FIGURE 6.

Pivotal role of cAMP-Epac in governing two parallel but converging signaling pathways during the late steps of the AR. SLO-permeabilized human sperm were treated for 10 min at 37 °C with 2 μg/ml PDE, 10 μm KH7, or anti-Epac (6.7 or 134 nm) antibodies (A) or anti-Rab3A (20 μg/ml), PTP1B D181A (300 nm), or NSF Y83E (186 nm) (B) followed by 0.5 mm CaCl₂ and an additional 15 min of incubation. To rescue the effect of the blockers, we added adenophostin (5 μm) and incubated for a further 10 min at 37 °C (black bars). Controls (gray bars) included the following: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium), and AR inhibition by 2 μg/ml PDE, 10 μm KH7, anti-Epac (6.7 or 134 nm), and anti-Rab3A (20 μg/ml) antibodies, 300 nm PTP1B D181A, and 186 nm NSF Y83E. Sperm were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized as described under “Experimental Procedures.” C, SLO-permeabilized spermatozoa were loaded with 6.7 nm anti-Epac antibodies for 10 min at 37 °C to selectively block the signaling pathway leading to IP₃-sensitive calcium mobilization. AR was subsequently initiated by adding 0.5 mm CaCl₂. After 10 min of incubation at 37 °C to allow exocytosis to proceed to the Epac-sensitive step, sperm were treated with antibodies that recognize Rab3A (20 μg/ml) or NSF (1:300) and incubated for an additional 10 min at 37 °C. Finally, we added 5 μm adenophostin to rescue the anti-Epac antibody block and incubated as before (black bars). We ran several controls in parallel (gray bars) as follows: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium), and the inhibitory effect of the antibodies when present throughout the experiment (anti-Epac → anti-Rab3A/NSF → calcium → adenophostin). Sperm were fixed, and AR was measured by FITC-PSA binding as described under “Experimental Procedures.” The data represent the mean ± S.E. of at least three independent experiments.