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

Abstract

In the starfish, Asterias amurensis, 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-Ca²⁺ 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_i) and Ca²⁺ ([Ca²⁺]_i), while ARIS slightly elevated the basal level of [Ca²⁺]_i. However, a sustained elevation of [Ca²⁺]_i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca²⁺]_i elevation and acrosome reaction. The sustained [Ca²⁺]_i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni²⁺, specific blockers of the store-operated Ca²⁺ channel (SOC). These results suggest that sustained [Ca²⁺]_i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca²⁺]_i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pH_i was artificially increased to more than 7.7. Asterosap increased pH_i from 7.6±0.1 to 7.7±0.1. Furthermore, the sustained [Ca²⁺]_i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pH_i. Taking these results into account, we suggest that an asterosap-induced pH_i elevation is required for triggering the ARIS-induced sustained [Ca²⁺]_i elevation and consequent acrosome reaction.