科研之友
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摘要
We have previously shown that the preovulatory LH surge downregulates estrogen receptor-beta (ER beta) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ER beta mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ER beta gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERP gene transcription. In contrast, when ER beta mRNA levels were estimated in granulosa cells that were cultured in the presence of,5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ER beta mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agents that mimic LH actions in granulosa cells, also showed similar effects. Thus, these results suggest that LH decreases ER beta mRNA expression in the granulosa cells of preovulatory follicles, primarily by destabilizing the preexisting ER beta mRNA. We next determined the decay rate of the ERP mRNA in granulosa cells that were cultured in the presence of DRB and additional hCG, FSK, or TPA for various time periods, by estimating ER beta mRNA levels, using semiquantitative RT-PCR assays and subsequent linear regression analyses. The half-life of the ER beta mRNA in the presence of vehicle was 17.87 +/- 1.2 h (n = 4), hCG dramatically decreased the half-life of the ER beta mRNA (4.85 +/- 0.49 h, n = 4). Similarly, both FSK and TPA decreased the half-life of the ER beta mRNA to 3.57 +/- 0.31 h and 4.02 +/- 0.13 h, respectively. We extended these findings by examining whether the LH-induced down-regulation of the ER beta mRNA is cycloheximide-sensitive. When granulosa cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor, the inhibitory effects of hCG, FSK, and TPA on ER beta mRNA levels were abolished. Similar results were obtained in the presence or absence of DRB, indicating that the hormone-induced destabilization of the ER beta mRNA is coupled with translation processes. Taken together, our results demonstrate that LH decreases ER beta mRNA expression, predominantly at the posttranscriptional level, in a cycloheximide-sensitive manner.
- 出版日期2001-6
