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塑料板，其中连接了六种在烷基链末端具有单糖的糖脂类似物。向这些平板中加入猪或牛精子。洗涤平板后，计数残留精子数。结果表明，猪精子主要与糖脂类似物的β-半乳糖残基结合，而牛精子主要与α-甘露糖残基结合。2）猪和牛精子与卵透明质酸的结合精子与卵透明质酸的结合具有种属特异性。在这里，我们研究了猪和牛的精子是否结合到异源卵的透明质酸。结果表明，与异源精子结合的精子数与同源精子几乎相同，反映了精子透明带表面糖链的分布。3）重组透明带糖蛋白与精子的结合活性在Sf 9细胞中表达了ZPA、ZPB、ZPC及其组合，并分析了重组子与猪和牛精子的结合活性。由于高甘露糖型链通过该表达系统连接到蛋白质上，因此重组体不与猪精子结合，而是与牛精子结合。任何单一组分均不与牛精子结合，而ZPB/ZPC共表达的糖蛋白与精子结合。4）妊娠期表达糖蛋白的糖结构测定测定了牛妊娠相关糖蛋白的N-连接主链的结构。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.