Release of Ca2+ from Intracellular Stores and Entry of Extracellular Ca2+ Are Involved in Sea Squirt Sperm Activation

Abstract

A rise in intracellular free Ca2+ concentration ([Ca2+]i) is required to activate sperm of all organisms studied. Such elevation of [Ca2+]i can occur either by influx of extracellular Ca2+ or by release of Ca2+ from intracellular stores. We have examined these sources of Ca2+ in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca2+-sensitive dye fura-2 to monitor [Ca2+]i by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca2+], as well as by the presence of high [K+]o or the Ca channel blockers pimozide, penfluridol, or Ni2+, but not nifedipine or Co2+. The accompanying rise in [Ca2+]i was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca2+ entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca2+]i increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca2+]i could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca2+ from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP3) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca2+ and that thimerosal stimulates release of Ca2+ from IP3-sensitive intracellular stores.