SPERM GLYCINE RECEPTOR AND ZONA-INITIATED EXOCYTOSIS

说明(改编自《调查员摘要》)：此修订版 Proposal Addresses研究了一个假定的甘氨酸受体(GlyR)的作用 在哺乳动物的精子表面。顶体反应是一种胞吐反应 涉及质膜与下层顶体融合的事件 膜，然后释放顶体内容物。它是必需的，用于 受精。人们认为透明带是一种生理诱导物。 顶体反应，但控制胞吐的机制不是 很好理解。Ca~(2+)和Cl~-助熔剂是诱导细胞生长所必需的。 透明带的顶体反应。私家侦探S的最新研究 实验室研究表明，GlyR/Cl-通道参与了小带介导的 途径，尽管还不确定这样的受体是否存在以及 它在顶体反应中可能扮演的角色的性质。这 应用验证GlyR激活产生氯离子外流的假设 由此产生的去极化激活了电压敏感的钙离子 频道。 P.I.打算描述精子GlyR的特征并确定其在 顶体反应。这项研究将在人类和 猪系统。在AIM 1中，将使用共聚焦显微镜来定位 BODIPY-士的宁，一个可能的GlyR的探针，在活体和固定的人身上 精子。AIM 2将监测人类精子中依赖甘氨酸的氯离子通量 MEQ荧光探头。某些GlyR激动剂、拮抗剂、 人类精子中氯离子和顶体反应的增强剂将是 下定决心。目标4将确定GlyR介导的氯-外流 通过电压敏感的钙通道激活钙离子内流。内部 单个精子对甘氨酸刺激的Ca~(2+)反应将通过 定量荧光显微镜，与Nuccitelli合作。 更多的研究将确定钙通道拮抗剂对 这样的回应。目标5-7涉及生化和功能 猪GlyR的特性研究在目标5中，私家侦探将会溶解 猪精子膜，亲和层析分离受体。 将对分离的受体进行生化表征。

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.