Signaling Mechanisms of Polyspermy Block

受精是自然界中最基本的过程之一，但我们对这一重要过程的理解存在重大差距。之一 成功繁殖的最早和最普遍的障碍是一个卵子被一个以上的精子受精，或多精受精。所有有性生殖物种的卵子都面临这个共同问题，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仅由一个精子受精的分子机制，从而允许正常的胚胎发育。在许多生物的卵中，受精引起了延长的膜去极化，这是多精受精的快速阻断。快速多精受精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身份尚不清楚。在许多物种中，包括青蛙，氯离子通道可能介导这一过程。巧合的是，受精诱导的钙离子增加也发生在快速多精受精阻滞之前。这两个事件之间的一个可能的联系是最近发现的钙激活的Cl-通道编码的TMEM 16 a基因。在具体的目标一，我将确定来源的Ca 2+所需的去极化受精。具体目标2中概述的实验将揭示TMEM 16 a通道在快速多精受精阻断中的作用。沿着受精诱发的Ca 2+增加，受精也伴随着磷脂酰肌醇4，5-二磷酸（PIP 2）的两倍增加。这种升高的PIP 2在受精后最初几分钟可能发挥的作用尚不清楚。因为PIP 2是一种已知的结构多样的离子通道的调节器，并且因为受精诱发的PIP 2升高发生在快速多精受精阻滞的时间范围内，所以我假设PIP 2调节受精诱发的去极化。我将在具体目标中检验这一假设 第三，确定PIP 2的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理解受精的生物学，并为生殖医学的未来发展提供基础。 公共卫生相关性：受精是自然界最基本的过程之一，但该过程中涉及的许多过程尚不清楚。利用尖端的实验技术，该项目将揭示确保每个卵子仅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命的事件。

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.