title: Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. morrill@aecom.yu.edu
authors.forename: G A
authors.initials: GA
authors.lastname: Morrill
authors.forename: G
authors.initials: G
authors.lastname: Ma
authors.forename: A
authors.initials: A
authors.lastname: Kostellow
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019852:Diacylglycerol Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D004075:Diglycerides / Q000737:chemistry* / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects; D005990:Glycerol / Q000032:analysis; D020404:Glycerophospholipids / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008540:Meiosis / Q000187:drug effects; D008745:Methylation; D008938:Mitosis / Q000187:drug effects; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000032:analysis; D010716:Phosphatidylinositols / Q000032:analysis; D011374:Progesterone / Q000378:metabolism* / Q000494:pharmacology; D011418:Prophase / Q000187:drug effects; D011894:Rana pipiens
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D005227:Fatty Acids; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; C051521:phosphatidylethanol; D000431:Ethanol; D011374:Progesterone; D019852:Diacylglycerol Kinase; D005990:Glycerol