Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) protonATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that mus t be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membr anes of three metabolically different types of ATPases. The modifications i nduced during in vitro capacitation of rabbit spermatozoa by the specific i nhibition of V-type H+-ATPases with bafilomycin A were studied. We used chl ortetracycline binding to rabbit spermatozoa to monitor capacitation, and t he coomassie brilliant blue method to identify acrosome-reacted sperm cells . There was a significant difference between the percentage of epididymal ( 66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, af ter a 6-h incubation period in the presence of Ca2+ without ATPase inhibito r. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16 +/-8%, epididymal and ejaculated spermatozoa, respectively) and e liminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reac tions induced by the addition of A23187 (12 +/-5, 23 +/-6 and 31 +/-5 after 15, 30 and 45 min). The presence of 0.2 mu mol l(-1) bafilomycin during th e capacitation incubation induced a significant decrease in the acrosome re action percentages (4 +/-2, 8 +/-3 and 14 +/-4 after 15, 30 and 45 min). Th e addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate th at vacuolar ATPases play an important role during rabbit sperm capacitation . However, once the spermatozoa have been capacitated, V-type ATPases do no t have a significant participation during the acrosome reaction.