EXOCYTOTIC SIGNALING THROUGH SPERM RECEPTORS FOR EGGS

我们的长期目标是对受精的分子基础有更清晰的基本理解，以便控制这一过程，要么促进受精，要么阻止受精。在识别ZP3方面已经取得了很大进展，ZP3是哺乳动物卵衣中的一种蛋白质，它结合精子并刺激顶体反应，顶体反应是精子穿透卵衣必须发生的顶体小泡的释放。尽管取得了这一进展，但精子上ZP3受体的身份一直难以捉摸。一个强有力的候选者是β-1，4-半乳糖基转移酶，这种酶最初是在体细胞的高尔基体中发现的，但后来在精子表面发现了。β-1，4-半乳糖基转移酶结合ZP3，但不结合其他蛋壳蛋白。除了在配子粘连中的作用外，β-1，4-半乳糖基转移酶还可以触发精子中的顶体反应。顶体反应可以被β-1，4-半乳糖基转移酶的抗体刺激，该抗体作为ZP3的模拟物与1，4-半乳糖基转移酶结合和交联。有证据表明，结合β-1，4-半乳糖基转移酶刺激精子内至少一些与ZP3刺激相同的信号系统，导致有人提出，β-1，4-半乳糖基转移酶可能是信号转导顶体反应的主要ZP3受体。定向缺失β-1，4-半乳糖基转移酶基因的小鼠，尽管可以生育，但产生的精子顶体反应和穿透卵衣的能力严重受损。在这个方案中，将研究由β-1，4-半乳糖基转移酶激活的信号系统。目的是确定结合β-1，4-半乳糖基转移酶是否能完全复制ZP3结合激活的所有已知的信号系统，研究β-1，4-半乳糖基转移酶突变体以确定信号传递所必需的结构特征，并鉴定与胞浆中发现的β-1，4-半乳糖基转移酶部分相互作用的蛋白质。这些研究将阐明卵壳的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.