Uterine Estrogen Receptor Levels Research Articles

Photoperiod-induced differences in uterine growth in Phodopus sungorus are evident at an early age when serum estradiol and uterine estrogen receptor levels are not different

Sexual development is inhibited in Siberian hamsters ( Phodopus sungorus) in short days (SD), and a small uterus is an obvious indicator of photo-inhibition. The small uterus in SD is presumably due to the delayed onset of estrous cycles. However, in an earlier study, the investigators reported that serum estradiol (E2) concentration was significantly higher in young females raised in SD than in long days (LD), with the highest concentrations measured in SD at 4 weeks of age. These seemingly contradictory findings were investigated in the present study. First, uterine mass and body mass were measured in SD- and LD-reared hamsters from 1 to 12 weeks of age. Uterine mass was significantly greater in LD than in SD by 3 weeks of age and onward. Thereafter, our investigation focused on 4-week-old hamsters. Serum E2 concentrations in LD and in SD were not significantly different and there were no significant LD–SD differences in uterine estrogen receptors (ER), as measured by immunohistochemistry and quantitative real-time RT-PCR. Therefore, alternative explanations for the photoperiodic difference in uterine size in young Siberian hamsters are considered.

Modulation of Estrogen Receptor Levels in Mouse Uterus by Protein Kinase C Isoenzymes

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKCα, -βI, -βII, -δ, and -ζ are expressed in the uterus, only PKCα and -βI are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Modulation of estrogen receptor levels in mouse uterus by protein kinase C isoenzymes.

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKC alpha, -beta1, -betaII, -delta, and -zeta are expressed in the uterus, only PKC alpha and -beta1 are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Dose-Response Relationships in Mice Following Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo- p-dioxin: CYP1A1, CYP1A2, Estrogen Receptor, and Protein Tyrosine Phosphorylation

The dose-response relationships for different endpoints in different tissues were compared in response to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) treatment. TCDD was administered 5 days a week for 13 weeks at doses ranging from 1.5 to 150 ng/kg/day to female B6C3F1 mice. Ethoxyresorufin- O-deethylase (EROD) activity, a marker for CYP1A1, was increased in liver, lung, and skin at doses as low as 1.5 ng/kglday. EROD activity did not attain maximal induction. Liver acetanilide-4-hydroxylase activity, a marker for CYP1A2, was significantly induced at 1.5 ng/kglday and reached maximal induction at 45 ng/kg/day. TCDD treatment significantly increased the amount of phosphorylated forms of three phosphotyrosyl proteins (pp32, pp34, and pp38) in liver S-20 fractions. Changes in these phosphotyrosyl proteins occurred at 1.5 ng/kg/day and reached maximal induction at 4.5 ng/kg/day. No changes in phosphotyrosyl proteins were observed in skin. Hepatic and uterine estrogen receptor levels were not altered at any of the doses tested. These data indicate that induction of CYP1A1, CYP1A2, and the increases in phosphorylated forms of pp32, pp34, and pp38 are sensitive indicators of TCDD exposure. The dose-response curves for increases in CYP1A1, CYP1A2, and phosphoylated pp32, pp34, and pp38 in liver were different from each other. TCDD produces multiple effects with multiple dose-response curves suggesting that there are events in addition to receptor binding that are endpoint specific, leading to different dose-response relationships.

Regulation of uterine estrogen receptors (ER) by beta-adrenergic stimulation in immature rats.

The effects of a 3-day intramuscular (i.m.) administration of clenbuterol (25 micrograms/Kg), propranolol (12 mg/kg), clenbuterol (25 micrograms/kg) plus propranolol (12 mg/Kg) and estradiol (0.5 microgram) upon the female reproductive system were investigated in immature Sprague-Dawley rats. Clenbuterol and estradiol treatments induced a significant increase in uterus weight and in relative uterus weight, whereas in the groups treated with propranolol and clenbuterol plus propranolol no differences were detected versus controls. The uterine estrogen receptor levels were significantly increased by clenbuterol administration. In the rats dosed with propranolol and clenbuterol plus propranolol, no modifications occurred in estrogen receptor concentrations when compared with control values. Uterine progesterone receptors were never significantly affected by any of the considered treatments. Data obtained indicate that clenbuterol treatment induces an increase in uterus weight and in estrogen receptor levels and that these effects are regulated by acute beta-adrenergic stimulation, as the contemporaneous administration of high doses of a beta-blocker inhibit such effects.

Restoration of the DNA binding activity of estrogen receptor in MRL‐lpr/lpr mice by a polyamine biosynthesis inhibitor

Diverse data link estrogen influences to both the frequency and severity of systemic lupus erythematosus in humans and to murine lupus. A fundamental mechanism of action of estrogen involves the interaction of the hormone with its receptor protein, which is then transformed into the DNA binding form. We measured the concentration of uterine estrogen receptor and its DNA binding in normal BALB/c mice, lupus-prone MRL-lpr/lpr mice, and MRL-lpr/lpr mice that had been treated with 1% difluoromethylornithine (DFMO). Uterine estrogen receptor levels in 20-week-old mice from the 3 groups were not significantly different. In contrast, DNA binding activity was significantly higher in BALB/c mice (mean +/- SD 775 +/- 100 fmoles/mg of DNA) than in untreated MRL-lpr/lpr mice (80 +/- 16 fmoles/mg of DNA) (P less than 0.001). Treatment with 1% DFMO was associated with an increase in uterine estrogen receptor DNA binding (1,100 +/- 218 fmoles/mg of DNA) in MRL-lpr/lpr mice (P less than 0.001). Polyamine levels were 2-6-fold higher in the uterine tissues of untreated MRL-lpr/lpr mice compared with the BALB/c mice and were significantly reduced by DFMO treatment. Our results link uterine polyamine production to a dysfunction of the estrogen receptors in MRL-lpr/lpr mice. Reduction of the polyamine level by the irreversible inhibition of ornithine decarboxylase with DFMO restores estrogen receptor function.

Effects of postnatal des treatment on uterine growth, development, and estrogen receptor levels

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1–5 and 1–25). Treatment on days 1–5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10–25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1–25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1–5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin on hepatic and uterine estrogen receptor levels in rats

Administration of a single dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 20 or 80 μg/kg) resulted in significantly decreased hepatic and uterine estrogen receptor (ER) levels in 25-day-old female Long-Evans rats. By contrast, estradiol (5 and 15 μg/kg) administration increased hepatic and uterine ER levels, while a combination of 2,3,7,8-TCDD plus estradiol resulted in uterine and hepatic ER levels which were similar or lower than those observed after treatment with only 2,3,7,8-TCDD. In addition, 2,3,7,8-TCDD significantly decreased the effects of estradiol on uterine wet weight increase. Competitive binding studies showed that estradiol did not bind to the aryl hydrocarbon (Ah) receptor and that 2,3,7,8-TCDD did not bind to the ER. The effects of structure on the activity of polychlorinated dibenzo- p-dioxin congeners on their activity to down-regulate hepatic and uterine ER levels were determined by using 2,3,7,8-TCDD, 1,2,3,7,8-pentachlorodibenzo- p-dioxin (PeCDD), 1,3,7,8-TCDD, and 1,2,4,7,8-PeCDD. Both 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD exhibit high affinities for the Ah receptor and at dose levels of 80 μg/kg the hepatic ER levels were decreased 42 and 41%, respectively, and uterine ER levels were decreased 53 and 49%, respectively. 1,3,7,8-TCDD and 1,2,4,7,8-PeCDD bind less avidly to the Ah receptor and at dose levels of 400 μg/kg these compounds decreased hepatic ER levels 36 and 40%, respectively, and uterine ER levels 21 and 24%, respectively. These results support a role for the Ah receptor in the down-regulation of uterine and hepatic ER levels in the female rat by 2,3,7,8-TCDD and this effect may be associated with the decrease in spontaneous mammary and uterine tumors observed in female rats treated with 2,3,7,8-TCDD.

Role of the Ah receptor in mediating the down regulation of uterine and hepatic estrogen receptor levels in rats

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to 21 day old female Long Evans rats resulted in a dose-dependent decrease in uterine and hepatic estrogen receptor (ER) levels. At a dose level of 80 ug/kg of 2,3,7,8-TCDD, uterine and hepatic ER levels were 54 and 41% of values observed in untreated animals. 2,3,7,8-TCDD also antagonized the estrogenicity of estradiol in the 21 day old rat. For example, administration of estradiol (15 ug/kg) resulted in a 194 and 155% increase in cytosolic and uterine ER levels and a 42% increase in uterine wet weight 48 hours after treatment. Pretreatment of the rats with 2,3,7,8-TCDD (80 ug/kg) prior to administration of estradiol (15 ug/kg) completely blocked the estrogenic effects of estradiol. In fact after cotreatment the uterine and hepatic ER levels were 51 and 54% lower than observed in the controls and the uterine wet weight was 20% lower than in the control animals. The comparative effects of 2,3,7,8-TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,3,7,8-TCDD and 1,2,4,7,8-PeCDD on the down regulation of uterine and hepatic ER levels were structure-dependent. There was an excellent correlation between the binding affinities of these congeners for the Ah receptor and their activity as antiestrogens and the results support a role for the Ah receptor in the down regulation of uterine and hepatic ER levels in the rat. (Supported by the Texas Agricultural Experiment Station.)

Estrogen action in the mouse uterus: the influence of the neuroendocrine-adrenal axis.

Estrogen receptor levels were measured in uteri from ovariectomized-hypophysectomized mice. Cytosolic estrogen receptor levels had a range of 1.65-3.83 X 10(-13) mol/100 micrograms DNA and varied significantly with time after surgery. Nuclear receptor levels also varied with time and had a range of 0.26-1.19 X 10(-13) mol/100 micrograms DNA. Microsomal receptor levels varied from 0.24-0.72 X 10(-13) mol/100 micrograms DNA. Concurrent temporal changes in uterine or vaginal epithelial histology were not observed. Binding properties of the estrogen receptor from the ovariectomized-hypophysectomized mouse uterus were determined; Scatchard analysis of saturation binding data showed one class of binding sites (Kd = 1.32 nM; n = 276 fmol/mg protein) with values similar to those obtained from ovariectomized-adrenalectomized mouse uterus. The ability of the ovariectomized-hypophysectomized mouse uterus to respond to estrogen stimulation was determined in uterine wet weight and DNA synthesis assays; uteri from ovariectomized-hypophysectomized mice responded to estrogen in the same way as uteri from ovariectomized mice. Uteri from ovariectomized-adrenalectomized mice showed a similar growth response to estrogen stimulation. The temporal pattern of estrogen receptor distribution was determined in uteri from ovariectomized-adrenalectomized mice; there were two peaks of nuclear receptor translocation at 1 and 8 h, but the rate of cytosol receptor replenishment was slower than that previously seen in ovariectomized mouse uteri. Circulating estrogen and progesterone levels were measured in ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice at different times after surgery. There was no significant difference in systemic estradiol levels among the groups. However, there were significant differences in progesterone levels; mean values from ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice were 6.5, 2.1, and 0.029 ng/ml, respectively. The differences in progesterone levels correlate with previously reported differences in uterine estrogen receptor levels. Thus, data from this system suggest that adrenal-derived progesterone may play a greater role in regulating estrogen receptor levels in target tissues than was previously thought.

An ovarian independent population of uterine estrogen receptors

Estrogen receptor levels in uteri from ovariectomized mice varied markedly with time after surgery; these changes were not due to nuclear translocation. Receptor cyclicity was abolished in ovariectomized-adrenalectomized mice, and receptor levels remained low. This indicates that the adrenals significantly influenced uterine estrogen receptor levels. However, these adrenal-mediated changes did not alter uterine responsiveness to estrogen stimulation.

Estrogen Action in the Mouse Uterus: Adrenal Modulation of Receptor Levels

Adrenal regulation of estradiol receptor levels was demonstrated in the ovariectomized mouse uterus. After ovariectomy, cytosol receptor levels initially followed a cyclic pattern, with a periodicity of approximately 5 days and a range of range of 2.25-4.80 x 10(-13) mol estrogen receptor/100 micrograms DNA. Nuclear receptor levels showed marked fluctuations initially, but stabilized after day 15 at approximately 0.5 x 10(-13) mol/100 micrograms DNA. Microsomal receptor levels also changed with time, but at a lower level than the cytosol and nuclear fluctuations. Concurrent cyclic changes in uterine or vaginal histology were not observed. After simultaneous ovariectomy and adrenalectomy, cytosol estradiol receptor levels remained low (less than or equal to 1.5 x 10(-13) mol/100 micrograms DNA) from 5-24 days, with no fluctuations. Nuclear and microsomal receptor levels changed, but to a smaller extent than those in ovariectomized mice. Concurrent histological changes were not observed. The residual cytosolic estrogen receptor remaining in the ovariectomized-adrenalectomized mouse uterus was characterized. Scatchard analysis of saturation binding data showed one class of binding sites with a Ka of 6.56 nm-1 (n = 350 fmol/mg protein). This cytosol receptor sedimented at 8S on low salt sucrose density gradients. Receptor specificity was assessed by competitive equilibrium binding. The rank order of ligands was 17 beta-estradiol = diethylstilbestrol greater than nafoxidine. Progesterone and 5 alpha-dihydrotestosterone showed no appreciable binding activity. After the injection of estradiol (10 micrograms/kg), 80-90% of the total available estrogen receptor translocated to the nuclear compartment. This translocated receptor could stimulate uterine hypertrophy and hyperplasia, as assessed in 1- and 3-day bioassays, and was able to initiate progesterone receptor synthesis. From these results, we conclude that the adrenal can modulate uterine estrogen receptor levels.

The effects of estradiol on uterine estrogen receptor and thymidine kinase in immature and spayed-adult rats (author's transl)

Uterine estrogen receptor (ER) and thymidine kinase (TK) activity were investigated in response to an injection of estradiol (E2) (1.43microgram/100g body weight) in SD-strain immature female rats aged 21 days and spayed-adult rats aged 104 days (14 days after castration). In immature rats, uterine cytosol ER decreased abruptly after the injection of E2 and increased gradually from 4 hours later. On the other hand, nuclear ER increased rapidly and shaped a peak 1 hour after the injection of E2 and decreased gradually afterward. Uterine TK activity began to increase rapidly from 18 hours and shaped a peak 24 hours after the injection of E2 and was reduced to the same level as the controls 48 hours later. No differences in behavior of uterine cytosol and nuclear ER after the injection of E2 between immature and spayed-adult rats were noticed. Although uterine TK activity in immature rats was stimulated 30 times 24 hours after the injection of E2, that of spayed-adult rats increased by only twice. Uterine TK was separated into 3 fractions (A, B and C) by means of a DEAE-cellulose column chromatography. In immature rats, 95% of the great TK activity induced by E2 was involved in fraction A, which was not inhibited by dCTP. In contrast, uterine TK isozymes in spayed-adult rats were at fraction B and C, whose activities were inhibited by dCTP, without relation to the administration of E2. The molecular weight of fraction A was estimated to be approximately 125,000 and that of B was 100,000 by the method of gel filtration on a Sepharose CL-6B column. No differences in uterine ER levels between immature and spayed-adult rats were found, but E2-induced different TK isozymes seem to depend on the stage of uterine growth.

Control of Uterine Estrogen Receptor Levels by Progesterone

The mechanism by which progesterone antagonizes estrogenic stimulation of uterine growth was examined in the immature rat. Rats received daily injections of 2.5 mug estradiol (E) for 2 days and on day 3 either 2.5 mug E or 2.5 mug E plus 2.5 mg of progesterone (P). The quantity of nuclear and cytoplasmic estrogen receptor was determined by [3H]estradiol exchange at various intervals after injection of E or E + P. In both groups, nuclear receptor estrogen complex (RnE) increased dramtically one hour after injection and showed a gradual decline from 4 to 24 h after injection. The quantity of cytoplasmic receptor, Rc, decreased to low levels by one hour and began a gradual increase from 4 to 8 h in both groups. However, between 8 and 24 h after injection, the level of Rc continued to increase in the E treatment group (2.39 +/- 0.21 pmol/uterus at 24 h) but remained at the 8 h level in the E + P group (1.09 +/- 0.04 pmol/uterus at 24 h). This observation suggests that two seperate processes are involved in the replenishment of Rc and that progesterone inhibits the second phase of replenishment. The binding affinity and specificity of Rc for estrogens following E + P pretreatment were identical to those of the E pretreatment group. Therefore, P does not alter the binding properties but rather the intrauterine level of Rc. Treatment with E on day 4, when Rc levels differ between E and E + P groups, stimulated uterine weight and protein content on day 5 in the E pretreatment group. However, minimal stimulation was observed in the E + P pretreatment group. The quantity of RnE and the time of nuclear retention of RnE following E injection on day 4 was greater in the E group than in the E + P group. The effect of progesterone on Rc replenishment was dose-dependent (range, 0.1-2.5 mg; 1/2 maximal, 0.5 mg). Injection of testosterone propionate (1.0 mg), a weak estrogen antagonist, with E on day 3 resulted in slightly reduced levels of Rc on day 4. This reduction also correlated with a reduced sensitivity to treatment with E on day 4. These data, together with previous studies from our laboratory, suggest that progesterone and other estrogen antagonists such as nafoxidine and testosterone propionate inhibit estrogen action by interfering with the replenishment of Rc, thereby reducing the number of receptor estrogen complexes that are translocated and retained by uterine nuclei.