MECHANISMS OF MAMMALIAN FERTILIZATION

描述(申请人提供)：透明带(ZP)是一种厚厚的细胞外覆盖物，包围着所有哺乳动物的卵。小鼠卵子ZP由三种糖蛋白组成，称为mZP1-3。编码糖蛋白的基因在2-3周内由生长的小鼠卵母细胞专门表达，新生的糖蛋白组装成组成ZP的交联丝。其中一种糖蛋白mZP3，当它们与未受精卵结合时，可以通过自由游泳、顶体完整的精子以物种特有的方式识别。与mZP3的结合诱导精子经历顶体反应，这是一种细胞胞吐作用。只有顶体反应的精子才能穿透顶体并与卵质膜融合形成合子。在本申请中，描述了继续解决有关ZP糖蛋白和鸡蛋ZP的重要问题的实验。这项建议的具体目的是利用生长中的小鼠卵母细胞、转基因小鼠和转基因哺乳动物细胞来评估mZP2和mZP3多肽一级结构与这些糖蛋白分泌和组装成细丝的关系。目前认为ZP糖蛋白的分泌和组装受rnZP2和mZP3多肽的特定结构域调控。由于在前一批赠款期间取得的成果和发展的方法，许多拟议的实验都是可能的。这些实验包括使用突变的和野生型的mZP基因，将各种带有表位标记的mZP基因构建物显微注射到生长中的小鼠卵母细胞，携带突变的转基因基因的转基因小鼠，以及转基因的哺乳动物细胞。这些实验结果将为ZP糖蛋白一级结构、生长中卵母细胞分泌ZP糖蛋白和将ZP糖蛋白组装成长丝之间的可能关系提供重要的分子信息。鉴于小鼠和人类ZP糖蛋白之间以及小鼠和人类受精之间的巨大相似性，这里获得的信息很可能也适用于人类。此外，这项研究可能对开发新的可逆避孕方法防止人类卵子发生过程中ZP的形成具有重要影响。

DESCRIPTION (provided by applicant): The zona pellucida (ZP) is a thick extracellular c0at that surrounds all mammalian eggs. The mouse egg ZP consists of three glycoproteins, called mZP1-3. Genes encoding the glycoproteins are expressed exclusively by growing mouse oocytes during a 2-3 week period and the nascent glycoproteins are assembled into crosslinked filaments that constitute the ZP. One of the glycoproteins, mZP3, is recognized in a species-specific manner by free-swimming, acrosome-intact sperm when they bind to unfertilized eggs. Binding to mZP3 induces sperm to undergo the acrosome reaction, a form of cellular exocytosis. Only acrosome-reacted sperm can penetrate the ZP and fuse with egg plasma membrane to form a zygote. In this application, experiments are described that continue to address significant questions about ZP glycoproteins and the egg ZP. The Specific Aim of this proposal is to evaluate relationships between mZP2 and mZP3 polypeptide primary structures and secretion and assembly of these glycoproteins into filaments using growing mouse oocytes, transgenic mice, and transfected mammalian cells. It is proposed that secretion and assembly of ZP glycoproteins is regulated by specific domains of rnZP2 and mZP3 polypeptides. Many of the experiments proposed are possible because of results obtained and methodolgy developed during the previous grant period. The experiments include the use of mutated and wild-type mZP genes, a variety of epitope-tagged mZP cDNA constructs microinjected into growing mouse oocytes, transgenic mice carrying mutated transgenes, and transfected mammalian cells. Results of these experiments should provide important molecular information about possible relationships between ZP glycoprotein primary structures, secretion of ZP glycoproteins from growing oocytes, and assembly of ZP glycoproteins into long filaments. In view of the great similarities between mouse and human ZP glycoproteins and between fertilization in mice and humans, it is very likely that information obtained here will apply to humans as well. Furthermore, the research could have important consequences for development of novel reversible contraceptive methods that prevent ZP formation during oogenesis in humans.