Figure 4.

Capacitation-associated tyrosine phosphorylation induced by 3-isobutyl-1-methylxanthine (IBMX)-dibutyryl cAMP (dbcAMP) is differentially affected by PRDXs inhibitors added at different times of incubation. Percoll-selected human spermatozoa were capacitated with 0.1 mM IBMX+ 1 mM dbcAMP in BWW medium in the absence or presence of 10 μM TSP or MJ33 at 37°C for 4 h. PRDXs inhibitors were added after 0, 30, 45, 60 and 120 min of incubation. Sperm suspensions were supplemented with SDS-PAGE sample buffer and solubilized proteins from 0.1 × 10⁶ spermatozoa were loaded in each well, electrophoresed, electrotransferred and immunoblotted using the anti-P-Tyrosine antibody (1:10 000). Equal loading was determined by using an anti-α-tubulin antibody (1:10 000). The immunoblots presented are representative of four experiments (n = 4 different donors). Relative intensity of protein tyrosine phosphorylation was normalized to the relative intensity of tubulin of each sample and expressed as mean ± SEM. *P < 0.05 versus non-capacitated controls (ANOVA and Bonferroni test; n = 4).