FIGURE 4.

EGFR mediates α7nAChR-activated acrosome reaction. Sperm from WT or α7-null mice (B and D, MUT) were incubated in capacitation medium for 1.5 h. A, AG1478 (AG, 10 μm) was added for the last 30 min, and then ZP (∼7.5 ZP/μl) or PNU282987 (5 μm) was added for an additional 30 min. B, after 1.5 h of incubation in capacitation medium, EGF (1 ng/ml) was added for an additional 30 min. C, α-BgT (100 nm) or MLA (100 nm) was added for the last 30 min of incubation in capacitation medium, and then EGF (1 ng/ml) was added for an additional 30 min. Sperm samples were smeared on a slide for acrosome reaction determination. The data represent the mean ± S.D. of duplicates from at least three experiments. D, sperm from WT or α7-null mice were incubated in capacitation medium. After 30 min Fluo-4/AM was added for 1 h, and AG1478 (10 μm) was added for the last 30 min. The samples were washed in Ca²⁺-free medium and finally were resuspended in a medium that contained Ca²⁺; then they were loaded on 96-wells plate. EGF (1 ng/ml) or ZP (∼7.5 ZP/μl) was added, and the fluorescence was measured utilizing the TECAN plate reader as described under “Experimental Procedures.” Control treatments (cont) were also conducted giving the values of 30% of control for ionomycin (10 μm) and 90% for Triton X-100 (0.1% v/v); when EGTA (1 mm) was used there was no effect on intracellular calcium levels. The data represent the mean ± S.D. of duplicates from at least three experiments. * or **, significant difference from the corresponding control, p < 0.05.