HUMAN SPERM CAPACITATION INDUCED BY BIOLOGICAL-FLUIDS AND PROGESTERONE, BUT NOT BY NADH OR NADPH, IS ASSOCIATED WITH THE PRODUCTION OF SUPEROXIDE ANION

Recent evidence indicated that human sperm capacitation is associated with an increased production of superoxide anion (O-2(.-)). To further study the role and importance of O-2(.-) in capacitation,we investiga ted whether the O-2(.-) generation is a general feature of capacitatin g spermatozoa, irrespective of the inducer used, and is correlated wit h capacitation levels and increased tyrosine phosphorylation of two sp erm proteins (p105/p81). We also studied the time courses of O-2(.-) p roduction and action. Percoll-washed human spermatozoa were incubated in Ham's F-10 medium, supplemented or not supplemented with various ca pacitation inducers and in the presence or absence of superoxide dismu tase (SOD). Sperm capacitation was measured by induction of the acroso me reaction with lysophosphatidylcholine, O-2(.-) production was measu red by chemiluminescence, and tyrosine phosphorylation was measured by immunodetection after electrophoresis and western blotting of sperm p roteins. Progesterone and ultrafiltrates of human fetal cord serum, fo llicular fluid, and seminal plasma individually promoted sperm generat ion of O-2(.-), tyrosine phosphorylation of p105/p81, and capacitation . Fetal cord serum ultrafiltrate triggered a five-fold higher O-2(.-) production than the other inducers (1,700 +/- 300 and 300 to 400 mV/10 s/8 x 10(6) cells, respectively), a phenomenon possibly associated wit h the higher potency of this fluid to promote sperm hyperactivation. T he production of O-2(.-) by spermatozoa was rapid and transient. SOD p revented sperm capacitation triggered by the inducers mentioned above, but only when SOD was added at the beginning of incubation, and not a fter 30 minutes, indicating that the O-2(.-) initiates a chain of earl y events leading to sperm capacitation. NADH and NADPH (5 mM) triggere d sperm capacitation and phosphorylation of p105/p81, but these proces ses were not prevented by SOD or catalase, nor were they associated wi th an increased O-2(.-) production. Therefore, these cofactors appeare d to act by mechanisms different from those used by the other inducers studied. The sperm enzyme responsible for the O-2(.-) generation may be very different from the NADPH oxidase of neutrophils.