| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
In early phase of mammalian fertilization, sperm binds in species-specific manner to the homologous eggs through the sperm receptor which is a glycoprotein of zona pellucida surrounding the oocyte. Molecular mechanism, however, of this sperm-egg interaction has not been fully understood. In this study, we performed the experiments directed toward the elucidation of the active region of porcine sperm receptor and determination of its chemical structure. In the first year of this project, we analyzed the structure of sugar moieties of the glycoproteins of porcine zona pellucida. Since the zona in the ovary already retains the sperm binding activity, we developed a new method for preparation of the zona in large quantities from ovaries by the use of the stainless meshes and Percoll. Heat-solubilized zonae were separated into three families (PZP1-3) by gel-filtration HPLC, and PZP3 was found to have sperm receptor activity. N-Glycosidically linked sugar chains of PZP3 obtained by hydrazino
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lysis were separated into the acidic and neutral groups. After fractionation by reverse-phase HPLC, the structures of the major neutral chain were determined by NMR and HPLC. They were shown to have mainly bi-antennary and complex-type structure. Furthermore it was found that various amounts of the partially sulfated and sialylated lactosamines are linked to the non-reducing ends of the neutral chains, forming heterogeneous acidic chains.
In the second year, the sperm receptor region was defined by competition assay measuring the inhibition activity of sperm-egg binding. As the results, the neutral chains of the alpha-glycoprotein in PZP3 family was shown to have the receptor activity. Accordingly, some of the neutral chains of which structures have been determined must be the receptor, The removal of protein moiety decreased the activity, suggesting that the protein core of the glycoproteins participates in the sperm-egg interaction by providing the proper steric arrangement of the active sugar chain. Less
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