Sperm inner acrosomal membrane proteins: their ontogeny and role in fertilization

在哺乳动物受精过程中，精子与透明带接触引起顶体反应后，精子如何结合并穿透卵子的胞外膜(透明带)，这是哺乳动物受精过程中有待回答的重要问题。流行的观点认为，由于顶体内吞作用和顶体内容物的释放，顶体内膜(IAM)暴露，使其能够直接与顶体带相互作用，这是随后IAM引导精子穿过顶体带的强制性步骤。Wassarman及其同事对这一二级结合步骤的记录(1988；1991)刺激了对IAM上与小带相互作用的蛋白质受体的搜索，但在我们发表之前(Yang等人，2006年)，许多候选方案都没有达到所需的标准。我们对IAM最重要的外周蛋白IAM38的表征支持它在次生带结合中的潜在和普遍作用。在目前的建议中，我们计划通过进行体外受精(IVF)抑制试验，利用来自IAM38的特定阻断肽与ZP2相互作用来确认这一作用。ZP2是负责与IAM结合的带状配体(目标1)。IAM38可能与一个完整的膜蛋白IAM32结合。我们已经确定IAM32是一种热休克蛋白，并旨在表征其与外周IAM蛋白外壳(IAMC)以及潜在的核周膜(目标2)的明显联系。我们的第三个目标是在顶体生物发生过程中在超微结构水平上解决IAM32和IAM38的发育起源和组装以及它们之间的关系(目标3)。高盐和洗涤剂提取物表明，除了IAM38外，IAM的外周涂层还含有几种丝氨酸蛋白酶，它们对酪蛋白酶谱的蛋白分解作用可以被胰酶抑制剂抑制。由于胰酶抑制会阻止试管受精过程中精子带的穿透，这些丝氨酸蛋白酶可能是这一过程的主要候选者。我们将使用蛋白质组学方法来鉴定这些候选蛋白，并进行竞争性抑制体外受精试验，以测试这些蛋白酶在精子带穿透中的作用(目标4)。

Important questions that remain to be answered in mammalian fertilization are how the spermatozoan binds to and penetrates the extracellular coat (zona pellucida) of the egg after the acrosomal reaction is induced by sperm-zona contact. The prevailing idea is that as a consequence of acrosomal endocytosis and the release of the acrosomal contents, the inner acrosomal membrane (IAM) becomes exposed allowing it to directly interact with the zona, a compulsory step for subsequent IAM-directed sperm penetration through the zona. The documentation of this secondary binding step by Wassarman and colleagues (1988; 1991) stimulated a search for the protein receptor on the IAM that interacts with the zona but until our publication (Yang et al., 2006) none of the many candidates proposed met the criteria needed. Our characterization of the most prominent peripheral protein of the IAM, IAM38, supported its potential and universal role in secondary-zona binding. In this current proposal we plan to confirm this role by performing in vitro fertilization (IVF) inhibition trials utilizing specific blocking peptides derived from IAM38 that interact with ZP2, the zona-ligand responsible for binding to the IAM (Aim 1). IAM38 may be bound to an integral membrane protein, IAM32. We have identified IAM32 as a heat shock protein and aim to characterize its apparent association with the peripheral IAM protein coat (IAMC) as well as with the underlying perinuclear theca (Aim 2). Our third aim is to resolve the developmental origin and assembly of IAM32 and IAM38 and their relationship with each other, at the ultrastructural level, during acrosomal biogenesis (Aim 3). High salt and detergent extracts have revealed that in addition to IAM38 the peripheral coat of the IAM contains several serine proteases whose proteolytic action on casein zymograms can be inhibited by trypsin inhibitors. Since trypsin inhibition blocks sperm-zona penetration during IVF, these serine proteases could be prime candidates in this process. We will use a proteomic approach to identify these candidates and competitive inhibition IVF trials to test the involvement of these proteases in sperm-zona penetration (Aim 4).