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Transmembrane Regulation of Intracellular Calcium by a Plasma Membrane Sodium/Calcium Exchanger in Mouse Ova¹

^a Department of Obstetrics and Gynecology, Women and Infants Hospital, Brown University, Providence, Rhode Island 02905 ^b Marine Biological Laboratory, Woods Hole, Massachusetts 02543

Regulation of cytoplasmic free calcium concentration ([Ca²⁺)]_i) 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.

¹ This work was supported in part by NIH KO8 01099-01 to D.L.K.

² Correspondence: J.R. Pepperell, Department of Obstetrics and Gynecology, Women and Infants Hospital, Brown University, 101 Dudley St., Providence, RI 02905. FAX: 401 453 7599; jpeppere{at}wihri.org

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