Hormonal Regulation of Estrogen Receptor α and β Gene Expression in Human Granulosa-Luteal Cells in Vitro1
Authors: Chiang Chi-Hsin , Cheng Kwai Wa , Igarashi Shigeo , Nathwani Parimal S. , Leung Peter C. K. 
Published: January 2014

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 ERα and ERβ in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERα and ERβ messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERα is expressed at a relatively lower level than ERβ. Basal expression studies indicated that ERα mRNA levels remain unchanged, whereas ERβ mRNA levels increased with time in culture in vitro, suggesting that ERβ is likely to play a dynamic role in mediating estrogen action in hGLCs.

The regulation of ERα and ERβ expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERα (45%; P &lt; 0.01) and ERβ (40%; P&lt; 0.01) mRNA levels. The hCG-induced decrease in ERα and ERβ expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10μ 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 ERα and ERβ mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P &lt; 0.001) and 60% (P &lt; 0.001) decreases in ERα and ERβ mRNA levels, respectively, were observed after treatment with 0.1 μmol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHainduced down-regulation of ERα and ERβ 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 ERα and ERβ proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 μmol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 μmol/L), or phorbol 12-myristate 13 acetate (10 μmol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17β-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 μmol/L GnRHa for 24 h before 17β-estradiol administration.

In summary, we observed a differential expression of ERα and ERβ mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ERα and ERβ 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 ERα and ERβ expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.