Biochimk'a et Biophysica Acta, ! 109 (lqq2) 65-73 ~ i992 Elsevier Science Publishers B.V. All rights reserved 0005-2736 /92 /$05.01) t~5 BBAMEM 75714 The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa Benny Spira and Haim Breitbart DeiJartm,'nt o]'Lifi" Sciences. Bar-lhm Unirersity. Ramat-Gan (Israel) (Received 9 Januar~ 1992) (Revised manuscript received 6 May 1992) Key words: Anion channel; Acrostmlc reaction; Spermatozoon; J]icarbonale trarsport: cyclic AMP The involvement of anion channels in Ille mechanism of the acmsomc reaction (AR) was investigated. The AR was induced by Ca -~ or by addition of the Ca"' ionophore A23187. The occmrcncc of AR was determined by following lhc rclea,,,c of acrosin fl'om 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'-disulfimic acid). Other blockers, like SITS (4-acctamido-4'-isothicyanoslilbcnc-2,2'-dis- ulfonic acid), furoscmidc, probcncid 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 ICst , of DIDS and SITS in the presence of A23187 is 0.15 and I).22 mM, respectively. Tributyltin chloride (TBTC), an CI-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCOj is required for a maximal acrosin release and Ca:+-uptakc, in the presence or absence of A23!87. h is known that HCO~ activates adenylate cyclase and therefore, increases the intrace!!ular level of cAMP. Tlae inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCOa- is no longer required while clevatir, g the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO~ on Ca-' '-uptake is completely inhibi!cd by DIDS. Wc conclude that DIDS inhibits AR by blocking anion channels, including tho:;c that transport HCOj inio the cell. Introduction The acrosomc reaction is a prerequisite for success- ful fertilization in mammals, defined as a Ca2+-depen - dent exocytotic event in sperm, in which membrane fusion takes place between the outer acrosomal mem- brane and the overlying plasma membrar~e, thereby allowing release oi the acrosomal contents [1,2]. The involvement of cAMP in acrosome reaction was suggested by Hyne and Garbers [3] and later on they showed that bicarbonate is required for the Ca'+-in - duced elevatiorrs of cAMP in guinea-pig spermatozoa [4]. Stengel and Han~mne [5] observed the activation of ram sperm adenylate cyclase by bicarbonate but not by Ca 2÷, ana Garty and Saiomon [6] reported that bicar- bonate directly act'~vatcd partially purified bull sperm adenylate cyclase, rcsults which ale consist,,,t with the Correspondence to: H. Breitbart, Department of Life Sciences, Bar- llan University, Ramat-Gan 52900, Israel. Abbreviations: DiDS 4,4'diisothiocyaaostilbene-2,2'-disuifonic acid; SITS, 4-acetamid~-4'-isothicyanostilbene-2,2'-uJsulfonic acid; TBTC, tributyltin chloride; AR, acrosome reaction. findings of Okamura et al. [7]. Although the role of . . . . . . . . ~ ,,,,,,lie m~cleotides in the sperm acro- Oll.~liUOlidt~., uA.,,,.~ ,,... some reaction has been widely accepted, the regulation of their metabolism is not, clearly understood. The existence of a bicarbonate transport system in porcine spermatozoa was demonstrated by Okamura et al. [8]. In a more recent report., Visconti et al. [9] have shown that blockers of anion channels inhibit the bi- carbonate-dependent phorbol ester enhancing effect on cAMP in hamster spermatozoa, but they did not find any effect on acmsome reaction. In an earlier paper, Hyne [10] demonstrated a requirement of HCO.~ in monensin ((Na+/H + exchanger) induced acrosome reaction in guinea-pig sperm, which was inhibited by low concentrations of the anion-channel blocker di- isothiocyanatestiibene-2.2'-disulfonic acid (DIDS). This blocker also prevented the occurrence acrosome reac- tien in starfish spermatozoa [11]. In porcine epididymal sperm, DIDS enhanced bicarbonate-induced elevation in cAMP content and this effect was not ob:,:crvt, d in the absence of bicarbonate [12]. In the present report, we were investigating the role of the anion channel in the mechanism of ejaculated