Immunostaining Of ERα Research Articles

Young coconut juice can accelerate the healing process of cutaneous wounds

BackgroundEstrogen has been reported to accelerate cutaneous wound healing. This research studies the effect of young coconut juice (YCJ), presumably containing estrogen-like substances, on cutaneous wound healing in ovairectomized rats.MethodsFour groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. Two equidistant 1-cm full-thickness skin incisional wounds were made two weeks after ovariectomy. The rats were sacrificed at the end of the third and the fourth week of the study, and their serum estradiol (E2) level was measured by chemiluminescent immunoassay. The skin was excised and examined in histological sections stained with H&E, and immunostained using anti-estrogen receptor (ER-α an ER-β) antibodies.ResultsWound healing was accelerated in ovx rats receiving YCJ, as compared to controls. This was associated with significantly higher density of immunostaining for ER-α an ER-β in keratinocytes, fibroblasts, white blood cells, fat cells, sebaceous gland, skeletal muscles, and hair shafts and follicles. This was also associated with thicker epidermis and dermis, but with thinner hypodermis. In addition, the number and size of immunoreactive hair follicles for both ER-α and ER-β were the highest in the ovx+YCJ group, as compared to the ovx+EB group.ConclusionsThis study demonstrates that YCJ has estrogen-like characteristics, which in turn seem to have beneficial effects on cutaneous wound healing.

Effects of long-term tibolone treatment on nuclear sex steroid hormone receptors and G-protein–coupled estrogen receptor-1 expression in the macaque uterus

Tibolone is an alternative hormone treatment for managing climacteric symptoms in postmenopausal women. The aim of this study was to determine the effects of long-term tibolone (TIB) treatment in comparison with conventional hormone therapy on the expression and distribution of estrogen receptor (ER) α, ER-β, G-protein-coupled ER-1 (GPER), progesterone receptor (PR) A, PRB, androgen receptor (AR), and syndecan-1 in the macaque uterus. Eighty-eight cynomolgus macaques (Macaca fascicularis) were ovariectomized and treated orally with TIB, a combination of conjugated equine estrogens (CEE) and medroxyprogesterone acetate (MPA), CEE alone, or vehicle for 2 years. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in the monkey uterus. In the TIB group, immunostaining of ER-α and GPER was higher in the luminal and glandular epithelium, respectively, as compared with the CEE + MPA group. PRA and PRB protein expression was increased in the stroma and myometrium of the TIB group as compared with the controls. AR immunoreactivity was also up-regulated by TIB treatment in the stroma as compared with no treatment. Furthermore, epithelial immunoreactivity of AR was higher after TIB treatment as compared with CEE + MPA treatment. TIB treatment influences the protein expression of sex hormone receptors in monkey endometrium differently from that observed after conventional hormone therapy. We suggest that the observed differences in AR expression by TIB as compared with combined treatment may be of importance for endometrial atrophy as well as for the beneficial bleeding profile associated with this treatment.

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.

Steroidogenic Pathway Proteins Change with Estrous Cycle and Photoperiod, but Are Not Affected by Inhibition of Matrix Metalloproteinases in Siberian Hamster Ovaries.

Restoration of ovarian function as a result of photostimulation corresponds with differential regulation of a family of proteases called matrix metalloproteinases (MMPs) in Siberian hamsters. Blocking MMP action with the in vivo inhibitor GM6001 impedes recrudescence: unlike vehicle treated controls, photostimulation of regressed animals treated with GM6001 fails to increase antral follicle numbers, ovulation, and plasma estradiol concentrations. Because normal recrudescence requires a return of estradiol synthesis and because inhibiting MMPs prevented the normal increase in plasma estradiol, we hypothesized that MMPs may be important in restoring steroidogenesis during photostimulated recrudescence. To test this hypothesis, we examined potential changes in steroidogenic enzymes 3β hydroxysteoid dehydrogenase (3βHSD), 17β hydroxysteoid dehydrogenase (17βHSD), aromatase, as well as estrogen receptor α in: stages of normal cycling hamsters (proestrus-P, estrus-E, diestrus I-DI, diestrus II-DII), hamsters exposed to long-days (LD; 16L:8D), regressed hamsters exposed to short days (SD; 8L:16D) for 14 wks, and regressed hamsters transferred to LD for 2 wks to stimulate recrudescence (PT). Hamsters in LD and PT groups were administered vehicle, IP GM6001, or no treatment daily for 2 wks. Immunoreactivity for 3β HSD protein was observed throughout the estrous cycle, with declines in extent, but not intensity of staining occurring at DII as compared to P, E, and DI (P < 0.05). No changes in 3βHSD immunostaining were noted in LD, SD, or PT ovaries, regardless of treatment with vehicle or GM6001 (P > 0.05). Both extent and intensity of 17β HSD immunostaining declined in DII as compared to other estrous cycle stages (P < 0.05). No changes in 17βHSD immunostaining were noted among LD ovaries, regardless of treatment; however, SD photoperiod decreased extent of immunoreactivity as compared to all groups regardless of treatment (P < 0.05). Aromatase immunostaining was noted throughout the estrous cycle, with declines in intensity and extent in DII as compared to other stages (P < 0.05). Aromatase was present in LD ovaries, and administration of vehicle or GM6001 did not alter intensity/extent of immunostaining (P > 0.05). In contrast, extent of aromatase immunoreactivity declined with SD exposure as compared to all other photoperiod groups (P < 0.05), although administration of vehicle or GM6001 did not affect a return of aromatase immunostaining in PT ovaries (P > 0.05). Finally, extent of immunostaining for ERα peaked in P as compared to DII (P < 0.05). Immunoreactivity for ER was observed in LD at low levels, regardless of treatment. In contrast to steroidogenic enzymes, intensity and extent of ER immunostaining peaked in SD, although administration of vehicle or GM6001 did not alter immunodetection. Our findings confirm and extend the cyclic nature of steroidogenic proteins, with most peaking in the active stages of the estrous cycle. Additionally, the significant decline in plasma estradiol that occurs with SD exposure is concomitant with decreases in later enzymes (17βHSD, aromatase) of steroidogenesis. Finally, although MMPs influence photostimulated return of ovarian function, they do not directly affect the return of ovarian steroidogenesis, as inhibiting MMP action did not affect these steroidogenic enzymes in either cycling LD females, or females undergoing recrudescence. Research supported by NIH 1SC3GM089611 (K.A.Y.) and CSUPERB-Howell Award (J.J.S.). (poster)

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%)

Sex steroid and thyroid hormone receptor expressions in the thyroid of the American alligator (Alligator mississippiensis) during different life stages

The expression of estrogen receptors, ESR1 (ERα) and ESR2 (ERβ), and androgen receptors (AR) in the thyroid gland has been reported in few vertebrate species other than a few mammals. This study reports the presence of sex steroid hormone receptors and thyroid receptors (ERα, ERβ, AR, TRα, and TRβ) in the thyroid gland of the American alligator at several life stages. It provides a semiquantification and distribution of ERα in the thyroid follicle cells using an immunohistochemical approach as well as reports quantitative differences in mRNA expression of ERα, ERβ, TRα, TRβ, and AR in the same tissue using quantitative real time-PCR (Q-PCR) with primers designed specifically for alligators. The thyroid tissue of the American alligator expresses ERα, ERβ, and AR at all of the life stages examined here although no statistically significant differences were observed between male and female in thyroid mRNA expression for any of the genes analyzed. No sexual dimorphism was observed in ERα immunostaining. No statistical analysis across life stages were performed due to confounding factor of season.

Differential expression of estrogen receptor alpha gene in the ampullae and isthmus regions of the rabbit oviduct during early pregnancy

We characterized the expression pattern of estrogen receptor α (ERα) gene in two regions of the oviduct, ampullae and isthmus, of the rabbit ( Oryctolagus cuniculus) during early pregnancy (1–4 days) by RT-PCR and immunohistochemistry. In both regions of the oviduct, ERα mRNA was increased ( P < 0.01) in pregnant rabbits as compared with non-pregnant animals (NG). In the ampullae, the greatest amount of ERα mRNA was detected on the third day of pregnancy (1.01 ± 0.02 relative amount), followed by a decrease on Day 4. In the isthmus, an increase in ERα mRNA was observed during the first 4 days of pregnancy, with the greatest amount on Day 3 (1.49 ± 0.3 relative amount). A marked ERα immunostaining was detected in epithelial, stromal and smooth muscle cells of the ampullae on the second and third days of pregnancy as compared with the NG group. In contrast, a significant increase in ERα immunostaining was observed on the first and second days of pregnancy with a reduction on the third and fourth days in the epithelial, stromal and smooth muscle cells of the isthmus. The overall results suggest there is differential expression pattern for the ERα gene in the ampullae and isthmus during early pregnancy of the rabbit, and that these variations are related to specific functions of ERα in the reproductive tract during early pregnancy.

Effect of Estradiol Supplementation on Induced Diabetic Changes in the Vagina of Albino Rat: An Experimental Immunohistochemical Study

Background: Estrogen hormone and receptors (ER) play an important role in maintaining vaginal health. Therefore, their disruption may adversely affect vaginal structure and function. Limited studies are available investigating the effects of diabetic complications on ER expression and distribution in the vaginal wall. Aim of the Work: This work aimed to study the effects of diabetes-induced changes on the vaginal structure, the expression of ERα as well as to determine whether the supplementation of estradiol can ameliorate these changes or not. Material and Methods: Thirty female albino rats were divided into 3 equal groups, 10 rats each. The first was the control received the vehicle only, the second was the diabetics, received a single intraperitoneal injection of alloxan (150mg/kg), and the third was diabetic/estradiol treated. Eight week-diabetic animals were injected subcutaneously with estradiol 20μg/kg/day dissolved in peanut oil for 8 weeks. By the age of 16 weeks, the animals were sacrificed, blood samples were collected to estimate the serum estradiol level and the vagina was removed and processed for paraffin sections at 5μm thick. For routine histopathological assessment H and E was used. Masson’s trichrome used for collagen fibers and estrogen immunoperoxidase stains for ERα. Results: Diabetic rats showed highly significant decline in the serum estradiol level (19.6 ± 8.4 pcg/ml) compared to the controls (126.6 ± 7.6 pcg/ml). Histopathological examination revealed thinning of the vaginal epithelial layers, increase in the collagen deposition in the submucosa, marked atrophy in the muscularis layer and decrease in ERα immunostaining. Treatment of diabetic animals with estradiol for eight weeks led to its increase to a sub-physiological level (35.1 ± 5.7pcg/ml) and marked hypertrophy of the muscularis layer and re-stratification of the vaginal epithelium. Moreover, there was marked reduction in the nuclear and cytoplasmic ERα immunostaining in the epithelium and increase in its expression in the stroma of the lamina propria and in the muscularis layer as compared to the control group. Conclusion: Diabetes-induced structural changes in the vagina may be a consequence of decreased levels of estrogen. The increase in the estradiol level, even at a sub-physiologic level, can ameliorate these atrophic effects.

Endometrial Population of Oestrogen Receptors Alpha and Beta and Progesterone Receptors A and B During the Different Phases of the Follicular Wave of Llamas (Lama glama)

The aim of this study was to characterize the distribution of oestrogen receptor (ER)α and ERβ as well as both progesterone receptors isoforms progesterone receptor (PR) A and PRB in the luminal and glandular epithelia and stroma of the endometrium during the different phases of the follicular wave in llamas. Six llamas were examined by transrectal ultrasonography, and a transcervical biopsy was obtained when a follicle at the growing, plateau and regressing phase was recorded. Blood samples were collected at the time of biopsy for hormone determinations. An immunohistochemical technique was used to study receptor populations. Total positive area was evaluated in the different cell types by Image Analysis. Mean diameter measurements of the largest follicle were 6.9, 8.5 and 5.1 mm (p < 0.001) and mean plasma oestradiol-17β concentrations were 27.9 ± 3.26; 30.0 ± 2.79 and 24.0 ± 1.78 pmol/l (p = 0.32) during the growing, plateau and regressing phases, respectively. Immunostaining of ERα was higher in the luminal epithelium during the plateau and regressing phases (p < 0.05) than during the growing phase. More positive cells to ERβ were observed in the glandular epithelium of the growing and plateau phases (p < 0.05) than during the regressing phase. A higher percentage of cells positive to PRB was recorded in the luminal and glandular epithelia during the plateau phase (p < 0.05), while the PRA immunostaining was similar among phases. In brief, this study showed an increased population of ERα and PRB in the luminal epithelium, and only of PRB in the glandular epithelium at the time when an ovulatory follicle is present. The physiological importance of these changes in llamas remains to be elucidated.

Viral Vector–mediated Delivery of Estrogen Receptor-α to the Hippocampus Improves Spatial Learning in Estrogen Receptor-α Knockout Mice

Estrogen, which influences both classical genomic and rapid membrane-associated signaling cascades, has been implicated in the regulation of hippocampal function, including spatial learning. Gene mutation studies suggest that estrogen effects are mediated by estrogen receptor-α (ER-α); however, because gonadal steroids influence the organization of the hippocampus during development, it has been difficult to distinguish developmental effects from those specific to adults. In this study we show that lentiviral delivery of the gene encoding ER-α to the hippocampus of adult ER-α-knockout (ER-αKO) mice restores hippocampal responsiveness to estrogen and rescues spatial learning. We propose that constitutive estrogen receptor activity is important for maintaining hippocampus-dependent memory function in adults.

Estrogenic activity of environmental polycyclic aromatic hydrocarbons in uterus of immature Wistar rats

Polycyclic aromatic hydrocarbons (PAHs) are an important group of environmental pollutants, known for their mutagenic and carcinogenic activities. Many PAHs are aryl hydrocarbon receptor (AhR) ligands and several recent studies have suggested that PAHs or their metabolites may activate estrogen receptors (ER). The present study investigated possible estrogenic/antiestrogenic effects of abundant environmental contaminants benzo[ a]pyrene (BaP), benz[ a]anthracene (BaA), fluoranthene (Fla) and benzo[ k]fluoranthene (BkF) in vivo, using the immature rat uterotrophic assay. The present results suggest that BaA, BaP and Fla behaved as estrogen-like compounds in immature Wistar rats, when applied for 3 consecutive days at 10 mg/kg/day, as documented by a significant increase of uterine weight and hypertrophy of luminal epithelium. These effects were likely to be mediated by ERα, a major subtype of ER present in uterus, as they were inhibited by treatment with ER antagonist ICI 182,780. BaA, the most potent of studied PAHs, induced a significant estrogenic effect within a concentration range 0.1–50 mg/kg/day; however, it did not reach the maximum level induced by reference estrogens. The proposed antiestrogenicity of the potent AhR agonist BkF was not confirmed in the present in vivo study; the exposure to BkF did not significantly affect the uterine weight, although a weak suppression of ERα immunostaining was observed in luminal and glandular epithelium, possibly related to its AhR-mediated activity. The PAHs under study did not induce marked genotoxic damage in uterine tissues, as documented by the lack of Ser-15-phoshorylated p53 protein staining. With the exception of Fla, all three remaining compounds increased CYP1-dependent monooxygenation activities in liver at the doses used, suggesting that the potential tissue-specific antiestrogenic effects of PAHs mediated by metabolization of 17β-estradiol also cannot be excluded. Taken together, these environmentally relevant PAHs induced estrogenic effects in vivo, which might affect their toxic impact and carcinogenicity.

Detection of aromatase and estrogen receptors (ERα, ERβ1, ERβ2) in human Leydig cell tumor

A Leydig cell tumor is a rare neoplasm, deriving from the interstitial cells, whose pathogenesis has not been still defined. Leydig cells of normal adult testis are known as physiological targets for estrogens. However, some studies on transgenic rodents suggest a role of estrogens in the development of Leydig cell hyperplasia and Leydig cell tumor. Therefore, with the aim to evaluate a possible link between estrogens and testicular tumorigenesis, this study investigated the expression of aromatase and estrogen receptors (ERalpha, ERbeta(1), ERbeta(2)) in testes from two patients with Leydig cell tumor. A strong immunoreactivity for aromatase, ERbeta(1), and ERbeta(2), together with a detectable ERalpha immunostaining, was revealed in tumoral tissues. These findings were confirmed by western blot analysis of tumor extracts detecting a 55 kDa P450arom, a 67 kDa ERalpha band, a 59 kDa ERbeta(1) band, and a 53 kDa ERbeta(2) band. The pattern of ER expression in neoplastic cells appears different from that of control Leydig cells exhibiting only ERbeta(1) and ERbeta(2) isoforms. The authors hypothesize how the high estrogen production could play a role in the neoplastic transformation of Leydig cells, while the exclusive presence of ERalpha in tumoral cells could amplify estradiol-17beta signaling contributing to the tumor cell growth and progression.

Effects of (R,S)/(S,R)-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazolines and (R,S)/(S,R)-2,3-bis(2-chloro-4-hydroxyphenyl)piperazines on estrogen receptor alpha level and transcriptional activity in MCF-7 cells

4,5-Diaryl-2-imidazolines (Ims) and 2,3-diarylpiperazines (Pips) belong to the type II class of estrogens. These compounds enhance ERα-mediated transcription of ERE-driven reporter genes in MCF-7 cells but do not compete with [3H]estradiol (E2) for receptor binding, because of distinct anchoring modes. The present study examined whether the estrogenic action of Ims and Pips is associated with a down regulation of ERα, as reported for conventional agonists. Im and Pip derivatives displaying a large spectrum of activities in three distinct ERE-dependent transactivation systems were selected for that purpose. ERα immunostaining as well as Western blotting analysis revealed that both classes of compounds down regulated ERα with an efficiency closely related to their transactivation potency. MG-132 abrogated this down regulation, pointing to a proteasomal degradation process. Ims and Pips with strong transactivation potency also altered [3H]E2 binding parameters, leading to a progressive decrease of cellular estrogen binding capacity. This property occurred largely before ERα down regulation and persisted even in presence of MG-132, indicating that it did not result from ERα breakdown but rather from a conformational change of the receptor. The additional finding that the most active agonist tested in this study enhanced the capacity of a purified ERα recombinant to recruit LxxLL co-activators, while its inactive counterpart failed to do so confirmed this hypothesis. Altogether, our data indicate that the association of Ims and Pips with ERα elicits similar responses to conventional agonists, even if they interact with distinct residues of the binding pocket.

Expression of hormone receptors ERα, ERβ and PgR in HER2-positive breast cancers

10525 Background: HER-2/neu gene is amplified and overexpressed in 15–20% of invasive breast cancers. HER2-positive breast cancers have a worse prognosis than HER2-negative tumors and distinctive clinical features. They express hormone receptors for estrogen (ERα) and for progesterone (PgR) less frequently than HER2-negative tumors. The identification of the other human estrogen receptor, receptor beta (ERβ), raises a question of ERβ occurrence in HER2-positive breast cancer. Patients and methods: Formalin-fixed, paraffin embedded tissues from 90 patients with invasive HER2-positive breast cancer and from 99 patients with HER2-negative breast cancer were used in this study. The HER2 status was analyzed using HercepTest TM (IHC), and IHC 2+ results were confirmed with FISH test. Immunostaining for ERα, ERβ and PgR was performed using monoclonal antibodies against ERα, PgR (DakoCytomation) and against ERβ (CHEMICON). The EnVision detection system was applied. The data were analyzed using nonparametric Fisher-Freeman-Halton test; the statistical significance was considered when p &lt; 0.5. Results: Only 33% of the HER2-positive breast cancers were ERα-positive compared with 63% in the HER2-negative group (p &lt; 0.001). The expression of ERβ protein was observed in almost equal frequency in both groups (57% of HER2-positive breast cancers and 57.7% of HER2-negative tumors, p = 0.889). The expression of PgR was observed in 30% of HER2-positive breast cancers and in 68.7% of HER2-negative tumors (p &lt; 0.001). Conclusion: The expression of ERβ (unlike that of ERα and PgR) was similar in HER2-positive and in HER2-negative breast cancers. Thus, ERβ may be a potential target in future endocrine therapy for women with HER2-positive breast cancers. No significant financial relationships to disclose.

Immunolocalization of estrogen and androgen receptors and steroid concentrations in the stallion epididymis

The presence of steroids and their receptors throughout development, specifically androgen receptor (AR), estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), in the epididymis of a high estrogen producing species like the stallion has not been determined. Epididymal and testicular samples were collected for analysis of testosterone and estradiol-17β (E2) concentrations and for immunolocalization of AR, ERα and ERβ. The concentration of testosterone in the testis and epididymis were not different among age groups (P>0.05). AR was localized in the principal cells of the caput, corpus and cauda in all four age groups. This lack of change in testosterone concentration and receptor localization suggests that testosterone is important for both development and maintenance of epididymal function. There was an age-related increase in E2 concentrations in all regions of the epididymis (P<0.05), suggesting that E2 is also important for adult function. ERβ was localized in the principal cells of the caput, corpus and cauda in all four age groups, but the localization of ERα was regional and age dependent. In peri-pubertal animals, ERα immunostaining was most prominent and estradiol was similarly present in all three epididymal regions; this suggests that estradiol also plays a key role in the maturation of the stallion epididymis during the pubertal transition when sperm first arrive in the epididymis. In conclusion, these results suggest that the stallion epididymis is regulated by both androgens and estrogens throughout development and that estradiol is more important to epididymal function in the stallion than previously believed.

Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

Expression of estrogen receptors alfa and beta (ERa and ERb) in hereditary breast cancer

550 Background: Breast cancers (BC) in women carrying mutations in BRCA1 gene are more frequently estrogen receptor negative than the nonhereditary BC. However, tamoxifen has been found to have a protective effect in preventing contralateral tumors in BRCA1 mutation carriers. The identification of the second human estrogen receptor, ERβ, raised a question of its role in hereditary breast cancer. Patients and Methods: Formalin-fixed, paraffin embedded breast cancer tissues from 46 patients with mutations in BRCA1 gene were used in this study. A mutation analysis was carried out by a multiplex PCR assay. Only 3 common founder mutations in Poland in the BRCA1: 5382insC, 4153 delA, C61G were tested. Immunostaining for ERα, ERβ and PgR (progesterone receptor) was performed using monoclonal antibodies against ERα, PgR (DakoCytomation), and against ERβ (CHEMICON). The EnVision detection system was applied. The study population comprised a control group of 65 BC patients operated successively in 2002. The data were analyzed using a nonparametric Fisher-Freeman-Halton test; the statistical significance was considered when p<0,05. Results: BRCA1 mutation carriers were more likely to have ERα-negative BC than those in the control group. Only 17% of BRCA1-related cancers were ERα-positive compared with 58% in the control group (p=0,0001). The expression of ERβ protein was observed in 37% of BRCA1-related tumors. The expression of ERβ protein in ERα-negative tumors was twice as frequent in the BRCA1-related group. This was particularly striking at the age ≥ 50 where 33% of BRCA1 mutation bearing patients were exclusively ERβ positive vs. 13% in the control group. The results obtained in a similar analysis for patients below 50 showed 25% and 16%, respectively. The expression of PgR was observed in 17% of BRCA1-related BC and in 68% of tumors in the control group. Conclusions: In BRCA1-associated tumors the expression of ERβ was higher than the expression of ERα. This may, in part, explain the protective effect of tamoxifen in preventing contralateral tumors in BRCA1 mutation carriers. Testing patients with BRCA1 mutation for the presence of both ERα and ERβ may help to identify those who might benefit from endocrine therapy. No significant financial relationships to disclose.

Morphological Studies of Prolactin-Secreting Cells in Estrogen Receptor α and Estrogen Receptor β Knockout Mice

Estrogens play a major role in the regulation of prolactin (PRL) secretion through activation of pituitary and hypothalamic estrogen receptors (ERs). In order to evaluate the relative role of ERα and ERβ in the control of PRL density in the pituitary gland, we performed immunocytochemical localization of PRL and ERs in pituitaries of wild-type (WT), ERα knockout (KO) and ERβKO mice. In WT and ERβKO anterior pituitaries, the vast majority of secretory cells contained ERα immunoreactivity, while no ERα immunostaining could be found in ERαKO pituitaries. No ERβ immunoreactivity could be detected in pituitaries of WT, ERαKO or ERβKO mice. At the light microscopic level, a large number of cells staining for PRL were present in pituitaries of female WT, while in female ERαKO pituitaries, the density of PRL cells was much lower. In WT male pituitaries, the density of PRL cells was lower than observed in female WT, while PRL staining was markedly decreased in male ERαKO as compared to male WT. In ERβKO mice of both sexes, the results were identical to those observed in WT animals. At the electron microscopic level, in WT mice of both sexes, type 1 PRL cells exhibited a well-developed Golgi apparatus and a large number of strongly stained large mature and immature secretory granules. Type 2 PRL cells were also present in the pituitary. Type 2 PRL cells contain small poorly labelled granules. In ERαKO mice of both sexes, type 1 PRL cells were atrophied with poorly developed Golgi apparatus, and no type 2 PRL cells could be observed. In ERαKO pituitaries, typical gonadectomy cells were found. No ultrastructural changes were observed in PRL cells of ERβKO mice. The present data strongly suggest that the positive regulation of PRL expression at the pituitary level by estrogens is mediated by ERα and does not involve ERβ activation.

Mammary gland development in adult mice requires epithelial and stromal estrogen receptor alpha.

Complete mammary gland development takes place following puberty and depends on the estrogen receptor (ER)alpha and the progesterone receptor (PR) and is tightly regulated by the interaction of the mammary epithelium with the stromal compartment. Studies using mammary tissues of immature mice have indicated that stromal but not epithelial ER alpha is required for mammary gland growth. This study investigates whether these same tissue growth requirements of neonate tissue are necessary for mammary development and response in adult mice. Mammary epithelial cells were isolated from adult mice with a targeted disruption of the ER alpha gene (alpha ERKO) or from wild-type counterparts and injected into epithelial-free mammary fat pads of 3-wk-old female alpha ERKO or wild-type mice. Ten weeks after cell injection, analysis of mammary gland whole mounts showed that both stromal and epithelial ER alpha were required for complete mammary gland development in adult mice. However, when the mice were treated with high doses of estradiol (E2) and progesterone, stromal ER alpha was sufficient to generate full mammary gland growth. Surprisingly, ER alpha-deficient epithelial cells were able to proliferate and develop into a rudimentary mammary ductal structure in an ER alpha-negative stroma, indicating that neither stromal nor epithelial ER alpha are required for the mammary rudiment to form in the adult mouse, as confirmed by the phenotype of the alpha ERKO mammary gland. Use of this in vivo model system has demonstrated that neonatal and adult mammary tissues use a different tissue-specific role for ER alpha in mammary response. Immunostaining for ER alpha and PR in the mammary outgrowths supported the view that both stromal and epithelial ER alpha, in cooperation with epithelial PR, govern mammary gland development in adult mice.