Effect of extracellular Ca2+ depletion on the human zona pellucida-induced intracellular [Ca2+] increase in spermatozoa. Capacitated, Fura-2-loaded spermatozoa were incubated in normal BWW medium (upper curve) or in BWW medium lacking calcium (lower curve). One minute before adding hZP, EGTA (500 μM) was added to the Ca2+ free BWW, to ensure that the extracellular medium was devoid of Ca2+. Solubilised hZP (2/μl) was added (arrow) to the medium for each incubation. Results are expressed as the fluorescence excitation ratio (340/380 excitation wavelengths, in arbitrary fluorescence units (AFU)) as a function of time (min). A typical experiment is shown.