Douglas C. Eckery

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.

Effects of a Gonadotropin-Releasing Hormone Agonist Implant on Reproduction in a Male Marsupial, Macropus eugenii

Abstract 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 anti-Müllerian hormone mRNA during gonadal and follicular development in the brushtail possum (Trichosurus vulpecula)

The ontogeny of anti-Müllerian hormone (AMH) gene expression in the brushtail possum during formation of the ovary and growth of follicles was examined using in situ hybridization. For comparative purposes, the expression pattern of AMH was also examined in the developing testis. In the female, AMH mRNA was observed in the ovary of 50% (3/6) of pouch young collected around the time of sexual differentiation of the gonad (Days 1-5): the signal was predominately localized to the inner-cortical and outer-medullary region of the ovary. Thereafter, AMH mRNA was not observed in the developing ovary until Days 78-113 of postnatal life when follicles first formed at the cortical-medullary boundary. At this time, AMH mRNA was observed in the cuboidal granulosa cells of some early growing (i.e. transitional) follicles and in the granulosa cells of primary follicles. Thereafter, AMH mRNA was present in granulosa cells at all subsequent stages of follicular growth (i.e. primary through antral), but not in preovulatory follicles. In all cases, once follicles had formed, AMH mRNA was limited to the granulosa cells and was not observed in the surface epithelium, stromal cells, oocytes, theca, corpus luteum, medullary cords, rete or interstitial glands. In the possum testis, Sertoli cells strongly expressed AMH around the time of sexual differentiation of the gonad, but expression decreased to very low levels in adults, suggesting that AMH plays a similar role in brushtail possums to that observed in other mammalian species. In conclusion, localization of mRNA for AMH exclusively to granulosa cells of growing follicles in the brushtail possum is consistent with a central role for this hormone in control of granulosa cell function in marsupials. In addition, expression of AMH in the developing ovary around the time of morphological sexual differentiation raises intriguing questions regarding the possible role of AMH at this time.

Gene Expression of the Tyrosine Kinase Receptor c-kit During Ovarian Development in the Brushtail Possum (Trichosurus vulpecula)

Abstract Ovarian development and function have been extensively studied in eutherian species, with stem cell factor and its receptor, c-kit, having been shown to play key roles at various stages of these processes. In contrast, relatively little is known regarding ovarian development in marsupials. The aims of this study were, first, to establish the timing of key events during germ cell maturation and follicular development and, second, to determine the timing and cellular localization of gene expression for c-kit in the ovaries of a marsupial, the brushtail possum (Trichosurus vulpecula). For this study, ovaries were collected from possums ranging in age from Day 1 after birth to adult. Using stereology, the number of germ cells was found to increase rapidly during the first 60–100 days of life. This was followed by a sharp decline in number, wherein almost 90% of germ cells had disappeared by Day 180. From histological examinations, the time of initiation of meiosis, follicular formation, and follicular growth were determined to occur on Days 35, 50, and 60, respectively. Using in situ hybridization, c-kit gene expression was localized to germ cells and somatic cells during the first 15 days of life; however, after Day 30 and into adult life, c-kit expression was exclusive to germ cells. Results from this study suggest that the pattern of ovarian development is similar in marsupials to eutherians, and that c-kit may play a key role in germ cell development at various stages throughout life.

Ovarian Expression of Messenger RNA Encoding the Receptors for Luteinizing Hormone and Follicle-Stimulating Hormone in a Marsupial, the Brushtail Possum (Trichosurus vulpecula)

Abstract Both LH and FSH play a central role in controlling ovarian function in mammals. However, little is known about the type of ovarian cells that are responsive to LH and FSH in marsupials. We determined, using in situ hybridization, the localization of mRNA encoding the receptors (R) for LH and FSH in ovaries of brushtail possums. The mRNA encoding FSH-R was observed in granulosa cells of healthy follicles containing at least two complete layers of cells. The mRNA encoding LH-R was first observed in granulosa cells at the time of antrum formation. Cells of the theca interna expressed LH-R mRNA but not FSH-R mRNA. Neither FSH-R nor LH-R mRNA was detected in atretic follicles. Both FSH-R and LH-R mRNAs were observed in luteal tissue, but only LH-R mRNA was observed in interstitial cells. Granulosa cells from follicles of various sizes (0.5 to >2 mm in diameter) responded to LH and FSH treatment with an increase in cAMP synthesis. In contrast, luteal tissue did not respond to either FSH or LH treatment. In conclusion, expression of FSH-R in the brushtail possum ovary was similar to that observed in many eutherian mammals. However, active LH-R was expressed in granulosa cells much earlier in follicular development than has been previously observed. In addition, although mRNAs for both FSH-R and LH-R were observed, neither FSH nor LH treatment stimulated cAMP synthesis in luteal tissue.

Expression of the FcRn receptor (α and β) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young

Abstract The neonatal IgG transporter FcRn consists of two chains, FcRn α and β (also known as β 2 microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG α and β chain mRNA levels were investigated in the gut of brushtail possum ( Trichosurus vulpecula ) pouch young (PY) using Northern hybridisation. FcRn α 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 β 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 α and β 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 α and β 2 microglobulin genes were cloned and analysed for predicted cis -acting transcription control elements. Both the FcRn α and β 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 α and β 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 α and β 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 α and β 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.

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

The Australian brushtail possum (Trichosurus vulpecula) is a nocturnal, arboreal marsupial. It has become a pest of significant ecological and economic importance in New Zealand, and thus a renewed interest in understanding the reproductive biology of this species has been generated. The corpus luteum (CL) in possums is a largely autonomous gland in that it does not rely on pituitary hormones to function and is not responsive to luteolytic agents for its demise. Its importance in regulating the oestrous cycle and pregnancy has been established; however, little is known regarding the mechanisms involved in its function. Interstitial tissue (IT) is a prominent feature found throughout the ovarian stroma, yet little is known regarding the origin or function of these cells. Based on histological examinations, our data support the hypothesis that interstitial tissue arises from a unique cell type called medullary cords during early ovarian development. Using possum-specific probes for proteins involved in steroidogenesis, receptors for pituitary hormones and members of the TGF-beta superfamily we have initiated studies investigating the expression of genes that may be important in the function and regulation of the CL and interstitial tissue. Results show that both tissues are steroidogenic and that both express receptors for prolactin and luteinising hormone (LH). Collectively these findings suggest that prolactin and LH may be important in the regulation of steroidogenesis in the CL and interstitial tissue in possums.

Determination of Steroidogenic Potential of Ovarian Cells of the Brushtail Possum (Trichosurus vulpecula)

Abstract The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase/Δ5,Δ4 isomerase (3βHSD), cytochrome P45017αhydroxylase (P45017αOH), and P450 aromatase (P450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain P450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed P450arom and 3βHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, P450scc, 3βHSD, and P45017αOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed P450arom, indicating that this tissue also has the potential to secrete estrogens in this species.

Ontogeny and pathway of formation of 5α-androstane-3α,17β-diol in the testes of the immature brushtail possum Trichosurus vulpecula

The testicular androgen 5α-androstane-3α,17β-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with 3H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Δ4-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5α-reduced androgen (androsterone) were synthesised by testes from Day 87–200 males and these appeared to be formed from the 5α-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, 3H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.

The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.