Fertilization-induced ionic conductances in eggs of the frog, Rana pipiens

Abstract

Fertilization of the frog egg (Rana pipiens) elicits a positive-going shift in membrane potential (fertilization potential) that lasts 10-20 min and functions as a fast block to polyspermy. We examined the ion conductances underlying the fertilization potential, using the voltage-clamp technique. We measured the membrane capacitance during the fertilization potential by applying an alternating current. We also determined the intracellular K and Cl concentrations in the egg, using ion-selective micro-electrodes. The conductance is largest in the first 2 min after fertilization. Regardless of whether the stimulus is provided by one or by more than one sperm or by artificial activation, the size of the conductance increase is the same, reaching a maximum of about 40 microseconds. Two separate conductances are involved at fertilization: Cl and K. [K]i = 121 mM and [Cl]i = 44 mM. The natural external medium is pond water (approximated in our experiments by 10% Ringer solution); therefore, an increase in K and Cl conductances leads to an efflux of both ions. The equilibrium potential of the fertilization current is between the Cl and K equilibrium potentials (ECl and EK), closer to ECl. 10 mM-external tetraethylammonium (TEA) brings the equilibrium potential close to ECl and reduces the maximum conductance by about half. The Cl conductance is not blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). The time courses of the K and Cl conductances are similar. The TEA-resistant conductance (primarily Cl conductance) activated at fertilization increases as the membrane potential becomes more positive. A voltage-sensitive Na conductance present in the unfertilized egg disappears after fertilization. During fertilization this conductance is too small to contribute significantly to the fertilization potential. The membrane capacitance increases by an average of 1.9 times in the first 2 min following the rise of the fertilization potential, during the period of cortical vesicle exocytosis. Capacitance then gradually decreases; at 1 h after fertilization, capacitance is 82% of the value in the unfertilized egg. The conductance increase precedes the capacitance increase by several seconds. Therefore the initial appearance of Cl and K channels cannot be accounted for by addition of membrane by cortical vesicle exocytosis. The conductance subsequently decreases, suggesting that the disappearance of the Cl and K channels is not caused by membrane removal.