Regulation of Calcium Signaling during Oogenesis

描述（由申请人提供）：“一切开始的地方”。“大觉醒”，这些是用来描述受精时卵子激活的一些术语。它们突出了围绕主题的魅力。事实上，受精时发育的激活是生殖和发育生物学中最有趣的领域之一，因为它具有医学，社会和经济意义。Ca 2+是所有有性生殖物种（从植物到人类）受精时卵子激活的通用信号。重要的是，受精诱导的Ca 2+信号具有专门的空间和时间动力学，这对卵激活和胚胎发生的启动至关重要。卵子在卵母细胞成熟过程中获得产生这种专门的Ca 2+信号的能力。然而，尽管Ca 2+信号分化的基本作用，其分子调控仍然知之甚少。本研究旨在阐明非洲爪蟾卵母细胞成熟过程中钙信号通路分化的分子调控机制。非洲爪蟾为拟议的研究提供了一个特殊的系统，因为它是脊椎动物受精的良好模型，并且因为非洲爪蟾卵母细胞是研究Ca 2+信号传导的有利细胞。非洲爪蟾的受精诱导持续几分钟的Ca 2+上升，这对卵的激活至关重要。我们的初步研究表明，卵中持续Ca 2+升高的两个最重要的决定因素是：IP 3受体的延长开放和质膜Ca 2 +-ATP酶（PMCA）从细胞膜上的去除。因此，定义这两个Ca 2+效应子在卵母细胞成熟过程中的调节对于我们理解Ca 2+信号在成熟过程中如何分化以准备受精变得至关重要。目标1中提出的研究将使用功能性Ca 2+成像、磷酸肽图谱和膜片钳方法来确定IP 3受体的延长开放是否是由于MPF的磷酸化。目的2提出了实验，将阐明介导PMCA的内吞作用在卵母细胞成熟的机制。使用的方法包括功能敲除、质谱和结构-功能突变分析。这些研究将极大地提高我们对卵子如何调节Ca 2+信号效应子以在受精时产生所需的专门Ca 2+瞬变的理解。这些知识是非常重要的，因为受精诱导的Ca 2+瞬变是卵激活所必需的，卵激活决定了胚胎的发育能力。

DESCRIPTION (provided by applicant): 'Where it all begins'... the great awakening', these are some of the terms that have been used to describe egg activation at fertilization. They highlight the fascination surrounding the subject. Indeed, the activation of development at fertilization is one of the most intriguing areas of reproductive and developmental biology, because of its medical, social and economic implications. Ca2+ is the universal signal for egg activation at fertilization in all sexually reproducing species, from plants to humans. Importantly, the fertilization-induced Ca2+ signal has specialized spatial and temporal kinetics that are vital for egg activation and the initiation of embryogenesis. Eggs acquire the ability to produce this specialized Ca2+ signal during oocyte maturation. However, despite the fundamental role of Ca2+ signaling differentiation its molecular regulation remains poorly understood. This proposal aims to elucidate the molecular mechanisms regulating the differentiation of Ca2+ signaling pathways during oocyte maturation in Xenopus. Xenopus offers an exceptional system for the proposed studies, because it is a good model for vertebrate fertilization, and because the Xenopus oocyte is a favored cell to study Ca2+ signaling. Fertilization in Xenopus induces a sustained Ca2+ rise that lasts for several minutes, and is essential for egg activation. Our Preliminary Studies show that the two most important determinants of the sustained Ca2+ rise in eggs are: prolonged opening of IP3 receptors, & removal of the plasma membrane Ca2+-ATPase (PMCA) from the cell membrane. Therefore, defining the regulation of these two Ca2+ effectors during oocyte maturation becomes essential to our understanding of how Ca2+ signals differentiate during maturation in preparation for fertilization. Studies proposed in Aim 1 will determine whether the prolonged opening of IP3 receptors is due to phosphorylation by MPF, using functional Ca2+ imaging, phosphopeptide mapping and patch clamping approaches. Aim 2 proposes experiments that will elucidate the mechanisms mediating PMCA endocytosis during oocyte maturation. Approaches to be used include functional knockdown, mass spectrometry and structure-function mutational analyses. Together the proposed studies will greatly improve our understanding of how eggs modulate Ca2+ signaling effectors to produce the required specialized Ca2+ transient at fertilization. Such knowledge is fundamentally important because the fertilization-induced Ca2+ transient is essential for egg activation, which defines the developmental competence of the embryo.