A critical investigation of NADPH oxidase activity in human spermatozoa

It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to p romote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the su peroxide (O-2(-)) probe, lucigenin, We confirmed these observations, but de monstrated that lucigenin increases NADPH consumption by spermatozoa and st imulates artefactual O-2(-) production via a diphenyleneiodonium (DPI) sens itive flavoprotein, In the absence of cytochrome c, DPI-inhibitable NADPH o xidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directl y reduce cytochrome c by a flavoprotein dependent mechanism making this O-2 (-) assay also unreliable in sperm suspensions. We were unable to observe O -2(-) production by 40 x 10(6) spermatozoa/ml using electron paramagnetic r esonance spectroscopy but could identify O-2(-) generation from 2000 4 gamm a -phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spec trophotometry, we did not detect the reduced cytochrome b(558) component of the neutrophil NADPH oxidase in human spermatozoa, No hydrogen peroxide ge neration was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and tha t the mechanism by which NADPH promotes capacitation must be re-evaluated.