This proposal focuses on purification and chemical characterization of complementary molecules present on the opposite gametes which initiate sperm-egg recognition and binding in mammalian species. This focus originates from our recent finding indicating the presence of an a-D- mannosidase on the surface of sperm from the rat, hamster, and mouse. Unlike mammalian sperm acrosomal """"""""acid"""""""" mannosidase, the newly- discovered a-D-mannosidase of rate spermatozoa is an intrinsic plasma membrane component and is localized mainly on the head region of caudal sperm. Several published reports in three species, including the rat, suggest that the sperm plasma membrane mannosidase may have a ligand- like role in binding to mannose saccharides that are likely present on zona pellucida glycoproteins. The studies proposed here are designed to characterize the putative ligand and to understand the molecular mechanism of its interaction with the complementary glycoproteins of the zona pellucida. The specific objectives of this proposal are; 1) Purification of the newly-discovered a-D-mannosidase to a state of homogeneity for the purpose of chemical characterization and production of polyclonal antibody. 2) Immunohistochemical localization of the antigen on germ cells and spermatozoa from various regions of the epididymis. 3) Determining stage-specific synthesis and modification of the antigen during spermatogenesis and sperm maturation. 4) Investigation of the role of the newly-discovered enzyme in recognition and binding to zona pellucida glycoproteins. 5) Identification, purification, and chemical characterization of sperm receptors present on the surface of zona pellucida, 6) Determination of the structure of oligosaccharide units present on the ligand and receptor molecule(s). Rats will be the primary species used in this study. However, if the anti-rat sperm a-D-mannosidase-antibody shows cross-reactivity with sperm enzyme from other species (i.e., mouse and hamster), we will study sperm-egg interactions in these species. Basic information form the proposed studies should not only disclose the mechanism of sperm-egg interactions in the rat, but may also provide insight into the reasons for the high degree of species-specificity observed in mammalian fertilization.
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