Effects of the Immunocontraceptive Gonacon on Pregnancy in Brucella-Seropositive American bison (Bison bison).

The purpose of this study was to determine if the number of pregnancies in naturally infected Brucella abortus-positive bison (Bison bison) cows would be reduced over a period of 5 yr after one treatment with 3000 µg gonadotropin-releasing hormone immunocontraceptive (GonaCon) compared to a similar group of naturally infected B. abortus-positive bison cows not treated with GonaCon. In each of the 5 yr, GonaCon-treated cows produced fewer offspring in relation to number of cows than the nontreated cows. Fisher's Exact test comparing offspring produced during the first reproductive season showed a significant difference between the two groups (P=0.0028). Differences in number of calves produced in GonaCon-treated and control groups were also noted in remaining years, but statistics were not applied because of data constraints. These data indicate that one treatment with GonaCon in brucellosis-seropositive female bison reduced pregnancies over five reproductive years. Thus, immunocontraception could potentially be used to manage brucellosis in affected herds.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

Long-term effect of a GnRH-based immunocontraceptive on feral cattle in Hong Kong.

Increasing human-wildlife conflicts worldwide are driving the need for multiple solutions to reducing "problem" wildlife and their impacts. Fertility control is advocated as a non-lethal tool to manage free-living wildlife and in particular to control iconic species. Injectable immunocontraceptives, such as GonaCon, stimulate the immune system to produce antibodies against the gonadotrophin-releasing hormone (GnRH), which in turn affects the release of reproductive hormones in mammals. Feral cattle (Bos indicus or Bos taurus) in Hong Kong are an iconic species whose numbers and impacts on human activities have increased over the last decade. Previous studies have proven that a primer vaccination and booster dose of GonaCon in female cattle are safe and effective in reducing pregnancy levels one year post-treatment. The aims of this project were 1. to evaluate the longevity of the effect of GonaCon in feral cattle up to four years post-vaccination; and 2. to assess if a second booster dose of GonaCon, administered at either two or four years post-vaccination, extends the contraceptive effect in this species. Vaccination with GonaCon, administered as a primer and booster dose, was effective in causing significant infertility in free-living cattle for at least three years post-vaccination, with the percentage of pregnant animals in the vaccinated group decreasing from 76% at vaccination to 35%, 19% and 7% in years 2, 3 and 4 post-vaccination, compared with 67% at vaccination to 50%, 57% and 14% respectively in the control group. A second booster dose of GonaCon administered either 2 or 4 years after vaccination rendered 100% of the Treated cattle infertile for at least another year. These results suggested that vaccination with GonaCon can reduce feral cattle population growth and that a second booster dose can extend the longevity of the contraceptive effect.

Short Term Safety, Immunogenicity, and Reproductive Effects of Combined Vaccination With Anti-GnRH (Gonacon) and Rabies Vaccines in Female Feral Cats.

Overpopulation of free-roaming cats is a major problem leading to negative impacts on animal health and welfare, public nuisance, transmission of zoonotic diseases, and well-documented harm to wildlife. Surgical sterilization had failed to provide a practical solution to free-roaming cats' overpopulation under field conditions; therefore, efficient and safe non-surgical immunocontraception methods are aspired. Rabies is a deadly virus that may infect people and animals. However, the safety and efficacy of combined vaccination with anti-GnRH and rabies vaccines in feral cats, which often suffer from disrupted health conditions and experienced high stress level, has never been studied. Therefore, our objective was to examine the short-term safety and efficacy of anti-GnRH vaccine (Gonacon), in combination with rabies vaccine in female feral cats. Mature feral female cats were captured and divided into the following groups: (I) GonaconX1-Rabies: queens vaccinated with both Gonacon and rabies (n = 5); (II) GonaconX2-Rabies: queens vaccinated twice with Gonacon (3 weeks apart) and with Rabies (n = 4); (III) OVx-Rabies: queens ovariohysterectomized and vaccinated with rabies (n = 4); (IV) Intact-Rabies: queens vaccinated against rabies and remained intact (n = 3). Comprehensive veterinary examinations and blood tests were performed every 2 weeks for 14 weeks. Data were analyzed by Repeated-Measures-ANOVA or Fisher-Exact-Test. There were neither systemic nor local adverse reactions at the vaccination sites. Blood count (PCV, TS, RBC, HGB, HCT, WBC) and chemistry (Total protein, Total globulin, Albumin, Urea, Creatinine, Creatine kinase, Bilirubin, GGT, ALT, AST) analyses revealed no differences among groups. There were no differences in serum rabies antibodies titers among groups, and queens kept a protective titer (>0.5 IU/mL) starting at 2-4 weeks after vaccination. Anti-GnRH antibodies were detected in all Gonacon-vaccinated queens, excluding one queen (GonaconX2-Rabies group). Anti-müllerian hormone serum concentrations reduced significantly after ovariohysterectomy, as well as gradually following vaccination with Gonacon, but it remained high in intact queens. Evaluation of vaginal cytology and ovarian histology suggested that reproductive cyclicity was suppressed in Gonacon-vaccinated queens. Our results support the conclusion that in the short term, the combined vaccination with Gonacon and rabies is safe and effective in female feral cats. However, further long-term studies are warranted to test this immunologic regimen in feral cats.

Fertility control for managing free-roaming feral cattle in Hong Kong.

Human-wildlife conflicts are increasing worldwide. For instance, growing numbers of free-roaming feral cattle in Hong Kong are causing traffic accidents and damaging crops. Public antipathy towards lethal methods to manage wildlife has promoted research into alternative options, such as fertility control. The aims of this study were to assess the potential side effects and effectiveness of the injectable immunocontraceptive vaccine GonaCon on free-roaming feral cattle in Hong Kong. Sixty female cattle were captured and randomly assigned to treatment or control groups. Treatment animals were administered one dose of GonaCon, followed by a booster dose 3-6 months later. Control animals were administered an equivalent dose of a saline solution. The side effects of GonaCon were assessed by monitoring injection site, body condition and body weight at vaccination, at the booster stage and one year after initial vaccination. At the same times, blood samples were collected to quantify antibodies to the vaccine and to assess pregnancy status. GonaCon did not affect the body weight or body condition of cattle and had no adverse side effects such as injection site reactions, limping or abnormal behaviour. GonaCon did not appear to interrupt ongoing pregnancies but reduced fertility significantly: the proportion of pregnant animals in the GonaCon-treated group decreased from 76% at initial vaccination to 6% one year after vaccination, compared to 67% and 57% respectively in the control group. There was no difference between antibody titres at the booster stage or one year post vaccination, suggesting the booster dose maintained antibody levels. This study confirmed that GonaCon is safe and effective in inducing infertility in feral cattle, with a booster dose critical for maintaining infertility.

Reimmunization increases contraceptive effectiveness of gonadotropin-releasing hormone vaccine (GonaCon-Equine) in free-ranging horses (Equus caballus): Limitations and side effects.

Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (Equus caballus) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009-2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (n = 29) or a saline control group (n = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (P = 0.03) and third (P = 0.08) post-treatment foaling seasons but was similar (P = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (P <0.001) than that for control mares for three consecutive years and ranged from 0.0-0.16. The only detectable adverse side effect of vaccination was intramuscular swelling at the vaccination site. Regardless of vaccine treatment (primary/secondary), approximately 62% (34/55) of immunized mares revealed a visible reaction at the vaccine injection site. However, none of these mares displayed any evidence of lameness, altered gait or abnormal range of movement throughout the 8 years they were observed in this study. Our research suggests that practical application of this vaccine in feral horses will require an initial inoculation that may provide only modest suppression of fertility followed by reimmunization that together could result in greater reduction in population growth rates over time.

Effects of immunization against bone morphogenetic protein-15 and growth differentiation factor-9 on ovarian function in mares.

Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n = 10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a control of phosphate buffered saline and adjuvant. Horses received vaccinations at weeks 0, 6, 12, and 18. Ovarian activity and estrous behavior were evaluated 3 days a week via ultrasonography and interaction with a stallion. The study was initiated on March1, 2016. Upon evaluation of ovulation rate, the GDF-9 group did not have a difference (P = 0.66) in ovulation rate when compared to controls (10.8 and 10.0 ovulations, respectively), but the number of ovulations in the BMP-15 group was less (P = 0.02; 4.9 ovulations). Average follicle size prior to ovulation was less (P < 0.0001) in both treatment groups compared to controls. Estrous behavior was altered in both the BMP-15 and GDF-9 groups compared to controls after the second vaccination (P = 0.05 and 0.03, respectively). Although further research is required to determine the continued effects of vaccination against GDF-9 on ovulation rates, these results indicate that vaccination against BMP-15 and GDF-9 could serve as a contraceptive in wild horse populations.

Evaluation of antibody response to an adjuvanted hapten-protein vaccine as a potential inhibitor of sexual maturation for farmed Atlantic salmon.

An experimental contraceptive vaccine was evaluated in Atlantic salmon (Salmo salar). A peptide derived from the beta subunit of luteinizing hormone (LH) was conjugated to two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and formulated with one of four immunostimulants in a water-in-oil emulsion. Specific antibody responses to the peptide and each carrier protein were evaluated. While the antibody response to KLH was stronger than the response to BSA, both carrier proteins stimulated comparable antibody responses to the LH peptide. The immunostimulant proved to be more important for enhancing the LH peptide antibody response than the carrier protein selection; vaccines containing a combination of Aeromonas salmonicida and Vibrio anguillarum stimulated significantly greater LH peptide antibody production than any of the other three immunostimulants evaluated at 12 weeks post-vaccination. This study provides proof-of-concept for specific antibody production against a hapten-carrier protein antigen in Atlantic salmon and reinforces the importance of vaccine immunostimulant selection.

The Role of Dog Population Management in Rabies Elimination-A Review of Current Approaches and Future Opportunities.

Free-roaming dogs and rabies transmission are integrally linked across many low-income countries, and large unmanaged dog populations can be daunting to rabies control program planners. Dog population management (DPM) is a multifaceted concept that aims to improve the health and well-being of free-roaming dogs, reduce problems they may cause, and may also aim to reduce dog population size. In theory, DPM can facilitate more effective rabies control. Community engagement focused on promoting responsible dog ownership and better veterinary care could improve the health of individual animals and dog vaccination coverage, thus reducing rabies transmission. Humane DPM tools, such as sterilization, could theoretically reduce dog population turnover and size, allowing rabies vaccination coverage to be maintained more easily. However, it is important to understand local dog populations and community attitudes toward them in order to determine whether and how DPM might contribute to rabies control and which DPM tools would be most successful. In practice, there is very limited evidence of DPM tools achieving reductions in the size or turnover of dog populations in canine rabies-endemic areas. Different DPM tools are frequently used together and combined with rabies vaccinations, but full impact assessments of DPM programs are not usually available, and therefore, evaluation of tools is difficult. Surgical sterilization is the most frequently documented tool and has successfully reduced dog population size and turnover in a few low-income settings. However, DPM programs are mostly conducted in urban settings and are usually not government funded, raising concerns about their applicability in rural settings and sustainability over time. Technical demands, costs, and the time necessary to achieve population-level impacts are major barriers. Given their potential value, we urgently need more evidence of the effectiveness of DPM tools in the context of canine rabies control. Cheaper, less labor-intensive tools for dog sterilization will be extremely valuable in realizing the potential benefits of reduced population turnover and size. No one DPM tool will fit all situations, but if DPM objectives are achieved dog populations may be stabilized or even reduced, facilitating higher dog vaccination coverages that will benefit rabies elimination efforts.

Immunocontraception for managing feral cattle in Hong Kong.

Conflicts between human interests and feral cattle in Hong Kong derive from growing numbers of free-roaming cattle. Public antipathy towards lethal population control led the local authorities to consider fertility control to reduce cattle numbers. This study assessed the potential side effects of the immunocontraceptive GonaCon on individual female cattle and established the effectiveness of GonaCon to induce infertility. We evaluated GonaCon in 34 captive cattle assigned to four groups: Control administered a sham solution; Webbed (surgically sterilized through removal of the oviducts), administered one dose of GonaCon; Webbed, administered one dose of GonaCon and a booster dose three months later, and Treated, administered one dose of GonaCon. The side effects of GonaCon were assessed by monitoring injection site, body weight, body condition, size of lymph nodes, body temperature, and feeding behaviour 1 week and 1, 3, 6, 9 and 12 months after vaccination and by haematological and biochemical variables at vaccination and three months post-vaccination. The effectiveness of GonaCon to cause infertility was monitored by quantifying anti-GnRH antibody titres and by using kits to detect cycling and pregnancy. GonaCon-treated cattle showed no injection site reaction, limping, or abnormal behaviour. No differences were observed in all physiological and welfare indicators between control and vaccinated cattle. All control cattle and 4 of the 12 cattle in the Treated group became pregnant. Cattle administered a booster dose had higher anti-GnRH antibody titres than cattle that received one dose. We concluded that GonaCon does not compromise the animals' welfare and is effective in reducing fertility in cattle. A booster dose is likely to increase the duration of infertility. Further studies are required to assess the feasibility and costs of immunocontraception for controlling free-roaming cattle populations.

Twenty years of immunocontraceptive research: lessons learned.

The National Wildlife Research Center (NWRC) began immunocontraception vaccine research by testing porcine zona pellucida (PZP) on white-tailed deer (Odocoileus virginianus). Early PZP research demonstrated that PZP induced infertility; however, increased length of the rut was observed in PZP-treated deer. An alternative vaccine using a keyhole limpet hemocyanin-gonadotropin-releasing hormone (KLH-GnRH) conjugate formulated with modified Freund's adjuvant was developed at NWRC. Suppression of GnRH has reduced reproduction in both sexes but is most effective in females. This vaccine was effective in preventing contraception in female deer for several years after a prime and boost. Due to adverse side effects of Freund's adjuvant, NWRC developed a new adjuvant called AdjuVac, a mineral oil/surfactant adjuvant with the addition of Mycobacterium avium as an immunostimulant. The price of KLH prompted a search for a more economical hemocyanin carrier protein for the GnRH peptide. Blue protein, derived from the mollusk Concholepas concholepas, proved to be a successful option. Formulation improvements resulted in a vaccine that can be effective as a single injection for multiple years, now called GonaCon. GonaCon is registered with the Environmental Protection Agency (EPA) for use in white-tailed deer in urban/suburban areas and for wild horses (Equus caballus) and burros (Equus asinus). Future GonaCon applications may include reducing reproduction to manage populations of other wildlife species, such as prairie dogs (Cynomys ludovicianus) in urban areas and suppressing reproduction to reduce the spread of venereal diseases such as brucellosis. Research is being conducted to develop a GnRH vaccine used in combination with the rabies vaccine to control population growth in free-roaming dogs, with the secondary effect of managing the spread of rabies. The EPA would regulate all these uses. Research is also ongoing on a GnRH vaccine to delay the onset of adrenocortical disease in pet ferrets (Mustela putorius), a use regulated by the United States Department of Agriculture.

The hypothalamic-pituitary-ovarian axis and manipulations of the oestrous cycle in the brushtail possum.

The main purpose of this review is to provide a comprehensive update on what is known about the regulatory mechanisms of the hypothalamic-pituitary-ovarian axis in the brushtail possum, and to report on the outcomes of attempts made to manipulate by hormonal means, these processes in the possum. Over the last 15 years, several unique features of possum reproductive physiology have been discovered. These include an extended follicular phase despite elevated concentrations of FSH during the luteal phase, and early expression of LH receptors on granulosa cells of small antral follicles, suggesting a different mechanism for the selection of a dominant follicle. The use of routine synchronisation protocols that are effective in eutherians has failed to be effective in possums, and so the ability to reliably synchronise oestrus in this species remains a challenge.

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.

Expression of mRNAs encoding oestrogen receptor (ER) alpha and ERbeta, androgen receptor and progesterone receptor during gonadal and follicular development in the marsupial brushtail possum (Trichosurus vulpecula).

The objective of the present study was to determine which ovarian cells express mRNAs for oestrogen (ERalpha and ERbeta), androgen (AR) and progesterone (PR) receptors during ovarian and follicular development in the brushtail possum. Expression of ERalpha and/or ERbeta mRNA was observed from birth, initially in cells of the blastema, then in the medullary cords from Day 20. ERalpha was expressed in the oocytes and granulosa cells of secondary and antral follicles. Preovulatory follicles did not express ERalpha mRNA, although their oocytes were not examined for any gene. ERbeta mRNA was observed in oocytes at all follicular stages examined, but was not consistently observed in granulosa or theca cells. Expression of AR mRNA before Day 40 was very faint; thereafter, expression was observed in the medullary cords, peaking between Days 60 and 120. Oocytes, granulosa cells and theca of secondary and antral, but not preovulatory, follicles expressed AR mRNA. PR mRNA was expressed throughout the gonad by Day 20. Granulosa cells of some secondary and antral follicles and theca of antral follicles expressed PR mRNA. Thus, the expression of mRNAs encoding steroidogenic receptors in a time- and cell-specific manner supports a role for steroids in the process of ovarian follicular formation and growth.

Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula).

The present study investigated the effects of slow-release implants containing the gonadotrophin-releasing hormone (GnRH) agonist deslorelin on reproduction in the common brushtail possum (Trichosurus vulpecula). Captive female brushtail possums were assigned to control (placebo implant), low dose (4.7 mg deslorelin) or high dose (9.4 mg deslorelin) groups; males were assigned to control or high dose (9.4 mg deslorelin) groups. The acute effects of deslorelin treatment at the level of the pituitary gland were similar between the two sexes, where a transient rise in luteinising hormone concentration was induced over the first 24 h. In females, this was associated with the disruption of the normal oestrous cycle and mating within 2-10 days in some treated individuals, but no young were subsequently detected. By 3 weeks after treatment, treated females became anoestrus and remained infertile for at least one breeding season. The effects of treatment were reversible in a subset of females that had their implants removed, although the time taken to produce offspring was variable. Paradoxically, male brushtail possums remained fertile during chronic deslorelin exposure. Despite significant declines in basal follicle-stimulating hormone and testosterone concentrations, as well as an inability to respond to a GnRH challenge, treated males sired as many offspring as control males and there was no evidence of testicular regression. In conclusion, there is potential to control reproduction in female brushtail possums by using chronic GnRH agonist treatment.

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

The testicular androgen 5alpha;-androstane-3alpha,17beta-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 (3)H-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 Delta(4)-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5alpha-reduced androgen (androsterone) were synthesised by testes from Day 87-200 males and these appeared to be formed from the 5alpha-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, (3)H-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.

Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula).

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), 3beta-hydroxysteroid dehydrogenase/Delta5,Delta4 isomerase (3betaHSD), cytochrome p45017alphahydroxylase (p45017alphaOH), 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 3betaHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, p450scc, 3betaHSD, and p45017alphaOH 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.

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.

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.

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.