Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals.

Immunisation of the male tammar wallaby (Macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilisation rate.

Immunocontraception has been proposed as an effective and humane means of controlling overabundant kangaroo populations in Australia. We have examined the feasibility of using a sperm-based vaccine for this purpose using a model macropod species, the tammar wallaby (Macropus eugenii). This study has demonstrated immunocontraception in a marsupial species following immunisation of males with homologous spermatozoa.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection.

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula).

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids.

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii).

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.

Immune response of the tammar wallaby (Macropus eugenii) to sperm antigens.

In the present study, male and female tammar wallabies were immunised with whole tammar wallaby sperm in adjuvant. An assay for sperm antibodies using a live sperm ELISA has been developed to detect sperm surface antigens and used to validate an assay using a 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (CHAPS) membrane extract of whole tammar wallaby sperm. The tests were used to monitor the immune response to whole sperm in both male and female tammar wallabies.

Timing and ultrastructure of events following intracytoplasmic sperm injection in a marsupial, the tammar wallaby (Macropus eugenii).

The aim of the present study was to determine the timing of oocyte activation, sperm decondensation and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the tammar wallaby and to determine the fate of sperm structures at an ultrastructural level. Metaphase II-stage tammar wallaby oocytes were injected with spermatozoa and cultured for 1 (n = 15), 2 (n = 24), 4 (n = 30), 6 (n = 14), 8 (n = 32), 10 (n = 25), 12 (n = 29) or 19 h (n = 12). Oocytes were assessed using light, fluorescence and electron microscopy.

The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii).

The distribution of T- and B-cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3- and CD5-positive T-cells were first observed at day 12 postpartum whilst rare B-cells were first detected at day 23. Both T- and B-lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes.

Effects of repeated superovulation and surgical oocyte collection on ovarian response and natural breeding ability of the tammar wallaby (Macropus eugenii).

The aim of this study was to assess the response of a marsupial, the tammar wallaby (Macropus eugenii) to repeated superovulation and surgical oocyte collection and monitor any effects on subsequent natural breeding ability. Animals (n = 5 per group) were superovulated once, twice or three times with pig FSH (pFSH; 6 mg administered twice per day for 4 days) followed by 4 mg pig LH (pLH). There was an interval of either 5-6 weeks (n = 9) or 12 weeks (n = 1) between the first and second superovulation and 13-17 weeks (n = 5) between the second and third superovulation.

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial, the brushtail possum (Trichosurus vulpecula).

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E.

The gut-associated lymphoid tissues of the northern brown bandicoot (Isoodon macrourus).

The gut associated lymphoid tissues (GALT) of a juvenile bandicoot has been examined using histological and immunohistochemical techniques. The mesenteric lymph nodes were hyperfollicular and had well defined paracortical and medullary areas. Lymphocytes were densely packed throughout the cortex and paracortex and the mantles of the follicles.

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums.

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro.

Induction of sperm maturation in vitro in epididymal cell cultures of the tammar wallaby (Macropus eugenii): disruption of motility initiation and sperm morphogenesis by inhibition of actin polymerization.

A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days.

Hormonal profiles in the tammar wallaby, Macropus eugenii, following FSH/LH superovulation.

This study investigated the effect of superovulation with exogenous porcine FSH/LH on the normal hormonal milieu of the tammar wallaby (Macropus eugenii). During seasonal and lactational quiescence, groups of 6 females were treated with either multiple doses of porcine follicle-stimulating hormone (FSH) (8 x 6 mg i.m.

Actin polymerisation during morphogenesis of the acrosome as spermatozoa undergo epididymal maturation in the tammar wallaby (Macropus eugenii).

In the tammar wallaby (Macropus eugenii), post-testicular acrosomal shaping involves a complex infolding and fusion of the anterior and lateral projections of the scoop-shaped acrosome into a compact button-like structure occupying the depression on the anterior end of the sperm nucleus. The present study has generated cytochemical and histological evidence to demonstrate that the occurrence of actin filaments (F-actin, labelled by Phalloidin-FITC) in the acrosome of tammar wallaby spermatozoa is temporally and spatially associated with the process of acrosomal shaping in the epididymis, through a pool of monomeric actin (G-actin, labelled by Rh-DNase I) present in the acrosome throughout all stages of epididymal maturation. F-actin was not detected in the acrosome of testicular spermatozoa, but was found in the infolding and condensing acrosome of caput and corpus epididymal spermatozoa.

Sperm binding and penetration of the zona pellucida in vitro but not sperm-egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation.

Secretory proteins from the female reproductive tract of the brushtail possum (Trichosurus vulpecula): binding to sperm and effects on sperm survival in vitro.

Previous studies have demonstrated that co-culture of brushtail possum epididymal spermatozoa with oviduct epithelial cell monolayers prolongs sperm survival and results in the re-orientation of the sperm head and tail to the T-shape (thumbtack) configuration. Transformation of sperm to thumbtack orientation is believed to be associated with marsupial sperm capacitation. Here we report that incubation in oviduct-conditioned media also significantly prolongs sperm survival and results in the transformation of sperm to the thumbtack orientation.

Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii).

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42-48 h in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 microg mL(-1) porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4-8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18-30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro.

Genetic analysis of the mating system of the common brushtail possum (Trichosurus vulpecula) in New Zealand farmland.

We examined male reproductive success in a common brushtail possum population in New Zealand farmland. Paternity was assigned to 66 of 91 pouch young (maternity known), using a likelihood approach applied to genotypes at six microsatellite loci having an overall average exclusion probability of around 99%. The distribution of number of offspring per male was L-shaped with a standardized variance of 1.

In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture.

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers.

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination.

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus).

cDNA cloning of lymphotoxin alpha (LT-alpha) from a marsupial, Macropus eugenii.

Lymphotoxin (LT) is a proinflammatory cytokine with a broad spectrum of immunological activities. While the 'classic' form of the molecule is a secreted homotrimer, now referred to in the literature as LT-alpha3, it has more recently been recognised that a membrane-bound form of LT exists on activated T lymphocytes and that this represents a complex between LT-alpha and a closely related type II membrane protein, LT-beta. Together with another related cytokine, tumour necrosis factor alpha (TNF-alpha), these molecules have been extremely well studied in eutherian mammals but not in any other group.

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head.

Cloning and characterisation of a zona pellucida 3 cDNA from a marsupial, the brushtail possum Trichosurus vulpecula.

The mammalian zona pellucida (ZP) is an extracellular glycoprotein coat that plays vital roles throughout fertilisation and preimplantation development. Like that of eutherian mammals the brushtail possum ZP is composed of three glycosylated proteins of 137 kDa, 92 kDa and 62 kDa. The 62 kDa protein is a ZP3 orthologue based on its nucleotide and deduced amino acid sequence.

Time of ovulation in the brushtail possum (Trichosurus vulpecula) following PMSG/LH induced ovulation.

This study aimed to determine the timing of ovulation in response to a new induced ovulation regime for the monovulatory brushtail possum (Trichosurus vulpecula). Ovarian stimulation was achieved by a single subcutaneous injection of 15 i.u.