Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca²⁺ and following acrosome reaction in starfish spermatozoa

<jats:p>In the starfish, <jats:italic>Asterias amurensis</jats:italic>, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca<jats:sup>2+</jats:sup> or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pH<jats:sub>i</jats:sub>) and Ca<jats:sup>2+</jats:sup> ([Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>), while ARIS slightly elevated the basal level of [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>. However, a sustained elevation of [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevation and acrosome reaction. The sustained [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevation and acrosome reaction were highly susceptible to SKF96365 and Ni<jats:sup>2+</jats:sup>, specific blockers of the store-operated Ca<jats:sup>2+</jats:sup> channel (SOC). These results suggest that sustained [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pH<jats:sub>i</jats:sub> was artificially increased to more than 7.7. Asterosap increased pH<jats:sub>i</jats:sub> from 7.6±0.1 to 7.7±0.1. Furthermore, the sustained [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pH<jats:sub>i</jats:sub>. Taking these results into account, we suggest that an asterosap-induced pH<jats:sub>i</jats:sub> elevation is required for triggering the ARIS-induced sustained [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevation and consequent acrosome reaction.</jats:p>