Changes in Rat Sperm Chromatin Integrity and Protamination Levels Following Chronic Treatment with the Chemotherapeutic Cocktail Used to Treat Testicular Cancer.

Abstract

The incidence of testicular cancer has increased dramatically over the past 50 years such that it has become the most common cancer among young men of reproductive age. Advances in the treatment of this disease, that includes the co-administration of bleomycin, etoposide and cis-platinum (BEP), have brought the cure rate to over 90 percent. This high cure rate, in conjunction with the young age of patients, increases the likelihood that, after treatment, these men desire fathering children. Thus, the impact of the treatment on reproductive function, fertility, and progeny outcome are of particular importance. Results previously published from our laboratory have demonstrated that chronic exposure to BEP for nine weeks in male rats severely affected sperm chromatin integrity and resulted in early postnatal mortality in progeny sired by these animals. We hypothesize that DNA damage resulting from a chronic nine-week exposure to BEP will be repaired following cessation of treatment, such that the subsequent wave of spermatogenesis will produce newly divided germ cells that are unaffected. To test our hypothesis, male Brown Norway rats were treated with 1.8 mg/kg cis-platinum and 9.0 mg/kg etoposide daily by oral gavage and 0.9 mg/kg bleomycin weekly via an intraperitoneal injection for 9 weeks. Control animals were treated in the same fashion with 0.9% saline. Animals were divided into two groups such that one group of animals was euthanized at the end of the treatment period while the other group was euthanized nine weeks post-treatment, after the completion of one entire cycle of spermatogenesis post-treatment. Both the susceptibility of DNA to denaturation, evaluated by the sperm chromatin structure/acridine orange assay, and the number of single and double DNA strand breaks, as measured by the COMET assay, were significantly increased in mature cauda sperm following nine weeks of treatment with BEP. However, cauda sperm from animals nine weeks post-treatment did not show any significant DNA damage, indicating that DNA damage was repaired in the following wave of spermatogenesis. Interestingly, the protamination level in these post-treatment sperm, assessed by the chromomycin A3 (CMA3) assay, revealed a significant decrease in protamine content as demonstrated by an increase in CMA3 fluorescence that was not seen following nine weeks of treatment. These findings demonstrate that the chromatin integrity of cauda sperm is not completely repaired nine weeks post-treatment; we suggest that persistent damage may result in adverse effects on progeny outcome. Supported by CIHR and the REDIH CIHR training program. (platform)