Figure 4.

Sequestration of sperm Rab3 prevents the subsequent function of NSF and calcium mobilization from the acrosome: results obtained with the reversible pair anti-Rab3A antibody (inhibitor 1)/recombinant Rab3A (inhibitor 1 rescue). (A-E) SLO-permeabilized spermatozoa were loaded with 70 nM anti-Rab3A antibody. The AR was subsequently initiated with 0.5 mM CaCl₂. Next. sperm were treated with 10 μM KH7 (B), anti-NSF antibodies (whole rabbit serum diluted 1:300) (C), 134 nM anti-Rap1 antibodies (D), or 10 μM BAPTA-AM (E). Last, we added 140 nM GST-Rab3A (unprenylated and not loaded with nucleotides) to rescue the anti-Rab3A antibody block and incubated as before (black bars). When indicated, 5 μM adenophostin A (AdA) was added at the end of the series. Samples were incubated for 8 min at 37°C after each addition. We run several controls in parallel (grey bars): background AR (control), AR stimulated by 0.5 mM CaCl₂ (calcium); inhibition by anti-Rab3A, anti-NSF, and anti-Rap1 antibodies, and BAPTA-AM; rescue of the anti-Rab3A antibody by Rab3A and of anti-Rap1 antibodies by adenophostin A; lack of rescue of KH7 by adenoposthin A; and the inhibitory effect of the blockers when present throughout the experiment (anti-Rab3A → calcium → KH7/anti-NSF/anti-Rap1/BAPTA-AM → Rab3A). Sperm were fixed and AR was measured as described. The data represent the mean ± SEM of at least three independent experiments. (F) SLO-permeabilized sperm were treated with 100 μM 2-APB and 10 μM KH7 before initiating the AR with 0.5 mM CaCl₂ or 50 μM 8-pCPT-2'-O-Me-cAMP (8pCPT). Incubations were for 15 min at 37°C after each addition. Samples were processed for Rab3-GTP immunodetection as described. Shown is the percentage of cells immunodecorated in the acrosomal region with the anti-GST antibodies. The data represent the mean ± SEM of at least three independent experiments. Different letters indicate statistical significance (P < 0.001).