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

参与哺乳动物受精和发育的糖蛋白的结构和功能分析

基本信息

批准号：
18570103
负责人：
NAKANO Minoru
金额：
$ 2.55万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570103/
关键词：
Zona Pellucida Mammalian Ovum Glycoprotein Glycolipid In vitro fertilization Sperm-egg binding Placenta Pregnancy Associated Glycoprote 免疫回避精子・卵子間結合

项目摘要

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.

1)猪和牛精子与糖脂类似物的结合我们制备了附着有六种在烷基链末端具有单糖的糖脂类似物的塑料板，在清洗板后，向这些板中添加猪或牛精子。通过该方法对残留精子的数量进行计数，结果表明猪精子主要与糖脂类似物的β-半乳糖残基结合，而牛精子则主要与糖脂类似物的β-半乳糖残基结合。此外，两种物种的顶体反应精子都与α-甘露糖残基结合。2)猪和牛精子与卵透明带的结合普遍认为，精子物种特异性地与透明带结合。在这里，我们研究了猪和牛的精子是否与异源卵子的透明带结合。结果表明，与异源卵子结合的精子数量几乎相同。 3）重组透明带糖蛋白与精子的结合活性透明带糖蛋白的三种成分（ZPA、ZPB、ZPC）及其组合在 Sf9 细胞中表达，并分析了重组体与猪和牛精子的结合活性，因为高甘露糖型链附着在蛋白质上。通过该表达系统，重组子不与猪精子结合，而是与牛精子结合，没有与牛精子结合，并且ZPB/ZPC共表达被结合。4)妊娠结构中表达的糖蛋白的碳水化合物结构的测定。牛妊娠相关糖蛋白的主N-连接链被确定为具有GalNAc的不寻常结构。