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

精子内顶体膜蛋白：它们的个体发育和在受精中的作用

• 批准号: 192093-2007
• 负责人: Oko, Richard
• 金额: $ 2.7万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=476562
• 关键词: Sperm; inner; acrosomal; membrane; proteins

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).

哺乳动物受精过程中尚待回答的重要问题是精子与卵细胞接触后，精子如何结合并穿透卵细胞外膜（透明膜）。流行的观点是，作为顶体内吞作用和顶体内容物释放的结果，顶体内膜（IAM）变得暴露，使其能够直接与顶体相互作用，这是随后IAM引导的精子穿透顶体的必要步骤。Wassarman及其同事（1988; 1991）对该二级结合步骤的记录刺激了对IAM上与该受体相互作用的蛋白质受体的搜索，但直到我们的出版物（Yang et al.，2006年）提出的众多候选人中没有一个符合所需的标准。我们对IAM最突出的外周蛋白IAM 38的表征支持了其在继发性免疫球蛋白结合中的潜在和普遍作用。在目前的提案中，我们计划通过利用来自IAM 38的特异性阻断肽进行体外受精（IVF）抑制试验来确认这一作用，该肽与ZP 2相互作用，ZP 2是负责与IAM结合的带状配体（Aim 1）。IAM 38可以结合至整合膜蛋白IAM 32。我们已经确定了IAM 32作为热休克蛋白，并旨在表征其与外周IAM蛋白外壳（IAMC）以及与潜在的核周膜（Aim 2）的明显关联。我们的第三个目标是解决顶体生物发生过程中，在超微结构水平上，IAM 32和IAM 38的发育起源和组装以及它们之间的关系（目标3）。高盐和去污剂提取物表明，除了IAM 38外，IAM的外周涂层还含有几种丝氨酸蛋白酶，其对酪蛋白酶谱的蛋白水解作用可被胰蛋白酶抑制剂抑制。由于胰蛋白酶抑制作用会阻止IVF期间精子透明带的渗透，因此这些丝氨酸蛋白酶可能是该过程中的主要候选蛋白酶。我们将使用蛋白质组学的方法来确定这些候选人和竞争性抑制IVF试验，以测试这些蛋白酶在精子穿透中的参与（目的4）。