MECHANISMS OF MAMMALIAN FERTILIZATION

透明细胞膜（ZP）是一层厚厚的细胞外被膜， 哺乳动物卵 小鼠卵ZP由3种糖蛋白mZP 1 -3组成。 其中一种糖蛋白，mZP 3，在一个物种特异性的 当精子与ZP结合时， 在受精过程中。 精子识别并结合特定的Ser/Thr- 位于mZP 3结合位点的O-连接寡糖 找精子 与mZP 3结合诱导精子经历顶体 反应，一种细胞胞吐作用。 顶体反应的精子， 穿透ZP，到达卵周间隙，与卵融合 质膜 在本申请的支持下， 描述了解决有关mZP 3方面的重要问题 结构和功能。 这些方面包括基本的 精子mZP 3结合位点的残基和分子基础 哺乳动物精卵相互作用的物种特异性。 一个共同 这里的实验主题是使用胚胎癌（EC）细胞 稳定转染突变的mZP 3基因和转基因小鼠 携带mZP 3无效突变 在我们的实验室中， 实验室通过使用同源重组的靶向诱变， 小鼠胚胎干细胞（ES细胞）。 来自纯合子（mZP 3-/-）雌性的卵 缺乏ZP，动物是不育的。 另一方面，在一项研究中， 杂合雌性（mZP 3 +/-）和纯合雄性（mZP 3-/-）表现出 野生型表型并且是可育的。 这里描述的实验使用 突变的mZP 3基因，在EC细胞和mZP 3无效突变小鼠中评价 解决哺乳动物受精的基本问题。研究 提出的是我们实验室以前工作的逻辑扩展， mZP3。 实际上所有的实验技术 近年来，在我们的实验室里， 考虑到 关于小鼠和人ZP 3的相关性，预期 这项调查将极大地影响我们对 人类受精的基本方面。

The zona pellucida (ZP) is a thick extracellular coat that surrounds mammalian eggs. The mouse egg ZP consists of 3 glycoproteins, mZP1-3. One of the glycoproteins, mZP3, is recognized in a species-specific manner by free-swimming, acrosome-intact sperm when they bind to the ZP during fertilization. The sperm recognize and bind to specific Ser/Thr- linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm. Binding to mZP3 induces sperm to undergo the acrosome reaction, a form of cellular exocytosis. Acrosome-reacted sperm then penetrate the ZP, reach the perivitelline space, and fuse with egg plasma membrane. In this application for support, experiments are described that address important questions about aspects of mZP3 structure and function. These aspects include the nature of essential residues at the mZP3 combining site for sperm and the molecular basis of species-specificity of mammalian sperm-egg interactions. A common experimental theme here is the use of embryonal carcinoma (EC) cells stably transfected with mutated mZP3 genes and of transgenic mice bearing an mZP3 null mutation. Null mutation mice were produced in our laboratory by targeted mutagenesis using homologous recombination in mouse embryonic stem (ES) cells. Eggs from homozygous (mZP3-/-) females lack a ZP and the animals are infertile. On the other hand, heterozygous females (mZP3+/-) and homozygous males (mZP3-/-) exhibit a wild-type phenotype and are fertile. Experiments described here use mutated mZP3 genes, evaluated in EC cells, and mZP3 null mutation mice to address fundamental aspects of mammalian fertilization. The studies proposed are a logical extension of previous work in our laboratory on mZP3. virtually all of the experimental techniques have been successfully in recent years in our laboratory. In view of the relatedness of mouse and human ZP3 it is anticipated that results of this investigation will impact significantly on our understanding of fundamental aspects of fertilization in human beings.