MECHANISMS OF MAMMALIAN FERTILIZATION

透明带(ZP)是一层厚厚的细胞外衣，包裹在 哺乳动物的蛋。小鼠卵母细胞ZP由3种糖蛋白mZP1-3组成。 其中一种糖蛋白mZP3在一种特定的物种中被识别 自由游动、顶体完整的精子与ZP结合时的方式 在受精过程中。精子识别并结合特定的丝氨酸/苏氨酸- 位于mZP3结合位点的连接(O-连接)低聚糖 为了精子。与mZP3结合诱导精子经历顶体 反应，一种细胞胞吐作用。然后顶体反应的精子 穿透ZP，到达卵周隙，并与卵子融合 质膜。在这个支持应用程序中，实验是 描述了解决有关mZP3各方面的重要问题 结构和功能。这些方面包括必要的性质 精子mZP3结合部位的残基及其分子基础 哺乳动物精子-卵子相互作用的物种特异性。一种常见的 实验主题是使用胚胎癌细胞(EC) 稳定转染突变的mZP3基因及转基因小鼠 携带mZP3零突变。在我们的实验室中产生了零突变小鼠 利用同源重组进行靶向突变的实验室 小鼠胚胎干细胞。纯合子(mZP3-/-)雌性卵子 缺乏ZP，动物就不能生育。另一方面， 杂合子女性(mZP3+/-)和纯合子男性(mZP3-/-)表现出 一种野生型的表型，并且是可育的。此处描述的实验使用 突变的mZP3基因，在EC细胞和mZP3缺失突变小鼠中进行评估 以解决哺乳动物受精的基本问题。这些研究 建议对我们实验室以前的工作进行合理的扩展 MZP3.几乎所有的实验技术都是 近几年在我们实验室取得了成功。鉴于 小鼠与人ZP3的亲缘关系预计结果 这项调查将对我们对 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.