EXOCYTOTIC SIGNALING THROUGH SPERM RECEPTORS FOR EGGS

我们的长期目标是对受精的分子基础有一个更清晰的基本认识，这样就可以控制这个过程来促进或阻止受精。在鉴定ZP3方面已经取得了很大进展，ZP3是哺乳动物卵膜中的一种蛋白质，它能结合精子并刺激顶体反应，顶体囊泡的释放是精子穿透卵膜所必须发生的。尽管取得了这些进展，但精子上ZP3受体的身份一直是难以捉摸的。一个强有力的候选者是β 1,4-半乳糖转移酶，这种酶首先在体细胞的高尔基体中发现，后来在精子表面发现。β a1,4-半乳糖转移酶与ZP3结合，但不与其他蛋壳蛋白结合。在精子中，β 1,4-半乳糖转移酶除了在配子粘附中起作用外，还能引发顶体反应。顶体反应可由β a1,4-半乳糖转移酶抗体刺激，该抗体模拟ZP3结合并交联β a1,4-半乳糖转移酶。有证据表明，结合β a1,4-半乳糖转移酶刺激精子内至少一些与ZP3刺激相同的信号系统，从而提出β a1,4-半乳糖转移酶可能是ZP3受体介导顶体反应的主要信号。靶向缺失β 1,4-半乳糖转移酶基因的小鼠，虽然具有生育能力，但产生的精子顶体反应和穿透卵子外壳的能力严重受损。本研究将对β 1,4-半乳糖转移酶激活的信号系统进行研究。目的是确定结合beta1,4-半乳糖转移酶是否能够特异性地完全复制所有由ZP3结合激活的已识别的信号系统，研究beta1,4-半乳糖转移酶突变体以识别信号传递所需的结构特征，并识别与细胞质中发现的beta1,4-半乳糖转移酶部分相互作用的蛋白质。这些研究将阐明精子受体在顶体反应中的作用，顶体反应是受精过程中的一个必要步骤。

Our long-range objective is to develop a clearer fundamental understanding of the molecular basis of fertilization so that the process can be controlled to either promote fertilization or block it. Much progress has been made to identify ZP3, the protein in the mammalian egg coat that binds sperm and stimulates the acrosome reaction, the release of the acrosomal vesicle that must occur for sperm to penetrate the egg coat. Despite this progress, the identity of the ZP3 receptor on sperm has been elusive. One strong candidate is beta1,4-galactosyltransferase, an enzyme first discovered in the Golgi apparatus of somatic cells but later found on the surface of sperm. Beta1,4-Galactosyltransferase binds ZP3 but not other egg coat proteins. In addition to its role in gamete adhesion, beta1,4- galactosyltransferase acts to trigger the acrosome reaction in sperm. The acrosome reaction can be stimulated with antibodies to beta1,4-galactosyltransferase that act as mimics of ZP3 to bind and crosslink beta1,4-galactosyltransferase. There is evidence that binding beta1,4-galactosyltransferase stimulates at least some of the same signaling systems inside sperm that ZP3 stimulates, leading to the proposal that beta1,4- galactosyltransferase may be the major ZP3 receptor signaling the acrosome reaction. Mice with a targeted deletion of the beta1,4-galactosyltransferase gene, although fertile, produce sperm with severely compromised ability to acrosome react and penetrate the egg coat. In this proposal, the signaling systems that are activated by beta1,4-galactosyltransferase will be studied. The goals are to determine if binding beta1,4-galactosyltransferase specifically can completely reproduce all of the identified signaling systems activated by ZP3 binding, to study beta1,4-galactosyltransferase mutants to identify structural features necessary for signaling, and to identify proteins that interact with the portion of beta1,4- galactosyltransferase found in the cytosol. These studies will clarify the role of a sperm receptor for the egg coat in the acrosome reaction, a necessary step in the process of fertilization.