ACROSOMAL MATRIX, EXOCYTOSIS AND MAMMALIAN FERTILIZATION

性状（申请方提供）：有受精能力的精子可以获能，以特定方式与卵子的透明质酸（ZP）结合，并完成顶体胞吐作用。不能执行这些功能会导致不育。虽然ZP和覆盖精子顶体的质膜之间的相互作用被认为是重要的精卵识别和信号，最近的结果促使重新评估目前的范式有关这些相互作用。这项提议的广泛而长期的目标是了解顶体在受精中的功能。一般的假设是，顶体胞吐导致暴露的顶体基质蛋白，成为一个事实上的细胞外基质（ECM）的表面上的精子头部，和这个新暴露的精子ECM与卵子ECM（ZP）的动态相互作用支配精卵识别和精子渗透的ZP。具体目标1是研究顶体胞吐作用，使用新的，灵敏的分析方法来监测OAM和PM融合活精子的事件。将检查自发顶体胞吐在获能过程中启动的可能性，获能是一种定义不清的成熟过程，通常发生在雌性生殖道内，但可以通过实验模拟。假设顶体胞吐作用在获能期间启动，并且在缺乏刺激因子的情况下，是缓慢的、不连续的和Ca依赖性的过程。然而，在刺激因素（例如，ZP），顶体胞吐作用以加速的速率完成。具体目标2是表征哺乳动物卵的顶体基质与透明带的相互作用。将研究AM的组分，以确定它们是否具有配体结合特性，使它们能够与未受精卵的ZP结合。具体目标3是确定AM组分的结构特性和胞吐相关加工，例如，SP 56，影响其功能。将研究sp 56的结构决定因素，以了解哪些结构域是必不可少的精子结合ZP糖蛋白通过检查突变的重组sp 56蛋白的结构-功能关系。此外，将创建sp 56基因缺失的突变小鼠，以确定sp 56是否是受精所必需的。这些实验将为研究顶体胞吐作用的分子机制提供新的技术。这些实验的结果可能提供新的方法来解决一些人类不育患者精子的ZP结合不良的问题，并可能通过破坏有目的的精子-ZP结合来提供避孕的新途径。

DESCRIPTION (provided by applicant): Sperm competent for fertilization can become capacitated, bind to the zona pellucida (ZP) of an egg in a specific manner, and complete acrosomal exocytosis. Failure to carry out these functions results in infertility. Although the interactions between the ZP and the plasma membrane overlying the sperm acrosome have been considered important for sperm-egg recognition and signaling, recent results have prompted a reassessment of current paradigms concerning these interactions. The broad, long-term objective of this proposal is to understand how the acrosome functions in fertilization. The general hypothesis is that acrosomal exocytosis leads to the exposure of acrosomal matrix proteins that become a de facto extracellular matrix (ECM) on the surface of the sperm head, and that the dynamic interactions of this newly-exposed sperm ECM with the egg ECM (the ZP) govern sperm-egg recognition and sperm penetration of the ZP. Specific Aim 1 is to investigate acrosomal exocytosis using novel, sensitive analytical approaches to monitor OAM and PM fusion events in live sperm. The possibility will be examined that spontaneous acrosomal exocytosis is initiated during capacitation, a poorly defined maturation process that normally takes place within the female reproductive tract but can be mimicked experimentally. The hypothesis is that acrosomal exocytosis is initiated during capacitation and, in the absence of stimulatory factors, is a slow, discontinuous and Ca- dependent process. However, in the presence of stimulatory factors (e.g., ZP), acrosomal exocytosis is completed at an accelerated rate. Specific Aim 2 is to characterize the interactions of the acrosomal matrix with the zonae pellucidae of mammalian eggs. Components of the AM will be studied to determine if they possess ligand-binding properties that enable them to bind to the ZP of unfertilized eggs. Specific Aim 3 is to determine how structural properties and exocytosis-associated processing of AM components, e.g., sp56, affect their function. Structural determinants of sp56 will be studied to learn which domains are essential for binding of sperm to ZP glycoproteins by examining the structure-function relationships of mutated recombinant sp56 proteins. In addition, mutant mice null for the sp56 gene will be created to determine if sp56 is essential for fertilization. The proposed experiments will develop new technologies for the study of molecular mechanisms of acrosomal exocytosis. Results from these experiments may provide new ways to address the poor ZP binding of sperm from some human infertility patients and may offer new avenues for contraception through the disruption of purposeful sperm-ZP binding.