Regulation of Ca2+ influx in mouse oocytes and eggs during maturation and fertilization to improve assisted reproductive technologies and modulate fertility

在介导Ca 2+内流的分子和机制方面，我们的知识存在持续的空白 转化成卵母细胞和卵子所有哺乳动物的卵激活和胚胎发育都需要Ca 2+内流， 包括人类。因此，我们知识中的这一差距是一个严重的障碍，直到 我们不能生理调节Ca 2+内流或客观地诊断和治疗不孕症， 与Ca 2+稳态紊乱有关。长期目标是了解Ca 2 + 在卵母细胞和卵子中调节稳态，并鉴定其分子效应物。这里的目标是 鉴定在成熟和受精过程中介导Ca 2+内流的Ca 2+通道，并表征 其监管机制。小鼠卵母细胞/卵子是一个很好的模型，因为它们显示出独特的 Ca ~（2+）在卵成熟和受精过程中进入，Ca ~（2+）的释放是卵激活所必需的。中央 假设是血浆上欠定Ca 2+通道表达和/或不同的调节 膜是卵母细胞中Ca 2+内流的基础，其在成熟过程中的失活和成熟后的恢复 受精这一假设是根据大量的初步数据提出的。这样做的理由 研究表明，一旦确定了通道， 卵母细胞的成熟和受精。这里的发现也有可能转化为 来帮助这个国家的不孕夫妇。我们计划验证我们的核心假设 通过以下具体目标：1）鉴定介导卵母细胞中Ca 2+内流的Ca 2+通道 在成熟之前和成熟期间;以及2）识别支持在成熟之后振荡的Ca 2+流入通道。 受精在目标1下，Ca 2+成像、药理学、条件性基因敲除小鼠和电生理学 将用于鉴定活性通道并评估Ca 2+流入对成熟的影响; 申请人和合作者最近发现的TRPM 7样电流的作用将密切相关。 考察在目标2下，受精刺激Ca 2+内流的信号传导机制和受精刺激Ca 2+内流的信号传导机制被认为是一个重要的信号传导机制。 将确定贡献信道。一种克服Ca 2+失活的新方法 卵子的流入将促进这些研究。遗传模型和电生理学将证实其功能 在受精过程中，这些通道。这项应用的研究是创新的，因为它结合了 电生理学，药理学和新的KO线，方法，已用于确定两个新的 通道，包括TRPM 7，其整体缺失在E7.5时是胚胎致死的。的 拟议项目的贡献是重要的，因为它预计将允许生理调节 卵母细胞和卵子中Ca 2+进入的新的条件和激活方案， 诊所这也将扩大我们对Ca 2+稳态对卵母细胞能力影响的理解 和不孕症。

There is a persistent gap in our knowledge of the molecules and mechanisms that mediate Ca2+ influx into oocytes and eggs. Ca2+ influx is required for egg activation and embryo development in all mammals, including human beings. This gap in our knowledge therefore represents a serious impediment, as until it is filled we cannot physiologically modulate Ca2+ influx or objectively diagnose and treat infertility associated with disturbances in Ca2+ homeostasis. The long-term goal is to understand how Ca2+ homeostasis is regulated in oocytes and eggs and identify its molecular effectors. The objective here is to identify the Ca2+ channel(s) that mediate Ca2+ influx during maturation and fertilization and characterize their regulatory mechanisms. Mouse oocytes/eggs are a great model because they display distinctive Ca2+ entry during maturation and fertilization, and Ca2+ release is required for egg activation. The central hypothesis is that expression and/or distinct regulation of underdetermined Ca2+ channels on the plasma membrane underlies Ca2+ influx in oocytes, its inactivation during maturation and its recovery after fertilization. This hypothesis was conceived based on extensive preliminary data. The rationale for this research is that once the channels are identified, a better understanding of the molecular determinants of oocyte maturation and fertilization will be gained. The findings here also have the potential to translate into therapeutic methods to assist infertile couples in this country. We plan to test our central hypothesis by pursuing the following specific aims: 1) Identify the Ca2+ channel(s) that mediate Ca2+ influx in oocytes prior to and during maturation; and 2) Identify the Ca2+ influx channel(s) that support oscillations after fertilization. Under Aim 1, Ca2+ imaging, pharmacology, conditional knockout mice, and electrophysiology will be used to identify the active channel(s) and to assess the impact of Ca2+ influx on maturation; the role of a TRPM7-like current recently discovered by the applicant and collaborators will be closely examined. Under Aim 2, the signaling mechanism(s) whereby fertilization stimulates Ca2+ influx and the contributing channel(s) will be determined. A novel approach that overcomes the inactivation of Ca2+ influx in eggs will facilitate these studies. Genetic models and electrophysiology will confirm the function of these channel(s) during fertilization. The research in this application is innovative because it combines electrophysiology, pharmacology and new KO lines, approach that has served to identify two new channels in oocytes, including TRPM7, whose global deletion is embryonic lethal at E7.5. The contribution of the proposed project is significant because it is expected to allow physiological modulation of Ca2+ entry in oocytes and eggs that will produce new conditions and activation protocols for use in the clinic. It will also expand our understanding of the impact of Ca2+ homeostasis on oocyte competence and infertility.