The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca²⁺ or by addition of the Ca²⁺ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). Other blockers, like SITS (4-acetamido-4′-isothicyanostilbene-2,2′-disulfonic acid), furosemide, probeneid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC₅₀ of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributylin chloride (TBTC), an Cl^-/OH^- exchanger, partially overcomes DIDS inhibition of the AR. HCO^-₃ is required for a maximal acrosin release and Ca²⁺-uptake, in the presence or absence of A23187. It is known that HCO^-₃ activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO^-₃ is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO^-₃ on Ca²⁺-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO^-₃ into the cell.