Progesterone-induced phospholipid N-methylation and sphingomyelin synthesis in the amphibian oocyte plasma membrane: a second source of the 1,2-diacylglycerol second messenger associated with the G 2/M transition

Abstract

The effects of progesterone and GTPγS on phospholipid N-methylation and sphingomyelin synthesis were studied in plasma-vitelline membranes isolated from amphibian ( Rana pipiens) oocytes. Plasma-vitelline membranes were preincubated with S- adenosyl- l [methyl- 3 H] methionine for w min at 20°C and total phospholipids extracted at 0, 15, 30 and 60 s after addition of progesterone and/or GTPγS. Progesterone levels (3 μM) that induce meiosis in the intact oocyte stimulated [ 3H-methyl]incorporation into phosphatidylmonomethylethanolamine (PME) 9–10-folrd over the first 6 s with smaller increases in phosphatidyldimethylethanolamine (PDE) and phosphatidylcholine (PC). [ methyl- 3H] labeling of sphingomyelin (SM) rises after 30 s, approaching that of [ methyl- 3H]PME by 60 s. 17β-Estradiol, a noninducer of meiosis, was inactive. When oocytes were prelabeled with [ 3H]palmitic acid, it was found that a fall in [ 3H]ceramide coincides with the transient increase in [ 3H]SM, indicating that the end product of N-methylation (PC) undergoes a transfer reaction with ceramide to form SM and 1,2-DG. GTPγS levels previously reported to stimulate PC-specific phospholipase C activity in oocyte plasma membranes (5 μM) also stimulated both [ methyl- 3H]PME and [ methyl- 3H]SM formation. An inhibitor of phospholipid N-methylation, 2-(methyl-amino)ethanol, blocked stimulation of [ methyl- 3H]SM synthesis by both progesterone and GTPγS as well as induction of meiosis by progesterone. Progesterone thus acts at the oocyte plasma membrane to stimulate PE N-methyltransferase and SM synthase. The finding that GTPγS mimics progesterone suggests that N-methyltransferase is mediated by G-protein(s). The transient increase in 1,2-DG which we had previously reported to occur within 1–2 min following progesterone stimulation of the Rana oocyte appears to arise from PC by two different pathways: SM synthesis and hydrolysis of PC by phospholipase C.