engleski

Serum-free versus serum-supplemented in vitro culture of gastrulating rat embryo - effects of teratogen 5-azacytidine

Gastrulating rat embryo, devoid of extraembryonic parts, when cultivated for two weeks in vitro, gives rise to various differentiated tissues in serum-supplemented and even in serum-free chemically defined culture media. This model was used before to investigate various developmental signals (Bulić-Jakuš et al. International Journal of Developmental Biology 34, 275-279, 1990 ; Vlahović, M. et al. Periodicum Biologorum 96, 65-66, 1993). The aim of the present study was to compare the acivity of teratogen 5-azacytidine (5azaC) in serum-free and serum-supplemented culture conditions. Also a more precise investigation of developmental parameters (growth and differentiation) was done. Fisher rat egg-cylinders 9, 5 days old were microsurgically isolated and their embryonic parts cultivated for two weks in protein-free Eagle's Minimal Essential Medium (MEM) as well as in MEM enriched with 50 ľg/ml transferrin or 50% rat serum. Light microscopy, transmission electron microscopy and immunohistochemical detection of Proliferating Cell Nuclear Antigen (PCNA) showed that terminal differentiation of some tissues (e.g. epidermis, neural tissue) has occured, but also that some cells were still retained in the cycling compartement. In serum-free media one dose of 5azaC was able to impair survival, growth and differentiation of embryos, while in serum-supplemented medium only repeated application of 5-azacytidine impaired growth. Moreover, in serum-supplemented medium differentiation with 5azaC was even better than in controls, probably because of its DNA demethylating property which causes gene activation. The above results demonstrated that in chemically defined media 5azaC exhibited its effect promptly and in concordance with its known embryotoxic activity.

serum-free culture; serum-supplemented cultre; rat embryo; 5-azacytidine

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