Calcium removal from the medium promptly reduces human sperm motility and induces a Na⁺-dependent depolarization that is accompanied by an increase in intracellular sodium concentration ([Na⁺]_i) and a decrease in intracellular calcium concentration ([Ca²⁺]_i). Sodium loading activates a Na⁺/K⁺-ATPase.

Membrane potential (Vm) and [Ca²⁺]_i were simultaneously detected in human sperm populations with the fluorescent probes diSC₃(5) and fura 2. [Na⁺]_i and was measured independently in a similar fashion using sodium-binding benzofuran isophthalate. Motility was determined in a CASA system, ATP was measured using the luciferin-luciferase assay, and cAMP was measured by radioimmunoassay.

Human sperm motility reduction after calcium removal is related to either Na⁺-loading or Na⁺-dependent depolarization, because, under conditions that inhibit the calcium removal-induced Na⁺-dependent depolarization and [Na⁺]_i increase, sperm motility was unaffected. By clamping sperm Vm with valinomycin, we found that the motility reduction associated with the calcium removal was related to sodium loading, and not to membrane potential depolarization. Mibefradil, a calcium channel blocker, markedly inhibited the Na⁺-dependent depolarization and sodium loading, and also preserved sperm motility. In the absence of calcium, both ATP and cAMP concentrations were decreased by 40%. However ATP levels were unchanged when calcium removal was performed under conditions that inhibit the calcium removal-induced Na⁺-dependent depolarization and [Na⁺]_i increase.

Human sperm motility arrest induced by external calcium removal is mediated principally by sodium loading, which would stimulate the Na⁺/K⁺-ATPase and in turn deplete the ATP content.