Immunisation of female brushtail possums against infertility - relevant peptides derived from a possum zona pellucida protein (ZP2) : A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours at Lincoln University

Abstract

In New Zealand, the brushtail possum (Trichosurus vulpecu/a) is a major vertebrate pest and research to develop a possum-specific method of immunologically-based fertility control (immunocontraception) is currently under way. This study examined the ability of three ZP2 peptide vaccines (ZP2(A), ZP2(8) and ZP2(C)) to elicit immune responses in possums and to interfere with their fertility and ovarian follicular dynamics. Female brushtail possums were randomly assigned to one of four treatment groups (control, ZP2(A), ZP2(B) and ZP2(C): n=20). Treated possums were injected subcutaneously with a vaccine containing the relevant peptide, conjugated to haemocyanin from the keyhole limpet (KLH), and Freund's complete adjuvant. Animals were given booster injections of Freund's incomplete adjuvant 3 and 6 weeks later. Control animals received either adjuvant emulsified with phosphate buffered saline solution (Control; n=10) or KLH plus adjuvant emulsified with phosphate buffered saline solution (Control+KLH; n=10). Blood, to provide serum, was collected from the tail vein on Week 0, 3, 6, 8 and on completion of the fertility trial. After the second booster injection, animals were superovulated with PMSG/LH (Week 8). Possums were artificially inseminated, the reproductive tract was flushed and the ovaries were inspected to determine number of recent ovulation sites. Numbers of eggs (immature and mature) and embryos (2-cell and 8-cell) recovered from flushed oviducts and (or) uteri were recorded. Follicular fluid was collected from large follicles in the left ovary of each animal for antibody analysis. Right ovaries from 8 of the Control group animals and 4 each from two of the immunised groups (ZP2(A) and ZP2(8)) were stained, serial sectioned and assessed for numbers of small, medium, large and atretic follicles and number of abnormal luteal cells. Left ovaries from all animals in the study were serial sectioned and assessed for fluorescence. Antibody binding in serum and follicular fluid was determined by an ELISA. The results showed that immunisation with ZP2(C) peptide reduced fertility. Animals in this treatment group had significantly (P<0.05) fewer embryos (mean = 0.5/possum) than their corresponding controls (mean = 2.4/possum) and relatively fewer of these females had embryos (5/20 versus 7/10, respectively, P<0.05). In contrast, there was no effect on fertility following immunisation with ZP2(A) or ZP2(B) peptides. Immunisation with all three ZP2 peptides stimulated antibody production, which was detected in serum and follicular fluid, but there was no antibody bound to native ZP in ovaries, as indicated by an absence of detectable fluorescence. A trend that immunisation reduced the number of follicles and abnormal luteal cells in ovaries was observed, but this was statistically significant only for the number of small and total follicles following immunisation with ZP2(A) peptide. However, peptide ZP2(C) has yet to be assessed for this effect. These results confirm that immunisation based on targeting antigens of the possum zona pellucida can impair fertility and disrupt ovarian function. Thus, immunocontraception has potential for application in the control of possum populations.