Inoue Kyoko, Nakamura Kazuto, Abe Kazuko, Hirakawa Takashi, Tsuchiya Megumi, Oomori Yuki, Matsuda Hiroko, Miyamoto Kaoru, Minegishi Takashi
Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan.
Biol Reprod. 2003 Oct;69(4):1238-44. doi: 10.1095/biolreprod.102.014753. Epub 2003 Jun 11.
The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) beta on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFbeta (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFbeta on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5'-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta. Transforming growth factor beta stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFbeta-induced FSH-R mRNA. Therefore, the profile of the TGFbeta effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.
本研究旨在确定转化生长因子（TGF）β对大鼠颗粒细胞中促卵泡激素受体（FSH-R）作用的潜在机制。与对照组相比，用TGFβ（10 ng/ml）处理颗粒细胞可使FSH-R mRNA转录本（5.5和2.4千碱基）呈时间依赖性增加，在48小时时最大增加约2倍。然后我们研究了TGFβ对FSH-R mRNA水平的影响是否是转录增加和/或mRNA稳定性改变的结果。为了确定FSH-R 5'侧翼区域是否在指导FSH-R mRNA表达中起作用，将FSH-R 5'侧翼区域的近端区域插入到一个表达载体pGL-Basic中，该载体含有荧光素酶作为报告基因，然后将所得质粒瞬时转染到大鼠颗粒细胞中。促卵泡激素（30 ng/ml）显著增强了FSH-R 5'侧翼区域1862个碱基对的活性，但用TGFβ处理并未显著影响促卵泡激素诱导的活性。另一方面，原代颗粒细胞中FSH-R mRNA转录本的衰变曲线显示，添加TGFβ后半衰期显著增加。转化生长因子β以剂量和时间依赖性方式刺激卵泡抑素mRNA积累的表达。如报道的那样，用激活素处理可使FSH-R mRNA水平大幅增加。尽管同时用卵泡抑素处理不影响TGFβ诱导的FSH-R mRNA，但同时用卵泡抑素处理可中和激活素对FSH-R mRNA的这种作用。因此，TGFβ对颗粒细胞中FSH-R mRNA的影响可能是由于FSH-R mRNA稳定性增加以及对卵泡抑素不敏感所致。
