Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca2+ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca2+ increase in Ca2+ concentration ([Ca2+) in the cytosol is due to Ca2+ release from the endoplasmic reticulum (ER), Ca2+ influx from outside is required for Ca2+ oscillations to persist. In this study, we investigated how Ca2+ influx is interrelated to the cycle of Ca2+ release and uptake by the intracellular Ca2+ stores during Ca2+ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca2+] with fura-2, the influx rate was evaluated using Ma(2+) quenching technique, and the change in [Ca2+] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca2+ release and then diminished gradually to the basal level, and demonstrated that the ER Ca2+ stores once depleted by Ca2+ release were gradually refilled until the next Ca2+ transient to be initiated. Experiments altering extracellular [Ca2+] in the middle of Ca2+ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca2+ influx, indicating the crucial role of Ca2+ influx in determining the intervals of Ca2+ transients. As for the influx pathway supporting Ca2+ oscillations to persist, STIM1/Orai1-mediated store-operated Ca2+ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate. (C) 2012 Elsevier Inc. All rights reserved.