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，1-10% 的受精卵变成三倍体（并死亡），最常见的原因是多个精子受精。这通常可以通过释放卵皮质颗粒（CG）来预防，其内容物会改变透明带（ZP）。该目标将确定排卵前卵母细胞 CG 释放失败的生化基础，以及细胞内钙 (Ca2+) 振荡在成熟小鼠卵中 CG 释放和细胞周期恢复中的作用，如下。 1) 在成熟卵中，将测试以下假设：CG 胞吐作用和细胞周期恢复具有编码在 Ca2+ 振荡参数中的不同 Ca2+ 需求。尽管已知 Ca2+ 振荡会在受精时引起 CG 释放、ZP 修饰和细胞周期恢复（通过 H1 和 MAP 激酶的减少），但尚不清楚这些事件是否需要不同数量或类型的振荡来激活和/或计时。将通过实验改变振幅、频率或振荡次数，并量化上述卵子激活事件的变化来测试三个假设。 2) 在排卵前的卵母细胞中，CG 分泌途径中被阻断的步骤将被识别。将研究三个假设：(A) CG 从皮质到质膜易位失败，(B) 正常易位随后 CG 对接失败，以及 (C) 易位/对接后膜融合失败。对于阻断步骤（A、B 或 C），将比较有分泌能力的成熟卵和无能力卵母细胞中的相关结构和调节蛋白的蛋白质含量、定位和磷酸化状态，以开始识别导致失败的生化缺陷胞吐作用（包括分泌机器蛋白、钙调蛋白深度激酶 II、突触蛋白和相关细胞骨架蛋白）。意义（长期目标）：目标 1：精确的 Ca2+ 需求（本研究）与 Ca2+ 效应器的识别（目标 2 和其他研究）相结合，将提供有关 Ca2+ 引起卵子激活事件的分子机制的基本信息负责哺乳动物发育的开始。使用该提案中的方法，可以在没有精子的情况下测试体外成熟的人卵母细胞的激活能力（本文未应用）。目标 2：鉴定参与分泌的蛋白质和负责 CG 释放的（排卵前）生化变化将为卵母细胞成熟提供有价值的标记，以评估体外或体内成熟动物和人类卵母细胞的新兴方案。