Hormonal regulation of estrogen receptor α and β gene expression in human granulosa-luteal cells in vitro

Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ER alpha and ER beta in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ER alpha and ER beta messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ER alpha is expressed at a relatively lower level than ER beta. Basal expression studies indicated that ER alpha mRNA levels remain unchanged, whereas ER beta mRNA levels increased with time in culture in vitro, suggesting that ER beta is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ER alpha and ER beta expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ER alpha (45%; P < 0.01) and ER beta (406; P < 0.01) mRNA levels. The hCG-induced decrease in ER alpha and ER beta expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 mu mol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ER alpha and ER beta mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ER alpha and ER beta mRNA levels, respectively, were observed after treatment with 0.1 mu mol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHa-induced down-regulation of ER alpha and ER beta expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ER alpha and ER beta proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 mu mol/L), 8-bromo cAMP (1 mmol/L), forskolin (10 mu mol/L), or phorbol 12-myristate 13 acetate (10 mu mol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17 beta-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 mu mol/L GnRHa for 24 h before 17 beta-estradiol administration. In summary, we observed a differential expression of ER alpha and ER beta mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ER alpha and ER beta gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ERa and ERP expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.