THE MEMBRANE BLOCK TO POLYSPERMY IN MAMMALIAN EGGS

描述（由申请人提供）：多精子，或一个卵子有超过1个精子受精，被认为是导致人类自然流产至少5%的原因。为了抑制额外精子的受精，卵子已经形成了一种被称为多精阻滞的预防机制。在许多不同的动物物种中，卵细胞外被层水平的阻滞已被很好地表征，而在一些非哺乳动物物种中，卵质膜水平的阻滞已被理解。然而，尽管50-90年前的数据为其存在提供了证据，但人们对哺乳动物卵子中膜阻滞多精现象几乎一无所知。我们最近的数据表明，精子诱导的Ca2+信号和卵细胞肌动蛋白骨架是参与受精后改变的两个成分，这种改变将卵膜从支持受精的形式转变为阻止受精的形式。这个项目广泛的、长期的目标是阐明膜阻断到多精的机制，从启动膜阻断建立的受精相关信号到阻止额外受精的卵膜和皮层的变化。为了实现这一目标并在我们最近公布的初步数据的基础上取得进展，我们在本提案中的研究将涉及以下具体目标。特异性Aim 1将确定精子和精子诱导的Ca2+信号诱导膜阻断建立的机制。这一目标将确定哪些精子成分参与了膜阻断的建立，以及精子诱导的Ca2+信号如何影响膜阻断的特征。特异性Aim 2将通过鉴定参与建立多精子膜阻断的Ca2+依赖性效应分子来表征Ca2+途径的下一步。最后，Specific Aim 3将重点关注该途径的后期步骤，最终导致膜阻滞到多精，检查卵子膜和皮层的具体变化。本研究旨在验证以下假设：受精后，皮质颗粒胞吐、内吞和受精后皮质成分和膜顺序的变化有助于下调卵膜对精子的接受性。这些关于精子诱导信号、钙、受精后膜和皮质动力学的研究将为哺乳动物膜阻断多精的细胞和分子机制提供重要的见解，促进我们对生殖和发育生物学中这一基本问题的认识。

DESCRIPTION (provided by applicant): Polyspermy, or fertilization of an egg by more than 1 sperm, is believed to be the cause of at least 5% of spontaneous pregnancy loss in humans. To inhibit fertilization by additional sperm, eggs have developed preventative mechanisms known as blocks to polyspermy. The block at the level of the egg extra cellular coat has been well characterized in many different animal species, and the block at the level of the egg plasma membrane is understood in some non-mammalian species. However, virtually nothing is known about the membrane block to polyspermy in mammalian eggs, despite data dating back 50-90 years that provide evidence for its existence. Our recent data demonstrates that sperm-induced Ca2+ signaling and the egg actin cytoskeleton are 2 components involved in this post-fertilization change that transforms the egg membrane from a form that supports fertilization to 1 that prevents it. The broad, long-term goal of this project is elucidating the mechanism of the membrane block to polyspermy, from the fertilization-associated signaling that initiates membrane block establishment to the changes in the egg membrane and cortex that prevent additional fertilization. To make progress toward this goal and to build on our recent published and preliminary data, our studies in this proposal will address the following specific aims. Specific Aim 1 will determine the mechanisms by which sperm and sperm-induced Ca2+ signaling induce the establishment of the membrane block. This aim will identify what sperm component(s) is involved in membrane block establishment and how sperm-induced Ca2+ signaling affects characteristics of the membrane block. Specific Aim 2 will characterize the next step in the pathway following Ca2+ by identifying the Ca2+-dependent effector molecules that are involved in the establishment of the membrane block to polyspermy. Finally, Specific Aim 3 will focus on the later steps of the pathway culminating in the membrane block to polyspermy, examining specific changes in the egg membrane and cortex. This aim will test the hypotheses that cortical granule exocytosis, endocytosis, and post-fertilization changes in cortex composition and membrane order contribute to the down-regulation of egg membrane receptivity to sperm that follows fertilization. These inquiries into sperm-induced signaling, calcium, and post-fertilization membrane and cortical dynamics will provide important insights into the cellular and molecular mechanisms underlying the mammalian membrane block to polyspermy, advancing our knowledge of this fundamental question in reproductive and developmental biology.