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 an d ER beta variants in granulosa cells have been determined using several in tegrated 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 ac tivity, and estradiol (E) regulation of target genes in granulosa cells. Sp ecifically, the studies presented herein document that messenger RNAs (mRNA s) encoding ERP and its splice variants, as well as mRNA encoding ER alpha, are expressed in granulosa cells of immature rats before and during cultur e in senum-free medium. The results also provide the first documentation th at functional (DNA binding and transcriptionally active) ER is present in c ultured granulosa cells and that its ability to bind consensus estrogen res ponse element (ERE) oligonucleotide and to transactivate an ERE promoter-re porter construct is associated with the level (type?) of receptor protein a s 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 electr ophoretic mobility shift assays) in extracts of granulosa cells cultured ov ernight (0 h) in defined medium alone. When the cells were cultured with FS H and testosterone (T) to stimulate their differentiation, ER beta binding activity, as well as immunoreactive ER beta as determined by Western blot a nalyses, 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 immunoreactiv e ER alpha were detected by Western blot analyses. Immunofluorescent analys es documented that ER beta, as well as ER alpha, were localized to granulos a cell nuclei and that the intensity of nuclear staining was related to ago nist stimulation and differentiation: forskolin increased, whereas E decrea sed immunostaining for ER beta and ER alpha at 48 h. When an ERE-E1b lucife rase vector was transfected into granulosa cells of unprimed rots, basal lu ciferase 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 th e same vector was transfected into differentiated granulosa cells (cultured for 48 h with FSH/T), forskolin alone increased activity. Collectively, th ese results show that ER beta protein is preferentially expressed in immatu re 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 endog enous gene encoding c-Jun.