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

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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).