Structural and Functional Analysis of glycoproteins participating in the mammalian fertilization and development

1)猪和牛精子与糖脂类似物的结合我们制备了六种糖脂类似物的塑料板，烷基链末端有一个单糖。在这些培养皿中加入了猪或牛的精子。洗涤后，计数剩余精子的数量。结果表明，猪精子主要与糖脂类似物的β-半乳糖残基结合，牛精子主要与α-甘露糖残基结合。此外，两种物种的顶体反应精子都与α-甘露糖残基结合。猪和牛精子与卵细胞透明带的结合是公认的精子物种特异性结合在透明带上。本文研究了猪和牛精子是否能与异源卵子的透明带结合。结果表明，与异体带结合的精子数量与同源精子的数量几乎相同，这反映了碳水化合物链在带表面的分布。3)重组透明带糖蛋白与精子的结合活性在Sf9细胞中表达了3种透明带糖蛋白组分（ZPA、ZPB、ZPC）及其组合，并分析了重组透明带糖蛋白与猪和牛精子的结合活性。由于高甘露糖型链通过这种表达系统附着在蛋白质上，因此重组体不是与猪精子结合，而是与牛精子结合。未发现任何单一组分与牛精子结合，ZPB/ZPC共表达。4)测定妊娠期表达的糖蛋白的碳水化合物结构测定了牛妊娠相关糖蛋白的主n链结构。n -聚糖具有不寻常的结构，具有GalNAc。

1) Binding of porcine and bovine sperm to glycolipid analogues We prepared plastic plates to which six kinds of glycolipid analogues having a monosaccharide at the end of alkyl chain are attached. To these plates, porcine or bovine sperm were added. After washing the plates, the numbers of residual sperm were counted. By this method, it was revealed that porcine sperm mainly bind to β-galactose residue of the glycolipid analogue, while bovine sperm to α-mannose residue. Furthermore, the acrosome-reacted sperm of both species bind to α-mannose residue.2) Binding of porcine and bovine sperm to the egg zona pellucida It is universally accepted that sperm species-specifically bind to the zona pellucida. Here we investigated whether porcine and bovine sperm bind to the zona pellucida of heterologous egg. As a result, it was shown that almost the same number of the sperm bound to the heterologous zona as that of the homologous sperm did, reflecting the distribution of the carbohydrate chains on the surface of the zona.3) The binding activity of recombinant zona pellucida glycoproteins to sperm Three zona glycoprotein components (ZPA, ZPB, ZPC) and their combinations were expressed in Sf9 cells and the binding activities of the recombinants to porcine and bovine sperm were analyzed. Since the high-mannose type chains are attached to the proteins by this expression system, the recombinants were bound not to porcine sperm but to bovine sperm. Any single component was not bound to bovine sperm and ZPB/ZPC coexpressed was bound.4) Determination of the carbohydrate structure of glycoproteins expressed at pregnancy The structure of main N-linked chain of bovine pregnancy associated glycoproteins was determined. The N-glycan has an unusual structure having GalNAc.