Summary. Intravenous administration of PGF-2α or an analogue stimulated uterine contractility in gravid uteri during seasonal quiescence and late pregnancy. The exogenous PGF-2α was metabolized to 13,14-dihydro-15-keto-PGF-2α (PGFM) which was assayed in peripheral plasma by a radioimmunoassay. PGFM was cleared from the circulation with a half life of 8–15 min. Blood samples were taken approximately every 6 h from Day 26 to Day 30 from tammars which were expected to give birth on Day 27– 28. Basal levels of PGFM were <400 pg/ml, and only occasional samples contained higher PGFM concentrations. Therefore, if uterine PGF-2α release is elevated at parturition in tammars, the corresponding rise of circulating PGFM is either small or of short duration.
Summary. Nulliparous female tammar wallabies during the non-breeding season and adult male wallabies were treated with PGF-2α at doses of 0·008, 0·04, 0·2 and 1·0 mg/kg. All the male and female wallabies responded to the three highest doses by showing parturient behaviour. At the lowest dose 4/4 males and 1/4 females responded. The peak concentrations of PGF-2α metabolite (PGFM) in the peripheral plasma after administration of 0·008, 0·04 and 0·2 mg PGF-2α/kg were 0·70 ± 0·08, 3·02 ± 0·37 and 8·48 ± 0·76 ng/ml (mean ± s.e.m.). Since the peak plasma concentrations of PGFM at normal parturition are reported to be 2·5 ± 0·9 ng/ml, parturient behaviour can be induced by physiological concentrations of exogenous PGF-2α. The effectiveness of PGF-2α in males indicates that parturient behaviour is probably a result of a direct action of PGF-2α on the brain, rather than a response to uterine or vaginal contractions. These experiments confirm that PGF-2α is an important behavioural hormone in the tammar wallaby.
Keywords: PGF-2α; parturition; behaviour; tammar wallaby; PGFM
D Mattiske, G Shaw, and JM Shaw
Ovaries from a marsupial, the tammar wallaby (Macropus eugenii), were grafted into a eutherian recipient at known stages of development to ascertain whether normal development would occur. Xenografted ovaries from pouch young < 20 days old, before the onset of meiosis, retained few germ cells and developed tubule-like structures reminiscent of seminiferous cords. Ovaries from 50-day-old pouch young, which contain primordial follicles, developed into antral follicles and corpora lutea within the eutherian host, and produced hormones that stimulated the reproductive tract of the host. The timing of onset of antrum formation and the progress of follicle development were advanced relative to the timing of events in ovaries in situ. Frozen-thawed ovaries from 50-day-old donors developed into preantral follicles, but at a reduced rate and number. This finding shows that gonads of a marsupial species can develop as xenografts in a eutherian, forming large antral follicles. Accelerated follicular development in xenografts provides a potentially valuable model for studying the factors that control follicle development. Assisted reproduction of endangered marsupials may also be feasible using follicles from pouch young grown as xenografts in a eutherian host.
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.
G. Shaw and Marilyn B. Renfree
Summary. Oestradiol-17β concentrations were measured by radioimmunoassay in peripheral blood samples from 10 tammar wallabies after their pouch young were removed to terminate embryonic diapause. Oestradiol concentrations rose from 8·3 ± 1·2 pg/ml on Days 3 and 4 to a peak of 15·8 ± 2·9 pg/ml on Day 5, coincident with an increase in 'progesterone' concentrations, and then fell to 10·5 ± 2·7 pg/ml on Day 7. No changes in oestradiol concentrations were associated with parturition. Five females came into oestrus and mated 9·8 ± 6·1 h post partum; peak concentrations of plasma oestradiol (20·9 ± 2·1 pg/ml) occurred around the time of mating. None of the females that did not mate up to the end of the experiment at Day 30 had a rise in plasma oestradiol concentrations. Corpora lutea contained 20-100 pg oestradiol during pregnancy. The highest ovarian oestradiol content (> 1200 pg) was measured in whole ovaries containing Graafian follicles from full-term pregnant females. The rise in oestradiol concentrations at Day 5 may be important in the termination of diapause. The post-partum increase in plasma oestradiol concentrations coincides with oestrus. The source of this oestrogen appears to be the preovulatory follicle.
D Coveney, G Shaw, and MB Renfree
This study reports the effect of oestrogen treatment on the development of the genital ducts, prostate gland, testicular descent and inguinal canal closure in male tammar wallaby young treated with oestrogen over four time spans during the first 25 days of pouch life (days 0-10, 10-15, 15-25 and 0-25) and sampled at day 50. In control males, the Mullerian ducts had regressed and the Wolffian ducts had developed into the vas deferens and epididymis. The prostate gland had formed epithelial buds extending from the ventral, lateral and posterior walls of the urethra. The testes were in the neck of the scrotum and the gubernaculum and processus vaginalis were present at the base of the scrotum. In most males treated with oestradiol from day 0 to day 25, the testes had failed to descend by day 50. The gubernaculae were long and thin. The retained Mullerian ducts formed a lateral vaginal expansion like that of normal day 50 females. The Wolffian ducts of the males treated on days 0-25 were regressed, but were present in males in the other three treatment groups. The prostate glands were hyperplastic and epithelial budding was highly invasive. Some treated males from the day 10-25 and 0-25 groups had inguinal hernias. These results demonstrate that oestrogen treatment has profound effects on the development of the internal genitalia of a male marsupial, preventing inguinal closure and interfering with testicular descent. Therefore, the tammar wallaby may provide a useful experimental model animal in which to investigate the hormonal control of testicular migration and closure of the inguinal canal.
S-L. Cox, J. Shaw, and G. Jenkin
Sixteen-day-old fetal mouse ovaries were slowly frozen in 1.5 mol dimethylsulfoxide ml−1 and subjected to one of two thawing procedures – fast thaw or slow thaw. Fresh and frozen–thawed fetal ovaries were transplanted orthotopically (to the bursal cavity) to either bilaterally or unilaterally ovariectomized adult female recipients. Fresh fetal ovaries were also transplanted heterotopically (under the kidney capsule) to intact, bilaterally or unilaterally ovariectomized adult females. Transplantation of fetal ovaries to bilaterally ovariectomized adult recipients resulted in restoration of cyclic activity within 20.5 ± 4.7 (mean ± sem) days or 23.4 ± 0.8 days in orthotopic and heterotopic groups, respectively. Developing follicles and corpora lutea were observed within 4 weeks after transplantation of fetal ovaries to heterotopic sites and within 6 weeks after transplantation to orthotopic sites. After orthotopic transplantation, 33% of the recipients became pregnant. Orthotopic or heterotopic transplantation to intact of unilaterally ovariectomized recipients resulted in quiescence of the fetal ovary. After cryopreservation, transplantation of fetal ovaries to bilaterally ovariectomized recipients resulted in restoration of cyclic activity within 19.3 ± 2.1 days and 23.4 ± 5.1 days after transplantation in slow thaw and fast thaw groups, respectively. Fertility was restored to 86% of fast thawed and 25% of slow thawed fetal ovary transplants to bilaterally ovariectomized adult recipients. No ovarian tissue was observed on the side of the fetal graft in unilaterally ovariectomized recipients that received frozen–thawed fetal ovaries. These results demonstrate that cryopreserved fetal ovarian tissue can be transplanted to adult recipients with subsequent restoration of fertility and that this process is dependent on the absence of the ovaries of the recipients.
H. J. Shaw and G. R. Foxcroft
Summary. Blood samples were collected from primiparous sows via indwelling jugular cannulae at 15-min intervals for 12 h before and for 24 h (2 sows) or 48 h (10 sows) after weaning and then every 4 h until behavioural oestrus. Weaning to oestrus intervals ranged from 3 to 10 days and 2 sows showed no signs of oestrus and had not ovulated by Days 11 and 16 after weaning.
Prolactin concentrations in plasma decreased significantly (P < 0·001) and reached basal levels 1–2 h after weaning in all sows whilst plasma progesterone concentrations remained basal until ~30 h after the preovulatory LH surge in sows that ovulated. Elevated concentrations of prolactin or progesterone during the post-weaning period were, therefore, not responsible for delayed restoration of cyclicity.
Overall, mean LH concentrations rose significantly (P < 0·001) from 0·22 ± 0·02 during the 12-h period before weaning to 0·38 ± 0·03ng/ml during the 12-h post-weaning period. After weaning, pulsatile and basal LH secretions were markedly increased for sows that showed an early return to oestrus (≤ 4 days) compared with sows showing a longer weaning to oestrus interval but a correlation did not exist between either of these LH characteristics and the time taken to resume cyclicity. Mean LH concentrations before weaning were, however, inversely related (r = − 0·649; P < 0·05) to the weaning to oestrus interval.
Overall, mean FSH concentrations rose significantly (P < 0·001) from 151·1 ± 6·2 (s.e.m.) ng/ml in the 12-h period immediately before weaning to 187·7 ± 9·7ng/ml in the subsequent 12-h period but there was no correlation between FSH concentrations, before or after weaning, and the interval from weaning to oestrus. However, a significant correlation was apparent between ovulation rate and peak concentrations of the rise in FSH after weaning (r = 0·746; P < 0·05) and overall mean FSH values (r = 0·645; P < 0·05).
It is concluded that both LH and FSH concentrations in peripheral blood rose in response to removal of the suckling stimulus at weaning. The increase in LH pulse frequency associated with weaning was not directly related to the weaning to oestrus interval although a specific pattern of LH secretion was observed in sows showing an early return to oestrus ( ≤ 4 days). It is proposed that the characteristics of LH secretion after weaning may reflect the ovarian status at the time of weaning and that this may, in part, be dependent on steroid feedback. The weaning-associated rise in FSH may be involved in the determination of ovulation rate.
D Coveney, G Shaw, JM Hutson, and MB Renfree
Androgens are essential for testicular descent in eutherian mammals, but little is known about its hormonal control in marsupials. This study reports the effects of daily treatment with the anti-androgen flutamide (10 mg kg(-1)) from day 9 to day 75 after birth on the descent of the testis and inguinal closure in tammar wallabies. By day 75 after birth, the testes of control males had descended and the prostate gland was well developed. The testes of all flutamide-treated males had passed through the inguinal canal and were situated in the base of the scrotum. Three of the nine flutamide-treated males had unilateral inguinal hernias. The size of the inguinal canal, regardless of whether a hernia was present, was significantly wider than that of control males. Development of the prostate gland was significantly inhibited. By day 75 after birth, the phallus was significantly longer in control males than in females, whereas the phallus of flutamide-treated males was similar to that of control females. In flutamide-treated males, the lumbar 1 dorsal root ganglia was feminized and significantly fewer cell bodies expressed calcitonin gene- related peptide. As the anti-androgen treatment resulted in a reduction in the number of calcitonin gene-related peptide-positive cell bodies in the dorsal root ganglion supplying the genitofemoral nerve, the process of inguinal closure in tammar wallabies may be mediated by calcitonin gene-related peptide via the genitofemoral nerve, as indicated in humans. Flutamide treatment inhibited development of the prostate gland and phallus, which are both androgen-dependent structures, but it did not affect the normal descent of the testis, indicating that testicular descent can proceed when the action of androgens is blocked.
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.