FIGURE 2.

Calcium and 8-pCPT-2′-O-Me-cAMP activate Rap1 during sperm exocytosis. A, proteins from whole sperm homogenates (100 × 10⁶ cells) were partitioned in Triton X-114 and precipitated with organic solvents as described under “Experimental Procedures.” Proteins from the aqueous and detergent phases (normalized by initial cell counts) were resolved in 10% Tricine/SDS-polyacrylamide gels and immunoblotted with the custom-made anti-Rap1 antibody. Molecular mass markers are indicated on the left. B, top, SLO-permeabilized human sperm were treated with 20 μg/ml of an anti-Rap1 polyclonal antibody (Santa Cruz Biotechnology) for 15 min at 37 °C. Acrosomal exocytosis was subsequently initiated by adding 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT, black bar) and incubating for a further 15 min at 37 °C. Bottom, SLO-permeabilized human sperm were incubated for 15 min at 37 °C with 20 μg/ml of the commercial or the custom-made (CM) anti-Rap antibodies (black bars). Asterisks indicate that the antibodies had been pretreated with 205 μg/ml recombinant Rap (anti-Rap* → calcium) or 453 μg/ml Rap peptide (CM-anti-Rap* → calcium). Sperm were challenged with 0.5 mm CaCl₂ and incubated for an additional 15 min at 37 °C. Control condition (gray bars) include the following: background AR in the absence of any stimulation (control), AR induced with 0.5 mm CaCl₂ (calcium), or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT); lack of effect on the AR of recombinant Rap1 and 200 μm 6-Bnz-cAMP alone; and unaffected exocytosis by 20 μg/ml anti-Rab11 and nonimmune antibodies (anti-Rab11/IgG → calcium). Cells were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized as described under “Experimental Procedures.” C, sperm suspensions (20 × 10⁶ cells) were treated (+) or not (−) with 50 μm 8-pCPT-2′-O-Me-cAMP for 10 min at 37 °C in the presence of 100 μm 2-APB to prevent protein loss due to exocytosis. Cells were disrupted; whole cell lysates were subjected to pulldown assays using GST-Ral-GDS-RBD-Sepharose, and the levels of GTP-bound Rap1 were determined as described under “Experimental Procedures.” Shown is a blot representative of three repetitions. D, permeabilized spermatozoa were incubated with 1 μg/ml Ral-GDS-RBD or 1 μg/ml GST for 15 min at 37 °C. Acrosomal exocytosis was then initiated by adding 0.5 mm CaCl₂ (calcium) or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) and a further 15-min incubation at 37 °C. Sperm were stained and the AR scored as in B. The data represent the mean ± S.E. of at least three independent experiments.