Science Collected Works - Theses

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Type: PhD thesis × Start date: 2000 × End date: 2014 × Subject: Embryology -- Marsupials ×

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    A marsupial shell coat protein (CP4) and its potential as an immunocontraceptive target in the common brushtail possum, Trichosurus vulpecula
    Menkhorst, Ellen Melaleuca. (University of Melbourne, 2007)
    Egg and embryonic coats surround the vertebrate conceptus during early development. A uterine-secreted embryonic shell coat surrounds the marsupial conceptus for 60 to 80 % of gestation. Marsupial shell Coat Protein 4 (CP4), is the first protein identified that contributes to a vertebrate uterine-secreted embryonic coat. Dot blot hybridisation identified genes homologous to cp4 in all major vertebrate groups except amphibians, but the strongest identity to cp4 was found in mammalian species known to have uterine-secreted additions to the embryonic coats, suggesting that CP4 was incorporated into the mammalian embryonic coats associated with loss of the shell crust. cp4 was cloned from a second marsupial species, Sminthopsis macroura. CP4 transcription and secretion was identified in S. macroura by semi-quantitative RT-PCR and immunohistochemistry, respectively. It began during the pre-ovular period and fell to low levels during the unilaminar (secretion) and bilaminar (transcription and secretion) blastocyst stages. CP4 transcription and secretion resumed during the trilaminar blastocyst stage and continued during the embryonic and fetal stages of development. In vitro, cp4 transcription increased after incubation with progesterone, and further, the in vivo biphasic transcription pattern was correlated with the plasma progesterone profile characterised in this study. New evidence of conceptus-maternal feedback during pregnancy was identified in a polyovular marsupial, S. macroura: 1. ovarian progesterone concentration was correlated with conceptus number on the day prior to implantation; 2. The apical glandular epithelial mitotic index was significantly higher on Day 3 in pregnant animals; and 3. The daily body weight profile predicted the length of pregnant luteal phases. A previously unidentified final pulse of mitotic activity in the uterine epithelium was identified in pregnant S. macroura on the day prior to implantation, presumably associated with uterine preparation for implantation, and was observed just after the equivalent time of implantation in a pregnant cycle in non-pregnant S. macroura and in the contralateral uterus of pregnant T. vulpecula, possibly initiating the shedding of the epithelium. In vitro investigations into the time taken for oocytes to transit through the oviduct in S. macroura, indicated that it could be as short as 50 min, and, including four ovulations from the one ovary, the entire ovulation event could take as little as 4 hr. Light microscopy identified a period of intense glandular epithelial remodelling, initiated in the basal region of the glandular epithelium, during ovulation in S. macroura. This remodelling was possibly associated with the incorporation of new cells from the stroma into the glandular epithelium. These cells appeared to have arisen from stromal blood vessels, possibly indicating a blood stem cell progenitor for the uterine glandular epithelium. T. vulpecula immunised against GST-CP4 showed a significant reduction in fertility, because the immune response against GST-CP4 targeted the shell coat, the conceptus and the uterine glandular epithelium, preventing secretion of the shell coat and nutrients prior to implantation. This study provides further insight into the homology, expression and function of the marsupial shell coat, and illustrates the potential of uterine-secreted proteins as immunocontraceptive targets in mammals.
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