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Expression of apoptotic markers in fetal rat testis after exposure to 5-azaC

DNA methylation is important mechanism of gene regulation in embryogenesis and gametogenesis. 5-azacytidine (5-azaC) is cytosine based analogue, which causes DNA hypomethylation if incorporated during the process of cell proliferation. Weekly administration of this drug disturbs spermatogenesis of adult rat or mice while its teratogenic effects were also described, mostly caused by a single administration of the pregnant animal. We have studied the effect of single 5-azaC administration on the development of fetal rat testis. Pregnant female Fisher rats have received single intraperitoneal injection of 5-azaC in dose of 5mg/kg on 13th, 14th, 15th or 16th day of pregnancy. Fetal testes were isolated on the day 20 and processed for routine histology and immunohistochemistry. Apoptotic-like cells were found in testes of all experimental groups with the highest incidence of apoptotic cells in tubules of the group treated on day 15. Electron microsopy revealed cells in various stages of apoptosis which are presumed to be gonocytes. Moreover, TUNEL analysis and staining against early apoptotic marker γ -H2AX (Asp175) (Cell Signaling Technology) were done on semi-thin Tokuyasu cryosections. TUNEL positive cells were mostly detected in testes which were exposed to the drug on 15th and 16th day of pregnancy. On the other hand, less γ -H2AX positive cells were found, with the γ -H2AX foci in the nucleus and the morphology of early-to-middle apoptotic stage. Testes of control fetuses were negative for the presence of apoptotic cells or any apoptotic marker. Described results suggest that 5-azaC disturbs cell homeostasis in fetal rat testis by causing apoptosis, with the most effect when applied on 15th day of pregnancy. Moreover, most of apoptotic cells were in late stage with few of them in earlier stage, where γ -H2AX foci were found.

5-azacytidine; testis; rat; apoptosis; H2AX; TUNEL

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