Cellular localization of estrogen receptor-alpha (ERα) and -beta (ERβ) mRNA in the boar testis

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes α and β (ERα, ERβ). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERα and ERβ gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERα and ERβ mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERα mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V–VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERβ mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERα nor ERβ mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERβ and germ cell formation via ERα.     

Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Aging of Brain: Role of Estrogen

The brain undergoes many structural and functional changes during aging. Some of these changes are regulated by estrogens which act mainly through their intracellular receptors, estrogen receptor ERα and ERβ. The expression of these receptors is regulated by several factors including their own ligand estrogen, and others such as growth hormone and thyroid hormone. The levels of these factors decrease during aging which in turn influence estrogen signaling leading to alterations in brain functions. In the present paper, we review the effects of aging on brain structure and function, and estrogen action and signaling during brain aging. The findings suggest key role of estrogen in the maintenance of brain functions during aging.     

Immunohistochemical localization of estrogen receptors ERα and ERβ in the spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary during postnatal development

This study was designed to determine the expression pattern of estrogen receptor (ER) subtypes in the Acomys cahirinus ovarian cells during its postnatal development. Immunohistochemical studies revealed the presence of ERα and ERβ in germinal epithelium cells and interstitial tissue. Both these ER subtypes were also seen in granulosa cells and oocytes of growing follicles, however, the level of ERβ expression was higher in comparison with ERα. In contrast to ERβ, ERα protein was also present in theca cells. The expression of ERs increased with animals’ age, but it decreased during follicular maturation. Moreover, the immunolocalization of ER subtypes in luteal cells showed that not ERβ, but ERα expression is up-regulated throughout corpus luteum development. These immunohistochemical studies demonstrate, for the first time, that ERα is also expressed in the mouse granulosa cells and it may be a mediator of estrogen action in granulosa cells proliferation and differentiation.     

Estrogen receptor beta yield from baculovirus lytic infection is higher than from stably transformed Sf21 cells

The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERβ than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERβ decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERβ expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERβ production than stable-transformation of Sf21 cells.     

Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

Background  

Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.     

Gender- and region-specific variations of estrogen receptor α and β expression in the growth plate of spine and limb during development and adulthood

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and β in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and β expression were shown in the growth plates. Mainly nuclear staining of ERα and β immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and β expression and of region on temporal ERα/ERβ expression ratio. However, spatial differences of region-related ERα and β expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and β immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and β gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated.     

Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERα and ERβ by immunohistochemistry and in situ hybridization. ERβ, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERβ-positive cells increased significantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17β-estradiol (E₂), but not E₂ + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a significant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy.     

Regulation of intracellular calcium release and PP1α in a mechanism for 4-hydroxytamoxifen-induced cytotoxicity

Treatment with tamoxifen, or its metabolite 4-hydroxytamoxifen (4OHT), has cytostatic and cytotoxic effects on breast cancer cells in vivo and in culture. Although the effectiveness of 4OHT as an anti-breast cancer agent is due to its action as an estrogen receptor-alpha (ERα) antagonist, evidences show that 4OHT is also cytotoxic for ERα-negative breast cancer cells and can be effective therapy against tumors that lack estrogen receptors. These findings underscore 4OHT signaling complexities and belie the most basic understandings of 4OHT action and resistance. Here, we have investigated the effects of 4OHT on Ca²⁺ homeostasis and cell death in breast cancer cells in culture. Measurement of Ca²⁺ signaling in breast cancer cells showed that 4OHT treatment altered Ca²⁺ homeostasis and was cytotoxic for both an ERα+ and an ERα- cell line, MCF-7 and MDA-MB-231, respectively. Further investigation lead us to the novel discovery that 4OHT-induced increase of ATP-dependent Ca²⁺ release from the endoplasmic reticulum correlated with 4OHT-induced upregulation of protein phosphatase 1α (PP1α) and the inositol 1,4,5-trisphosphate receptor (IP3R). Blocking 4OHT-induced PP1α upregulation by siRNA strategy reduced the effects of 4OHT on both Ca²⁺ signaling and cytotoxicity. Results from these investigations strongly suggest a role for PP1α upregulation in a mechanism for 4OHT-induced changes to Ca²⁺ signaling that ultimately contribute to the cytotoxic effects of 4OHT. Aliccia Bollig, Liping Xu- contributed equally to this work     

Ontogeny of estrogen receptor alpha,estrogen receptor beta and androgen receptor,and their co-localization with Islet-1 in the dorsal root ganglia of sheep fetuses during gestation

The aims of the present study were to detect the ontogeny of estrogen receptor (ERα and ERβ) and androgen receptor (AR) expressions and their co-localization with Islet-1 in the developing dorsal root ganglia (DRG) of sheep fetuses by immunohistochemistry. From the single staining results, the ERα immunoreactivity (ERα-ir), ERβ immunoreactivity (ERβ-ir) and AR immunoreactivity (AR-ir) was first detected at days 90, 120 and 90 of gestation, respectively. From days 90 to 120, ERα and AR were consistently detected in the nuclei of DRG neurons and the relative percentage (approximately 60%) of ERα-ir or AR-ir cells did not change significantly. Moreover, there was no change in ERα expression, while a dramatic loss of AR expression was observed at birth. From day 120 of gestation to birth, very few neurons (approximately 8%) showed nuclear ERβ immunoreactivity. The dual staining results showed that Islet-1 was co-localized with ERα, ERβ or AR in the nuclei of DRG neurons with various frequencies, and over 70% ERα-ir, ERβ-ir or AR-ir cells contained Islet-1. These results imply that ERs, AR and Islet-1 may be important in regulating the differentiation and functional maintenance of some phenotypes of DRG neurons after mid-gestation in the sheep fetus.     

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage

 Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)₂D₃ and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)₂D₃. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation. Accepted: 4 November 1998     

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) α and β was assessed, an induction of ERβ by DES was found predominantly in PRL cells. However, since these effects were abolished in ERα knockout mice, DES appears to act primarily through ERα. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10–15 days, and Pit-1-positive cells were increased at 5–15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5–10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.     

Phylogenetic Analyses Reveal Ancient Duplication of Estrogen Receptor Isoforms

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERβ), we examined the phylogenetic relationship of ERβ to the well-known α isoform (ERα) and other steroid receptors. Our phylogenetic analyses traced the origin of ERβ to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH₂ terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERα and ERβ dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERα and ERβ share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction. Received: 19 January 1999 / Accepted: 26 May 1999