Fertilization involves two phases: (1) excitation (transduction of a signal from the sperm to the egg), and (2) activation (explosive release of Ca2+ and elevation of inositol triphosphate levels), which triggers the onset of development. The steps causing excitation and those which link excitation to activation are not known. Long term objectives are to determine the mechanism underlying sperm-induced excitation and the coupling of excitation to activation in the egg. The following will be determined: (1). the extent to which Na+, K+, H+ and Ca 2+ ions are responsible for the membrane currents during excitation and activation. Shifts of the reversal potentials caused by systematic ion substitutions and monitored electrophysiologically will allow us to determine the relative permeability for each ion. (2). the extent to which Na+, K+, H+, Ca 2+ ions affect excitation and/or the coupling of excitation to activation. Shifts in the voltage dependence of excitation, activation and sperm entry caused by systematic ion substitution will allow us to pinpoint ions which specifically affect these events. (3). Whether or not the sperm-egg fusion event precedes activation or even excitation. Membrane fusion may permit the diffusion of an excitatory molecule from the cytoplasm of the sperm to the egg. Three methods, will be used, namely, transfer of fluorescent markers between the gametes; patch clamp capacitance measurements; and electron microscopy of serial sections using improved methods. (4) Whether or not a sperm surface ligand-egg receptor interaction contributes to excitation of the egg. Bindin, the species specific, sperm-egg adhesive protein, as well as other sperm components will be examined for their ability to cause the same conductance changes as observed during excitation. The proposed research could ultimately provide information basic to the design of new contraceptive agents or to the understanding of certain types of infertility. The research is directed toward understanding what causes a quiescent cell to undergo activation, cell division and growth, and thus may be related to carcinogenesis. A combination of electrophysiology (membrane potential, resistance, and capacitance measurements, current and voltage clamp analysis, and patch clamp techniques), image processing methods, microinjection, and electron microscopy will be used.
