STEROID-INITIATED EXOCYTOSIS IN SPERM

描述(改编自《调查者摘要》)：这项研究 三次修订的提案旨在了解分子步骤 参与人类精子顶体反应的启动。 卵丘卵泡细胞分泌的黄体酮 通过与精子相互作用被认为是体内顶体反应的启动者 膜受体触发钙离子内流和氯离子外流。在……里面 此外，黄体酮或3a-羟基黄体酮代谢物可结合或 激活一个独特的人精子GABAA样受体Cl-通道以及 与钙离子通道的受体结合。存在这样一种可能性： 黄体酮与一个受体/通道结合，然后激活另一个受体/通道 通过串扰实现接收器/通道。该应用程序包括一系列 关于受体、通道和离子相互作用的研究 黄体酮通过它启动人类精子顶体反应。这个 被提议的特定目标将首先决定类似于GABAA的精子 受体/氯离子通道参与孕酮介导的氯离子外流 使用将确定精子之间差异的药理学试剂 神经元GABAA样受体/氯离子通道。第二个目标是确定 黄体酮介导的胞浆氯离子在精子头部的哪个区域 通过类固醇受体/氯离子通道的外流开始于顶体 反应。第三个目标是使用可用的单抗来对抗 细胞内孕酮受体结构域和神经元GABAA样蛋白 受体/氯通道亚基用于识别和进一步表征人类 精子孕酮受体(S)。第四个目标是比较 孕酮立体异构体对氯离子和钙离子通量的测定 不同的孕激素受体可产生离子通量。第五个目标 是为了确定人类精子是否能将黄体酮转化为 3A-羟基代谢物可激活神经元GABAA样受体/Cl- 并能够启动人类顶体反应。第六个目标是 确定GABAA样受体/氯-是否介导氯-外流 通道控制孕酮介导的钙内流。最后，第七个目标 将使用免疫电子显微镜来确定 人孕酮受体(S)与人精子GABAA样受体/Cl- 高分辨率的通道。

DESCRIPTION (Adapted from the Investigator's Abstract): Research in this three-times revised proposal is aimed at understanding the molecular steps involved in the initiation of the human sperm acrosome reaction. Progesterone secreted by follicular cells of the cumulus oophorus, is regarded as an in vivo acrosome reaction initiator by interacting with sperm membrane receptors to trigger an influx of Ca2+ and an efflux of Cl-. In addition, progesterone or a 3a-OH progesterone metabolite may bind or activate a unique human sperm GABAA-like receptor Cl- channel as well as bind to a receptor for a Ca2+ channel. The possibility exists that progesterone binds to one receptor/channel site and then activate the other receptor/channel by cross-talk. The application includes a series of studies concerned with the receptors, channels and ionic interactions through which progesterone initiates the human sperm acrosome reaction. The proposed specific aims will determine first whether a sperm GABAA-like receptor/Cl- channel is involved in progesterone-mediated Cl- efflux by using pharmacological agents which will determine differences between sperm and neuronal GABAA-like receptor/Cl-channels. A second aim is to determine which region of the sperm head the progesterone-mediated cytosolic Cl- efflux through the steroid receptor/Cl-channel begins during acrosome reaction. A third aim is to use available monoclonal antibodies against domains of the intracellular progesterone receptor and neuronal GABAA-like receptor/Cl- channel subunits to identify and further characterize the human sperm progesterone receptor(s). A fourth aim is to compare the effect of progesterone stereo isomers on Cl- and Ca2- flux to determine whether two separate progesterone receptors operate to elicit ion fluxes. A fifth aim is to determine whether human sperm can convert progesterone into an 3a-hydroxy metabolite known to activate neuronal GABAA-like receptor/Cl- channels and able to initiate the human acrosome reaction. A sixth aim is to determine whether Cl- efflux mediated by the GABAA-like receptor/Cl- channel controls progesterone-mediated Ca2+ influx. Finally, a seventh aim will use immunoelectron microscopy to determine the distribution of the human progesterone receptor(s) and human sperm GABAA-like receptor/Cl- channel at a high resolution level.