Mammalian Ova and One-Cell Embryos Do not Incorporate Phosphate into Nucleic Acids

The incorporation of [32P]Phosphate by unfertilized mouse ova and 1-cell embryos has been studied. [32P]Phosphate was incorporated into material insoluble in cold trichloroacetic acid by unfertilized ova throughout the period 1–6 h after ovulation with the lowest level of incorporation occurring 2–4 h after ovulation. Incorporation of label by 1-cell embryos was highest soon after fertilization but rapidly fell to a minimum within 2 h before rising to a second maximum at about the time of commencement of pronuclear DNA synthesis. A variety of extraction procedures failed to liberate labeled RNA from ova or embryo lysates and less than 10% of the incorporated label was soluble in chloroform/methanol. The 32P-labeled material precipitated from embryo lysates by ethanol/sodium acetate, pH 5 was resistant to snake venom phosphodiesterase before or after digestion with pronase. However, 32P-labeled material insoluble in cold trichloroacetic acid was hydrolysed by hot alkali to 32Pi and by 6 M HCl to 32P-labeled serine and threonine phosphates and 32Pi thus showing that [32]phosphate was incorporated solely into protein. Paper electrophoresis and thin-layer chromatography showed that 32P-labeled diphosphates and triphosphates but not monophosphates were present in the 32P-labeled soluble fraction from embryo lysates. 32P-labeled nucleoside monophosphates were also absent from the snake venom phosphodiesterase digest of the labeled soluble fraction but 32Pi and 32PPi were products of hydrolysis. Those results show that the mouse ovum does not contain a store of nucleosides, that pathways for mononucleotide synthesis are not functional in the 1-cell embryo, and suggest that the ovum contains a large endogenous store of nucleotides.