IP3r-1 Regulation and Egg Activation

描述(申请人提供)：本申请的长期目标是阐明哺乳动物物种中卵子激活的控制机制。哺乳动物卵子在第二次减数分裂中期停止排卵。卵子的激活需要退出mIL，并完成一系列形态和生化变化，这些变化启动了有丝分裂，从而完成了发育程序。受精通过引起细胞内游离钙浓度([钙]i)的周期性变化来诱导卵子激活，这种变化称为[钙]i振荡。肌醇1，4，5-三磷酸受体(IP3R-1)是一种配体门控通道，在受精过程中调节细胞内钙离子浓度的升高。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[Ca~(2+)]i反应的时空组织所必需的。尽管IP3R-1发挥着关键作用，但人们对其在卵子中发挥作用的分子机制知之甚少。在这里，我们假设IP3R-1构成了卵子激活过程中钙释放组织的关键调控基因。为了进一步阐明控制IP3R-1功能的机制，我们建议：1)研究IP3R-1的降解在激活过程中是如何调节的，以及它是如何影响精子启动的[Ca~(2+)]i振荡的；2)确定细胞周期相关蛋白的磷酸化是否介导了所观察到的细胞周期、IP3R-1电导率和[Ca~(2+)]i振荡之间的关联；3)研究IP3R-1重新分布的定位和机制(S)，以及IP3R-1的分布/皮质簇形成如何影响钙的释放。为了解决这些问题，我们将进行[Ca~(2+)]i监测、免疫印迹、免疫荧光、mRNAs显微注射、笼状化合物的释放、IP3R-1的突变和标记，然后在卵子/体细胞中表达和共聚焦显微镜可视化。相关性：鉴于IP3R-1功能在成熟过程中的不同和精确的调节，在排卵/成熟后评估这些参数可能提供评估激素刺激/体外成熟条件对卵母细胞质量的影响的标志物。此外，由于排卵后老化的卵子会出现异常的钙释放，这可能会导致胚胎碎裂，因此阐明控制IP3R-1功能的机制有望促进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.