FIGURE 8.

Activation of Epac by 8-pCPT-2′-O-Me-cAMP stimulates a GEF for Rab3A during sperm exocytosis. A, permeabilized spermatozoa were incubated for 10 min at 37 °C with 4 μm GDI (in all the experiments using GDI, the buffer was supplemented with 0.7 mm MgCl₂ and 0.2 mm GTP) followed or not by 300 nm Rab3A-GDP and further incubation for 10 min at 37 °C. Acrosomal exocytosis was initiated by adding 50 μm 8-pCPT-2′-O-Me-cAMP and incubating for an additional 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 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT), and lack of effect on the AR of 300 nm Rab3A-GDP and 4 μm GDI plus 300 nm Rab3A-GDP. Sperm were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized (mean ± S.E.) as described under “Experimental Procedures.” B, SLO-permeabilized spermatozoa were incubated for 10 min at 37 °C with 5 μg/ml RIM-(11–398) followed or not by 300 nm Rab3A-GDP and further incubation for 10 min at 37 °C. Acrosomal exocytosis was initiated by adding 0.5 mm CaCl₂ or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) and incubating for an additional 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 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT), and lack of effect on the AR of 300 nm Rab3A-GDP. Samples were processed, and the AR was scored as in A. C, capacitated human sperm were permeabilized with SLO, and the onset of the AR was prevented by 100 μm 2-APB. Sperm suspensions were subsequently incubated with (right) or without 20 μg/ml anti-Rap1 antibodies or with 1 μg of His₆-Rab3A (top left). AR inducers 50 μm 8-pCPT-2′-O-Me-cAMP (top left and right) and 0.5 mm CaCl₂ (bottom left) were added and incubations proceeded as described under “Experimental Procedures.” Whole sperm lysates were subjected to pulldown assay using GST-RIM-Sepharose, and the levels of GTP-bound Rab3A were determined by Western blot with an anti-Rab3A antibody. Shown are experiments representative of three repetitions; right, quantification (carried out with Image J, freeware from National Institutes of Health) is depicted above the immunoblot as mean ± S.E. from all replicates; different letters indicate statistical significance (p < 0.05).