Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown

Abstract

It has been known that the intracellular Ca2+ level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase–anaphase transition. Comparable intracellular Ca2+ increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus, and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca2+ increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca2+ increase at the time of polar body extrusion, a similar Ca2+ increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca2+ response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca2+ response to 1-MA, which is independent of external Ca2+ and takes a form of intracellular Ca2+ wave traveling three times as fast as that in the fertilized eggs, this late stage Ca2+ response comprised a train of numerous spikes representing Ca2+ influx. These Ca2+ spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca2+ spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca2+ spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2h after the fertilization in natural seawater. Specific inhibitors of L-type Ca2+ channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca2+ spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca2+ response. When added immediately after the hormone-dependent period, LAT-A inhibited the occurrence (frequency) of the spikes in a dose-dependent manner, but the amplitude of the prevailing Ca2+ spikes itself was rather significantly increased. These results suggest that the cortical actin cytoskeleton and some nuclear factors may play a role in regulating ion channel activities during this stage of meiotic progression.