Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 micromol/kg wet weight. Within 3-5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with 14C] or 3H]glycerol and 14C] or 3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1-2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The 3H]oleic acid/14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.