Structural Study on the Sperm Receptor of Porcine Zona Pellucida

在哺乳动物受精的早期阶段，精子通过卵母细胞周围的透明膜糖蛋白精子受体以种属特异性的方式与同源卵结合。然而，这种精卵相互作用的分子机制尚未完全了解。本研究旨在阐明猪精子受体的活性区并测定其化学结构。在本计画的第一年，我们分析了猪透明质酸糖蛋白的糖基结构。由于卵巢中的精子已经保留了精子结合活性，我们开发了一种新的方法，通过使用不锈钢网和Percoll从卵巢中大量制备精子。凝胶过滤HPLC将热溶的透明带分为3个家族（PZP 1 -3），发现PZP 3具有精子受体活性。通过肼基获得的PZP 3的N-糖苷连接糖链 ...更多信息 裂解分为酸性和中性组。通过反相HPLC分级分离后，通过NMR和HPLC确定主要中性链的结构。结果表明，它们主要具有双触角和复杂型结构。此外，还发现不同量的部分硫酸化和唾液酸化的乳糖胺连接在中性链的非还原端，形成异质性酸性链。第二年，通过测定精卵结合抑制活性的竞争试验确定了精子受体区。结果表明，PZP 3家族α-糖蛋白的中性链具有受体活性。因此，一些结构已确定的中性链必须是受体。蛋白质部分的去除降低了活性，表明糖蛋白的蛋白质核心通过提供活性糖链的适当空间排列参与精卵相互作用。少

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