Evidence for an androgen receptor in the seminiferous tubules of the human testis

Androgen receptors (AR) were studied in seminiferous tubule cytosol and testicular nuclear extracts prepared from testes of previously untreated elderly men undergoing orchiectomy as therapy for prostatic carcinoma. Cytosol exhibited high affinity (Kd = 0.8 nM), saturable binding of [3H]methyltrienolone; however, the synthetic progestin, promegestone was a stronger competitor for MT binding sites than were 5 alpha-dihydrotestosterone (DHT) or testosterone (T), suggesting the presence of progesterone-like binding sites. Addition of triamcinolone acetonide (TA) produced the usual relative steroid specificity for AR binding and reduced the measured AR binding capacity by 19 +/- 8% (Mean +/- SD, n = 3). The umber of MT binding sites was 30-40 fmol/mg protein, or an average of 65 fmol/g testis, and the equilibrium dissociation constant at 0 degrees C was 0.6-1.4 nM. In the presence of sodium molybdate, binding was stable for 40 h at 0 degrees C and the half-time of dissociation of the MT-AR complex was 12-16 h. The binding of salt extractable (600 mM KCl) nuclear sites to MT was saturable and was specific for androgens. The number of binding sites in nuclear extracts was 170 fmol/g testis and the apparent equilibrium dissociation constant was 4.2 nM. Thus, the binding of MT to human seminiferous tubule cytosol and testicular nuclear extract exhibits properties which are nearly identical to those of the prostate AR. Further study of this androphilic protein may provide insight into the role of androgen in normal and abnormal spermatogenesis in man.     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.     

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

Estrogen and androgen receptors in the liver of cynomolgus monkeys (Macaca fascicularis)

Estrogen receptors (ER) and androgen receptors (AR) were evaluated in the hepatic cytosol from cynomolgus macaques to determine if there were differences associated with gender and endogenous hormone secretion. Saturable, high affinity binding (Kd = 0.2-0.8 nM) was demonstrated for both ER and AR from either male or female monkeys. Displacement of tritiated estradiol from the ER was estrogen specific (including ethinyl estradiol). Both androgens and the synthetic progestins (levonorgestrel and norethindrone) displaced tritiated mibolerone from the AR. Both 8S and 4S molecular forms of ER and AR were demonstrated on 5-20% sucrose density gradients. The ER levels were higher in females in the follicular phase of the menstrual cycle (40.5 +/- 1.9 fmol/mg protein) than levels in males (26.4 +/- 4.8 fmol/mg protein; P less than 0.01) or levels in luteal phase females (31.8 +/- 2.4 fmol/mg protein; P less than 0.05). AR levels were not different between females during different phases of the menstrual cycle (65.8 +/- 4.6 and 69.5 +/- 4.3 fmol/mg protein, follicular and luteal, respectively), but there was a tendency (P less than 0.10) for the levels in males (54.4 +/- 6.6 fmol/mg protein) to be lower than female levels. The demonstration of saturable, high affinity binding of androgens and estrogens in liver tissue of these primates, along with differences associated with gender and the stage of the menstrual cycle, suggests that hepatic receptors are functional and may play an important role in hepatic protein secretion.     

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.     

Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate

Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms (Ka = 0.5-2.9 x 10(10) M-1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates.     

Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone (3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 +/- 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 +/- 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 +/- 0.6 nM) on the other hand was not different from that (1.7 +/- 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.     

Tamoxifen decreases progesterone and nuclear androgen receptors in the human prostate

Androgen (AR) and progesterone (PR) receptors were measured in resected prostate tissues of patients with benign prostatic hypertrophy. One group of patients received an anti-estrogen, tamoxifen (Tm 20 mg b.i.d.) for 10 days prior to prostate resection; a second group served as controls and were untreated. Plasma levels of Tm were 200-500 pmol/ml at the time of surgery. Statistically significant decreases (P less than 0.05) were found in cytosol PR (154 fmol/mg DNA +/- 33 SE in 14 Tm-patients vs 266 +/- 40 SE in 13 untreated patients) and in nuclear AR (103 fmol/mg DNA +/- 70 SE in 18 Tm-patients vs 257 +/- 62 SE in 17 controls). Cytosol AR was not significantly different in Tm-treated patients (257 fmol/mg DNA +/- 79 SE in 15 Tm-patients vs 346 +/- 130 SE in 17 controls, P greater than 0.6). Although receptor recycling is one of several possible explanations, these decreases in progesterone and nuclear androgen receptors in Tm-treated patients suggest that estrogen has a role in the biological regulation of steroid receptors in the human prostate.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.     

Interaction of dexamethasone-receptor complexes with rat liver nuclei and DNAs]

The role of DNAs in the nuclear binding of dexamethasone-receptor complexes (DRC) was studied. The cytosolic receptors from rat liver have a sedimentation coefficient of about 7S, the Stock's radius--of about 50 A and possess a high affinity to dexamethasone (Kas = 2,6 X 10(8) M-1). Their capacity is 3 X 10(-13) and 5.5--7.0 X 10(-12) mole of dexamethasone per mg cytosolic protein and mg DNA, respectively. DRC has the ability to bind to the nuclei of rat liver. DRC binding to nuclei is increased approximately 3-fold by temperature activation of cytosol. The nuclear acceptor sites are saturated at the level of 16.2 pmoles of bound DRC per mg nuclear DNA. Free DNA has the ability to compete with nuclei for binding with DRC. Temperature-activated DRC can bind both with homo- and heterologous DNAs. Secondary DRC-DNA complexes were isolated by means of gel filtration on Sepharose 4B. Thermal denaturation of DNA decreases its ability to bind DRC approximately 2-fold. DNAs of a similar nucleotide composition, i.e. DNA from rat liver (GC = 43 mole%) and DNA from Photobacterium belozerskii (GC = 44 mole%), have a close DRC-binding ability. At the same time, these DNAs bind about 1.5-fold less DRC, as compared to DNA from Pseudomonas aeruginosa (GC = 67 mole%) and about 1.5-fold more, than does DNA from T2 phage (GC = 35 mole%). Thus the positive correlation between the GC composition of DNA and its DRC-binding ability was established. Unique sequences (Cot greater than 600) bind several times less DRC than the reiterated sequences (also denaturated) (Cot = O--600) of rate liver DNA. Thus, DNA can be considered as a nuclear acceptor of DRC. It is assumed, that DRC is able to recognise in DNA certain short GC-rich sequences, distributed in the rate genome in a non-random fashion.     

Phosphorylation and nuclear processing of the androgen receptor.

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Mineralocorticosteroid receptor of the chick intestine. Oligomeric structure and transformation

The binding of [3H]aldosterone in the chick intestine cytosol was analyzed in terms of affinity and specificity. In this tissue, aldosterone binds to the mineralocorticosteroid receptor, with a high affinity (Kd approximately 0.3 nM) and low capacity (approximately 50 fmol/mg protein), and to the glucocorticosteroid receptor. The selective labeling of the mineralocorticosteroid receptor was achieved by incubating the cytosol with [3H]aldosterone in the presence of RU 486. This synthetic steroid completely inhibited the binding of [3H]aldosterone to the glucocorticosteroid receptor and did not bind to the mineralocorticosteroid receptor. The oligomeric structure of the mineralocorticosteroid receptor was studied by using BF4, a monoclonal antibody which reacts with the 90-kDa heat shock protein (hsp 90), a nonhormone-binding component of nontransformed steroid receptors. The mineralocorticosteroid receptor sedimented at 8.5 +/- 0.4 S (n = 8) in a 15-40% glycerol gradient. This peak was shifted to 11.2 +/- 0.6 S (n = 5) after incubation with BF4, indicating that, in the cytosol, hsp 90 was associated with the mineralocorticosteroid receptor. Dissociation of the complex was observed on gradients containing 0.4 M KCl, as judged by the absence of displacement by BF4 of the 4.3 +/- 0.4 S (n = 10) peak. The effect of molybdate and tungstate ions, and of dimethyl pimelimidate, an irreversible cross-linking agent, on the stability of the hsp 90-receptor complex was investigated. Complexes recovered in the presence of 20 mM molybdate ions dissociated on gradients containing 0.4 M KCl (5.2 +/- 0.6 S (n = 4), whereas complexes prepared in the presence of 20 mM tungstate ions sedimented at 8.5 +/- 0.4 S (n = 7). Similarly, complexes prepared in the presence of molybdate ions dissociated during high pressure liquid chromatography (HPLC) gel filtration analysis performed in 0.4 M KCl (RS (Stokes radius) = 3.9 +/- 0.5 nm (n = 3) versus 7.3 +/- 0.2 nm (n = 3) in the presence of 20 mM molybdate ions), whereas complexes prepared in the presence of tungstate ions did not dissociate (RS = 6.9 +/- 0.2 nm (n = 3]. As observed for the tungstate-stabilized receptor, the cross-linked receptor dissociated neither on gradient containing 0.4 M KCl (9.5 +/- 0.1 S (n = 3] nor during HPLC performed in 0.4 M KCl (RS = 6.5 +/- 0.3 (n = 4]. Furthermore, the cross-linked receptor was more resistant to the inactivating effect of urea on aldosterone binding than the noncross-linked receptor prepared in the presence of either molybdate or tungstate ions.     

Prolactin binding in the developing rat fetal liver

The binding of prolactin by fetal rat liver cell membrane fractions from 17 to 21 days gestation was studied. Particulate liver membranes were prepared in Dulbecco's Phosphate Buffered Saline (PBS) by ultracentrifugation and incubated at 22 degrees C for 16 hours with [125I] iodo-human growth hormone (hGH). Non-specific binding was assessed by parallel incubations in the presence of a 2000-fold excess ovine prolactin. Specific prolactin binding sites were detected only at 21 days gestation (2932 +/- 401 cpm/mg protein) in freshly prepared membranes. On freezing at -20 degrees C for 24 to 48 hours, the membranes of 20 days gestation animals were able to specifically bind prolactin (1295 +/- 239 cpm/mg protein). Freezing led to a 45 +/- 7% increase (4270 +/- 701 cpm/mg protein) in prolactin binding at 21 days gestation. No hormonal binding was detected from 17 through 19 days gestation in either fresh or freeze-thawed membranes. Scatchard analysis revealed a high affinity binding site with a Ka of approximately 1.4 X 10(8)M-1 in both fresh and freeze-thawed membrane preparations. The data show that 1) prolactin receptors appear in liver only during late fetal life and that 2) freezing of membranes may unmask binding sites that are initially unavailable to specifically bind prolactin.     

A cytoplasmic oestrogen-binding component in chicken liver.

A macromolecular component in the liver cytosol from laying hens as well as roosters, protein in nature and sedimenting at 4S, was shown to bind oestradiol. The dissociation constant (Kd) of the complex is approximately 5 X 10(-6)M. No binding component with a higher affinity for oestradiol was detectable in the cytosol. The binding is specific for the tissue and hormone, with the exception that progesterone also shows some affinity for this 4S component. The number of binding sites is about 330 pmol/mg cytosol protein. This number is not altered significantly after treatment of a rooster with oestrogen (24 h) or with cycloheximide (3 h). The cytoplasmic complex (oestradiol-4S-component) does not enhance the binding of oestradiol to the chromatin from rooster liver. The nuclear complex (oestradiol bound to the soluble nuclear receptor seems to be more effective in doing so.     

Cytosol androgen receptor (AR) in human skin fibroblasts: characterization of the binding reaction and differentiation from androgen binding molecules of lower affinity

Androgen binding was studied in cytosol of human fibroblasts at 4 degrees C. When 5 alpha-dihydrotestosterone (DHT) was the ligand, a curvilinear Scatchard plot was seen, which was resolved into two components: I the androgen receptor (AR), Kd = 0.12-0.44 nM, and II a low affinity species, Kd = 6.3-28 nM. The same cytosol demonstrated only type I binding for 3H-methyltrienolone (MTr), Kd = 0.10-0.40 nM. The AR, i.e., 3H-MTr binding activity, eluted at 440,000 d by gel filtration chromatography in pre-labeling and post-labeling experiments. When the ligand was 3H-DHT, binding activity in the 10,000-45,000 d range was seen in addition to AR. Thus, saturable nonreceptor steroid binding was seen for DHT but not for MTr. The latter is the preferred ligand for the study of the AR in this system.     

Estrogen binding component in the pilosebaceous tumor developed in Suncus murinus

We transplanted a pilosebaceous tumor developed on the sidegland of Suncus murinus to male nude athymic (BALB/c-nu/nu) mice. This tumor can be transplanted to female hosts as well, with a lower rate of graft-taking and slower growth rate. In this study we demonstrated the presence of macromolecules which specifically bind to estrogen. Measurement of 17 beta-estradiol (E2) binding by a dextran-coated charcoal assay revealed that the number of binding sites and the dissociation constant were 22.3 +/- 4.6 fmol/mg protein and 1.4 +/- 0.24 X 10(-9) M, respectively. This binding was specific for E2 and diethylstilbestrol (DES). Sucrose gradient centrifugation of the [3H]E2-labeled cytosol yielded a sharp peak of radioactivity at 3.5S-4S under high salt conditions and a 9S peak with a shoulder at 3.5S under low salt conditions. This 3.5S shoulder was due to dissociation of [3H]E2 from the 9S peak during the centrifugation, since only the 9S peak was obtained by postlabeled density gradient analysis. An assay of the in vivo binding of [3H]E2 showed significant radioactivity in the nuclear extract from the tumor. This nuclear uptake was markedly decreased by simultaneous administration of 100-fold excess of E2. In tumor-bearing castrated nude mice, 1-100 micrograms/day of E2 did not affect tumor growth, whereas it counteracted the stimulative effect of testosterone propionate.     

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

1. Binding of l-tri-[(125)I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25 degrees C. 2. The l-tri-[(125)I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100 degrees C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at -38 degrees C for several months. 3. It displayed saturability, high affinity (apparent K(d) 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3'-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[(125)I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304+/-0.11mug/mg of cytosol protein (mean+/-s.d., n=4) was obtained; the mean concentration in plasma was 0.309+/-0.07mug/mg of plasma protein (mean+/-s.d., n=3). 7. The K(a) value of 6.3x10(8)m(-1) of l-tri-[(125)I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[(125)I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.