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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD018201-07
Application #
3315221
Study Section
Reproductive Biology Study Section (REB)
Project Start
1983-04-01
Project End
1994-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
7
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
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Kiefer, Susan McLeskey; Saling, Patricia (2002) Proteolytic processing of human zona pellucida proteins. Biol Reprod 66:407-14
Tomes, C N; Carballada, R; Moses, D F et al. (1998) Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation. Mol Hum Reprod 4:17-25
Carballada, R; Saling, P M (1997) Regulation of mouse epididymal epithelium in vitro by androgens, temperature and fibroblasts. J Reprod Fertil 110:171-81
Tomes, C N; McMaster, C R; Saling, P M (1996) Activation of mouse sperm phosphatidylinositol-4,5 bisphosphate-phospholipase C by zona pellucida is modulated by tyrosine phosphorylation. Mol Reprod Dev 43:196-204
Leyton, L; Tomes, C; Saling, P (1995) LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm: evidence that the antigen recognized is not hexokinase. Mol Reprod Dev 42:347-58
Burks, D J; Carballada, R; Moore, H D et al. (1995) Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. Science 269:83-6
Beebe, S J; Leyton, L; Burks, D et al. (1992) Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction. Dev Biol 151:48-54
Wolf, D E; McKinnon, C A; Leyton, L et al. (1992) Protein dynamics in sperm membranes: implications for sperm function during gamete interaction. Mol Reprod Dev 33:228-34
Saling, P M (1991) How the egg regulates sperm function during gamete interaction: facts and fantasies. Biol Reprod 44:246-51

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