FUCOSYLTRANSFERASE DURING SPERMATOGENESIS

精子发生过程中的岩藻糖基转移酶

• 批准号: 2200522
• 负责人: CLARKE F MILLETTE
• 金额: $ 10.66万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2200522
• 关键词: Sertoli cells; affinity chromatography; antibody; bioassay; cell adhesion; cell cell interaction; cell membrane; egg /ovum; enzyme substrate; epididymis; fertilization; fucose; glycosylation; hexosyltransferase; high performance liquid chromatography; laboratory mouse; sperm; spermatogenesis

The overall goal of this project is the biochemical and physiological analysis of cell-surface associated fucosyltransferase (FT) during mammalian spermatogenesis. We will test the hypothesis that a family of related FT enzymes, each with a different glycosidic specificity, is involved in Sertoli-germ cell interactions, sperm maturation and fertilization. Preliminary studies have identified the spermatogenic stage-specific expression of FT in the mouse and have implicated this enzyme in sperm-egg recognition. In contrast to most somatic cells, a substantial fraction of the spermatogenic cell FT is associated with the cell surface as an integral plasma membrane ecto-enzyme. It appears that a family of FT enzymes, each with differing glycosidic specificities is involved. This application proposes five Specific Aims designed to extend our understanding of the role FT has in regulating spermatogenesis. These Aims are (1) To determine the glycosidic specificity of FTs during spermatogenesis and sperm maturation. Methods used here will include newly developed thin layer chromatographic micropartitioning procedures as well as the introduction of neoglycolipid analysis for the determination of FT specificity. (2) To purify and characterize germ cell surface FT. Isolated plasma membrane from spermatogenic cells and intact purified germ cell classes will be used as source materials for affinity chromatographic enzyme purifications, using HPLC techniques. Comparisons will be made with FTS isolated by previously published protocols from sources other than the testis. (3) To examine the role of cell surface FT in modulating germ cell-Sertoli cell interactions. An in vitro cell-cell binding assay already developed by this laboratory will be employed here. This assay permits quantification of cell-cell binding using purified populations of spermatogenic cells. (4) To examine the function of spermatozoon cell surface FT in sperm-egg interactions. These experiments will entail in vitro binding studies between sperm and the zona pellucida. (5) To identify and characterize the chemical structure of endogenous testicular acceptors for germ cell surface FT. This final Specific Aim will involve HPLC fractionation of germ cell plasma membranes as well as biochemical analysis of (a) cell surface glycoproteins, (b) fucosylated lipids and (c) novel disaccharide glucosyl-fucose, linked O-glycosidically to polypeptides. Results from these studies will related directly to the molecular mechanisms underlying cell differentiation and movement within the mammalian seminiferous epithelium and during initial binding of sperm to the egg and its protective vestments at fertilization. In addition, these studies should provide new data on the interaction of the maturing sperm plasma membrane with epithelial cell-derived macromolecules during sperm epididymal transit.

该项目的总体目标是生化和生理学 分析细胞表面相关的岩藻糖基转移酶（FT） 哺乳动物的精子发生。 我们将检验一个假设，即 相关的FT酶，每种具有不同的糖苷特异性，是 参与Sertoli-Germ细胞相互作用，精子成熟和 受精。 初步研究已经确定了精子 FT在鼠标中的特定阶段表达，并牵涉到这一点 精子识别中的酶。 与大多数体细胞相反， 精子生成细胞的大量部分与 细胞表面作为整体质膜酶 - 酶。 看来 一个FT酶家族，每个酶具有不同的糖苷特异性是 涉及。 该应用程序提出了五个旨在扩展的特定目标 我们对FT在调节精子发生中的作用的理解。 这些 目的是（1）确定FT的糖苷特异性 精子发生和精子成熟。 这里使用的方法将包括新的 开发的薄层色谱微分配程序 作为用于确定FT的Neoglycolipid分析的引入 特异性。 （2）纯化和表征生殖细胞表面ft。 从生精细胞中分离的质膜和完整的纯化生殖 细胞类将用作亲和力色谱的源材料 酶净化，使用HPLC技术。 比较将与 由以前发表的协议隔离的FT与来自其他来源 睾丸。 （3）检查细胞表面ft在调节胚芽中的作用 细胞 - 辛硫代细胞相互作用。 体外细胞 - 细胞结合测定 该实验室已经开发的将在这里使用。 这个测定法 允许使用纯化群体定量细胞细胞结合 精子生成细胞。 （4）检查精子细胞的功能 精子相互作用中的表面ft。 这些实验将需要 精子和Zona pellucida之间的体外结合研究。 （5）到 识别和表征内源睾丸的化学结构 生殖细胞表面ft的受体。 这个最终的特定目标将涉及 生殖细胞质膜和生化的HPLC分馏 分析（a）细胞表面糖蛋白，（b）岩藻糖基化脂质和（c） 新型的二糖葡萄糖葡萄糖，以O-糖苷的链接 多肽。 这些研究的结果将直接与分子有关 细胞分化和运动的机制 哺乳动物的生精上皮和精子初始结合期间 鸡蛋及其保护性服装。 另外，这些 研究应提供有关成熟精子相互作用的新数据 精子期间具有上皮细胞衍生的大分子的质膜 附子转运。