MECHANISMS OF MAMMALIAN FERTILIZATION/POLYSPERMY BLOCK

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Fertilization of mouse eggs initiates a cascade of signaling events that include a transient rise in intracellular Ca2+, the stimulation of cortical granule exocytosis that results in modifications of the zona pellucida, recruitment of maternal mRNAs, and completion of the meiotic cell cycle and conversion to a mitotic cell cycle. These events constitute a process termed "egg activation". By analogy to differentiated somatic cells, the sperm may initiate these events of egg activation using G protein-coupled signal transduction pathways. the initial events of sperm-egg plasma membrane interaction that ultimately lead to intracellular signaling may involve adhesion and membrane fusion processes mediated by a sperm ligand and an egg receptor. One aim of this proposal will examine the role of the potential downstream effector for Ca2+ action, the calmodulin-dependent protein kinase II, on specific events of egg activation. This will be done by using a constitutively active form of this kinase, as well as a battery of specific inhibitors of this enzyme. Another aim will address directly the role of G proteins in sperm-induced egg activation by using an antisense approach to ablate the function of specific egg G protein subunits and then assessing this effect on events of egg activation. The last aim will use a sperm- specific protein called fertilin that has been implicated as a specific ligand/adhesion molecule involved in sperm-egg plasma membrane interactions to identify by chemical crosslinking experiments the egg binding protein for fertilin. Since fertilin contains a disintegrin domain an egg integrin may serve as the binding protein for fertilin. The role of an egg integrin in mediating this process will be examined using a transgenic antisense approach in which specific egg integrins are targeted and the effects on sperm binding and egg activation are assessed. Results of these studies focus on the molecular basis of sperm-egg interaction and egg activation and will translate directly to the clinical setting of assisted reproductive technologies. Moreover, these studies address fundamental questions in cell biology, namely, cell adhesion-induced signaling between heterologous cells that relate to questions of regulated exocytosis, cell cycle, and translational control.