Reactive oxygen species in human sperm suspensions: production by leukocytes and the generation of NADPH to protect sperm against their effects.

In men from couples consulting for infertility, 72% of washed sperm preparations produced detectable amounts of reactive oxygen species (ROS) compared to only 25% of preparations from a population of fertile donors. High ROS production was associated with oligozoospermia and poor sperm function. The rate of ROS production was closely correlated with the concentration of leukocytes (r = 0.826) and the degrees of stimulation by N-formyl-methionyl-leucyl-phenyl alanine (NFMLP) and by phorbol 12-myristate 13 acetate (PMA) were similar (r = 0.923). The removal of leukocytes with 'Dynabeads' either abolished or substantially decreased ROS production but in a few cases ROS production that could be stimulated by PMA but not NFMLP was observed in leukocyte-free preparations. When sperm preparations which produced ROS were incubated under 95% O2 there was a rapid 40% decrease in the number of sperm that could be stimulated to acrosome react although the acrosome reaction was unaffected by incubation under 95% N2 for up to 6 h. The harmful effect of oxygen was not seen in preparations that produced no ROS and could be prevented by removing leukocytes from the suspension or by adding superoxide dismutase and catalase. We conclude that leukocytes are the predominant source of ROS in human sperm preparations and that the ROS they produce are harmful to sperm. On the other hand these data confirm that highly purified sperm can produce ROS albeit in smaller amounts. We have demonstrated that flux through the pentose phosphate pathway (PPP) in purified sperm preparations increases in response to oxidative stress. This is required to make reduced glutathione available for glutathione peroxidase and we suggest that measurement of PPP flux provides an index of the capacity of glutathione peroxidase to protect sperm against oxidation.