Effect of pubertal development on estrogen receptor levels and stromal morphology in the guinea pig prostate

Using an immunocytochemical assay (ERICA) with a monoclonal antibody (H222Sp gamma) to the human estrogen receptor, we have demonstrated a stromal localization of the estrogen receptor in the dorsolateral prostate of the guinea pig. Specific staining of estrogen receptor in the guinea pig prostate was confined to the nuclei of periacinar and interacinar stromal cells. In comparison with prepubertal tissues, estrogen receptor staining intensity was markedly reduced in postpubertal prostatic tissues. No immunoreactive estrogen receptor was detected in the acinar epithelial cells irrespective of the developmental stage of the guinea pig prostate. Electron microscopic examination of the guinea pig prostate showed that the stromal component consists predominantly of smooth muscle cells, which, during pubertal development, undergo marked cytological changes and increase in size. These changes in the prostatic stroma were associated with a greater than fivefold reduction in levels of cytosolic and nuclear estrogen receptor determined by either a radioligand binding assay or an enzyme immunoassay (EREIA) and expressed relative to soluble protein. Morphometric analysis of the prostatic stromal cell density (SCD: nuclei/mm2 interacinar stroma), which is inversely proportional to stromal cell size, indicated that the SCD decreased approximately threefold during pubertal development. Furthermore, cytosolic estrogen receptor levels in mechanically separated prostatic stromal fractions were found to vary concordantly with the SCD during pubertal development. To determine whether estrogen influences normal development of the guinea pig prostate, the effect of various hormonal manipulations on stromal development was examined. Castration of prepubertal animals prevented the threefold decrease in SCD that is characteristic of pubertal development. Treatment of prepubertal castrates with estradiol and 5 alpha-dihydrotestosterone (DHT) in combination over a period equivalent to the transpubertal growth phase resulted in a stromal cell density similar to that seen in prostatic sections from intact postpubertal animals. In contrast, treatment of prepubertal castrates with either estradiol or DHT alone resulted in a prostatic stromal cell density intermediate between that observed in intact prepubertal and postpubertal animals. These findings suggest that both estrogen and androgen are required for the normal development of the guinea pig prostatic stroma.     

Expression of estrogen receptor-alpha and -beta in anterior vaginal walls of genuine stress incontinent women.

Our objective was to study the expression of estrogen receptor (ER) isoforms, ER-alpha and ER-beta, in the anterior vaginal wall of menopausal and fertile women with genuine stress incontinence (SI) by immunohistochemistry and Western blot analysis. Eighteen menopausal women with SI who either were or were not taking estrogen/progestin replacement therapy and 14 fertile women with SI who either were or were not taking contraceptives were enrolled in the study. Biopsies from the suburethral anterior vaginal wall were obtained at tension-free vaginal tape (TVT) operation. Monoclonal antibody to ER-alpha and polyclonal antibody to ER-beta were used to stain frozen sections of vaginal tissue. The receptor expressions were scored based on percentage of positive cells. ER-alpha was detected in vaginal epithelial, stromal and smooth muscle cells. In menopausal SI women ER-alpha was detected significantly more frequently in the vaginal walls of estrogen/progestin-treated patients than in those who were untreated. Fertile SI women had significantly higher expression of ER-alpha than menopausal SI women. ER-alpha was not observed in vaginal blood vessels. ER-beta was detected in epithelial and vascular smooth muscle cells of the vagina. No significant difference in ER-beta expression was observed between different groups of patients. The expression of ER-alpha was not correlated with that of ER-beta. Both ER-alpha and -beta were detected, indicating a potential role for both types of estrogen receptor in the human vaginal wall. The expression of ER-alpha, but not of ER-beta, in menopausal SI women was regulated by estrogen/progestin replacement therapy. The presence of ER-beta in vaginal vascular smooth muscle cells raises the possibility of vascular effects of estrogen on the human vaginal wall.     

Expression of estrogen receptor alpha and beta mRNAs in prostate cancers treated with leuprorelin acetate

OBJECTIVE: The discovery of a novel estrogen receptor (ER), ER-beta, has given rise to new possibilities regarding estrogen's roles in the prostate. Although ER-beta is reported to be expressed preferentially in the rat prostate, its expression in the human prostate and relationship to cancer development has not been investigated. Thus the purpose of the study was to examine mRNA levels of ER-alpha and ER-beta in benign prostatic hyperplasia and prostate carcinoma. METHODS: Samples of 15 prostate cancers obtained at radical prostatectomy were examined. All the patients had been maintained on androgen withdrawal therapy for at least 3 months. ER-alpha and ER-beta mRNAs were measured with a competitive PCR technique. RESULTS: Both ER-alpha and ER-beta mRNAs were detected in all of the prostate cancer tissues examined, as well as in PC3 and LNCap cells, although the levels varied among specimens. Interestingly, both types were significantly decreased in cases with lymph node metastasis. However, there was no correlation between ER mRNA levels and any other clinicopathological parameters. CONCLUSIONS: (1) Both ER-alpha and ER-beta mRNAs are expressed in prostate cancer and (2) expression of ER mRNA may not be related to cancer progression but may be negatively correlated with metastasis.     

Immunolocalization of estrogen receptor alpha and beta in human fetal prostate

PURPOSE: We examined the immunolocalization of estrogen receptor (ER)alpha and ERbeta in the human fetal prostate. MATERIALS AND METHODS: Tissue sections from human fetal prostates at 7 to 22 weeks of gestation were stained with antibodies to ERalpha, ERbeta, and cytokeratin 10 and 14. RESULTS: ERalpha expression was not detected until 15 weeks of gestation with sparse staining in the utricle. By 19 weeks increased ERalpha expression was seen in the luminal cells of the ventral urogenital epithelium (UGE), basal cells of the dorsal UGE, utricle, distal periurethral ducts, peripheral stroma and posterior prostatic duct. K14 was detected in basal cells of the UGE and in several posterior acini. At 22 weeks ERalpha expression was more intense in all of these areas. ERbeta was expressed throughout the UGE, ejaculatory ducts, müllerian ducts and entire stroma at 7 weeks. Intense ERbeta staining was observed in these areas and in the prostatic buds by 8 weeks with persistent intense staining through 22 weeks. CONCLUSIONS: To our knowledge we report the first immunolocalization of ERalpha in the human fetal prostate and the earliest demonstration of ERbeta expression in the prostate at 7 weeks of gestation. ERbeta expression is intense during ductal morphogenesis, suggesting a role in normal glandular growth and proliferation. The induction of squamous metaplasia in the UGE, distal periurethral ducts and utricle is associated with ERalpha expression in these areas, while the induction of squamous metaplasia in peripheral prostatic acini is associated with peripheral stromal ERalpha expression. This study suggests estrogen signaling pathways in the human fetal prostate via ERalpha that involve epithelial-epithelial and epithelial-stromal interactions.     

Semiquantitative morphology of human prostatic development and regional distribution of prostatic neuroendocrine cells

BACKGROUND: The neuroendocrine cells of the human prostate have been related to proliferative disorders such as prostatic cancer. Their origin, distribution, and development have therefore been studied and discussed in terms of current stem cell concepts in the prostate. METHODS: Prostatic tissue specimens (n = 20) from human fetuses (n = 8), prepubertal and pubertal children (n = 8) and mature men (n = 4) were studied immunohistochemically using antibodies directed against neuroendocrine, epithelial as well as secretory markers. Semiquantitative computer-assisted evaluation of different epithelial and stromal components based on stereological principles was performed on azan-stained sections representative of all developmental stages. RESULTS: By the end of gestational Week 9, neuroendocrine (NE) cells appear in the epithelium of the urogenital sinus and are subsequently closely associated with the formation of urethral prostatic buds. The fetal and postnatal distribution pattern of NE cells within the gland is characterized by a relatively constant number of cells per gland similar to prostatic smooth muscle cells. Likewise, a density gradient exists with the highest density in the large collicular ducts and almost no NE cells in subcapsular peripheral acini. In peripheral ducts, the distribution is random. Maturation of the NE cells precedes that of the secretory cells by about 10-16 years. CONCLUSIONS: A second prostatic stem cell lineage, different from the urogenital sinus (UGS)-lineage is hypothesized originating from immature neuroendocrine cells. Being morphologically indistinguishable from the UGS-derived prostatic secretory cell lineage, it gives rise to neuroendocrine cells. Their presence is apparently important for proliferation regulation of the UGS-derived lineage of the prostate.     

Involvement of estrogen receptors in prostatic diseases

Accumulating evidence shows that estrogens participate in the pathogenesis and development of benign prostatic hyperplasia and prostate cancer by activating estrogen receptor α. In contrast, estrogen receptor β is involved in the differentiation and maturation of prostatic epithelial cells, and thus possesses antitumor effects in prostate cancer. However, the natural ligands of estrogen receptor β are not fully understood, and its mode of action according to its ligands and the binding sites located in the promoter regions of downstream genes remains to be elucidated. Here, we review recent experimental investigations of estrogen receptors and their urological relevance. Estrogen receptor‐mediated signaling in the prostate is essential together with the androgen receptor‐mediated pathway, providing a new therapeutic target for prostatic diseases.     

Expression of estrogen receptor (ER-alpha and ER-beta) mRNA in human prostate cancer.

OBJECTIVE: The distribution of the two estrogen receptors (ER-alpha, ER-beta) in human prostate tissue have not been fully clarified, so the present study investigated the mRNA expression of the receptors to explain the broad spectrum of estrogen activity in prostate cancer. MATERIALS AND METHODS: Four human prostate cancer cell lines (LNCap, JCA-1, DU-145 and PC-3) and 24 pairs of untreated prostate cancer tissue and noncancerous tissue from resected prostate glands were subjected to RT-PCR testing. RESULTS: Both LNCap and JCA-1 expressed the mRNA of both receptors, but DU-145 and PC-3 only expressed ER-beta mRNA. In the human prostate tissue samples, 20 of the 24 prostate cancer tissues expressed ER-alpha, and 23 of the 24 expressed ER-beta. Of the 24 noncancer tissues, 14 expressed ER-alpha mRNA and 17 expressed ER-beta mRNA. The incidence of ER-beta mRNA expression between the paired cancer and noncancer tissues was statistically significantly different (p<0.05). CONCLUSIONS: A higher incidence of ER-beta mRNA expression in untreated prostate cancer tissues was observed. Furthermore, the absence of ER-alpha mRNA and the presence of ER-beta mRNA expression in hormone-independent and/or untreated prostate cancer cells leads to a tentative speculation of the mechanism of the hormone refractory feature of prostate cancer.     

Differential expression of estrogen receptor-related receptor alpha and estrogen receptors alpha and beta in osteoblasts in vivo and in vitro.

The orphan nuclear estrogen receptor-related receptor (ERR) alpha is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-alpha in bone formation, we compared its expression to that of the estrogen receptor (ER) alpha and ER-beta in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-alpha is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-alpha was localized similarly but at lower levels and ER-beta, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-alpha appeared to be more highly expressed than either of the ERs. Interestingly, ERR-a also is highly expressed in RBM cells, while ER-alpha and ER-beta mRNA is expressed, but at lower levels. Moreover, we found that ERR-alpha, ER-alpha, and ER-beta were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-alpha may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions.     

Inhibition of SULT2B1b expression alters effects of 3beta-hydroxysteroids on cell proliferation and steroid hormone receptor expression in human LNCaP prostate cancer cells

BACKGROUND: Sulfation is an important steroid inactivation in human tissues. Sulfotransferase (SULT) 2B1b selectively conjugates 3beta-hydroxysteroids and is expressed in epithelial cells of normal and cancerous prostate tissues. Dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (Delta(5)-Adiol) sulfation prevents their conversion to more potent androgens and estrogens in tissues although both compounds may also be biologically active. METHODS: SULT2B1b expression and activity were inhibited >85% in human LNCaP prostate adenocarcinoma cells using short interference RNA (siRNA). The effects of treating control and SULT2B1b-deficient LNCaP cells with DHEA, Delta(5)-Adiol, and 5alpha-androstane-3beta-17beta-diol (Anstane-diol) on cellular proliferation, estrogen receptors (ERs), androgen receptor (AR), and prostate specific antigen protein levels were examined. RESULTS: Physiological concentrations of DHEA and Delta(5)-Adiol increased proliferation of control cells and the proliferative effects were significantly increased in SULT2B1b-siRNA cells. DHEA, but not Delta(5)-Adiol increased AR levels at concentrations >/=1,000 nM in SULT2B1b-siRNA cells but not in control LNCaP cells. ER-alpha levels were not affected with any of the compounds tested. Physiological concentrations of DHEA and Delta(5)-A-diol decreased ER-beta levels in control cells and had significantly greater effects in SULT2B1b-siRNA cells. In contrast, Anstane-diol had no effect on AR or ER-alpha levels but induced more elevation of ER-beta levels in SULT2B1b-siRNA cells at concentrations >/=1,000 nM. CONCLUSIONS: SULT2B1b is involved in regulating prostate cell responsiveness to DHEA and Delta(5)-Adiol. Inhibition of SULT2B1b increased cell proliferation and ER-beta repression after treatment with physiological levels of DHEA and Delta(5)-Adiol indicating that SULT2B1b has an inhibitory effect on DHEA and Delta(5)-Adiol activity.     

Stromal and epithelial growth of the prostate during puberty

In 30 male Chacma baboons, prepubertal, pubertal, and adult animals have been identified by their testicular and prostatic weights. The volume of the stromal and epithelial components of caudal prostate were calculated from morphometric determination of the percentage volume of each component and the total caudal prostate volume. Both stromal and epithelial volumes increased at puberty and plateaued at maturity. These increases could be fitted to logistic curves that support the conclusion that stromal pubertal growth precedes epithelial pubertal growth. The stromal contribution to prostates of increasing size followed a model distinct to that of the epithelial contribution, thus suggesting that the factors (e.g., androgens) controlling stromal and epithelial growth are not identical. Stromal growth may be a prerequisite for pubertal epithelial development of the prostate.     

Declining estrogen receptor-beta expression defines malignant progression of human breast neoplasia

It has been shown that the risk of breast cancer developing in certain morphologically identifiable benign breast lesions correlates with expression of estrogen receptor alpha (ER-alpha). Although ER-alpha and ER-beta genes share a large degree of homology, it is generally thought that their distribution and functions are substantially different in many tissues. Recent development of reliable antibodies to ER-beta has provided this first opportunity to test the hypothesis that the likelihood of malignant transformation in morphologically benign breast lesions can be accurately defined by the distribution and level of ER-beta expression relative to that of ER-alpha. Using a monoclonal antibody, ER-beta protein expression has been analyzed in 53 normal breasts and compared with a cohort of histologically distinct breast lesions of different prognostic risk (54 hyperplasia of usual type, 35 ductal carcinoma in situ, and 141 invasive cancers). All of these tissues were also assessed for ER-alpha. Expression of ER-beta protein was also analyzed in an additional spectrum of benign breast lesions with low or negligible risk of progression to malignancy. The median proportion of cells expressing ER-beta was highest in normal breast lobules (median 94.33%, interquartile range 78.25-99.00) but declined significantly through usual ductal hyperplasia (median 76.67, interquartile range 49.17-95.00, P = 0.002) and ductal carcinoma in situ (median 70.00, interquartile range 59.00-85.00, P = 0.009) to invasive cancer (median 60.00, interquartile range 50.00-80.00, P < 0.001). An appreciable proportion (33.81%) of ER-alpha-negative invasive cancers expressed ER-beta. A high but variable level of ER-beta expression occurred in the benign lesions. The data from the intact histologic tissues were evaluated with respect to the relative expression of ER-alpha and ER-beta in five mammary cell lines of different behavioral phenotype (MCF7, ZR-75, T47D, MDAMB231, HUMA121). The highly significant differences in expression and distinct tissue distributions of ER-alpha and ER-beta within the histologic lesions of defined risk, together with the data from the cell lines, support the original hypothesis that the tissue concentration, relative occurrence, and/or interaction of these two types of estrogen receptor may play an important role in modulating mammary tumorigenesis.     

Effect of the addition of estrogen to medical castration on prostatic size, symptoms, histology and serum prostate specific antigen in 4 men with benign prostatic hypertrophy.

A total of 4 men with benign prostatic hypertrophy who underwent medical castration therapy with a long-acting gonadotropin-releasing hormone agonist (leuprolide) for more than 6 months elected to add an estrogen transdermal patch (0.05 mg. to the skin biweekly) to the leuprolide regimen. The average prostatic size (transrectal ultrasound), serum prostate specific antigen (PSA) levels and symptoms of prostatism were dramatically decreased with leuprolide alone. The addition of estrogen for 6 months did not result in any change in prostate size, symptoms or serum PSA levels over that seen with leuprolide alone. The development of squamous metaplasia was noted in 1 man with leuprolide alone and in 1 man after the addition of estrogen. Immunohistochemical staining with anticytokeratin 903 antibodies reveals that squamous metaplasia appears to arise from prostatic basal cells. We postulate that the target cell for estrogen action in the prostate is the prostatic basal cell. In the absence of androgen the only direct effect of estrogens is the induction of squamous metaplasia.     

Two different pathways for the maintenance of trabecular bone in adult male mice.

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER-alpha and ER-beta. The aim of this study was to investigate the relative roles of ER-alpha, ER-beta, and AR in the maintenance of trabecular bone in male mice. Seven-month-old male mice, lacking ER-alpha (ERKO), ER-beta (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 microg/mouse per day of 17beta-estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER-a nor ER-beta is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild-type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen-induced activation of ER-alpha. These results indicate that AR and ER-alpha are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER-beta is of no importance for the regulation of trabecular bone in male mice.     

Antagonism of estrogen-mediated cell proliferation by raloxifene in prevention of ageing-related prostatic hyperplasia

Estrogen has important roles in the initiation and development of benign prostatic hyperplasia （BPH）. Regulators of the estrogen receptor （ER） are tissue- and cell-specific. We evaluated the effect of estrogen antagonist, raloxifene （Ral）, on the prevention and treatment of BPH by investigating its effect on the proliferation of two different prostate cell lines： a stromal cell line, WPMY-1, and a benign prostatic hyperplasia epithelial cell line, BPH-1. We additionally evaluated its effect on prostatic hyperplasia induced by estrogen and androgen in a rat model. The effect of Ral on the prevention of prostatic hyperplasia was analyzed by haematoxylin and eosin staining and quantitative immunohistochemistry （IHC） for proliferating cell nuclear antigen and α-smooth muscle actin. In vitro and in vivo, tamoxifen （Tam）, another anti-estrogen drug, and finasteride （Fin）, a drug for the clinical treatment of BPH, served as efficacy controls. The in vitro data showed that neither Ral nor Tam alone affected the proliferation of WPMY-1 and BPH-1, but both antagonized the effect of oestradiol in promoting the proliferation of the two cells. Results from the IHC staining of the rat prostates indicated that, similar to Tam and Fin, Ral inhibited the proliferation of stromal cells in vivo. Interestingly, in contrast to Tam, both Ral and Fin inhibited the proliferation of epithelial cells. Furthemore, Ral treatment much strongly decreased the number of prostatic acini and the surrounding layers of smooth muscle cells than Fin （P 〈 0.05）. Our data showed for the first time that Ral may have a role in the response of the rat prostate to selective ER modulators.     

Proliferative activity and branching morphogenesis in the human prostate: a closer look at pre- and postnatal prostate growth

BACKGROUND: To gain further insight into the molecular cell biologic features of prostate development, we investigated the proliferative activity of prostate epithelial and stromal cells and their topographic relationship with neuroendocrine (NE) cell distribution and regional heterogeneity. METHODS: Consecutive sections from 43 prostates taken during autopsy representing fetuses (12-38 weeks of gestation), infants, prepubertal males and adults were double stained for chromogranin A and MIB-1. MIB-1 labeling index (LI) was calculated in the budding tips, forming acini, major collecting ducts, adjacent and non-adjacent stromal compartments. Furthermore, the topographic relationship between proliferating cells and NE cells was evaluated. RESULTS: In the first half of gestation, cell proliferation as revealed by MIB-1 LI was significantly higher in epithelial structures and stroma than in older fetuses and other age groups. MIB-1 LI was higher in budding tips than in other epithelial regions. MIB-1 LI in stroma adjacent to budding tips was not higher than that adjacent to other epithelial branching segments. Co-expression of chromogranin A and MIB-1 staining was not observed. MIB-1 LI was lower in cells in the direct vicinity of chromogranin A positive NE cells than at a distance from NE cells. CONCLUSIONS: Prostate development in the first half of gestation is explosive. Thereafter, the prostate basically is a slow-growing organ. Budding tips are the major growth foci during early prostate development, while stromal growth is evenly distributed throughout the prostate, probably indicating that stromal-epithelial interactions do not manifest in enhanced proliferation at their interface. NE cells may have an inhibitory effect on proliferation of exocrine epithelial cells and are probably only associated with differentiation of prostate exocrine cells in the prostate.     

Expression of estrogen receptor in diseased human prostate assessed by non-radioactive in situ hybridization and immunohistochemistry

To understand the role of estrogen in the pathogenesis of benign prostatic hyperplasia, expressions of estrogen receptor (ER) mRNA and ER protein by in situ hybridization and by immunohistochemistry, respectively, were investigated in human prostatic tissues. In non-malignant region, ER mRNA and ER protein were found in cytoplasm and nucleus, respectively, of stromal cells, but not in glandular epithelial and basal cells. In benign regions, ER mRNA/ER protein positive cells were found in fibromyoadenomatous and myoadenomatous hyperplasia, but not in adenomatous hyperplasia. A striking feature was periacinar arrangement of ER mRNA/ER protein positive stromal cells in all prostate carcinoma treated with androgen withdrawal. The ER mRNA/ER protein positive cells were immunohistochemically identified as fibroblasts, myoblasts, and smooth muscle cells. These results indicate that stromal cells are the primary target of estrogen in prostate, and that androgen withdrawal upregulates the expression of ER gene. © 1995 Wiley-Liss, Inc.     

Developmental estrogenization and prostatic neoplasia

The association of estrogens with benign prostatic hyperplasia and prostatic cancer has been widely studied, but no conclusive evidence exists for a role of estrogens in prostatic disease. This paper reviews the literature and describes studies which have sought to show a correlation of estrogens and alterations in the prostates of humans and experimental animal models. Using the developmentally estrogenized mouse model, we propose an alternative role for estrogens as a predisposing factor for prostatic diseases: estrogen exposure during development may initiate cellular changes in the prostate which would require estrogens and/or androgens later in life for promotion to hyperplasia or neoplasia. Thus, the critical time for estrogen action would be during the development of the prostatic tissue. We further suggest that estrogen-sensitive cells may remain in the prostate and be more responsive to estrogens later in life or less responsive to the normal controlling mechanisms of prostatic growth. © 1994 Wiley-Liss, Inc.