Analysis of Mn2+/Ca2+ influx and release during Ca2+ oscillations in mouse eggs injected with sperm extract

Repetitive Ca2+ release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn2+ and Ca2+ during Ca2+ oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn2+-quenching of intracellular Fura-2 after adding Mn2+ to external medium. Mn2+/Ca2+ influx was detected at the resting state. A marked Mn2+/Ca2+ influx occurred during the first Ca2+ release upon SE injection, and persistently facilitated Mn2+/Ca2+ influx was observed during steady Ca2+ oscillations. As intracellular Mn2+ concentration ([Mn2+](i)) increased progressively, periodic [Mn2+](i) rises appeared, corresponding to each Ca2+ transient but taking a slower time course. A numerical simulation based on continuous Mn2+/Ca2+ influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn2+ oscillations calculated from experimental data, strongly suggesting that repetitive Mn2+ release develops after Mn2+ entry and uptake into the ER. In other experiments, a marked Mn2+ influx occurred upon Mn2+ addition to Ca2+-free medium after depletion of the ER using an ER Ca2+ pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn2+ influx was induced by injection of SE, InsP(3), or Ca2+, when Ca2+ release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca2+ influx is activated during the initial large Ca2+ release possibly by a capacitative mechanism and kept facilitated during steady Ca2+ oscillations. The finding that repetitive Mn2+ release is caused by continuous Mn2+ entry suggests that continuous Ca2+ influx may play a critical role in refilling the ER and, thereby, maintaining Ca2+ oscillations in mammalian fertilization. (C) 2001 Harcourt Publishers Ltd.