SPERM GLYCINE RECEPTORS AND ZONA-INITIATED EXOCYTOSIS

超出所提供的空间。哺乳动物顶体反应（AR）是受精过程中必需的精子头部胞吐事件。神经元甘氨酸受体/Cl通道（GlyR）存在于哺乳动物精子中，并且对于由卵透明质（ZP）蛋白ZP 3启动的AR是必需的。这项资助的总体目标是增加我们对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C1流出至少部分负责去极化，导致AR必需的Ca 2+内流，GlyR的激活涉及PKA介导的磷酸化。本研究采用体外AR试验，采用成像、光度法和拮抗剂法，分别研究小鼠ZP和人重组人ZP 3（rhZP 3）对小鼠精子和人精子的影响。以下是具体目标。1.测定人和小鼠精子GlyR是否介导AR过程中rhZP 3、小鼠ZP或甘氨酸引起的细胞内Ca 2+（Ca 2 +i）升高。2.确定人和小鼠精子GlyR是否参与甘氨酸、rhZP 3或小鼠ZP引起的膜电位变化。3.确定蛋白激酶A（PKA）对GlyR的磷酸化是否参与甘氨酸、小鼠ZP或rhZP 3引发的AR。4.确定PKA是否对GlyR介导的膜电位变化和甘氨酸、小鼠ZP和rhZP 3引起的Ca 2 +i增加重要。5.确定在来自具有GlyR突变的小鼠的精子中小鼠ZP介导的膜电位和Ca 2 +i变化是否将不存在或降低。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。如果没有，确定这些精子在ZP上是否被孕酮刺激AR和受精？能够比较人类精子和rhZP 3获得的结果与小鼠精子和小鼠ZP体内合成的结果将有助于我们确定是否涉及哺乳动物物种的共同机制。性能现场=

EXCEED THE SPACE PROVIDED. 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 C1- 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. PERFORMANCE SITE ========================================Section End===========================================