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- W2018539006 abstract "Objective: Seventeen-βhydroxysteroid dehydrogenase type 1 (17β-HSD1) and type 7 (17β-HSD7) are responsible for the high level of estradiol formation in the human ovary. Steroidogenic factor-1 (SF-1) is an essential transcriptional factor for steroidogenesis, as well as for the development of the primary steroidogenic organs. The aim of this study was to examine the role of SF-1 on expression of 17β-HSD1 and 7 in immortalized human granulosa cells.Design: Immortalized human granulosa cell culture model.Materials/Methods: We established an immortalized human granulosa cell line (GC1a) derived from developing follicles by SV40 large T antigen transfection. GC1a cells showed no steroid hormone biosynthesis and no detectable expression of SF-1, steroidogenic acute regulatory protein (StAR) and the cytochrome P450 enzymes, P450scc and P450arom. We transfected the mouse SF-1 expression vector pIND/SF-1 into GC1a cells using the ecdysone-inducible expression system. Total RNA was extracted from pIND/SF-1 transfected GC1a cells after 0–72 hours of incubation with ecdysone. For quantitative analysis of 17β-HSDs, we employed TaqMan reverse transcription-polymerase chain reaction (RT-PCR) system. The amount of 17β-HSDs mRNA at time 0 (the time of addition of ecdysone) assigned a value of 100%, and all other values at different time-points were expressed a percent of the time 0 value. To determine whether SF-1 regulates 17β-HSDs expressions, cycloheximide (20μg/ml), protein synthesis inhibitor, was added for 24 hours.To study the enzymatic activity of 17β-HSDs, estrone (50ng/ml) was added in the cultured medium as the substrate for estradiol.Results: SF-1 elicited the expression of the genes encoding StAR, P450scc and P450arom. RT-PCR showed that the transcripts for 17β-HSD1 and 7 were found in the steady state of GC1a cells, and were not changed by SF-1. A linear relationship between the threshold cycle and the log of the starting RNA copy number was demonstrated (R∧2 > 0.95) in TaqMan RT-PCR. In pIND/SF-1 transfected GC1a cells, levels of 17β-HSD7 mRNA were exceeded approximately one thousand times of 17β-HSD1 mRNA. Expression levels of both 17β-HSD1 and 7 were increased about threefold at 72 hours after the addition of ecdysone. The mRNA levels of 17-HSD1 and 7 were augmented with cycloheximide treatment fivefold and threefold by SF-1 respectively. Estrone was converted to estradiol by the action of SF-1.Conclusions: 17β-HSD7 is a dominant enzyme for the activation of estrogen in the immortalized human granulosa cells. SF-1 concomintantly elicits synchronous induction of the genes encoding the proteins for ovarian steroidogenesis and augments the activity of 17β-HSD1 and 7 in the immortalized human granulosa cells.Supported by: None. Objective: Seventeen-βhydroxysteroid dehydrogenase type 1 (17β-HSD1) and type 7 (17β-HSD7) are responsible for the high level of estradiol formation in the human ovary. Steroidogenic factor-1 (SF-1) is an essential transcriptional factor for steroidogenesis, as well as for the development of the primary steroidogenic organs. The aim of this study was to examine the role of SF-1 on expression of 17β-HSD1 and 7 in immortalized human granulosa cells. Design: Immortalized human granulosa cell culture model. Materials/Methods: We established an immortalized human granulosa cell line (GC1a) derived from developing follicles by SV40 large T antigen transfection. GC1a cells showed no steroid hormone biosynthesis and no detectable expression of SF-1, steroidogenic acute regulatory protein (StAR) and the cytochrome P450 enzymes, P450scc and P450arom. We transfected the mouse SF-1 expression vector pIND/SF-1 into GC1a cells using the ecdysone-inducible expression system. Total RNA was extracted from pIND/SF-1 transfected GC1a cells after 0–72 hours of incubation with ecdysone. For quantitative analysis of 17β-HSDs, we employed TaqMan reverse transcription-polymerase chain reaction (RT-PCR) system. The amount of 17β-HSDs mRNA at time 0 (the time of addition of ecdysone) assigned a value of 100%, and all other values at different time-points were expressed a percent of the time 0 value. To determine whether SF-1 regulates 17β-HSDs expressions, cycloheximide (20μg/ml), protein synthesis inhibitor, was added for 24 hours.To study the enzymatic activity of 17β-HSDs, estrone (50ng/ml) was added in the cultured medium as the substrate for estradiol. Results: SF-1 elicited the expression of the genes encoding StAR, P450scc and P450arom. RT-PCR showed that the transcripts for 17β-HSD1 and 7 were found in the steady state of GC1a cells, and were not changed by SF-1. A linear relationship between the threshold cycle and the log of the starting RNA copy number was demonstrated (R∧2 > 0.95) in TaqMan RT-PCR. In pIND/SF-1 transfected GC1a cells, levels of 17β-HSD7 mRNA were exceeded approximately one thousand times of 17β-HSD1 mRNA. Expression levels of both 17β-HSD1 and 7 were increased about threefold at 72 hours after the addition of ecdysone. The mRNA levels of 17-HSD1 and 7 were augmented with cycloheximide treatment fivefold and threefold by SF-1 respectively. Estrone was converted to estradiol by the action of SF-1. Conclusions: 17β-HSD7 is a dominant enzyme for the activation of estrogen in the immortalized human granulosa cells. SF-1 concomintantly elicits synchronous induction of the genes encoding the proteins for ovarian steroidogenesis and augments the activity of 17β-HSD1 and 7 in the immortalized human granulosa cells. Supported by: None." @default.
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- W2018539006 title "Regulation of 17β-hydroxysteroid dehydrogenases activity in the immortalized human granulosa cells" @default.
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