engleski

DNA-demethylating drug acts as “time-bomb” in damaging mammalian embryonic germ cells

Background: DNA methylation is an important epigenetic mechanism involved in normal and disturbed spermatogenesis or carcinogenesis in adults (Jaiswal et al., 2015 ; Buljubasic et al., 2018). A DNA demethylating epigenetic drug 5- azacytidine (5-azaC), used for treatment of malignancies such as the myelodysplastic syndrome also negatively affected development of various tissues (Bulic-Jakuset al., 2016 ; Serman et al. 2009). Objective: The aim of our present research was to investigate the stage-specific effect of DNA-demethylation on spermatogenesis in mammalian embryonic testis. Methods: 5-azaC was intraperitoneally injected to Fisher dame rats on different days of gestation (day 13-day 16)and controls were sham treated by PBS. Embryonic testes were isolated prior to birth and analyzed by the light histology and TEM. Cleaved-caspase 3, and cH2AX were used to for detection and staging of apoptosis. Global DNA methylation levels at cytosines in CpG dinucleotides of repetitive elements (LINE-1 and Satellite regions) were assessed by pyrosequencing. Results: More cells with middle to late apoptotic morphology were found in prospermatogonia of day 20 testes treated with 5-azaC in comparison to the controls. The apoptotic index in embryonic testes showed the correlation to the time of treatment, with the peak when treated with 5-azaC on day 15. To exclude a possible cytotoxic effect the apoptotic index was determined in several days after treatment and showed the rise of apoptotic cells at day 19. Epigenetic analysis has showed also a rise in global methylation at day 19 in normal testis. On the other hand in treated group, a global and satellite DNA hypomethylation was discovered, starting already from the day 18. We may conclude that day 15 of rat gestation seems to be the most sensitive stage to the DNA demethylation activity of5-azaC. Because 5-aza-C replaces cytidine during DNA synthesis in cycling cells it is possible that its incorporation prevented de novo DNA methylation process that is necessary for epigenetic stability of prospermatogonia prior to birth. Therefore, this activity of 5-azacytidine maybe compared to the “time-bomb”. Conclusion: This basic investigation done with a single dose of a DNA demethylating drug during the gestation might be of importance for further understanding of epi-genetic disturbances important for the pathology of the testis.

vidaza ; epigenetics ; DNA-methylation

nije evidentirano