Voltage-dependent calcium influx in human sperm assessed by simultaneous optical detection of intracellular calcium and membrane potential

Abstract

There are several physiological and pharmacological evidences indicating that opening of voltage dependent calcium channels play a crucial role in the induction of the acrosome reaction in mammalian sperm. In mature sperm, physiological inductors of the acrosome reaction such as ZP3, a zona pellucida protein, and the steroid hormone progesterone, induce depolarization and calcium influx, which are required for the acrosome reaction. In this paper, we describe a voltage-dependent calcium influx present in human sperm. We report an experimental procedure that allows measurement of intracellular calcium and membrane potential simultaneously using the fluorescent dyes DiSC 3(5) and Fura-2. We found that in human uncapacitated sperm, depolarization induces a nifedipine-insensitive calcium influx that, in most cases, was transient. Calcium influx was observed in the range of −60 to −15 mV (the range tested). At resting membrane potential (around −40 mV), potassium addition depolarized and induced calcium influx, but when the depolarization was preceded by a hyperpolarization (induced with valinomycin), calcium influx was remarkably enhanced, suggesting that at −40 mV, channels are in a putative inactivated state. When sperm was incubated in medium without calcium, calcium restoration caused calcium influx that depended on voltage, and decayed between 1 and 2 min after depolarization. Unlike ram, mouse or bovine sperm, in which an alkalinization is required to induce calcium influx with potassium, the voltage-dependent calcium influx observed in human sperm did not require an increase in internal or external pH. However, we observed that ammonium, which increases intracellular pH, enhanced the voltage-dependent calcium influx about 90%. Furthermore, depolarization by itself caused a small increase in intracellular pH suggesting that pH can be regulated by membrane potential in human sperm.