Morphometric estimate of gas-exchange tissue in the new-born tammar wallaby,Macropus eugenii

The lung of the new-born marsupial is at the terminal air sac stage of development. The maturational status of the lung of new-born tammar wallaby was assessed using established morphometric techniques and the results were compared with data from a morphometric study of the lung of the rat. Volume densities of the parenchyma and non-parenchyma, conducting airways and blood vessels, the relative volumes of airspace and tissue, the thickness and the composition of the septa differed between the two species. In addition the volume of capillaries and the surface area of the effective gas-exchange tissue was greater in the new-born rat than in the new-born tammar pouch young. The lung of the new-born tammar appears to be at an earlier phase of the terminal air sac stage than that of the new-born rat. Lung development up to birth appears to be commensurate to the metabolic needs of the organism at birth.     

Thyroid hormones during development of a marsupial, the tammar wallaby, Macropus eugenii

The levels of thyroid hormones in the plasma and the activities of 5'-deiodinase activity in liver and kidney were determined in the tammar wallaby, Macropus eugenii, from early pouch life to adulthood. The total concentration of plasma thyroxine (T4) was below 15 nmol/l before day 75 of pouch life, rose to about 75 nmol/l at day 160, and then decreased to about 12 nmol/l in the adult. The total concentration of plasma tri-iodothyronine (T3) was below 0.4 nmol/l before day 120, increased to 3 nmol/l by about day 220 and then decreased to 1.0 nmol/l in adults. Concentrations of free T4 and free T3 followed a similar pattern but peaked at 45 and 160 pmol/l respectively. Concentrations of reverse T3 (rT3) were extremely variable, ranging from 0 to 1 nmol/l at day 100, and from 0 to greater than 2 nmol/l at day 180. After about day 230, rT3 levels fell rapidly and were below 0.3 nmol/l in adults. Liver and kidney 5'-deiodinase activities, which were undetectable before day 80, reached adult levels by day 220. Half-maximal activity of both these enzymes occurred at about day 205, mid-way between the peaks of T4 and T3. These findings suggest that the systems supporting synthesis and release of hormones from the thyroid gland are probably mature by about day 160 of pouch life in the tammar, while peripheral deiodinase activity, which is a major factor in the production of T3 in the plasma, matures by about day 220. These events thus precede the development of physiological independence of the young tammar from its mother.     

Steroid metabolism in the yolk sac placenta and endometrium of the tammar wallaby, Macropus eugenii

Yolk sac and endometrial tissue were obtained from tammar wallabies between 11 and 25 days after the removal of pouch young. Tissues were examined histologically and steroid-metabolizing enzymes were identified by incubation for 3 h at 37 degrees C in Medium 199 containing labelled steroid precursors. Yolk sac membrane (YSM) incubated with labelled pregnenolone produced a small amount of progesterone and pregnanediols; 80.5 +/- 8.4 (S.E.M.) % of the original substrate remained unmetabolized. Labelled androstenedione was metabolized to 5 alpha-androstane-3,17-dione and androsterone, and only 5.8 +/- 3.8% of the original substrate remained at the end of incubation. Incorporation of androstenedione or dehydroepiandrosterone (DHA) into phenolic compounds was low (0.5 +/- 0.1%). There was no evidence for the enzymes, arylsulphatase or sulphotransferase, in YSM. Endometrial tissue from the same animals metabolized pregnenolone, DHA and androstenedione, converted progesterone to androstenedione, and produced aqueous-soluble steroid conjugates. The results demonstrated that YSM contains enzymes associated predominantly with steroid catabolism and with incipient progesterone synthesis. The findings are discussed in relation to the histological appearance of the tissues and compared with placental steroid synthesis in eutherian mammals.     

Developmental profile of thyroid hormone distributor proteins in a marsupial, the tammar wallaby Macropus eugenii

The ontogeny of thyroxine distributor proteins in serum of the marsupial Macropus eugenii (tammar wallaby) was investigated from day 3 after birth until adulthood. The thyroxine distributor proteins in the serum of adult M. eugenii are transthyretin and albumin. Northern analysis of RNA prepared from liver showed that transthyretin mRNA levels were initially high (about adult levels at the earliest ages tested), reduced to about 60% adult levels (between days 50 and 150), and then steadily increased to adult levels (by days 200 to 250). Albumin mRNA levels were initially about 50% of adult levels (day 3) and steadily rose to 90% of adult levels by days 175 to 220. A globulin, "wallaby thyroxine-binding protein" (W-TBP), bound [(125)I]thyroxine from day 3 until about day 200. Of the protein-bound thyroxine, the proportion bound by transthyretin had a similar pattern to the transthyretin mRNA levels. From day 26 onward, about half of the protein-bound thyroxine was bound to albumin. On day 3, less than 10% was bound to W-TBP and the proportion steadily increased to a maximum of about 46% by about day 120 and then reduced to undetectable levels by around day 250. The developmentally regulated W-TBP was present throughout pouch life, when the pouch young is dependent on obtaining thyroxine required for normal growth and development from the mother. After the young tammar wallaby leaves its mother's pouch, a time when it has reached a level of physiological development approximately equivalent to that at the time of birth in precocious eutherian mammals such as cattle and sheep, W-TBP was no longer detected as a thyroxine distributor protein in serum.     

Prolactin-dependent accumulation of alpha-lactalbumin in mammary gland explants from the pregnant tammar wallaby (Macropus eugenii)

The minimal hormonal requirements for the in-vitro accumulation of alpha-lactalbumin have been investigated in a marsupial, the tammar (Macropus eugenii). Mammary gland explants from 24-day pregnant tammars cultured in medium containing bovine insulin, cortisol and ovine prolactin showed a progressive increase in accumulation of alpha-lactalbumin during 4 days of incubation. No increment was observed if prolactin was omitted from the medium. However, a similar rate of increase was observed after 3 days of culture in medium containing prolactin alone. This induction of alpha-lactalbumin was maximal at a prolactin concentration of approximately 0.02 mg/l, which corresponds to physiological levels during pregnancy and early lactation. The absence of an effect of bovine insulin on tammar explants is not due to a general unresponsiveness to this hormone since insulin-stimulated DNA synthesis and amino acid uptake was evident after 3 days of culture. The inclusion of tri-iodothyronine and raised concentrations of cortisol in culture media have been shown to modulate alpha-lactalbumin synthesis in eutherian mammals but were without effect in the tammar. In addition, increased levels of progesterone did not inhibit the induction of alpha-lactalbumin, confirming an earlier in-vivo study suggesting that progesterone withdrawal may not be the lactogenic trigger in this species. Thus the pregnant tammar is the only species yet described in which alpha-lactalbumin is induced maximally in vitro in response to a single hormone.     

Metabolic effects of infusion of cortisol and adrenocorticotrophin in the tammar wallaby (Macropus eugenii Desmarest).

Infusion of cortisol (1 mg/kg of body weight infused over 1 hr) into the tammar wallaby, Macropus eugenii Desmarest, was followed by an immediate increase in plasma corticosteroid concentration. Plasma glucose concentration began to rise 45 min later and remained above control levels for 3 hr. There were no changes in the concentrations of amino acids, free fatty acids, or urea in the plasma. It is concluded that cortisol affects plasma glucose concentration directly rather than via protein catabolism. Intravenous infusion of 0.45 IU of porcine ACTH/kg of body weight over 1 hr was followed by an immediate increase in plasma corticosteroid concentration and a rise in plasma glucose level 15 min later. The plasma glucose response to ACTH was advanced about 1 hr compared with that to cortisol. It is concluded that the effects of ACTH cannot be mediated wholly by an increase in plasma corticosteroid concentration. There were no changes in the concentration of amino acids or urea in the plasma, but the free fatty acid concentration was increased after ACTH infusion. This is consistent with the lipolytic effects of ACTH in other mammalian species.     

The effects of age,season, and gender on serum cortisol levels in the tammar wallaby,Macropus eugenii

Serum cortisol levels were measured in a total of 73 tammar wallabies maintained in a captive population at Macquarie University, NSW, Australia. Previous studies of corticosteroids in marsupials have generally involved low sample numbers, a diverse array of analytical techniques, and a variety of sampling conditions. We have conducted a substantive, longitudinal study of serum cortisol levels using a radioimmunoassay protocol, and data have been analysed with respect to age, sex, and seasonality. There were no apparent effects of age or sex on serum cortisol levels, although an inverse but non-significant relationship was observed between males and females. However a significant difference in serum cortisol levels was observed between seasons, with mean serum cortisol significantly higher in summer than in autumn. These data will serve as a reference for 'normal' ranges of serum cortisol levels, particularly in the female tammar wallaby. As deviations from these values can indicate compromised animal health and well-being, this information will assist wildlife managers in assessing and monitoring the health status of individuals in captive and free-ranging populations.     

Prolactin receptors in the mammary gland, corpus luteum and other tissues of the tammar wallaby, Macropus eugenii.

Specific binding of radio-iodinated ovine prolactin to subcellular tissue fractions of the tammar wallaby (Macropus eugenii) was investigated. Specific binding was found, in order of decreasing binding activity, in the lactating mammary gland, corpus luteum, corpus albicans, adrenal gland and ovary. Specific binding was absent in kidney, liver, brain and inactive mammary gland. The mean association constant (Ka at 23 degrees C) was determined as 0.90 x 10(9), 2.20 x 10(9), 2.44 x 10(9), 3.38 x 10(9) and 10.98 x 10(9) l/mol for mammary gland, adrenal, corpus albicans, corpus luteum and ovary respectively. The mean receptor concentration (N) varied from 92.87 x 10(-14) mol/mg protein for the mammary gland to 1.03 x 10(-14) mol/mg protein for the ovary. The concentration in the corpus luteum varied between tissue pools collected at different times of the annual breeding cycle. The specificity for prolactin was shown in the mammary gland and corpus luteum by the failure of ovine FSH, LH, GH and TSH to displace 125I-labelled ovine prolactin, whereas it was displaced readily by both ovine and bovine prolactin.     

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.     

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.     

Seasonal patterns of circulating progesterone and prolactin and response to bromocriptine in the female tammar Macropus eugenii

Concentrations of prolactin and progesterone in the plasma of female tammars (Macropus eugenii) were measured during lactational quiescence and seasonal quiescence and during the period of natural resumption of reproduction after the summer solstice in December. Prolactin concentrations were consistently low (less than 40 ng/ml) during the period of declining day length and consistently elevated (greater than 40 ng/ml) during the period of increasing day length. Basal levels of progesterone were lowest (118.9 +/- 9.1 pg/ml) at the winter solstice and highest (244.1 +/- 21.0 pg/ml) at the summer solstice. Treatment with bromocriptine (CB154) did not depress prolactin levels at either time of the year but during February to June a single injection of 5 mg/kg body wt induced development of the quiescent corpus luteum (CL) and the diapausing embryo was reactivated. In February and March the level of progesterone increased in association with the developing CL, but after treatment at the winter solstice in June plasma progesterone did not increase although pregnancy was successfully completed. From September through November only 1 of 50 females responded to bromocriptine. None responded to bromocriptine given at the summer solstice in December, but all these animals spontaneously reactivated 2 weeks later. The difference in response of female tammars to bromocriptine treatment in the two halves of the year suggests that different endocrine controls operate in lactational and seasonal quiescence and that the latter is more complex.     

Metabolic effects of ACTH in the tammar wallaby, Macropus eugenii: The role of the adrenal medulla

Infusion of ACTH into bilaterally adrenalectomised tammar wallabies (Macropus eugenii) has no effect on blood glucose levels, whereas infusion of cortisol or adrenaline results in increases in blood glucose concentrations. Infusion of cortisol into intact tammars does not change the rate of turnover of blood glucose, while during infusion of ACTH or adrenaline, blood glucose turnover increases. These findings, together with those previously reported (Cooley and Janssens, 1977), are consistent with the hypothesis that adrenaline is involved in early metabolic responses to ACTH administration in the tammar. Bilaterally adrenalectomised tammars can be maintained for at least 2 months with daily injections of 0.2 mg/kg deoxycorticosterone. Tammars die within 48 hr of cessation of hormone injection even if they are offered saline to drink.     

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.     

Effect of gonadectomy, season and the presence of female tammar wallabies (Macropus eugenii) on concentrations of testosterone, luteinizing hormone and follicle-stimulating hormone in the plasma of male tammar wallabies

Concentrations of FSH, LH and testosterone in plasma were measured in groups of adult male tammar wallabies before and after gonadectomy, and during the breeding and non-breeding seasons. Gonadectomy resulted in a rapid fall in plasma testosterone to undetectable levels by day 2, and significant increases in plasma LH and FSH levels. The concentrations of FSH, LH and testosterone did not change significantly between the non-breeding and breeding seasons in groups of male wallabies maintained in the absence of females. However, when male wallabies were associated with sexually mature females there were significant three- to fourfold increases in concentrations of LH and testosterone in plasma at the commencement of the breeding season. The observed increases in LH and testosterone were highly synchronized in the eight animals studied and occurred approximately 2 weeks before the synchronous onset of mating. Concentrations of FSH did not change significantly at this time.     

Seasonal Changes in the Circadian Plasma Melatonin Profile of the Tammar, Macropus eugenii

The circadian plasma melatonin profile of a marsupial, the tammar, was determined at various stages of the annual reproductive cycle. At 6-14 days after each of the solstices and equinoxes, six females were exposed to a photoperiod equivalent to the natural day length at these times. Serial blood samples were taken 8 days later at 2-4-hourly intervals, and plasma melatonin concentrations were measured by radioimmunoassay. Melatonin concentrations were elevated during the dark phase of each photoperiod, and there were significant changes between the profiles in each season. The amplitude of the nocturnal rise was significantly higher (P less than 0.05) during the breeding season after the summer solstice (peak 259.5 +/- 26.8 pg/ml, mean +/- SEM) and autumnal equinox (287 +/- 53.2 pg/ml) compared to those during the nonbreeding season after the winter solstice (111.5 +/- 10.5 pg/ml) and vernal equinox (154.5 +/- 10.4 pg/ml). The duration of the nocturnal rise was significantly correlated (r=0.996, P less than 0.01) with the length of the dark phase and so was shortest after the summer solstice and longest after the winter solstice. Either of these changes in amplitude or duration might provide the photoperiodic information that regulates the annual reproductive cycle of the tammar.     

Mammary gland lactose, plasma progesterone and lactogenesis in the marsupial Macropus eugenii

Mammary gland lactose concentrations in pregnant tammar wallabies remained low at 115 +/- 24 (S.E.M.) micrograms/g wet weight of tissue until immediately before parturition, then increased to 1274 +/- 262 micrograms/g after birth. Concentrations in non-pregnant cyclic animals were generally low (143 +/- 36 micrograms/g), but were raised in three animals around the time of oestrus. Removal of the corpus luteum on day 18 of pregnancy or the oestrous cycle caused an increase in lactose concentrations in both lutectomized and sham-operated animals. This occurred despite a significant lowering of peripheral plasma progesterone concentration in only the lutectomized group. Plasma cortisol concentrations were high in some of these animals, but showed no consistent relationships with the raised lactose concentrations. The increased peripartum lactose concentration normally coincides with a sharp fall in peripheral plasma progesterone concentration, but artificial maintenance of high progesterone levels had no effect on the increase of mammary gland lactose at parturition. Mammary gland lactose concentrations in tammar wallabies are therefore a useful indicator of biosynthetic activity and as an index of lactogenesis but the role, if any, of progesterone withdrawal in lactogenesis remains unclear.