The corpus luteum and interstitial tissue in a marsupial,the brushtail possum (Trichosurus vulpecula)

Proline-rich tyrosine kinase 2 (Pyk2) expression in prostate epithelium inversely correlated with degree of malignancy of prostate cancers, thus the role of Pyk2 in the regulation of prostate cells proliferation and differentiation was investigate in PC3 cells. Pyk2 can be activated by canonic stimuli such as tumor necrosis factoralpha and lysophosphatidic acid (LPA) in PC3 cells, in addition, LPA stimulated Pyk2 phosphorylation also induced extracellular signal-regulated kinase 1 and 2 activation in these cells. Proliferation of PC3 cell clones (PC3-PKM) expressing a dominant negative kinase-defective Pyk2 mutant is consistently decreased in respect to that of wild type PC3 cells. In addition, PC3-PKM clones underwent total block cell proliferation upon treatment with dibutyryl cAMP. Finally, in the presence of sustained levels of intracellular cAMP, PC3-PKM cells, but not wild type PC3 cells, acquired a neuron-like morphology. Taken together our results suggest that Pyk2 plays a role in the regulation of prostate cell proliferation and, more interestingly, its expression may represents a sensitive marker of prostate state of differentiation.     

Plasma gonadotropin concentrations in the cyclic female brushtail possum (Trichosurus vulpecula).

Changes in plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and their relationship to antral follicle development and ovulation, were determined in female brushtail possums (Trichosurus vulpecula) in experiments in which pouch young were removed (RPY) from lactating females to promote ovarian activity. In Experiment 1 (n = 8), the development of preovulatory follicles and ovulation was monitored by laparoscopy. In Experiment 2 (n = 15) estrus and mating were monitored by cytology of urine. Ovulation occurred in 4/8 (Experiment 1) and 9/16 (Experiment 2) possums, and in these animals, plasma FSH concentrations fell progressively over the period of preovulatory follicle development and returned to pretreatment levels after ovulation. With the exception of samples taken at the time of the preovulatory gonadotropin surge, mean plasma LH levels remained basal. In those possums that failed to ovulate, plasma FSH concentrations were elevated while plasma LH concentrations were low; these patterns remained unchanged throughout the sampling period. It was not possible to distinguish between animals that would ovulate and those that would not ovulate after RPY on the basis of gonadotropin profiles at the time of RPY. A further group of possums (Experiment 3, n = 10) were blood-sampled at hourly intervals for 48 h to characterize preovulatory gonadotropin surges, using laparoscopy to monitor preovulatory follicular development and predict ovulation. A preovulatory LH surge (max. conc. 10.2-43.5 ng/ml, duration 7-9 h) was recorded in 4 animals, with a coincident preovulatory FSH surge (max. conc. 1.4-21.4 ng/ml, duration 3-11 h) observed in 3 of these possums. The patterns of gonadotropin secretion in the cycling brushtail possum conform to those reported for eutherians that ovulate spontaneously and appear to be regulated by similar mechanisms.     

Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula)

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.     

Weekly variations in body weight and plasma testosterone concentrations in the captive male possum, Trichosurus vulpecula

The possum is a seasonally breeding marsupial which gives birth in Queensland from March through to September. To ascertain whether this seasonality in breeding is manifest in the male reproductive system, body weight and plasma testosterone concentrations were examined in five possums. Plasma testosterone concentrations fluctuated throughout the year and a seasonal cycle was observed, with a peak in testosterone concentration in March and a nadir in September. Body weights fluctuated in a similar manner. Statistical analysis suggested that the annual plasma testosterone profile correlated well with the rate of change of daylength.     

Prolactin in the brushtail possum (Trichosurus vulpecula): development of homologous radioimmunoassay using recombinant possum prolactin

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.     

The GH/IGF-I axis in the brushtail possum (Trichosurus vulpecula) pouch young

Plasma and pituitary GH concentrations and liver GH receptor (GHR), IGF-I and IGF-binding protein-3 (IGFBP-3) mRNA expression were determined 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-partum but then increased rapidly to a maximum at 50 days post-partum 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.     

Immunocytochemical location of oxytocin and mesotocin within the hypothalamus of two Australian marsupials, the bandicoot Isoodon macrourus and the brushtail possum Trichosurus vulpecula

The presence of oxytocin and mesotocin in the hypothalamus of two Australian marsupials, the bandicoot (Isoodon macrourus) and the brushtail possum (Trichosurus vulpecula), was examined by immunocytochemistry. Tissue was fixed in paraformaldehyde in phosphate buffer and immunoreactive cells were detected using highly specific rabbit antioxytocin and sheep anti-mesotocin as primary antisera. Immunoreactive oxytocin cells were demonstrated in the paraventricular and supraoptic nuclei of bandicoot and possum hypothalami, with greater density being observed in paraventricular nuclei. Immunoreactive mesotocin cells were also found in both hypothalamic nuclei of the possum but not of the bandicoot. The same cells appeared to stain for both peptides.     

The prolactin receptor from the brushtail possum (Trichosurus vulpecula): cDNA cloning, expression and functional analysis

A full length, prolactin receptor cDNA clone has been isolated from the brushtail possum (Trichosurus vulpecula). This clone encodes a 625 amino acid protein which shares 60-70 and 54% sequence identity with prolactin receptor (long form) sequences from mammalian and avian species, respectively. Sequence similarity was highest in the extra-cellular, hormone-binding domain and in specific regions of the intracellular domain which regulates prolactin receptor signalling in cells. Prolactin receptor mRNA was detected in a wide range of possum tissues and in the mammary gland the PRL-R gene was differentially expressed during lactation with peak mRNA levels being detected during the first 6 days of lactation and after day 115 throughout late lactation. This pattern of PRL-R mRNA expression in the mammary gland is similar to that observed for circulating prolactin in the lactating possum. In CHO cells transiently transfected with the possum prolactin receptor, expression of a beta-lactoglobulin promoter/reporter gene construct was increased 3-fold by adding prolactin. The possum prolactin receptor is therefore capable of binding ovine prolactin and activating the Jak2/Stat5 signalling cascade. This provides evidence for the highly conserved nature of the prolactin signalling pathway in mammalian evolution.     

Expression of mRNA encoding growth differentiation factor 9 and bone morphogenetic protein 15 during follicular formation and growth in a marsupial,the brushtail possum (Trichosurus vulpecula)

The oocyte derived growth differentiation factor (GDF) 9 and bone morphogenetic protein 15 (BMP15; also known as GDF9b) are essential for normal follicular growth. However, little is known about expression of these factors during ovarian development. Therefore, we determined the ontogeny of expression of GDF9 and BMP15 mRNA in the developing ovary of the brushtail possum. Ovaries were collected from pouch young (n=3-5 per group) around times of key developmental events namely: (1) morphological sexual differentiation (i.e. days 1-5 following birth), (2) after sexual differentiation (i.e. days 10-15), (3) before and during initiation of germ-cell meiosis (i.e. days 22-45), (4) shortly after initiation of follicular growth (i.e. days 78-85), (5) during preantral follicular growth (i.e. days 96-113) and (6) during antral follicular growth (i.e. days 155-190). Ovaries were also collected from three juvenile and four adult animals and gene expression was determined by in situ hybridization. The mRNAs encoding GDF9 and BMP15 were first observed in oocytes of newly-formed primordial follicles (i.e. days 78-85). Expression of both mRNAs was restricted to the oocyte and was present in follicles irrespective of whether they were non-growing primordial follicles or undergoing preantral or antral development. Thus, since the mRNAs encoding GDF9 and BMP15 were not observed until follicular formation, it is unlikely that these proteins have any role in early germ cell development. Nevertheless, the findings that the mRNAs encoding both proteins were observed in oocytes from the primordial stage of follicular formation suggest a possible role for these proteins in the maintenance of primordial follicles as well as a key role during follicular development. These results highlight important species differences in the ontogeny of expression of GDF9 and BMP15 between possums and other species such as the human, sheep or rat.     

The control of adrenocortical secretion in the brush-tailed possum, Trichosurus vulpecula

Circulating levels of corticosteroids and androgens have been studied in the brush-tailed possum (Trichosurus vulpecula) under conditions of tropic hormone stimulation. In both males and anestrous females, levels of androgen (considered to consist largely of testosterone) were elevated by 3 days of treatment with PMS. Further treatment with ACTH (Synacthen) subsequently reduced the level of testosterone in the females to values significantly below the control values, and in the male to a value intermediate between control and PMS stimulated levels. Levels of corticosteroid (considered to consist largely of cortisol) were unaffected by gonadotropin treatment, but significantly increased with ACTH. Dexamethasone treatment greatly decreased cortisol levels, but androgen levels were unaffected in both sexes.In vitro androgen production by the definitive adrenal cortex of the female was significantly decreased by dexamethasone pretreatment. Androgen production by a special hypertrophied adrenocortical zone was unaffected. Corticosteroid formation was reduced in both types of tissue.It is concluded that the adrenal cortex contributes to circulating testosterone levels in the female possum. This is supported by gonadotropin, whereas ACTH has a double effect of stimulating overall steroidogenesis, but switching the relative proportions of the steroids by decreasing the synthesis of androgen and stimulating the corticosteroid pathway. The possible physiological role for this phenomenon and the function of the special hypertrophied zone in the female adrenal cortex are discussed.     

Plasma concentrations of prolactin in brushtail possums (Trichosurus vulpecula) in different physiological states

Prolactin (Prl) has been implicated in reproduction in many mammalian species and is illustrated by the distinctive patterns of secretion during the breeding season, the oestrous cycle and lactation. The recent development of a homologous RIA for measuring the circulating Prl concentrations in brushtail possums has facilitated the reliable measurement of Prl in plasma during different physiological states in this species for the first time. Determination of Prl concentrations during lactation involved the collection of weekly blood samples from eight female possums from the time of parturition through either one or two consecutive lactational cycles. Prl was at baseline levels during early lactation (weeks 0-14 post-partum), and then increased markedly to maximum concentrations at weeks 19-21 before returning to nadir levels at a time coincident with the weaning of pouch young (weeks 23-27). The profile of Prl secretion over the oestrous cycle and in particular at the time of the preovulatory LH surge was obtained from 14 possums during the reproductive cycle, in which preovulatory follicle development and ovulation were monitored by laparoscopy. There was no distinct daily pattern of Prl secretion during the oestrous cycle; however, in 3/4 possums in which a typical preovulatory LH surge was measured, a biphasic preovulatory Prl surge was also observed. The preovulatory Prl surge commenced 2-6 h prior to, and had returned to baseline close to the onset of, the preovulatory LH surge, and a second surge of Prl occurred concomitantly with the delayed preovulatory FSH surge. Seasonality of Prl levels was established from weekly blood samples collected from six barren female possums, and concentrations of Prl were lower during the breeding season compared to the non-breeding season. Additionally, a circadian pattern of Prl secretion was evident in both female and male possums, with Prl levels higher in the morning compared to the afternoon. In conclusion, interpretation of endogenous secretory patterns suggests that Prl may be important during late lactation and at impending ovulation, but the involvement of the circannual rhythm of Prl in the regulation of seasonality in the brushtail possum remains to be determined.     

Prolactin acts on the hypothalamic-pituitary axis to modulate follicle-stimulating hormone gene expression in the female brushtail possum (Trichosurus vulpecula)

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic-pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 μg) resulted in an 18-fold stimulation (P < 0.05) of progesterone (P₄) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n = 10) or saline (n = 8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P < 0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P < 0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P₄ concentrations were not different (P = 0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.     

Increased protection against bovine tuberculosis in the brushtail possum (Trichosurus vulpecula) when BCG is administered with killed Mycobacterium vaccae

SETTING: The Australian brushtail possum is the major wildlife reservoir for Mycobacterium bovis infection in New Zealand. Development of an effective tuberculosis vaccine for possums will reduce the spread of infection to cattle and farmed deer. OBJECTIVES: To determine whether killed M. vaccae can improve the efficacy of vaccination with M. bovis bacillus Calmette Guerin (BCG) against bovine tuberculosis in the possum. DESIGN: Groups of possums (n=6-8) were vaccinated via intranasal and intraconjunctival routes with BCG alone or BCG in combination with heat-killed M. vaccae. Controls were non-vaccinated or vaccinated with heat-killed M. vaccae alone. After challenge with virulent M. bovis, protection was assessed by a reduction in loss of body weight and bacterial counts in lungs and spleens. Blood lymphocyte proliferative responses to M. bovis purified protein derivative were monitored throughout. RESULTS: The earliest lymphocyte responses following vaccination were from animals inoculated with BCG plus 100 microg heat-killed M. vaccae. Loss of body weight was significantly reduced in all BCG-vaccinated groups compared control groups. Spleen bacterial counts were significantly lower in animals vaccinated with M. vaccae plus BCG compared to the non-vaccinated group. Furthermore, vaccination with 100 microg M. vaccae plus BCG significantly reduced spleen bacterial counts compared to vaccination with BCG alone. CONCLUSION: The possum infection model is one of the first to show that novel vaccine strategies may offer better protection against tuberculosis than BCG alone.     

Peripheral androgen levels in the male brush-tail possum (Trichosurus vulpecula)

Radioimmunoassays were established for the measurement of total androgens and the specific measurement of testosterone and 5 alpha-dihydrotestosterone in peripheral plasma of the brush-tail possum. Androgen concentrations were measured in blood collected from indwelling jugular cannulae to determine whether the normal pattern of androgen secretion in this species was episodic and to attempt to relate total androgen and the pattern of testosterone secretion to the changes previously reported in prostatic, but not epididymal, weight in the breeding season. Blood was collected from restrained animals at varying time-intervals during daylight hours and darkness. Despite an apparent good adaptation to the sampling procedure there was generally a progressive decline in plasma androgen level during the collection period. This was true for animals bled during or out of the breeding season. There was no significant seasonal effect on the androgen concentration in the initial blood sample. When less restraint was used, two of three animals showed fluctuations in androgen levels over the 7-h sampling period. Testosterone levels in blood obtained by cardiac puncture were four- to nine-fold higher than those of 5 alpha-dihydrotestosterone but levels of these androgens in samples obtained during the breeding season were not significantly different from those obtained out of season. The results do not argue for a pulsatile release of testosterone in the possum but do demonstrate a marked capacity for changes in the peripheral androgen concentration. There was a poor correlation between testosterone and 5 alpha-dihydrotestostosterone levels and prostatic weight.     

Corticosteroid binding by plasma proteins and the effects of oestrogens in the marsupial brush-tailed opossum, Trichosurus vulpecula (Kerr).

Using the techniques of equilibrium dialysis at 36 degrees C and gel filtration at 4 degrees C, a high-affinity, transcortin-like, corticosteroid binding system has been demonstrated in the blood plasma of the marsupial Trichosurus vulpecula. The affinity constant for non-albumin binding was 4-018 +/- 1-032 X 10(7) (S.C.) 1/mol for males and 4-046 +/- 0.981 X 10(7) for females. The concentration of non-albumin binding sites was 1-82 +/- 0.76 X 10(-7) mol/1 in males and 1-86 +/- 0-57 X 10(-7) mol/1 for females. Oestrogen administration, sufficient to cause marked hypertrophy of the genital tract in the femlaes, had no effect on the affinity constant or the concentration of the non-albumin binding sites in either males or females. The general condition of the animals deteriorated during oestrogen administration and there were significant falls in the concentrations of albumin and cortisol in the blood plasma. In one animal which died during oestrogen treatment, the adrenal glands were significantly hypertrophied.