Androgen-binding Activity Research Articles

Receptors for androgen and estrogen in the rat lung.

By the use of sucrose density gradient and protamine sulfate analyses, the cytosol fraction of rat lung has been found to contain binding activities for both androgenic and estrogenic steroids. These activities are present in lungs of rats of both sexes and at all ages examined. Both activities interacted stereospecifically with, and possessed high affinity for, the respective ligand, and sedimented in the 8-9S region of low salt sucrose density gradients. The androgen-binding activity appeared to bind 5alpha-dihydrotestosterone (DHT) and testosterone with similar affinities, but in vitro, whole lung incubation studies demonstrated that DHT is the perdominant androgen found in lung nuclei. In the immature male rat, removal of endogenous androgen by castration-adrenalectomy had no effect on DHT-binding activity in lung cytosol. In the adult, however, this procedure led to a marked cycloheximide-sensitive increase in binding activity. During postnatal development in male rats, relatively constant levels of both androgen- and estrogen-binding activities were found in the lung until the peripubertal period, from which time a striking increase in DHT binding and a decline in that of estradiol were observed. In female rats, cyclic changes in the level of both binding activities were evident during the estrous cycle. When considered in the light of evidence that sex steroids influence certain aspects of lung biochemistry, these data suggest that the demonstrated binding activities represent hormone receptor activities which may mediate a variety of biological events in the lung.

Androgen-Binding Activity in the Human Testis and Epididymis

The quantitation of androgen-binding activity in human testes, epididymides, and serum is reported and compared with that of similar tissue in the rat. By using steady-state polyacrylamide gel electrophoresis and a tritiated 0.5 nM testosterone label, values for specific binding activity, reported as picomoles per milligram of protein, were found to be 0.21 +/- 0.01 in normal human, epididymides, 0.03 in testes, and 0.44 +/- 0.07 in human serum. Tissues from men receiving exogenous estrogen were also similarly analyzed and found to possess significantly higher androgen-binding activity: 4.10 +/- 0.48 pmoles/mg of protein (epididymides), 4.01 (testes), and 5.63 +/- 1.15 (serum). Since man possesses a sex steroid-binding globulin, attempts were made to differentiate Ion-exchange chromatography, steroid specificity, and dissociation half-time determinations were suggestive but did not conclusively demonstrate the existence of two distinctly different androgen transport macromolecules in man.

The Coexistence of Androgen and Glucocorticoid Receptors in the DDT1Cloned Cell Line1

The hamster ductus deferens cloned tumor cell line (DDT1) has been shown to contain both androgen and glucocorticoid binding activity. The androgen receptor binding site concentration is 1.07 x 10(-13) mol of testosterone/mg protein, and testosterone (T) binds with a Kd of 4.3 x 10(-10) M. Dihydrotestosterone (DHT) is also bound to the receptor with a Kd of 2.99 x 10(-10) M and the binding site concentration is 1.33 x 10(-13) mol/mg protein. The order of steroid binding affinity is DHT greater than T greater than Estradiol greater than Progesterone. Cortisol, dexamethasone, and triamcinolone acetonide do not inhibit the androgen binding in vivo or in vitro. In a cell free system antiandrogens inhibit the binding of DHT. The DDT1 cells have a separate receptor for cortisol which binds at saturation 3.44 x 10(-13) mol cortisol/mg protein and has a Kd of 4.54 x 10(-9) M. These studies provide evidence that these endocrine target cells contain specific high affinity receptors for more than one type of steroid. The glucocorticoid receptor may be important for maintaining essential undifferentiated functions while the DHT receptor gives the specific characteristics of sex hormone responsive tissues.

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.

Some properties of androgen-binding activity in rat testis

High-affinity ( K a ≅ 5 × 10 8 M −1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant ( t 1 2 = 3 min , 0 °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 °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 ( R f 0.54 ) from albumin ( R f 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 receptor in human skin fibroblasts characterization of a specific 17β-hydroxy-5α-androstan-3-one-protein complex in cell sonicates and nuclei

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2- 3H-testosterone, 1,2- 3 H-17β-hydroxy-5α-androstan-3-one (dihydrotestosterone,DHT) and 1,2- 3H-5α-androstane-Sα, 17β-diol. This receptor had a high affinity (Kd = 0.2–1.6 nM) and a high degree of specificity for DHT, It was measured as a 3H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4° and 37°. Saturation analysis indicated that there were 1250–18,600 binding sites per whole cell. By sucrose gradient centrifugatlon the receptor had a sedimentation coefficient (S 20, w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3H-DHT binding. Within 15 hours puromycin (20 μg/ml) in serum-free medium caused a 40–60 percent decrease in binding for the same cell lines. Although the highest levels of 3H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro .

Androgen binding to cytosol prepared from epididymides of sexually mature castrated rabbits: Evidence for a cytoplasmic receptor

The presence of androgen-binding activity in cytosol prepared from the major anatomical segments (caput, corpus, and cauda) of the epididymis of castrated sexually mature rabbits has been demonstrated. A portion of this binding activity is likely to be the epididymal androgen receptor. When epididymal cytosol from adult castrated rabbits is analyzed on low-ionic strength (0.01 M KC1) sucrose gradients, two peaks of macromolecular binding could be detected, one ∼- 4.6S and one ∼- 8S. On gradients containing 1.0 M KC1, only one sedimenting form ∼- 4.6S could be demonstrated, suggesting that the 8S component is composed of aggregates. If cytosol was preincubated with labeled androgen, followed by an incubation with unlabeled androgen, and subsequently analyzed for binding on low-ionic strength gradients, only the ∼- 8S peak could be detected, indicating that most of the binding in the ∼- 4.6S region was rapidly dissociable. This suggests that binding in this region was to moieties other than receptor. Since androgen binding proteins (ABP) of testicular origin would have been cleared from the epididymis at the timepoints that we concentrated on for most of these studies, the 4.6S binding probably represents the association of androgen with plasma testosterone binding globulin (TeBG). The binding of androgen to the receptor can be inhibited by cyproterone, while this antiandrogen does not inhibit binding to either ABP or TeBG at the concentration used.

The effect of testosterone propionate on the concentration of testicular and epididymal androgen binding activity in the hypophysectomised rat

The effect of testosterone propionate on the level of androgen binding activity in both testis and epididymis was investigated by both a dextran coated charcoal (DCC) method and by equilibrium polyacrylamide gel electrophoresis (PAGE) which measures specifically androgen binding protein. There was a decrease in androgen binding activity in both testis and epididymis after hypophysectomy. Testosterone propionate treatment (1 mg/day), begun at one day posthypophysectomy, maintained androgen binding activity above the level in testes from hypophysectomised rats for 30 days (PAGE) or 60 days (DCC) and 60 days in epididymides (PAGE). The ability of testosterone propionate treatment (250, 500 and 750 μg/day) to restore androgen binding activity in testis and epididymis of chronically hypophysectomised rats (30 days) was tested using a 3-day treatment period. These doses restored testicular androgen binding activity to 60, 36 and 75% of the levels in control animals, respectively. The degree of restoration of androgen binding activity in epididymis in this time interval was inferior to that in the testis at all doses. Prolonged treatment with testosterone propionate (1 mg/day for 10, 30 and 60 days) resulted in complete restoration of androgen binding activity in testis, expressed per mg protein (PAGE and DCC), and over 50% restoration in the epididymis (PAGE). It was concluded that testosterone propionate is able to maintain and restore testicular and epididymal androgen binding activity and, in particular, androgen binding protein, after hypophysectomy and that this hormone may, in the short term, influence testicular rather than epididymal levels of binding activity.

Hormonal Influences on the Level of Testicular Androgen Binding Activity: Effect of FSH Following Hypophysectomy

Concentrations of high affinity (Ka equals 5.5 plus or minus 0.83 times 10-8M-1) androgen binding activity in carbonextracted rat testicular supernatants have been determined by Scatchard plot analysis under a variety of hormonal situations: (a) 0-90 days following hypophysectomy; (b) 0-60 days of daily injection of NIH-FSH-P1 (150 mug) beginning 1 day after hypophysectomy; and (c) 0-60 days of daily FSH treatment beginning 30 days after hypophysectomy. The high affinity binding component declined from 0.32 pmoles/mg protein intact adults to 0.28, 0.17, and less than 0.08 (limit of detectability) pmoles/mg protein at 11, 16 and 31 days, respectively. FSH treatment beginning immediately after surgery slowed this decline, giving values of 0.30, 0.17, and 0.12 pmoles/mg protein after 11, 29, and 54 days of treatment, respectively. Expressed as pmoles per testis this represented 96, 61, and 40% of the intact control level. Similar effects on the level of androgen binding protein (Rf 0.54) were measured by steady-state polyacrylamide gel electrophoresis (PAGE). The concentration in both testis and epididymis declined gradually to nondetectable levels by 30 days after surgery. FSH treatment for 11, 29, and 54 days, respectively, resulted in 0.37, 0.38, and 0.03 pmoles of sites/mg protein in testis compared to 0.34 in intact controls and 5.7, 2.6, and 0.2 pmoles/mg protein in epididymis compared to 2.8 in intact controls. Doses of 80, 150, and 300 mug FSH/rat/day for 3 days beginning 30 days after hypophysectomy when postmeiotic elements of the germinal epithelium had degenerated caused graded increases in both testicular and epididymal levels of androgen binding protein. Prolonged FSH treatment (150 mug) under these conditions resulted in an increase in binding activity to a level of 0.12, 0.19, 0.17, and 0.20 pmoles/mg protein after 3, 11, 25, and 56 days of treatment. On a per testis basis this represented less than 20% of intact control levels. PAGE estimates were comparable except at the long treatment intervals. These results indicate that FSH treatment influences the level of androgen binding protein in adult testis and epididymis. This may reflect a direct influence on synthesis, degradation, and transport and/or indirect effects on general maintenance and responsiveness of the pertinent cell types.

Androgen binding in the testis: in vitro production of androgen binding protein (ABP) by Sertoli cell cultures and measurement of nuclear bound androgen by a nuclear exchange assay.

Androgen binding activity in the testis has two components. One component, ABP, has been shown to be produced by Sertoli cell cultures for at least 9 days in the absence of exogenously added hormones. FSH (10-100 microgram/ml) markedly enhances the secretion of ABP. MIX has a potentiating effect after long treatment intervals (7 days). In order to study the second component, intracellular androgen receptor, a nuclear exchange assay was developed. Competition for exchange activity using 3H-dihydrotestosterone was significant for a 500 fold excess of testosterone, dihydrotestosteron, progesterone, and cyproterone acetate. The exchange activity was increased 2-10 fold by prior treatment in vitro or in vivo with testosterone. Significant exchange activity was found in long-term hypophysectomized adult and immature animals and in tubule and germ cell fractions. In isolated germ cell fractions, the highest concentration of exchange activity was associated with the most mature elements. These data suggest that androgen exchange activity may exist in both Sertoli cell and germ cell fractions and suggest that the mechanism of action of androgens in the testis is quite complex.

Tissue androgens and androphilic proteins in rat epididymis during sexual development

The appearance of the epididymal 8s cytoplasmic receptor in the rat during sexual development was followed and correlated with the endogenous concentrations of three biologically active androgens in the epididymis, testosterone (T), dihydrotestosterone (DHT)and 5α-androstane-3α, 17β-diol (Diol). The results indicate that receptors could not be evidenced in the 10-day-old animal in which androgens were undetectable. The 8s receptor was first detected in the 20 day-old rat, coincidently with a rise in the concentration of DHT (0,24 ng/ epididymis) while T and Diol remained too low to be measured. At the 26th day of life a peak of androgen binding activity, sedimenting at approximately 4S, was seen and probably corresponds to ABP. This pattern of two binding peaks (8 and 4S) remained constant through adulthood. The association constant (Ka) of the 8s receptor from 35-, 45- and 60-day-old rats was found to be similar. T became detectable in the epididymides of the 35 day-old rat (0.03 ng/ep.) and increased with sexual maturation. However, DHT concentrations were higher than those of T at all ages studied and Diol could not be detected at any age. These results raise the possibility that the synthesis or availability of receptors in the developing animals is under androgenic control.

Hormonal control of mammalian spermatogenesis.

Studies of the hormonal control of the spermatogenic process are reviewed. Most of the investigations used the techniques of ablation and replacement therapy and were performed on the rat. Results from the studies indicate that changes in the metabolism of proteins lipids and carbohydrates take place in the testes under the control of hormones. However it may be that the observed biochemical changes which occur are a reflection of the changes in cellular composition of testicular tissue rather than the result of metabolic changes within a specific cell type in response to the effect of a hormone. A hypothesis concerned with the qualitative aspects of hormonal control of spermatogenesis in the rat has been formed. It is proposed that the multiplication and growth of gonocytes prenatally and in the first few days after birth and formation of type A spermatogonia are under the control of testosterone. The formation of type A and B spermatogonia and primary spermatocytes and progression of the meiotic prophase may be influenced by growth hormone. Reduction division is under control of testosterone. The early steps of spermatid formation may be under no hormonal control or may require testosterone while the late stages of spermatid maturation require the presence of FSH.