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

1)猪、牛精子与糖脂类似物的结合我们制备了六种烷基末端含单糖的糖脂类似物的塑料平板。在这些盘子里，加入了猪或牛的精子。清洗培养皿后，计数剩余精子数。结果表明，猪精子主要结合糖脂类似物的β-半乳糖残基，而牛精子主要结合α-甘露糖残基。此外，两种顶体反应的精子与α-甘露糖残留物结合。2)猪和牛精子与卵子透明带的结合。人们普遍认为，精子物种与透明带特异结合。在这里，我们研究了猪和牛的精子是否与异种卵子的透明带结合。结果表明，与异源透明带结合的精子数量与同源精子几乎相同，反映了透明带表面糖链的分布。3)重组透明带糖蛋白与精子结合活性的研究在Sf9细胞中得到表达，并对重组蛋白与猪、牛精子的结合活性进行了分析。由于高甘露糖型链通过该表达系统连接到蛋白质上，因此重组子不是与猪精子结合，而是与牛精子结合。4)孕期表达糖蛋白碳水化合物结构的测定测定了牛妊娠相关糖蛋白的主要N-链链结构。N-糖链具有一种不寻常的结构，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.