Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova

Abstract

Regulation of cytoplasmic free calcium concentration ([Ca2+)](1)) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+](i) is known to undergo oscillations, but it is unknown how basal [Ca2+](i) or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+](i) of metaphase II-arrested mouse oocytes (ova), Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 ± 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 μM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+](i). The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+](i). In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+](i).