Figure 5.

cAMP agonists rescued the inhibitory effects on cAMP/PKA pathway and Em caused by NBC inhibitor S0859. Human sperm were incubated in medium that support capacitation in the presence or absence of 5 μm S0859 and cAMP analogs (1 mm BrcAMP + 0.2 mm IBMX). Aliquots from each condition were processed for Western blotting with anti-pPKA (A) or anti-pTyr (B) antibodies (upper panel) and then membranes were reblotted with an anti-β-tubulin antibody for loading control (lower panel). Blots displayed in Fig. 1, A and B, were quantified and values represent the mean ± S.E. of at least 6 experiments (bottom panel). **, p < 0.01. C, human sperm were incubated in medium that support CAP in the absence or presence of 5 μm S0859. Subsequently, aliquots from each condition were processed to evaluate Em with DISC₃(5) and with BCECF-AM to estimate viability by flow cytometry. cAMP analogs (1 mm Br-cAMP and 0.2 mm IBMX, dotted line) rescued the decrease in Em produced by S0859 (5 μm, solid line) (upper panel). Fluorescence median of DISC₃(5) was normalized with respect to CAP condition (bottom panel). Values represent the mean ± S.E. of 5 experiments. ***, p < 0.001 versus CAP condition.