SPERM GLYCINE RECEPTOR AND ZONA-INITIATED EXOCYTOSIS

精子甘氨酸受体和透明带引发的胞吐作用

• 批准号: 2883145
• 负责人: STANLEY MEIZEL
• 金额: $ 22.63万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2883145
• 关键词: affinity chromatography; calcium flux; chloride channels; chloride ion; exocytosis; fertilization; glycine; glycine receptors; human subject; male; protein structure function; reproductive system pharmacology; sperm; swine; zona pellucida glycoproteins

DESCRIPTION (Adapted from the Investigator's Abstract): This revised proposal addresses examines the role of a putative glycine receptor (GlyR) on the mammalian sperm surface. The acrosome reaction is an exocytotic event involving fusion of the plasma membrane with the underlying acrosomal membrane, followed by release of acrosomal contents. It is required for fertilization. The zona pellucida is believed to be a physiological inducer of the acrosome reaction, but the mechanisms that control exocytosis are not well understood. Ca2+ and Cl- fluxes are required for the induction of the acrosome reaction by the zona pellucida. Recent studies from the P.I.'s laboratory indicate that a GlyR/Cl- channel is involved in the zona-mediated pathway, although it is not certain whether such a receptor is present and what the nature of its role in the acrosome reaction might be. This application tests the hypothesis that GlyR activation produces Cl- efflux and that the resulting depolarization activates voltage-sensitive Ca2+ channels. The P.I. intends to characterize a sperm GlyR and to determine its role in the acrosome reaction. Studies will be carried out in both the human and pig systems. In Aim 1 confocal microscopy will be used to localize BODIPY-strychnine, a probe for the putative GlyR, on live and on fixed human sperm. Aim 2 will monitor Gly-dependent Cl- fluxes in human sperm using the MEQ fluorescent probe. The effects of certain GlyR agonists, antagonists, and potentiators on Cl- and acrosome reactions in human sperm will be determined. Aim 4 will determine whether the GlyR-mediated Cl- efflux activates a Ca2+ influx through voltage-sensitive Ca2+ channels. Internal Ca2+ responses to Gly stimulation will be determined in single sperm by quantitative fluorescence microscopy, in collaboration with Nuccitelli. Additional studies will determine the effects of Ca2+ channel antagonists on this response. Aims 5-7 address the biochemical and functional characterization of the porcine GlyR. In Aim 5, the P.I. will solubilze boar sperm membranes and isolate the receptor by affinity chromatography. Biochemical characterization of the isolated receptor will be performed.

描述（改编自研究者的摘要）：本修订版 提案地址检查了假定的甘氨酸受体 (GlyR) 的作用 哺乳动物精子表面。 顶体反应是一种胞吐反应 涉及质膜与下面的顶体融合的事件 膜，然后释放顶体内容物。 需要用于 施肥。 透明带被认为是一种生理诱导剂 顶体反应，但控制胞吐作用的机制不是 很好理解。 感应需要 Ca2+ 和 Cl- 通量 透明带顶体反应。 P.I. 最近的研究 实验室表明 GlyR/Cl- 通道参与透明带介导的 途径，尽管不确定是否存在这样的受体并且 它在顶体反应中的作用可能是什么？ 这 应用测试了 GlyR 激活产生 Cl- 流出的假设 由此产生的去极化会激活电压敏感的 Ca2+ 渠道。 P.I.打算表征精子 GlyR 并确定其在 顶体反应。 研究将在人类和 养猪系统。 在目标 1 中，将使用共焦显微镜来定位 BODIPY-士的宁，一种针对活体和固定人体的假定 GlyR 的探针 精子。 目标 2 将使用以下方法监测人类精子中依赖于甘氨酸的 Cl 通量： MEQ 荧光探针。 某些 GlyR 激动剂、拮抗剂的作用， 以及人类精子中 Cl- 和顶体反应的增效剂 决定。 目标 4 将确定 GlyR 介导的 Cl 流出是否 通过电压敏感 Ca2+ 通道激活 Ca2+ 流入。 内部的 Ca2+ 对 Gly 刺激的反应将通过以下方式确定单个精子中的 Ca2+ 反应： 定量荧光显微镜，与 Nuccitelli 合作。 其他研究将确定 Ca2+ 通道拮抗剂对 这个回应。 目标 5-7 涉及生化和功能 猪 GlyR 的表征。 在目标 5 中，P.I.会溶解 公猪精子膜并通过亲和层析分离受体。 将进行分离受体的生化表征。