Chronic treatment of female tammar wallabies with deslorelin implants during pouch life: effects on reproductive maturation.

The present study reports on attempts to delay puberty in a model marsupial species using the gonadotrophin-releasing hormone (GnRH) agonist deslorelin. Female tammar wallaby pouch young received deslorelin (5 mg) or placebo implants (n=8/group) when they were 193±2 days old. Sexual maturity was significantly delayed in deslorelin-treated animals, with the first successful production of offspring in treated and control animals occurring at 813±62 and 430±42 days of age, respectively. This delay was associated with a period of retarded pouch and teat development. Progesterone concentrations remained at basal levels throughout the first breeding season, indicating the absence of luteal cycles in treated females. Recovery and maturation of the hypothalamic-pituitary axis was a gradual process. Treated animals failed to respond to GnRH challenge at 12 months of age and had a reduced LH response at 18 months of age, before attaining full responsiveness by 24 months of age. Despite this apparent pituitary recovery by 24 months of age, as evidenced by complete teat eversion and LH responsiveness to GnRH, the time to first parturition was significantly delayed beyond this time in three females. This suggests that there may be longer-lasting effects at the level of the ovary and/or on FSH secretion. The significant delay in the onset of sexual maturation in response to chronic GnRH agonist treatment in this model marsupial species may be of practical significance to the management of fertility in captive and semi-free range marsupial populations.

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 (P(4)) 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 P(4) 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.

Gene expression and secretion of LH and FSH in relation to gene expression of GnRH receptors in the brushtail possum (Trichosurus vulpecula) demonstrates highly conserved mechanisms.

In eutherian mammals, the gonadotrophins (LH and FSH) are synthesized and stored in gonadotroph cells under the regulation of multiple mechanisms including GnRH. Very little is known about the regulation of gonadotrophin secretion and storage in pituitary glands of marsupials. This study revealed, using quantitative PCR and heterologous RIA techniques, that LHB mRNA expression levels remained constant over the oestrous cycle, regardless of the presence of a preovulatory LH surge, which is characteristic of a hormone secreted under regulation. Our sampling regime was unable to detect pulses of LH during the follicular phase, although GNRHR mRNA levels had increased at this time. Pulses of LH were, however, detected in the luteal phase of cycling females, in anoestrus females and in males. There was a positive correlation between gene expression of FSHB and plasma levels of FSH at different stages of the oestrous cycle and no pulses of FSH were detected at any time; all characteristics of a hormone secreted via the constitutive pathway. Using in situ hybridisation and immunohistochemistry methods, we determined that mRNA expression of LHB and FSHB, and protein storage of gonadotrophins exhibited a similar pattern of localisation within the pituitary gland. Additionally, sexual dimorphism of gonadotroph populations was evident. In summary, these findings are similar to that reported in eutherians and considering that marsupial evolution diverged from eutherians over 100 million years ago suggests that the regulation of gonadotrophins is highly conserved indeed.

Chronic treatment of male tammar wallabies with deslorelin implants during pouch life: effects on development, puberty, and reproduction in adulthood.

The present study evaluated the effects of chronic GnRH agonist (deslorelin) treatment on sexual maturation in the male tammar wallaby. Slow-release deslorelin or placebo implants were administered to male pouch young (n = 10/group) when they were between 180 and 200 days old, to determine if disruption of the pituitary-testicular axis during development altered the timing of sexual maturation or had long-term effects on adult reproductive function. Deslorelin treatment caused retardation of testicular growth and reduced the serum FSH and testosterone concentrations between 12 and 24 mo of age. Maturation of the hypothalamic-pituitary-testicular axis was also delayed in treated animals at 13 and 19 mo of age. Despite these alterations in the pattern and timing of neuroendocrine development, sexual maturation was not permanently blocked in these animals and deslorelin-treated animals reached sexual maturity at the same age as treated animals, as evidenced by a fully functional pituitary-testicular axis and proven fertility at 25 mo of age. The ability of the treated animals to reach puberty at the same time as control animals, despite delayed maturation of the hypothalamic-pituitary-testicular axis, suggests that puberty in the male tammar wallaby is additionally regulated by other, gonadotropin-independent factors.

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 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.

Long-term effects of deslorelin implants on reproduction in the female tammar wallaby (Macropus eugenii).

The contraceptive and endocrine effects of long-term treatment with implants containing the GnRH agonist deslorelin were investigated in female tammar wallabies (Macropus eugenii). Fertility was successfully inhibited for 515 +/- 87 days after treatment with a 5 mg deslorelin implant (n = 7), while control animals gave birth to their first young 159 +/- 47 days after placebo implant administration (n = 8). The duration of contraception was highly variable, ranging from 344 to 761 days. The strict reproductive seasonality in the tammar wallaby was maintained once the implant had expired. This inhibition of reproduction was associated with a significant reduction in basal LH concentrations and a cessation of oestrous cycles, as evidenced by low progesterone concentrations. There was evidence to suggest that some aspect of either blastocyst survival, luteal reactivation, pregnancy or birth may be affected by deslorelin treatment in some animals. These results show that long-term inhibition of fertility in the female tammar wallaby is possible using slow-release deslorelin implants. The effects of deslorelin treatment were fully reversible and there was no evidence of negative side effects. Slow-release GnRH agonist implants may represent a practicable method for reproductive management of captive and semi-wild populations of marsupials.

Oral and intraperitoneal immunization with rotavirus 2/6 virus-like particles stimulates a systemic and mucosal immune response in mice.

We examined the use of a virus-like particle (VLP) as an immunogen by analysing the IgA and IgG response generated in serum, intestinal (fecal), pulmonary and uterine samples. The particle comprised two rotavirus capsid proteins (simian VP2 and murine VP6) generated using recombinant baculovirus expression of the two capsid proteins, which self-assembled into particulate VLP2/6. Mice were immunized orally or intraperitoneally (i.p.) with 0 or 100 microg VLP2/6 with or without 5 microg cholera toxin adjuvant. The results showed a systemic and mucosal immune response to VLP2/6 when administered i.p. and, to a lesser extent, when delivered orally which was not dependent on adjuvant use and further proves the concept of VLP2/6 as an effective immunogen.

Effects of a gonadotropin-releasing hormone agonist implant on reproduction in a male marsupial, Macropus eugenii.

This study evaluated the potential of slow-release GnRH agonist (deslorelin) implants to inhibit reproductive function in the male tammar wallaby. The specific aim was to measure the effects of graded dosages of deslorelin on testes size and plasma LH and testosterone concentrations. Adult male tammar wallabies were assigned to four groups (n = 6 per group) and received the following treatment: control, placebo implant; low dose, 5 mg deslorelin; medium dose, 10 mg; high dose, 20 mg. All dosages of deslorelin induced acute increases (P < 0.001) in plasma LH and testosterone concentrations within 2 h, with concentrations remaining elevated during the first 24 h but returning to pretreatment levels by Day 7. Thereafter, there was no evidence of a treatment-induced decline in plasma testosterone concentrations. There was no detectable difference in basal LH concentrations between treated and control animals, nor was there a significant change in testes width or length (P > 0.05). These results suggest that the male tammar wallaby is resistant to the contraceptive effects of chronic GnRH agonist treatment. Despite the maintenance of testosterone secretion, the majority of male tammars (10 of 17) failed to respond to a GnRH challenge with a release of LH between Days 186 and 197 of treatment. The failure of animals to respond to exogenous GnRH suggests a direct effect of deslorelin on the pituitary, resulting in a level of desensitization that was sufficient to inhibit a LH surge but insufficient to inhibit basal LH secretion. The variation between animals is believed to result from earlier recovery of some individuals, in particular those that received a lower dose, or individual resistance to the desensitization process.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

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.

Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles.

In sheep, growth and development of ovarian follicles beyond 2 mm in diameter is acutely dependent on gonadotropin support. As a consequence, following hypophysectomy (HPX) or hypothalamic-pituitary stalk disconnection (HPD), growth of follicles beyond 2 mm is arrested and all follicles > 2 mm undergo atresia. Although administration of exogenous gonadotropins stimulates follicular growth and ovulation in HPD ewes, follicles in HPX ewes remain unresponsive unless growth hormone (GH) is also given. To determine whether the difference in follicular sensitivity to gonadotropins after HPD (gonadotropin sensitive) or HPX (gonadotropin insensitive) is related to the distribution and quantity of binding sites for FSH, LH, and/or insulin-like growth factor I (IGF-I), binding sites for these hormones were localized and quantified using topical autoradiography in healthy follicles from control (pituitary-intact), HPD, and HPX ewes. In addition, in situ hybridization was performed to localize mRNA for GH and FSH receptors. Irrespective of treatment, binding of FSH and mRNA for FSH receptor were greatest (p < 0.05) in the membrana granulosa; LH binding was greatest (p < 0.05) in the theca interna; and IGF-I binding was greatest (p < 0.05) in the theca externa. Although the relative number of binding sites for LH did not differ among treatments, those for FSH and IGF-I were lower (p < 0.05) in HPD and HPX ewes compared to controls. Attempts to quantify binding sites for GH were unsuccessful due to high nonspecific binding. However, mRNA for GH receptor was most abundant (p < 0.05) in the membrana granulosa and oocytes of small antral and preantral follicles. Compared to levels in controls and HPD ewes, the level of GH receptor mRNA was lower (p < 0.05) in follicles obtained from HPX ewes. On the basis of these data, failure of small antral follicles in HPX ewes to respond to exogenous gonadotropins is not due to a reduction in receptors for FSH, LH, or IGF-I. The observed reduction of mRNA for GH receptor in the membrana granulosa of follicles from HPX ewes provides evidence that GH may play an important role in early stages of folliculogenesis and that it is involved in the maintenance of sensitivity to gonadotropins.

Effects of luteinizing hormone and growth hormone on luteal development in hypophysectomized ewes.

To test the hypothesis that growth hormone (GH) as well as luteinizing hormone (LH) is required for normal luteal growth and function, 16 western range ewes were hypophysectomized (HPX) on day 5 of the estrous cycle. Ewes were randomly assigned to receive saline (S), LH, GH, or LH + GH (n=4 per group) from the time of HPX until collection of corpora lutea 7 days after HPX (day 12). Corpora lutea were also collected from pituitary-intact ewes on days 5 (day 5 control,n=4) and 12 (day 12 control,n=4) of the estrous cycle. To assess luteal function, concentrations of progesterone in sera, luteal weights and luteal concentrations of mRNA encoding cytochrome P450 side-chain cleavage enzyme (P450(scc)) and 3ß-hydroxysteroid dehydrogenase/Δ5,Δ4 isomerase (3ß-HSD) were determined. Concentrations of progesterone in sera and luteal weights increased between days 5 and 12 of the estrous cycle in control ewes, but not in HPX + S ewes. In HPX ewes treated with LH, concentrations of progesterone in sera and luteal mRNA for P450(scc) and 3ß-HSD increased but luteal weights were unaffected. Treatment with GH increased luteal weight and luteal concentrations of mRNA encoding P450(scc) but did not increase concentrations of mRNA encoding 3ß-HSD compared to HPX + S ewes. Concentrations of progesterone in sera of GH-treated, HPX ewes were similar to those of day 12 control ewes but not significantly different from those in HPX + S ewes. Treatment of HPX ewes with LH + GH increased all parameters of luteal function measured to values similar to those in day 12 controls. In conclusion, both GH and LH are necessary for normal luteal development in the ewe.

Recombinant bovine somatotropin does not improve superovulatory response in sheep.

Although treatment of heifers and ewes with recombinant bovine somatotropin (rbST) does not increase ovulation rate, data for heifers indicate that the number of small antral follicles is approximately doubled. Accordingly, the objectives of this study were to determine whether 1) treatment of ewes with rbST would increase the number of small antral follicles, thereby increasing the number of follicles that could potentially respond to superovulation treatment, and 2) superovulatory responses could be improved in ewes with "synchronized" populations of follicles. Twenty-four ewes were divided into four groups: control, control+rbST, hypothalamic-pituitary stalk disconnected (HPD), and HPD+rbST. Beginning on d 5 of the estrous cycle, ewes were injected once daily for 13 d with either rbST (3 mg) or saline. The superovulatory regimen consisted of a single dose of PMSG followed by twice-daily injections of FSH for four consecutive days. After ovariectomy, ovulation sites and follicles were counted. Twice-daily blood samples were assayed for somatotropin (ST) and IGF-I. The concentrations of ST in rbST-treated ewes were greater (P < .05) than those in controls. Treatment with rbST increased (P < .05) the mean serum concentration of IGF-I in control but not in HPD ewes. There was no increase in ovulation rate or number of small antral follicles in response to rbST. Synchronizing follicle populations also failed to increase ovulation rate or reduce variability of response. We conclude that supplementation with rbST and synchronization of follicles does not increase the superovulatory response in sheep.