Regulation of Calcium Signaling during Oogenesis

描述（由申请人提供）：“一切开始的地方”……“大觉醒”，这是一些用来描述受精时卵子激活的术语。它们突出了这个主题的魅力。事实上，受精时的发育激活是生殖和发育生物学中最令人感兴趣的领域之一，因为它具有医学、社会和经济意义。Ca2+是所有有性繁殖物种（从植物到人类）受精时卵子激活的普遍信号。重要的是，受精诱导的Ca2+信号具有特殊的空间和时间动力学，这对于卵子激活和胚胎发生的开始至关重要。卵子在卵母细胞成熟过程中获得产生这种特殊Ca2+信号的能力。然而，尽管Ca2+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Ca2+信号通路分化的分子调控机制。非洲爪蟾为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，因为非洲爪蟾卵母细胞是研究Ca2+信号的有利细胞。爪蟾受精诱导持续几分钟的Ca2+上升，这对卵子激活是必不可少的。我们的初步研究表明，卵子中Ca2+持续上升的两个最重要的决定因素是：IP3受体开放时间延长，以及细胞膜上Ca2+- atp酶（PMCA）的去除。因此，确定这两种Ca2+效应物在卵母细胞成熟过程中的调节对于我们理解Ca2+信号在成熟过程中如何分化以准备受精至关重要。Aim 1中提出的研究将利用功能性Ca2+成像、磷酸肽定位和贴片夹持方法，确定IP3受体的延长开放是否由于MPF的磷酸化。目的2提出的实验将阐明在卵母细胞成熟过程中介导PMCA内吞作用的机制。方法包括功能敲除、质谱分析和结构功能突变分析。总之，提出的研究将大大提高我们对卵子如何调节Ca2+信号效应产生所需的特殊Ca2+瞬态受精的理解。这些知识是至关重要的，因为受精诱导的Ca2+瞬态对卵子激活至关重要，卵子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.