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G. Shaw and M. B. Renfree

Summary. Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2–3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9.

The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration.

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T. P. Fletcher and M. B. Renfree

Summary. The quiescent corpus luteum of female tammars was reactivated by removal of the pouch young (RPY). The reactivated corpus luteum was ablated 3 days after RPY. Plasma progesterone and oestradiol concentrations were measured by radioimmunoassay in these and in sham-operated controls. Excision of the CL abolished the rise in progesterone seen at Day 5–6 in the sham-operated animals (130·7 ± 56·6 vs 452·4 ± 176·0 pg/ml, mean ± s.d.). By contrast, oestradiol-17β values increased within 6–16 h of CL excision to 16·3 ± 6·9 pg/ml and remained high for 1–3 days while in the sham-operated animals there were less sustained and more variable peaks of 10–20 pg/ml between Days 3 and 5 (mean 12·0 ± 3·6 pg/ml at Day 4–5). We conclude that the early transient increase in peripheral plasma of progesterone is of luteal origin but the source of the oestradiol remains unknown.

Keywords: corpus luteum; progesterone; oestradiol-17β; early pregnancy; marsupial

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S. C. Nurse and M. B. Renfree

In the female tammar wallaby, Macropus eugenii, which has a highly seasonal breeding pattern, teat eversion and enlargement of the pouch occur at puberty, about 40 weeks after birth. The most obvious sign of puberty is teat eversion: 22 of 23 wild caught, and 23 of 24 captive postpubertal animals had fully everted or everting teats. Full eversion of the teats took on average two to three weeks after puberty. The pouch opening enlarged at puberty, and the rate of enlargement from 2 weeks before puberty to 2 weeks after puberty was significantly greater than the rate before puberty. In a group of pouch young ovariectomized at 5–10 weeks of age, no such changes in either teats or pouch were observed by 46 weeks of age. However, after treatment with oestradiol (0.5 μg kg−1 body mass), four of five young showed teat eversion within 3–4 weeks. Progesterone (2 mg kg−1) had no effect on inverted teats. In these ovariectomized females oestradiol treatment caused a significant increase in the rate of growth of the pouch opening. During progesterone injections the size of the pouch remained the same. Thus, at puberty the teats and pouch of the tammar wallaby undergo rapid developmental changes and growth. Ovariectomy at an early stage of gonadal differentiation disrupts these normal changes, but treatment of these animals with physiological doses of oestradiol at the age when puberty would normally have occurred can restore teat and pouch maturation. Teat eversion and pouch enlargement can therefore be used as markers for puberty. Both of these events appear to be under the control of ovarian oestradiol secretion.

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R. V. Short, A. P. F. Flint and M. B. Renfree

Summary. Pouch young were removed from 10 wallabies in lactational diapause, and half the animals were injected intravenously with 8 ml normal wallaby serum or 8 ml GnRH antiserum every 3rd day from the day of removal of pouch young until Day 30. Laparotomies were performed on Day 24 to monitor luteal size, follicular development and uterine enlargement. The pouches were examined daily for the presence of newborn young from Days 26 to 30, and all the animals were killed on Day 30.

The corpora lutea had hypertrophied in all the animals of both groups by Day 24, but none of the antiserum-treated animals showed any signs of follicular development (follicles < 1 mm diam.), whereas all the control animals had large follicles (mean 3·5 mm diam.). In each group 4/5 animals were visibly pregnant at laparotomy, and parturition occurred normally in 3 of the experimental animals and 1 of the controls.

At autopsy, none of the antiserum-treated animals showed any evidence of follicular development or post-partum ovulation, whereas 3 of the controls had new corpora lutea and the other 2 had large preovulatory follicles. These differences were reflected in the weights of the lateral vaginae; the treated animals showed no evidence of oestrogenic stimulation (4·9 ± 0·2 g), whereas the controls showed the hypertrophy characteristic of oestrus (9·5 ± 1·2 g). Lactogenesis, as measured by an increase in mammary gland lactose concentrations, was established in all animals, regardless of treatment.

These results indicate that passive immunization with a GnRH antiserum reduced pituitary FSH and LH secretion, thereby inhibiting follicular development during pregnancy and preventing post-partum oestrus and ovulation. Since parturition occurred normally in 3 of the 4 pregnant treated animals, follicular development in late gestation cannot be important in the initiation of labour, nor is it required for normal lactogenesis. Passive immunization failed to prevent hypertrophy of the corpus luteum after removal of the pouch young, confirming that neither FSH nor LH are likely to be involved in this process.

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P. Williamson, T. P. Fletcher and M. B. Renfree

Summary. Testicular growth and maturation of the hypothalamic–pituitary–testicular axis were assessed in male tammars from 12 to 25 months of age to establish the time of sexual maturity. The testicular dimensions and body weights of 20 male tammars, ∼12 months of age at the beginning of the study, were measured monthly for 1 year. Groups of 3 animals were castrated at 13, 19 and 25 months of age and their testes sectioned for histological examination. Testicular volume increased between 12 and 24 months of age and was highly correlated with body weight (r = 0·91). In the 13-month group the seminiferous tubules were closed with few mitotic figures. Spermatogenesis had begun in 2 of the 19-month animals. All stages of spermatogenesis were present in the other 19-month male, and in all of the 25-month males.

Basal FSH concentrations increased with the age of the animal (21·0 ± 32·48, 94·40 ± 55·18 and 193·05 ± 40·21 ng/ml (mean ± s.d.) at 19, 20 and 25 months respectively) while basal LH concentrations were similar at 20 months and 25 months (0·43 ± 0·18 and 0·58 ± 0·25 ng/ml respectively). Basal testosterone concentrations were also similar 0·11 ± 0·04, 0·35 ± 0·16 and 0·22 ± 0·10 ng/ml in 13-, 19- and 25-month-old animals.

LHRH injection in tammars at 13, 19 and 25 months of age induced release of both LH and testosterone 10#x2013;30 min after injection. The hormone concentrations increased in both magnitude and duration with increasing age. At 13, 19 and 25 months, peak testosterone values of 1·13 ± 0·27, 1·90 ± 0·45 and 6·58 ± 0·91 ng/ml (mean ± s.d.), respectively, were measured by 90–120 min after injection and peak LH values at 13, 19, 20 and 25 months of age were 2·46 ± 0·82, 8·31 ± 2·02, 9·0 ± 2·18 and 7·86 ± 1·41 ng/ml, respectively, when measured 30 min after injection. By contrast, LHRH injection did not increase FSH concentration at 20 or 25 months of age above the basal concentration.

Taken together, the morphological and endocrinological data show that puberty in male tammars begins around 19 months and is complete by 25 months of age.

Keywords: testis, testosterone; LH; FSH; LHRH; spermatogenesis; marsupial; puberty

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P. A. Towers, G. Shaw and M. B. Renfree

Summary. The urogenital vasculature of the tammar comprises 4 major paired arteries and veins: the ovarian, the cranial urogenital, the caudal urogenital and the internal pudendal artery and vein. The ovarian artery and vein and their uterine branches which supply the ovary, oviduct and uterus, ramify extensively. Each anterior urogenital artery and vein supplies the caudal regions of the ipsilateral uterus, lateral and median vagina and cranial parts of the urogenital sinus. The caudal urogenital arteries and veins supply the urogenital sinus and caudal regions of the bladder. The internal pudendal artery and vein vascularize the cloacal region, with some anastomoses with branches of the external pudendal vessels. Anastomoses connect the uterine branch of the ovarian artery with the uterine branch of the cranial urogenital and cranial branches of the caudal urogenital arteries, and connect the caudal urogenital and the internal pudendal arteries. Anastomotic connections between the left and right arterial supply also occur across the midline of the cervical regions of the uteri and the anterior lateral vaginae. Similar connections are seen in the venous system.

The uterine branch of the ovarian artery ramifies extensively very close to the ovary, giving a plexiform arrangement with the ovarian veins, and also with the uterine venous system on the lateral side of each uterus. This plexiform structure provides an anatomical arrangement which could allow a local transfer of ovarian hormones from ovarian vein into the uterine arterial supply, and thence to the ipsilateral uterus. Progesterone concentrations in plasma from the mesometrial side of the uterine branch of the ovarian vein are markedly higher than in tail vein plasma, especially during the 'Day 5 peak' early in pregnancy, and also at full term. There is also a marked decrease in progesterone concentration from all sites immediately before birth as previously reported for peripheral plasma. These results support the suggestion of a countercurrent transfer mechanism, at least for progesterone, and possibly other hormones, between the ovarian vein and uterine artery. Such a local transfer could explain the different morphological responses of the endometria of the two adjacent uteri during pregnancy in macropodid marsupial species.

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M. B. Renfree, L. J. Parry and G. Shaw

The oxytocin receptor antagonist [1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn]-oxytocin (Atosiban) is a specific antagonist of both mesotocin- and oxytocin-induced myometrial contractions in late pregnant tammars in vitro. Continuous intravenous infusion of Atosiban (1 mg kg−1 day−1) for 3 or 7 days from day 24 of the 26.5 day gestation significantly delayed births. In both the 3 day and 7 day infusion groups, all 15 control animals were pregnant and gave birth within the normal time (day 26.75 ± 0.20, mean ± sem), during the infusion of saline. The neonates weighed 387 ± 8 mg. Deliveries were observed in 15 Atosiban-treated animals significantly (P < 0.05) later than in the controls (day 27.85 ± 0.19; neonate weight 413 ± 9 mg). All pouch young were successfully suckled, even in the continued presence of Atosiban. Baseline plasma concentrations of the prostaglandin F metabolite (PGFM) in pregnant tammars were < 200 pg ml−1. A surge in plasma PGFM occurred at birth (811 ± 116 pg ml−1), followed by a rapid fall to baseline concentrations within 1 h after birth. This was observed both in saline- and in Atosiban-treated animals that gave birth during the observation period, and did not differ significantly between the treatment groups. Plasma progesterone concentrations in the control and the Atosiban-treated animals showed the normal pattern of luteolysis immediately after birth. Thus, infusion of an oxytocin receptor antagonist at the end of gestation delays birth, the peripartum surge in prostaglandin release, and the fall in progesterone, suggesting that mesotocin is an important part of the hormonal cascade associated with delivery in this marsupial.

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K. Gordon, T. P. Fletcher and M. B. Renfree

Summary. Pouch young of wallabies presumed to be carrying diapausing blastocysts were removed from the teat for times varying between 24 and 96 h and then returned to the same teat. The mothers were monitored for termination of diapause and checked for births or oestrus. In this way we were able to determine the critical time required to reactivate the quiescent corpus luteum and diapausing blastocyst after withdrawal of the sucking stimulus. When pouch young were removed from the teat for 76–96 h the corpus luteum and blastocyst were reactivated, with birth and/or oestrus occurring in 10/11 animals. When pouch young were removed for 72 h or less (n = 22) reactivation did not take place.

We conclude that it takes longer than 72 h for the maternal endocrine system to become committed to reactivation. The precise sequence of endocrine events which precede blastocyst reactivation still remains to be determined.

Keywords: corpus luteum; sucking; tammar; diapause; blastocyst

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T. P. Fletcher, A. E. Jetton and M. B. Renfree

Summary. Forty tammar wallabies, presumed to be carrying quiescent blastocysts, were injected with progesterone and oestradiol alone, or in combination, during seasonal quiescence when the corpus luteum is inactive. Plasma progesterone concentrations were increased to values equivalent to those of late pregnancy for the duration of the treatment in progesterone-treated groups but otherwise remained at values equivalent to seasonal quiescence. Tammars treated with low doses of oestradiol showed no measurable increase in plasma oestradiol concentrations but in those treated with high doses plasma concentrations were increased to oestrous levels. At autopsy on Day 18 after the start of treatment the embryos and reproductive tracts were assessed. While progesterone alone caused reactivation of about 50% of the embryos, blastocysts in tammars treated with oestradiol alone remained in diapause (low dose) or disappeared from the uterus (high dose): 2 blastocysts collapsed after some slight expansion. No synergistic effect on pregnancy was noted in tammars receiving both oestradiol and progesterone. We conclude that oestrogen alone is not capable of stimulating normal growth of blastocysts, and its role during early pregnancy in tammars remains unclear.

Keywords: progesterone; oestradiol-17β blastocyst; seasonal diapause; marsupial

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M. B. Renfree, G. Shaw and T. P. Fletcher

Female tammar wallabies were treated prepartum with the prostaglandin synthase inhibitor indomethacin, with or without the dopamine agonist bromocriptine, to suppress the peripartum pulses of plasma prostaglandin and prolactin. The animals were observed continuously to detect birth, and a series of blood samples taken to define the hormonal profiles before and immediately after parturition. Birth was observed in ten of twelve control animals but not in the six animals treated with indomethacin alone or the six animals treated with indomethacin and bromocriptine. Indomethacin disrupted the normal profile of PGF metabolite 13,14-dihydro-15-keto-prostaglandin F (PGFM) concentrations, and in the females treated with bromocriptine plus indomethacin the pulse of prolactin normally seen at parturition was completely abolished. Plasma progesterone concentrations fell slowly in treated animals, whereas in control animals they fell steeply immediately after parturition. Postpartum oestrus was delayed or absent in treated and most control animals, suggesting that the frequent blood sampling and disturbances in the peripartum period interfered with these endocrine processes. We conclude that prostaglandin is essential for normal birth. Prolactin, in the apparent absence of a prostaglandin peak, does not induce birth or rapid luteolysis. Prostaglandin release may synchronize the rapid fall in progesterone concentrations associated with birth, but in the absence of this signal, the corpus luteum undergoes a less rapid, autonomous decline.