The proposal focuses on identification, purification, and chemical characterization of complementary molecules present on the opposite gametes which initiate sperm-egg recognition and binding in mammalian species. This has been our focus since we characterized a novel alpha-D- mannosidase on spermatozoa of several species. The enzyme is an intrinsic plasma membrane (PM) component and its catalytic domain is oriented towards the outer surface of the intact spermatozoa. Several reports, including our recent study in the mouse, suggest that the enzyme has a receptor-like role in the recognition and binding to high mannose/hybrid type oligosaccharide (OS) chains which we have demonstrated on mouse zona components. A polyclonal antibody raised against an isoform of sperm surface mannosidase cross-reacts with the mannosidase activity present in the detergent solubilized sperm PM. This antibody will be used to examine the chemical nature of the sperm surface mannosidase and evaluate its role in sperm-egg binding and fertilization. This proposal is designed to determine the molecular mechanisms of the interaction of sperm surface mannosidase with the complementary molecules present on the rat zona pellucida (ZP) glycoconjugate(s). The specific objectives of the proposal are: l) Examination of kinetic properties, chemical nature, and localization of the alpha-D-mannosidase. 2) Examination of its membrane topology and stage-specific synthesis during spermatogenesis and sperm maturation. 3) Evaluation of the role of the sperm surface mannosidase in sperm-egg binding and fertilization. 4) Identification and characterization of cDNA encoding sperm PM alpha-D-mannosidase. 5) Identification and characterization of ligand-like molecule(s) from the rat ZP. 6) Determination of the chemical nature, number, and structure of the OS chains on ligand molecule(s). Rats will be the primary species used in this study. However, if the antibody to alpha-D-mannosidase shows cross-reactivity with the sperm enzyme from other species, we will examine the role of the sperm mannosidase in other species. The proposed studies will provide information on the chemical nature of the sperm surface mannosidase and its complementary molecule(s) on the zona pellucida. Combined, the studies will further our understanding of the molecular mechanisms of sperm-egg interactions and fertilization in the rat, and will provide insight into the reasons for the high degree of species-specificity observed in mammalian species.
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