An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions

An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.     

Differential effects of aging on estrogen receptor dynamics in hypothalamus, pituitary and uterus of the C57BL/6J mouse

Estrogen receptor (ER) dynamics and content were measured in the hypothalamus (HYPO), pituitary (PIT) and uterus (UT) of aging mice because of their potential importance to age-related changes in sensitivity to estrogen. Young (3-6 months), and old (22-24 months) C57BL/6J mice were injected with a dose of E2 (0.05 micrograms/10 g body wt) sufficient to achieve maximal levels of nuclear ER (ERn) in all tissues, and the rise and fall of ERn and the depletion and replenishment of cytosolic ER (ERc) were measured 0, 1, 2, 4, 8, 12 and 24 h later. Integrated areas under the ERn profiles in old HYPO, PIT and UT were reduced 34, 28 and 19%, respectively. These reductions were due to (1) lower levels of ERn throughout the profiles, (2) delays in attainment of peak ERn in UT and PIT, and (3) accelerated loss of peak ERn in HYPO. ERc levels were also reduced in old mice, and replenishment of ERc was delayed in old HYPO and PIT, but not in UT. Reductions in total ER (ERn + ERc) were sufficient to account for all reductions and altered dynamics of ERn, except for the delayed attainment of peak ERn in UT. These results indicate that levels and dynamics of nuclear ER are altered during aging, and that most of these changes are secondary to alterations in ER content and turnover rather than a reduced ability of ER to bind to nuclear sites.     

Age-related alterations in estrogen receptor dynamics are independent of cycling status in middle-aged C57BL/6J mice

The objective of this study was to determine whether changes in estrogen receptor (ER) levels and dynamics that were previously observed in old acyclic mice were present in middle-aged mice and whether the cycling status of the mice influenced those changes. Young (3-6 months) regularly cycling and middle-aged (12-14 months) C57BL/6J mice that were either acyclic or still cycling regularly were injected with a dose of E2 (0.05 microgram/10 g body wt) sufficient to achieve maximal levels of nuclear ER (ERn) in all tissues examined: hypothalamus (HYPO), pituitary (PIT), and uterus (UT). The rise and fall of ERn and the replenishment of cytosolic ER (ERc) were measured 0, 1, 2, 4, 8, 12, and 24 h later. Cycling status did not affect ER binding profiles in middle-aged tissues. Therefore, data from cycling and acyclic subgroups were pooled for comparison with young mice. The increase in ERn following E2 injection, measured as the integrated area under the ERn profile, was reduced 33, 23, and 17%, respectively, in HYPO, PIT, and UT of middle-aged mice. In addition, the duration of elevated ERn was selectively reduced in middle-aged HYPO. ERc levels were reduced in middle-aged HYPO and UT, but replenishment rates were not altered. Reductions in total ER (ERn + ERc) were sufficient to account for the decline in ERn in middle-aged HYPO and UT, but factors in addition to ER loss appear to contribute to reduced ERn in middle-aged PIT. These results indicate that alterations in ER levels and dynamics occur prior to the transition to acyclicity, that these alterations are not secondary to hormonal or other changes associated with acyclicity, and that receptor loss appears to account for most of the age-related reduction in nuclear ER binding.     

Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse Leydig cell

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Continuous estrogen exposure in the rat does not induce loss of uterine estrogen receptor

Cytosol estrogen receptor (ERc) and nuclear estrogen receptor (ERN) levels were investigated in rat uteri under different conditions of hormonal exposure. The amount of directly assayable receptor was closely related to the serum concentration of 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2). A double injection technique was established to maintain serum levels of [3H]E2 which were sufficient to saturate receptor sites. Under these conditions, stable ERC and ERN levels are maintained throughout the study period. 30% of the total ER remains cytoplasmic in localization despite continuous hormonal exposure. Properties of ERC and ERN after 6 h of continuous hormonal exposure were investigated and found to be different from receptors found in these subcellular compartments 30 min after hormone injections. ERC from uteri 30 min after injection showed a faster sedimentation coefficient than ERC prepared 6 h after hormone treatment. ERC after 6 h of hormonal exposure showed a reduction of binding to calf thymus DNA adsorbed on cellulose in a cell-free system. ERC 30 min after [3H]E2 treatment had a biphasic dissociation pattern consistent with two different receptor populations, whereas uterine ERC obtained after 6 h of in vivo exposure to estradiol showed virtually no dissociation at 22 and 28 degrees C. In contrast to ERC, ERN 6 h after hormone injection sedimented faster than ERN obtained 30 min after treatment. KCl extractable ERN obtained either at 30 min or 6 h posthormone treatment showed biphasic dissociation kinetics at 22 and 28 degrees C, whereas KCl nonextractable ERN showed virtually no dissociation. Virtually all of the specifically bound ligand in cytosol and nuclear preparations was proven to be authentic E2. We conclude that total cellular receptor is quantitatively conserved during 6 h of continuous hormonal treatment. Nuclear receptor loss is not a requisite for receptor-mediated steroid function, although important time-dependent changes in receptor properties in both cytoplasmic and nuclear compartments do occur.     

Effects of copper on cytoplasmic and nuclear binding of 17 beta-estradiol in the rat uterus

Interference of Cu++ with the initial events in estrogen action was tested by determining Cu++ effects on estradiol-receptor interactions. When immature rat uteri were incubated in vitro with [3H] estradiol ([3H]E2), steroid was bound in cytoplasmic fractions and rapidly accumulated in the nuclear fraction in a manner which was dependent upon time and hormone concentration. Uteri which were preincubated with 2 X 10(-4) M CuCl2 for 40-60 min and then exposed to [3H]E2 were found to have a 30-50% decrease in the amount of steroid bound in the cytoplasmic and nuclear fractions. When copper-treated uteri were exposed to [3H]E2 for variable times, the quantity of steroid bound in the cytoplasmic fraction was markedly depressed and the rate of nuclear accumulation of [3H]E2 was significantly decreased. These results show that Cu++ can inhibit [3H]E2 binding to tissue cytoplasmic receptors in vitro and thereby interfere with hormone delivery to target cell nuclei.     

Neonatal castration of male and female rats affects hypothalamic and pituitary estrogen nuclear and progestin cytosol receptors

We compared the effects of neonatal or adult castration (7 days) and 2 or 7 days of estrogen treatment on the concentrations of estradiol cystolic (ERc) and nuclear (ERn), and progestin cytosolic receptors (PRc) in the hypothalamus, amygdala and pituitaries of adult rats. Two days of estradiol (E2) treatment greatly increased ERn levels, but no further concentration changes occurred by Day 7 in any of the tissues. Long- and short-term castrated males and females had comparable ERn concentrations on Day 2 versus Day 7. Tissue ERn levels were significantly lower in short-term males compared to short-term females or neonatally castrated males and females. In a second study, ERn levels were compared in E2-treated short-term castrated males and females on Day 2. A sex difference was observed, with females having greater ERn levels in most areas. Estrogen significantly increased PRc levels in pituitary (PIT) and hypothalamus, and these levels were comparable in Day 2 and Day 7 animals. Thus, the ability of estrogen to induce PRc synthesis is somewhat refractory in long-term castrated rats.     

Role of porcine endometrial estrogen sulfotransferase in progesterone mediated downregulation of estrogen receptor

Estrogen sulfotransferase (EST) is a progesterone (Pg) induced secretory endometrial enzyme which may effect estrogen receptor levels by esterifying estradiol-17 beta (E2) to an inactive, sulfate form. The effects of this enzyme were studied using specific inhibitors of EST that do not bind to estrogen receptor (ER): 4-nitroestrone 3-methyl ether and 4-fluoroestrone 3-methyl ether. A 1 h pulse with 4 nM E2 caused ERn (i.e. E2-bound, chromatin-bound receptor) to increase 40% in incubations of proliferative gilt endometrium (no EST activity), while the same E2 treatment of secretory endometrium (high EST activity) caused no increase in ERn. ERn accumulation was completely restored in these experiments by preincubating secretory endometrium with 4 microM 4-fluoroestrone 3-methyl ether. Gilt endometrial explants cultured 7 days with 1 nM E2 plus 1 microM Pg (which induced EST activity) possessed half the ERn as explants devoid of EST activity which were cultured in E2 alone. The addition of 10 microM 4-nitroestrone 3-methyl ester to the cultures of secretory endometrium restored ERn to the levels seen in minces cultured with E2 alone. Furthermore, ovariectomized gilts injected daily with 250 micrograms E2 plus 25 mg Pg had much lower ERn (0.06 fmol/micrograms DNA) than gilts injected with E2 only (0.21 fmol/microgram DNA). ERn was restored completely by supplementing the E2 plus Pg injections with 0.5 g 4-nitroestrone 3-methyl ether administered by vaginal suppositories.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oestradiol and progesterone receptors in the pig oviduct during the oestrous cycle

The concentrations of the tissue receptors for oestradiol (E) and progesterone (P) in the porcine oviduct at different stages on the oestrous cycle have been investigated by in vitro binding and exchange methods. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The concentrations of ERc and ERn were two-fold higher in the ampulla as compared to the isthmus. The amount of ERc in the isthmic portion of the oviduct did not vary throughout the oestrous cycle. However, the ampullar ERc concentrations increased during prooestrus, showed a maximum at standing oestrus, thereafter decreasing. Significant variations in the amount of oviductal ERn were observed. Despite the differences in ERn amounts between segments, the concentration of ERn increased significantly during late prooestrus, attaining a three-fold elevation and remaining elevated during the period of standing oestrous and early luteal phase (days 3-4), thereafter returning to basal levels. No significant variations in the amount of isthmic PRc were found throughout the period studied. The ampulla, however, showed a significant increase in PRc concentrations during standing oestrus, thereafter decreasing. The concentrations of PRn in isthmus and ampulla were of about the same magnitude and varied significantly during the oestrous cycle, increasing in concentration from standing oestrous onwards. The temporal relationships between the variations in levels of oestradiol and progesterone receptors in oviductal tissues and those of the circulating plasma levels were established. The data obtained in this study suggest a relationship between the changes in the levels of oestradiol and progesterone oviductal binding during the first days of the oestrous cycle, and the gamete and embryo transport throughout the oviduct in the porcine species.     

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells.Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid.Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed.Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.     

Making sense of cross-talk between steroid hormone receptors and intracellular signaling pathways: who will have the last word?

In classical models of nuclear steroid hormone receptor function, ligand binds receptor, heat shock proteins dissociate, and receptor dimers enter or are withheld in the nucleus and interact with coregulatory molecules to mediate changes in gene expression. The footnotes, "receptors become phosphorylated" and "dynamic nucleo-cytoplasmic shuttling occurs" describe well-accepted, but less well-understood aspects of receptor action. Recently, the idea that several protein kinases are activated in response to steroid hormone binding to cognate cytoplasmic or membrane-associated receptors has become fashionable. However, the precise role of steroid hormone receptor phosphorylation and our understanding of which cytoplasmic kinases are activated and their functional significance remain elusive. This review provides an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using the human progesterone receptor (PR) as a model. The functional consequences of PR phosphorylation by protein kinases classically activated in response to peptide growth factors and novel extranuclear or nongenomic functions of PR as potential independent initiators of signal transduction pathways are discussed. Intracellular protein kinases are emerging as key mediators of steroid hormone receptor action. Cross-talk between steroid receptor- and growth factor-initiated signaling events may explain how gene subsets are coordinately regulated by mitogenic stimuli in hormonally responsive normal tissues, and is suspected to play a role in their cancer biology.     

Characterization of uterine sex steroid receptors in the pig and their variation during the oestrous cycle

The present study establishes and validates an in vitro binding and exchange assay for tissue receptors for oestradiol (E) and progesterone (P) in pig uterus. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The relative concentrations of the receptors were measured in dissected samples from endometrium and myometrium obtained at late prooestrus, oestrus, and luteal phases of the oestrous cycle. The Scatchard analysis of the oestradiol and R 5020-receptor complex displayed linearity and indicated a single class of high affinity, low capacity binding sites. Significant variations were seen in the binding of E and P to their cytosolic and nuclear receptors, following the changes in the circulating levels of the hormones in blood plasma during the oestrous cycle. Both tissue components, i.e. endometrium and myometrium followed a similar pattern when related to the stage of the oestrous cycle considered. The ERc increased from prooestrus, reaching a maximum at standing oestrus, thereafter decreasing. The concentration of ERn increased from prooestrus towards the early luteal phase, with a significant reduction by day 8 of the cycle. The amounts of PRc were maximal at standing oestrus, remaining high during the early luteal phase, while the PRn showed a linear increase from oestrus onwards throughout the luteal phase.     

蜕皮激素与其受体EcR-USP的转录调控机制

蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone, 20E)是一种典型的类固醇激素, 主导调控昆虫的蜕皮、变态、生殖等重要生理过程。20E受体EcR-USP已被鉴定近20年, 20E与其受体复合物的转录调控机制也有了许多重要突破。已有研究表明： （1）20E受体由核受体EcR和USP形成; （2）EcR-USP异源二聚体在分子伴侣蛋白复合物的协助下获得DNA结合活性; （3）20E通过解除共阻遏因子和募集共激活因子来激活EcR USP异源二聚体并启动下游基因的转录; (4）20E-EcR-USP配体-受体复合物引发20E初级应答基因的表达, 由20E初级反应基因编码的转录因子诱导表达的20E次级应答基因级联放大20E信号, 从而调控昆虫蜕皮、变态、生殖等生理过程。     

Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.     

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Induction of porcine uterine estrogen sulfotransferase activity by progesterone

Studies have been carried out which were designed to examine the hormonal requirement for the appearance of estrogen sulfotransferase activity in porcine uteri. Mature, ovariectomized (OVX) gilts were housed for 3 weeks before being treated with various regimens of estradiol-17 beta (E2) and progesterone (P). Uteri were then removed, minced, incubated for 2 h with [3H] E2 (10(-8) M) and Na2 35SO4 (10(-4) M) and the labeled metabolic products were extracted and analyzed. Endometrial samples were also taken for the determination of E2 and P cytoplasmic and nuclear receptors (R). It was found that 4 daily injections of 250 micrograms of E2 was sufficient to bring plasma E2 concentrations to that representative of a normal estrous cycle (approx. 30 pg/ml) and to induce cytoplasmic PR to high levels (7000--19000 fmol/mg DNA). Estrogen sulfotransferase activity, which was negligible in OVX and E2-treated pigs, increased to near normal secretory levels (4 pmol product/h per 0.4 g tissue) only in pigs primed with E2 and subsequently treated with E2 and P (25--250 mg/day, 3 days). This treatment also brought about the translocation of PR to the nuclear compartment. The steroid alcohol sulfotransferase activity in these tissues decreased upon ovariectomy and remained unaffected by the hormone treatments. Endometria from treated and untreated pigs were cultured for a period up to 7 days. During this time E2 (10(-8) M) induced and/or maintained PR and P (10(-6) M) was shown to stimulate estrogen sulfurylation concomitant with the translocation of PR to the nucleus. These studies have demonstrated that, in OVX pigs and endometrial cultures, P stimulated uterine estrogen sulfotransferase activity to a level normally found in secretory uteri. In order for P to bring about elevated levels of estrogen sulfurylation it was necessary that the endometrium contain adequate concentrations of cytoplasmic PR (which required E2 priming of the system) and the P receptor complex must display nuclear translocation.     

Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.     

Progesterone-binding components of chick oviduct. VIII. Receptor activation and hormone-dependent binding to purified nuclei.

A cell-free system prepared from the estrogen-primed chick oviduct was developed and used to study the uptake of cytoplasmic progesterone-receptor complex by isolated nuclei. The receptor and purified nuclei were shown to be stable at 25 degrees, but not at 37 degrees. Thus, nuclear incubations were routinely performed at 25 degrees. Such incubations revealed greater nuclear uptake of the cytoplasmic hormone-receptor complex as compared to control incubations performed at 0 degrees. The uptake process showed a quantitative preference for oviduct nuclei. No net uptake occurred during 0 degrees incubations when the nuclei were preincubated in the absence of cytoplasmic components at 25 degrees. In contrast, the temperature requirement was partially removed by preincubation of the hormone-receptor complex at 25 degrees prior to incubation with nuclei at 0 degrees. Nuclear uptake was not accompanied by measurable alterations in the sedimentation properties of the progesterone receptor. The activation and nuclear uptake of receptor was clearly dependent upon prior binding of steroid hormone to the receptor indicating that the active nuclear form of the receptor could not be generated in the absence of the hormone. Receptor precipitation with ammonium sulfate also partially removed the temperature requirement for nuclear binding. In contrast to temperature activation, ammonium sulfate precipitation activated the receptor in the absence of hormone. It thus seemed likely that temperature and salt activation of receptor occurred via different mechanisms. Although we were able to destroy up to 60% of the nuclear DNA content by treatment with DNase prior to nuclear incubation, some 80 to 85% of the receptor-binding capacity was still present in the treated nuclei. Thus, chick progesterone receptors apparently bind to a relatively DNase-resistant portion of the oviduct genome. The properties of this system indicate its value for further investigation into the initial events of progesterone action in the chick oviduct.