Immunodetection of estrogen receptor in epithelial and stromal tissues of neonatal mouse uterus (original) (raw)
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Biology of Reproduction, 1989
Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to I) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 1 713-estradiol (E2), estriol (E3), and diet hylstilbe strol (DES) exhibited abnost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (pos fixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCI, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCI or 0.5 M NaCI, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of iminunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 mm with PBS, 0.4 M KC1, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.
Molecular and Cellular Endocrinology, 2002
Ontogenetic expression of progesterone receptor (PR) and effect of estrogens on PR expression in the fetal female rat reproductive tract were investigated. To evaluate ontogenetic PR expression, female reproductive tract from untreated fetuses was examined on gestational days (GD) 15.5, 17.5, 19.5 and 21.5. To evaluate estrogen effects, pregnant rats were injected once per day with oil, 17bestradiol (E 2) or diethylstilbestrol (DES) from GD15 through 21. Female fetuses were prepared for real-time reverse-transcription polymerase chain reaction (RT-PCR) or immunohistochemistry for PR. Increase in PR mRNA expression was detected in the Mü llerian duct on GD21.5 compared to that on GDs15.5 and 17.5 in untreated fetuses (P B/0.05). Prenatal administration of E 2 or DES increased Mü llerian PR mRNA levels by GD21.5 compared with oil controls (P B/0.01). To identify cell and region in which PR was expressed and up-regulated by E 2 and DES, localization was evaluated within three regions along the Mü llerian duct axis which differentiate into oviduct, uterus and upper vagina in immunohistochemistry. In untreated fetuses, Mü llerian epithelial PR immunoreactivity was weak on GDs15.5 and 17.5, but then became moderate on GDs 19.5 and 21.5 in all three regions. These fetuses exhibited faint signals in Mü llerian mesenchymal PR immunoreactivity during gestational monitoring. Critically, Mü llerian mesenchymal PR staining became intense after E 2 exposure in all three regions by GD21.5, but no change was observed in Mü llerian epithelial PR. Similarly, DES dramatically induced Mü llerian mesenchymal PR in all regions by GD21.5, and also enhanced proximal epithelial PR. On the other hand, middle and caudal epithelial PRs were reduced by DES. These affected mesenchymal and epithelial cells by DES were ERa immunopositive in the Mü llerian duct, except for middle Mü llerian epithelium. These findings clearly demonstrate cell-specific PR localization and region-specific effect of DES on PR in the developing rat Mü llerian duct, and provide fundamental information critical for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen receptor agonists.
Progesterone receptor repression by estrogens in rat uterine epithelial cells
The Journal of Steroid Biochemistry and Molecular Biology, 1997
Measurements performed using cell lines or animal tissues have shown that the progesterone receptor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of estrogens on the PR levels in the individual celi types of the target organs uterus and breast. In the uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and surrounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture emerged when intact rats were treated with the pure estrogen receptor antagonist, ZM 182780 (10 mg/kgld). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myometrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we found that in the epitlaelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced mammary carcinom~s the PR expression was induced by estrogens and was blocked by the pure antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diverging effects on PR expression in different cell types even within the same organ.
Characterization of rat uterine estrogen receptors in vivo
The Journal of Steroid Biochemistry and Molecular Biology, 1993
In vivo binding of [3H]estradiol ([3H]E2) in the rat uterus was performed by an intraluminal perfusion of the ligand for different time periods. In this way the binding takes place in the intact organ before processing the tissue. In 10 min, with 10 nM [3H]E 2 apparent saturation or steady state incorporation of the [3I-I]E2 was achieved with a similar distribution of the label between cytosol and nuclear fractions. In vitro, the subcellular localization of the estrogen receptor (ER) is influenced by the extent of tissue damage. With the intact organ the ER subcellular distribution approaches that of the/n vivo perfusion. With increasing [3H]E2 in the perfusate it was possible to obtain a "saturation" curve and to derive the kinetic parameters. For cytosol: Kd 16 nM; Bm~ 235 fmol/mg prot. For nucleus: Kd 2.7 nM; Bm~ 103 fmol/mg prot. To follow the time course of the ER movement/n vivo, "pulse and wait" experiments were designed. Both uterine horns were peffused for 1 min. One of the horns was immediately processed (0 time) and the other was left in place after the peffusion for different periods. At 0 time 90% of the bound label appeared in the cytosol. At 5, 15 and 30 min, the label in the cytosol decreased and that of the nucleus increased approx, to 50%. Thus, translocation of the bound label from cytosol to nucleus was apparent. The role of the cytoplasm-nucleus ER traffic in the regulation of gene transcription by estrogens is discussed.
2006
In this study, we compared the uterine tissue of estrogen receptor (ER)beta(-/-) mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERbeta(-/-) mice, but E-cadherin and cytokeratin 18 expression was lower in ERbeta(-/-) mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERbeta(-/-) mice. Sixteen hours after injection of 17beta-estradiol (E(2)), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERbeta(-/-) mice. Although ERalpha was abundant in intact mice, after ovariectomy, ERalpha could not be detected in the luminal epithelium of either WT or ERbeta(-/-) mice. In both untreated and E(2)-treated mice, ERalpha and ERbeta wer...
Journal of Veterinary Medical Science, 2003
The action of estrogen on target organs has been actively studied with the discovery of estrogen receptor (ER) β. This study was carried out to examine the expression of ERα and ERβ in the uterus and the vagina of immature Sprague-Dawley rats treated with 17-ethinyl estradiol (EE). Twenty days old rats were subcutaneously treated with EE at the doses of 0 (vehicle control), 0.03, 0.3, 1.0, 3.0, and 10.0 µg/kg/day for three consecutive days. The treatment of EE at the doses of 0.3, 1.0, 3.0 and 10.0 µg/kg/day significantly increased the weights of the uterus and vagina of rats (p<0.01) and retained fluid in the uterus of rats. At the high doses of 3.0 and 10.0 µg/kg/day, the treatment of EE caused an increase in the uterine height, hypertrophy, and a decrease in the expression of ER α and ERβ in the uterine luminal and glandular epithelium. The treatment of EE at the doses of 3.0 and 10.0 µg/kg/day also caused cornification and a decrease in the expression of ERα and ERβ in the vaginal epithelium. These results suggest that the EE treatment decrease the expression of ERα and ERβ in the uterus and vagina of immature rats and that may be associated with the morphological changes such as increase in the uterine height, hypertrophy of the uterine epithelium, and cornification of the vagina. KEY WORDS: estrogen receptor α and β, 17-ethinyl estradiol, uterus, vagina.
Domestic Animal Endocrinology, 1998
Objectives were to establish conditions for preparation, growth, and maintenance of a primary culture cell model of fetal uterine cells, and to determine whether cells maintained under those conditions would maintain their capacity to respond to estrogen stimulation in vitro. Fetal uteri (n ϭ 19) were enzymatically dispersed and grown on Type 1 collagen in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Fetal-uterine cells appeared fibroblast-like and exhibited positive immunostaining for both vimentin and cytokeratin. Effects of gestational age (GA), passage number (p), and hormonal treatment on appearance of specific mRNAs were determined by RT-PCR; relative concentrations of products determined by densitometry were analyzed as the ratio of target cDNA to the GAPDH loading control. Cells expressed mRNAs for estrogen receptor (ER), TGF-, EGF-R, PRL-R, IL-1 ␣, and IL-6. ER mRNA was greater at 185-200 than at 100-110 d GA (P Ͻ 0.01). All specific mRNAs examined were greater in p5 cells than p2 at both 100-110 (P Ͻ 0.01) and 185-200 d GA (P Ͻ 0.02). There was no effect of estradiol on these specific mRNAs in cells from 100-110 d GA; at 185-200 d GA, there was an estradiol (1.0 nM) effect both at 6 hr (P Ͻ 0.001) and 24 hr (P Ͻ 0.02). Overall, there was an effect of 8-br-cAMP (1 mM; 6 h) on specific mRNAs in cells at both 100-110 (P Ͻ 0.001) and 185-200 d GA (P Ͻ 0.001). In p5 cells from Day 185-200 GA, there was increased cell proliferation (P Ͻ 0.001) in response to estradiol (1 nM; 24 hr). These data suggest that primary fetal uterine cells retain their age-specific and hormone-responsive phenotype under these in vitro conditions.
The Journal of Steroid Biochemistry and Molecular Biology, 2013
The exposure to endocrine disrupters and female reproductive tract disorders has not been totally clarified. The present study assessed the long-term effect of perinatal (gestation + lactation) exposure to diethylstilbestrol (DES) on the rat uterus and the effect of estrogen replacement therapy. DES (5 g/kg bw/day) was administered in the drinking water from gestational day 9 until weaning and we studied the uterus of young adult (PND90) and adult (PND360) females. To investigate whether perinatal exposure to DES modified the uterine response to a long-lasting estrogen treatment, 12-month-old rats exposed to DES were ovariectomized and treated with 17-estradiol for 3 months (PND460). In young adult rats (PND90), the DES treatment decreased both the proliferation of glandular epithelial cells and the percentage of glandular perimeter occupied by ␣-smooth muscle actin-positive cells. The other tissue compartments remained unchanged. Cell apoptosis was not altered in DES-exposed females. In control adult rats (PND360), there were some morphologically abnormal uterine glands. In adult rats exposed to DES, the incidence of glands with cellular anomalies increased. In response to estrogens (PND460), the incidence of cystic glands increased in the DES group. We observed glands with daughter glands and conglomerates of glands only on PND460 and in response to estrogen replacement therapy, independently of DES exposure. The p63 isoforms were expressed without changes on PND460. Estrogen receptors ␣ and  showed no changes, while the progesterone receptor decreased in the subepithelial stroma of DESexposed animals with estrogen treatment. The long-lasting effects of perinatal exposure to DES included the induction of abnormalities in uterine tissues of aged female rats and an altered response of the adult uterus to estradiol.
DiVerential immunolocalization of estrogen receptor Æ and ‚ in rat ovary and uterus
2000
In order to investigate the localization of estrogen receptor (ER) Æ and ER‚ in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERÆ antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ER‚ antibody detected a 55 kDa band in rat ovary, uterus