Ca2+ OSCILLATIONS AND KINASES REGULATE EGG ACTIVATION

Ca2 振荡和激酶调节卵子激活

• 批准号: 6678420
• 负责人: THOMAS W DUCIBELLA
• 金额: $ 29.16万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678420
• 关键词: artificial fertilization; calcium flux; calmodulin dependent protein kinase; cell cycle; cell cycle proteins; enzyme activity; fertilization; gonadotropins; in vitro fertilization; kinase inhibitor; laboratory mouse; mammalian embryology; mitogen activated protein kinase; nuclear membrane

DESCRIPTION (provided by applicant): Fertilization is a fundamentally important event in which signaling pathways are activated that stimulate the initiation of embryonic development. In mammals, fertilization-associated oscillations of egg intracellular calcium (Ca2+) are necessary and sufficient to cause egg activation, including the block to polyspermy, cell cycle resumption for the completion of meiosis, changes in cell cycle regulatory kinases, and alterations in protein expression. Fertilization failure, abnormal early development, and polyspermy represent significant problems in humans and animals. The mechanisms by which Ca2+ and its oscillations stimulate the events of egg activation are not well understood and represent important gaps in our knowledge of how fertilization activates early development. Supported by preliminary data and recent publications, the Aims will determine the role(s) and Ca2+-dependence of an important Ca2+ sensor, CaMKII, in the initiation of mouse development. AIM 1 will determine if crucial events of egg activation are CaMKII-dependent in fertilized eggs, including the decrease in activity of the cell cycle kinase MAPK, pronuclear formation, and changes in protein expression. AIM 2 will test the hypothesis that CaMKII activity oscillates as a function of Ca2+ oscillations. The experimental strategy will be to directly correlate CaMKII activity with the intracellular Ca2+ level in the same egg. This innovative approach will demonstrate how CaMKII activity responds to Ca2+ oscillations in a living cell and may be the first demonstration directly correlating Ca2+ levels and kinase activity in the same cell. AIM 3 will determine how CaMKII activity and CaMKII-dependent events are regulated by individual parameters of Ca2+ oscillations. By experimentally controlling these Ca2+ parameters (amplitude, frequency, number, etc.), it will be determined how CaMKII activity varies as a function of these oscillation parameters in vivo. In addition, the dependency of downstream cell cycle kinases (e.g., MPF, MAPK) on Ca2+ and CaMKII will be determined. These studies will significantly increase our knowledge of the signaling pathways downstream of the elevation of Ca2+, provide a more comprehensive biochemical mechanism for the initiation of individual events during the onset of mammalian development, and have implications for evaluating existing clinical (but artificial) egg activation procedures and for animal cloning.

描述（由申请人提供）：受精是一个非常重要的事件，其中信号通路被激活，刺激胚胎发育的启动。在哺乳动物中，受精相关的卵细胞内钙 (Ca2+) 振荡对于引起卵激活是必要且充分的，包括阻止多精受精、恢复细胞周期以完成减数分裂、细胞周期调节激酶的变化以及蛋白质表达的改变。受精失败、早期发育异常和多精受精是人类和动物面临的重大问题。 Ca2+ 及其振荡刺激卵子激活事件的机制尚不清楚，这代表了我们在受精如何激活早期发育的知识中的重要差距。在初步数据和最近出版物的支持下，该目标将确定重要的 Ca2+ 传感器 CaMKII 在小鼠发育启动中的作用和 Ca2+ 依赖性。 AIM 1 将确定受精卵中卵子激活的关键事件是否依赖于 CaMKII，包括细胞周期激酶 MAPK 活性的降低、原核形成和蛋白质表达的变化。 AIM 2 将测试 CaMKII 活性作为 Ca2+ 振荡函数振荡的假设。实验策略是将 CaMKII 活性与同一卵中的细胞内 Ca2+ 水平直接相关。这种创新方法将演示 CaMKII 活性如何响应活细胞中的 Ca2+ 振荡，并且可能是第一个直接关联同一细胞中 Ca2+ 水平和激酶活性的演示。 AIM 3 将确定 CaMKII 活性和 CaMKII 依赖性事件如何通过 Ca2+ 振荡的各个参数进行调节。通过实验控制这些 Ca2+ 参数（幅度、频率、数量等），将确定 CaMKII 活性如何随着体内这些振荡参数的函数而变化。此外，还将确定下游细胞周期激酶（例如 MPF、MAPK）对 Ca2+ 和 CaMKII 的依赖性。这些研究将显着增加我们对 Ca2+ 升高下游信号通路的了解，为哺乳动物发育开始期间个体事件的启动提供更全面的生化机制，并对评估现有的临床（但人工）卵子激活具有重要意义。程序和动物克隆。