CTP:phosphocholine cytidylyltransferase activity in the preimplantation mouse embryo.

Abstract

Cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 microgram lysophosphatidylethanolamine ml-1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 +/- 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min-1 (mean +/- SEM). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 +/- 0.05 fmol CDP-choline produced per embryo min-1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by alpha-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.