FIGURE 5.

Rab3A-GTP-γ-S requires cAMP, Epac, Rap1, and an active PLC to induce exocytosis. A, SLO-permeabilized human sperm were treated for 15 min at 37 °C with 10 μm KH7, 2 μg/ml PDE, or 10 μm H89 before initiating acrosomal exocytosis with 300 nm GTP-γ-S-bound Rab3A (Rab3A) and incubating at 37 °C for 15 min. Sperm were fixed; the AR was measured by FITC-PSA binding, and the data were normalized as described under “Experimental Procedures.” B, SLO-permeabilized spermatozoa were incubated with 6.7 or 134 nm anti-Epac antibodies, 20 μg/ml anti-Rap antibodies (Santa Cruz Biotechnology), 1 μg/ml Ral-GDS-RBD, or 15 μm U73122 for 15 min at 37 °C. Acrosomal exocytosis was evaluated by FITC-PSA binding after an additional 15 min of incubation at 37 °C in the absence (control) or presence of 0.5 mm CaCl₂ (calcium) or 300 nm GTP-γ-S-bound Rab3A (Rab3A). Plotted results represent the mean ± S.E. of at least three independent experiments.