Function of a voltage-sensitive phosphatase in the regulation of sperm-egg fusion

描述（由申请人提供）：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，从而使正常的胚胎发育发生。在许多动物物种中，防止多精的一个重要组成部分是卵膜的电去极化，这是对第一个精子融合的反应，它阻止了其他精子的融合。在卵子中检测电压变化的传感器已知位于精子中，但尚未在分子水平上确定。拟议的研究将探讨这个问题，重点是最近发现的电压激活的磷脂酰肌醇脂质磷酸酶，该酶在精子中表达。激活磷酸酶的电压和抑制精卵融合的电压之间的密切联系支持了这一假设。具体目的一是研究电压敏感磷酸酶（VSP）在精子膜中的定位。定位在头部区域，即发生精卵融合的地方，将支持VSP在这一过程中的调节作用。另外，在尾部区域的定位将支持其在精子运动调节中的作用。具体目标二将研究VSP的功能，使用转基因青蛙，其精子表达点突变形式的蛋白质，其中磷酸酶在更负的电压下激活。这些功能实验的方向将由目标一的结果决定。如果VSP定位于精子头部，转基因精子将被应用于电压固定的卵子，以测试修改后的VSP的存在是否会改变抑制精子-卵子融合的电压。或者，如果VSP定位于精子尾部，则将研究修饰后的VSP对精子活力的影响。通过利用一个独特的生物系统，其中精子-卵子融合是电压依赖的，这些研究将提供磷脂酰肌醇脂在调节融合过程中的可能功能的见解，并将测试电压敏感磷酸酶在精子中这一和其他生理调节机制中的作用。这些发现将有助于了解受精的生物学，并将为生殖医学的未来发展提供基础。

DESCRIPTION (provided by applicant): This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing normal embryonic development to occur. In many animal species, one essential component of polyspermy prevention is an electrical depolarization of the egg membrane in response to fusion of the first sperm, which prevents the fusion of additional sperm. The sensor that detects the voltage change in the egg is known to be located in the sperm, but has not been identified on a molecular level. The proposed studies will investigate this question, focusing on a recently discovered voltage-activated phosphatidylinositol lipid phosphatase that is expressed in sperm. A close correlation between the voltage that activates the phosphatase and the voltage that inhibits sperm-egg fusion supports this hypothesis. Specific aim one will investigate the localization of the voltage-sensitive phosphatase (VSP) in the sperm membrane. Localization in the head region, where sperm-egg fusion occurs, would support a role for the VSP in the regulation of this process. Alternatively, localization in the tail region would support its role in the regulation of sperm motility. Specific aim two will investigate the function of the VSP, using a transgenic frog whose sperm express a point-mutated form of the protein in which the phosphatase activates at a more negative voltage. The direction of these functional experiments will be determined by the results of aim one. If the VSP is localized in the sperm head, transgenic sperm will be applied to voltage-clamped eggs to test whether the presence of the modified VSP shifts the voltage that inhibits sperm-egg fusion. Alternatively, if the VSP is localized in the sperm tail, the effect of the modified VSP on sperm motility will be investigated. By taking advantage of a unique biological system in which sperm-egg fusion is voltage dependent, these studies will provide insights into the possible function of phosphatidylinositol lipids in regulating the fusion process, and will test the role of a voltage-sensitive phosphatase in this and other physiological regulatory mechanisms in sperm. The findings will contribute to understanding of the biology of fertilization, and will provide a basis for future advances in reproductive medicine. PUBLIC HEALTH RELEVANCE: This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing normal embryonic development to occur. The findings will contribute to understanding of the biology of fertilization, and will provide a basis for future advances in reproductive medicine.