Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.     

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

Effect of aminoglutethimide on extraglandular metabolism of exogenous testosterone

Aminoglutethimide (AG), an inhibitor of steroid biosynthesis, seems to have an extraglandular site of action on steroid catabolism. To study this effect, five males with peripheral hypogonadism were first given testosterone propionate and then the same dose was repeated combined with AG and urinary testosterone, and its metabolites were measured. AG was shown to have a very evident effect on the peripheral degradation of exogenous testosterone. This may be responsible for a few signs of virilization and fetal masculinization in women taking AG.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

In vitro inhibition of aldosterone biosynthesis by 17-hydroxylated steroids in rat adrenal

The in vitro effect of the addition of non-17-hydroxylated and 17-hydroxylated steroids on the production of aldosterone has been studied in the rat. The biosynthesis of deoxycorticosterone, corticosterone, and aldosterone was increased when progesterone or pregnenolone was added to the incubation media, but neither cortisol nor 11-deoxycortisol was produced. The addition of 17α-hydroxyprogesterone or 17α-hydroxypregnenolone to the incubation media resulted in the biosynthesis of cortisol and 11-deoxycortisol by the rat adrenal; concomitantly, a decrease in aldosterone production occurred. However, no decrease in aldosterone production resulted when cortisol and 11-deoxycortisol were added directly to the incubation media. These studies show that neither cortisol nor 11-deoxycortisol inhibits the in vitro biosynthesis of aldosterone and suggest that other mechanisms are involved.     

Interaction of glucocorticoids and androgens with skeletal muscle.

This review is a discussion of recent developments in our understanding of the metabolic changes that occur in skeletal muscle following treatment with glucocorticoid and androgenic steroid hormones. Exposure of muscle to glucocorticoids results in protein wasting due to a reduction in protein synthesis and an increase in intracellular proteolysis. The protease involved in this response appears to be located in the myofibrillar fraction and its activity is increased by glucocorticoid treatment as well as starvation, diabetes, and tumor-induced cachexia. Treatment with androgens causes an anabolic response in skeletal muscle, mainly due to enhancement of protein-synthesizing machinery. These metabolic changes are reviewed in the light of our current knowledge of the functional role of receptor proteins in steroid hormone action. In rat muscle, two classes of receptors are present that bind cortisol and the synthetic steroids dexamethasone and triamcinolone acetonide, respectively. The cortisol-binding macro-molecule resembles cortisol-binding glubulin or transcortin in several of its physicochemical properties. Attempts to identify specific receptors for androgens in rat skeletal muscle have not been successful. However, in this tissue androgens compete, both in vivo and in vitro, with the receptor proteins that bind dexamethasone. Thus the antagonism of glucocorticoid-induced muscle wasting by concomitant androgen administration may be a consequence of the competition between these steroids for the same site on a receptor in muscle which mediates the catabolic action of glucocorticoids. Current progress in the area of muscle response to steroidal hormones depends on the development of procedures for the measurement and identification of proteolytic enzymes in muscles that are involved in the muscle-wasting diseases. A new approach to the study of muscle protein degradation is based on evidence that 3-methylhistidine is a unique amino acid insofar as it is present in skeletal muscle in relatively high concentrations. The methylation of histidine to form this derivative takes place only after histidine is incorporated into protein. Since the methyl derivative is not reutilized for protein synthesis, the rate of release of 3-methylhistidine from muscle serves as a marker for protein degradation. Future studies are also required to assess the role of normal and abnormal concentrations of blood glucocorticoids in muscle metabolism, and to delineate the sequence of events occurring in these target cells following cytoplasmic binding of the hormones and culminating in the catabolic response.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

Dissociation between cortisol-induced pycnosis and inhibition of [3H]uridine incorporation in rat thymocytes.

Treatment of rat thymocytes with cortisol induced an inhibition of [3H]uridine incorporation after 30-90 min, an accumulation of pycnotic cells after 90 min, and a decrease in cell viability after several hours. No cortisol-resistant cells could be distinguished, and dose-response curves for a number of glucocorticoids showed a correlation to the saturation of the glucocorticoid receptors. The pycnotic effect of cortisol increased between pH 5.2--7.0 in parallel with a stimulation of the spontaneous development of pycnotic cells. The cortisol-induced accumulation of pycnotic cells and inhibition of [3H]uridine incorporation varied independently as a function of the cell density, and in a glucose-salt medium only the pycnotic effect of cortisol became inhibited. The inhibition of [3H] uridine incorporation is therefore not an integral part of the pycnotic change of the cells. The glucocorticoid sensitivity was found to increase with the age of the animals, before the onset of thymus involution.     

Activation of the progesterone receptor of rabbit uterus

The influence of several parameters on the kinetics of activation of the progesterone receptor in the cytosol of rabbit uterus is described. The estimation of the proportion of activated receptor is based on the differential affinity of the activated and non-activated forms of the receptor for phosphocellulose. Under appropriate conditions binding to phosphocellulose can be used as a test of activation and gives results similar to those obtained with DNA--cellulose, or isolated cell nuclei. The kinetics of receptor activation is temperature-dependent and compatible with a first-order reaction at all temperatures tested. The thermodynamic activation energy of this reaction is 67.8 kcal mol-1. The progesterone receptor can be activated to various extents by increased ionic strength or by dilution of the cytosol with buffers of low ionic strength, and in all cases the activation follows apparent first order kinetics. At a concentration of 0.4 M NaCl, 70--80% of the receptor can be converted into the activated form. The activated and non-activated forms of the receptor appear to be in equilibrium. Salt-activated and heat-activated receptor can be transformed to a non-activated form by decreasing either the salt concentration, or the temperature of incubation. The rate of dissociation of the steroid from the activated form of the receptor is indistinguishable from that observed with the non-activated form, but the activated receptor is more thermolabile. Upon centrifugation on sucrose gradients there are no major differences in the sedimentation behaviour of the two forms of the receptor.     

Year-to-year differences in plasma levels of steroid hormones in pre-spawning chum salmon

Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Evidence suggesting 11 beta-hydroxylase inhibition during aminoglutethimide administration

Steroid hormone excretion during amino-glutethimide administration was studied in a patient with Cushing's syndrome due to bilateral adrenocortical hyperplasia. Plasma and urinary 17-OHCS showed a persistent decrease, as did urinary total 17-KS. Chromatographic fractionation of urinary 17-KS demonstrated a dramatic reduction of 11-oxy-17-KS, while 11-deoxy-17-KS, after a transient fall, tended to recover. Urinary THS showed an absolute increase, while pregnanetriolone diminished. Δ⁵-pregnenetriol and pregnanetriol, after initial decreases, showed a gradual trend upward. The sum of these data, and particularly the increase in THS excretion, suggests that, in this case, aminoglutethimide exerted an inhibitory effect not only on the early steps of adrenocortical steroidogenesis but also on 11 β-hydroxylation.     

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.     

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.     

The impact of radioimmunoassay on our understanding of human adrenal physiology

Radioimmunoassay has only been used for a relatively short time to study the human hypothalamic-pituitary-adrenal system, but the findings have already contributed immeasurably toward a better understanding of the physiology of this system. In some instances classical concepts have been confirmed and extended. Other findings have led to revisions of old concepts and the formulation of new ones. These physiologic studies have produced new and better laboratory tests for the pituitary-adrenal function that are simple and reliable enough to be used not only in clinical research but in the routine practice of medicine. Even greater contributions from radioimmunoassay can be expected in the future.     

Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

Mice lacking Mrp1 have reduced testicular steroid hormone levels and alterations in steroid biosynthetic enzymes

The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ABC active transporter family that can transport several steroid hormone conjugates, including 17β-estradiol glucuronide, dehydroepiandrosterone sulfate (DHEAS), and estrone 3-sulfate. The present study investigated the role that MRP1 plays in maintaining proper hormone levels in the serum and testes. Serum and testicular steroid hormone levels were examined in both wild-type mice and Mrp1 null mice. Serum testosterone levels were reduced 5-fold in mice lacking Mrp1, while testicular androstenedione, testosterone, estradiol, and dehydroepiandrosterone (DHEA) were significantly reduced by 1.7- to 4.5-fold in Mrp1 knockout mice. Investigating the mechanisms responsible for the reduction in steroid hormones in Mrp1−/− mice revealed no differences in the expression or activity of enzymes that inactivate steroids, the sulfotransferases or glucuronosyltransferases. However, steroid biosynthetic enzyme levels in the testes were altered. Cyp17 protein levels were increased by 1.6-fold, while Cyp17 activity using progesterone as a substrate was also increased by 1.4- to 2.0-fold in mice lacking Mrp1. Additionally, the ratio of 17β-hydroxysteroid dehydrogenase to 3β-hydroxysteroid dehydrogenase, and steroidogenic factor 1 to 3β-hydroxysteroid dehydrogenase were significantly increased in the testes of Mrp1−/− mice. These results indicate that Mrp1−/− mice have lowered steroid hormones levels, and suggests that upregulation of steroid biosynthetic enzymes may be an attempt to maintain proper steroid hormone homeostasis.     

Immunolocalization of 3beta-HSD and 17beta-HSD in the testis of the spotted ray Torpedo marmorata

Using polyclonal antibodies, we examined the localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as markers of the site of steroidogenetic activity during the spermatogenesis of Torpedo marmorata. These enzymes play a central role in the biosynthesis of steroid hormones, including androgen and oestrogen production. We demonstrated that in the spotted ray testis, Sertoli and Leydig cells, as well as spermatogonia, show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies. In particular, we demonstrated that Sertoli cells show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies in cysts containing spermatogonia and spermatozoa, while Leydig cells present a positive reaction only when they are located between cysts containing meiotic cells. This study strongly suggests that, as hypothesised in our previous study [Prisco, M., Liguoro, A., D'Onghia, B., Ricchiari, L., Andreuccetti, P., Angelini, F., 2002. Fine structure of Leydig and Sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata. Mol. Reprod. Dev. 63, 192-201.], Sertoli and Leydig cells are differently involved in the hormonal control of spermatogenesis: Sertoli cells before the beginning of meiosis and after spermiation, Leydig cells only during meiosis phase. Moreover, the present paper deals with the possibility that also spermatogonia are engaged in the production of androgen hormones, as they are characterized by the presence of 3beta-HSD and 17beta-HSD enzymes, and show the ultrastructural features of steroid hormone-producing cells.     

Role of the hypophysis in estrogen-mediated induction of enzymes in the regenerating liver from castrated male rats.

Perfusions with corticosterone, of isolated regenerated livers from adult male rats, subjected to castration, partial hepatectomy and hypophysectomy with or without estradiol treatment during parenchymal regeneration, yielded very similar patterns of biliary steroid metabolites. The degree of steroid conjugation was lower than that seen in livers from normal, untreated, adult male rats. In operated animals, with or without estradiol benzoate treatment, ring A-reduced 20-keto metabolites constituted about 20%, whereas metabolites with a 20-hydroxy group made up approximately 80% of the corticosterone metabolites formed. Furthermore, no 15-hydroxylated metabolites derived from corticosterone, quantitatively the most important compounds in bile from female rats, could be detected in bile from these treated male animals. However, livers from male rats which had been castrated, hepatectomized and treated with estradiol benzoate during liver regeneration, produced 15-hydroxy-tetrahydrocorticosterone to the same extent as female rat livers, when perfused with corticosterone. The results obtained indicate that the effects of estradiol on the induction and differentiation of steroid metabolizing enzymes in the regenerating liver are pronounced and manifested only in the presence of an intact hypophysis.