Past studies from my laboratory have shown that surface GalTase mediates selected cellular interactions by binding to its specific glycoside substrate on adjacent cell surfaces and/or in the extracellular matrix. The purpose of this renewal application is to continue our analysis of surface GalTase function during fertilization in the mouse. During previous funding periods, we have shown that initial gamete recognition is mediated, at least in part, by the binding of sperm surface GalTase to its specific substrate on the zona pellucida. We have also examined the expression and function of surface GalTase during spermatogenesis, in vitro capacitation and the acrosome reaction. During the current funding period we have: 1) shown that sperm GalTase behaves as an integral plasma membrane protein, 2) purified sperm surface GalTase to apparent homogeneity and characterized its substrate specificity, 3) shown that affinity purified sperm GalTase competitively inhibits sperm-egg binding, 4) identified the substrate, or ligand, for sperm GalTase as specific oligosaccharides on the zona pellucida glycoprotein ZP3, 5) shown that the sperm binding activity of ZP3 is dependent upon its interaction with sperm GalTase, 6) shown that sperm GalTase no longer recognizes ZP3 after fertilization, consistent with ZP3's loss of sperm binding activity, 7) shown that crosslinking GalTase residues within the sperm plasma membrane induces the acrosome reaction, 8) shown that sperm GalTase no longer binds ZP3 after the acrosome reaction, consistent with the inability of acrosome-reacted sperm to bind ZP3,9) defined the expression of GalTase mRNA during wild-type and T/t-mutant spermatogenesis, 10) mapped the locus within the T/t-complex that regulates GalTase expression, 11) shown that GalTase is preferentially localized on the plasma membrane of a variety of other mammalian sperm, 12) synthesized non-hydrolyzable substrates for sperm GalTase that offer distinct advantages for the study of GalTase function, and 13) cloned GalTase cDNA and made antibodies against the recombinant protein expressed in bacteria. As a result of these studies we have identified the complementary interacting receptors that mediate sperm-egg binding in the mouse (i.e., sperm GalTase and ZP3), and therefore, are in a unique position to dissect the molecular pathway for associated steps of the fertilization process. During the next funding period, we will address specific aspects of sperm binding, zona penetration and polyspermy prevention through six new specific aims. 1) To examine more rigorously the involvement of sperm GalTase in sperm-egg binding using antibodies against the recombinant murine GalTase and binding of labeled ZP3 to cells transfected with murine cell surface GalTase cDNA. 2) To define the oligosaccharide structure of the ZP3 substrate for sperm GalTase. 3) To define the mechanism whereby crosslinking surface GalTase induces the acrosome reaction. 4) To define the function o...