Ca2+ OSCILLATIONS AND KINASES REGULATE EGG ACTIVATION

Ca2 振荡和激酶调节卵子激活

• 批准号: 6898800
• 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/6898800
• 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）：受精是一个从根本上重要的事件，在该事件中，激活信号通路，刺激胚胎发育的启动。在哺乳动物中，必要且足以引起卵子激活的卵子内钙（Ca2+）的受精相关的振荡，包括到多性体经体的障碍，细胞周期恢复的减数分裂，细胞周期调节性激酶的变化以及蛋白质表达的变化。受精衰竭，异常早期发育和多植物代表了人类和动物的重大问题。 Ca2+及其振荡刺激卵子激活事件的机制尚不清楚，并且在我们了解受精如何激活早期发育的知识中代表了重要差距。在初步数据和最新出版物的支持下，目的将确定重要的Ca2+传感器CAMKII的作用和Ca2+依赖性在小鼠发育的启动中。 AIM 1将确定鸡蛋激活的关键事件是否依赖于受精卵，包括细胞周期激酶MAPK的活性降低，核形成和蛋白质表达的变化。 AIM 2将检验CAMKII活性随CA2+振荡的函数振荡的假设。实验策略将是将CAMKII活性与同一卵中的细胞内Ca2+水平相关联。这种创新的方法将证明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将Ca2+水平和激酶活性在同一细胞中的演示。 AIM 3将确定CAMKII活性和CAMKII依赖性事件如何受CA2+振荡的各个参数调节。通过实验控制这些CA2+参数（幅度，频率，数量等），将确定CAMKII活动如何随体内这些振荡参数的函数而变化。另外，将确定下游细胞周期激酶（例如MPF，MAPK）对Ca2+和Camkii的依赖性。这些研究将显着提高我们对CA2+升高下游信号通路的了解，为哺乳动物发育开始期间开始单个事件提供了更全面的生化机制，并对评估现有的临床（但人工）卵子活化程序和动物施加具有影响。