Gastrulating Rat Embryo in a Serum-free Culture Model: Changes of Development Caused by Teratogen 5-Azacytidine

Abstract

Developmental processes in gastrulating rat embryos were investigated by using an original, serum-free, chemically defined model system. 9.5-day-old rat embryos, without extraembryonic membranes, were cultivated at the air–liquid interface in a serum-free medium, with and without a protein supplement, for 2 weeks. A teratogenic, demethylating agent, 5-azacytidine, was added to serum-free and protein-free culture medium and to serum-free medium supplemented with human transferrin. A single dose of 5-azacytidine impaired the survival, growth and differentiation of embryos in protein-free medium and serum-free medium with transferrin. In contrast, repeated exposure to 5-azacytidine was required to impair growth in serum-supplemented medium. It was concluded that the activity of 5-azacytidine was easier to detect in a simple, chemically defined medium than in a serum-supplemented medium. This serum-free in vitro method could be useful in screening for teratogenic or embryotoxic substances during gastrulation, the most critical stage of mammalian development.

Influence of hyperthermal regimes on experimental teratoma development in vitro

“…Obviously, cell cycle dynamics were affected, similarly as in other in vitro studies on rat embryos. 55 Based on the previous research with the metabolically poor protein-free medium and an embryotoxic substance, 26,27,29,32 our results with the 3 most extreme regimes discussed below corroborate the idea that in our model a simple simultaneous assessment of survival and overall growth endpoints can efficiently screen for the most deleterious conditions of the microenvironment.…”

“…Therefore, this in vitro model seems to be as sensitive to this externally applied physical factor as it was to various biologically active substances, among which a DNAdemethylating agent (5-azacytidine) used today in the therapy of myelodysplastic syndrome. 26,27 We may suppose that the similar effects may be obtained at least in the experimental teratoma model in vivo that originates from transplanted embryos to an ectopic environment. 11 In 2011, Yarmolenko et al reviewed CEM43°C and thermal threshold damage data (≥20 -1.5 9 10 4 minutes) mostly in rodent models and found that they are different for various tissues and species.…”

“…20 In the whole rat in vitro culture (WEC) used as an alternative to in vivo experiments in animals, 21 malformations of the brain, face and eyes were found and the temperature thresholds were defined during the short-term (2-day) culture in vitro. [22][23][24][25] In an original experimental teratoma model in vitro, it was possible to screen for embryotoxic or teratogenic substances during rat development, 11,26,27 and also reduce the need for screening experiments in living animals accordingly to the 3Rs of Russel and Burch. 21 In this model, the development starts at the most critical period of development (gastrulation) when the embryo consists of the 3 germ layers containing only stem cells for the 3 basic cell lineages (ectodermal, mesodermal, endodermal).…”

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

“…When applied in serum-free conditions to the gastrulating rat embryo-proper (consisting of ectoderm, mesoderm, and endoderm), 5azaC impaired survival, growth, and differentiation (11) but in serum-supplemented conditions it promoted differentiation of muscle (12). In a culture of a younger, pre-gastrulating embryo (consisting of epiblast and hypoblast), it promoted differentiation of muscle, cartilage, blood islands, and neural tissue (13).…”

Impact of 5‐azacytidine on rat decidual cell proliferation

“…For each investigated group, the orientation/pilot stereological measurement was carried out in order to define the number of fields to be tested (Bulic‐Jakus et al . ). The numerical density of PCNA‐positive cells was determined by the point counting method.…”