Expression and function of estrogen receptor subtypes in granulosa cells: Regulation by estradiol and forskolin

dThe expression and function of estrogen receptor ER alpha/beta subtypes and ER beta 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 ERP and its splice variants, as well as mRNA encoding ER alpha, are expressed in granulosa cells of immature rats before and during culture in senum-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 ER beta and ER alpha, we show that ER beta but not ER alpha 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, ER beta binding activity, as well as immunoreactive ER beta as determined by Western blot analyses, decreased progressively from 24 to 48 h and was undetectable by 72 h. ER beta mRNA was low, and ER beta binding activity was not observed in luteinized granulosa cells. ER alpha DNA binding activity was not observed in any of the granulosa cell cultures, although low levels of immunoreactive ER alpha were detected by Western blot analyses. Immunofluorescent analyses documented that ER beta, as well as ER alpha, 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 ER beta and ER alpha at 48 h. When an ERE-E1b luciferase vector was transfected into granulosa cells of unprimed rots, basal luciferase activity was low but increased by forskolin (3-4X) and by E (2x), 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), forskolin alone increased activity. Collectively, these results show that ER beta protein is preferentially expressed in immature granulosa cells, is functionally active (binds DNA), can transactivate (either as a homodimer or heterodimer with ER alpha) ERE-containing promoter constructs, and might be associated with increased expression of the endogenous gene encoding c-Jun.