Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology

Genomic methylation patterns originate during gametogenesis and are postulated to be involved in important developmental events, including gene regulation, embryogenesis, and genomic imprinting. In previous work, treatment of male rats with 5-azacytidine, a drug that blocks DNA methylation, resulted in abnormal embryo development when germ cells were exposed throughout spermatogenesis, encompassing mitotic, meiotic, and postmeiotic development, but not if they were only exposed postmeiotically. To explore the mechanisms underlying the effects of 5-azacytidine on sperm function, we determined the effects of the drug on testicular morphology, assessed whether exposure of meiotic spermatocytes resulted in abnormal pregnancy outcome, and examined the role of germ cell genomic demethylation in mediating the effects of 5-azacytidine on spermatogonia and spermatocytes. Male Sprague Dawley rats were treated three times a week with saline or 5-azacytidine (2.5 and 4.0 mg/kg) for 6 weeks (meiotic and postmeiotic germ cell exposure) and 11 weeks (mitotic, meiotic, and postmeiotic exposure). Six weeks of paternal treatment with the highest dose of 5-azacytidine resulted in an in-crease in preimplantation loss (corpora lutea minus implantation sites) without affecting testicular morphology or altering sperm DNA methylation levels. Eleven weeks of 5-azacytidine treatment at doses that cause preimplantation loss resulted in severe abnormalities of the seminiferous tubules, such as degeneration and loss of germ cells, atrophy of seminiferous tubules, presence of multinuclear giant cells, and sloughing of immature germ cells into the lumen, and a 22-29% decrease in genomic methylation levels in epididymal sperm. On closer evaluation of testicular histology using terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling detection in situ, both 6 and 11 weeks of 5-azacytidine treatment resulted in an increase over the control value in the number of apoptotic germ cells in the seminiferous tubules. Analysis of DNA methylation levels in isolated germ cells of treated males indicated that spermatogonia were more susceptible to the hypomethylating effects of 5-azacytidine than were spermatocytes. These studies provide evidence of an association between demethylation of germ cell DNA and alterations in testicular histology.