Glycosyltransferase function during fertilization

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DESCRIPTION (provided by applicant): My laboratory is interested in the molecular basis of cellular interactions during mammalian fertilization. During the past 20 years, we have used a variety of biochemical and molecular approaches to characterize an unusual sperm surface receptor (Beta-1,4-galactosyltransferase I, GaIT) for glycoside ligands in the egg zona pellucida. With the exception of some early work, HD 23479 has supported all of our studies of GaIT function during fertilization. GaIT is traditionally viewed as a biosynthetic enzyme in the Golgi complex, although a small amount also appears on the surface of somatic cells. Sperm are unusual in that all of their GaIT is expressed on the sperm surface, where it selectively binds to the egg coat glycoprotein, ZP3. In response to ZP3-induced aggregation, GaIT activates, directly or indirectly, a pertussis toxin-sensitive G-protein cascade, contributing to the acrosome reaction. Our recent studies have examined the consequences of altering GaIT expression, both positively and negatively, on heterologous cells and on mouse sperm. In this regard, Xenopus oocytes expressing murine GaIT bind ZP3, and aggregation of GaIT by ZP3 or anti-GaIT antibodies induces G-protein activation and subsequent activation of the Xenopus oocyte. Similarly, sperm that overexpress GaIT bind more ZP3 and undergo accelerated G-protein activation and acrosome reactions. In contrast, sperm that are null for GaIT do not bind ZP3 and do not undergo ZP3-dependent acrosome reactions. These results show clearly that Gall functions as a ZP3 receptor and is capable of activating a pertussis toxin-sensitive G-protein cascade. Despite the fact that GaIT-null sperm do not bind ZP3, they still bind to the zona pellucida, although they do not undergo subsequent zona-induced acrosomal exocytosis. This implies that non-GalT:non-ZP3 receptor:ligand interactions mediate initial sperm-zona adhesion, and that GalT-ZP3 interactions participate in subsequent acrosomal exocytosis. The specific Aims of this proposal will examine candidates for these newly appreciated sperm receptors and egg coat ligands. In the first Aim, we will test the hypothesis that the egg coat ligand responsible for sperm adhesion is a ZP3-independent, peripherally associated component of the egg coat. The second Aim will test whether the sperm protein p47 functions as a binding protein for the egg coat. Finally, in the third Aim, we will continue our analysis of GalT-mediated signal transduction leading to the acrosome reaction.