CONTROL MECHANISMS FOR EGG ACTIVATION/CORTICAL GRANULES

卵子激活/皮质颗粒的控制机制

• 批准号: 6044987
• 负责人: THOMAS W DUCIBELLA
• 金额: $ 24.57万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6044987
• 关键词: calcium flux; calmodulin dependent protein kinase; cell cycle; cytoskeletal proteins; egg /ovum; egg proteins; exocytosis; female; fertilization; granule; laboratory mouse; mitogen activated protein kinase; phosphorylation; protein binding; protein localization; protein structure function; protein transport; synapsins; tissue /cell culture

In humans and other mammals, 1-10% of fertilized eggs become triploid (and die) most often due to fertilization by more than one sperm. This is normally prevented by the release of egg cortical granules (CGs) whose contents modify the zona pellucida (ZP). The Aims will determine the biochemical basis for the failure of CG release in pre-ovulatory oocytes, and the roles of intracellular calcium (Ca2+) oscillations in CG release and cell cycle resumption in mature mouse eggs, as follows. 1) In mature eggs, the hypothesis will be tested that CG exocytosis and cell cycle resumption have different Ca2+ requirements encoded in Ca2+ oscillation parameters. Although Ca2+ oscillations are known to cause CG release, ZP modifications, and cell cycle resumption (via decreases in H1 and MAP kinases) upon fertilization, it is not known whether these events require different numbers or types of oscillations for their activation and/or timing. Three hypotheses will be tested by experimentally changing the amplitude, frequency, or number of oscillations, and quantifying changes in the aforementioned events of egg activation. 2) In pre ovulatory oocytes, the blocked step in the CG secretory pathways will be identified. Three hypothesis will be investigated: (A) Failure of CG translocation from the cortex to the plasma membrane, (B) normal translocation followed by a failure in CG docking, and (C) failure of membrane fusion after translocation/docking. For the blocked step (A, B, or C), relevant structural and regulatory proteins will be compared in secretion-competent mature eggs and incompetent oocytes for protein amount, localization, and phosphorylation status to begin to identify the biochemical deficiency responsible for the failure of exocytosis (including secretory-machinery proteins, calmodulin-depth kinase II, synapsin, & relevant cytoskeletal proteins). Significance (of long-range objectives): Aim 1: The precise Ca2+ requirements (this study) coupled with the identification of the Ca2+ effectors (Aim 2 & other studies) will provide fundamental information about the molecular mechanism by which Ca2+ causes egg activation events responsible for the onset of mammalian development. Using the methods in the proposal, in vitro matured human oocytes could be tested for activation competence without sperm (not applied for herein). Aim 2: Identification of the proteins involved in secretion and the (pre-ovulatory) biochemical change(s) responsible for CG release will provide valuable markers of oocyte maturation to evaluate emerging protocols to mature animal and human oocytes in vitro or in vivo.

在人类和其他哺乳动物中，有1-10％的受精卵变成三倍体（并死亡），通常是由于超过一个精子的施肥而导致的。通常，这是通过释放卵皮质颗粒（CGS）的释放来预防的，这些卵皮质颗粒（CGS）会改变Zona pellucida（ZP）。目的将确定卵巢前卵母细胞中CG释放失败的生化基础，以及细胞内钙（CA2+）振荡在CG释放和成熟小鼠卵中细胞周期恢复中的作用，如下所示。 1）在成熟的卵中，将检验假设CG胞吐作用和细胞周期恢复具有不同的Ca2+要求，以Ca2+振荡参数编码。尽管已知CA2+振荡会导致CG释放，ZP修饰和细胞周期恢复（通过H1和MAP激酶减少），但尚不清楚这些事件是否需要不同数量或类型的激活和/或时间振荡。将通过实验更改振幅，频率或数量来测试三个假设，并量化上述鸡蛋激活事件的变化。 2）在排卵前卵母细胞中，将确定CG分泌途径中的阻塞步骤。将研究三个假设：（a）CG从皮质转移到质膜的失败，（b）正常易位，随后在CG对接中发生故障，以及（c）易位/docking后膜融合的失败。对于被阻塞的步骤（A，B或C），将在具有分泌能力的成熟卵和无能的蛋白质量，定位和磷酸化状态的能力的成熟卵和无能的卵母细胞中比较相关的结构和调节蛋白，从细胞骨架蛋白）。意义（远程目标）：目标1：确切的Ca2+需求（本研究），再加上CA2+效应子的识别（AIM 2＆其他研究）将提供有关CA2+导致Egg激活事件的分子机制的基本信息，从而导致哺乳动物发育的发作。使用提案中的方法，可以在没有精子的情况下测试体外成熟的人类卵母细胞的激活能力（此处不适用）。目标2：鉴定涉及分泌物的蛋白质以及负责CG释放的（卵巢前的）生化变化（S）将提供有价值的卵母细胞成熟标志物，以评估体外或体内成熟动物和人类卵母细胞的新兴方案。