LP3R-1 Regulation and Egg Activation

LP3R-1 调节和卵子激活

基本信息

批准号：
7260244
负责人：
Rafael Antonio Fissore
金额：
$ 29.28万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-07 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7260244
关键词：
Actins Address Affect Antibodies Assisted Reproductive Technology Biochemical Calcium Cell Cycle Cell Cycle Stage Condition Confocal Microscopy Consensus Cytoskeleton Development Embryo Embryonic Development Endoplasmic Reticulum Evaluation Experimental Models Fertilization Goals Hormonal Human Imagery Immunoblotting Immunofluorescence Immunologic Immunoprecipitation In Vitro Inositol Inositol 1,4,5-Trisphosphate Interphase Laboratories Lead Ligands Location Mammals Mediating Meiosis Messenger RNA Metaphase Microfilaments Microinjections Microtubules Mitosis Mitotic Molecular Monitor Mus Mutation Oocytes Ovulation Pathway interactions Pattern Periodicals Phospholipase Phosphoproteins Phosphorylation Phosphotransferases Production Regulation Research Personnel Role Series Signal Transduction Site Somatic Cell Staging Work aged egg human PLK1 protein improved kinase inhibitor ligand gated channel oocyte maturation programs receptor response sperm cell success zygote

项目摘要

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.

描述（由申请人提供）：本申请的长期目标是阐明哺乳动物物种中控制卵子活化的机制。哺乳动物卵在第二次减数分裂中期(MI1)停止排卵。卵激活需要退出MI1并完成一系列启动有丝分裂并因此启动发育程序的形态和生化变化。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）来诱导卵子活化。肌醇 1, 4, 5-三磷酸受体 (IP3R-1) 是一种配体门控通道，介导受精时 [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 反应的时间和空间组织所必需的。尽管 IP3R-1 发挥着关键作用，但人们对控制其在鸡蛋中功能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 Ca2+ 释放组织的关键调节位点。为了进一步阐明控制 IP3R-1 功能的机制，我们建议：1）研究 IP3R-1 降解在激活过程中如何受到调节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的 IP3R-1 磷酸化是否介导观察到的细胞周期、IP3R-1 电导率和 [Ca2+]i 振荡之间的关联； 3) 检查负责 IP3R-1 重新分布的定位和机制以及 IP3R-1 分布/皮质簇形成如何影响 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 显微注射、笼中化合物的释放以及 IP3R-1 的突变和标记，然后在卵/体细胞中表达，并进行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，排卵/成熟后对这些参数的评估可能提供标记物，用于评估激素刺激/体外成熟条件对卵母细胞质量的影响。此外，由于排卵后老化的卵子中会发生异常的 Ca2+ 释放，这可能会导致胚胎破碎，因此阐明控制 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。