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: 2025701
• 负责人: STANLEY MEIZEL
• 金额: $ 21.93万
• 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/2025701
• 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（改编自调查员的摘要）：此修订 提案探讨了假定的甘氨酸受体（GLYR）的作用 在哺乳动物的精子表面。 Adrosom反应是胞吐 涉及质膜融合与潜在的Aromal融合的事件 膜，然后释放杂色含量。 这是必需的 受精。 Zona Pellucida被认为是生理诱导剂 ACROSOM体反应，但控制胞吐作用的机制不是 理解。 Ca2+和Cl-磁通量是需要诱导的 Zona Pellucida的Acrosom体反应。 P.I.的最新研究 实验室表明Glyr/Cl-通道参与了Zona介导的 途径，尽管不确定是否存在这样的受体，并且 其在高级反应中的作用的性质可能是什么。 这 应用测试了Glyr激活产生Cl-Offlux的假设 所产生的去极化激活电压敏感的Ca2+ 频道。 P.I.打算表征精子，并确定其在 高级反应。 研究将在人类和 猪系统。 在AIM 1中，共聚焦显微镜将用于本地化 Bodipy-strychnine，对假定的Glyr的探测，现场和固定人类 精子。 AIM 2将使用人类精子中的Gly依赖性cl胞 MEQ荧光探针。 某些Glyr激动剂，拮抗剂的影响 和人类精子中Cl-和Acrosom反应的增强剂将是 决定。 AIM 4将确定Glyr介导的Cl-流出是否 通过对电压敏感的Ca2+通道激活Ca2+涌入。 内部的 Ca2+对Gly刺激的反应将在单个精子中确定 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解决生化和功能 猪的特征。 在AIM 5，P.I。会溶性 公猪精子膜和通过亲和色谱分离受体。 将进行分离受体的生化表征。