A comparative study of estrogen receptors alpha and beta in the rat uterus.

The uterus is an important target organ for steroid hormones. The effects of these hormones are mediated via specific receptors. The aim of this study was to compare the expression, distribution, and regulation of estrogen receptor (ER) alpha and beta in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E(2)), progesterone (P(4)), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ERalpha is the dominating subtype in the rat uterus. E(2) seemed to increase the ERalpha mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P(4) reduced ERalpha expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E(2) or P(4) did not alter the expression of ERbeta, on either the mRNA or protein level. In conclusion, the distribution and regulation of ERalpha and ERbeta differ in the different compartments of the rat uterus. The complex uterine responses to E(2) and P(4) are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERbeta probably plays a minor role in the regulation of uterine physiology.     

Interaction of tetrahydrocrysene ketone with estrogen receptors alpha and beta indicates conformational differences in the receptor subtypes

Estrogen receptors (ER) alpha and beta bind estradiol (E2) and other estrogenic ligands with different affinities. To measure the rate of E2 association with ERa and ERbeta, we employed tetrahydrocrysene ketone (THCK), a fluorescent ligand that is an agonist with ERalpha and an antagonist with ERbeta. We report that THCK binds E2-liganded and unliganded ERalpha and ERbeta, indicating a THCK binding site(s) other than the E2 binding pocket. THCK fluorescence was greater for ligand-occupied ERbeta than ERalpha, suggesting differences in the microenvironment of the THCK binding site(s). THCK fluorescence was also significantly greater for E2-, 4-hydroxytamoxifen-, and tamoxifen aziridine-liganded versus unliganded ER, allowing calculations of E2 association rate constants (ka) of 7.60 +/- 0.75 and 5.12 +/- 0.30 x 10(5) M(-1) s(-1) for E2-ERalpha and E2-ERbeta, respectively. THCK did not affect ERalpha binding to estrogen response element (ERE) DNA, but decreased ERbeta-ERE binding. We conclude that THCK binding site(s) on ERalpha versus ERbeta are different and important for ER function.     

Quantitative analysis of estrogen receptor proteins in rat ovary

The mRNAs of estrogen receptor beta (ERbeta), and its splice variant, ERbeta2, are abundant in granulosa cells in the ovary. With the use of antibodies, ERbeta protein has also been shown to be abundantly expressed, but to date no ERbeta2 protein has been demonstrated in the ovary. ERbeta2 has a peptide, 18 amino acids in length, inserted into its ligand-binding domain, resulting in a reported 35-fold reduction in its affinity for estrogen (E2). ERalpha, ERbeta1 and ERbeta2 were quantified by Western blotting and by RT-PCR and their cellular localization in the ovary was examined by immunohistochemistry. In 3- and 5-week-old virgin, pregnant, lactating and post-lactating rats, the level of ERalpha protein ranged between 1.6 and 3.8 fmol/microg total protein. That of ERbeta was 8.8-11.2 and of ERbeta2, in the same samples, 4.1-5.9 fmol/microg total protein. ERbeta2 and ERbeta1 proteins were, therefore, present in approximately equal amounts in the ovary throughout the various reproductive stages. The major ERbeta proteins in rat ovary, detected by their molecular weights on Western blots, were ERbeta1-530 and ERbeta2-548 (530+18 amino acids (aa)). Immunohistochemical staining revealed that ERbeta and ERbeta2 were expressed predominantly in granulosa cells of growing follicles, while ERalpha was found only in theca cells. In some theca cells, both ERalpha, ERbeta2 were expressed. The data suggests that in theca cells, where it is co expressed with ERalpha, ERbeta2 could function as a repressor of ERalpha. However, in granulosa cells where no ERalpha is detectable, and where E2 levels are high, ERbeta2, with its low affinity for E2, could be an important sensor through which E2 exerts regulatory control.