The DDX4/VASA gene plays an important role in germ cell development in animals. We cloned and characterized a marsupial DDX4/VASA homolog (TvDDX4, 2,769 bps) from the possum and examined its expression in adult tissues at mRNA and protein levels. The isolated cDNA had a deduced 704 amino acid residues with significant homology to DDX4 from other animals, including mouse (86%) and human (87%). The DDX4 transcript was detected in the ovary and testis, and was undetectable in somatic tissues. The recombinant possum DDX4 protein (TvDDX4) was successfully produced in a bacterial expression system and used in polyclonal antibody generation. The recombinant TvDDX4 was detected by antibody against human DDX4 and mouse antibody against TvDDX4, but native possum TvDDX4 was only recognized by the possum antibody in the ovary and testis. Our results suggest a structural and functional conservation of DDX4 in marsupials and in mammals in the therian branches.

The marsupial conceptus is surrounded by a uteri ne-secreted shell coat for 60-80% of gestation. Coat protein 4 (CP4) is the only marsupial shell coat protein characterized and it has only been identified in one species, the Common Brushtail Possum. In this possum, uterine transcription and secretion of cp4 during the oestrous cycle is biphasic and associated with the stage of conceptus development. Here we cloned cp4 (sm-cp4) from a distantly related species, the Stripe-faced Dunnart (Sminthopsis macroura). Transcription of sm-cp4 and secretion of smCP4 were identified by semi-quantitative RT-PCR and immunohistochemistry, respectively. The effect of reproductive hormones on sm-cp4 transcription was investigated in vitro by treatment of uterine explant cultures with oestrogen and/or progesterone. In vivo uterine, expression of smCP4 was biphasic and associated with conceptus development. Uterine smCP4 expression (transcription and secretion) began during the pre-ovulatory period and continued post-ovulation during cleavage stages. Transcription of sm-cp4 continued during the unilaminar blastocyst stage, but smCP4 secretion was reduced during this stage. During the bilaminar blastocyst stage, both transcription of sm-cp4 and secretion of smCP4 were low before they both resumed during the trilaminar blastocyst stage, and continued during the embryo and fetal stages. In vitro uterine transcription of sm-cp4 increased after incubation with progesterone.

We report the first immunocontraceptive trial in mammals using a uterine-secreted protein, the marsupial shell coat protein 4 (CP4). The marsupial shell coat, which surrounds the conceptus for 60-80% of gestation, is secreted by the uterine epithelium. Following immunization against glutathione S-transferase (GST)-CP4, the fertility of female common brushtail possums (n=6) was significantly reduced (P=0.000), and this reduction in fertility was positively correlated with the maximum GST-CP4 humoral immune response (P=0.025). Ultrastructural examination of the reproductive tract indicated that the cell-mediated immune response against GST-CP4 targeted the shell coat, the shell-free conceptus and the uterine glandular epithelium, thus preventing normal conceptus development and uterine secretion of shell coat proteins and nutrients. These results show that uteri ne-secreted proteins are promising immunocontraceptive targets, especially in pest mammal species, e.g. possum, rabbit and horse, that have uteri ne-secreted additions to embryonic coats, or that have late implantation requiring uterine nutrient provisioning from secretions.

Ovarian-based immunological research is currently restricted to proteins of the zona pellucida. This study examined the immunocontraceptive potential of a novel vesicle-associated protein, VAP1, previously isolated from the vesicle-rich hemisphere of the brushtail possum oocyte. Seven female possums were immunized against recombinant glutathione S-transferase-VAP1 fusion protein. Control animals (n=3) received antigen-free vaccinations. Following immunization, regular blood sampling determined the level and duration of immune response. Animals were monitored daily, pre- and post-immunization, to determine estrous cycling activity and the percentage of reproductive cycles yielding viable young. The reproductive tracts and somatic organs of VAP1-immunized (n=7), control-immunized (n=3) and non-immunized (n=5) animals were collected and examined by histology and transmission electron microscopy. VAP1 immunization caused a strong and sustained immune response. Elevated levels of VAP1 antibody binding were detected in sera following initial injections, and immune titers rose as boosters were administered. Immunization had no adverse effect upon animal behavior or body condition. Immunized females demonstrated no major change in annual estrous cycling activity; however, the percentage of reproductive cycles resulting in pouch young decreased significantly (P<0.05) by 40%. Histological and ultrastructural analyses revealed an abundance of lipid-like degradation bodies within the ooplasm of developing oocytes and the cytoplasm of failing uterine zygotes. Active macrophage invasion of enlarged endometrial glands was observed in the uteri of two females. Reproductive tract changes are discussed in relation to observed fertility decline. The results of this study indicate that VAP1 has exciting potential as an immunocontraceptive target for possum control in New Zealand.

A reverse transcription technique using RNA templates combined with polymerase chain reaction (RT–PCR) was used to clone the cDNA fragment encoding the amino acid sequence of mature LIF protein of the marsupial, the brushtail possum, Trichosurus vulpecula. A PCR product with expected size, of 546 bp, and termed tvLIF, was obtained using cDNA reverse-transcribed from total RNA isolated from possum uterus. A genomic DNA fragment (about 650 bp) between the specified primers was also amplified, indicating the similarity in structure and organization of this gene and LIF genes from studied eutherian species, although the full-length of its cDNA and genomic DNA needs to be further clarified. The deduced amino acid sequence of tvLIF shows a high level of sequence identity and similar molecular characteristics to eutherian LIF, which suggests similar biological actions of this molecule in this marsupial. Because the expression of LIF gene in other mammalian species has been found to be at very low levels and its transcripts cannot be detected by Northern hybridization analysis, the expression pattern of tvLIF in adult tissues and reproductive tracts during early development was investigated using the RT–PCR technique. Resultant products of the RT–PCR were further analyzed by Southern hybridization using tvLIF as a probe. tvLIF transcripts were detected in most of the adult tissues and in the reproductive tracts of pregnant females. These results lend support to the idea that LIF contributes to the maintenance of pregnancy in this marsupial.