Structure and biochemistry of the Sertoli cell.
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Androgen-Binding Protein↗
Biomedical subjects
D J Tindall
Publications and source records attributed to D J Tindall.
Affinity labeling of the androgen receptor in rat prostate cytosol with 17 beta-[(bromoacetyl)oxy]-5 alpha-androstan-3-one.
An androgen affinity label, 17 beta-[(bromoacetyl)-oxy]-5 alpha-androstan-3-one, has been synthesized in both radioactive and nonradioactive forms. The affinity label (170 Ci/mmol) was characterized and found to have a high degree of purity. Affinity labeling of the androgen receptor from rat ventral prostate was androgen specific and appeared to be directed at the steroid binding site of the protein. Covalent binding was achieved at 0 degrees C; however, heat treatment at 23 degrees C for 30 min enhanced covalent binding by 31%. The covalently bound steroid was resistant to extraction with organic solvents and precipitation with trichloracetate. The Stokes radius (4.2 nm) and sedimentation coefficient (4.5 S) were identical with those found for receptor bound noncovalently to dihydrotestosterone. Gel electrophoresis of the affinity-labeled receptor under denaturing conditions revealed a molecular weight of 86000. The same molecular weight was observed for the receptor from rat seminal vesicle. This affinity label will be useful in future studies on the structure and function of androgen receptors.
Affinity Labels↗
Physicochemical characterization of the androgen receptor from hyperplastic human prostate.
In this manuscript we have characterized the androgen receptor from human benign prostatic hypertrophied (BPH) tissue. BPH tissue was obtained fresh from surgery, ground, and frozen in liquid nitrogen. Tissue was homogenized in 10 mM Tes buffer (pH 7.4 at 25 degrees C) containing 0.25 M sucrose, 20 mM Na2MoO4, 12 mM monothioglycerol, and 1.5 mM EDTA. Cytosol was labeled with (3H)-R1881 (0.5-10 nM) +/- a 100-fold excess of R1881 and a 500-fold excess of triamcinolone acetonide to identify specific androgen receptor sites. An exchange assay was developed, whereby maximum binding was achieved by raising the temperature to 30 degrees C for 15 min. A high-affinity binding protein was detected (Kd = 1.3 +/- 0.8 nM) which had a binding capacity of 15 +/- 7 fmol/mg protein. Binding was specific for androgens with the following Ki (nM) values: R1881 (0.09), dihydrotestosterone (6), testosterone (50), progesterone (92), estradiol (100), epitestosterone (860), and cortisol (greater than 1,000). Under high ionic conditions the sedimentation coefficient of the receptor was 4.1 S. Higher S values were not observed in the presence of the following protein inhibitors: leupeptin (20 or 150 microM), iodoacetamide (10 mM), DFP (5 mM), PMSF (1 mM), bacitracin (0.1 mM), antipain (0.1 mM), aprotinin (1 TIU/ml). Gel filtration analysis revealed a Stokes radius of 6.5 nm. These data indicate a Mr of 120,000 and a f/fo of 2.01. The receptor eluted from a chromatofocusing column at a pH of 6.8. The receptor bound to phosphocellulose and DNA cellulose after being heat-treated for 15 min at 30 degrees C. These results suggest that BPH tissue contains an androgen receptor which is similar to receptors in other androgen-responsive tissues.
Binding, Competitive↗
Exchange assay for androgen receptors in the presence of molybdate.
Several reports have shown that sodium molybdate stabilizes steroid hormone receptors. We have utilized these observations to develop an exchange assay for the androgen receptor at elevated temperatures. Exchange was found to be complete after 30 min at 30 degrees C. Receptor degradation was negligible during this treatment. Scatchard analysis indicated that the dissociation constant of the androgen receptor was similar both in the absence (Kd = 3.9 nM) and presence (Kd = 2.9 nM) of molybdate. Steroid specificity of the androgen receptor was unaltered by this treatment. The exchange procedure was reproducible, with an interassay variation of 2.45% and intraassay variation less than 10.0%. Using this assay, highest concentrations of androgen binding were measured in androgen target tissues of the rat (Dunning R3327 tumor, prostate and seminal vesicle; 23.37, 20.20 and 19.84 fmol/mg protein respectively). Lower concentrations were observed in other tissues (lung, brain, heart, spleen, liver and kidney; 9.06, 5.63, 3.50, 2.42, 2.33 and 1.36 fmol/mg protein respectively). These results demonstrate that molybdate stabilization of the androgen receptor allows efficient steroid exchange without significant alteration of the receptor's steroid binding properties. Furthermore, this exchange assay can be used to obtain a reasonable measurement of receptor concentrations in different androgen target tissues.
Animals↗
Androgen antagonists in androgen target tissues.
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Abnormalities, Drug-Induced↗
Molecular properties of the androgen receptor in rat ventral prostate.
Results from these studies demonstrate that we have purified a protein from rat prostate cytosol that is similar to the beta-protein (complex II) but different from the alpha-protein (complex I) reported by Liao et al. The purified receptor was different from androgen binding protein (ABP) in that ABP has a faster dissociation rate (6 min), a lower pI value (4.6), and requires higher concentrations of ammonium sulfate for precipitation (40-50%) than the prostatic androgen receptor. It is not likely that we have purified a serum sex-steroid binding protein since no such protein is found in rat serum. This report presents a rapid and efficient procedure for the purification of androgen receptor from rat ventral prostate. However, the present procedure only allowed us to obtain a limited quantity of purified receptor from each preparation. It is obvious that we need to scale up the purification of the receptor in order to study in detail its physicochemical properties and to produce monospecific antibodies against the protein. This work is in progress. In addition, we have demonstrated that two affinity labels can be used to bind covalently to the androgen receptor. Most importantly, these compounds can be used to characterize androgen receptors under both nondenaturing and denaturing conditions and represent useful tools for future work with androgen receptor proteins and androphilic proteins in general.
Affinity Labels↗
Effects of sodium molybdate on the androgen receptor from the R3327 prostatic tumor.
The present study describes the effects of sodium molybdate on several physicochemical properties of the androgen receptor from the Dunning R3327 prostatic tumor. Molybdate was found to stabilize the steroid-binding activity of the receptor. Maximum binding activity was found with concentrations of 10 mM molybdate or greater. Density gradient and gel filtration analyses of the receptor revealed an 8.5-9.0S, 68A form (mol wt, approximately 265,000-275,000) in either the presence or absence of molybdate (20 mM) when determined under low ionic conditions. Under high ionic conditions (400 mM KCl), the receptor was maintained in a similar form (8.5-9.0S; 72-73A; mol wt, approximately 275,000-295,000) in the presence of molybdate; however, it disaggregated to a smaller 4.4S, 61 A form (mol wt, approximately 120,000) in the absence of molybdate. Affinity labeling of the receptor with 17 beta-[(bromoacetyl)oxy]-[1,2,4,5,6,7,16,17-3H8]5 alpha-androstan-3-one showed a mol wt of 118,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Molybdate inhibited the salt-induced transformation of the receptor to a DNA-binding state, but did not inhibit the DNA binding of the receptor transformed previously in the absence of molybdate. These studies suggest that sodium molybdate stabilizes the steroid-binding activity of the androgen receptor in an aggregated nontransformed state.
Animals↗
Purification and characterization of the androgen receptor from rat ventral prostate.
The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.
Androgens↗
Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts.
Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.
Binding, Competitive↗
Physicochemical characterization of the androgen receptor in rat uterine cytosol.
In this manuscript a number of physicochemical and DNA-binding properties of the androgen receptor from rat uterus were characterized. The dissociation of [3H]testosterone from the receptor followed first order kinetics, with a half-time dissociation rate of approximately 48 h at 0 C. Maximum binding was maintained at pH 7. Ammonium sulfate precipitated the receptor at concentrations between 25-40% saturation. Gel filtration and sucrose gradient analysis revealed a 62 A, 6S binding component (mol wt, 167,000; frictional ratio, 1.71) in both the absence and presence of the protease inhibitor leupeptin. Isoelectric focusing revealed an isoelectric point of 5.9. The receptor bound to DEAE-agarose, heparin-Sepharose, and hydroxylapatite. Increased receptor binding to phosphocellulose and DNA-cellulose was observed after heat treatment at 24 C for 30 min. These results demonstrate that the uterine androgen receptor has many properties similar to those of androgen receptors in male target tissues.
Ammonium Sulfate↗
Purification and characterization of androgen receptor from steer seminal vesicle.
The androgen receptor has been purified from steer seminal vesicle cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. The procedure produced about 3 micrograms of receptor protein from 35 g of steer seminal vesicle, with a yield of 48%. The receptor protein, as a complex with unlabeled testosterone, was purified approximately 540000-fold. A single band, migrating at 60000 daltons, was observed following electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate (NaDoDSO4). This was confirmed by affinity labeling of the partially purified receptor with both 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one and 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8)-5 alpha-androstan-3-one 17-(2-bromoacetate), which showed a peak of radioactivity migrating at 60000 daltons by NaDoDSO4 gel electrophoresis. The receptor had an estimated Stokes radius of 35 A and a sedimentation coefficient of 3.8 S in the presence of 0.3 M NaCl. The calculated molecular weight and frictional ratio for the androgen binding activity were 57000 and 1.42, respectively. Chromatofocusing of the purified receptor protein revealed an isoelectric point of 6.6. [3H]Methyltrienolone, bound to the purified receptor, was displaced with methyl-trienolone greater than testosterone greater than 5 alpha-dihydrotestosterone much greater than 3 beta-hydroxy-delta 5-androsten-17-one much greater than 5 alpha-androstane-3 alpha,17 beta-diol. The physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol. These results demonstrate that the androgen receptor from steer seminal vesicle was substantially purified without significant modification of its physicochemical or steroid binding properties.
Affinity Labels↗
Characterization of human Sertoli cells in vitro.
The Sertoli cell is thought to play a significant role in the hormonal regulation of spermatogenesis within the rat testis. Little, however, is known about Sertoli cell function in man, largely because of the difficulties associated with the isolation of pure cell populations from human tissue. We have now developed a rapid and reproducible technique for establishing a human Sertoli cell monolayer culture. This has involved mechanical separation of the tissue, sequential trypsin and collagenase enzyme digestion, and final disruption of tubules by passage through a wire mesh grid. Using this technique, primary cultures can be maintained for up to 45 days. Ultrastructural studies of these cells have demonstrated the presence of the perinucleolar spheres, cell to cell junctional complexes, abundant lipid droplets, and smooth endoplasmic reticulum, all characteristic of Sertoli cells. Furthermore, biochemical markers of animal Sertoli cells, androgen-binding protein and gamma-glutamyl transpeptidase, have also been identified in these human cells. Concentrated media electrophoresed on nondenaturing gels containing 2 nM [3H]dihydrotestosterone produced a single peak of bound activity which coelectrophoresed with rat androgen-binding protein. This binding activity persisted despite media changes, thus ruling out contamination by serum binding proteins; fresh media lacked demonstrable binding activity. Using a colorimetric assay, these cells were also found to contain significant gamma-glutamyl transpeptidase activity compared to human foreskin fibroblasts and Leydig cells. Enzyme activity increased in a characteristic dose-response fashion in the presence of FSH (0.05-0.5 microgram/ml) and dibutyryl cAMP (0.1-1 microgram/ml), but not with LH or testosterone. These data offer the first demonstration of human Sertoli cells in monolayer culture and their production of a marker specifically regulated by FSH.
Androgen-Binding Protein↗
Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP).
The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding to SBP was assessed. Binding was enhanced by reduction of the delta 4 double bond to 5 alpha-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14,15. Affinity was always reduced by modifications of the C-17 beta hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17 alpha-ethinyl had limited effects on binding; whereas, other modifications such as 7 alpha-methyl or 17 alpha-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5 beta steroids had reduced affinity.
Androgen-Binding Protein↗
Estradiol-17 beta inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: a comparison with cyproterone acetate.
The effects of estradiol-17 beta on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 beta inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 beta and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 beta than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 beta; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 beta, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 beta and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 beta may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 alpha reduction of testosterone as well as binding to the androgen receptor.
Animals↗
Factors affecting Sertoli cell function in the testis.
The Sertoli cell is the primary target for FSH action in the mammalian testis. These cells contain the majority of testicular plasma membrane receptors for this hormone. Receptor occupancy is directly correlated with a stimulation of adenylyl cyclase and a decrease in the activity of a cytoplasmic Ca++-sensitive cAMP phosphodiesterase. Regulation of these two enzymes allows increased intracellular accumulation of cAMP, activation of cAMP-dependent protein kinase and phosphorylation of a variety of protein substrates. All of these events occur within the first 30 min following exposure of isolated Sertoli cells to FSH. RNA and protein synthesis are also enhanced by FSH. Previous studies have suggested that this gonadotropin may augment the overall cellular synthesis of proteins. Our results reveal that protein kinase inhibitor (PKI) is selectively elevated by FSH both in vivo and in vitro. PKI thus becomes the initial intracellular protein whose synthesis is under FSH control. In addition to effects on protein synthesis, FSH also positively modulates the secretion of several specific proteins. One of the proteins in this latter category is androgen binding protein (ABP). Again, regulation can be observed both in vivo and in vitro. Elevated synthesis of PKI occurs prior to demonstrable secretion of ABP. Both of these events occur subsequent to the effects of FSH on cAMP metabolism. Indeed cAMP (or any of several nonhydrolyzable derivatives) can substitute for FSH in vitro. The temporal sequence of events subsequent to hormone binding and cAMP production are identical, but occur more rapidly. Together these data support the hypothesis that most of the biochemical steps leading to the synthesis and secretion of proteins by FSH are regulated by elevated levels of cAMP.
Adenylyl Cyclases↗
Regulation of the testis sertoli cell by follicle stimulating hormone.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Epididymal androgen binding protein in protein-calorie malnourished rats.
Androgen binding protein (ABP) was measured at five-day intervals from 20 to 50 days of age in the epididymides of rats fed a low protein diet. The results were compared to findings made in age-matched controls fed a standard laboratory diet. ABP was consistently lower in the caputs of the malnourished rats throughout the entire study. Moreover, no significant changes were noted in caput ABP concentrations as these rats aged. In contrast, this protein was observed to increase during the course of the month-long experiment in those animals fed the standard laboratory diet. These findings indicate that dietary protein deficiency has a marked debilitative effect on Sertoli cell function.
Aging↗
Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.
Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.
Androgens↗