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The measurement of oestrogen receptors in the nuclei of cells of human breast cancer is becoming increasingly important for patient management. However, the steroid-binding properties of the oestrogen nuclear receptor of human cells under different conditions of temperature and ionic strength have received little attention despite the relevance of the receptor for interpretation of assay data. This paper reports a study on the influence of temperature and ionic strength on the exchange rate of [³H]oestradiol from human breast and endometrial nuclear receptor.

When the oestrogen–nuclear receptor complex was bound to intact or sheared nuclei, the displacement of bound [³H]oestradiol into buffer containing excess unlabelled oestradiol increased with temperature but was significant over 24 h even at 4 °C for nuclei from both breast and endometrium. The use of protease inhibitors combined with relabelling of nuclear receptor after incubation confirmed that the observations at 4 °C represented exchange of hormone rather than degradation of the hormone–receptor complex. Degradation was seen at higher temperatures.

Measurement of the on-rate of [³H]oestradiol onto nuclear receptor prefilled with unlabelled oestradiol showed that on-rate was also significant over 24 h at 4 °C The displacement of oestradiol from salt-extracted, hydroxylapatite-precipitated receptor also increased with temperature although, in this case, no displacement could be detected until temperatures above 4 °C were reached.

Only 40% of total oestrogen receptor detectable in intact human nuclei was solubilized by the standard method of salt extraction in 0·6 mol KC1/1. As the salt concentration was raised (0–0·6 mol KC1/1), an increase in the stripping of oestradiol from the hormone–receptor complex was observed. Such stripping of nuclear receptor during salt extraction would lead to a false impression of the proportion of 'empty' nuclear receptors.

The results show that filled oestrogen nuclear receptor from human tumour tissue may be assayed by exchange at 4 °C over 24 h. This eliminates the protease degradation of receptor which occurs at higher assay temperatures.

ABSTRACT

The correlation between the occurrence of Graves' ophthalmopathy and Graves' hyperthyroidism may indicate a role for tri-iodothyronine (T₃) hormone in the pathogenesis of Graves' ophthalmopathy. In Graves' ophthalmopathy the recti eye muscles are greatly enlarged whereas skeletal muscles seem unaffected. The distribution of the nuclear T₃ receptor was studied in normal human and rat eye and skeletal muscles with immunohistochemistry using mouse (monoclonal) antibodies, and by in-situ hybridization for the detection of mRNA encoding the T₃-receptor protein.

Nuclear staining with T₃-receptor antibodies was found in all types of tissues studied. Cytoplasmic staining occurred predominantly in the muscle fibres of the orbital layer of the eye muscles and was generally absent or very low in skeletal muscle fibres and hepatocytes. Immunostaining could be inhibited by preabsorbing the antibodies with bacterially expressed T₃-receptor protein, implying specificity. The presence of nuclear and cytoplasmic hormonefree T₃ receptor sites was indicated after preincubation of sections with T₃ hormone: T₃-receptor immunostaining decreased and T₃-hormone staining increased. In-situ hybridization clearly revealed the presence of α-1 and β-1 forms of the T₃-receptor mRNA in liver, skeletal muscles, and orbital and intermediate layers of the eye muscles.

The data demonstrate the presence of T₃ hormone-receptor molecules in the extraocular and skeletal muscles. The different susceptibilities of these muscles to Graves' hyperthyroidism may relate to the quantitative differences in T₃ hormone-receptor distribution.

Journal of Endocrinology (1992) 133, 67–74

ABSTRACT

Previous studies have shown that progesterone rapidly inhibits retention of uterine nuclear oestrogen receptor in several mammalian species. This effect of progesterone may constitute a general mechanism by which progesterone modulates oestrogen action. The objective of the present study was to examine the temporal pattern of progesterone inhibition of retention of occupied nuclear oestrogen receptors in the rat uterus at various sustained serum concentrations of progesterone. Silicone elastomer implants (1 cm) packed with crystalline oestrogen were placed s.c. in the flank region of ovariectomized adult rats. Twenty-four hours after placement of the implants, animals were either injected s.c. with 5 mg progesterone in corn oil every 24 h, treated with 2 × 5 cm implants of progesterone, or treated with 1 × 5 cm silicone elastomer implants of progesterone. Serum concentrations of progesterone at the time of necropsy were 0·47 ± 0·02, 0·18 ± 0·02 and 0·10 ± 0·01 μmol/l respectively. Control animals were given oestrogen implants alone and had a serum progesterone level of 0·03 ± 0·01 μmol/l. Occupied nuclear oestrogen receptor and cytosolic oestrogen and progesterone receptor levels (pmol/uterus) were measured between 0 and 48 h following progesterone treatment. Cytosolic progesterone receptor levels were suppressed similarly in all progesterone-treated groups compared with controls given oestrogen alone throughout the 48-h test period. Cytosolic oestrogen receptor levels were significantly suppressed at 12 h following progesterone treatment in all groups. Except for the highest (pharmacological) serum progesterone concentration, cytosolic oestrogen receptor exhibited a replenishment phase between 12 and 48 h. Although pharmacological levels of serum progesterone (0·47± 0·02μmol/l) significantly suppressed the level of occupied nuclear oestrogen receptor at 12 h, the level increased subsequently at 24 and 36 h and was equivalent to the control value (oestradiol alone) by 48 h. At a mean serum concentration of 0·18 ± 0·02 or 0·10 ± 0·01 μmol/l, the level of occupied nuclear oestrogen receptor was also suppressed significantly at 12 h, but to a lesser degree. These results show that progesterone decreases the concentration of occupied nuclear oestrogen receptor in the rat uterus and that the duration and extent of suppression of occupied nuclear oestrogen receptor is related to the serum progesterone concentration.

Journal of Endocrinology (1989) 121, 101–107

SUMMARY

A method has been developed which allows the estimation of occupied and unoccupied androgen receptor sites in both cytoplasmic and nuclear fractions of rat ventral prostate. The procedure involves precipitation of receptor proteins and incubation of precipitates with labelled 5α-dihydrotestosterone. Uptake of ³H-labelled steroid at 0–4 °C gives an indication of free receptor, whereas binding at a raised temperature (15 °C) allows estimation of occupied receptor. Non-specific binding was measured in the presence of a 100-fold excess of unlabelled 5α-dihydrotestosterone. The exchange method was specific for androgens, and specific binding was detected only in fractions of androgen-dependent tissues. The method can be applied to cytosol, whole nuclei, chromatin and salt-extractable and salt-resistant protein preparations from nuclear fractions, and gives a reliable estimate of total receptor sites when occupied as compared with control measurements of unoccupied sites.

ABSTRACT

Rat liver nuclear thyroid hormone receptor lost 3,5,3′-tri-iodo-l-thyronine (T₃)-binding activity with a half-life of 14 days, 4 h, 139 min, 62 min, 16 min or 6 min at 0, 36, 38, 40, 43 or 45 °C respectively, when present in crude nuclear extracts. Glycerol increased the half-life of the receptor during heat inactivation. Protection was reversible by removing the glycerol. The receptor was unstable at a pH below 6·0 or above 10·0. We also found a loss of the receptor activity during the separation of bound and free hormone using the resin test. Of several conditions tested for the separation of bound and free hormone, the addition of heated nuclear extract gave the most accurate estimation of bound hormone when using the resin test. Using these characteristics of the receptor, we purified the receptor to 1220 pmol T₃-binding capacity/mg protein with a final yield of 14·6 μg/4 kg rat liver.

Journal of Endocrinology (1989) 120, 237–243

ABSTRACT

Previous work has demonstrated that thyroid hormones influence testis development. Specific receptors for tri-iodothyronine (T₃) have been demonstrated in Sertoli cells. The aim of the present study was to examine the possible effect of thyroid hormone on its own receptor during pubertal development by evaluating the influence of thyroid status on T₃-binding capacity, -binding affinity and receptor occupancy in nuclei isolated from immature rat testes. The binding capacity for T₃ of nuclei from rat testis significantly decreased during pubertal development, being 375±32, 117±15 and 44±7 fmol/mg DNA in 7-, 21-and 35-day-old rats respectively, whereas the affinity of binding, as evaluated by the dissociation constant (K _d), did not change. Early induced hypothyroidism significantly affected the time-course of the postnatal decline of nuclear T₃ receptors in the testis. At 21 days of age, the binding capacity for T₃ in the testis of methimazole-treated rats was significantly higher with respect to euthyroid controls, being 173 ±21 and 117 ± 15 mol/mg DNA respectively, while the K _d was unaffected. T₃ replacement therapy completely prevented changes in T₃ receptor number induced by hypothyroidism without modifying the K _d. Our results indicate that nuclear T₃ receptors in the developing rat testis are modulated by thyroid hormone.

Journal of Endocrinology (1993) 136, 277–282

The binding of [³H] 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to progesterone receptors in cytosol and nuclear extracts (0·6 m-KCl) of the pregnancy-dependent, TPDMT-4 mouse mammary tumour was measured at various stages of pregnancy. Compared with conventional dextran-coated charcoal (DCC) assays, a hydroxylapatite assay with DCC pretreatment and precharging of the cytosol with unlabelled R5020 (4 × 10⁻⁸ mol/l, for 3–4 h at 4 °C) showed the highest level of binding. The DCC treatment markedly increased the level of R5020 binding in both cytosol and nuclear extracts by allowing the receptor to bind to hydroxylapatite. The DCC pretreatment apparently removed a heat-stable and non-dialysable factor which prevented the receptor from binding to the hydroxylapatite. Using this assay R5020 binding reached a steady state in 24 h at 4 °C, with complete exchange of radioactive for non-radioactive ligand by 20 h. Nuclear extracts did not require precharging and complete exchange was more rapid. Scatchard analysis (without precharging) disclosed a single class of binding sites with a dissociation constant for cytosol of 3·2 ± 0·8 (s.e.m.) × 10⁻⁹ mol/l (n = 3) and 4·7 ± 0·6 × 10⁻⁹ mol/l (n = 5) for the nuclear extract. Binding was hormone-specific and progesterone translocated binding from the cytoplasm to the nucleus both in vivo and in vitro. Translocation, however, led to a substantial loss of total (nuclear + cytoplasmic receptors.

During pregnancy, cytoplasmic progesterone receptor levels were unchanged and low compared to nuclear progesterone receptors which increased by sevenfold from days 1 to 11 and then decreased at day 16. Compared with recent data on cytoplasmic progesterone receptors in normal mammary gland, our results suggested that this tumour may have a reduced sensitivity to the down-regulatory activity of progesterone. This lesion may, in part, account for the failure of the tumour to differentiate during pregnancy.

ABSTRACT

In the ovariectomized adult rat uterine oedema induced by 0·01 and 0·1 μg oestradiol-17β/100 g body weight increased further in the presence of theophylline. Nuclear retention of oestrogen-receptor complexes also increased in response to theophylline both in vivo and in vitro. Theophylline decreased the number of eosinophils in the blood and concurrently decreased oestrogen-induced uterine eosinophilia at doses of 0·001, 0·01, 0·1, 1, 10 or 30 μg oestradiol/100 g body weight, through a mechanism independent of glucocorticoids. There was, therefore, no correlation between changes in the number of uterine eosinophils and changes in uterine wet weight induced by theophylline and oestrogen. It is suggested that the presence of oestrogen-receptor complexes in the nucleus for at least 4 h is a prerequisite for the induction of uterine oedema and growth in the presence of theophylline and oestradiol-17β.

J. Endocr. (1985) 105, 397–403

The present paper describes studies conducted to detect and characterize the nuclear receptor for oestrogen in the baboon endometrium. Only 10% of the [³H]oestradiol nuclear receptor complexes were extracted with a 0·5 m-KCl solution. This solubilized receptor migrated as a 4·4S peak during 5–20% sucrose gradient centrifugation. The oestrogen receptor was not bound to oestrogen in the nuclei under normal physiological conditions. Using an unlabelled competitor addition technique with intact nuclei the variation in oestrogen-receptor concentration of baboon endometrium during the menstrual cycle was measured. This concentration increased slightly during the first week of the cycle, being maximal on day 7 before ovulation (2500 molecules/cell), then decreasing gradually, reaching the lowest level (300 molecules/cell) on day 5 after ovulation, where it remained until the end of the cycle.