Hepatic estrogen receptors and plasma estrogen-binding activity in the Atlantic salmon

Livers of male and female immature Atlantic Salmon (Salmo salar) contain specific high-affinity [3H]estradiol binding sites in cytosol (Kd 2-4 nM, concentration about 0.6 pmol/g liver). Low levels of high-affinity binding are detectable in salt extracts of nuclei of untreated fish, but injections of estradiol result in transient depletion of the cytosol binder and in accumulation of high levels of binding sites in nuclear salt extracts (Kd 5-6 nM; concentration about 6 pmol/g liver). Both the cytosol and nuclear binding sites are temperature sensitive and are optimally assayed by incubation at 2 degrees. Both are specific for estradiol and diethylstilbestrol (DES) and no significant competition by dihydrotestosterone (DHT), progesterone, or hydrocortisone is seen. The triphenylethylene nonsteroidal antiestrogen, 4-hydroxytamoxifen, exhibits an affinity comparable to that of estradiol. The nuclear binding activity sediments with a coefficient of 3.6 S in salt-containing sucrose density gradients, and is stable on storage at -20 degrees for several months. The cytosol binder on the other hand is not stable on sucrose density gradients or on prolonged storage. Salmon plasma contains two [3H]estradiol binding components, one with a relatively high affinity for [3H]estradiol (kd 13 nM) and the other having a much lower affinity but present in high concentrations. The high-affinity plasma binder exhibits distinctive specificity with no affinity for DES or 4-hydroxytamoxifen but some affinity for DHT and progesterone. These properties serve to distinguish the plasma activity from the intrahepatic estrogen binders. The salmon liver estrogen receptor system has many features in common with typical estradiol receptors from other vertebrates. Immature salmon liver appears to be the richest source of hepatic estrogen receptor so far found for any vitellogenic species.     

Evidence for an androgen receptor in porcine Leydig cells

Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using [3H]methyltrienolone as labelled ligand. Androgen receptors in Leydig cells had high affinity for [3H]methyltrienolone and steroid binding specificity typical of an androgen receptor. The mean receptor concentrations were 76 fmol/mg protein and 210 fmol/mg DNA for cytosol and nuclei, respectively. In sucrose gradients, cytosol androgen receptors sedimented in the 4 S region. The cells maintained androgen receptors under culture conditions. Exposure of cultured cells to [3H]methyltrienolone (10 nmol/l) resulted in accumulation of androgen receptors in the nuclei with maximal uptake by 1 h. We conclude that methyltrienolone binding sites with characteristics of androgen receptors were identified in both cytosol and nuclei of porcine Leydig cells.     

The testicular estrogen receptor system in two strains of mice differing in susceptibility to estrogen-induced Leydig cell tumors.

Cytosol obtained from cryptorchid testes of tumor-susceptible BALB/c and resistant C3HBi (Z) mice both bound 17 beta-estradiol (E2) and diethylstilbestrol (DES) specifically. The dissociation constant (Kd) of this binding component (RE) for E2 was determined to approximate 5 x 10(-9) M. Gel filtration of cytosols resulted in a significant increase in the binding constant (Kd approximately 3 x 10(-10) M) with the majority of the complex migrating in the 7-8S area after sucrose gradient centrifugation. Incubation of either untreated or gel-filtered cytosol with [3H]DES resulted in considerable nonspecific binding appearing in the 4S region in a low salt sucrose gradient. This 4S binding of [3H]DES was not inhibited by the addition to the incubation mixtures of a 100-fold excess of either E2 or DES, while the lesser peak at 7-8S as well as the major 7-8S peak formed with E2 were inhibited by both. In vitro translocation of the cytosol RE to the nucleus was demonstrated in both mouse strains using either estrogen. Quantitation of the in vivo translocation, employing the exchange method after a single injection of 2.5 micrograms E2/mouse, revealed a rapid increase in cytoplasmic receptor content accompanied by a concomitant increase in nuclear receptor content. Greater nuclear receptor content was identified in nuclei from BALB/c mice than in those from Z animals 45 min after injection of E2. The binding behavior of E2-RE complexes to nuclei was studied by the KCl extraction method. The percent extracted from the nuclei in the Z strain was significantly greater than that in the BALB/c at all concentrations of KCl tested. Essentially 100% of the RE was extracted from nuclei of Z animals at 0.4 M KCl, while nuclei of BALB/c mice retained 35-40% even in 2 M KCl. Cross-over experiments in a cell-free system suggested that the difference in binding was due to differences in chromatins rather than in nuclear estrogen-receptor complexes. The greater nuclear receptor content and stronger binding of nuclear receptor to chromatin might explain why estrogen-induced phenomena, including neoplastic transformation occur to a much greater degree in the BALB/c strain than in the Z strain of the mouse.     

Catecholamine synthesis inhibitors acutely modulate [3H]estradiol binding by specific brain areas and pituitary in ovariectomized rats

Drugs known to alter endogenous levels of catecholamines were administered to adult ovariectomized rats to assess catecholaminergic effects on estradiol (E2) uptake and binding in nuclear and supernatant fractions of pituitary and specific brain regions and on cytoplasmic E2 receptor numbers and affinities. Specific (i.e. diethylstilbestrol-blockable) binding in vivo was measured 1 h after the iv injection of [3H]E2 (1 micrograms/kg). Administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (alpha MPT) 2 h before [3H]E2, to reduce levels of dopamine (DA), norepinephrine (NE), and epinephrine (Ep), decreased total and specific [3H]E2 binding by 36-56% in the nuclear fraction of the anterior pituitary, basal hypothalamus, and anterior hypothalamus. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDC), administered 2 h before [3H]E2 to reduce levels of only NE and Ep, increased the total and specific uptake of [3H]E2 by 62-140% in nuclear and supernatant fractions of the anterior pituitary and also increased uptake in several brain areas. In vitro analysis of hypothalamic and pituitary cytoplasms showed that in vivo administration of DDC increased E2 binding. Scatchard analysis showed that DDC increased receptor numbers 18-29%, with no change in the dissociation constant in pituitary cytoplasms. At the same time, plasma PRL levels were reduced by DDC treatment, indicating that DDC had increased DA output. Phenoxybenzamine (a blocking agent at alpha 1 postsynaptic binding sites) and a high dose of clonidine (a pre- and postsynaptic alpha-receptor agonist) did not significantly alter specific uptake in the cell nuclear fraction of any tissue, suggesting that postsynaptic alpha-receptors do not play a major role in modulating [3H]E2 uptake. No drug altered plasma levels of radioactivity. Because alpha-methyl-p-tyrosine and DDC both inhibit synthesis of NE and Ep, it is suggested that their opposite effects on uptake of [3H]E2 are related to their opposite effects on DA output. This interpretation is compatible with our previous observations that DA agonists increase [3H]E2 uptake in brain and pituitary in ovariectomized rats.     

Receptors for 17beta-estradiol in prolactin-secreting rat pituitary cells

Estrogens stimulate prolactin (PRL) synthesis by GH3 cells, a clonal strain of rat pituitary cells grown in culture. At 4 degrees C the binding of [3H]17 beta-estradiol to monolayer cultures of GH3 cells was specific and of limited capacity, with half-maximal and maximal binding after 1--2 h and 12 h, respectively. Scatchard analysis showed one single class of binding sites with Kd = 3.1 X 10(-10) M and n = 309 X 10(-15) mol 17 beta-estradiol/mg cell protein, calculated to give approx. 25,000 binding sites per cell. At 4 degrees C less than 10% of the specifically bound [3H]17 beta-estradiol was found in the nuclear fraction. When the incubation temperature was raised to 37 degrees C, the amount of radioactivity in the nucleus increased to 25% within 30 min with a corresponding reduction in the cytoplasm. The cytosol fractions from monolayer cultures as well as from tumors of GH3 cells contained specific 17 beta-estradiol binding proteins, having a sedimentation constant close to 8S in a salt-free buffer and 4S in the presence of 0.5 M KCl. scatchard analysis showed one single class of binding sites with Kd = 3.6 X 10(-10) M and n = 258 X 10(-15) mol 17 beta-estradiol/mg cytosol protein (GH3 tumor tissue). Thus, GH3 cells grown in culture and in the intact animal have similar binding characteristics as judged from the data for binding affinity, capacity and specificity. After the in vivo administration of [3H]17 beta-estradiol to GH3 tumor-bearing rats, radioactivity could be extracted (0.5 M KCl) from purified nuclei bound to 4.5S macromolecules. We suggest that the action of 17 beta-estradiol on GH3 cells involves an initial binding of the steroid to specific receptors in the cytoplasm, followed by transport of a fraction of the hormone-receptor complexes to the nucleus involving a temperature-sensitive step.     

Characterization and partial purification of androgen receptors from ram seminal vesicles

An androgen receptor has been demonstrated in the cytosol and in the nuclear fraction of ram seminal vesicles.The cytosol receptor was stabilized by sodium molybdate and 2 distinct [³H]methyltri-enolone-binding proteins, one sedimenting at 9S and one sedimenting at 3S, could be demonstrated by sucrose-gradient centrifugation in the presence of 50 mM molybdate. The slower sedimenting form could be partially purified by ADP-sepharose chromatography. The purified receptor still sedimented at 3S after centrifugation on sucrose gradients containing either 0.6 M KCl or 50 mM molybdate. The receptor was destroyed by heating at 50°C for 30 min and its complex with [³H]methyltrienolone dissociated slowly at low temperatures. The apparent equilibrium-dissociation constant (K_D) for the purified receptor was: 3.8 × 10^?10 M. The relative affinities for different steroids decreased in the following sequence: 5α-dihydrotestosterone ? methyltrienolone > testosterone > estradiol > R5020 > progesterone > diethylstilbestrol.The nuclear androgen receptor sedimented at 3S on sucrose gradients containing 0.6 M KCl. At pH 7.4 it behaved as an acidic protein with an electrophoretic mobility towards the anodic region of the agar gel.Because of the relatively large content of cytoplasmic and nuclear androgen receptors and the availability of large amounts of tissue the ram seminal vesicles could be a suitable source for large-scale purification of these receptors.     

Human androgen insensitivity mutation does not alter oligonucleotide recognition by the androgen receptor-DHT complex

We studied the binding of dihydrotestosterone-receptor complexes (DHT-R) from human genital skin fibroblasts to oligodeoxyribonucleotides and DNA. Following incubation of fibroblasts with 2 nM [3H]DHT (45 min, 37 degrees C), DHT-R were prepared as total fibroblast sonicates (sonication of cells in 0.5 M KCl), intact fibroblast cytosol (100000 X g supernatant) or intact fibroblast nuclear extract (sonication of nuclei in 0.5 M KCl). DHT-R were also prepared by incubation of fractionated fibroblast cytosol with 4 nM [3H]DHT (4 h, 0 degrees C). Optimal conditions were established for binding of DHT-R from total fibroblast sonicates to oligo-dT cellulose: 60 min, 0 degrees C, low salt (0.05-0.10 M KCl), linearity with DHT-R concentration, and nucleotide saturation. With total fibroblast sonicates the rank order of DHT-R binding was oligo-dT approximately equal to -dG greater than DNA greater than -dC greater than or equal to -dA approximately equal to -dI. Intact fibroblast cytosol displayed a similar preference of DHT-R binding to oligo-dT and -dG but the binding was quantitatively higher than for total fibroblast sonicates, the binding for fractionated fibroblast cytosolic DHT-R formed at 0 degrees C being quantitatively lower. However, binding of DHT-R from cytosol (0 degrees C) to DNA-cellulose was equal to that for DHT-R from cytosol (37 degrees C). Binding of DHT-R from intact fibroblast nuclear extracts was lower than for total fibroblast sonicates. Preparations from cells of patients with receptor-negative, complete androgen insensitivity lacked both DHT-R formation and specific oligonucleotide binding. Binding of oligonucleotides to DHT-R from cells of patients with receptor-positive, complete androgen insensitivity could not be distinguished from that of normal cells. These results suggest: (a) androgen receptor-steroid complexes recognize and bind to certain preferred deoxyribonucleotides; (b) various factors affect the quantitative binding of DHT-R from different cellular preparations to deoxyribonucleotides; and (c) neither qualitative nor quantitative abnormalities for DHT-R of complete androgen-insensitive patients were detectable from oligonucleotide or DNA binding.     

3H]-Thyroliberin (TRH) binding to nuclei isolated from a pituitary clonal cell line (GH3).

[3H]Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and nucleus. We studied the [3H]-TRH binding capacity of a highly purified nuclear fraction isolated by an original procedure from GH3 cells. The nuclei still presented their double nuclear envelope. They are able to bind [H]-TRH to the same extent as nuclei isolated from GH3 cells previously exposed to [3H]-TRH. The equilibrium of binding was reached after 2--5 min incubation at 25 degrees or 35 degrees C. The binding is stable at 4 degrees C and partially (50%) dissociated within 15 min at 25 degrees C. 50% of the binding was inhibited by large excess of unlabelled TRH. Nuclei obtained from a variant GH3 cell which has lost its responsiveness to TRH presented only the noncompetitive binding compartment. The binding was found dose dependent and not saturable. Two apparent dissociation constants were evaluated: 1.5--2.5 x 10--8M and 2.10--6M, respectively, for high and low doses of [3H]-TRH. The first one was identical to that previously found for intact GH3 cells. The present data show the existence of specific nuclear binding sites for TRH, establish their characteristics and suggest a possible nuclear site of action for that peptide hormone.     

The androgen receptor of the human endometrium

Available androgen binding to soluble proteins from the cytosol of human endometrium was studied using the dextran coated charcoal adsorption method and sucrose density centrifugation analysis. Specific binding of [3H]-5 alpha-dihydrotestosterone ([3H]-DHT) was observed with both methods. The apparent dissociation constant (Kd), for DHT binding is 1.3 +/- 0.2 (SEM) nM and the binding capacity 177 +/- 42 (SEM) fmol/mg protein. Sucrose density ultracentrifugation identifies specific [3H]-DHT binding that sediments at 4S and 8S. The stability of the androgen receptor in human endometrium is increased by the addition of 10% glycerol to the homogenization buffer. The addition of trypsin or pronase and heating at 60 degrees C reduces specific binding which demonstrates that the specific [3H]-DHT binder is a protein. The uptake of [3H] DHT in endometrial tissue minces indicated that 20% of the bound radioactivity was nuclear. Steroid specificity suggests that the binding protein from the uterus is specific for androgens. These observations indicate that androgen binding protein in the human uterus has the characteristics of the androgen receptor.     

The estrogen cytosol receptor of female ovine pituitary.

The biochemical parameters of estrone and estradiol binding to the cytosol fraction of ovine anterior pituitary were investigated. When increasing amounts of [3H]estrone or [3H]estradiol were incubated with the 105,000 g fraction from the pituitary, both hormones bound to a receptor with the same apparent KD (mean +/- S.E., estrone = 1.40 +/- 0.30 X 10(-10) M, estradiol = 1.03 +/- 0.11 X 10(-10)M) and the same concentration of binding sites (estrone = 3.22 +/- 0.58 X 10(-14) moles/mg protein, estradiol = 3.92 +/- 0.19 X 10(-14)). No conversion of [3H]estrone to [3H]estradiol under the experimental conditions used could be demonstrated. The receptor-estrogen complex exhibited identical sedimentation coefficients (7-8 S) with either hormone. The receptor was specific only for estrogens; neither 500-fold excess of testosterone nor progesterone affected binding. Competitive inhibition using increasing amounts of non-radioactive estrone or estradiol with [3H]estrone or [3H]estradiol resulted in parallel displacement of the radioactive hormone. These results strongly suggest that both hormones bind to the same pituitary cytosol receptor.     

Effects of gonadectomy and hormone replacement on steroid hormone receptors and 5 alpha-reductase activity in pituitaries of male rhesus macaques

We measured androgen, estrogen, and progestin receptors and 5 alpha-reductase activity in the anterior and posterior pituitary gland of intact and 6-week castrate adult male rhesus monkeys and castrate males which were treated with testosterone (T) or estradiol (E) from time of surgery. Saturation analysis of anterior pituitary tissues from monkeys receiving various treatments revealed an apparent mean dissociation constant (Kd) of 0.53 +/- 0.17 (+/- SE) X 10(-10) mol/L (n = 3) for [3H]R1881 (androgen) binding to cytosol, 2.6 +/- 0.50 X 10(-10) mol/L (n = 3) for [3H]R2858 (estrogen) binding to cytosol, 1.7 X 10(-10) mol/L (n = 2) for [3H]R5020 (progestin) binding to cytosol, and 6.2 X 10(-10) mol/L (n = 2) for [3H )R1881 binding to cell nuclear extract. The highest levels of nuclear androgen receptor (AR) were found in intact males [37.1 +/- 3.5 (+/- SE) fmol/mg DNA; n = 7] and castrated males treated with T for 6 weeks (89.7 +/- 30.2 fmol/mg DNA; n = 5). High levels of nuclear AR corresponded to serum T levels and low serum LH levels. Nuclear AR was undetectable in castrated males and castrated males treated with E. Significantly greater levels of cytosolic AR were detected in intact males (27.5 +/- 1.6 fmol/mg protein) compared to all other groups (P less than 0.05). T or E treatment had no effect on cytosolic AR. Increased levels of cytosolic progestin receptor were found in intact monkeys and after E or T treatment compared to levels in untreated castrates. No differences in 5 alpha-reductase activity were found between any treatment groups. These data indicate that anterior pituitary nuclear androgen receptor is correlated with serum LH levels and support the hypothesis of a direct action of T on anterior pituitary LH secretion. In addition, it appears that cytosolic progestin receptor, but not AR, is regulated by estrogen in intact male rhesus monkeys. In the posterior pituitary, AR dynamics followed a profile in which cytosolic AR increased after castration and decreased after T treatment. Nuclear AR decreased after castration and increased after T treatment. The presence of a dynamic AR system in the posterior pituitary suggests hormonal regulation of its function by androgens.     

The nuclear accumulation of [3H]testosterone and [3H]estradiol in the brain of the female primate: evidence for the aromatization hypothesis

To identify the metabolites of estradiol (E2) and testosterone (T) in nuclei obtained from the female primate brain and, hence, to investigate the mechanism of their actions on behavior, 9 ovariectomized adult rhesus monkeys were studied. Two of these females were injected with 5.5 mCi [3H]T, and 30 min later, samples of 14 brain areas, pituitary gland, and peripheral tissues were removed and homogenized. Purified cell nuclei and a crude cytosol fraction were prepared, extracted with ether, and fractionated by HPLC to identify steroid metabolites. In nuclei from the hypothalamus, preoptic area, and amygdala, [3H]E2 formed locally was the major form of radioactivity. In nuclei from the clitoris, [3H]dihydrotestosterone was the major form of radioactivity, and in nuclei in all other brain samples and in the pituitary gland and uterus, [3H]T predominated. Two females (controls) were pretreated for 5 days with oil sc, injected with 1 mCi [3H]E2, and killed 60 min later. In these females, elevated nuclear concentrations of [3H]E2 were found in the hypothalamus, preoptic area, amygdala, pituitary gland, and uterus. Similar results were obtained in 2 females that were pretreated for 5 days with 2 mg/day dihydrotestosterone propionate, sc, and then injected with 1 mCi [3H]E2. In 3 females that were pretreated for 5 days with 2 mg/day T propionate, sc, and then injected with 1 mCi [3H]E2, levels of [3H]E2 were reduced by 100% (P less than 0.01) in nuclei from preoptic area and amygdala compared with control values and by 78% (P less than 0.05) in nuclei from the hypothalamus. There were no comparable reductions in steroid levels in cerebral cortex, pituitary gland, or uterus. This is the first direct evidence in the brain of a female primate that the actions of T and E2 involve the same receptor systems.     

Localization and role of transcortin-like molecules in the anterior pituitary

The uptake and binding of [³H]dexamethasone and [³H] corticosterone in the anterior pituitary has been studied with special reference to the transcortin-like protein and compared with the uptake and binding in the hippocampal region of the rat brain, which lacks this protein.Isolated pituitary cells of adrenalectomized rats contained both glucocorticoid receptors and transcortin-like molecules.A collagenase treatment was used for isolation of the intact pituitary cells. Exposure of cytosol from broken cells of the anterior pituitary to this enzyme destroyed nearly all binding activity for the corticosteroids. The results indicate an intracellular localization of the transcortin-like molecules in the anterior pituitary, although the possibility that plasma transcortin adheres to the cell surface cannot be excluded.Estradiol treatment increased the level of transcortin in the plasma and of transcortin-like molecules in the anterior pituitary. The concentration of the glucocorticoid receptors in the anterior pituitary and the hippocampus remained unaffected.Subcutaneous injection of doses up to 50 μg corticosterone per 100 g body weight did not compete for the binding in vitro of [³H]dexamethasone to glucocorticoid receptors in anterior pituitary cytosol. The same doses of corticosterone reduced binding of the synthetic steroid in cytosol of the hippocampus by 40%.Endogenous corticosterone competed poorly for the uptake of [³H]dexamethasone in cell nuclei of the anterior pituitary. In cytosol 71% of the receptor sites were still available for [³H] dexamethasone binding in vitro.It is proposed that transcortin-like molecules in the anterior pituitary, whether intra- or extracellularly, modulate the effect of corticosterone on pituitary-adrenal activity by mediating the extent of interaction of the steroid with the glucocorticoid receptor.     

Nuclear [3H]4-hydroxytamoxifen (4-OHTAM)- and [3H]estradiol (E2)-estrogen receptor complexes in the MCF-7 breast cancer and GH3 pituitary tumor cell lines

Nuclear [3H]4-OHTAM-ER complexes extracted by 0.6 M KCl from the MCF-7 human breast cancer and the GH3 rat pituitary tumor cell lines, sedimented as a 5S form on sucrose gradients after 1 to 24 h exposure to ligand (20 nM). The nuclear [3H]4-OHTAM-ER from MCF-7 cells increased from 2 +/- 0.2 pmoles/mg DNA at 1 h to approximately 4 +/- 0.1 pmoles/mg DNA at 6 and 24 h. In the GH3 cells the nuclear binding of [3H]4-OHTAM increased rapidly, and there was no significant difference in the receptor levels at 1 and 6 h (10 +/- 1.3 and 8.9 +/- 1.1 pmoles/mg DNA, respectively). At 24 h there was a decrease in [3H]4-OHTAM-ER levels (7.0 +/- 0.2 pmoles/mg DNA); however, this was due primarily to an increase in DNA synthesis, which occurred in these cells by 24 h. It appears that in both cell lines there is no processing of nuclear [3H]4-OHTAM-ER, and it is not associated with changes in sedimentation coefficient of the complex. When both cell lines were incubated with [3H]E2 (20 nM), processing of the nuclear [3H]E2-ER occurred over 6 h. In the MCF-7 cells there was a decrease in receptor content within 6 h from 3.2 +/- 0.2 to 0.9 +/- 0.2 pmoles/mg DNA. The nuclear [3H]E2-ER in the GH3 cells decreased from 7.3 +/- 0.5 to 2.8 +/- 0.3 pmoles/mg DNA. Although these cell lines appeared to process the [3H]E2-ER complex, the nuclear forms of [3H]E2-ER were different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin

We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.     

Cytosol and nuclear [3H]oestradiol binding in the foetal tissues of guinea pig.

The formation of [3H]oestradiol macromolecule complexes was studied in vivo and in vitro in the kidney, lung and liver of intact foetal guinea pig. Specific binding of [3H]oestradiol was demonstrated in the cytosol and nuclear fractions of foetal kidney. Intensive binding was found in the cytosol of foetal lung but most of the bound radioactive material (70 %) was [3H]oestrone. Some binding was found in the cytosol of foetal liver but not in the nucleus of this tissue. In the in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22 % of the plasma radioactivity was bound of which 67 % was identified as oestrone sulphate. Oestradiol sulphate represented 7% and unconjugated oestradiol only 0.6 % (0.1 % of the total plasma radioactivity). On the other hand, 2-3 % of the foetal plasma radioactivity was found as unbound [3H]oestradiol. In the kidney, the formation of [3H]oestradiol complexes in the cytosol fraction does not depend on temperature while nuclear [3H]oestradiol complexes increase with increasing temperature. Maximal formation of [3H]oestradiol complexes in the cytosol fraction and the 0.1 M TRIS and 0.3 M NaCl nuclear extracts was reached after 15 min but binding in the 1 M NaCl nuclear extract continued to increase up to 30 min. After incubation of purified nuclei of foetal kidney with 1.1 x 10(-7) M [3H]oestradiol, specific binding was found in the different nucelar fractions. Specific binding was also detected in isolated nuclei previously extracted by 0.1 M TRIS and 0.3 M NaCl before being incubated with 1.1 x 10(-7) M [3H]oestradiol. The Kd of binding of [3H]oestradiol in the renal cytosol fraction is 2.5 x 10(-10) M with n = 4.5 x 10(-14) moles/mg protein. Incubation of isolated 1 M NaCl nuclear extract from foetal kidney with [3H]oestradiol gives a Kd of 3.3 x 10(-10) M with n = 2.5 x 10(-14) moles/mg protein. It is concluded that the nuclear complexes in the foetal kidney could be formed either through an intermediate cytosol complex or that the "two step" mechanism could take place in the nucleus. Furthermore, direct binding of [3H]oestradiol with high affinity was observed in the 1 M NaCl nuclear extract.     

Androgen receptor specificity and growth response of a human cell line (NHIK 3025)

Growth of cultured NHIK 3025 cells stemming from a carcinoma of the human uterine cervix can be stimulated by the androgens 4-androstene-3β,17β-diol and testosterone, but is not influenced by oestradiol. In the cytosol fraction of these cells testosterone and 5α-dihydrotestosterone could be bound specifically to a steroid receptor with limited capacity (8 fmol/ mg cytosol protein), but no specific binding of oestradiol or of the synthetic progestagen R-5020 could be demonstrated. The specificity of the binding was studied by agar-gel electrophoresis and sucrose density gradient centrifugation. In the cytosol no specific binding of 4-androstene-3β,17β-diol could be demonstrated and addition of this steroid to cytosols containing [³H]testosterone did not interfere with [³H]testosterone binding. Receptor-bound steroid could be extracted from the nuclei after incubation of intact NHIK 3025 cells with ³H-labelled 4-androstene-3β,17β-diol, testosterone or 5α-dihydrotestosterone. However, considerable metabolism of 4-androstene-3β,17β-diol occurred and the radioactivity recovered from the bound fraction represented mainly testosterone and 5α-dihydrotestosterone. In addition to the androgen receptor the NHIK 3025 cells also contain a glucocorticoid receptor (190 fmol [³H]dexamethasone bound/mg cytosol protein).     

Progesterone modulation of the luteinizing hormone surge: regulation of hypothalamic and pituitary progestin receptors

We have investigated the possible role of hypothalamic and pituitary progestin receptors (PR) in modulation of the estradiol-induced LH surge by progesterone in the immature rat. Rats (28 days old) that received Silastic implants containing estradiol in oil at 0900 h had LH surges approximately 32 h later. Progesterone implants were inserted concurrently with estradiol capsules or 24 h later, leading to inhibition or facilitation of the LH surge, respectively. Cytoplasmic and nuclear PR were measured by in vitro exchange assays, using near-saturating concentrations of [3H]R5020 (3H-labeled promegestone; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), 1, 8, 24, and 32 h after insertion of progesterone or blank implants. Kd values of complexes between [3H]R5020 and PR were 0.5-1 nM (cytoplasmic), and 2-3 nM (nuclear). The sedimentation rates of these complexes in sucrose gradients were 7-8S (cytoplasmic) and 3-4S (nuclear). In rats treated concurrently with estradiol and progesterone for 1 or 8 h, cytoplasmic PR were depleted to 40-60%, and this was accompanied by slight increases in nuclear PR. In control rats treated with estradiol and blank implants, there was no induction of either cytoplasmic or nuclear PR in the hypothalamus-preoptic area for up to 48 h; however, in the pituitary and uterus of these animals, PR increased significantly in both compartments (2- to 3-fold at 24 h, 3- to 5-fold at 32 h, and 4- to 7-fold at 48 h). Administration of progesterone either to inhibit or facilitate LH surges almost completely blocked the inductive effect of estradiol on cytoplasmic PR, but the absence of PR from the cytosol could not be accounted for by their presence in the nucleus. In the hypothalamus-preoptic area of estradiol-treated control rats, neither cytoplasmic nor nuclear PR increased significantly for up to 48 h. The low levels of specific [3H]R5020 binding in pituitary and uterine cytosols from progesterone-treated rats appeared to be due mainly to a decrease in the number of binding sites, rather than to an effect on binding affinities. The 7-8S peak of cytoplasmic PR was considerably reduced in rats treated for 48 h with estradiol and 24 h with progesterone. These results are consistent with the notion that hypothalamic and pituitary PR are involved in modulation of the LH surge by progesterone and point primarily to a pituitary site of action for progesterone facilitation.     

Androgen binding in nuclear matrix of human genital skin fibroblasts from patients with androgen insensitivity syndrome

Specific sex steroid-binding sites are associated with the salt-insoluble nuclear matrix from which lipids, histones, and chromatin have been extracted. In intact cultured normal human genital skin fibroblasts incubated for 1 h at 37 C with a saturating concentration (2 nM) of [3H]dihydrotestosterone [( 3H]DHT), approximately 50% of the total intracellular androgen receptor-steroid complexes were found in the nucleus. Within isolated nuclei from such cells, 28-49% of the specific androgen receptor binding was associated with the nuclear matrix. The antiandrogen cyproterone acetate inhibited DHT binding within the nuclear matrix. Cultured genital skin fibroblasts from two unrelated patients with receptor-positive complete androgen insensitivity (CAIS, AR+), had normal (approximately 50%) nuclear binding of DHT, and 35% and 45% of it was localized to the nuclear matrix. Genital skin fibroblasts from a patient with receptor-negative complete androgen insensitivity (CAIS, AR-) had no specific DHT binding in isolated nuclei or nuclear matrix. Scatchard analysis of specific DHT binding in the nuclear matrix isolated from cells of normal subjects after an in vitro exchange assay (0 C; 24 h) revealed the presence of saturable (maximum binding, approximately equal to 200 fmol/mg nuclear DNA), high affinity (Kd approximately equal to 1.0 nM) binding sites. By contrast, in the nuclear matrix isolated from cells of a patient with CAIS, AR+, the binding affinity for DHT was 3-fold lower (Kd approximately equal to 3.0 nM). When cytosolic androgen receptor-DHT complexes prepared from cells preincubated at 37 C for 1 h with [3H]DHT were incubated at 0 C for 1 h with isolated nuclei and nuclear matrix in the presence of 0.15 M KCl, 40-60% of specific nuclear binding was associated with the nuclear matrix. In these cell-free in vitro experiments, radiolabeled DHT-receptor complexes prepared from normal or mutant cells were mixed with isolated nuclei and nuclear matrix prepared from cells of normal subjects or patients with CAIS, AR+ or CAIS, AR-. Under these conditions, specific DHT binding in nuclei and nuclear matrix was quantitatively similar in the presence of a mutant (CAIS, AR+) receptor-steroid complex or in the presence of nuclei or nuclear matrix from the mutant cells (CAIS, AR- or AR+) when compared simultaneously with the same subcellular fractions prepared from the cells of normal subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mechanism of 17 beta-estradiol uptake into prolactin-producing rat pituitary cells (GH3 cells) in culture

Estrogens stimulate PRL synthesis in cultured GH3 cells, which are clonal strains derived from the rat pituitary gland. This model system was used to study the mechanism by which 17 beta-estradiol (E2) enters target cells. The cellular uptake of [3H]E2 was rapid at 37 C and reached half-maximal values within 10-15 sec. Maximal uptake was observed in less than 2 min at 37 C. The initial rates of E2 uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]E2 in intact cells and the binding to cytosol studied at different temperatures resulted in linear Arrhenius plots, and the energies of activation were 39.0 and 33.5 kJ mol-1 degree-1, respectively. Purified GH3 cells membrane fractions, which showed specific binding sites for [3H]TRH, displayed the same maximal binding of [3H]E2 in the absence or presence of cold hormone. The amount of membrane-associated [3H]E2 increased linearly with temperature and extra-cellular hormone concentration. The effect of temperature on binding of E2 to membrane fractions occurred gradually without phase transitions and was not saturable. We suggest that the mechanism by which E2 is taken up by target cells at physiological temperature involves instantaneous dissolution in the cell membrane from where it diffuses passively into the cell. E2 binds thereafter to specific receptors in an energy-dependent step.