Time-course of the intracellular Ca2+ modifications induced by human zona pellucida in spermatozoa. Capacitated, Fura-2-loaded spermatozoa (15 × 106/ml) were incubated at 37°C in BWW medium. After stabilisation of the fluorescent signal, solubilised hZP (2/μl final concentration) (heavy line) or the same volume of solvent (light line) was added to the sperm suspension (arrow). Results are expressed as the fluorescence excitation ratio (340/380 excitation wavelengths, in arbitrary fluorescence units (AFU)) as a function of time (min). Two patterns of change in calcium concentration were observed in response to the addition of hZP to sperm samples: biphasic (Pattern I A and B) or monophasic (Pattern II C and D).