Extensive in situ activation of nuclear estrogen receptors after exposure of murine uteri to [3H]estradiol or [3H]4-hydroxytamoxifen

Estrogen receptor activation has been examined in murine uteri by characterizing binding to ATP-Sepharose. Determinations were performed under conditions in which specific binding to estrogen receptors was demonstrated by both agonist and antagonist without participation by nonreceptor antiestrogen-binding components. Cell-free activation of estrogen receptors in cytosol was more effectively promoted by [3H]estradiol estradiol than by [3H]4-hydroxytamoxifen or [3H]tamoxifen aziridine. However, when in situ activation was examined after intact uteri were exposed to [3H]estradiol or [3H]4-hydroxytamoxifen, virtually all extracted nuclear receptors demonstrated activated binding to ATP-Sepharose within 20 min of hormone exposure. Profiles of nuclear receptor activation were remarkably similar after exposure to either agonist or antagonist. Estrogen receptors in cytosol prepared after exposing intact uteri to 3H-labeled ligands were characterized by much less ability to bind to ATP-Sepharose than nuclear receptors. After uteri were exposed to [3H]estradiol, the activated receptor fraction in the cytosol progressively increased in contrast to preparations obtained after uteri were exposed to [3H]4-hydroxytamoxifen, which demonstrated a constant level of activation. Thus, even when activation has occurred within the intact uterus, agonists and antagonists may be characterized by different apparent levels of receptor activation in the cytosol fraction. These differences in the cytosol, however, are considerably overshadowed by the extensive activation occurring within the nuclear fraction, which we have observed to be similar with agonist and antagonist. Since estrogen receptors appear to act within chromatin, and cytosol receptors may be produced by receptor redistribution during preparation, we interpret these observations to indicate that in situ receptor activation is very similar after exposure to either agonist or antagonist. Consequently, antagonism does not appear to be associated with antiestrogens that impede receptor activation within intact murine uteri.     

Effects of rat alpha-fetoprotein administration on estradiol free fraction, the onset of puberty, and neural and uterine nuclear estrogen receptors.

The cause of the onset of puberty in the rat or any other mammalian species is unknown. According to one theory, puberty is initiated through switching of the brain "gonadostat." It is hypothesized here that puberty in the rat is the consequence of the appearance of free, and therefore physiologically active, estrogen in the circulation. To test this, the unbound fraction of estradiol in serum of immature female rats was measured in relation to the nuclear receptor occupancy of estradiol in the hypothalamus, preoptic area, and uterus at various times after birth. In addition, an attempt was made to alter the free fraction of estradiol by injection of the estradiol-binding protein alpha-fetoprotein (AFP) into immature female rats. The free fraction of estradiol was low (less than 1%), but began rising at about 20 days of age, and a significant increase in nuclear bound estradiol was observed in 23-day-old rats (P less than 0.001). By day 30, unbound levels attained adult values (3.99 +/- 0.15%). At this time, nuclear bound estradiol in all tissues examined fell (P less than 0.01), but by day 40, these were greatly increased in rats in estrus (P less than 0.001), being trebled in the preoptic areas and doubled in the hypothalamus. Injection of AFP into immature female rats extended the period of low free estradiol (1.22 +/- 0.08%), while in albumin-injected rats, the free fraction was 4.44 +/- 0.1%. Injection of AFP resulted in levels of nuclear-bound estradiol that were less than half those measured in nuclei from AFP-injected animals (P less than 0.001), and AFP delayed puberty. The affinity of the reaction between estradiol and nuclear receptors in brains of immature and mature rats was not significantly different; the Kd fell within the range of 0.05-0.08 nM. It is suggested that in the rat, puberty is the result of the appearance in the circulation of physiologically active estradiol after day 20.     

Antagonism to estradiol in the mouse: reduced entry of receptors complexed with 4-hydroxytamoxifen into a Mg2+-soluble chromatin fraction

Antagonism to estradiol has been examined in murine uteri. When tamoxifen was administered simultaneously with estradiol (0.05 microgram/mouse), it was able to act as an antagonist over the dosage range 0.05-50 micrograms/mouse. The metabolite 4-hydroxytamoxifen (4OH-tamoxifen) had high affinity for estrogen receptors and was a slightly better antagonist over the dosage range 0.005-1 microgram/mouse. After uteri were exposed to either [3H] estradiol or [3H]4OH-tamoxifen, receptors complexed with [3H] estradiol penetrated a chromatin region, which was released as the Mg2+-soluble chromatin fraction after DNAase I treatment more readily than receptors complexed with [3H]4OH-tamoxifen. [3H]4OH-tamoxifen-receptor complexes could not be driven into the Mg2+-soluble chromatin fraction by increasing the ligand concentration during translocation. Relative to [3H]estradiol, significantly more [3H]4OH-tamoxifen was observed to associate with uterine cells and to penetrate the nucleus so that neither restricted entry nor extranuclear partitioning could explain the failure of [3H]4OH-tamoxifen-receptor complexes to enter the Mg2+-soluble chromatin. Bleomycin, an agent that interrupts DNA continuity, did not interfere with the appearance of estrogen receptor activity in the Mg2+-soluble chromatin fraction. Preincubation of intact uteri in the presence of molybdate (20 mM) did inhibit the appearance of receptor activity in this chromatin fraction; however, this effect did not occur through inhibition of receptor activation, but, rather, through the lowering of receptor activity in all chromatin fractions. In the studies reported here, the chromatin positioning of estrogen receptors complexed with estradiol appeared to be distinct from the positioning of receptors complexed with 4OH-tamoxifen. These observations suggest an additional basis from which the mechanisms separating the actions of estrogen agonists and antagonists can be approached.     

Impaired conversion of rat uterine estradiol receptors during aging

We have examined the effects of aging on the capacity of rat uterine estradiol receptors to be transformed from 8S to 4S and 5S species. Cytosol receptors from mature (6-month-old) rats or senescent (24-month-old) rats have been exposed to various KCl concentrations, ammonium sulfate precipitation and 25 degrees C heating. Estradiol receptors of both the mature and senescent age groups exist in an 8S form on linear 5-20% sucrose gradients in the absence of KCl and are converted to a 4S molecule in the presence of 0.4 M KCl. At intermediate salt concentrations a greater portion of mature receptors was converted to the 4S species. At 0.15 M KCl 62.3% +/- 2.8 of the mature receptors are converted to 4S versus 41% +/- 1.9 of the senescent receptors, and at 0.2 M KCl 79.6% +/- 3.2 of the mature receptors are converted to the 4S versus 58.2% +/- 2.1 of the senescent. Ammonium sulfate treatment in the presence of 0.3 M KCl converted about 80% of the receptors from the 4S to the 5S form, while only about half of the old receptors are affected. When ammonium sulfate precipitates were heated to 25 degrees C all to mature receptors were converted to the 5S species, while only two thirds of the senescent receptors were sedimented at 5S under the same conditions. Inclusion of 20 mM molybdate during preparation blocks conversion of about 15% of the senescent receptors from the 8S to the 4S form but does not affect the mature preparations. Similarly, molybdate treatment does not affect the conversion of the mature estradiol receptors to the 5S form but increases the percentage of senescent receptors remaining in the 4S form from 30 to 45%. Such qualitative differences in receptor conversion may be related to age associated deterioration of estradiol stimulated uterine responsiveness.     

Displacement of estradiol from estrogen receptors by simple alkyl phenols

Simple alkyl phenols have been tested for their ability to prevent the binding of [3H]estradiol and to displace the prebound hormone from estrogen receptors of uterine cytosols. Tetrahydronaphthol, an analog of the A and B rings of estradiol, is highly effective in preventing the forward bindng of estradiol. p-sec-Amyl phenol (pSAP) with a flexible alkyl chain corresponding to the B ring of estradiol is highly effective at 0 C in displacing estradiol which had been prebound by the receptor. Both compounds were more effective at 0 C than at 23 C. The data are discussed in terms of sequential conformational changes which might be required for the binding and release of the natural hormones and their possible relevance to receptor action.     

Specificity of oligodeoxynucleotide binding of mouse uterine cytosol estradiol receptors.

The relative capacities of oligodeoxynucleotides, covalently linked to cellulose, to bind estradiol receptor complexes (E2R) of mouse uterine cytosol have been shown to follow the order oligo(dG) > oligo(dT) greater than or equal to oligo(dC) > oligo(dA). The E2R . oligo(dT)-cellulose-binding reaction is more sensitive to Cibacron blue F3GA than is E2R . oligo(dG)-cellulose or oligo(dC)-cellulose binding. Preformed E2R . oligo(dT)- or oligo(dC)-cellulose complexes are dissociated more readily by lower concentrations of KCl or Cibacron blue F3GA than is the E2R . oligo(dG)-cellulose complex. Preincubation of E2R at 37 degrees C results in a rapid loss of binding ability towards oligo(dT)- and oligo(dC)-cellulose, while up to 90% of the binding ability to oligo(dG)-cellulose is retained. On the basis of the differential sensitivities of E2R to temperature and the inhibition by Cibacron blue F3GA of the binding reaction, it is suggested that the polynucleotide-binding domain consists of two types of subsites, one of which has a higher affinity for oligo(dG) residues and the other of which recognizes oligo(dT), oligo(dC), and, to a lesser extent, oligo(dA).     

Age-associated changes in nuclear binding of rat uterine estradiol receptor complexes

Nuclear binding of cytoplasmic estrogen receptors was measured in an in vitro cell-free system, using various mixtures of cytosols and nuclei from uteri of mature (6-9 month old) and senescent (24-25 month old) Wistar rats. Both nuclei and cytoplasmic receptors from senescent uteri were 25-35% less efficient in supporting nuclear binding than those obtained from mature tissues as evidenced by the concentrations of occupiable nuclear acceptor sites. No age differences in association or dissociation constants were observed for the nuclear binding reaction. However, the apparent inability of some aged receptors to bind to the full complement of mature nuclear acceptor sites may indicate a qualitative deficiency in the cytosols of senescent uteri.     

Strain differences in the ontogeny of estrogen receptors in murine uterine epithelium

The expression of estrogen receptor (ER) in the reproductive tracts of neonatal mice was examined using immunocytochemical and autoradiographic methods. Two strains of mice used in previous studies that reported contradictory results showed different rates of uterine epithelial development. In the inbred strain, BALB/c, the epithelium was devoid of receptor from birth through 5 days of age, while uterine epithelial cells of the outbred strain, CD-1, expressed ER as early as 3 days of age. Oviductal epithelium and cervical epithelium expressed ER on the day of birth in CD-1 mice. Glandular ontogeny in the uteri of CD-1 animals was also advanced by 3 days compared to that of BALB/c mice. These observations reconcile the conflicting reports of ER ontogeny in the neonatal mouse. More importantly, these results confirm our earlier observations, indicating that the cells lining uteri of 2- and 4-day-old BALB/c mice lack ER at a time when estrogen induces their proliferation.     

Effect of estradiol on insulin secreting INS-1 cells overexpressing estrogen receptors

BACKGROUND: Estrogen has been shown to have profound effects on insulin and glucose metabolism in vivo. Indeed, estrogens were recently shown to modulate ion channel and secretory activities in endocrine cells. DESIGN AND METHODS: To investigate whether estrogenic influences are caused by direct effects on pancreatic beta-cells, we equipped INS-1 insulinoma cells with estrogen receptors and monitored insulin content and Ca(2+) fluxes as well as basal and stimulated insulin secretion upon different stimuli including glucose, the Ca(2+) ionophore ionomycin, the Ca(2+) channel agonist BayK8644, the protein kinase C activator TPA, and the adenylate cyclase activator forskolin. RESULTS AND CONCLUSION: Our data reveal that estradiol has no significant direct effect on proliferation rate, insulin content, basal and stimulated insulin output as well as Ca(2+) fluxes of insulin secreting cells in vitro, indicating that in vivo responses to estrogen on insulin and glucose metabolism result from indirect betacytotropic effects.     

Studies on the activation of the oestrogen receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384 by using three monoclonal antibodies

Three monoclonal antibodies, H222, H226 and D547, which provided evidence of the structural transformation and change in exposure of the functional domains of the oestrogen receptor from fetal guinea-pig uterus upon activation, were used to study the receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384. No differences in the structure of non-activated 4-hydroxytamoxifen- and ICI 164,384-receptor complexes, as compared with the oestradiol-receptor complex, were detected by the three monoclonal antibodies. When heated at 28 degrees C, both anti-oestrogen-receptor complexes became capable of binding the D547 antibody, which reacts selectively with the activated receptor; however, this binding was lower than that of the oestradiol-receptor complex. The interaction with the H226 antibody showed that anti-oestrogens can induce receptor dimerization, but to a lesser extent than oestradiol. In addition, both anti-oestrogen-receptor complexes can bind to DNA-cellulose and are retained in nuclei from intact cells at 28 degrees C, but less efficiently than the oestradiol-receptor complex. On the other hand, the nuclear receptor seems to have a similar dimeric structure when bound to either anti-oestrogens or oestradiol, as detected by the three monoclonal antibodies. The data suggest that 4-hydroxytamoxifen and ICI 164,384 induce and impaired activation of the oestrogen receptor; this difference, although quantitative rather than qualitative, might be related to the partial agonistic action of these anti-oestrogens in the fetal guinea-pig uterus.     

Studies on the interrelationship among serum estrogen levels, estrogen receptors (unbound cytoplasmic, bound cytoplasmic and bound nuclear), RNA syntheses and protein syntheses in the normal endometrial tissues (author's transl)

The physiological effects of estrogen on normal and human endometrial tissues were investigated. In the experimental animal uterus, it was well established that the 1st response to estrogen required RNA synthesis which then led to protein synthesis. The aim of this investigation was to study the interrelationships between serum estrogen levels, (ER) estrogen receptors (unbound cytoplasmic, bound cytoplasmic, and bound nuclear), RNA syntheses, and protein syntheses in normal human endometrial tissues. The unbound cytoplasmic ER were measured by the D.C.C. method, and bound cytoplasmic and nuclear ER were measured by the protamine exchange method. The following results were obtained. 1) The unbound and bound ER concentrations of the cytoplasmic fraction of normal human endometrial tissues were progressively increased throughout the proliferative phase, and were at their highest during the secretory and late secretory phases of the cycle. 2) The bound ER concentrations of the nuclear fraction were highest during the late proliferative phase and lowest during the early secretory phase, but increased throughout the secretory phase. 3) RNA syntheses were increased progressively throughout the proliferative phase and were highest in the late proliferative phase and lowest in the late secretory phase. 4) Protein syntheses were also highest in the late proliferative phase and lowest in the early secretory phase, but increased throughout the secretory phase. 5) The serum estrogen levels displayed a positive correlation with unbound cytoplasmic receptors (r=0.85), bound cytoplasmic receptors (r=0.79), and unbound nuclear receptors (r=0.70) in the proliferative phase, and a low positive correlation with RNA syntheses (r=0.86) and protein syntheses (r=0.84) in the proliferative phase, but no correlation was found in the secretory phase. 7) RNA syntheses in the endometrium have a positive correlation with unbound cytoplasmic receptors (r=0.88), bound cytoplasmic receptors (r=0.80), and bound nuclear receptors (r=0.79) in the proliferative phase, but in the secretory phase these have a positive correlation with unbound cytoplasmic receptors (r=0.61), bound cytoplasmic receptors (r=0.68), and bound nuclear receptors (r=0.83) in the proliferative phase, and a positive correlation only with bound nuclear receptors (r=0.61) in the secretory phase. (Author's modified)     

Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle

Estrogen receptors (ER) and progesterone receptors (PgR) were studied immunohistochemically using specific antireceptor monoclonal antibodies in uterine tissue samples from 33 women in various stages of the menstrual cycle. Immunohistochemical localization was quantified as to intensity of staining and tissue distribution in glandular epithelium, stroma, and myometrium, and the results were compared with those of standard ligand binding assays. In all samples ER and PgR localized within the nuclei of target cells. The maximal concentrations of ER and PgR occurred in the mid- to late proliferative phase of the menstrual cycle. ER content declined throughout the secretory phase. In contrast, PgR content underwent unexpectedly complex and dyssynchronous fluctuations during the secretory phase of the menstrual cycle. Specifically, the glandular epithelium had diminished PgR content, while the stroma and myometrium maintained a significant PgR content. PgR and perhaps ER are not concordant in different cell types within the uterus. Segregation of function through alteration of receptor content may be an important mechanism in steroid-dependent growth and differentiation of target tissues.     

Selective availability of protein bound estrogen and estrogen conjugates to the rat kidney

Estrogen glucuronides are selectively cleared by the kidney as compared to estrogen sulfates. The selective trafficking of circulating serum estrogen conjugates to kidney and urine may arise from differential transport properties of the various estrogen conjugates in the renal microcirculation. In the present study, the effects of glomerular and peritubular permeability barriers, and plasma protein binding on the influx of unconjugated and conjugated estrogens into rat kidney were studied. Experiments were carried out utilizing an in vivo double isotope, single injection, timed tissue sampling technique. The extractions of these steroids by the renal cortex were examined utilizing inulin as the reference substance, as it is freely permeable through the glomerular and tubular capillary permeability barriers, but not extracted by tubular epithelial cells. The method was validated by studying the extraction of para-amino hippuric acid (PAH) before and after probenecid treatment. In the absence of plasma proteins, all the estrogens and estrogen conjugates readily diffused through both the glomerular and peritubular capillary permeability barriers and were extracted by tubular epithelia. The addition of 4% albumin to the injection solution led to significant inhibitions of extraction of estradiol (E2) and estrone sulphate (E1-S) only. The extraction of E1-S was reduced to a value less than that of inulin; the extraction of E2 was less than that of control value but significantly more than that of inulin. The addition to the injection solution of human pregnancy sera containing sex hormone binding globulin and albumin was associated with a reduction in the extraction of all estrogens and estrogen conjugates except estriol and E1-S to values approximating that of inulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exclusive nuclear location of estrogen receptors in Squalus testis.

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.     

Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium

Estradiol-17β (E₂) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E₂ elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E₂ or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [³H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E₂ induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E₂ despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E₂-induced uterine epithelial proliferation. Instead, E₂ induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.     

Ligands differentially modify the nuclear mobility of estrogen receptors alpha and beta

Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor alpha (ERalpha) and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible because ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, whereas ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERbeta retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.