PLASMA MEMBRANE CHANGES IN FERTILIZATION

尽管我们对大脑的结构和功能的理解有了很大的进步， 对于体细胞中的受体， 参与受精时细胞间的相互作用。 工作 在之前的一个资助期内， 确定受体是蛋白酶敏感的， 导致精子失去与卵子结合并使其受精的能力。 此外，确定蛋白水解释放的片段 以物种特异性的方式抑制受精。 其后依次为 表明完整的受体是一个复杂的蛋白聚糖样分子。 还获得证据表明，受体的碳水化合物链 作为分子的粘附元件，而多肽 链定义了结合过程的物种特异性。 鉴于 极不溶性和表观高分子量的完整 受体，程序被开发出来分离它的片段， 保持物种特异性。 其中一块碎片，被认为是 纯化受体的胞外结构域的一部分， 通过凝胶过滤和阴离子交换色谱法测定均一性， 是70-kDa糖基化肽。 这种受体片段与精子结合 具体的物种。 目前的建议有五个目标。 第一，初级结构 完整的受体，以及连接位点和结构 其寡糖链将被定义。 第二，建立了 受体分子的基本结构特征，多种方法 将被用来部分降解它，以确定结构基础 的物种特异性，特别是在其作用方面， 多肽主链和寡糖链。 三、潜力 将鉴定该受体的其它配体。 虽然 很明显，来自精子顶体的结合蛋白是一种配体， 与卵子受体的相互作用，不知道是否其他精子 组件涉及。 第四，卵子的表观活化 70-kDa受体片段的抗体将与多种 精子诱导的"早期激活事件"。 微量注射 已知信号转导途径组分的抑制剂或活化剂， 应该可以确定这些组件是否涉及 精子受体系统 如果一级结构分析表明 受体是一种跨膜蛋白，它将被重组成 青蛙卵母细胞或海星卵母细胞质膜的显微注射， 它的mRNA。 或者，将完整的受体纯化，引入 磷脂脂质体，然后与细胞质膜融合， 海星卵母细胞 第五，S.紫质受体将 与其他海胆物种相比。 最初，这将是 在基因水平上完成，这将提供关于 主要结构。 此外，还将进行比较研究 与抗体的集合，以及与卵受体水解酶， 似乎是物种特异性的。 受体结构的比较 各种海胆物种应该提供详细的洞察力， 精子和卵子物种特异性相互作用的分子基础。

Despite great advances in our understanding of the structure and function of receptors in somatic cells, relatively little is known about receptors involved in the cell-cell interactions that occur at fertilization. Work in a prior grant period on the sea urchin egg receptor that binds sperm established that the receptor was protease-sensitive and that its release resulted in loss of ability of sperm to bind to and fertilize these eggs. Furthermore, it was established that proteolytically-released fragments inhibited fertilization in a species-specific manner. Subsequently, it was shown that the intact receptor was a complex proteoglycan-like molecule. Evidence was also obtained that the carbohydrate chains of the receptor serve as the adhesive element of the molecule, whereas the polypeptide chain defines the species specificity of the binding process. Given the extreme insolubility and apparent high molecular weight of the intact receptor, procedures were developed to isolate fragments of it that retained species specificity. One of these fragments, presumed to be a portion of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kDa glycosylated peptide. This receptor fragment binds to sperm species specifically. The current proposal has five objectives. First, the primary structure of the intact receptor, and the sites of attachment and the structure(s) of its oligosaccharide chains will be defined. Second, having established the basic structural features of the receptor molecule, a variety of methods will be used to partially degrade it so as to define the structural basis of its species specificity, especially with respect to the role of the polypeptide backbone and the oligosaccharide chains. Third, potential additional ligands for the receptor will be identified. Although it is clear that bindin from the sperm acrosome is one ligand involved in interaction with the egg receptor, it is not known if other sperm components are involved. Fourth, the apparent activation of eggs by antibody to the 70-kDa receptor fragment will be compared with a variety of "early activation events" induced by sperm. By microinjection of inhibitors or activators of known signal transduction pathway components it should be possible to determine if these components are involved in the sperm receptor system. If primary structure analysis indicates that the receptor is a transmembrane protein, it will be reconstituted into the plasma membrane of frog oocytes or starfish oocytes by microinjection of its mRNA. Alternatively, the intact receptor will be purified, introduced into phospholipid liposomes and then fused with the plasmic membrane of starfish oocytes. Fifth, the structure of the S. purpuralus receptor will be compared with that of other sea urchin species. Initially, this will be accomplished at the level of the gene, which will provide information about primary structure. In addition, comparative studies will be carried out with a collection of antibodies, and with an egg receptor hydrolase that appears to be species specific. A comparison of the receptor structure in various sea urchin species should provide detailed insight into the molecular basis of the species-specific interaction of sperm and egg.