RESUMO
Oocyte maturation in medaka is induced by the maturation-inducing hormone (MIH) via its membrane receptor. The most likely candidates for the membrane receptor are membrane progestin receptors (mPRs). In order to characterize the mPRα subtype of medaka, a human cell line expressing the mPRα gene of medaka was established and its steroid binding property was assessed. The α subtype exhibited high binding affinity for 17,20ß-DHP, the MIH in medaka. Treatment with a morpholino antisense oligonucleotide to mPRα blocked oocyte maturation in vivo. These results suggest that the medaka mPRα protein acts as an intermediary during MIH-induced oocyte maturation in medaka in a manner similar to that described previously for fish species.
Assuntos
Oócitos/fisiologia , Oryzias/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Progesterona/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , Feminino , Humanos , Camundongos , Oryzias/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Progesterona/genética , TransfecçãoRESUMO
Previously, we established cell lines stably producing goldfish membrane progestin receptor α (goldfish mPRα) proteins, which mediate steroidal nongenomic actions. In this study, we transfected these cell lines (MDA-MD-231) with cDNAs encoding a recombinant luciferase gene (GloSensor). These cells can be used for monitoring the effects of ligands that bind to mPR by means of luminescence, the intensity of which reflects intracellular cyclic adenosine monophosphate (cAMP) levels. Luminescence intensity of the cells increased significantly when cells were treated with forskolin, strong activator of adenylyl cyclase. Then, we established a strategy to measure changes in luminescence that correlated with the actions of the ligands. The actions of ligands were measurable by the prevention of stimulation caused by forskolin after ligand stimulation. The studies using these cell lines indicated that cAMP concentrations were decreased specifically by the mPR ligands 17α,20ß-dihydroxy-4-pregnen-3-one, diethylstilbestrol and progesterone. Furthermore, pertussis toxin inhibited the decrease in cAMP levels caused by mPR ligands. These results support evidence from previous results that mPRα is coupled to an inhibitory G protein.
