GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE DURING SPERMATOGENESIS & FERTILIZATION

精子发生过程中的甘油醛 3-磷酸脱氢酶

• 批准号: 6440534
• 负责人: Deborah A. O'Brien
• 金额: $ 17.42万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440534
• 关键词: Sertoli cells; cell differentiation; cofactor; enzyme activity; enzyme biosynthesis; enzyme linked immunosorbent assay; enzyme mechanism; enzyme substrate; fertility; genetically modified animals; glyceraldehyde 3 phosphate dehydrogenase; glycolysis; histology; laboratory mouse; male; radioimmunoassay; reproductive system; sperm capacitation; sperm motility; spermatogenesis; synthetic enzyme; tissue /cell culture; western blottings

The long-term goal is to identify mechanisms regulating glycolysis druing spermatogenesis and sperm function. Isolated spermatids are unacble to maintain ATP levels when glucose is their oonly energy substrate, while sperm require glucose for in vitro fertilization, suggesting that unique mechanisms regulate glycolysis in male germ cells. Glyceraldehyde 3- phosphate dehydrogenase (GAPD) appears to be the rate limiting enzyme for glycolysis in spermatids. A gene (Gapd-s) encoding a unique form of this enzyme (GAPD-S) is expressed only in spermatids and may serve a pivotal role in regulating glycolysis in these cells. Competitve inhibitors of NAD coenzyme binding to this enzyme suppress glycolysisin spermatids, and competitive inhibitors or substrate(glyceraldhyde 3 phosphate) binding are male reproductive toxicants. GAPD-S also has unique sequence features that may be involved in regulating its activity. It is hypothesized that glycolysis is suppressed in late spermatids due to inhibition of GAPD-S, but that GAPD-S is activated in sperm where glycolysis provides energy required for fertilization. The specific aims of this study are to: 1) Determine when GAPD-S is synthesized and where it is localized in spermatozoa. Specific antibody probes will be used to define when the GAPD-S protein is expressed during spermatid differentiation (spermiogenesis) and determine whether this enzyme binds to sperm structural components. 2) Define the role of GAPD-S during spermiogenesis, sperm maturaltion and fertilization. In vitro studies will determine when GAPD-S is activation during the development and function of sperm and define its role in providing energy required fertilization. Target disruption of the Gapd-s gene by homogous recombination will be used to determine whether GAPD-S is required for spermiogenesis and/or fertility in mice. 3) Identify the molecular mechanisms that regulate GAPD-S in spermatids and sperm. To determine how GAPD-S is regulated, activities of recombinant GAPD-S and GAPD will be compared in cell-free assays in the presence of competitive inhibitors of coenzyme and substrate binding. The studies will determine whether the regulation of energy production in spermatogenic cells is due to unique structural and functional features of GAPD-S, and will provide fundamental information on the role of GAPD-S in fertilization. Mutations in the Gapd-s gene or inhibition of the GAPD-S enzyme by environmental agents may cause infertility in men, and the GAPD-S enzyme may be a feasible target for a highly specific contraceptive agent.

长期的目标是确定糖酵解的调节机制， 精子发生和精子功能。 分离的精子细胞不能 当葡萄糖是它们唯一的能量底物时，维持ATP水平， 精子体外受精需要葡萄糖，这表明 调节雄性生殖细胞糖酵解的机制。 甘油醛3- 磷酸脱氢酶（GAPD）似乎是 精子细胞的糖酵解。 一个基因（Gapd-s）编码一种独特的形式， GAPD-S仅在精子细胞中表达，可能是一种关键的 在调节这些细胞的糖酵解中起作用。 竞争性抑制剂 NAD辅酶与这种酶结合抑制精子细胞的糖酵解， 竞争性抑制剂或底物（甘油醛3磷酸）结合 是男性生殖毒素 GAPD-S还具有独特的序列 可能参与调节其活性的特征。 是 假设糖酵解在晚期精子细胞中受到抑制， 抑制GAPD-S，但GAPD-S在精子中被激活， 糖酵解提供受精所需的能量。 具体目标 本研究的目的是：1）确定何时合成GAPD-S，以及在何处合成 它位于精子中。 将使用特异性抗体探针 为了确定GAPD-S蛋白在精子细胞中何时表达， 分化（精子生成），并确定这种酶是否结合 精子的结构成分。 2)定义GAPD-S在以下过程中的作用： 精子发生、精子成熟和受精。 体外研究 将决定在开发过程中何时激活GAPD-S， 精子的功能并定义其在提供所需能量方面的作用 受精同源重组靶向破坏Gapd-s基因 重组将用于确定是否需要GAPD-S 小鼠精子发生和/或生育力。 3)识别分子 调节精子细胞和精子中GAPD-S的机制。 以确定 如何调节GAPD-S，重组GAPD-S和GAPD的活性将 在存在竞争性抑制剂的情况下在无细胞测定中进行比较 辅酶和底物的结合。 这些研究将确定能源生产的监管是否 是由于生精细胞独特的结构和功能 GAPD-S的功能，并将提供有关角色的基本信息 GAPD-S在受精中的作用。 Gapd-s基因突变或抑制 环境因素对GAPD-S酶的影响可能会导致不育， 男性，GAPD-S酶可能是一个可行的目标，高度特异性 避孕药