engleski

Effects of the prolonged heat shock on postimplantation embryo-proper in a two-week in vitro culture

Influence of the heat shock applied during the two-week in vitro culture on mammalian postimplantation embryo was investigated. Fisher rat egg-cylinders 9, 5-days-old were isolated and the extraembryonal part was discarded. Embryos were cultivated on a lens paper at the air-liquid interface supported by a steel grid for 14 days. Chemically defined Eagle's Minimum Essential Medium was supplemented with 50% rat serum or 50μ g/ml transferrin. Experimental samples were kept at different temperatures (430C, 420C or 40, 50C) for various periods of time (10 minutes to 14 days). Medium was changed every other day and minor and major axes of embryos were measured during culture period. Embryos were fixed in St. Marie's fixative and processed for routine histology or immunohistochemistry. Antibodies against proliferating cell nuclear antigen (PCNA) (Dako) and nestin (Rat 401, Hybridoma Bank) were applied and detected by Dako ARK Animal Research Kit and Dako EnVision Doublestain System. In the serum-supplemented cultures overall growth and expression of PCNA and nestin in embryos seemed neither to be affected with 420C applied for 15 minutes at the beginning of culture, only nor when temperature of 430C was applied for 30 minutes every day for the first 7 days. Growth was affected with 430C when embryos were treated for 24 hours. At temperature of 40, 50C applied throughout the culture period overall growth was more affected in serum-free than in serum-supplemented culture conditions. It can be concluded that rat embryo-proper in this in vitro model of postimplantational development is sensitive to heat shock of longer duration.

Heat shock; Embryo culture; Proliferating Cell Nuclear Antigen; Nestin

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