Promotion of human sperm capacitation by superoxide anion.

Abstract

Capacitation of spermatozoa is an essential procedure for fertilization. Capacitated spermatozoa have an increase in the intracellular cAMP and acrosome reaction (AR) occurs immediately. The effect of exogenous superoxide anion (O2-) on the level of intracellular cAMP and the percentages of both spontaneous AR and lysophosphatidylcholine-induced AR (LPC-AR) were studied using semen samples collected from 10 healthy and fertile volunteers working or studying in Lanzhou Medical College. Spermatozoa were separated by Percoll and incubated at 37 degrees C in Ham's F-10 medium with O2- generation system: xanthine+xanthine oxidase +catalase+diethylenetriaminepentaacetic acid+ sodium formate. The intracellular cAMP was determined by (3H)-cAMP radioimmunoassay at 3 h of incubation, and the percentages of AR and LPC-AR were evaluated by the triple-stain technique at 3.5 h of incubation. The effects of SOD with different concentration were also determined. The results showed:the level of intracellular cAMP (pmol/10(8) spermatozoa) of spermatozoa increased from 14.0 +/- 1.3 to 23.2 +/- 2.5 (P < 0.01), and the percentages of AR and LPC-AR increased from 4.5 +/- 1.1% and 14 +/- 1.9% to 16 +/- 2.0% and 32.5 +/- 1.7%, respectively (P < 0.01 in both comparisons). SOD inhibited these processes concentration dependently. To investigate the source of O2- during in vivo sperm capacitation, female genital tract fluids collected from 6 healthy nonpregnant donors of reproductive age, and seminal plasma, capacitated and noncapacitated spermatozoa from 10 fertile volunteers were investigated by spin trapping method. The results showed: A typical electron paramagnetic resonance spectrum for O2- spin adduct was exhibited only in capacitated spermatozoa but not in vaginal or cervical secretions, uterine and fallopian tubal fluids, nor in seminal plasma and noncapacitated spermatozoa. These results suggested that only capacitated spermatozoa themselves are able to generate O2- which stimulated their capacitation in turn. Furthermore, on the basis of these data, we propose that it may be possible to utilize the inhibitory effect of SOD on sperm capacitation so as to regulate fertilization.