IP3r-1 Regulation and Egg Activation

描述（由申请人提供）：本申请的长期目标是阐明控制哺乳动物物种卵子激活的机制。哺乳动物的卵子在第二次减数分裂（MIl）的中期被抑制。卵子激活需要从MIl中退出，并完成一系列形态和生化变化，这些变化启动了有丝分裂，从而启动了发育程序。受精通过引起细胞内游离钙（[Ca2+]i）浓度的周期性变化（称为[Ca2+]i振荡）诱导卵子激活。肌醇1,4,5 -三磷酸受体（IP3R-1）是一个配体门控通道，介导受精时的[Ca2+]i升高。有证据表明，IP3R-1的质量、分布和电导率在成熟和受精过程中受到调节，这是建立受精时[Ca2+]i响应的时空组织所必需的。尽管IP3R-1具有如此关键的作用，但人们对其在卵子中控制其功能的分子机制知之甚少。在这里，我们假设IP3R-1是卵子激活过程中Ca2+释放组织的关键调控位点。为了进一步阐明控制IP3R-1功能的机制，我们建议：1)研究在激活过程中如何调节IP3R-1降解以及它如何影响精子启动的[Ca2+]i振荡；2)确定细胞周期相关激酶对IP3R-1的磷酸化是否介导了观察到的细胞周期、IP3R-1电导率和[Ca2+]i振荡之间的关联；3)研究IP3R-1再分布的定位和机制，以及IP3R-1分布/皮质簇的形成如何影响Ca2+释放。为了解决这些问题，我们将进行[Ca2+]i监测，免疫印迹，免疫荧光，mrna的显微注射，笼化化合物的释放，IP3R-1的突变和标记，随后在卵子/体细胞中表达和共聚焦显微镜可视化。相关性：考虑到成熟过程中IP3R-1功能的多样化和精确调节，在排卵/成熟后评估这些参数可能为评估激素刺激/体外成熟条件对卵母细胞质量的影响提供标记。此外，由于异常Ca2+释放发生在排卵后老化的卵子中，这可能导致胚胎破碎，阐明控制IP3R-1功能的机制有望改善胚胎发育和辅助生殖技术的成功。

DESCRIPTION (provided by applicant): The long-term goal of this application is to elucidate the mechanisms that control egg activation in mammalian species. Mammalian eggs are ovulated arrested at the metaphase stage of the second meiosis (MIl). Egg activation entails exit from MIl and the completion of a series of morphological and biochemical changes that initiate the mitotic and, consequently, the developmental program. Fertilization induces egg activation by evoking periodical changes in the intracellular concentration of free calcium ([Ca2+]i) known as [Ca2+]i oscillations. The inositol 1, 4, 5-trisphosphate receptor (IP3R-1), a ligand-gated channel, mediates the [Ca2+]i rises at fertilization. Evidence shows that the mass, distribution and conductivity of IP3R-1 are regulated during maturation and fertilization and that this is required to establish the temporal and spatial organization of the [Ca2+]i responses at fertilization. Despite this pivotal role of IP3R-1, little is known about the molecular mechanisms that control its function in eggs. Here, we hypothesize that IP3R-1 constitutes a key regulatory locus for the organization of Ca2+ release during egg activation. To further elucidate the mechanisms that control IP3R-1 function we propose to: 1) investigate how IP3R-1 degradation is regulated during activation and how it impacts sperm-initiated [Ca2+]i oscillations; 2) determine whether IP3R-1 phosphorylation by cell cycle-associated kinases mediates the observed association between the cell cycle, IP3R-1 conductivity and [Ca2+]i oscillations; 3) examine the localization and mechanism(s) responsible for IP3R-1 redistribution and how IP3R-1 distribution/cortical cluster formation affects Ca2+ release. To address these questions we will perform [Ca2+]i monitoring, immunoblotting, immunofluorescence, microinjection of mRNAs, release of caged compounds, and mutations and tagging of IP3R-1 followed by expression in eggs/somatic cells and confocal microscopy visualization. Relevance: Given the diverse and precise regulation of IP3R-1 function during maturation, evaluation of these parameters after ovulation/maturation may provide markers with which to assess the impact of hormonal stimulation/in vitro maturation conditions on oocyte quality. Also, since abnormal Ca2+ release occurs in eggs aged after ovulation and this may lead to embryo fragmentation, elucidation of the mechanisms that control IP3R-1 function promises to improve embryo development and the success of Assisted Reproductive Technologies.