CAPACITATION-ASSOCIATED PRODUCTION OF SUPEROXIDE ANION BY HUMAN SPERMATOZOA

Previous results demonstrating the role of the superoxide anion in hum an sperm capacitation indirectly suggest that the superoxide anion is not only essential to this process, but is also generated by spermatoz oa themselves. Our aim was to provide direct evidence for production o f superoxide anion by capacitating spermatozoa. Three chemiluminescenc e probes were tested to detect the superoxide anion. Luminol- and luci genin-amplified chemiluminescence of capacitating spermatozoa did not differ from that of background level. However, significant SOD-inhibit able chemiluminescence was observed when the Cypridina luciferin analo g, MCLA, was used as probe. The chemiluminescence associated to capaci tation of spermatozoa incubated in Ham's F-10 supplemented with fetal cord serum ultrafiltrate was 1270 +/- 77 mV/10 s (with 8 X 10(6) cells /ml; n = 37), and corresponded to levels of sperm hyperactivation (12 +/- 1%) and capacitation (17 +/- 2%) that were significantly different from those of control spermatozoa (4.9 +/- 0.8% and 6 +/- 1%, respect ively). MCLA was cell impermeant and superoxide specific. The level of capacitation-associated chemiluminescence was directly related to spe rm concentration up to 30 X 10(6) cells/ml. Sperm production of supero xide anion started at the beginning of incubation in capacitating cond itions and was sustained over more than 4 h. This is the first direct evidence for the generation, by human spermatozoa, of a sustained leve l of superoxide anion that is associated with the progressive developm ent of hyperactivation and capacitation.