Hormonal Regulation of Estrogen Receptor a and b 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 receptormediated 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 ERa and ERb in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERa and ERb messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERa is expressed at a relatively lower level than ERb. Basal expression studies indicated that ERa mRNA levels remain unchanged, whereas ERb mRNA levels increased with time in culture in vitro, suggesting that ERb is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ERa and ERb expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERa (45%; P , 0.01) and ERb (40%; P , 0.01) mRNA levels. The hCG-induced decrease in ERa and ERb expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 mmol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 39,59cyclic 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 ERa and ERb 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 ERa and ERb mRNA levels, respectively, were observed after treatment with 0.1 mmol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHainduced down-regulation of ERa and ERb 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 ERa and ERb proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 mmol/L), 8-bromocAMP (1 mmol/L), forskolin (10 mmol/L), or phorbol 12-myristate 13 acetate (10 mmol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17b-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 mmol/L GnRHa for 24 h before 17b-estradiol administration. In summary, we observed a differential expression of ERa and ERb mRNA in hGLCs in vitro. The demonstration of hCGand GnRHainduced down-regulation of ERa and ERb 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 ERb expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively. (J Clin Endocrinol Metab 85: 3828–3839, 2000) I IS WELL documented that estrogen plays an important role in regulating reproductive function. Until recently, the classical estrogen receptor (now known as ERa) was thought to be the only form of nuclear receptor that binds estradiol and mediates its hormonal effects. However, the recent cloning of second form of estrogen receptor (ERb) in rat (1), mouse (2), and human (3) has opened a new era in the study of estrogen signaling. Studies using in situ hybridization and immunohistochemistry have revealed that ERa is localized primarily in the ovarian stromal cells and thecal cells of the rat (4), whereas ERb is concentrated predominately in the granulosa cells of small, developing, and preovulatory follicles in rat and bovine ovary (4–6). The development of ERa knockout (aERKO) mice (7) and ERb knockout (bERKO) mice (8) provides an excellent model to study ERaand ERb-mediated events. Female aERKO mice are anovulatory and infertile even in the presence of significant levels of ERb expression (7, 9), whereas ovarian follicular development and ovulation are partially compromised in bERKO mice (8). The observation that the aERKO ovary contains primary and secondary follicles implicates a role for ERb in early follicular development. On the other hand, final follicle maturation is hampered in the aERKO, suggesting an interaction between ERa and ERb in controlling the later phases of folliculogenesis. Although both ERa and ERb have been detected in the rat corpus luteum (CL) (5, 10, 11), the precise role of estrogen and its receptor in the CL remains unclear. Several studies have suggested that estrogen may regulate ovarian function in humans, including modulation of steroidogenesis from human granulosa-luteal cells (hGLCs) (12), inhibition of 3b-hydroxysteroid dehydrogenase in luteal cells (13), and inhibition of luteal progesterone producReceived December 30, 1999. Revision received June 28, 2000. Accepted July 7, 2000. Address all correspondences and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30-4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: [email protected]. * This work was supported by the Medical Research Council of Canada, a postdoctoral fellowship from the Chang Gung Memorial Hospital (Taipei, Taiwan; to C.-H.C.), a postdoctoral fellowship from Gunma University (Gunma, Japan; to S.I.), and a studentship award from British Columbia Research Institute of Children’s and Women’s Health (to P.S.N.). † C.-H.C. and K.W.C. contributed equally and should be considered as first authors. ‡ Career investigator with the British Columbia Research Institute for Children’s and Women’s Health. 0021-972X/00/$03.00/0 Vol. 85, No. 10 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright © 2000 by The Endocrine Society