The expression of growth arrest genes during preimplantation embryogenesis
The nutritional and hormonal requirements of preimplantation development are poorly understood and the in vitro culture of mouse embryos retards growth and decreases preimplantation viability. This study was carried out to identify the molecular factors responsible for compromised development. The effect of inappropriate culture conditions was examined in F9 embryonal carcinoma (EC) cells, which have a similar phenotype to the inner cell mass cells of the preimplantation embryo. After exposure to all-trans retinoic acid, F9 EC cells differentiate to primitive endoderm, with an increase in alkaline phosphatase (ALP) activity. Growth arrest induced by lysine deficient or low serum media did not prevent a 3-8 fold increase in ALP activity. This showed that differentiation was unaffected despite severe growth retardation. The expression of genes associated with growth arrest was examined in F9 cells. Culture in lysine deficient and low serum media increased gas5 and gadd153 expression. An elevation of gas6 mRNA transcripts was observed during serum arrest, however expression during culture in low lysine medium was below the threshold of detection using Northern analysis. Expression of gas5 and gas6 genes was detected in the preimplantation mouse embryo. Semi-quantitative RT-PCR showed that the embryonic gas5:β-actin ratio decreases with advancing developmental stage. This was probably due to β-actin levels which rise with increasing embryo cell number, and suggests that gas5 expression remains constant during this period. The effect of culture conditions on embryonic gas5;β-actin ratios was examined by culturing mid morular embryos in M16 medium containing either 10% foetal calf serum (FCS) or 500μM ammonium chloride. These experiments showed that embryonic gas5:β-actin ratios are inversely related to the rates of developmental progress. Accelerated development resulted in a decrease in the gas5:β-actin ratio, while retarded development tended to increase the ratio.