ACROSOMAL MATRIX, EXOCYTOSIS AND MAMMALIAN FERTILIZATION

描述（由申请人提供）：能够受精的精子可以变得有能力，以特定的方式与卵子的透明带（ZP）结合，并完成顶体胞吐。不能执行这些功能会导致不孕症。尽管ZP和覆盖在精子顶体上的质膜之间的相互作用被认为对精子-卵子识别和信号传导很重要，但最近的研究结果促使人们对这些相互作用的当前范式进行重新评估。本研究的长远目标是了解顶体在受精过程中的作用。一般的假设是顶体胞外分泌导致顶体基质蛋白暴露在精子头部表面，成为事实上的细胞外基质（ECM），并且这种新暴露的精子ECM与卵子ECM （ZP）的动态相互作用控制着精子-卵子识别和精子穿透ZP。具体目的1是研究顶体胞吐使用新的，敏感的分析方法来监测OAM和PM融合事件在活精子。自发性顶体胞吐是在获能过程中开始的，这是一个定义不明确的成熟过程，通常发生在女性生殖道内，但可以通过实验模拟。假设顶体胞吐是在获能过程中开始的，在没有刺激因素的情况下，是一个缓慢的、不连续的和钙依赖的过程。然而，在刺激因子（如ZP）的存在下，顶体胞吐以加速的速度完成。具体目的2是表征顶体基质与哺乳动物卵透明带的相互作用。将研究AM的成分，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成分（如sp56）的结构特性和胞吐相关处理如何影响其功能。将研究sp56的结构决定因素，通过检查突变重组sp56蛋白的结构-功能关系，了解哪些结构域是精子与ZP糖蛋白结合所必需的。此外，将创建sp56基因缺失的突变小鼠，以确定sp56是否对受精至关重要。本实验将为顶体胞吐的分子机制研究提供新技术。这些实验结果可能为解决一些人类不孕症患者精子与ZP结合不良的问题提供新的方法，并可能为通过破坏精子与ZP的有目的结合提供新的避孕途径。

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.