This project focuses on the structure and function of the oligosaccharide moieties of the mouse zona pellucida (mZP) and how this extracellular glycocalyx that surrounds the egg interacts with complementary molecules on the surface of spermatozoa. Previous evidence strongly suggests that the sperm binding activity of mZP is associated with its oligosaccharide portion. Recent studies in the applicant's laboratory have demonstrated for the first time the presence of N-linked polylactosaminyl glycans and an O-linked trisaccharide that are thought to have important roles in sperm-egg recognition and binding. To assess the bioactivity of these oligosaccharides, the proposal has five specific aims: (1) to purify mZP2 and mZP3, and then cleave and radiolabel the N-linked glycans and O-linked trisaccharide, (2) to assess the functional role of the radiolabeled N-linked and O-linked glycans in sperm-egg recognition and binding, (3) to isolate and chemically characterize functional N-linked glycan(s), (4) to synthesize the O-linked trisaccharide, examine its function, and the mechanism underlying the formation of the sperm surface antigen-trisaccharide complex, and (5) to identify and chemically characterize the mouse sperm plasma membrane (PM) proteins recognized by the N-linked and/or O-linked glycans. When completed, this study will be the first published report on structure-function diversity of both N-linked and O-linked oligosaccharides. After the complementary molecules (i.e., the bioactive glycans on ZP glycoproteins and complementary receptors on sperm PM) are identified in the selected strain of experimental mice, it will be possible to address the question of interspecies and intraspecies relationships during interactions of opposite gametes. Collectively, these studies will further our understanding of the mechanism of sperm-egg interaction and fertilization in the mouse, and they will provide needed information for the regulation of the fertilization processes in other species, including humans.
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