (Moore, '78; Thornber et al., '81; Shaw and Renfree, '86). Glucose incorporation has also increased by day 5 RPY but is most significant between days 5 and 10 (Pike, '81). Recent evidence has demonstrated that glucose, pyruvate, and lactate use is low from days 0,2,3,4, and 5 but at day 10 is mark- edly increased (R. Spindler, D. Gardner, and M.B. Renfree, unpublished results). These data suggest that a change in uterine secretion by day 4 initi- ates embryonic reactivation by day 5 , which is also

the day of the progesterone pulse and which also coincides with a pulse of estradiol (Shaw and Renfree, '84, '861, so an earlier change must be responsible for triggering reactivation of the blastocyst either directly or indirectly via the uterus.

The blastocyst appears to be indirectly stimulated by the progestational stimulus on the uterine secretions and is under maternal control for at least the first 6 days. Exogenous estradiol and progesterone can both cause increased uterine secretion and an increase in RNA polymerase activity in the nuclei of blastocyst cells within 48 hr of injection (Shaw and Renfree, '86; Moore, '78). Similarly, exogenous steroids can reactivate tammar blastocysts and pregnancy proceeds to term (Berger and Shar- man, '69; Renfree and Tyndale-Biscoe, '73). Although both progesterone and estradiol can initiate development, only progesterone will sustain it (Fletcher et al., '88).

After melatonin injections, the dawn prolactin pulse is abolished by the third day (Tyndale-Biscoe et al., '86) and reactivation occurs as in removal of the sucking inhibition (Gordon et al., '88). If melatonin implants are given, the shortest time to birth is identical to that taken from RPY to birth (Renfree and Short, '84). Since the time from the progesterone pulse to birth is similar (melatonin treatment 21.6 days; photoperiod change 23.5 days; natural photoperiod change after summer solstice 23.0 days), the additional days taken after photoperiod changes before reactivation must reflect the time taken for the message to be acted upon, and relate to how quickly and by how much the melatonin concentrations change (Tyndale-Biscoe et al., '86). Clearly these data provide new and exciting information on how seasonal breeding is controlled in mammals.

The control of diapause in macropodid marsupials is a complex interaction between the sucking stimulus, the hypothalamus, the pituitary, corpus luteum, and uterus, resulting in the case of the tammar, in an 11 month cessation of growth of the 100 cell blastocyst. Photoperiodic signals are superimposed on this system in the second half of the year when days are lengthening. Most of the steps on the hypothetical pathways have now been filled in (Fig. 31, but the precise sequence of events between day 3 and day 6 after RPY needs to be better studied. It also still remains to be demonstrated whether the blastocyst responds directly to pituitary or ovarian hormones, or whether it simply awaits a changed uterine milieu.

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Pineal Melatonin , gland

-_I yypothalamus SDinal

C o r d I (P IF

p i tu i tary I--- r o o t , *:-% Afferent arc

t t e r i n e secret ion

BIas t o c y s t

Fig. 3. Summary of the known and suggested pathways for the control of quiescence of Macropus eugenii. CL, corpus luteum; E,p, estradiol 17p; Prog, progesterone; PIF, prolactin- inhibiting factor; Prl, prolactin; SCG, superior cervical ganglion; SCN, suprachiasmatic nucleus; 1 , stimulation; I, inhibition. Redrawn from Renfree ('81).

PREGNANCY AND THE MARSUPIAL PLACENTA

Embryonic and fetal growth After the unilaminar blastocysts stage there is

a rapid expansion of the blastocyst through absorp- tion of fluid into the blastocoel across the trophoblast. A feature of marsupial embryogenesis is the relatively slow rate of embryonic development, which only increases during the latter stages of pregnancy (Qndale-Biscoe and Renfree, '87). The embryo remains unattached and free in the uterus for at least two-thirds of the active gestation period, with varying degrees of attachment or inva- siveness occurring relatively late in gestation (Renfree, '77; Hughes, '74, '84).

There are few complete, timed studies of embryonic development in marsupials, and the first, of

Fig. 4. Representative stages of development of the Ameri- can opossum, Didelphis uirgzntana, redrawn from McCrady ('38). (a) Primitive streak, Stage 21 on day 7 ofpregnancy. The complete vesicle has a diameter of 2.5 mm. (b) Four somite embryo, Stage 23. The mesodermal layer has extended to cover the top surface of the vesicle which has a diameter of around 4-5 mm, also on day 7 of pregnancy. (c) Hind limb club, Stage 32, at day 11 of pregnancy. (d) Full term fetus, Stage 35, at 12.5 days of pregnancy. Note the claws on the forelimb digits. No representation of the mucin layer or shell membrane is shown in this series. Detailed descriptions of the key developmental events are given in McCrady ('38).

the opossum Didelphis virginiana (Fig. 41, remains the most detailed today (McCrady, ,381, and together with what is known of the tammar (Tyndale-Biscoe and Renfree, '87) and the recent studies of Hughes and colleagues (Hughes, '82; Hughes and Hall, '84, '88; Hughes et al., '89) provide a useful guide to the significant developmental features of marsupial embryos.

After the formation of the primitive streak the embryo acquires a bilateral symmetry, with Hen- sen's node at the anterior end. The first somites are seen in vesicles between 4 and 8 mm diameter. Am- niogenesis occurs relatively late in marsupials, soon after the appearance of somites (Tyndale-Biscoe and Renfree, '87). Cervical flexure occurs when the em-

bryo has about 18-20 somites and the neural tube is closed along most of the length of the embryo.

It is about this time that the shell membrane, which has become more and more attenuated, rup- tures, thus allowing for a close and direct apposi- tion of the trophoblast to the uterine epithelium. Whereas all development up to this stage has been relatively slow and occupied two-thirds of the active gestation period, organogenesis is relatively rapid (Fig. 4). The time it takes is only a two- fold or so difference in very divergent species which may also have very different body sizes. In species of the family Dasyuridae the time taken from primitive streak to birth may be extremely rapid, and is only 64 hr in Sarcophilus (Hughes et al., '89).

Placental structure and function The placental membranes apposed to the uter-

ine wall consist of vascular and nonvascular por- tions of the yolk sac in most marsupials and yet control the transfer of materials from mother to fetus, as in any other functional placenta (Renfree, '73b). Constituents of fluids from the yolk sac, allantois, amnion, and fetal circulation are main- tained at different compositions from those of the maternal blood circulation and uterine secretions, and it is clear that active transport and selective synthesis occurs (Renfree, '73a,b). The placenta also has important morphogenetic effects on the endometrium, and there are significant unilateral bio- chemical and anatomical differences that occur in response to the feto-placental unit (Renfree, '72, %a). In most, only a small area of true chorion remains, and the allantois is enclosed in the fold of the yolk sac membrane, except in the Perameli- dae, where it forms an intimate attachment. In the tammar, the allantois never reaches the chorion, and as gestation proceeds it accumulates urea in the allantoic fluid, presumably via the patent Wolff- ian ducts draining the mesonephros (Renfree, '73b). A similar arrangement of fetal membranes has been described for a variety of macropodids and phalan- gers (Tyndale-Biscoe and Renfree, '87). In some species like Dasyurus and Sarcophilus there is an invasive yolk sac in the vascular region (see Tyndale- Biscoe and Renfree, '87; Hughes et al., '89; R.L. Hughes, personal communication). The bilaminar yolk sac is attached to the uterine endometrium. In the koala a chorioallantois and the bilaminar yolk sac fuse with the endometrium, but are not invasive. In Sminthopsis there is an invasive bilaminar yolk sac. As noted above, some marsupials have an allantoic placenta in addition to a yolk sac placenta, and the most elaborate form of allantoic

placentation is found in the bandicoots-Perameles nasuta, ?? gunnii, and Isoodon obesulus-where a true discoid chorioallantoic placenta is present in addition to the yolk sac placenta (Flynn, '23; Pady- kula and Taylor, '76, '77, '82). In this group, the uterine wall and the chorion thicken, in the region of the chorioallantoic placenta and the vascular yolk sac. Thus, among marsupials there are four types of placentation: (1) those in which the allantois never reaches the chorion, (2) those in which the allantois approaches the chorion but does not fuse with it, and in which a close attachment or invasion of the endometrium occurs, (3) those in which the allantois makes contact with the chorion to form a chorioallantoic placenta that attaches to the uterine epithelium, and finally (4) those in which there is extensive and invasive choriovitel- line and chorioallantoic placental attachment.

Endocrine functions of the placenta The placenta apparently provides a local endo-

crine stimulation to the adjacent uterus (Renfree, '72, '73a). This could explain why pregnancy continues to full term after ablation of the corpus luteum or ovariectomy in several marsupials, and there is now evidence that the placenta is far from inactive, despite the fact that the yolk sac placenta is only 3 cell layers thick in the vascular region, and is bilaminar for over half of its surface. The placentas of the quokka S. brachyurus and the tammar M . eugenii have incipient endocrine activity, and can convert pregnenolone to progesterone (Bradshaw et al., '75; Renfree and Heap, '77; Ren- free, '77). A more detailed study of the yolk sac membrane of M . eugenii showed that it can convert a range of steroid precursors into a variety of prod- ucts, although levels of conversion were generally low (Heap et al., '80). It is also possible that the marsupial placenta produces gonadotrophins, as preliminary results (cited in Tyndale-Biscoe and Renfree, '87) suggest that the tammar placenta has biological activity similar to that induced by au- thentic gonadotrophin. Finally, as we shall see be- low, the fetoplacental unit also influences the mother's physiology at parturition.

PARTURITION Until recently (Tyndale-Biscoe et al., '74; Tyn-

dale-Biscoe, '79), it was not acknowledged that marsupials had any endocrine recognition of pregnancy (reviewed in Tyndale-Biscoe and Renfree, '871, as it was assumed that such a small conceptus could not redirect maternal physiology. It is now clear that marsupials do have a maternal recognition of preg-

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nancy, and that the blastocyst (or trophoblast) does influence the secretory activity of the uterus (Ren- free, '72, '73a), that the fetus influences the duration of the luteal phase (Merchant, '79) and directs the time of parturition (Kirsch and Poole, '72; Poole, '75) by stimulating (directly or indirectly) the release of prolactin and the cascade of events which results in a sharp rise in prostaglandin and regres- sion of the corpus luteum (Tyndale-Biscoe et al., '83, '88; Lewis et al., '86; Fletcher et al., '90; Hinds, et al., '90). Marsupials are therefore now known to have a similar, and complex, control of parturition comparable to eutherians, and the wallaby pattern bears many similarities to that of the sheep.

Parturition requires the corpus luteum and both anterior and posterior pituitary, but as in many eutherian mammals, in the macropodids the fetus and or placenta may also determine the time of birth. In marsupials the corpus luteum has an essential role for normal parturition at least up to the last few days of gestation (Young and Renfree, '79; Bryant and Rose, '86). Parturition occurs via the median vagina, and relaxin of luteal origin may be necessary for loosening the connective tissue of the vagina and urogenital sinus.

The myometrium remains quiescent presumably under the influence of progesterone throughout pregnancy, but can become highly responsive to the effects of prostaglandin even while progesterone is

high (Renfree and Young, '79; Shaw, '83a,b). Par- turition is not invariably preceded by a fall in progesterone (Hinds and Tyndale-Biscoe, '82a), and when progesterone levels are artificially elevated by injections or implants, parturition still occurs at the normal time (Ward and Renfree, '84).

Although corpora lutea of tammars and brushtail possums contain high concentrations of bioassayable relaxin as well as progesterone (Tyndale-Biscoe, '66; '811, no convincing effect of relaxin on softening the cervix and vaginal canal has been demonstrated in either species (Tyndale-Biscoe, '66; Renfree and Young, '79). Progesterone treatment can also cause substantial loosening of the connective tissue of the urogenital strand in ovariectomized opossums (Risman, '471, in the brushtail possum (Sharman, '65a; mdale-Biscoe, '66) and in the tammar (Young, '78). It appears that both these hormones may be necessary for preparation of the birth canal.

Endocrine control of parturition For normal parturition to occur, the corpus luteum

needs to be present at least to day 23 (Young and Renfree, '79). Parturition goes to term but fails in five species of marsupials so far studied without corpora lutea (after ovariectomy or lutectomy), as it does in tammars reactivated by exogenous progesterone for 10 days which have corpora lutea that remain inactive (Renfree and Tyndale-Biscoe, '73).

Hormones throughout parturition in the tammar wallaby.

I I T T

- 4 8 - 2 4 0 2 4 4 8

HOURS FROM APPEARANCE O F NEONATE IN POUCH

Fig. 5. Hormonal changes at parturition in the tammar wallaby, Macropus eugenii. Data derived from Hinds and Tyndale-Biscoe ('82a,b), Tyndale-Biscoe et al. ('831, Shaw and Renfree ('84), Harder et al. ('841, and Lewis et al. ('86). Redrawn from Renfree et al. ('89).

Progesterone is high prior to birth in all species so far studied, and is the major steroid hormone of the tammar (Renfree et al., '79; Hinds and Tyndale- Biscoe, '82a; Shaw and Renfree, '84; Lewis et al., '86), and Bennett's wallaby (Walker and Gemmell, '83). Estrogens are also elevated at parturition before the postpartum estrus (Flint and Renfree, '82; Shaw and Renfree, '84; Harder et al., ,841, but es- trogen is clearly not essential in macropods for parturition, because estrus does not invariably follow parturition. The Graafian follicle is the main source of estradiol in the peripheral circulation in the tammar (Harder et al., '84, '85) and presumably in the other species as well. Gonadotrophins are not essential for parturition because animals im- munized against GnRH give birth, although fol- liculogenesis is inhibited and there are no Graafian follicles (Short et al., '85).

The sequence of hormonal changes at parturition of the tammar (Fig. 5 ) is now clearly defined from recent detailed studies (Flint and Renfree, '82; Shaw, '83a; Tyndale-Biscoe et al., '83; Shaw and Renfree, '84; Ward and Renfree, '84; Harder et al., '84, '85; Lewis et al., '86). Progesterone declines pre- cipitously coincident with or occasionally within 6 hr of birth. A sharp rise in estradiol occurs 8 hr after the progesterone drop, and estrus 10 hr later. The LH surge is dependent on the estradiol rise and follows it by 7 hr. Ovulation follows the LH surge by 24 hr. Estradiol levels are basal 24 hr after the progesterone fall.

In tammars sampled very frequently there is a marked but very short lived peak of 800-1200 pg/ml of prostaglandin metabolite if collected within 10 min of birth, with values declining to less than 200 pg/ml within 45 min, and being nondetectable by 2 hr postpartum (Lewis et al., '86). In bandicoots, prostaglandin is elevated for longer periods (Gemmell et al., 'SO). In the red-necked wallaby, Walker and Gemmell('83) could not detect a marked rise, but if the pattern of prostaglandin release is like that of the tammar, a more frequent blood sampling re- gime would be required to detect it. In pregnant females there is a peak of prolactin released in response to prostaglandin at parturition which is not observed in nonpregnant females (Qndale-Biscoe et al., '83; Hinds et al., '90; Shaw, '90).

Myornetrial activity in vivo and in vitro can also be stimulated by oxytocin, and the gravid uterus responds strongly (Shaw, '83b; Tyndale-Biscoe and Renfree, '87). Injections of oxytocin or prostaglandin cause massive contractions and expulsion of the fetus with considerable damage to it. In preliminary experiments with an oxytocin inhibitor we have

Fig. 6. Birth of a neonatal tammar. (a) The yolk sac fluid (ysf) precedes the emergence of the neonate from the urogenital opening. (b) Moments after birth the neonate is in the process of freeing itself from the fetal membranes, the yolk sac and amnion (am). The neonate weighs 400 mg at birth and has a crown-rump length of 16-17 mm. Photograph by Dr. D.D. Parer.

delayed parturition (M.B. Renfree, G. Shaw, and L. Parry, unpublished results), so it appears that oxytocin is important in the initiation of parturition.

Fetal role in parturition In all mammals maturation of the fetus is an im-

portant prerequisite for successful parturition, and in many eutherian species it is known that the fetus signals its readiness by influencing the maternal system. However, because of the similarity between the length of gestation and the estrous cycle in marsupials, it was assumed that there was no fetal role in the timing and onset of parturition (Sharman, '70). Since that time, a variety of studies indicate that macropodid marsupials do not dif- fer from eutherians in this regard, and even the similarity of gestation length to estrous cycle length

458 M.B. RENFREE

has been brought into question by the finding of consistent differences which also depend on the presence of the fetus (Merchant, '79; Merchant and Calaby, '81). However, there is no evidence at present of any fetal influence on pregnancy in the opossum (Harder and Fleming, '81; Fleming and Harder, '81a,b), or in any other nonmacropodid so far studied (Stewart and Qndale-Biscoe, '83). Hybrid kangaroo fetuses are born after gestation periods intermediate between that of the parental geno- types (Kirsch and Poole, '72; Poole, '751, and thus the timing of birth must be determined by the fetus. In the tammar, the interval from estrus to postpartum estrus is shorter than the interval from estrus to the next estrus in nonpregnant tammars, an effect ascribed to the presence of the fetus (Mer- chant, '79). This difference in timing and in hormonal profiles was confirmed by Tyndale-Biscoe et al. ('83). In particular, there is a pulse in prolactin coincident with a sharp fall in progesterone around the time of parturition (Tyndale-Biscoe et al., '83; Hinds and Tyndale-Biscoe, '82b, '85). The prepar- tum pulse in maternal prolactin is associated with the presence of the fetus and not the stage of the ovarian cycle, and it is also responsible for inducing luteolysis (Qndale-Biscoe et al., '88). Although there is a sharp but short-lived rise in prostaglandin coincident with parturition in this species which was thought to be luteolytic (Lewis et al., '86), it is known that prostaglandin causes the release of prolactin, which in turn depresses the progesterone concentration (Hinds et al., '90).

The physiology of parturition is a complex process in all mammals, but one which also involves appropriate maternal behaviour. Successful birth ultimately depends on a precise synchronization with the appropriate maternal and fetal behaviour. Maternal behaviour at birth is well defined in eutherians, but aside from Sharman's early studies (Sharman and Calaby, '64) little had been recorded for marsupials. However, the adoption of a characteristic and highly stereotyped behaviour must be essential for the birth of the tiny macropodid neonate and its successful journey to the pouch (Figs. 6, 7, and 8). In macropodid marsupials, about 24 hr before birth, females show an increased tendency to lick and clean the pouch and the urogenital opening (Sharman and Calaby, '64; Renfree et al., '89). Around 1-5 min before birth the female adopts the characteristic "birth posture," with the tail passed forward between the legs, sitting on her lower back with hips rotated upward, with the upper body hunched forward. The intensity of licking declines gradually from about 10 min after birth.

Fig. 7. A neonatal tammar climbing unassisted towards the pouch. Remnants of the fetal membranes and umbilical cord (u) are still within the urogenital sinus. Photograph by Dr. D.D. Parer.

Nonpregnant female wallabies injected with PGF-2a on days 23 and 26 of the cycle show this "birth posture" behaviour within minutes (Hinds et al., '90). Shaw ('90) has now clearly demonstrated that PGF-2a injection induces parturient behaviour in physiological doses, not only in adult females, but in nonbreeding nulliparous females and in males. This behaviour has now been confirmed in four other macropodid and one peramelid marsupial (R. Rose, personal communication). Shaw ('90) concludes that the action of the prostaglandin is a direct one on the brain.

In tammars PGF-2a injection also induces an immediate rise in plasma prolactin. However, this rise in prolactin cannot be responsible for the birth behaviour, since injection of prolactin alone has no obvious behavioural sequelae (Hinds et al., '90). These data together provide the first evidence that PGF-2a has an important function in inducing the adoption of the birth position and thus is instru-

limbs (Figs. 7 and 8) (Hughes et al., '89; Renfree et al., '89). Its lungs are functional, the nostrils open, and the olfactory centre of its brain is well developed (Gemmell and Rose, '89a,b). The mouth, tongue, and digestive system, including liver and pancreas, are sufficiently developed to cope with the change to a milk diet (Janssens and Ternouth, '87). By contrast, features such as the eyes, the hind limbs, and the gonads remain undifferentiated (Hughes and Hall, '88; 0 et al., '88); the scrotum is visible in embryos from day 22 RPY, but the pouch can be clearly seen only after about 8 days after birth (Renfree and Short, '88); the metanephros is differentiated but not functional immediately (Wilkes and Janssens, '88). Despite the fact that the entire period of differentiation is accomplished in such a short time, the neonate has perfectly adapted its growth and development to signal its mother's physiology to change from nurturing the young in its uterus to providing milk to sustain it.

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Fig. 8. The neonate attached to one of the four available teats (t) within the pouch (p). Mammary hairs cover the head of the pouch young. Photograph by Dr. D.D. Parer.

mental in controlling birth behaviour. The question which now must be raised is how the fetus might induce the prostaglandin release.

Production of steroids by the fetal adrenal is a common step in the early stages of initiation of parturition in many eutherian species. In the tammar, presumptive adrenal tissue is evident by day 21, and the cortex is differentiated by day 22 RPY (Renfree, '72; Call et al., 'SO), and corticosteroids can be identified in the plasma of day 24 fetus (Catling and Vinson, '76). Fetal cortisol might play an important role in the timing of parturition. By administration of the synthetic corticosteroid dex- amethasone we induced early parturition within 24 hr of injection (Shaw et al., ,921, providing strong evidence that hormone of the fetal adrenal may be important for the timing of parturition, and that cortisol may be the fetal trigger to parturition.

CONCLUSIONS The marsupial neonate is the product of a very

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