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

哺乳动物受精的重要问题是，精子与卵带接触引起顶体反应后，精子如何结合并穿透卵的细胞外膜（透明带）。普遍的观点认为，由于顶体内吞作用和顶体内容物的释放，顶体内膜（IAM）暴露，使其能够直接与带相互作用，这是随后由IAM引导的精子穿透带的必要步骤。Wassarman及其同事（1988;1991）对这一次级结合步骤的记录激发了对IAM上与带相互作用的蛋白质受体的研究，但在我们发表之前（Yang et al., 2006），提出的许多候选受体中没有一个符合所需标准。我们对IAM中最突出的外周蛋白IAM38的表征支持了其在次级区结合中的潜在和普遍作用。在目前的提案中，我们计划通过使用来自IAM38的特异性阻断肽进行体外受精（IVF）抑制试验来证实这一作用，这些肽与ZP2相互作用，ZP2是负责与IAM结合的区配体（Aim 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）以及潜在的核膜周围的明显关联（aim 2）。我们的第三个目标是在超微结构水平上解决顶体生物发生过程中IAM32和IAM38的发育起源和组装及其相互关系（目的3）。高盐和洗涤剂提取物表明，除了IAM38外，IAM的外周外壳还含有几种丝氨酸蛋白酶，它们对酪蛋白酶谱的蛋白水解作用可以被胰蛋白酶抑制剂抑制。由于胰蛋白酶抑制在体外受精过程中阻断精子带穿透，这些丝氨酸蛋白酶可能是这一过程的主要候选酶。我们将使用蛋白质组学方法来确定这些候选基因，并进行竞争性抑制试管婴儿试验，以测试这些蛋白酶在精子带穿透中的作用（目的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).