Fig. 5. Evaluation of ROS generation in human spermatozoa following WGA challenge. To investigate ROS generation following WGA challenge, spermatozoa recovered by Percoll fractionation (pellet) and those isolated by a swim up procedure (swim up) were incubated in (A) luminol-peroxidase or (B) lucigenin and chemiluminescence recorded for 20 minutes. WGA (0.5 µg/ml) was then added and chemiluminescence recorded for an additional 30 minutes. Each sample was then tested for leukocyte contamination through the addition of PMA. The resultant ROS generation profile for each treatment was graphed. Control treatments included spermatozoa incubated in the absence of WGA (control) and a cell-free medium control. The latter control generated only basal levels of chemiluminescence and in the interest of clarity is not shown in the figure. (C) The role of ROS generation was further examined by pre-incubating sperm in either SOD and/or catalase (300 and 3000 U respectively) for 1 hour prior to the addition of WGA (0.5 µg/ml). This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative data are presented.