SPERM GLYCINE RECEPTORS 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: 6731181
• 负责人: STANLEY MEIZEL
• 金额: $ 26.73万
• 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 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731181
• 关键词: SPERM; GLYCINE; RECEPTORS; ZONA; INITIATED

DESCRIPTION (provided by applicant): The mammalian acrosome reaction (AR) is a sperm head exocytotic event essential to fertilization. A neuronal glycine receptor/Cl channel (GlyR) is present in mammalian sperm and is necessary for the AR initiated by the egg zona pellucida (ZP) protein ZP3. The overall goal of this grant is to increase our understanding of the role of the GlyR in the ZP-initiated AR and the mechanisms involved in controlling its activation. We hypothesize that ZP activates the GlyR directly or via receptor cross-talk, that Cl- efflux through the GlyR is at least partly responsible for depolarization leading to Ca 2+ influx essential to the AR and that activation of the GlyR involves PKA-mediated phosphorylation. The effects of mouse ZP and human recombinant human ZP3 (rhZP3) on mouse sperm and human sperm respectively will be studied using imaging, photometry and antagonists in in vitro AR assays. The following are the Specific Aims. 1. Determination of whether the human and mouse sperm GlyR mediate the intracellular Ca 2+ (Ca2+i) increase elicited by rhZP3, mouse ZP or glycine during the AR. 2. Determination of whether the human and mouse sperm GlyR are involved in the membrane potential change elicited by glycine, rhZP3 or mouse ZP. 3. Determination of whether phosphorylation of the GlyR by protein kinase A (PKA) is involved in AR initiated by glycine, mouse ZP or rhZP3. 4. Determination of whether PKA is important to GlyR mediation of membrane potential change and to the Ca2+i increase elicited by glycine, mouse ZP and rhZP3. 5. Determination of whether mouse ZP-mediated membrane potential and Ca2+i changes would be absent or decreased in sperm from mice with GlyR mutations. 6. Determination of whether the AR of sperm from mice with GlyR mutations occur on the ZP of intact mouse eggs in vitro. If not, determination of whether the AR and fertilization be stimulated by progesterone in these sperm while they are on the ZP? Being able to compare results obtained with human sperm and rhZP3 and those obtained with mouse sperm and mouse ZP synthesized in vivo will help us determine whether common mechanisms are involved in mammalian species.

描述（由申请人提供）：哺乳动物顶体反应（AR）是受精所必需的精子头胞吐事件。神经元甘氨酸受体/Cl 通道 (GlyR) 存在于哺乳动物精子中，对于卵透明带 (ZP) 蛋白 ZP3 启动的 AR 是必需的。这项资助的总体目标是加深我们对 GlyR 在 ZP 启动的 AR 中的作用以及控制其激活的机制的理解。我们假设ZP直接或通过受体串扰激活GlyR，通过GlyR的Cl-流出至少部分负责导致AR必需的Ca 2+ 内流的去极化，并且GlyR的激活涉及PKA介导的磷酸化。将在体外 AR 测定中使用成像、光度测定和拮抗剂来研究小鼠 ZP 和人类重组人 ZP3 (rhZP3) 分别对小鼠精子和人类精子的影响。以下是具体目标。 1. 确定人和小鼠精子 GlyR 是否介导 AR 期间由 rhZP3、小鼠 ZP 或甘氨酸引起的细胞内 Ca 2+ (Ca2+i) 增加。 2.测定人和小鼠精子GlyR是否参与甘氨酸、rhZP3或小鼠ZP引起的膜电位变化。 3.确定GlyR被蛋白激酶A(PKA)磷酸化是否参与由甘氨酸、小鼠ZP或rhZP3引发的AR。 4.确定PKA对于GlyR介导的膜电位变化以及对于甘氨酸、小鼠ZP和rhZP3引起的Ca 2+i 增加是否重要。 5. 确定具有 GlyR 突变的小鼠精子中小鼠 ZP 介导的膜电位和 Ca2+i 变化是否缺失或减少。 6. 确定GlyR突变小鼠精子的AR是否发生在体外完整小鼠卵子的ZP上。如果不是，确定这些精子在 ZP 上时是否会受到黄体酮的刺激而发生 AR 和受精？能够将人类精子和 rhZP3 获得的结果与小鼠精子和体内合成的小鼠 ZP 获得的结果进行比较，将有助于我们确定哺乳动物物种中是否涉及共同机制。