MECHANISMS OF MAMMALIAN FERTILIZATION

描述（由申请人提供）：透明卵囊（ZP）是一种厚的细胞外囊，包围着所有哺乳动物的卵。小鼠卵ZP由三种糖蛋白组成，称为mZP 1 -3。编码糖蛋白的基因仅在2-3周期间通过生长的小鼠卵母细胞表达，并且新生的糖蛋白组装成构成ZP的交联丝。其中一种糖蛋白，mZP 3，在自由游动的顶体完整的精子与未受精的卵子结合时，以种属特异性的方式被识别。与mZP 3结合诱导精子进行顶体反应，这是一种细胞胞吐作用。只有顶体反应的精子才能穿透ZP与卵质膜融合形成合子。在本申请中，描述了继续解决关于ZP糖蛋白和卵ZP的重要问题的实验。本提案的具体目的是使用生长中的小鼠卵母细胞、转基因小鼠和转染的哺乳动物细胞评价mZP 2和mZP 3多肽一级结构与这些糖蛋白分泌和组装成细丝之间的关系。提出ZP糖蛋白的分泌和组装受rnZP 2和mZP 3多肽的特定结构域调节。由于在前一个资助期内获得的结果和开发的方法，提出的许多实验是可能的。实验包括使用突变和野生型mZP基因，各种表位标记的mZP cDNA构建体显微注射到生长中的小鼠卵母细胞，携带突变的转基因的转基因小鼠，和转染的哺乳动物细胞。这些实验的结果应该提供重要的分子信息ZP糖蛋白的一级结构，分泌的ZP糖蛋白从生长的卵母细胞，和装配成长丝的ZP糖蛋白之间可能的关系。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产生重要影响，这些方法可以防止人类卵子发生过程中ZP的形成。

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.