0013-7227/90/1276-3180$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 6 Printed in U.S.A.

Immunohistochemical Localization of the Androgen Receptor in Rat and Human Tissues* MADHABANANDA SAR, DENNIS B. LUBAHN, FRANK S. FRENCH, AND ELIZABETH M. WILSON Laboratories for Reproductive Biology and the Departments of Cell Biology and Anatomy (M.S.), Pathology (D.B.L.), Pediatrics (D.B.L., F.S.F., E.M.W.), and Biochemistry (E.M.W.), University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT. Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 ^im, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the


NDROGEN action in the male reproductive tract and other tissues requires the presence of the androgen receptor (AR) to mediate transcriptional activation. In general, the assessment of AR has been limited to radiolabeled androgen binding assays in solubilized tissue fractions and autoradiography. The cloning of rat and human AR provided the deduced amino acid sequence of the receptor protein, allowing the development of polyclonal antibodies to synthetic peptides with sequences corresponding to different regions of the AR protein. Two antibodies that react selectively with AR were raised in this laboratory using synthetic AR peptides common to rat and human AR (1-3). Both antibodies react with high titer against their respective peptide antigens in the enzyme-linked immunoadsorbent assay Received June 29,1990. Address all correspondence and requests for reprints to: Madhabananda Sar, Ph.D., Department of Cell Biology and Anatomy, 526 Taylor Hall CB 7090, University of North Carolina, Chapel Hill, North Carolina 27599. * This work was supported by NIH Grants NS-17479, HD-16910, HD-04466, and P30-HD-18968 (Histochemistry Core), the Andrew W. Mellon Foundation, and the Pew Scholars Program in the Biomedical Sciences.

testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness. (Endocrinology 127: 31803186,1990)

and against the native [3H]dihydrotestosterone-labeled AR, as shown by an increase in receptor sedimentation on sucrose gradients (1-3). Immunohistochemical localization of AR in a variety of androgen-sensitive tissues has not previously been determined. Biochemical studies demonstrated cytosolic and nuclear AR in reproductive organs (4-7), pituitary (8-10), and brain (8-12) of the male rat. Autoradiographic studies with [3H]testosterone and [3H]dihydrotestosterone indicated nuclear localization of androgens in a number of male reproductive organs, including brain and pituitary (13-16). The present study describes the cellular localization of AR in rat reproductive organs, pituitary, brain, and human prostate using antipeptide polyclonal antibodies. These antibodies reveal specific immunostaining of AR and reactivity selective for androgen-sensitive tissues.

Materials and Methods Antibody preparation A rabbit polyclonal antibody referred to as AR52 was raised against a synthetic peptide with sequence in common with rat


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NUCLEAR LOCALIZATION OF THE AR and human AR (1). The peptide sequence is positioned Nterminal to the DNA-binding domain and contained 15 amino acids derived from AR, followed by a 3-amino acid spacer for coupling to the protein keyhole limpet hemocyanin. The peptide sequence was Asp-His-Val-Leu-Pro-Ile-Asp-Tyr-Tyr-PhePro-Pro-Gln-Lys-Thr-Gly-Gly-Cys. A second polyclonal antibody, AR32, was raised against a peptide Arg-Val-Tyr-Pro-ArgPro-Pro-Ser-Lys-Thr-Tyr-Arg-Gly-Ala-Phe-Gln-Asn-LeuPhe-Gln-Cys, beginning 9 amino acids from the N-terminus and common to rat and human AR. Peptides were coupled to keyhole limpet hemocyanin through the C-terminal cysteine and used to immunize rabbits, as previously described (1). Immunoglobulin G (IgG) fractions of immune and preimmune sera were obtained by chromatography on protein-A-agarose (17). Both antibodies shifted the sedimentation of the 4.5S [3H] androgen-labeled receptor to 10S on sucrose gradients and recognized a 114-kDa protein on immunoblots (1, 3). Immunoblots of AR in extracts of prostate, epididymis, and spleen of 24-h castrate rats demonstrated a 114-kDa AR band in prostate and epididymis, but not in spleen (3). All 3 tissues contained in addition 74- and 118-kDa bands, which were not detected after peptide adsorption of AR32 antibody. It would appear that these latter 2 proteins do not react with the antibody in immunohistochemical reactions, since no immunostaining was observed with AR antibodies in spleen. We know from previous studies that rat spleen lacks AR, as determined by ligand binding and Northern blot analysis. AR was detected in ventral prostate from intact and 24-h castrate rats by both immunohistochemistry and immunoblotting. As described below, controls for nonspecific interactions using immunohistochemistry indicated low levels of background staining. Immunohistochemical localization of AR Intact male Sprague-Dawley rats, 2-3 months of age, were killed by decapitation. In experiments designed to determine the effect of androgen withdrawal, rats were castrated under ether anesthesia. Three to 72 h or longer after castration, testosterone, dihydrotestosterone (100 Mg/100 g BW, in ethanol) or vehicle only was administered ip, and the rats were killed by decapitation 15 min to 3 h after hormone treatment. Tissue sections were prepared from three to five rats for each experimental condition. Tissues from intact and castrated rats were removed rapidly, placed on fresh rat liver mounts, and frozen in isopentane precooled in liquid N2 (—180 C). Tissues were stored in liquid N2 until sectioned with a cryostat. Frozen sections of 6 or 8 ^m were cut at —30 C, air dried for 2-4 min, and fixed for 4-5 min at room temperature in 4% paraformaldehyde, 10% sucrose, and 0.1 M sodium phosphate, pH 7.2. Sections were washed twice with PBS for 5 min each time. For human prostate and brain, endogenous peroxidase activity was reduced by treating sections with 3% hydrogen peroxide in 0.1 M sodium phosphate buffer, pH 7.4, for 2-3 min at room temperature. Sections were blocked with 2% normal goat serum for 20 min at room temperature, as previously described (18), and incubated overnight at 4 C with preimmune IgG or immune IgG, either untreated or preadsorbed with uncoupled peptide. The concentration of IgG ranged from 1-5 /ig/ml, and immune IgG was preadsorbed with 1-5 ng peptide/ml diluted immune


IgG for 48 h at 4 C. Sections were washed with PBS for 5-10 min and treated with biotinylated goat antirabbit IgG secondary antibody (3.75 [ig/ml; 1:400 dilution) for 60 min at room temperature. This was followed by reaction with avidin-biotin peroxidase (ABC, Vector Laboraotries, Burlingame, CA; 1:400 dilution) for 60 min at room temperature. After a 5-min wash with PBS, the sections were treated for 10 min with a solution containing 75 mg diaminobenzadine and 8 /tl fresh 30% hydrogen peroxide in 100 ml 0.05 M Tris, pH 7.6. Some sections were counterstained with rapid hematoxylin (Cryoperm, Fisher Scientific, Orangeburg, NY) after washing with 0.05 M Tris, pH 7.6. The avidin-biotin peroxidase staining procedure has been described in detail previously (18).

Results The specificity of AR antipeptide antibodies in the immunohistochemical reaction was demonstrated by treating adjacent tissue sections with preimmune IgG and others with immune IgG adsorbed by the appropriate peptide or treatment with immune IgG. Preadsorption control experiments showed a low level of background staining with antipeptide antibody AR52 (Fig. IB) in epididymis and antipeptide antibody AR32 in testis (see Fig. 4C). All IgG fractions were used at dilutions of 1-5 /xg/ml. A control tissue, spleen, which lacked detectable AR by Northern (18) or immunoblot analysis (3), also lacked positive immunostaining for AR (data not shown). Tissue sections incubated with preimmune IgG (see Fig. 4A for testis) showed a similar low level of background staining. Below are described results using either of the two antipeptide antibodies. Essentially the same results were obtained with either antibody. In epididymis (Figs. 1A and 2A), ductus deferens (Fig.

FlG. 1. Localization of AR in rat epididymis. Frozen adjacent sections of rat epididymis were incubated with AR52 immune IgG (A) or immune IgG preadsorbed with the free peptide used in preparing the immunogen (B), as described in Materials and Methods. Background staining in B is due to the hematoxylin nuclear counterstain. Stromal staining of AR in A is also observed in the absence of the counterstain. Counterstained with hematoxylin; sections, 6 fim; magnification, X184.

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Endo • 1990 Vol 127 • No 6

F I G . 2. Localization of A R in rat epididymis and ductus deferens. T h e

epididymis (A) and ductus deferens (B) were immunostained using AR52, as described in Materials and Methods, yielding strong localization to acinar epithelial cell nuclei. Sections, 6 /*m; magnification: A X265; B X132.

FIG. 3. Localization of AR in rat ventral prostate and seminal vesicle. Frozen sections of ventral prostate (A) and seminal vesicle (B) were immunostained with AR52 IgG, as described in Materials and Methods. Sections, 6 /im; magnification, X270.

2B), ventral prostate (Fig. 3A), and seminal vesicle (Fig. 3B), acinar epithelial cells showed strong nuclear immunostaining with IgG AR52. In addition, stromal cell staining was evident in epididymis (Figs. 1A and 2A), but not in ventral prostate (Fig. 3A). In ductus deferens and seminal vesicle, nuclei of smooth muscle cells were also immunostained (Figs. 2B and 3B). Intense nuclear staining was present in interstitial cells, Sertoli cells, and peritubular myoid cells of the testis using AR32 (Fig. 4B). Although spermatogenic germ cells appeared devoid of immunostaining, it could not be ruled out that spermatogonia lacked AR due to the limited resolution of morphology in this region. Like the reproductive organs,

FIG. 4. Immunohistochemical localization of AR in rat testis. Frozen sections of rat testis were treated with AR32 IgG fraction of preimmune serum (A), immune serum (B), or immune serum preadsorbed with free peptide (C), as described in Materials and Methods. Immunostaining over background controls is predominantly in nuclei of Sertoli cells, peritubular cells, and interstitial cells. Sections, 8 /xm; magnification, X256.

epithelial cells of the submaxillary gland showed distinct nuclear staining (Fig. 5). Background staining in epididymis (Fig. 1) and pituitary (Fig. 6) included a weak

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FlG. 5. Immunohistochemical distribution of AR in rat submaxillary gland. Tissue sections were stained using AR52 IgG, as described in the text. Note the glandular epithelial cells of the rat submaxillary gland with intense immunostaining localizing AR to nuclei. Section, 6 /xm; magnification, X340.

nuclear counterstain, hematoxylin, that did not mask AR immunostaining. Cells throughout the rat anterior pituitary displayed nuclear immunostaining for AR, shown in Fig. 6 at two magnifications. Cells of the intermediate pituitary lobes lacked positive staining, but nuclear staining was present in certain cells of the posterior pituitary (not shown). Using both peptide antibodies, specific nuclear immunostaining was detected in cells of several areas of the brain, including hypothalamic and extrahypothalamic sites (Fig. 7, A-E). When brain sections were incubated with AR52 IgG that was preadsorbed with peptide 875, no immunostaining was detected (Fig. 7F). Accumulations of AR-containing neurons occurred in preoptic-


hypothalamic regions, including the nucleus preopticus medialis, nucleus arcuatus hypothalami, nucleus ventromedialis hypothalami, and nucleus premammillaris ventralis, and in extrahypothalamic sites, such as the CA-1 region of the hippocampus, the medial nucleus of the amygdala, and cortex. Regions lacking AR staining included the medial septal nucleus, anterior hypothalamic area and certain thalamic nuclear groups, and medial eminence. A more detailed mapping of AR neurons in the brain is currently under investigation. Human prostate epithelial cells in benign hyperplastic tissue showed uniformly intense nuclear immunostaining for AR (Fig. 8A). In contrast, sections of prostate cancer displayed a more heterogeneous nuclear staining of varying intensity. Staining in larger neoplastic nuclei was lighter than that in smaller nuclei of the more well differentiated cells (Fig. 8B). In benign prostatic hyperplasia with a predominance of epithelial cells, stromal cells were weakly immunostained; however, more intense AR staining was observed in stromal cells of fibromuscular hyperplasia (Fig. 9). In some cancer tissue sections, stromal staining was also evident. The immunohistochemical results described above for tissue sections from intact rats demonstrated nuclear AR with essentially no cytoplasmic staining using either antipeptide antibody. The influence of androgen withdrawal on AR immunostaining was investigated using tissue sections from castrated rats. In epithelial cells of ventral prostate from rats 12 and 24 h after castration, AR staining was nuclear but diminished in intensity relative to that in the intact rat. There was no significant AR staining in the cytoplasm at these time periods. At

FIG. 6. Immunostaining of AR in rat pituitary. A section of rat anterior pituitary was stained with AR52 IgG immune fraction and counterstained with hematoxylin, as shown, in 6-fim sections at magnifications of x64 (A) and x390 (B). The intense nuclear staining in a select population of cells indicates the presence of AR. The weak nuclear staining in B is due to the hematoxylin counterstain.

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Endo • 1990 Vol 127 • No 6

FIG. 7. AR localization in rat brain. Sections were immunostained with AR52 IgG fraction as described. Regions of brain that displayed the presence of AR include the nucleus preopticus medialis (A), nucleus ventromedialis hypothalami (B),

nucleus premammilaris ventralis

(C), CA-1 hippocampus (D), and nucleus amygdaloideus medialis (E). A control section of nucleus preopticus medialis is shown (F) stained with immune IgG preadsorbed with peptide. Sections, 6 ^m; magnification, X410.

48 h, staining was diminished further, and at 72 h or longer after castration, neither nuclear nor cytoplasmic immunostaining was detectable (Fig. 10A). Prostate sections from male rats 72 h or longer after castration which were administered an injection of testosterone or dihydrotestosterone (100 Mg/100 g body weight) 15 min (Fig. 10B) to 3 h (Fig. IOC) before tissue removal again showed strong nuclear staining of AR in epithelial cells.

Discussion Tissues from intact adult male rats immunostained with peptide-induced polyclonal antibodies AR52 or AR32 showed nuclear AR in epithelial cells of the prostate, seminal vesicle, testis, ductus deferens, epididymis, submaxillary gland, anterior pituitary, and neurons of the preoptic-hypothalamic nuclear groups. The immunolocalization of AR in reproductive organs reported here is in general agreement with the nuclear localization of 3H-labeled androgens in androgen-sensitive tissues

demonstrated by autoradiography (12-16). The intensity and distribution of AR varied among the tissues. For example, stromal AR was evident in epididymis, but was absent or weak in ventral prostate. Nuclear AR staining was strong in epithelial cells of ventral prostate, but moderate in epithelial cell nuclei of seminal vesicle. In epididymis, the highest level of nuclear AR was in caput epithelial cells, with lower levels toward the corpus and cauda. A similar regional distribution in epididymal epithelial cells was observed previously by autoradiography after the injection of [3H]dihydrotestosterone (19). Immunohistochemical localization of AR in Sertoli cells, interstitial cells, and peritubular cells confirms our earlier selective localization of androgen to these testicular cell types (20). Further studies with better preservation of cell morphology are required to establish whether other cell types, such as spermatogonia and/or early spermatocytes, contain AR. Autoradiographic studies using [3H]testosterone and 3 [ H]dihydrotestosterone demonstrated previously that

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FIG. 8. AR localized in cells of human benign prostatic hyperplasia and prostatic carcinoma. Frozen sections of human prostate, 8 nm in thickness, were obtained after radical prostatectomy from a patient with prostate adenocarcinoma (Gleason score 2+3). Shown are regions of benign prostatic hyperplasia (A) and adenocarcinoma (B). The two tissue sections were taken from different regions of the same prostate gland and immunostained using AR52 IgG. Counterstained with hematoxylin; magnification, X276.

FlG. 10. Immunostaining of AR in rat ventral prostate 12 days after castration with or without androgen replacement. Nuclear or cytoplasmic staining using AR62 IgG was undetectable in epithelial cells in the absence of androgen (A). Nuclear staining in epithelial cells was evident 15 min (B) and 3 h (C) after the administration of dihydrotestosterone. Weak staining was also detectable in stromal cells after androgen treatment. Sections, 6 /im; magnification, X334.

FlG. 9. AR localization in human fibromuscular benign prostatic hyperplasia. Frozen sections of human prostate, 8 nm in thickness, were stained using AR52 IgG. Note the immunostaining in nuclei of stromal cells. Magnification, X560.

certain regions of the brain contain AR. These include the septum, preoptic region, anterior and central hypothalamus, amygdala, and hippocampus (14, 16). Microdissection of rat brain and subsequent androgen binding studies indicate regions high in AR, i.e. the ventromedial nucleus of the hypothalamus and medial amygdala, and intermediate levels in the arcuate nucleus-median eminence, preoptic area, anterior hypothalamus, septum, and parietal cortex (12). In the present studies immunohistochemical staining with AR antibodies revealed AR localization in nuclei of cells of the preoptic-hypothalamic region, amygdala, hippocampus, and cortex.

These results are in agreement with autoradiographic and biochemical studies (12, 14, 16). The localization of AR in the brain corresponds with anatomical sites involved in the regulation of gonadotrophin secretion, reproductive behavior, and aggressive behavior (21-25). The topographical distribution of AR-containing cells in the pituitary also agrees with the distribution of androgen target cells as revealed by autoradiography. In castrated rats, nuclear immunostaining of AR in ventral prostate and epididymis diminished with time after castration, such that 3 days after castration, AR was no longer detectable. In agreement with the results of this study, Husmann et al. (26) detected nuclear immunostaining of AR in the intact and 24-h castrate rat prostates using polyclonal antibodies against AR N- and C-terminal synthetic peptides. The lack of immunostaining at longer time periods after castration is not understood at present. However, rapid recovery of nuclear immunostaining in the castrate within 15 min of andro-

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gen treatment suggests that the androgen-free receptor undergoes a marked increase in immunoreactivity after hormone binding. The effect of androgen on immunostaining was too rapid for receptor-mediated gene transcription and new protein synthesis. Moreover, androgen is known to down-regulate its own receptor (27). Another possible explanation for loss of AR immunostaining in the absence of androgen is that AR may be more loosely associated with cellular components and is lost during tissue preparation and fixation. Alternatively, the receptor free of hormone may be in a conformation that prevents antibody binding to its epitope. Additional antibodies to other regions of the AR sequence may clarify the intracellular distribution of AR in the absence of androgen. In conclusion, polyclonal antibodies raised against synthetic peptides with sequences derived from the deduced amino acid sequence of rat and human AR are suitable for immunodetection of AR. The antipeptide antibodies described here cross-react histochemically with AR from different species, including mouse, hamster, and guinea pig (Sar M., unpublished results) as well as primate and human.

Acknowledgments We thank Elizabeth D. Creesy and Jon Kemppainen for excellent technical assistance. We also thank Dr. James Mohler for providing surgical specimens of human prostate cancer tissue.

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8. Kato J 1976 Cytosol and nuclear receptors for 5 alpha-dihydrotestosterone and testosterone in the hypothalamus and hypophysis, and testosterone receptors isolated from neonatal female rat hypothalamus. J Steroid Biochem 7:1179-1187 9. McGinnis MY, Davis PG, Meaney MJ, Singer M, McEwen B 1983 In vitro measurement of cytosol and cell nuclear androgen receptors in male rat brain and pituitary. Brain Res 275:75-82 10. Handa RJ, Reid DL, Resko JA 1986 Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male. Biol Reprod 34:293-303 11. Handa RJ, Roselli CE, Horton L, Resko JA 1987 The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment. Endocrinology 121:233-240 12. Roselli CE, Handa RJ, Resko JA 1989 Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain. Neuroendocrinology 49:449-453 13. Sar M, Liao S, Stumpf WE 1970 Nuclear concentration of androgens in rat seminal vesicles and prostate demonstrated by drymount autoradiography. Endocrinology 86:1008-1011 14. Sar M, Stumpf WE 1972 Cellular localization of androgen in the brain and pituitary after injection of tritiated testosterone. Experentia 28:1364-1366 15. Sar M, Stumpf WE 1973 Pituitary gonadotrophs: nuclear concentration of radioactivity after injection of [3H]testosterone. Science 179:389-391 16. Sar M, Stumpf WE 1977 Distribution of androgen target cells in rat forebrain and pituitary after [3H]dihydrotestosterone administration. J Steroid Biochem 8:1131-1135 17. Goding JW 1976 Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods 13:215-226 18. Sar M 1985 Application of avidin-biotin complex technique for the localization of estradiol receptor in target tissues using monoclonal antibodies. In: Bullock GR, Petrusz P (eds) Techniques in Immunocytochemistry. Academic Press, New York, vol 3:43-54 19. Stumpf WE, Sar M 1976 Autoradiographic localization of estrogen, androgen, progestin and glucocorticosteroid in "target tissues." In: Pasqualini J (ed) Receptors and Mechanism of Action of Steroid Hormones. Marcell Dekker, New York, pp 41-84 20. Sar M, Stumpf WE, McLean WS, Smith AA, Hansson V, Nayfeh SN, French FS 1975 Localization of androgen target cells in the rat testis: autoradiographic studies. In: French FS, Hansson V, Ritzen EM, Nayfeh SN (eds) Hormonal Regulation of Spermatogenesis. Plenum Press, New York, pp 311-319 21. Barfield RJ 1984 Reproductive hormones and aggressive behavior. In: Flannelly KJ, Blanchard RJ, Blanchard DC (eds) Biological Perspective on Aggression. Liss, New York, pp 105-134 22. Larsson K 1979 Features of the neuroendocrine regulation of masculine sexual behavior. In: Beyer C (ed) Endocrine Control of Sexual Behavior. Raven Press, New York, pp 77-163 23. Kalra SP, Kalra PS 1983 Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4:311-351 24. Krey LC, MacLusky NJ, Davis PG, Lieberburg I, Roy EJ 1982 Different intracellular mechanisms underlie testosterone's suppression of basal and stimulation of cyclic luteinizing hormone release in male and female rats. Endocrinology 110:2159-2167 25. Roselli CE, Kelly MJ, Ronnekleiv OK 1990 Testosterone regulates progonadotropin-releasing hormone levels in the preoptic area and basal hypothalamus of the male rat. Endocrinology 126:1080-1086 26. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to nuclei of target cells in the rat and human prostate. Endocrinology 126:2359-2368 27. Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM 1990 Autologous down regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 4:22-28

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Immunohistochemical localization of the androgen receptor in rat and human tissues.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the ...
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