The nucleotide sequence for pituitary prolactin cDNA from the marsupial bandicoot (Isoodon macrourus) was determined by reverse transcription-polymerase chain reaction and 5'/3' rapid amplification of cDNA ends. The deduced amino acid sequence showed high sequence identity with brushtail possum prolactin (95%) and all of the expected structural features of a quadruped prolactin. A prolactin gene tree was constructed and rates of evolution calculated for bandicoot, possum, opossum and several mammalian and non-mammalian prolactins. Bootstrap analysis provided strong support for marsupials as a sister group with eutherian mammals and weak support for opossum and bandicoot as an independent grouping from the brushtail possum. The rates of molecular evolution for marsupial prolactins were comparable to the slow rate seen in the majority of quadruped prolactins that have been sequenced.

The mechanisms utilised by the newborn quoll to move from the uterus to the teat within the pouch are unknown. The ability to sense gravity and odour have been suggested and it is possible that temperature may also assist the young in this migration.2. An increasing temperature gradient was observed from the sinus at 28.98 degrees C increasing to 29.38 degrees C on the skin between the sinus and the pouch and further increasing to 30.96 degrees C within the pouch. This temperature gradient was not as apparent during lactation.3. Hairs may also play an important role in allowing the newborn to leave the gelatinous material emanating from the uterus and travel to the pouch. The hairs form a tunnel between the sinus and the pouch and may assist the young in the moving from uterus to the pouch. (c) 2005 Elsevier Ltd. All rights reserved.

Birth has been observed in a number of marsupial species and, in the studies to date, the newborn have crawled up to or across to the pouch. The method of birth in the quoll, a dasyurid, differs greatly from that observed in other marsupials. Births were recorded at normal speed using hand-held digital video cameras. Birth was heralded by a release of about 1 mL of watery fluid from the urogenital sinus followed by gelatinous material contained in either one or two tubes emanating from the sinus. The newborn, still encased in their placental membranes, were in the gelatinous material within a column. To exit this column, they had to grasp a hair and wriggle about 1 cm across to the pouch. In the pouch the newborn young had to compete for a teat. Although the quolls possessed 8 teats, the number of young in the pouch immediately after birth was 17, 16, 6, 16, 13 and 11 for each of the 6 quolls filmed. While birth has been described previously in another two dasyurids, the observers did not describe birth as reported here for the quoll. Nevertheless the movement of the newborn from the sinus to the pouch is so quick that this could have previously been missed. Filming birth from beneath and from the side allowed for a greater understanding of the birth process. Further studies are required to determine whether this use of a gelatinous material is part of the birth process in all dasyurids.

Plasma and pituitary GH concentrations and liver GH receptor (GHR), IGF-I and IGF-binding protein-3 (IGFBP-3) mRNA expression were deternimed in brushtail possum (Trichosurus vulpecula) pouch young aged 12-150 days post-partum and in adults. Mean plasma GH concentrations were highest, measuring around 150 ng/ml, from 12 to 100 days post-partum, and thereafter declined so that by 150 days post-partum levels were not significantly different from those in adults (10(.)8 +/- 1(.)8 ng/ml (S.E.M.)). In contrast to plasma levels, pituitary GH content increased markedly throughout pouch life, with an 87-fold increase between 12 and 150 days post-partum. However, when expressed per gram body weight, pituitary content was relatively constant between 25 and 150 days post-partum, indicating that the decline in plasma GH after 100 days post-partum was not due to decreased synthesis and/or storage of GH in the pituitary gland. Expression of GHR, IGF-I and IGFBP-3 mRNAs was determined by semi-quantitative RT-PCR. Liver GHR and IGF-I mRNA expression were low at 12 and 25 days post-partum and did not show sustained and significant increases (P<0(.)05) until 125 and 150 days post-partum. IGFBP-3 expression was also low at 12 days post-parturn but then increased rapidly to a maximum at 50 days post-parturn and thereafter declined. For all three mRNAs, liver expression at day 150 was not significantly different from that in adults. These patterns of gene expression for GHR and IGF-I suggest that the possum liver is resistant to the high plasma GH concentrations during early pouch life and in this way is similar to the fetal liver of some eutherian mammals.

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8+/-1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximately 65 kDa, similar to that identified in other mammalian species, and binding of (125)I-labelled human GH (hGH) was displaced by excess hGH (20 microg). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with (125)I-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K(a)) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96+/-0.2 x 10(9)/M, n=6). Binding capacity (B(max)) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4+/-0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

As in eutherians, maturation of the fetal pituitary and adrenal glands together with an increase in prostaglandin and mesotocin or oxytocin production initiates birth in marsupials. In this study, prostaglandin (Lutalyse) or oxytocin (Syntocinon) were administered to pregnant bandicoots at 05:00 h on the calculated day of birth and the resultant effects were filmed for analysis. The administration of prostaglandin caused the bandicoot to adopt the birth position several minutes after injection (n = 2). However, the bandicoot did not give birth for several hours. Birth occurred at a similar time of day to that observed for untreated bandicoots (n = 7), between 08:00 h and 12:00 h. After an injection of oxytocin, the bandicoot assumed the birth position and birth occurred within several minutes. The young were alive while still connected to their allantoic stalks. However, they were unable to attach to the teats and did not survive (n = 4). The induced young were the colour of venous blood and died soon after the umbilicus was separated, indicating that the cardiopulmonary system of these neonates was underdeveloped and inadequate to maintain life. The results from this study demonstrate that prostaglandin is required to prepare the bandicoot for birth, and mesotocin is required for contraction of the uterus and for birth to occur.

Birth is an event that allows the relatively immature marsupial to move from the internal environment of the uterus to the external environment of the pouch. The newborn marsupial passes down from the uterus to the urogenital sinus and then makes its way to the pouch and attaches to the teat at a very early stage of development. From the studies available, there appear to be three methods used by the newborn to move from the uterus to the pouch. In marsupials with a forward pouch such as the red kangaroo, tammar wallaby and the brushtail possum, the mother positions her urogenital sinus below the pouch and the newborn climb upward towards the pouch. The young climb with a swimming motion, moving the head from side-to-side and use the forearms in alternate strokes. In the bandicoot with a backward facing pouch, the mother positions the urogenital sinus above the pouch and the young slither down into the pouch. The young do not have a definite crawl, as seen with the macropodids and possum. The third method of birth has been observed in the marsupials without a definite pouch that have a mammary region that develops as the young grow in size. This type of pouch is observed in the dasyurids. The mother was noted to stand on four legs with her hips raised so that the urogenital sinus was above the pouch and the newborn young crawled downwards from the sinus to the pouch. In all species, birth was completed in 2-4 min.

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT–PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2 cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT–PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial.

Plasma concentrations of thyroxine (T4) and growth hormone (GH) were measured in the developing bandicoot from 21 through to 69 days post partum. Thyroxine concentrations increased from 7 ng mL(-1) at 21 days post partum to reach a maximum of around 40 ng mL(-1) approximately 40 days post partum. After this time, plasma T4 concentrations decreased until about 50 days post partum, when levels were not different from those in the adult (9.2 +/- 0.7 ng mL(-1), n = 10). In contrast, GH concentrations were greatest early in pouch life, with a maximum concentration of 88.2 ng mL(-1) at 24 days post partum, and thereafter declined to adult levels (4.9 +/- 0.9 ng mL(-1), n = 7) by about 60 days post partum. The temporal relationship between T4 and GH in the developing bandicoot is similar to that seen in developing eutherian mammals, but in the latter species, peak plasma T4 and the decline in GH occur before or soon after parturition, whereas in the bandicoot these events occur more than one month post partum. This comparison between eutherian mammals and a marsupial indicates that the timing of these endocrine changes correlates with key developmental or maturational changes rather than the time of parturition.

Birth has been observed and described in a number of marsupials. However, the ability of the newborn marsupial to move from the uterus to the pouch and locate the teat is still not fully understood. Birth and the path taken by the newborn from the urogenital sinus to the teat within the pouch were filmed in the brushtail possum, Trichosurus vulpecula. Prior to birth, females began to lick the pouch and urogenital sinus vigorously. The young took approximately 2 min to transfer from the urogenital sinus to the pouch and attached to the teat within 10–15 min. To determine the senses used by the newborn possum to reach the pouch, young were removed from the anaesthetised mothers immediately after birth and placed outside the pouch. From the subsequent observations, the newborn possum instinctively crawled upwards. However, when the newborn was in the vicinity of the pouch, odours emanating from the pouch presumably attracted the young. Thus, the senses of gravity and of olfaction were used by the newborn to reach the teat and probably the sense of touch, via the mechanoreceptor Merkel cells around the mouth, allowed the young to attach to the teat.

In marsupials testis determination requires the presence of a Y chromosome. The sex determining region on the Y gene (SRY) is necessary for testicular development in eutherians and it is assumed to play a similar role in marsupials. Relatively few studies have investigated the genetic basis of sexual development, and as yet there is no direct evidence that SRY is required for testis development in marsupials. Studies on intersexual marsupials have revealed a fundamental difference between marsupial and eutherian sex determination. The scrotum of marsupials is analogous, not homologous, to the eutherian scrotum and is under the control of X-linked genes not androgens. The current study describes two bandicoot (Isoodon macrourus) siblings. Both siblings had underdeveloped male reproductive tracts and testicular dysgenesis, one was ascrotal and the other had a diminutive scrotum. Their karyotypes were normal for this species which eliminates the Y chromosome from some somatic tissues. SRY was detected by Southern blotting. SRY, ubiquitin activating enzyme-1 on the Y (UBE1Y) and glucose 6-phosphate dehydrogenase (G6PD) gene expression were examined. UBE1Y was widely expressed in many tissues. SRY gene expression was much lower than normal in the abnormal siblings and may be responsible for their failure of testicular and epididymal development. The cause of their scrotal abnormalities is unknown. It is possible that the separate defects of scrotal and testis development in the two siblings, which had normal relatives, were due to a mutation in a gene common to both developmental pathways.

To determine the effect of relocation on the health of possums the body weights and hormone and immune responses of six male and nine female brushtail possums were monitored for 20 weeks following transfer from the environs of Armidale into enclosures in Brisbane. Over the first 6 weeks of captivity, male possums lost 11.0% of their original body weight and females lost 16.8%. The mean concentrations of plasma cortisol in the male and female possums were 14.5 and 29.4 ng/mL, respectively, and did not change over the 20-week period. Male and female possums displayed a similar pattern of thyroxine secretion over the 20 weeks, with low concentrations up to week seven (2.1 and 2.7 ng/mL, respectively) increasing to 6.9 and 5.8 ng/mL in weeks 7-12 (P < 0.005). This increase in the concentration of thyroxine corresponded with the increase in body weight. The number of white blood cells (WBCs) and the percentage of neutrophils increased from the capture to week 6-10. However, during the last 10 weeks of captivity the number of WBCs and the percentage of neutrophils decreased, indicating recovery of the immune system. This was in accord with the proliferative response of lymphocytes to the T cell mitogen PHA that increased from weeks 11-15 to weeks 16-20 in both male and female possums. The results above suggest that the Armidale possums, like the Brisbane possums, were stressed following their relocation; however, their immune systems were able to gradually recover as they adjusted to their new environment in Brisbane. The death rate of pouch young and of adult female possums after relocation was considerably higher in the Armidale possums compared to Brisbane possums. The mortality rate of Brisbane possums over the first 20 weeks of captivity was 8.3% and 19.6% for male and female possums, respectively, and for Armidale possums 16.6% and 47.1%, respectively. The possums transferred from the environs of Armidale into captivity in Brisbane were under greater stress than possums captured in Brisbane and placed in captivity in Brisbane.