Received for publication February 21, 2007.
Revised June 19, 2007.
Accepted for publication June 20, 2007.

Adverse effects of 5-aza-2’-deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation

Abstract

The anti-cancer 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 PCR. 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 non-functional 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.

Key words: 5-aza-2’-deoxycytidine, DNA methylation, Decitabine, Germ Cell, RLGS, Spermatogenesis

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