The proposed research is aimed at understanding the molecular details of mouse gamete interaction. We have generated monoclonal anti-sperm antibodies that have been used to identify proteins involved in two distinct fertilization events: the acrosome reaction and sperm-egg membrane fusion. In the present research plan, we will focus on sperm interaction with the zona pellucida (ZP). Several strategies will be used to identify the mouse sperm receptor(s) for zp. Monoclonal antibodies (mAbs) that block binding to zp will be sought by immunizing mice with membrane fractions from epididymal sperm. Three different approaches are outlined for membrane preparation; this material will be assayed for bioactivity, defined as the ability to compete with sperm for zp binding. Direct biochemical approaches using a) cross-linking regents and b) RGD containing peptides to identify the sperm's zp receptor(s) are also proposed. Depending upon the complexity of the preparations that result from these approaches, either conventional biochemical or immuno-affinity techniques will be used to isolate the receptor(s). A second major aim is to test the hypothesis that interaction between capacitated sperm and ZP3 results in receptor aggregation that itself is a primary event in initiating the cascade terminating in the acrosome reaction. A variety of experimental approaches to examine receptor aggregation and its possible physiological significance are proposed. In addition, bioactive membrane fractions, generated as part of the zp receptor studies, will be characterized for activities known to be involved in signal transduction, such as G proteins and endogenous protein kinases. MAbs developed in the zp receptor studies will be examined for inhibitory effects on AR triggering. Two of our existing mAbs, M42 and M5 are initial candidates to examine in detail. Sperm from the proximal epididymis are incapable of fertilization. We plan to examine biochemical changes that occur in specific sperm components during epididymal transit. We have already found that the M42 antigen is modified structurally in the epididymis; this modification coincides with acquisition of function. Co-culture of different epididymal sperm populations with epididymal epithelia in primary culture will be used to explore the biochemical maturation of relevant antigens in vitro.
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