Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract.
Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.
Mohri
T
T
Laboratory of Intracellular Metabolism, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan. tsmohri@nips.ac.jp
Shirakawa
H
H
Oda
S
S
Sato
M S
MS
Mikoshiba
K
K
Miyazaki
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002457:Cell Extracts; D005260:Female; D008297:Male; D008345:Manganese / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010063:Ovum / Q000378:metabolism / Q000502:physiology; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology
D002413:Cations, Divalent; D002457:Cell Extracts; D008345:Manganese; D002118:Calcium