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Calcium ionophore, A23187, induced a large depolarization of the membrane (referred to as the activation potential) of sea urchin Pseudocentrotus depressus eggs, which was very similar to the fertilization potential induced by a sperm. The egg membrane abruptly overshot to + 12 mV, then slightly fell, and again slowly polarized to about +10 mV, where the membrane sustained for about 10 sec. Peak values of this second component were found to be very sensitive to the external Na+ concentration and external K+ concentration. These features were very similar to the second component (component B) of the fertilization potential. On the other hand, even when removed Ca2+ from external artificial sea water, the activation potential was found to show essentially the same time course and magnitude as those in Ca2+ -containing artificial sea water. Thus the second component of the activation potential elicited by A23187 is probably induced by the increment of Na+ and K+ permeabilities, which might be triggered by the release of Ca2+ from the intracellular storing sites. These results suggest that component B of the fertilization potential is caused by the elevation of internal Ca2+ concentration. Fertilization of the sea urchin egg accompanies the transient change of membrane potential called the fertilization potential, which are considered to be important in facilitating the entry of the fertilizing sperm and in blocking the entry of supernumerary sperm (10, 12). In a previous paper (13), we revealed using Pseudocentrotus depressus that the fertilization potential involved three depolarizing components, of which peaks were 37 •} 4 mV at less than 10 sec (component A), 17 •} 3 mV at 43 •} 4 sec (component B) and -31 •} 5 mV at 182 •} 22 sec (component C) after the onset of the membrane potential change in standard artificial sea water (std ASW). Among them, component B was revealed to be originated from the increment of the permeability of egg membranes to both Na+ and K + . We, however, could not clarify what triggered the change of permeability to Na+ and K± at fer* Present address: Department of Anatomy , School of Medicine, Nagoya University, Tsurumai-cho, Showa-ku, Nagoya 466, Japan. ** To whom correspondence should be addressed . Abbreviations used: ASW, artificial sea water; EGTA, Ethylene glycol-bis(ƒÀ-aminoethyl ether) N,N,N',N'-tetraacetic acid; EPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid; std ASW, standard artificial sea water. 698 S. Obata and H. Kuroda tilization. Intracellular Ca2+ concentration ([Ca2+]i) in sea urchin eggs is known to increase transiently at the early stage of fertilization (4, 14, 20, 24) through the sequential events of the activation of GTP binding protein (21, 22), the increase in the turnover rate of polyphosphoinositides (23, 25), and, by the resultant inositol trisphosphate, the calcium release from intracellular storing sites (3). Artificial raise in [Ca2+]i by microinjecting inositol trisphosphate (25) or calcium buffer (7), or by applying calcium ionophore (18, 20) has been shown to induce the cortical reaction and fertilization membrane elevation (5, 26, for reviews). In the previous paper (13), we have shown that the elevation of fertilization membrane commenced at the early phase of development of component B of the fertilization potential. Moreover, calcium ionophore, A23187 or X-537A, has been demonstrated to induce the substantial membrane depolarization of unfertilized sea urchin eggs (2, 9, 18), and according to our preliminary experiments, the peak value of a transient depolarization elicited by A23187 was close to that of component B of the fertilization potential. The other interesting experimental result has been reported by Carroll and Heath (1) that A23187 induces the efflux of both K+ and Na+. Above mentioned observations suggest that the elevation of permeability to both Na+ and K+, which are responsible for component B of the fertilization potential, might be triggered by the rise in [Ca2+]i at the early phase of fertilization. The experiments reported below were designed to examine this possibility. We measured changes of the membrane potential provoked by A23187 at various extracellular Na+ concentration ([Na+] o) and external K+ concentration UK +10) in Ca2+-containing and Ca2+-free ASWs. MATERIALS AND METHODS Gametes. Eggs and sperms of Pseudocentrotus depressus were forced to be shed into filtered sea water by electrical stimulation (15-20 V). Jelly coats of eggs were removed by washing three times with standard ASW (std ASW) omitting CaC12 (see next section). These dejellied eggs were transferred to std ASW and stored at room temperature (18-22•Ž) until use. All experiments were performed using eggs shed within 4 hrs. Media. Composition of std ASW was as follows: 430 mM NaC1, 10 mM KC1, 10 mM CaC12, 50 mM MgC12, and 10 mM N-2-hydroxyethylpiperazine-N"-3-propanesulfonic acid (EPPS), pH 8.2. Media were continuously perfused during experiments. In order to alter the Nat concentration ([Nat]) in ASW, std ASW was mixed at an appropriate ratio with Nat-free ASW in which NaCl was replaced with choline chloride. Potassium concentration ([K+]) was altered by changing the mixing ratio of ASW containing 10 mM KCl to ASW containing 200 mM KCl. Sodium concentration was 100 mM in these ASWs instead of 430 mM in std ASW. Composition of Ca2±-free ASW was 440 mM NaCl, 10 mM KCl, 50 mM MgC12, 2 mM ethylene glycol bis(J3-aminoethyl ether) N,N,N",Nrtetraacetic acid (EGTA), and 10 mM EPPS. Alteration of [Nat] or [K+] in Ca2+-free ASWs was performed in the same way as described above. Ten mg of calcium ionophore, A23187, (Cal Biochem, La Jolla, CA, U.S.A.), was dissolved in 10 ml of ethanol and stored at 4•Ž. Just before use, one-hundredth volume of this A23187 solution was slowly added to the ASW with continuous stirring. Since A23187 did not dissolve completely at this concentration, the concentration was maximal 10 ƒÊg/ml. The final concentration of ethanol was 1%. At this concentration, ethanol was ascertained not to affect unfertilized and fertilized eggs in their resting membrane potential and the fertilization 699 Activation Potential by A23187 potential. Electrical Recording. Electrophysiological measurements were made by the method of our previous paper (13). Glass microelectrodes with 20-30 Mƒ¶ resistances were selected. All experiments were done at 22•Ž.

cell structure and function

Membrane potential change in a sea urchin egg induced by calcium ionophore A23187 has the same component that is involved in the fertilization potential.