Ca²⁺ influx-dependent refilling of intracellular Ca²⁺ stores determines the frequency of Ca²⁺ oscillations in...

On mammalian fertilization, long-lasting Ca(2+) oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca(2+) increase in Ca(2+) concentration ([Ca(2+)]) in the cytosol is due to Ca(2+) release from the endoplasmic reticulum (ER), Ca(2+) influx from outside is required for Ca(2+) oscillations to persist. In this study, we investigated how Ca(2+) influx is interrelated to the cycle of Ca(2+) release and uptake by the intracellular Ca(2+) stores during Ca(2+) oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca(2+)] with fura-2, the influx rate was evaluated using Mn(2+) quenching technique, and the change in [Ca(2+)] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca(2+) release and then diminished gradually to the basal level, and demonstrated that the ER Ca(2+) stores once depleted by Ca(2+) release were gradually refilled until the next Ca(2+) transient to be initiated. Experiments altering extracellular [Ca(2+)] in the middle of Ca(2+) oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca(2+) influx, indicating the crucial role of Ca(2+) influx in determining the intervals of Ca(2+) transients. As for the influx pathway supporting Ca(2+) oscillations to persist, STIM1/Orai1-mediated store-operated Ca(2+) 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.