title: Adverse effects of 5-aza-2'-deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation.
abstract: 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.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University-Montreal Children's Hospital Research Institute, 2300 Tupper St., Montreal, QC, Canada.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Oakes
authors.forename: Tamara L J
authors.initials: TL
authors.lastname: Kelly
authors.forename: Bernard
authors.initials: B
authors.lastname: Robaire
authors.forename: Jacquetta M
authors.initials: JM
authors.lastname: Trasler
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000009:adverse effects / Q000031:analogs & derivatives*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D005838:Genotype; D007248:Infertility, Male / Q000139:chemically induced; D008297:Male; D051379:Mice; D013091:Spermatogenesis / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*
chemical_list: D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse; D001374:Azacitidine