ROLE OF MAMMALIAN SPERM SURFACE MANNOSIDASE

这项建议的重点是提纯和化学表征 互补分子存在于相反的配子上，这些配子 哺乳动物的精卵识别和结合。这一焦点 源于我们最近的发现，表明存在a-d- 大鼠、仓鼠和小鼠精子表面的甘露糖苷酶。 与哺乳动物精子顶体的“酸性”甘露糖苷酶不同，这种新的- 发现率精子的α-D-甘露糖苷酶是一种内源性血浆 膜组分，主要定位于尾部头部 精子。在包括老鼠在内的三个物种中发表了几份报告， 提示精子质膜甘露糖苷酶可能有一个配体- 在与甘露糖结合中的类似作用，甘露糖可能存在于 透明带糖蛋白。这里提出的研究旨在 描述假定的配体并了解其分子 其与补体糖蛋白相互作用的机制 透明带。这项建议的具体目标是：1) 新发现的α-D-甘露糖苷酶的纯化 用于化学表征和生产的同质性 多克隆抗体。2)免疫组织化学定位 不同地区的生殖细胞和精子上的抗原 附睾症。3)确定特定阶段的合成和修饰 精子发生和精子成熟过程中的抗原。4) 新发现的酶在识别中的作用研究 与透明带糖蛋白结合。5)身份识别； 精子受体的提纯和化学特性研究 在透明带表面，6)结构的测定 低聚糖单位存在于配体和受体分子上(S)。 老鼠将是这项研究中使用的主要物种。但是，如果 抗大鼠精子α-D-甘露糖苷酶抗体与 来自其他物种(即小鼠和仓鼠)的精子酶，我们将进行研究 在这些物种中精子和卵子的相互作用。 建议研究的基本资料表格不应只披露 精子-卵子相互作用的机制，但也可能提供 洞察物种高度专一性的原因 在哺乳动物受精过程中观察到的。

This proposal focuses on purification and chemical characterization of complementary molecules present on the opposite gametes which initiate sperm-egg recognition and binding in mammalian species. This focus originates from our recent finding indicating the presence of an a-D- mannosidase on the surface of sperm from the rat, hamster, and mouse. Unlike mammalian sperm acrosomal "acid" mannosidase, the newly- discovered a-D-mannosidase of rate spermatozoa is an intrinsic plasma membrane component and is localized mainly on the head region of caudal sperm. Several published reports in three species, including the rat, suggest that the sperm plasma membrane mannosidase may have a ligand- like role in binding to mannose saccharides that are likely present on zona pellucida glycoproteins. The studies proposed here are designed to characterize the putative ligand and to understand the molecular mechanism of its interaction with the complementary glycoproteins of the zona pellucida. The specific objectives of this proposal are; 1) Purification of the newly-discovered a-D-mannosidase to a state of homogeneity for the purpose of chemical characterization and production of polyclonal antibody. 2) Immunohistochemical localization of the antigen on germ cells and spermatozoa from various regions of the epididymis. 3) Determining stage-specific synthesis and modification of the antigen during spermatogenesis and sperm maturation. 4) Investigation of the role of the newly-discovered enzyme in recognition and binding to zona pellucida glycoproteins. 5) Identification, purification, and chemical characterization of sperm receptors present on the surface of zona pellucida, 6) Determination of the structure of oligosaccharide units present on the ligand and receptor molecule(s). Rats will be the primary species used in this study. However, if the anti-rat sperm a-D-mannosidase-antibody shows cross-reactivity with sperm enzyme from other species (i.e., mouse and hamster), we will study sperm-egg interactions in these species. Basic information form the proposed studies should not only disclose the mechanism of sperm-egg interactions in the rat, but may also provide insight into the reasons for the high degree of species-specificity observed in mammalian fertilization.