Method for Detecting Toxin‐Induced Gamete DNA Damage in Male Rats

Despite the vast amount of literature on studies about the use of reproductive toxicants in laboratory rats as models, there are no reports documenting the detection of direct sperm DNA damage in rats as a result of exposure to a reproductive toxicant. These studies were initiated to develop methodology for detecting and scoring oxidative sperm DNA damage in Wistar rats. The first of four experiments used the basic Comet Assay to compare sperm collected from xanthotoxin‐treated rats with sperm collected from untreated rats to determine if oxidative sperm DNA damage was detectable. No differences were found between images of sperm from treated and non‐treated samples. During Experiment 2, fresh sperm samples from untreated rats were stored in PBS and then reacted with a 100mg/ml solution of xanthotoxin/acetone. Post‐reaction cells were subjected to mechanical sample grinding or incubation with trypsin in an attempt to disrupt the cell capsule. Again, no differences were recorded. Experiment 3 exposed treated and untreated sperm samples to proteinase K for varying time intervals (30 min – 3 hr) in another effort to disrupt the cell capsule. These proved to be promising, although at the time intervals used in Experiment 3, the entire cell was dissolved. A series of tests were then initiated during Experiment 4 to determine the best buffer and reaction time combination. All things were kept identical to Experiment 3 with the exception that samples were taken at more frequent intervals and a different buffer was used with each sample. The buffers used were PBS, TES, and PBS containing Chelex. The best results were obtained with TES at a 55‐minute time interval. The capsule surrounding the spermatatids proved to be very resilient to most digestive and mechanical agents. The enzyme proteinase K proved to be the best means for disrupting the cell capsule. Proteinase K in a TES buffer worked most effectively for detecting and scoring oxidative sperm DNA damage in Wistar rats.