Figure 10. LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one)–induced effect in intracellular Ca²⁺. (A) INDO-1/AM was incorporated into spermatozoa as described in "Materials and Methods." Spermatozoa were next diluted with Biggers-Whitten-Whittingham (BWW) medium (1 mmol of Ca²⁺ per liter). The evaluation of the intracellular free [Ca²⁺] was performed by flow cytometry. (A) The relative [Ca₂₊]_i represents the ratio between the intensity of fluorescence emitted by the Ca²⁺-bound (violet)/Ca²⁺-unbound (blue) INDO-1 probe according to the method described in Current Protocols in Cytometry (June et al, 1997). An experiment representative of 4 in which similar results were obtained is shown. The arrow indicates the time of LY294002 (30 µmol/L) injection. (B) INDO-1/AM–loaded spermatozoa were diluted with BWW medium (1 mmol of Ca²⁺ per liter) and then supplemented with a vehicle dimethylsulfoxide (DMSO), LY294002 (LY, 3 and 30 µmol/L), or wortmannin (W, 100 nmol/L). Results are expressed as the percentage of increase in relative [Ca²⁺]_i after subtraction of control (BWW medium) values of the same parameter. Values are mean ± SEM of 4 determinations performed independently on spermatozoa from different donors. ^*: Data are significantly different from those obtained in control (DMSO) spermatozoa.