Signaling Mechanisms of Polyspermy Block

说明(申请人提供)：受精是自然界中最基本的过程之一，但我们对这一基本过程的理解存在严重差距。其中之一 成功繁殖最早和最普遍的障碍是一个以上的精子或多精受精使一个卵子受精。所有有性繁殖物种的卵子都面临这个共同的问题，会导致严重的染色体缺陷，并导致胚胎死亡。该项目将研究确保每个卵子只由一个精子受精的分子机制，从而允许正常的胚胎发育。在许多生物的卵子中，受精引起长时间的膜去极化，这对多精受精起到了快速的阻碍作用。快速多精体阻断需要一个或多个离子通道的活性，但任何所需通道的分子特性尚不清楚。在包括青蛙在内的许多物种中，氯离子通道可能参与了这一过程。巧合的是，受精诱导的钙离子浓度升高也发生在快速多精受精阻断之前。这两个事件之间的一个可能的联系是最近发现的由TMEM16A基因编码的钙激活的Cl-通道。在特定的目标一中，我将确定化肥去极化所需的钙的来源。在特定目标二中概述的实验将揭示TMEM16A通道在快速多精受精区块中的作用。随着受精引起的钙离子浓度的增加，受精也伴随着磷脂酰肌醇4，5-二磷酸(PIP2)的两倍增加。这种升高的PIP2在受精后的第一分钟可能起到的作用尚不清楚。由于PIP2是已知的结构多样化的离子通道的调节器，并且受精诱导的PIP2升高发生在快速多精受精阻断的时间框架内，我假设PIP2调节受精诱导的去极化。我将在特定的目标中检验这一假设 三，并确定PIP2耗尽是否影响多精受精阻断。这些实验的结果将有助于我们对受精生物学的理解，并将为未来生殖医学的发展提供基础。 公共卫生相关性：受精是自然界中最基本的过程之一，但这一过程中涉及的许多过程都是未知的。利用尖端的实验技术，该项目将揭示确保每个卵子只由一个精子受精的分子机制。这些发现将有助于我们理解开始新生活的事件。

DESCRIPTION (provided by applicant): Fertilization is one of the most fundamental processes in nature, yet critical gaps exist in our understanding of this essential process. One of the earliest and most prevalent barriers to successful reproduction is the fertilization of an egg by more than one sperm, or polyspermy. This common problem, faced by the eggs of all sexually reproducing species, causes severe chromosomal defects and leads to embryonic mortality. This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing for normal embryonic development. In the eggs of many organisms, fertilization evokes a prolonged membrane depolarization, which acts as a fast block to polyspermy. The fast polyspermy block requires the activity of one of more ion channel, but the molecular identity of any required channel is not known. In many species, including frogs, Cl- channels likely mediate this process. Coincidentally, a fertilization-induced increase i Ca2+ also occurs prior to the fast polyspermy block. A possible connection between these two events is the recently identified Ca2+ activated Cl- channel encoded by the TMEM16a gene. In specific aim one, I will identify the source of Ca2+ required for the depolarization at fertilizatin. Experiments outlined in specific aim two will uncover the role of the TMEM16a channel in the fast polyspermy block. Along with a fertilization-evoked increase in Ca2+, fertilization is also accompanied by a two-fold increase in phosphatidylinositol 4,5-bisphosphate (PIP2). The role that this elevated PIP2 may play in the first minutes after fertilization is unknown. Because PIP2 is a known regulator of structurally diverse ion channels and because the fertilization-evoked PIP2 elevation occurs within the time frame of the fast polyspermy block, I hypothesize that PIP2 regulates the fertilization-evoked depolarization. I will test this hypothesis in specific aim three and determine if PIP2 depletion affects the polyspermy block. The results of these experiments will contribute to our understanding of the biology of fertilization, and will provide the basis for future advances is reproductive medicine. PUBLIC HEALTH RELEVANCE: Fertilization is one of the most fundamental processes in nature, yet many of the processes involved in this process are unknown. Using cutting-edge experimental techniques, this project will uncover the molecular mechanisms that ensure that each egg is fertilized by only one sperm. These findings will contribute to our understanding of the events that begin new life.