STEROID-INITIATED EXOCYTOSIS IN SPERM

描述（改编自研究者摘要）： 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。 由卵丘滤泡细胞分泌的孕酮， 通过与精子相互作用被认为是体内顶体反应引发剂 膜受体，以触发Ca 2+的流入和Cl-的流出。 在 此外，孕酮或3 α-OH孕酮代谢物可以结合或 激活一种独特的人类精子GABAA样受体Cl-通道， 与Ca 2+通道的受体结合。 可能的原因为 孕酮与一个受体/通道位点结合，然后激活另一个 受体/通道通过串扰。 该应用程序包括一系列 受体、通道和离子相互作用的研究 孕酮通过其启动人类精子顶体反应。 的 提出的具体目标将首先确定精子GABAA样 受体/Cl-通道参与甾体酮介导的Cl-流出， 使用药理学试剂来确定精子之间的差异 和神经元GABAA样受体/Cl-通道。 第二个目标是确定 精子头部的哪个区域是胆甾酮介导的细胞溶质Cl- 通过类固醇受体/Cl-通道的流出开始于顶体 反应 第三个目标是利用现有的单克隆抗体， 细胞内孕酮受体和神经元GABAA样结构域 受体/Cl-通道亚基，以鉴定和进一步表征人类 精子孕酮受体。 第四个目的是比较 孕酮立体异构体对Cl-和Ca 2-通量的影响，以确定两种 单独的孕酮受体起作用以引起离子流。 第五个目标 是为了确定人类精子是否能将孕酮转化为 已知激活神经元GABA A样受体的3 α-羟基代谢物/Cl- 通道，并能够启动人类顶体反应。 第六个目标是 确定GABAA样受体/Cl-介导的Cl-外排是否 通道控制孕酮介导的Ca 2+内流。 第七个目标 将使用免疫电子显微镜来确定 人孕酮受体和人精子GABAA样受体/Cl- 高分辨率的频道。

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.