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TOXICOLOGY

Adverse Effects of 5-Aza-2'-Deoxycytidine on Spermatogenesis Include Reduced Sperm Function and Selective Inhibition of de Novo DNA Methylation

Departments of Pharmacology and Therapeutics (C.C.O., B.R., J.M.T.), Human Genetics (T.L.J.K., J.M.T.), Obstetrics and Gynecology (B.R.), and Pediatrics (J.M.T.) and the Montreal Children's Hospital Research Institute (J.M.T., C.C.O., T.L.J.K.), McGill University, Montreal, Quebec, Canada

The anticancer agent, 5-aza-2'-deoxycytidine (5-azaCdR, decitabine), causes DNA hypomethylation and a robust, dose-dependent disruption of spermatogenesis. Previously, we have shown that altered testicular histology and reduced sperm production in 5-azaCdR-treated animals is associated with decreased global sperm DNA methylation and an increase in infertility and/or a decreased ability to support preimplantation embryonic development. The goal of this study was to determine potential contributors to 5-azaCdR-mediated infertility including alterations in sperm motility, fertilization ability, early embryo development, and sequence-specific DNA methylation. We find that although 5-azaCdR-treatment adversely affected sperm motility and the survival of sired embryos to the blastocyst stage, the major contributor to infertility was a marked (56–70%) decrease in fertilization ability. Sperm DNA methylation was investigated using Southern blot, restriction landmark genomic scanning, and quantitative analysis of DNA methylation by real-time polymerase chain reaction. Interestingly, hypomethylation was restricted to genomic loci that have been previously determined to acquire methylation during spermatogenesis, demonstrating that 5-azaCdR selectively inhibits de novo methylation activity. Similar to previous studies, we show that mice that are heterozygous for a nonfunctional Dnmt1 gene are partially protected against the deleterious effects of 5-azaCdR; however, methylation levels are not restored in these mice, suggesting that adverse effects are due to another mechanism(s) in addition to DNA hypomethylation. These results demonstrate that clinically relevant doses of 5-azaCdR specifically impair de novo methylation activity in male germ cells; however, genotype-specific differences in drug responses suggest that adverse reproductive outcomes are mainly mediated by the cytotoxic properties of the drug.

Address correspondence to: Dr. Jacquetta Trasler, McGill University-Montreal Children's Hospital Research Institute, 2300 Tupper St., Montreal, QC, Canada, H3H 1P3. E-mail: jacquetta.trasler{at}mcgill.ca

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