Abstracts Biological Sciences

Male mediated developmental effects of altering DNA methylation in the germ line

by Tonia Elaine. Doerksen

In this thesis, 5-azacytidine, a drug which is incorporated into DNA and blocks DNA methylation, was administrated to male rats to determine its effects on male germ cell development and function. Chronic treatment at doses of 0 to 5.0 mg/kg for four, six, eleven or sixteen weeks were designed to expose different populations of germ cells. Males were mated with normal females to evaluate progeny outcome. Treatment of rats for four weeks, which exposes postmeiotic germ cells only, resulted in no changes in male reproductive organ weights, or abnormal progeny outcome. Treatment for eleven and sixteen weeks which exposes germ cells throughout spermatogenesis, resulted in dose-dependent decreases in testis and epididymal weights, and sperm counts, and increased preimplantation loss when embryos were examined at day twenty of gestation. Examination of embryos at day two of gestation confirmed that embryos sired by males treated with 5-azacytidine were abnormal and died prior to implantation. Six weeks of treatment, which exposes meiotic and post-meiotic germ cells, had an effect on male reproductive organs and progeny outcome intermediate between that of four and eleven weeks of treatment. 5-Azacytidine exposure resulted in abnormalities in testicular histology after eleven, but not six weeks, however closer evaluation using terminal deoxynucleotidyl transferase-mediated nick end-labeling detection in situ showed that germ cells were undergoing apoptosis after both exposure times. Analysis of DNA methylation levels in isolated germ cells and sperm indicated that 5-azacytidine caused decreases in DNA methylation in spermatozoa after 6 and 11 weeks of treatment, and 6 weeks of treatment caused decreases in DNA methylation of round spermatids, but not pachytene spermatocytes. Preimplantation embryos sired by males treated with 5-azacytidine became developmentally arrested in culture. These embryos, however, were partially rescued by the addition of the RNA transcription