Effects of arachnotoxin on intracellular pH and calcium in human spermatozoa

ObjectiveTo determine the effect of arachnotoxin (ATx), a venom extracted from the Chilean spider Latrodectus mactans, on intracellular calcium ([Ca2+]i) and pH (pHi) in capacitated human spermatozoa.DesignSpermatozoa were collected from fertile adult men (n = 8). Mobile spermatozoa were collected by the “swim up” technique and stimulated with the crude extract of ATx and with progesterone (P).SettingHospital of the Federal University of São Paulo, São Paulo, Brazil.Main Outcome Measure(s)[Ca2+]i was measured in fura2-AM-loaded spermatozoa, and pHi was measured in spermatozoa loaded with the pH-sensitive dye [(2′,7′)-bis (carboxymethyl)-(5,6)-carboxyfluorescein]-AM (BCECF).Result(s)The ATx and P induced a biphasic change in [Ca2+]i consisting of a peak followed by a small but sustained elevation. The response to ATx was greatly reduced by pretreatment with P. The ATx caused intracellular acidification, whereas P induced alkalinization. Blockade of the NA+/H+ exchanger with ethylisopropylamiloride (EIPA) sharply increased ATx-induced acidification.Conclusion(s)Arachnotoxin increased [Ca2+]i through the opening of calcium channels and release of calcium from intracellular stores. The ATx reduced pHi in human sperm, possibly by inhibiting the Na+/H+ exchanger.