Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin1

• Published in: Endocrinology (Philadelphia) 1999-09, Vol.140 (9), p.4320-4334
• Main Authors: Sharma, S. Chidananda, Clemens, Jeffrey W, Pisarska, Margareta D, Richards, JoAnne S
• Format: Article
• Language: English
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.140.9.6965

The expression and function of estrogen receptor ERα/β subtypes and ERβ variants in granulosa cells have been determined using several integrated approaches: , Western blotting, indirect immunofluorescence, RT-PCR, and transient transfection assays. Each of these approaches has provided specific details concerning the dynamics of ER expression, ER functional activity, and estradiol (E) regulation of target genes in granulosa cells. Specifically, the studies presented herein document that messenger RNAs (mRNAs) encoding ERβ and its splice variants, as well as mRNA encoding ERα, are expressed in granulosa cells of immature rats before and during culture in serum-free medium. The results also provide the first documentation that functional (DNA binding and transcriptionally active) ER is present in cultured granulosa cells and that its ability to bind consensus estrogen response element (ERE) oligonucleotide and to transactivate an ERE promoter-reporter construct is associated with the level (type?) of receptor protein as well as the stage of granulosa cell differentiation. Using a labeled ERE consensus oligonucleotide and antibodies specific for ERβ and ERα, we show that ERβ but not ERα was detected (supershifted in electrophoretic mobility shift assays) in extracts of granulosa cells cultured overnight (0 h) in defined medium alone. When the cells were cultured with FSH and testosterone (T) to stimulate their differentiation, ERβ binding activity, as well as immunoreactive ERβ as determined by Western blot analyses, decreased progressively from 24 to 48 h and was undetectable by 72 h. ERβ mRNA was low, and ERβ binding activity was not observed in luteinized granulosa cells. ERα DNA binding activity was not observed in any of the granulosa cell cultures, although low levels of immunoreactive ERα were detected by Western blot analyses. Immunofluorescent analyses documented that ERβ, as well as ERα, were localized to granulosa cell nuclei and that the intensity of nuclear staining was related to agonist stimulation and differentiation: forskolin increased, whereas E decreased immunostaining for ERβ and ERα at 48 h. When an ERE-E1b-luciferase vector was transfected into granulosa cells of unprimed rats, basal luciferase activity was low but increased by forskolin (3–4×) and by E (2×), responses to both agonists being blocked by the ER antagonist, ICI. When the same vector was transfected into differentiated granulosa cells (cultured for 48 h with FSH/T), forsko