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SUMMARY
Oestrogen receptors were identified in both the cytoplasmic and the particulate fractions of the mammary glands of lactating rats. The quantities of cytoplasmic receptor and nuclear receptor—oestrogen complex in the particulate fraction were measured by the charcoal adsorption assay and the [3H]oestradiol exchange assay respectively. The quantity of cytoplasmic oestrogen receptor increased markedly between day 1 and day 10 and reached a maximum value on the 21st day of lactation. Although the quantity of cytoplasmic receptor increased markedly during lactation, the concentration of nuclear receptor—oestrogen complex that resulted from endogenous concentrations of blood oestrogen changed only slightly, indicating that the blood level of oestrogen was quite low during lactation. One hour after a subcutaneous injection of 25 μg oestradiol, the concentration of nuclear receptor—oestrogen complex increased dramatically and a translocation process similar to that for the uterine receptor—oestrogen complex was observed. The increased quantity of particulate receptor—oestrogen complex after the administration of exogenous oestradiol indicated that the low levels of endogenous nuclear receptor—oestrogen complex observed during lactation were not the result of failure of the translocation process. The concentration of cytoplasmic receptor on day 21 of lactation in rats that were ovariectomized on day 1 of lactation was not significantly different from the concentration in intact control animals. The translocation process was also similar in ovariectomized and intact rats on day 21 of lactation. These results suggest that the increased quantity of oestrogen receptors in lactating mammary tissue is not dependent on ovarian oestrogens.
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SUMMARY
Rat uterine nuclei containing unlabelled progesterone–receptor complexes were incubated at 0 °C with the synthetic progestogen [3H]R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione). Complete exchange of steroid bound to the receptor was observed after 5–6 h. For longer times (up to 26 h) there was no further change in the concentration of steroid–receptor complexes, but the non-specific binding was increased. Saturation of nuclear receptors was obtained with a concentration of 5–10 nm-[3H]R5020. Competition with unlabelled steroids showed that only progestogens inhibited the binding of [3H]R5020 to nuclei. Optimum experimental conditions to reduce the non-specific binding to nuclei were established, and a method for the assay of nuclear progesterone–receptor complexes was devised, based on these characteristics. The concentration of nuclear receptors was low in oestradiol-primed ovariectomized rats; adrenalectomy gave rise to slightly lower values. Injection of the rats with progesterone resulted in a 5·5-fold increase in the number of nuclear receptors and a parallel decrease in the number of cytosol receptors. Similar injections of corticosterone and testosterone were without effect. Nuclear receptors were also shown to be stable in uteri kept in liquid nitrogen for up to 3 weeks. This assay may be used to study the correlation of a biological response to progesterone with the extent of receptor occupation in the nuclei.
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This study describes the presence of a population of oestrogen receptors in cell nuclei from the pituitary gland and brain of untreated and oestradiol-treated ovariectomized rats. The receptors behaved as if they were not occupied by oestradiol. These 'unfilled' oestrogen receptors could be distinguished from occupied nuclear receptor sites on the basis of their ability to bind [3H]oestradiol at low temperatures (0–4 °C). Occupied receptors bound labelled [3H]oestradiol only under exchange conditions at an increased temperature (25 °C). Unfilled and occupied nuclear receptors were physicochemically similar in terms of sedimentation coefficients in sucrose density gradients containing 0·4 m-KC1 (4–5S), equilibrium dissociation constants for reaction with [3H]oestradiol (0·2–0·6 nmol/l) and ligand specificity. In ovariectomized rats, unfilled receptors constituted more than 75 % of the total nuclear receptor population. One hour after i.v. treatment with oestradiol (3·6 μg/kg), both total and unfilled nuclear receptor concentrations increased and then subsequently declined over the next 12 h. The increase in unfilled sites was, however, proportionately less than that occurring in the filled component; at 1 h after oestradiol injection unfilled sites constituted less than 20% of the receptors present in brain and pituitary cell nuclei. The physiological significance of unfilled nuclear oestrogen receptors remains unknown. The observations that they exist in various oestrogen target tissues and that their levels are influenced by oestradiol treatment suggest a possible role for these receptors in the mechanism of oestrogen action.
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actions of TZDs are likely mediated by their interaction with the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) ( Lehmann et al. 1995 , Hauner 2002 ). Adipose tissue is the major expression site for PPARγ ( Tontonoz et al. 1994
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Introduction
Retinoic acid and thyroid hormones regulate gene expression by binding to intracellular proteins that are members of the nuclear receptor superfamily of transcription factors. Members of this gene family activate transcription by binding to specific DNA sequences, termed response elements, that are generally located in the vicinity of target genes. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) can exert either of two effects on the transcription of target genes. In the absence of ligand, both the RAR and TR strongly repress transcription from promoters to which they bind. In the presence of an activating ligand, this repressive effect is relieved and transcription is markedly stimulated. As a result, a very large dynamic range in the level of transcriptional activity can be achieved. Recognition of DNA response elements by nuclear receptors is mediated by a central, highly conserved DNA binding domain, while ligand binding, dimerization and transcriptional activation
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The retinoids, vitamin D3 and thyroid hormone exert diverse and complex tissue-specific actions by a common mechanism within the cell nucleus. These hormones, like the classical steroid hormones, glucocorticoid and oestrogen, bind to nuclear receptor proteins and modify transcriptional activity of target genes. The receptors are members of the steroid/thyroid hormone nuclear receptor superfamily of structurally homologous ligand-responsive transcription factors which activate or repress expression of hormone-responsive target genes (Evans, 1988; Green & Chambon, 1988; Moore, 1990; O'Malley, 1990; Moore & Brent, 1991).
The receptors for 3,5,3′-l-tri-iodothyronine (T3Rs), 1,25(OH)2-vitamin D3 (VDRs) and all-trans retinoic acid (RARs) form a subclass of homologous and functionally related proteins within the steroid superfamily. The receptors can bind to DNA in the absence of ligand (Brent, Dunn, Harney et al. 1989; Graupner, Wills, Tzukerman et al. 1989), they reside in the nucleus and their response elements possess
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SUMMARY
The concentrations of progesterone receptors in uterine cytoplasm and nuclei were measured during the oestrous cycle of the rat. The concentration of cytoplasmic progesterone receptors was highest at pro-oestrus and declined at oestrus to reach lowest levels at metoestrus before rising at dioestrus. Similar changes were observed in the concentration of nuclear progesterone receptors, the highest levels being present at pro-oestrus and dioestrus. In addition, both activities A and B of RNA polymerase mirrored these alterations in nuclear receptor levels.
Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Department of Endocrinology, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 5TT, UK
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Introduction The cellular actions of glucocorticoids (GCs) are largely mediated through interactions with the glucocorticoid receptor (GR), a nuclear receptor that is found in its inactive form within the cytoplasm. Ligand
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ABSTRACT
The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH β and α subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary.
Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 μg on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen.
A marked increase in the concentrations of TSH β and α mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH β and α mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.
The influences of oestrogen on serum TSH and on TSH mRNA are consistent with augmentation of thyroid hormone effects; this influence may be mediated by an increase in the number of pituitary nuclear receptors for T3.
J. Endocr. (1987) 115, 53–59
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ABSTRACT
Female mice were used to examine the process of depletion and replenishment of cytosolic androgen receptors in submandibular glands, and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose-dependence of receptor depletion and replenishment in the cytosolic fraction, and that of receptor accumulation in the nuclear fraction were investigated. Almost 100% depletion was revealed 1 h after the injection of testosterone propionate at a dose of 500 or 50 μg testosterone/100 g body weight. With a 5 μg dose, depletion of cytosolic receptors was not complete and replenishment proceeded rapidly compared with that which occurred with the 50 or 500 μg dose. The nuclear receptor level increased 1 h after injection of testosterone, and the raised level was gradually reduced to the pretreatment level with all doses. However, the time required for this return to pretreatment level was dependent on the dose of testosterone. The change in the levels of cytosolic and nuclear androgen receptors following injection of testosterone was parallel to the level of circulating androgen. To determine the requirements for transcriptional and translational events in the replenishment process, cycloheximide and actinomycin D were given in vivo. The process of replenishment of cytosolic receptors was inhibited by the injection of cycloheximide. However, actinomycin D exerted no inhibitory effect on receptor replenishment. Neither cycloheximide nor actinomycin D had any effect on the nuclear receptor level until 6 h after the injection of testosterone. Cycloheximide or actinomycin D alone had no effect on the cytosolic or nuclear receptor level. These results suggest that receptor replenishment involves protein synthesis.
J. Endocr. (1987) 115, 411–418
