Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA

Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell.     

The binding of 3H-labelled androgen-receptor complexes to hypothalamic chromatin of neonatal mice: effect of sex and androgenization

The binding of 3H-labelled androgen-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105,000 g supernatant of hypothalamic cytosol, to hypothalamic chromatin of neonatal mice covalently coupled to cellulose was measured in vitro. Saturation binding was also determined after extraction of histones and the masking of acidic proteins with high molarities of guanidine hydrochloride. This investigation showed the presence of high-affinity, low-capacity acceptor sites for [3H]-testosterone-receptor complexes in male hypothalamic chromatin (Kd value = 0.39 x 10(-10) M and binding sites of 41 fmol per mg of DNA). Acceptor activity seems to be associated with the acidic protein fraction of chromatin. No specific acceptor sites of similar nature were found in chromatin taken from the hypothalami of female mice. On the basis of these results, it is suggested that the androgen-unresponsiveness of female mice is related to the absence of acceptors for the androgen-receptor in female mice hypothalami.     

Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.     

Binding of estradiol receptor complexes to isolated human breast chromatin

Summary The interaction of estradiol-receptor complexes and isolated human breast tumor chromatin was studied under equilibrium conditions.The estradiol-receptor complexes bound specifically to the chromatin of hormone dependent tumors and showed a single class of binding sites with a Ka of 0.96 × 10¹⁰ m ^–1 and a binding capacity of 1.5 pmoles/mg DNA. The binding was a temperature-dependent process and involved a transformation of the receptor protein.The heat-activated hormone-receptor com-plex was more active than the 8S form in the binding phenomenon. The specific interaction of estradiol-receptor complex with isolated chromatin was saturable and sensitive to conditions of temperature and ionic strength. Furthermore under optimal conditions no acceptor sites were detected in chromatin of hormone independent tumors.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

The physicochemical properties of the cytoplasmic androgen receptor in the kidneys of normal, carrier female (tfm/+) and androgen-insensitive (tfm/y) mice.

The physicochemical properties of the androgen-receptor complex in mouse kidney were determined. The sedimentation coefficient, 7.9S and Stokes radius of 82A, are compatible with an asymmetric protein [frictional ratio f/fo 1.98; axial ratio, assuming a prolate ellipsoid, 20] having a molecular weight of 270,000 daltons. The kidney receptor is a relatively acidic protein of esoelectric point (pI) 4.8 and readily precipitable with protamine sulfate or ammonium sulfate. Studies with protein-specific reagents suggest that both cysteine and tryptophan residues may be necessary for maintaining the functional configuration associated with androgen binding. The kidney receptor can promote the association of testosterone with purified DNA. These properties of the androgen receptor in mouse kidney are remarkably similar to those of male accessory sexual tissue. The receptor detected in carrier female mice (tfm/+ heterozygous for the gene for testicular feminization (tfm), has the same physical properties as that of normal mice. However, due to a decrease in receptor concentration, binding activity is only 69% that of normal. In cytosol from androgen-insensitive mice (tfm/+), a specific androgen receptor cannot be demonstrated by 5 different techniques.     

Steroid sulfates in testis tissue of prostatic cancer patients treated for six months with the gonadotropin releasing hormone agonist buserelin

In order to assess the extent of inhibition of testicular steroidogenesis during long-term treatment of prostatic cancer with GnRH agonist, we measured the intratesticular levels of 5 steroid sulfate conjugates in human testis tissue removed from patients after 6 months of intranasal treatment with buserelin. The most pronounced decreases were found in testosterone and pregnenolone sulfates, to 1.6 and 7.1%, respectively, of concentrations measured in testis tissue from primarily orchiectomized prostatic cancer patients. In contrast, clearly smaller decreases were found in three other steroid sulfates measured, those of dehydroepiandrosterone (to 26%), 17-hydroxyprogesterone (to 27%) and 5-androstene-3 beta, 17 beta-diol (to 62%). These results are in keeping with our previous analyses of unconjugated steroids in similar tissue samples, and indicate that testicular steroidogenesis per se is not totally blocked by long-term intranasal treatment with GnRH agonist. Testicular steroid sulfate conjugation may be specifically suppressed since the total concentration of these conjugates decreased more than free steroid levels in our earlier measurements.     

The use of deoxyribonucleic acid–cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid–receptor complexes

1. Two characteristic properties of the specific high-affinity steroid-binding proteins or receptors, their ability to bind to DNA-cellulose and their relatively acidic isoelectric point, have been exploited as a means of purification. These two fundamental properties distinguish the receptors from the steroid-binding proteins in serum and the non-specific low-affinity steroid-binding proteins in hormone-responsive cells. 2. A significant degree of purification of both cytoplasmic and nuclear steroid-receptor complexes can be achieved with practical facility by these procedures. The purity of the receptor complexes is sufficient to enable studies on their possible control of metabolic processes to be investigated in the future. 3. After extensive purification the physicochemical properties of the cytoplasmic androgen-receptor complex, such as sedimentation coefficient, were unchanged. Further, the purified complex fully retained at least one of its fundamental physiological properties, namely the ability to transfer 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) into chromatin in vitro. 4. The methods may also be employed for studying the changes in the structure and properties of the receptor complexes that are an essential prerequisite for the transfer of cytoplasmic receptor complexes into nuclear chromatin. The temperature-dependence of the binding of androgen-receptor complexes into chromatin is essentially due to a major change in cytoplasmic receptor complex before its attachment to nuclear chromatin. 5. The resolution of these analytical procedures was sufficient to enable a critical comparison of the receptor proteins from different male accessory glands to be undertaken. From these studies, no substantial evidence in support of the tissue specificity of androgen receptors could be established; rather the receptors from different androgen-dependent glands were remarkably similar in physicochemical properties. 6. Although the methods were initially developed for the partial purification of androgen-receptor complexes, they are equally suitable for the prompt and extensive purification of oestrogen-receptor and progesterone-receptor complexes.     

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1. A system has been developed for the specific transfer of [(3)H]dihydrotestosterone-receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [(3)H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [(3)H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.     

The binding of 3H-labelled oestradiol- and progesterone-receptor complexes to hypothalamic chromatin of male and female sheep.

Chromatin isolated from hypothalamic nuclei of sexually mature entire male and female sheep was linked to cellulose in u.v. light. The saturation binding of 3H-labelled oestrogen- and progesterone-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105000g supernatant of hypothalamic cytosol, was then measured in vitro in 0.15m-KCl. Saturation binding was also measured after extraction of histones and masking acidic proteins. Salt + urea was observed to be more effective than guanidine hydrochloride in unmasking receptor acceptor sites, and the binding of labelled receptor complexes to dehistonized unmasked chromatin was shown to be largely resistant to 0.4m-KCl extraction. Whereas extents of receptor-complex binding were similar to published values for comparable preparations of hen oviduct chromatin, no sex-related difference was observed. However, binding of progesterone-receptor to chromatin was greater than that of oestradiol-receptor. Binding also increased more after removal of histones and masking acidic proteins, suggesting the presence of a greater number of progesterone-receptor acceptor sites in hypothalamic chromatin than of estradiol-receptor acceptor sites. The failure to demonstrate a sex-related difference in oestradiol-receptor binding to hypothalamic chromatin in vitro is discussed.     

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

Partial purification and characterisation of the human skin fibroblast androgen receptor: detection of abnormal receptor complexes in cells from patients with androgen insensitivity syndromes

After incubation of hGSF with [3H]5 alpha-dihydrotestosterone, 17 beta-hydroxy-7 alpha, 17 alpha-dimethyl-4-estrene-3-one, or 17 beta-hydroxy-17 alpha-methyl-4,9,11-estrien-3-one, androgen-receptor complexes were extracted with 0.5 M KCl and precipitated by 35% ammonium sulphate. Receptor complexes from control hGSF sedimented at approximately 4S on linear 5-20% sucrose gradients. The 4S peak was diminished or absent in cells from androgen insensitive patients exhibiting absent, deficient or unstable binding of androgens in intact hGSF. This procedure may be a useful means of distinguishing quantitative and qualitative defects in androgen binding to receptor, since one cell line found to have normal levels of androgen receptor complexes in whole cell assays had a profile resembling that of receptor negative cells on sucrose gradients. The complexes from one patient with complete androgen insensitivity having normal androgen binding in intact hGSF were indistinguishable from control complexes after sucrose gradient analysis and ADP-Sepharose chromatography. Receptor complexes were eluted from the ADP-Sepharose between 0.5-1.0 M KCl. HPLC-gel filtration of androgen receptor complexes at 22 degrees C revealed two peaks, the larger had a Mr of 60-65K, Stokes radius of 3.16 nm and a frictional ratio between 1.21 and 1.43. The second peak, Mr of 15K, was believed to represent a fragment of the receptor containing the steroid binding domain. On gel filtration at 22 degrees C the complexes from a patient with partial androgen insensitivity, who showed a diminished 4S receptor peak on sucrose gradients, revealed only the small "meroreceptor" fragment, suggesting that the mutation in this individual might render the androgen receptor more susceptible to proteolysis in vitro.     

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium.

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 X 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.