Reactive oxygen species in human semen: investigations and measurements

Reactive oxygen species (ROS) in human semen, measured as luminol or lucigenin chemiluminescence (CL), originate from spermatozoa or leukocytes. To investigate the relative contribution of these cells to the overall CL signal, the leukocytes were separated by means of a magnetic separation system. Spermatograms according to WHO criteria were performed. The leukocytes were stained (o-toluidine) and counted in a counting chamber. A Berthold luminometer LB 953 was used to determine the luminol and lucigenin chemiluminescence at constant sperm concentrations of 10(7)/mL spermatozoa (MBSS as diluting agent). Then the leukocytes were separated by means of a magnetic cell separation system using different antibodies (MACS CD 14, 15, 16 or CD 45 RO microbeads). After these procedures, the mixture was filtered through a MACS column and the staining with o-toluidine was repeated. In the counting chamber no leukocytes could be found, indicating the complete separation of leukocytes. The CL counts in the spermatozoa fractions after separation of the leukocytes were significantly lower. The data show that leukocytes appear to be a major source for the CL in whole semen.