ROLE OF MAMMALIAN SPERM SURFACE MANNOSIDASE

该提案侧重于纯化和化学表征 互补分子存在于相反的配子上， 哺乳动物精子-卵子识别和结合。这一重点 源于我们最近的发现表明存在一种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"酸性"甘露糖苷酶不同， 发现精子的α-D-甘露糖苷酶是一种内源性的血浆 膜成分，主要位于尾侧的头部区域 精子几篇发表的关于三个物种的报道，包括老鼠， 表明精子质膜甘露糖苷酶可能有配体- 类似于结合可能存在于 透明珊瑚糖蛋白 这里提出的研究旨在 表征假定的配体并了解分子 它与互补糖蛋白的相互作用机制 透明的 本建议的具体目标是：1） 将新发现的α-D-甘露糖苷酶纯化至 用于化学表征和生产的均匀性 多克隆抗体。 2)免疫组织化学定位 抗原的生殖细胞和精子从不同地区的 附睾 3)确定阶段特异性合成和修饰 精子发生和成熟过程中的抗原。 四、 新发现的酶在识别中的作用研究 并与透明质酸糖蛋白结合。 5)身份证， 精子受体的纯化和化学表征 6）测定了透明质酸的结构 配体和受体分子上存在的寡糖单元。 大鼠将是本研究中使用的主要种属。 但如果 抗大鼠精子α-D-甘露聚糖酶抗体显示与 来自其它物种的精子酶（即，老鼠和仓鼠），我们将研究 精子和卵子的相互作用 基本资料表格建议的研究不应只披露 在大鼠精子-卵子相互作用的机制，但也可能提供 洞察高度物种特异性的原因 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.