Response in peripheral plasma melatonin to photoperiod change and the effects of exogenous melatonin on seasonal quiescence in the tammar, Macropus eugenii

During the light phase of each of 3 photoperiods tested, plasma melatonin concentrations were less than 16 to 62 pg/ml and during the dark phase they were 31 to 169 pg/ml. When the photoperiod to which the tammars were exposed was altered from 15 h light:9 h dark to 12L:12D the onset of the nocturnal rise in melatonin was advanced from the first day, thereby extending its duration, and the females gave birth 32 +/- 0.4 (mean +/- s.e.m.) days later. To test whether melatonin mediated this effect of photo-period change, tammars in a second group were injected s.c. with melatonin (400 ng/kg, N = 6) or the arachis oil vehicle (N = 6), 2.5 to 2.25 h before dark during 15L:9D for 15 days before exposure to 12.5L:11.5D. The melatonin injections mimicked the endogenous melatonin profile of 12L:12D and the melatonin-injected tammars gave birth 32 +/- 0.8 days after the start of injections, which was the same as the interval from photoperiod change in Exp. 1 but was significantly different (P less than 0.005) from the interval in the control group (46.0 +/- 1.1 days). These results show that exogenous melatonin given 2.5 to 2.25 h in advance of the endogenous rise fully mimics the response of the tammar to photoperiod change.     

Effects of progesterone on parturition in the tammar, Macropus eugenii

Tammar wallabies were treated with progesterone injections or implants during late pregnancy to determine whether progesterone withdrawal was essential for parturition. Neither physiological (implanted group) nor pharmacological (injected group) levels of circulating progesterone prevented parturition occurring at about the expected time in about two-thirds of animals that were pregnant. The neonates of both groups were normal in size and weight, but about a third of treated pregnant animals retained their fetuses or aborted. The retained fetuses were retarded in development. Therefore, progesterone treatment had no influence on the duration of gestation, or parturition, in the tammar wallaby, but high progesterone concentrations may interfere with the normal course of development and birth in a proportion of treated animals.     

Immunological aspects of gestation in the tammar wallaby, Macropus eugenii.

Sensitization to male histocompatibility antigens and repeated pregnancy to the same male were found to have little effect on fertility or length of gestation in the tammar wallaby, M. eugenii. However, in some sensitized females a long interval occurred between removal of pouch young and the next birth. In addition to studies on fertility, the immunological response of female tammars to their mate has been examined by one-way mixed leucocyte culture (MLC) carried out at the beginning and end of one breeding season. In virgin females, examined at the beginning of the breeding season, the MLC response to the prospective mate peaked on day 6. In contrast, by the end of the season, MLC responses were much lower and peaked earlier, on day 3 to day 5.     

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.     

Morning pulse of prolactin maintains seasonal quiescence in the tammar, Macropus eugenii

The pulse of prolactin, present in female tammars kept in an inhibitory photoperiod and absent 5 days after a change to a stimulatory photoperiod, was artificially maintained during stimulatory photoperiods (Exps 1A and 1B) or abolished during an inhibitory photoperiod (Exp. 2). In Exp. 1A, thyrotrophin-releasing hormone (TRH) was injected i.v. for 8 consecutive mornings and in Exp. 1B ovine prolactin was injected i.v. for 10 consecutive mornings beginning on the day the photoperiod was changed from 15L:9D to 12L:12D. In Exp. 1A, in 5 of 6 control females the prolactin pulse was present until the 3rd morning after the lighting was changed, and absent thereafter. In both experiments the early progesterone peak and the interval to birth in the treated females was significantly delayed compared to the control females; reactivation of the CL did not occur until treatment ceased. In Exp. 2, for 5 consecutive mornings, before the prolactin pulse was expected to occur, bromocriptine was injected i.m. In 2/4 treated animals the prolactin pulse was abolished and these animals showed an early progesterone peak and gave birth 28 and 29 days after treatment began. The remaining 2 animals and the controls (N = 4) did not show reactivation until after they experienced a stimulatory photoperiod. The results indicate that the prolactin pulse is the critical factor in the maintenance of the inhibition of the CL of the tammar during seasonal quiescence.     

Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) plays an essential role in the adaptive immune system of vertebrates through antigen recognition. Although MHC genes are found in all vertebrates, the MHC region is dynamic and has changed throughout vertebrate evolution, making it an important tool for comparative genomics. Marsupials occupy an important position in mammalian phylogeny, yet the MHC of few marsupials has been studied in detail. We report the isolation and analysis of expressed MHC Class I genes from the tammar wallaby, a model marsupial used extensively for the study of mammalian reproduction, genetics, and immunology. We determined that there are at least 11 Class I loci in the tammar genome and isolated six expressed Class I sequences from spleen and testes cDNA libraries, representing at least four loci. Two of the Class I sequences contain substitutions at sites known to be important for antigen binding, perhaps impacting their ability to bind peptides, or the types of peptide to which they bind. Phylogenetic analysis of tammar wallaby Class I sequences and other mammalian Class I sequences suggests that some tammar wallaby and red-necked wallaby loci evolved from common ancestral genes.     

Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii

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.     

Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)

Lactating tammars can provide two different milks simultaneously from adjacent glands to a young newborn (phase 2 of lactation) and an older animal at heel (phase 3 of lactation). Evidence that the two glands are controlled independently is shown by the capacity of mammary explants from these glands to synthesize different whey proteins and DNA and RNA at different rates. Prolactin is essential for the maintenance of milk synthesis, but its role in differential responses of the individual mammary glands remains to be established. Potential mechanisms for the control of milk synthesis are discussed.     

Regulation of the elemental composition of the epididymal fluids in the tammar, Macropus eugenii

Micropuncture, microanalytical and microelectrode techniques were used to study electrochemical aspects of 7 elements and fluid in the ductuli efferents and ductus epididymidis of the tammar. Rete testis fluid was isosmotic with blood and had a lower pH. It also contained lower concentrations of bicarbonate, sodium, calcium, magnesium, phosphorus and sulphur and higher concentrations of potassium and chloride than blood. The luminal fluid was acidified further during passage through the sperm ducts and all of the elements which were studied moved in or out of the lumen, usually against an electrochemical gradient. The ductuli efferents reabsorbed 87% of the fluid leaving the testis without changing the intraluminal concentrations of sodium, potassium and calcium, but the concentrations of magnesium, phosphorus and sulphur increased. The caput epididymidis reabsorbed about half the fluid entering it: sodium concentrations decreased and those of potassium and phosphorus increased. There was also some fluid reabsorption and an increase in the values of potassium and phosphorus in the corpus epididymidis. There was little net transport of fluid in the cauda epididymidis; sodium, chloride, magnesium and phosphorus concentrations decreased and potassium values increased. Studies involving filtration through a dialysis membrane of blood and fluid from the rete testis and cauda epididymidis showed that, whilst some of the calcium, magnesium, phosphorus and sulphur was associated with high molecular weight compounds in blood, the association was not significant in the reproductive fluids.     

Mammogenesis and changing prolactin receptor concentrations in the mammary glands of the tammar wallaby (Macropus eugenii)

All 4 mammary glands of the tammar wallaby showed a steady increase in weight and prolactin receptor concentration during the luteal phase of the oestrous cycle to reach a peak at oestrus. Removal of the corpus luteum abolished this mammogenesis , while pregnancy, which in this species is a day or so shorter than the oestrous cycle, had no effect. This provides an explanation for the previous finding that pregnancy is not a necessary pre-requisite for lactation in marsupials and that nonpregnant animals will lactate very successfully, provided the suckling stimulus is applied at the correct stage of the oestrous cycle. During lactation, only the gland supplying the teat to which the pouch young was attached developed and showed any further increase in prolactin receptors; the other 3 glands remained small and inactive. These results indicate the importance of the suckling stimulus and milk withdrawal on the initiation and maintenance of lactation.     

Testicular development and maturation of the hypothalamic-pituitary-testicular axis in the male tammar, Macropus eugenii

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, approximately 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 10-30 min after injection. The hormone concentrations increased in both magnitude and duration with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of photoperiod on embryonic diapause in the tammar wallaby,macropus eugenii

After inoculation of mycoplasmas via ductus deferens in male dogs with a vas deferens fistula the influence on gonads and general state of health was investigated using clinical, andrological, spermatological, microbiological and histological methods.On day 23 and 29 respectively after inoculation two animals showed local reactions within the fistulated gonads which were identified as orchitis and epididymitis. Clinical abnormalities correlated well with the decrease of sperm motility and significant increase (p < 0,05) of aberrant spermatozoa.The microbiological and histological findings are discussed in the light of the incidence of clinical symptoms and the influence of a mycoplasmal infection.     

Physiological and metabolic changes associated with weaning in the tammar wallaby,Macropus eugenii

Summary The tammar wallaby (Macropus eugenii) is a small macropodid marsupial in which the major part of weaning occupies the period between 28 and 36 weeks of pouch life. Before weaning the diet of the tammar is high in carbohydrate and low in lipid/volatile fatty acid whereas the reverse applies after weaning. The adult tammar is a forestomach fermenter. The aim of this study was to elucidate some of the physiological and metabolic changes associated with this major change in the diet.Hepatic glycogen content increased gradually early in development to a maximum of 7% of liver weight at 28–30 weeks of pouch life. It then fell precipitously to less than 1% of liver weight at 36 weeks before recovering to the adult level of about 3% liver weight. Plasma glucose levels were maintained at about 10 mM until 36 weeks, after which they fell gradually to adult values of about 4 mM. Hepatic hexokinase activity increased several-fold between 18 and 30 weeks of pouch life, remained high until 42 weeks, and then fell to the adult level. The hepatic activities of fructose-bisphosphatase and particulate phosphoenolpyruvate carboxykinase (PEPCK) were unchanged during development but soluble hepatic PEPCK activity, which was low until 28 weeks of pouch life, increased 3–4 fold between 30 and 36 weeks and then fell slightly to the adult level. Hepatic pyruvate kinase increased in activity up to 28 weeks and then fell to about half peak values at 36 weeks and 20% of peak activity in the adult. There was a greater than ten-fold increase in the ratio of soluble PEPCK activity to pyruvate kinase activity between 28 and 36 weeks of development. It has previously been reported that hepatic gluconeogenesis is inducible in pouch young but constitutive in adults. We conclude that the change in regulation of hepatic gluconeogenesis at the PEPCK/pyruvate kinase level is part of the weaning process.The urea content of the plasma changed little during development but plasma ammonia increased consistently through pouch life. Urine urea content was low until about 28 weeks of age and then increased rapidly to adult levels. Urine ammonia increased from about 20 mM early in pouch life to a maximum of more than 100 mM at 28 weeks. Thereafter, urine ammonia content fell rapidly to the adult value of about 20 mM. For the first 27 weeks of pouch life, urine pH was consistently between 4.4 and 5.7, but subsequently it rose and became more variable. Urine pH in adults was 8.1±0.3. The activities of the five enzymes of the ornithine-urea cycle increased 3–5 fold in activity between 28 and 36 weeks of pouch life.These findings indicate that there are major changes in metabolic regulation associated with weaning in the tammar. During weaning, glucose becomes essentially unavailable to the young animal and there is an increase in the rate of hepatic gluconeogenesis which is attributable primarily to increased activity of soluble PEPCK. Metabolism, which is acidotic before weaning, becomes alkalotic and there is a decrease in urinary ammonia content as proton excretion decreases. As ammonia excretion falls, the activity of the urea cycle increases and the concentration of urea in the urine rises. Weaning in the tammar is therefore a complex and well-orchestrated process which may be associated with the change in diet.     

The metabolic properties of spermatozoa from the epididymis of the tammar wallaby,Macropus eugenii

The utilization of various substrates by sperm from the cauda epididymidis of the tammar was examined because the major naturally occurring sugar in the semen of this species is N-acetyl-D-glucosamine (NAG) and not fructose, as in eutherian mammals. The sperm displayed a high level of endogenous respiration that supported motility for relatively prolonged periods of time in vitro. They also metabolised exogenous ¹⁴C-labelled glucose, NAG, sucrose, and acetate through glycolytic and/or oxidative processes to produce lactate and ¹⁴CO₂ at varying rates. The rate of uptake of NAG by tammar sperm was about four times greater than that of other substrates. Glucose and/or NAG stimulated the rate of oxygen consumption by about 20%, but acetate stimulated oxygen consumption by more than 40%. The most striking findings were that NAG almost completely inhibited the oxidation of glucose and sucrose by the sperm and depressed the uptake of glucose, 3-O-methylglucose, and sucrose. Acetate oxidation also was inhibited by NAG, but only by about 50%. Tammar sperm generated substantial amounts of free glucose during incubation with NAG, but this and the inhibitory effects of NAG on glucose oxidation were not mimicked by rat sperm. It is proposed that tammar sperm fail to oxidise glucose in the presence of NAG because of the rapid cellular uptake of NAG relative to glucose. Also, the intracellular glucose and acetate liberated from NAG would compete with exogenous glucose for processing in the Embden-Meyerhof and tricarboxylic acid (TCA) cycle pathways. It is also suggested that tammar sperm oxidise sucrose after extracellular hydrolysis into its glucose and fructose components. The biological implications of these metabolic and transport properties of tammar sperm have as yet to be determined. Mol. Reprod. Dev. 49:92–99, 1998. © 1998 Wiley-Liss, Inc.     

Structure of the pars distalis in the adult tammar wallaby (Macropus eugenii)

An immunohistochemical, light- and electron-microscopial study was made of the pars distalis in adult tammar wallabies (Macropus eugenii). The pars distalis of this marsupial mammal was divided into three regions, based on the distribution of cell types within the gland. Somatotropic, mammotropic, luteotropic, folliculotropic, corticotropic and thyrotropic cells were identified on the basis of their immunohistochemistry, cytology and ultrastructure. Non-granulated (folliculo-stellate) cells, identified in electron micrographs, were found throughout the pars distalis. Somatotropic cells were predominant in the posterior pars distalis in all animals examined. In the single male specimen and in the non-lactating females examined, small numbers of apparently inactive mammotropic cells were scattered throughout the pars distalis; the same cell type was apparently active and present in considerable numbers in lactating females. Only one morphological type of gonadotropic cell was evident; these cells were scattered throughout the pars distalis, but in largest numbers in the median region. Small numbers of thyrotropic cells were found, most commonly in the anterior pars distalis. Corticotrops were also observed in moderate numbers, predominantly in the anterior regions of the pars distalis.     

Faecal corticosteroid levels as an indicator of well-being in the tammar wallaby, Macropus eugenii

Faecal corticosteroid levels were measured in five female tammar wallabies, Macropus eugenii, at Macquarie University, NSW, Australia, to assess their reliability as indicators of well-being in this species. Animals were challenged with a change in conditions over the course of approximately 1 week, comprising movement from group yards to isolation in individual yards, in order to impose a disturbance to homeostasis ("stress"). Faecal samples were collected in 24-h intervals during the study period and analysed for corticosteroid concentration. The use of enzyme immunoassay for the measurement of corticosteroids in marsupial faecal pellets was validated. We observed a significant increase in faecal corticosteroids upon isolation and movement. Faecal corticosteroids remained above initial levels in all five animals throughout the study period, suggesting that faecal corticosteroid concentrations may be a useful indicator of a change in animal well-being. Faecal corticosteroid levels did not correlate with serum cortisol levels, implying that the use of noninvasive methods in a representative marsupial, the tammar wallaby, has the potential to provide information that is not readily apparent using blood-based protocols. Faecal corticosteroid analysis therefore has the potential for application in monitoring the well-being of captive and managed marsupial populations, as part of an integrated system of measures of animal health and well-being.