Structural Study on the Sperm Receptor of Porcine Zona Pellucida

猪透明带精子受体的结构研究

基本信息

批准号：
01540588
负责人：
NAKANO Minoru
金额：
$ 1.28万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01540588/
关键词：
Zona Pellucida Mammalian Egg Fertilization Carbohydrate Structure Cell Recognition Sperm-Egg Interaction Sperm Receptor

项目摘要

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 … More 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

在哺乳动物受精的早期，精子通过精子受体以特异性方式与同源卵结合，这是卵母细胞周围Zona pellucida的糖蛋白。然而，这种精子 - 卵子相互作用的分子机制尚未完全理解。在这项研究中，我们进行了针对猪群精子受体的活性区域的实验，并确定其化学结构。在该项目的第一年中，我们分析了猪Zona pellucida的糖蛋白糖蛋白的糖基结构。由于卵巢中的Zona已经保留了精子结合活性，因此我们通过使用不锈钢网格和Percoll开发了一种新的方法来从卵巢中大量制备Zona。通过凝胶过滤HPLC将热硫化的Zonae分离为三个家族（PZP1-3），并发现PZP3具有精子受体活性。通过Hydrozino获得的PZP3的N-糖苷连接的糖链，将更多的裂解分离为酸性和中性基团。通过反相HPLC分馏后，通过NMR和HPLC测定了主要中性链的结构。它们被证明主要具有双静脉和复杂型结构。此外，发现各种量的部分硫酸盐和硫化的乳糖胺与中性链的非还原末端有关，形成异质性酸性链。在第二年中，通过测量精子抑制活性的竞争分析来定义精子受体区域。结果，PZP3家族中α-糖蛋白的中性链被证明具有受体活性。据认为，确定结构的一些中性链必须是受体，去除蛋白质部分可以改善活性，这表明糖蛋白的蛋白质核心通过提供活性糖链的适当安排来参与精子 - 蛋的相互作用。较少的