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

Abstract

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+]irises 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 (InsP3). No significant increase in Mn2+influx was induced by injection of SE, InsP3, or Ca2+, when Ca2+release was prevented by pre-injection of an antibody against the InsP3receptor. 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.