Daily prolactin pulse inhibits the corpus luteum during lactational quiescence in the marsupial, Macropus eugenii.

The corpus luteum (CL) of the tammar wallaby is inhibited by prolactin during lactation and seasonal quiescence. In seasonal quiescence a daily transient pulse of prolactin (PRL) of less than 2h duration is sufficient to maintain inhibition. We investigated whether the same inhibition applies in lactation and, if so, how. Our results show that inhibition of the CL during lactation is maintained by a transient pulse of prolactin once a day. They also show that the minimum time without a PRL pulse for the CL to escape inhibition is more than 48 h and less than 72 h. Nevertheless, some animals had a longer refractory period than 72 h, which was reflected in a longer interval to the progesterone peak and birth. These results support the previous conclusion that PRL exercises its effect on a rate-limiting step in progesterone synthesis and secretion rate from the CL, which precedes any increase in its mass. Therefore, we conclude that the role of PRL is to act as a luteostatic agent, an effect that is in marked contrast to its luteotrophic effect in many eutherian species, including rodents.

Australasian marsupials--to cherish and to hold.

Considerable interchange of mammals between South America and Australasia occurred during the first half of the Tertiary, including the presence of placental mammals in Australia. This challenges the old assumption that the marsupial radiation in Australia was made possible by the absence of placental competition, and suggests that two properties of marsupial organization may have favoured their survival in the increasingly arid climates that developed after the separation of Australasia from Antarctica. The basal metabolic rates of marsupials are about 70% of equivalent placentals, so their maintenance requirements for energy, nitrogen and water are lower, whereas their field metabolic rates are about the same, which means that they have a greater metabolic scope to call on when active. This may have given marsupials an advantage in semi-arid environments. The lengthy and complex lactation of marsupials enables the female to exploit limited resources over an extended period without compromising the survival of the young. Both these properties of marsupials enabled them to survive the double constraints of low fertility soils and the uncertain climate of Australia throughout the Tertiary. The arrival of people was followed first by the extinction of the large marsupials and, much later, by the wholesale decline or extinction of the small-to-medium sized species. The common factor in both extinctions may have been the constraints of marsupial reproduction.

Australian and New Zealand mammal species considered to be pests or problems.

In New Zealand and Australia, 25 and 16 introduced mammals are viewed as pests, respectively, as well as a further 17 native mammals in Australia. Most introductions were deliberate and the deleterious effects became apparent later. These pests affect primary production, act as a sylvatic reservoir of disease, cause degradation of natural ecosystems, or threaten rare or endangered native animals and plants. Many species have multiple impacts. In Australia, some native mammals, particularly kangaroos and wallabies, are also controlled because of their adverse impacts on primary production. In both countries, current control depends largely on the use of poisons, shooting, the spread of disease (in the case of rabbits), trapping, habitat alteration, and commercial or recreational hunting. Methods of control by interfering with fertility (immunocontraception) are currently being investigated for rabbits, house mice, foxes, and kangaroos in Australia, and for the brushtail possum in New Zealand. If these methods prove effective, they may be applied to other mammal pests, but the need to tailor the particular approach to the ecology and behaviour of the species means that there will be a necessarily long lead time.

Levels of active and inactive reninlike enzymes in plasma and reproductive tract of the pregnant wallaby.

Active and total (acid-activated) levels of a reninlike enzyme (hereafter called renin) were measured in plasma, tissues, and yolk sac fluid of pregnant and postpartum wallabies. Plasma active renin generated angiotensin I (ANG I) from sheep angiotensinogen at 14 +/- 1.3 (SE) ng.ml-1.h-1, whereas acid-activated renin generated ANG I at 33.3 +/- 2.5 ng.ml-1.h-1, i.e., 44.2 +/- 3.7% of renin in plasma was active, and 58 +/- 3.7% was inactive. Inactive renin levels were highest in pregnant animals (P = 0.05). Uterine renin was mainly inactive (95%); levels were 5.1 +/- 1.1 times plasma levels. The levels of renin in nonpregnant uteri were the same as those in pregnant uteri from the same animals. Uterine renin levels did not change with gestation. Pooled acid-activated yolk sac fluid generated ANG I at low rates (0.7 and 1.6 ng.ml-1.h-1); the acid-activated supernatant of a homogenate of pooled fetal membranes generated ANG I at 15 ng.g wet wt-1.h-1. Yolk sac fluid was strikingly different in electrolyte composition from maternal plasma. Its lower osmolality suggests that the membranes separating it from maternal plasma have a low permeability to water. Thus, although eutherian and marsupial mammals diverged 136-164 million years ago, the wallaby, like many eutherian mammals, has inactive renin in blood, in the female reproductive tract, and in fetal membranes.

Virus-vectored immunocontraception of feral mammals.

The potential value of immunosterilization as a means to control species of wildlife that are widespread, numerous and undesirable is assessed. Key questions about the efficacy of fertility control and the means for delivering antigens expressed in recombinant viral vectors are discussed and the legal and social concerns that relate to its possible future use are raised.

The effects of bromocriptine on lactation and subsequent reproduction in grey kangaroos, Macropus fuliginosus and Macropus giganteus.

Both species of grey kangaroo were tested to determine whether a single exposure to bromocriptine in mid winter will terminate lactation, and whether females that cease to lactate will return to oestrus immediately and give birth early, or at the next summer breeding season. In Experiment 1, 11 lactating western grey kangaroos (Macropus fuliginosus) received injections either of saline (n = 2) or of bromocriptine at dose rates of 0.2, 1.0 or 5.0 mg kg-1 bodyweight (n = 3 per dose). Pouch young of females treated with the highest dose of bromocriptine showed either retarded growth or no growth, but they subsequently survived. In Experiment 2, lactating western (n = 12) and eastern grey kangaroos (M. giganteus) (n = 14) received a single intramuscular injection of saline or approximately 2 mg kg-1 or 4 mg kg-1 Parlodel LA (a long-acting form of bromocriptine) and their young were weighed. Within 3 weeks, the young of all 19 females treated with Parlodel LA had lost weight and 17 had died, whereas only one young, which had not lost weight, was lost from the seven control females. The western grey females did not give birth until the normal summer breeding season but the eastern grey females gave birth 2-4 months early. It is concluded that bromocriptine has potential for the humane control of grey kangaroo populations.

Production and secretion of progesterone in vitro and presence of platelet activating factor (PAF) in early pregnancy of the marsupial, Macropus eugenii.

The corpus luteum (CL) of the tammar is suppressed by lactation. Removal of the sucking pouch young induces reactivation of the CL which produces a peak in plasma progesterone concentration on Day 5, 6 or 7; reactivation of the embryo after diapause precedes by one day this transient peak of progesterone. This study examines progesterone production and secretion in vitro by the CL and the production of platelet activating factor (PAF) by the endometrium during this stage of pregnancy (Days 0 and 3-8). Blood samples were collected twice daily to determine for each animal (n = 28) at autopsy the relationship of PAF production to the day of the progesterone peak. Significant changes in basal plasma progesterone concentrations were used to define four groups according to the time the animals were killed: Group A, Day 0; Group B, other animals showing no significant change; Group C, peripheral progesterone concentration increasing; Group D, peripheral progesterone concentration decreasing after the peak. At autopsy on successive days, blood samples were taken from the heart, caudal vein and uterine branch of the ovarian vein, and CL and endometria were prepared for explant culture. Progesterone contents of pre- and post-incubation luteal tissues and of medium were determined at 4, 12 and 24 h to estimate production and secretion rates. The values for all progesterone parameters from animals in Group C were significantly higher than in the other groups. It is concluded that the progesterone peak results from a change in rate of production and secretion of progesterone by the CL, one day before the peak in peripheral plasma, and that this change could provide the first signal to the uterus. Endometrium was incubated for 24 h and the medium assayed for PAF. High concentrations of PAF were detected in half the animals in Groups B and C, that is, before or at the time progesterone was increasing. This is the first measurement of PAF in any marsupial and the first demonstration of the release of PAF by the endometrium. The detection of PAF provides a candidate for an endometrial signal that is responsive to the rising progesterone concentration in the utero-ovarian vein draining the CL-bearing ovary and that may stimulate reactivation of the blastocyst.

Components of the melatonin message in the response to photoperiod of the tammar wallaby (Macropus eugenii).

Female tammars experiencing long-day photoperiod (LD 15:9) are in a reproductive state termed seasonal quiescence. After a change to LD 12:12, a sequence of endocrine events, dependent on a melatonin message being interpreted during the next 3 days, leads to the disappearance of a morning pulse of prolactin on day 4, and to reactivation of the quiescent corpus luteum by day 8. Elements of the message were investigated in three experiments. In experiment 1, tammars on LD 15:9 were injected with melatonin 2.5 hr before lights out on 0-5 successive days. All those receiving four or five daily injections, and six of ten receiving two or three injections, reactivated. In experiment 2, tammars on LD 15:9 were injected with melatonin or oil on 5 consecutive days to achieve a lengthening of the period of elevated melatonin from 9.0 hr to 9.5 hr, 10 hr, 10.5 hr, and 11.5 hr respectively. One of five tammars experiencing 10 hr and seven of ten experiencing the longer periods reactivated. In experiment 3, tammars on LD18:6 received melatonin 6 hr, 6 + 3 hr or 3 hr before lights out for ten successive days. Between injections the melatonin concentrations returned to basal levels. The treatments thus provided a skeletal increase in the profile of melatonin from 6 to 9 hr or 6 to 12 hr. All those treated at 6 + 3 hr reactivated and three of five treated at 6 hr only reactivated. We conclude that the tammar stores information about the change in the maximum duration of elevated melatonin experienced each night and, after three successive periods in which the duration is greater than 10.5 hr, it reactivates. Since reactivation cannot occur until completion of the third dark phase, the processing center must be active during the fourth day, prior to the abolition of the prolactin peak.

Effects of the ovary, sucking stimulus and season on the pattern of LH and FSH release in the female tammar, Macropus eugenii.

The sequential effects of removal of the corpus luteum, removal of the non-luteal ovary and sucking stimulus and the effects of season of the year on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) have been determined in the tammar wallaby, Macropus eugenii. Plasma concentrations of LH and FSH were measured at 15 min intervals for 6 h in eight animals at four successive times in the breeding season (lactational quiescence); (A) while they were intact and lactating, (B) 10 days after removal of the ovary bearing the quiescent corpus luteum, (C) 21 days after bilateral ovariectomy and (D) 21 days after removal of the sucking stimulus, and (E) in the following non-breeding season (seasonal quiescence). Single blood samples were taken twice weekly during lactational quiescence. In the presence of ovarian tissue, basal concentrations of LH were low (0.94 ng mL-1) with pulses of low magnitude (1.3 ng mL-1) and low frequency (1.4 pulses per 6 h). There was no response to luteectomy but all three parameters increased after bilateral ovariectomy. Removal of the sucking stimulus affected the LH pulse frequency but seasonal differences were not evident. The pattern of release of FSH was not pulsatile. There was no response to luteectomy in basal concentrations of FSH but these rose significantly after bilateral ovariectomy (P less than 0.001) in lactational quiescence. There was no effect of removing the sucking stimulus but in seasonal quiescence concentrations were higher. The results indicate that non-luteal ovarian tissue is essential for the negative-feedback effects on LH and FSH secretion, that the pattern of release of LH, but not FSH, is pulsatile, and that there is no marked seasonal change in hypothalamo-hypophysial activity.

Blood oxygen carriage in the marsupial, tammar wallaby (Macropus eugenii), at prenatal and neonatal stages.

The measurements reported here are the first to be made on oxygen carriage of a prenatal marsupial. The oxygen equilibrium curves (OECs) of tammar wallaby blood 1-2 days before the due date of birth showed a high P50 (mean = 44 Torr at 36 degrees C at a PCO2 of 34 Torr), more than 1.5 times that of the mother. This was confirmed by measurements in red cell suspensions at controlled pH. The finding of a higher P50 than in adult is in contrast to the general finding in eutherian (placental) mammals. Also they showed interaction between O2 and CO2 carriage (expressed as delta log P50/delta log PCO2 between 34 and 64 Torr PCO2) about half the magnitude of that in adults. At high PCO2 this effect reversed in the lower part but not in the upper part of the OEC. The Hill plot of the OECs showed a bend in the middle range of saturation: in nearly all cases the Hill coefficient (nH) was greater than 4.0 above about 50% saturation suggesting aggregation of haemoglobin tetramers. These results are similar to those previously reported for neonatal tammars and confirmed by further measurements in this study. The prenatals all had four haemoglobin types, identical with those found in the neonates.

Effects of prostaglandin and prolactin on luteolysis and parturient behaviour in the non-pregnant tammar, Macropus eugenii.

In Exp. 1 non-pregnant female tammars were injected, on Day 26 (the day parturition would normally occur) after removal of pouch young, with saline, 200 micrograms ovine prolactin or 5 mg PG and changes in plasma concentrations of progesterone, prolactin, PGF-2 alpha metabolite (PGFM), oestradiol-17 beta and LH were determined. Luteolysis occurred in females treated with prolactin alone, while treatment with PG first induced a rapid rise in prolactin and subsequently a significant decrease in plasma progesterone. After prolactin treatment the oestradiol peak, oestrus and the LH surge were advanced significantly compared to the saline-treated females. In Exp. 2 the effects of the same treatments as used in Exp. 1 were determined on Day 23 and again on Day 26 after removal of pouch young in non-pregnant females. On Day 23 both prolactin and PG induced significant elevations in plasma progesterone, but luteolysis did not occur. On Day 26 the treatments initially induced significant elevations in plasma progesterone but these were followed by luteolysis within 8-12 h after treatment. PG treatment induced parturient behaviour in the non-pregnant females within 3-21 min and this persisted during the period that plasma concentrations of PGFM were elevated. The results show that PG induces birth behaviour and the release of prolactin, while prolactin first induces an elevation of plasma progesterone concentrations and, in the mature CL on Day 26, subsequently induces luteolysis.

The cytoskeletal system of mammalian primitive erythrocytes: studies in developing marsupials.

Seeking to resolve conflicting literature on cytoskeletal structure in mammalian "primitive" generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short-tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population at birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface-associated network (membrane skeleton), closely resembled that of non-mammalian vertebrate erythrocytes. By day 2 or 3, much smaller anucleate erythrocytes of "definitive" morphology, lacking marginal bands, appeared in abundance. These accounted for greater than 90% of the circulating population of both species by day 6-8. Non-nucleated erythrocytes of a different type, constituting 1-6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non-mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long-suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non-mammalian vertebrates.

A second form of gonadotropin-releasing hormone (GnRH), with chicken GnRH II-like properties, occurs together with mammalian GnRH in marsupial brains.

GnRH peptides in the hypothalami of marsupials (tammar wallaby, short-nosed bandicoot, and eastern quoll) and a monotreme (echidna) were investigated by reverse phase HPLC and RIA with region-specific antisera. In the wallaby hypothalamic extract, a single form of GnRH was present, which eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2- and COOH-termini of mammalian GnRH as well as by antibodies to the middle region. Two GnRH molecular forms were demonstrated in the bandicoot and quoll hypothalamic extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was quantified equally by two mammalian GnRH antisera. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Quantification of this immunoreactive peak with two chicken GnRH II antisera was not equal, suggesting that the peptide has similar properties to, but may not be identical to, chicken GnRH II. Immunoreactive GnRH was also detected in the echidna hypothalamic extract. These findings demonstrate that in some mammals more than one form of GnRH is present in the brain of a single species, as has previously been found in species from all nonmammalian vertebrate classes. The finding in marsupial brain of a peptide with properties of chicken GnRH II, which has previously been reported in species of Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early early evolved and conserved form of GnRH.

Influence of the immature testis on sexual differentiation in the tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia).

Reproduction in the tammar wallaby, Macropus eugenii (Desmarest), is highly seasonal in the females but not the males. This study was designed to determine whether the difference is established during early life as a result of exposure to the developing testes. At day 10 after birth, when the sex can be distinguished externally, testes were removed from males and placed under the flank skin of females, while other groups of males and females were subjected to surgery without interfering with the gonads. The testis grafts remained palpable for 3-6 months. Sex-chromosome constitution was confirmed by karyotyping. At 3 years of age, the body weights and dimensions of the grafted females were not significantly different from those of the sham-operated females, whereas those of the castrated males were significantly larger and were equal to those of the sham-operated males, indicating that there is genetical control of growth independent of the testis in this species of marsupial. During 5 years of observations, none of the grafted females ever produced young, whereas all of the sham-operated females produced young each year from the second year. The grafted females had a mixture of male and female reproductive structures. The pouch and mammary glands developed normally, as did the Mullerian duct derivatives, the vaginal complex, the uteri and the oviducts. The ovaries were either devoid of oocytes and follicles or had reduced numbers, the Wolffian ducts were retained to varying degrees, the urogenital strand had developed into a prostate indistinguishable in size and structure from that of intact males, and the genital tubercle had developed into a normal-sized penis with a crus penis and Cowper's glands. In the castrated males, the scrotum developed normally and contained the gubernaculum and vas deferens. There was no evidence of Mullerian duct derivatives, and the urogenital strand was a simple canal, as in females. There were no Cowper's glands and no penis or erectile tissue. In one hemicastrated male, there was no development of the penis, although the remaining testis occupied the scrotum and showed compensatory hypertrophy. These findings indicate that the testis, at day 10, has a profound influence on the early differentiation of the Wolffian ducts, prostate and penis but cannot influence the differentiation of the Mullerian duct derivatives. The testis does not have any effect on the development of the pouch, mammary glands or scrotum or on somatic growth, all of which are apparently under independent genetical control.

Oxygen carriage and carbonic anhydrase activity in the blood of a marsupial, the Tammar wallaby (Macropus eugenii), during early development.

Blood O2 transport and Hb type have been studied in pouch young and adult of a marsupial, the Tammar Wallaby. The O2-Hb equilibrium curves (at 35.5 degrees C and PCO2 = 34 Torr) had a high P50 in the first few days of life, up to 49 Torr. This fell to 32 Torr by 2 weeks of age. Also (delta log P50/delta PCO2) was low but it rose to adult levels by 2 weeks of age. The curves in these early pouch young showed a change in Hill coefficient (nH) at between 32 and 62% saturation, nH rising to more than 4.0 at higher O2 saturations. This indicates interaction between more than 4 Hb subunits. Model calculations showed that such curves could be produced by a mixture of 2 Hb components; one with a low P50 and low nH, and one with a high P50 and high nH. In this model the nH values were different from the nH values of either component. The temperature effect on P50 in early pouch young was higher than in adult Tammars and similar to that reported for adult eutherians. In the first 4 days all red cells were nucleated and four Hb types were present. Carbonic anhydrase activity in the blood before birth was about 30% of the adult levels. These levels remained until 2 days after birth, when a rapid rise in activity began, near-adult levels being reached at 5 days despite the animals being still very immature.

Fetal role in the control of parturition in the tammar, Macropus eugenii.

When female tammars carrying dormant blastocysts were injected with progesterone at the time of removal of their pouch young the development of the fetus was advanced and parturition occurred 5 days earlier than in the control tammars. In these tammars the prolactin pulse was also advanced by 5 days but the usually concomitant fall in progesterone was not. In non-pregnant tammars similar injections of progesterone did not advance the subsequent fall in progesterone, oestrus, or the LH pulse. In non-pregnant tammars injected with ovine prolactin on Day 26, to mimic the prolactin pulse, plasma progesterone was reduced to basal levels within 12 h, significantly earlier than in controls. Conversely, in 5 pregnant and 1 non-pregnant tammar injected with ovine prolactin on Day 23, to mimic the condition induced by advancing the time of parturition with progesterone, the decline in plasma progesterone was not advanced and the endogenous prolactin pulse, parturition, post-partum ovulation and the LH pulse all occurred after intervals similar to those of controls. The results support the view that the fetus is associated with the pre-partum prolactin pulse in maternal plasma and that a prolactin pulse at this stage is luteolytic in non-pregnant tammars.

Change in duration of elevated concentrations of melatonin is the major factor in photoperiod response of the tammar, Macropus eugenii.

In Exp. 1, 10 quiescent non-lactating tammars were exposed to 15L:9D (Days -41 to -1), 24L:0D (Days 0 to 14), 15L:9D (Days 15 to 34) and then to ambient increasing daylength from 13L:11D on Day 35. From Days 0 to 22 they received a s.c. injection of melatonin (400 ng/kg, N -5) on the arachis oil vehicle (N = 5) in the evening (19:30 h) 2.5 h before dark. Exposure to 24L:0D abolished the nocturnal plasma melatonin rise but this was reinstated by subsequent exposure to 15L:9D. Of 5 melatonin-treated tammars, 4 gave birth on Day 45, so had failed to respond to the melatonin injection alone but reactivated when this was combined with the endogenous melatonin rise during exposure to 15L:9D. Of 5 control tammars, 4 remained quiescent until reactivated by the decrease in daylength to 13L:11D, and gave birth significantly later (Day 63.7 +/- 2.2, mean +/- s.e.m., P less than 0.05). In Exp. 2, 6 tammars were exposed to 15L:9D (Days -15 to -1) and then to 12L:12D (Days 0 to 15) by extending the dark phase by 3 h in the morning. This extended the nocturnal melatonin rise by 2-3 h in the morning and all 6 tammars gave birth on Day 31.2 +/- 1.0. A transient pulse of peripheral plasma prolactin (81.5 +/- 31.0 ng/ml) was detected at dawn during 15L:9D in all 6 tammars but was not observed in any of them 5 days after exposure to 12L:12D. Together these results do not support the time of day hypothesis but indicate that increase in duration of the nocturnal melatonin rise mediates the effects of decreased daylength on reactivation of the corpus luteum, and that the first detectable result of this may be the abolition of a transient prolactin pulse at the end of the dark phase.