Regulation of Cellular Interactions by Extracellular Matrix Proteolysis

0111285 Lindsay卵细胞外膜在受精前后精卵相互作用的调节中起着至关重要的作用，因此是发育生物学中的一个重要课题。在最初的精子穿透和受精后，卵子被卵因子修饰，以保护卵子免受精子的侵害，以防止多精受精，这是一种对胚胎发育致命的情况。在这个复杂的“阻断多精受精”反应中，卵子包膜上的精子结合位点被灭活，包膜结构被改变，从而“硬化”以阻止精子穿透并保护发育中的胚胎。这些观察已经有将近世纪的历史了，但反应背后的机制仍然未知，主要是由于缺乏生物材料，特别是哺乳动物。一种假设是，鸡蛋蛋白酶参与的反应，因为蛋白水解的一个特定的鸡蛋包膜糖蛋白，ZPA，已观察到受精后。目前的研究项目旨在通过分离和表征负责的鸡蛋蛋白酶（命名为“ZPAase”）来验证这一假设，然后确定其对鸡蛋包膜物理特性和功能的影响。由于有大量的配子可供研究，因此使用两栖动物模型系统非洲爪蟾。先前资助的研究将ZPAase鉴定为金属蛋白酶，并对其进行了部分纯化。继续的研究将完成ZPA酶的纯化，并使用分子克隆技术来确定ZPA酶的氨基酸序列，以获得功能信息并与其他蛋白酶进行比较。将使用从卵子或通过蛋白表达获得的纯化蛋白酶来检验ZPA蛋白水解对精子与卵子包膜结合和包膜硬化的假设。这些研究的结果将对我们了解受精后调节精卵相互作用的复杂反应做出重大贡献，这些反应对正常动物发育至关重要。

0111285LindsayThe extracellular egg envelope plays a crucial role in regulating sperm-egg interactions both before and after fertilization, and is thus a major subject of interest in developmental biology. After initial sperm penetration and fertilization, the egg envelope is modified by egg factors to defend the egg against sperm to prevent polyspermy, a condition that is lethal to embryonic development. During this complex "block to polyspermy" reaction, sperm binding sites on the egg envelope are inactivated, and the envelope structure is modified so that it is "hardened" to impede sperm penetration and to protect the developing embryo. These observations are almost a century old but still the mechanisms behind the reactions are unknown, due primarily to the lack of biological material, especially in mammals. One hypothesis is that an egg protease is involved in the reaction, since proteolysis of a specific egg envelope glycoprotein, ZPA, has been observed following fertilization. The current research project is designed to test this hypothesis by isolating and characterizing the responsible egg protease, designated "ZPAase", followed by the determination of its effects on egg envelope physical properties and function. The amphibian model system Xenopus laevis is used due to the large amounts of gametes that are available for study. Previously funded research identified ZPAase as a metalloprotease and it was partially purified. Continuing studies will complete the purification of ZPAase, and use molecular cloning techniques to determine the amino acid sequence of ZPAase for functional information and comparison to other proteases. Purified protease, obtained from eggs or through protein expression, will be used test the hypothesis of ZPA proteolysis on sperm binding to the egg envelope and envelope hardening. The results of these studies will make a significant contribution to our knowledge of the complex reactions that regulate sperm-egg interactions following fertilization and are fundamental to normal animal development.