NSF Award
9630380
95-30380 Chambers The aim of the research is to identify the primary signals that lead to the sperm-induced excitation and activation of the egg. The nature of these signals is not understood. The specific ion fluxes responsible for generation of the sperm-induced phase 1 and phase 2 membrane currents in the voltage clamped egg will be investigated. The phase 1 current (~12 see) of abrupt onset, coincides with excitation of the egg, when no morphological changes can be detected. The phase 2 current (~18 see) coincides with activation, when exocytosis occurs. Whether a surface sperm-ligand-egg-receptor interaction has a role in initiating excitation of the egg will be investigated. This will be done by applying a suspension of bindin (soluble phase in equilibrium with the insoluble), or other sperm components, will be applied by micropipette to the surface of the homologous unfertilized eggs clamped at different membrane potentials. The ability of the applied bindin to initiate changes in membrane conductance similar to that induced by a sperm during the phase of excitation will be determined. Experiments will be conducted to determine when, and if, cytoplasmic coupling, analogous to gap junctions, between sperm and egg occurs relative to (a) the sperm-induced inward current Ion, and (b) the actual fusion event of sperm and egg plasma membranes. Evidence for the existence of gap junction type pores will be the passage of Lucifer Yellow from egg to sperm prior to the detection by electron microscopy of cytoplasmic bridge formation between the gametes at the site of their apposition. While an increase of cytosolic Ca2+ i is generally associated with activation, data obtained in the P.I.'s laboratory indicated that when this increase occurs prematurely at the site of sperm-egg interaction, activation, but not excitation, can be abrogated due to reversal of the sperm-egg fusion event. The "target" for this effect of an increase of Ca2 i is likely the mechanism responsible for dra wing the sperm into the egg. The possibility that this mechanism is an actin based myosin I motor will be investigated. The gametes of sea urchins will be used. The methods comprise a combination of membrane potential measurements, voltage clamp analysis, quantitative fluorescence imaging, microinjection, and electron microscopy.
