It has been known that DNA methylation is important during gametogenesis and therefore its changes could impair embryonic and gonadal development.Demethylating agent 5-azacytidine (5-azaC) was shown to disturb spermatogenesis in adult rat and mice when administered for several weeks. Additionally, the teratogenic effects on the rat embryo and placenta were reported, caused by single administration of 5-azaC to the pregnant female rats. The aim of the study was to investigate the effect of 5-azaC on the development of fetal rat testis. In separate experiments, single dose of 5-azaC was administered to a pregnant female Fisher rats by i.p. on 13th, 14th, 15th or 16th day of pregnancy. On the 20th day, testes of fetuses were isolated and processed for routine histology and immunohistochemistry or electron microscope analysis. Morphological and ultra structural analyses have pointed out the appearance of apoptotic-like gonocytes in fetal testes of all experimental groups at 20th day. However, in the group treated in utero on day 15 numerous middle to late stage-apoptotic cells, which are presumed to be gonocytes, were found in tubules. Moreover, semi-thin cryosections of control fetal testis and testis from the group treated on 15th day were stained with antibody against cleaved caspase-3 (Asp175) (Cell Signaling Technology) and detected by immunoflourescence. A strong signal of cleaved caspase-3 was detected in testes of fetuses treated with 5-azaC on 15th day of pregnancy. No apoptosis was observed in controls. Results of this study suggest the 15th day of pregnancy as the most sensitive period to the action of demethylating agent on development of gonocytes in fetal rat testis.

Temeljne medicinske znanosti