ERβ Immunostaining Research Articles

Immunohistochemical expression of CD117/KIT, HER2, and Erβ in schistosomal and non-schistosomal urothelial carcinoma of Egyptian patients

Bladder carcinoma is an endemic problem in Egypt with schistosomiasis being an additional risk factor. Due to gender disparities, Erβ investigation and its role in modulating chemosensitivity are studied. CD117/KIT expression is also considered since the emergence of the targets of the tyrosine kinase inhibitor imatinib mesylate (Gleevec). HER2 is one of the established therapeutic targets in many cancers. We aimed to investigate CD117/KIT immunoexpression in schistosomal and non-schistosomal urothelial carcinoma of Egyptian patients and its relationship with HER2 and Erβ expressions, correlating it with pertinent variables that will help to provide better treatment options of possible combined targeted and hormonal therapy that might be effective against this aggressive malignancy. Sixty cases of bladder carcinoma were tested. Depending on the schistosomiasis association status of each case, two groups have been established with 30 cases each. CD117/KIT, HER2, and ERβ immunostaining were done and correlated with clinico- immuno-pathological parameters. CD117/KIT expression was seen in 71.7% of cases that correlated significantly with schistosomiasis (P = 0.01). In addition, a positive correlation was detected between schistosomiasis association and the percentage of immunostained cells and intensity score of CD117/KIT with P = 0.027, 0.01, respectively. 30% and 61.7% of cases were positively stained with HER2 and Erβ, respectively, with no significant relation with schistosomiasis. Due to the high expression, we found further clinical trials are needed to offer individualized targeted therapeutic options in urothelial tumors using anti-CD117/KIT, HER2, and ERβ other than limited traditional chemo- and nontargeted therapies.

Paracrine roles of hormone receptors in Riehl’s melanosis: A quantitative analysis of oestrogen and progesterone receptor expression patterns

The incidence of Riehl's melanosis (RM) is most common in the fifth or sixth decade of life with a female preponderance. As the skin is regarded a non-reproductive organ on which sex steroid hormones act, a possible relationship between the pathogenesis of RM and sex steroid hormone receptors can be inferred. This study intended to evaluate the expression profile of oestrogen receptor (ER)β and progesterone receptor (PR) in RM. Twelve lesional and perilesional normal-appearing skin samples of RM patients and the skin of 12 healthy controls were retrieved for the analysis. Real-time PCR analysis and immunohistochemical studies were conducted for ERβ and PR, respectively. The lesional and perilesional normal-appearing skin of 12 patients with RM and the skin of 12 healthy controls were retrieved for the analysis. Interestingly, the dermal ERβ immunostaining intensity was increased more in lesional skin than in perilesional skin. When compared to healthy controls, increased expression of ERβ and PR mRNAs was observed in the lesional skin of patients with RM. Of note, epidermal and dermal ERβ and dermal PR expressions showed increased staining intensities in the lesional skin of RM patients compared with healthy controls. The altered expression of ERβ and PR in RM supports the possible role of these hormone receptors in the pathogenesis of RM.

Alterations of endometrial epithelial-mesenchymal transition and MAPK signalling components in women with PCOS are partially modulated by metformin in vitro.

Growing evidence suggests that epithelial-mesenchymal transition (EMT) and its regulator mitogen-activated protein kinase (MAPK) contribute to endometria-related reproductive disorders. However, the regulation of EMT and MAPK signalling components in the endometrium from polycystic ovary syndrome (PCOS) patients has not been systematically investigated and remains elusive. In humans, how metformin induces molecular alterations in the endometrial tissues under PCOS conditions is not completely clear. Here, we recruited 7 non-PCOS patients during the proliferative phase (nPCOS), 7 non-PCOS patients with endometrial hyperplasia (nPCOSEH), 14 PCOS patients during the proliferative phase (PCOS) and 3 PCOS patients with endometrial hyperplasia (PCOSEH). Our studies demonstrated that compared with nPCOS, PCOS patients showed decreased Claudin 1 and increased Vimentin and Slug proteins. Similar to increased Slug protein, nPCOSEH and PCOSEH patients showed increased N-cadherin protein. Western blot and immunostaining revealed increased epithelial phosphorylated Cytokeratin 8 (p-CK 8) expression and an increased p-CK 8:CK 8 ratio in PCOS, nPCOSEH and PCOSEH patients compared to nPCOS patients. Although nPCOSEH and PCOSEH patients showed increased p-ERK1/2 and/or p38 protein levels, the significant increase in p-ERK1/2 expression and p-ERK1/2:ERK1/2 ratio was only found in PCOS patients compared to nPCOS patients. A significant induction of the membrane ERβ immunostaining was observed in the epithelial cells of PCOS and PCOSEH patients compared to nPCOS and nPCOSEH patients. While in vitro treatment with metformin alone increased Snail and decreased Claudin 1, N-cadherin and α-SMA proteins, concomitant treatment with metformin and E2 increased the expression of CK 8 and Snail proteins and decreased the expression of Claudin 1, ZO-1, Slug and α-SMA proteins. Our findings suggest that the EMT contributes to the switch from a healthy state to a PCOS state in the endometrium, which might subsequently drive endometrial injury and dysfunction. We also provide evidence that metformin differentially modulates EMT protein expression in PCOS patients depending on oestrogenic stimulation.

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development

PurposeDisorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases.MethodsHomozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD. Additional cases with 46,XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-β (ER-β) was performed in an 8-week-old human male embryo.ResultsWe identified a homozygous ESR2 variant, c.541_543del p.(Asn181del), located in the highly conserved DNA-binding domain of ER-β, in an individual with syndromic 46,XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-β, were found in unrelated, nonsyndromic 46,XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-β immunostaining was positive in the developing intestine and eyes.ConclusionOur study supports a role for ESR2 as a novel candidate gene for 46,XY DSD.

Association between oestrogen receptor β immunoexpression and cause of culling, ovarian appearance and the existence of PRRS virus in the porcine ovary

The objective of this study was to determine the immunoexpression of oestrogen receptor beta (ERβ) in the gilt ovary and its association with cause of culling, ovarian appearance and the existence of porcine reproductive and respiratory syndrome (PRRS) virus in the ovary. In total, 62 ovaries were collected from culled replacement gilts. The gilts were classified according to the cause of culling (abnormal vaginal discharge, abortion, anoestrus, repeated service and non-reproductive causes), ovarian appearance (non-cycling and cycling) and the existence of PRRS virus in the ovary (not exist and exist). Ovarian ERβ expression was determined using immunohistochemistry. The proportion of ERβ immunostaining in the theca cells surrounding the follicles was evaluated using imaging software. The result revealed that ERβ immunostaining expressed in the theca interna cells of the follicles in the gilt ovary. The ERβ immunostaining was detected in 23.7 ± 19.8% (range 0–98.4%) of the theca cells. The gilts culled due to anoestrus had lower ERβ immunostaining (19.3%) compared to those culled due to non-reproductive causes (28.9%, P = 0.010) and repeated service (31.3%, P = 0.008). However, the expression of ERβ in the ovarian tissues with and without PRRS virus existence was not different (26.8 and 25.2%, respectively, P = 0.538). Likewise, ERβ immunoexpression in the follicles of non-cycling and cycling ovaries was not different (25.8 versus 26.3%, P = 0.851). In conclusion, the gilts culled due to anoestrus had relatively poor ovarian ERβ immunoexpression. Neither the existence of PRRS virus nor ovarian appearance was associated with ERβ immunoexpression in the gilt ovary.

Immunohistochemical evaluation of oestrogen receptors α and β in epithelium of the vaginal mucous membrane in women after oestrogen therapy.

Oestrogens act on target cells through α and β receptors (ERα and ERβ). Expression of oestrogen receptors is associated with the age and menopausal condition of women.The aim of the study was an immunohistochemical evaluation of ERα and ERβ receptors in epithelium of the vaginal mucous membrane of women subjected to different forms of hormonal therapy (HTM).Oestrogen receptors ERα and ERβ were identified using immunohistochemical methods and evaluated in smears of vaginal mucous membranes collected from 60 patients subjected to HTM (including 20 patients after oral therapy, 20 patients after transdermal therapy, and 20 patients after vaginal therapy). The results showed a significant change in immunoreactivity of both studied receptors after three months of hormone therapy. The biggest differences in the changes of intensity of ERα and ERβ reactions were observed in patients subjected to vaginal therapy. Immunostaining for α receptor showed differences between three types of hormone therapy. The highest increase in the overall intensity occurred after three months of topical therapy. Immunostaining for Erβ also varied for different types of hormone therapy.The results indicate that hormone therapy administered vaginally is the most effective in the treatment of urogenital ailments during menopause. In addition, topical therapy eliminates adverse effects of systemic oestrogen.

Expression of Estrogen Receptor Beta Predicts Oncologic Outcome of pT3 Upper Urinary Tract Urothelial Carcinoma Better Than Aggressive Pathological Features.

Upper urinary tract urothelial carcinoma (UT-UC) is rare and treatment options or prognostic markers are limited. There is increasing evidence indicating that urothelial carcinoma may be an endocrine-related cancer. The aim of this study was to analyze the prognostic effect of estrogen receptor beta (ERβ) on the outcome of UT-UC. From 2005 to 2012, this study included 105 patients with pT3 UT-UC. Perioperative factors, pathological features, and ERβ immunostaining were reviewed and prognostic effects were examined by multivariate analysis. This study divided patients into either the ERβ-high (n = 52) or ERβ-low (n = 53) group and analyzed their oncologic outcomes. All pathological features except infiltrating tumor architecture (significantly higher incidence in ERβ-low group, p = 0.004) are symmetric in both groups. Low ERβ expression was significantly correlated with local recurrence and distant metastasis in univariate analysis (p = 0.035 and 0.004, respectively) and multivariate analysis (p = 0.05 and 0.008, respectively). Cell line study also proved that knock down of ERβ cause less UTUC proliferation and migration. In addition, ERβ agonist also enhanced the cytotoxic and migration inhibition effect of cisplatin and ERβ antagonist cause the UTUC cell more resistant to cisplatin. This result may help identify patients in need of adjuvant therapy or develop potential targeted therapy.

Immunolocalization of estrogen and progesterone receptors in ewe mammary glands

Immunopresence of estrogen receptor α (ERα), β (ERβ) and progesterone receptor (PR) were examined in the ewe mammary gland from prepubertal stage to involution. Immunolocalization of ERα revealed a strong positive staining in nuclei of cells composing terminal ductular units (TDUs) in prepubertal ewes. A mild immunoreactivity was identified in early lactating gland. During late lactation immunoreactive product to ERα was observed in the cytoplasm of glandular epithelial cells in all alveoli. In mammary glands at involution ERα positivity was clearly nuclear, with weak to moderate cytoplasmic staining. Cytoplasmic strong immunostaining for ERβ was detected in cells of TDUs, whereas some stromal cells exhibited nuclear staining. A nuclear ERβ immunostaining was observed at early lactation, instead during late lactation, the positivity for ERβ showed only a moderate cytoplasmic distribution. At involution, ERβ positivity was very moderate and detected just in the cytoplasm of shrunken alveoli. Scattered nuclear staining of PR was observed just in mammary glands at early lactation. These results showed that in the mammary glands of sheep both estrogen receptor isoforms were displayed during lactation cycle and that PR appeared just at early lactation, reflecting their regulatory role in alveolar cells.

Total antioxidant status in plasma of breast cancer patients in relation to ERβ expression.

Aim of the studyThe aim of this pilot study was to evaluate the plasma total antioxidant capacity (TAS) in breast cancer patients in relation to ERβ expression.Material and methodsThe study group consisted of newly diagnosed consecutive female breast cancer patients (n = 41) and controls (n = 28) randomly selected from women with benign breast disease. TAS was determined with the ABTS reagent. Immunostaining for ERβ was performed using polyclonal antibodies. ERα, PgR and HER-2 were measured routinely (immunostaining for ERα and PgR with monoclonal antibodies and EnVision detection system; immunohistochemical method/FISH for HER-2 expression).ResultsThe plasma TAS was significantly decreased in the breast cancer patients in comparison to the controls independently of hormonal and lymph node status. The TAS level was not significantly different between breast cancer subgroups either in relation to the ERβ expression (ERβ+ vs. ERβ–) or considering the steroid receptor status (ERα+, ERβ+, Pg+ vs. ERα+, ERβ–, Pg+) even in the selected lymph node negative subgroup. Similarly, HER-2 expression did not significantly affect the TAS concentration. A tendency towards higher TAS level in all ERβ negative breast cancer subgroups was observed.ConclusionsThe results might confirm enhanced consumption of plasma antioxidants in breast cancer patients. The determination of ERβ isoforms along with parameters of redox status might enable better understanding of their mutual influence.

An Immunohistochemical Study on the Expression of Sex Steroid Receptors in Canine Mammary Tumors

Steroid hormones are found to play a major role in the genesis and progression of mammary tumors. The aim of this study was to immunohistochemically detect the presence of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and progesterone receptor (PR) and also to study the association between these markers in 29 cases of benign (11) and malignant (18) canine mammary tumors. ERα immunostaining was noticed in only one case of carcinosarcoma specifically in the nuclei of epithelial and a few myoepithelial cells. ERβ immunostaining was noticed in the nuclei and cytoplasm of epithelial cells and smooth muscles lining the blood vessels. Immunoexpression of ERβ was 82% in benign tumors and 78% in malignant tumors. PR immunostaining was expressed in the nuclei of epithelial cells in both benign and malignant tumors. Among the 15 PR+ cases, 6 (55%) were of benign type, and 9 (50%) were of malignant type. The most common group of hormone receptor was the ERα−/PR+/ERβ+ (46%) in benign tumors and ERα−/PR−/ERβ+ (38%) in malignant tumors. Although there was no significant association between ERα and PR with ERβ, the findings indicated that ERβ was consistently expressed in both benign and malignant tumors, irrespective of ERα and PR status.

Effects of selective estrogen receptor agonists on estrogen receptor expression in the uterus of ovariectomized rats

The aim of the present study was to investigate effects of short time treatment with estrogen receptor (ER) selective agonists on ERα, ERβ and G-protein coupled estrogen receptor-1 (GPER) expression in the uterus of ovariectomized rats. The rats were treated with either estradiol (E2), the specific ERα agonist PPT or the specific ERβ agonist DPN for 18 hrs. Uterine weights were higher after E2 or PPT treatment than after DPN or no treatment. ERα mRNA levels decreased significantly in PPT and DPN treated animals as compared to controls. Stromal ERα immunostaining was higher after E2 treatment than in controls. The ERβ mRNA level was lower in the E2 and PPT groups compared with controls. ERβ immunoreactivity was higher in the myometrium after DPN treatment than in controls. The GPER mRNA level was lower in the E2 and DPN groups as compared to the controls, whereas total protein levels did not display any change. The proliferation marker Ki-67 increased after PPT treatment in stroma and myometrium, as compared to controls. Thus, uterine growth and proliferation are predominately regulated via ERα. Also ERβ expression showed regulation via ERα, while GPER expression indicated control via ERβ. This short term treatment did not result in any regulation of the total protein level as determined by Western blot. However, treatment by E2 increased ERα immunostaining in the stroma and DPN augmented ERβ immunostaining in the myometrium. Thus, the estrogen receptors in the rat uterus are differently regulated depending on the ligand and tissue type.

Detection of estrogen receptor α and β in a relevant model of human invasive urinary bladder cancer.

e15160 Background: Each year in the US, >14,000 people die from invasive urinary bladder cancer (transitional cell carcinoma, InvTCC). Defining effective means to prevent InvTCC development and growth is crucial. Estrogen receptors (ERs) are present in InvTCC, but their role in InvTCC progression and potential value as targets for intervention are not clearly defined. Naturally occurring canine InvTCC closely mimics human InvTCC in molecular features and biological behavior. Gender and neuter status affect InvTCC risk in dogs. Canine InvTCC may offer a relevant in vivo system to study the role of ERs. ERα and β expression in canine InvTCC, with comparison to human InvTCC, were studied as an initial step to determine the role of ERs in InvTCC. Methods: ERα and β expression were determined by immunohistochemistry in canine and human InvTCC samples and normal bladder tissues. The majority of tissues were obtained from patients that had received previous treatment. Antibodies for ERα (SC-7207) and ERβ (SC-8974) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. The percentages of immunoreactive tumor cells and staining intensity in 10 HPFs were recorded by 2 independent reviewers. Tissues with immunoreactivity in greater than 10% of tumor cells were considered positive for ER expression. Results: Results of immunohistochemistry are outlined in the table. Paired tissues from primary and metastatic InvTCC in 17 dogs were evaluated. The ER expression between sites correlated in 17 of 17 cases for ERα, and in 12 of 17 cases for ERβ. ERα and β expression was similar between tissues from males and females analyzed to date. Conclusions: ERα and β were expressed in canine InvTCC, similar to that in human InvTCC. Canine InvTCC offers a relevant system to define the role of ERs in InvTCC development and progression. ERα immunostaining ERβ immunostaining Canine InvTCC 34/39 (87%) 29/39 (74%) Human InvTCC 7/15 (47%) 11/15 (73%) Canine lung metastases 12/14 (86%) 8/14 (57%) Canine lymph node metastases 9/11 (82%) 9/11 (82%) Canine normal bladder 7/7 (100%) 4/6 (67%) Human normal bladder 12/14 (86%) 9/14 (64%)

Effects of tibolone and conventional HRT on the expression of estrogen and progesterone receptors in the breast

ObjectiveThere is evidence that long-term hormone replacement therapy (HRT) is associated with an increased breast cancer risk. The aim of this study was to assess the effects of tibolone on estrogen and progesterone receptors in comparison to the effects of conventional HRT in the breast of surgically postmenopausal macaques. MethodSixty macaques were bilaterally ovariectomized 3 months before hormonal treatment was initiated. The animals were randomized into four treatment groups, including tibolone (TIB), conjugated equine estrogens (CEE), conjugated equine estrogens+medroxyprogesterone acetate (CEE+MPA) and control animals (C). After 2 years treatment, breast tissues were collected, fixed and paraffin embedded. Immunohistochemistry assays with monoclonal antibodies for estrogen receptors (ERα and ERβ) and progesterone receptors (PRA and PRB) were performed. ResultsThe expression of ERα was markedly decreased in the CEE+MPA group as compared to C and TIB groups. The TIB group was not different from the C and CEE groups. No significant differences were found for ERβ immunostaining. The expression of PRA was strongly increased in the TIB group as compared to the C and CEE+MPA groups. Immunostaining of PRB was increased in the CEE and TIB treated animals as compared to both C and CEE+MPA groups. ConclusionsTibolone increased the expression of both PRA and PRB, without affecting ERα and ERβ expression in the macaque breast. These findings indicate that the effects of tibolone in breast tissue could be mediated via differential regulation of PRA and PRB isoforms and therefore distinct from those observed with conventional HRT.

Estrogen receptor expression and neuronal nitric oxide synthase in the clitoris and prepucial gland structures of mice

Purpose ERα is the predominant form of estrogen receptor expressed in uterine and vaginal tissue of the murine female reproductive tract. Expression of both isoforms has been detected in vaginal tissue and in the ovary. Estrogen receptors were seen in basal and suprabasal cells of vaginal epithelium and epidermis of labia minora Understanding the locations of the different isoforms of the ER within genital structures may elucidate mechanisms of genital growth and development. It is well known that the sensory condition of the vulva epithelium is highly influenced by estrogen status with enlargement of the genitosensory field in response to this hormone. Estrogens may also prove crucial to the maintenance of urogential sensitivity. Their ability to promote the growth of neurons has been established. Abundant literature has reported on nitric oxide in the context of the internal female reproductive tract and penis, few studies have investigated the female external genital structure or the clitoris. The aim of this work is to study the presence of estrogen receptors (ER), as well as n-NOS in the mouse clitoris. Materials and methods Immunocytochemical studies specific for Estrogen Receptor-α, Estrogen Receptor-β, and nNOS were performed on a series of sections of the pelvic area including the prepucial glands and clitoris of ten mice. Selected sections were also stained with Masson's trichrome. Results ERα was expressed predominantly by the glandular epithelium as well as in the glandular tissue, preputial and apocrine glands. ERα was observed in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris. ERβ immunostaining was only detected in few cells located in the vascular lumen of the cavernous tissue of the clitoris. nNOS immunostaining was detected in the clitoris, the preputial glans and the connective tissue. Conclusions ERα, ERβ isoforms and neuronal NOS are present in the clitoris and prepucial glands of female mice in different cellular localizations and with differing levels of receptivity. Functional studies would further elucidate the role of receptor functions and their relationship to neuronal expression of nitric oxide.

Estrogen receptor beta (ERβ) mRNA and protein in serotonin neurons of macaques

This study used double in situ hybridization (ISH) to examine the colocalization of estrogen receptor beta (ERβ) mRNA in serotonin neurons of rhesus macaques (Macaca mulatta). In addition, immunocytochemistry (ICC) was used to examine the expression and regulation of ERβ protein in raphe neurons of the macaque midbrain. For double ISH, monkey specific riboprobes for ERβ incorporating radiolabeled-UTP and a riboprobe for the human serotonin reuptake transporter (SERT) incorporating digoxigenin were applied to midbrain sections from spayed rhesus macaques. ERβ mRNA hybridization signal was expressed in most cells containing SERT mRNA in the dorsal and median raphe and pons. There were also non-SERT neurons expressing ERβ mRNA. In addition, ERβ protein was detected with an affinity purified polyclonal antibody generated against a synthetic peptide corresponding to the D domain of human ERβ conjugated to bovine serum albumin (provided by Dr. Philippa Saunders, MRC, Edinburgh). Midbrain sections containing the dorsal raphe from spayed rhesus macaques with and without hormone replacement therapy were processed for ERβ immunostaining. ERβ protein was detected at a similar intensity and in a similar number of cells in the dorsal raphe neurons in all treatment groups. Thus, the expression of ERβ protein in the dorsal raphe was consistent with the expression of ERβ mRNA. In conclusion, ERβ mRNA is expressed by serotonin neurons and it is translated to protein. ERβ protein, like ERβ mRNA, is detected at similar levels in the presence or absence of ovarian hormones.

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

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), forskolin alone increased activity. Collectively, these results show that ERβ protein is preferentially expressed in immature granulosa cells, is functionally active (binds DNA), can transactivate (either as a homodimer or heterodimer with ERα) ERE-containing promoter constructs, and might be associated with increased expression of the endogenous gene encoding c-Jun.