Effect of metabolic inhibitors on macromolecular synthesis and early development in the mouse embryo

Abstract

Mouse embryos were cultured in vitro for various periods of time, during the interval from the 2-cell stage to the late blastocyst stage, in the continuous presence of actinomycin D or puromycin and labeled precursors. At various time intervals, the incorporation of 3H-uridine into total RNA and of 3H-leucine into protein, and the stage of development of the embryos were recorded. Actinomycin D at the concentration of 0.1 μg/ml caused a rapid and almost complete inhibition of the incorporation of 3H-uridine at all stages of development, and a rapid depression of the incorporation of 3H-leucine into protein until a level of about 50 % of the control incorporation which was attained after 12–16 h of incubation. Longer incubation with the antibiotic did not further depress the relative incorporation of 3H-leucine with respect to the control. The development of the embryos in culture was markedly depressed after continuous incubation with 0.01 and 0.1 μg/ml of actinomycin D, but was not completely arrested. Puromycin at the concentration of 50 μg/ml caused an immediate and complete inhibition of 3H-leucine incorporation and of development in culture. These results were interpreted to indicate that in the mouse embryo protein synthesis and normal development in culture from the 2-cell stage to the blastocyst stage are regulated by a continuous synthesis of RNA. These results correlate with previous biochemical evidence that in the mouse embryo, genes are transcribed very early during development. There is, however, a large fraction of protein synthesis which seems to be dependent on RNA molecules with very long half-life; this fraction of protein synthesis accounts probably for the partial development occurring after actinomycin treatment. The culture methods available at present do not allow to establish whether these stable messengers are synthesized during oogenesis cr after fertilization. The early dependence of embryonic development on gene activity in the mouse contrasts then markedly with the situation observed in sea urchin and amphibians, where embryonic development and protein synthesis until gastrulation are completely independent of simultaneous gene activity, but are probably fully regulated by ribosomes and stable RNA messengers synthesized during oogenesis and stored in the egg cytoplasm to be utilized after fertilization.