Expressions of androgen receptors in testicular and gubernaculum tissues in mice

Background: Sex steroids, estrogens and androgens, exert a number of effects on the morphological, biochemical and electrophysiological properties of the brain during its development. These effects are mediated through specific steroid receptors, estrogen receptor α (ERα), estrogen receptor β (ERβ) and androgen receptor (AR). Purpose: The aim of this study was to examine the expression of sex steroid receptors and correlate it to steriodogenesis during postnatal development of the brain. Methods: In the present study, expression of sex steroid hormone receptors and its correlation to 3β-hydroxydehydrogenase (3β-HSD) enzyme activity were examined in the mouse cerebral cortex at postnatal days (PD) 0, 7, 15, 30 and 45. Results: Immunofluorescence results revealed that the expression of both androgen receptor (AR) and estrogen receptors (ERα, ERβ) was lowest at PD0 and highest at PD45 in the cerebral cortex of male and female mice and the increase was evident from PD15. In parallel, the activity of 3-HSD enzyme continually decreased from PD0 to PD45 with no significant difference between male and female. Thus the expression of AR and ER is inversely related to 3-HSD activity in mouse cerebral cortex during postnatal development. Conclusion: These findings suggest the involvement of sex steroid hormone receptors in differentiation and establishment of neural circuitry during postnatal development of the brain. doi : 10.5214/ans.0972.7531.2009.160405 Competing interests: None. Source of Funding: CSIR, DBT, DST Received Date: 27 Aug 2009 Revised Date: 30 Sept 2009 Accepted Date: 12 Oct 2009

Androgen and androgen receptor (AR) play important roles in spermatogenesis, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR) and their littermate wild-type (WT) mice. Digital gene expression analysis identified 2,276 genes downregulated and 2,865 genes upregulated in the S-AR mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing ten times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/AR-regulated genes, including 164 up-regulated and 439 down-regulated, were found in both S-AR mice testis and TM4/AR cells.Ubiquitin-conjugating enzyme E2B (Ube2b) is one of the regulated genes from the digital gene expression analysis. The expression of UBE2B was decreased in the testes of the S-AR mice analyzed by quantitative RT-PCR (qRT-PCR) and immunofluorescence. The up-regulation of Ube2b gene by testosterone was further demonstrated by Western blot and qRT-PCR in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay validated that the ligand-bound AR activated Ube2b transcription via directly binding to the androgen-responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased UBE2B expression and activated H2A ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly down-regulated and 71 were up-regulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation.Our previous digital gene expression analysis data also showed that heat shock transcription factors 1 (HSF1) was regulated by androgen in mouse Sertoli cells. We found that the expression of Hsf1 was increased in the testes of S-AR mice compared with wild-type mice by quantitative real-time PCR and the expression of HSF1 in the S-ARSertoli cells was significantly increased as examined by immunofluorescence analysis. Besides, in vitro study showed that testosterone repressed the expression of Hsf1 in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to Hsf1 promoter. Our experiment also demonstrated that testosterone downregulated the expression of heat shock protein HSP105 and HSP60 by inhibiting the transcription of Hsf1. Taken together, these results reveal that HSF1 is a novel target of androgen receptor in mouse Sertoli Cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting HSF1 expression.

Objective To explore the relationships among the occurrence ofteaticular dysgenesis syndrome with hypospadias and cryptorchidism,the polymorphic androgen-receptor CAG repeat and associated perinatal factors.Methods Blood samples were collected from 60 hypospadias,32 cryptorchidism (unilatcral 29 and bilateral 3,6 of them combined with hypospadias) and 70 normal boys as control between May 2009 and Dec 2010.CAG repeat lengths of androgen receptor were measured.The perinatal data collected included birth weight and gestational age; mother's age,history of tocolysis in early trimester (the use of progesterone),assisted reproduction,mother's occupation,habitation and history of gestational hypertension.All data were statistically analyzed.Results The CAG repeat length of hypospadias and cryptorchidism were significantly longer than the control (P =0.008 and 0.028 respectively).Low birth weight of infants (P =0.003),mother's age (P =0.007) and the use of progesterone (P =0.000) were found to be risk factors for hypospadias.Mother's age (P =0.003)and agriculturalactivities (P =0.017) were risk factors for cryptorchidism.Conclusions The occurrence oftesticular dysgenesis syndrome is associated with multiple factors.The AR gene CAG repeat length,low birth weight,young mother,especially exposure to estrogen,anti-androgen substances,such as progesterone and pesticide all contribute to an increased risk of the occurrence of testicular dysgenesis syndrome. Key words: Testis; Hypospadias; Cryptorchidism; Androgen receptors; Perinatal factor

This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities. Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas. In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis. In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group). To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis). It is concluded that 1) reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume; 2) these changes are largely absent with a lower dose of DES (1 microg); 3) treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES; 4) a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.

Objective To explore the effect of neonatal exposure to different doses of Bisphenol A (BPA) on the hypothalamic-pituitary-testis axis in toddler and adolescent male rats. Methods Neonatal male Sprague-Dawley rats were randomly divided into 5 groups through random digital table method: control group, vehicle group, low-dose BPA [25 μg/(kg·d)] group, medium-dose BPA[50 μg/(kg·d)] group and high-dose BPA [250 μg/(kg·d)] group.The rats were subcutaneously injected with respective agents on postnatal days 1-7 (PND 1-7). Pups were sacrificed on PND 22 and PND 50.The hypothalamus and testis were taken and weighed.The hypothalamic Kiss-1 mRNA and the testis androgen receptor (AR) mRNA were tested by using real-time fluorescence quantitative and the levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) were tested by using radio immunity method, and inhibin B was measured by using enzyme linked immunosorbent assay. Results Compared with the controls, the level of serum FSH [(1.610 0±0.693 2) IU/L, (1.574 3±0.675 0) IU/L vs (2.362 9±0.580 3) IU/L](F=3.314, P=0.026), LH [(3.876 3±0.908 0) IU/L, (3.603 8±1.350 2) IU/L vs (5.302 5±0.768 4) IU/L](F=3.139, P=0.027) and T [(0.383 8±0.177 8) μg/L, (0.442 5±0.214 1) μg/L vs (0.782 5±0.282 1) μg/L](F=5.106, P<0.01) of medium and high-dose BPA groups, were decreased in PND 22, and the organ coefficient of testis [(0.952 90±0.049 15)%, (0.969 20±0.045 82)% vs (1.022 80±0.011 08)%](F=10.326, P<0.01)and serum T [(1.758 6±0.369 6) μg/L, (1.718 8±0.395 7) μg/L vs (3.357 5±0.749 8) μg/L](F=13.799, P=0.012) were significantly decreased in PND 50.In high-dose BPA group of PND 22, the expression of hypothalamic Kiss-1 mRNA (0.068 80±0.011 79) was increased compared with the other groups(F=272.125, P<0.01), while in PND 50, compared with control group, the Kiss-1 mRNA(0.002 00±0.000 25, 0.001 90±0.000 48 vs 0.001 40±0.000 17) of medium- and high-dose BPA groups was decreased(F=191.826, P<0.01). Conclusions Neonatal exposure to the medium and high-dose BPA may impair the function of testis in toddler and adolescent male rats, and affect the hypothalamus-pituitary-testis axis.Neonatal exposure to the low-dose BPA does not have a significant influence on the hypothalamus-pituitary- testis axis. Key words: Bisphenol A; Hypothalamic-pituitary-testicle axis; Neonatal male rat

Objective To evaluate the expression of Testosterone、Androgen Receptor and FGF8 in genital tubercle (GT) of hypospadiac male rats following in utero exposure to DBP,to investigate molecular mechanism of DBP on androgen related FGF8 pathway.Methods Twenty pregnant rats were randomly divided into 2 groups and given DBP by gastric intubation at a dose of 0,750 mg/kg from gestation day(GD) 14 to GD18.On postnatal day (PND) 1,male pups were examined for hypospadias.On PND7,the body weight of male pups were measured and the gross image of hypospadiac genitalia were checked.Real-time quantitative PCR and WesternBlot were used to evaluate expression of AR and FGF8 in the GT.Radio-immunoassay was used to evaluate testosterone concentration in the serum of hypopadiac rats.Results The number and body weight of live pups in the DBP group (9.10 ± 0.99;9.53 ± 0.12 g) was significantly lower when compared with the control group(12.60 ±1.26; 11.93 ± 0.15 g) ( P ＜ 0.05 ),and the incidence of hypospadias was 37.2 ％.Significantly decreased expression of AR and FGF8 were also found in both mRNA and protein levels in the DBP group(AR:0.404 ± 0.040; FGF8:0.036 ± 0.004),when compared with the control group(AR:1.669± 0.124; FGF8:0.168 ± 0.004) ( P ＜ 0.005 ).The testosterone concentration of DBP treating group (41.85 ± 8.38 ng/L) was also lower than control group( 107.40 ± 24.28 ng/L)(P＜0.05).Conclusions Toxic effects of DBP to pregnant rats were seen.The occurrence of hypospadias may be related to the interference on androgen related FGF8 pathway by DBP. Key words: Di-n-butyl phthalate; Hypospadias; Testosterone; Androgen receptors; Fibroblast

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

Objective To investigate androgen receptor(AR) expression in triple negative breast cancer(TNBC) patients and its predictive value for clinical outcomes. Methods Five hundred and sixty-eight primary breast cancer patients who received surgery treatment from January 2008 to January 2010 were included in this study.Tissue sections of surgical resection specimens were underwent estrogen receptor(ER), progesterone receptor(PR), and human epidermal growth factor receptor 2(HER2) immunohisto chemical staining, according to the judgment for triple negative breast cancer and non triple negative breast cancer.Data on age, family history, menstruation, tumor size, tumor stage, tumor grade, pathological type and lymph node metastasis were collected for analysis.Immunohistochemical analysis of AR was done in tissue sections of triple negative breast cancer patients, Kaplan-Meier method was used to compare survival between AR(+ ) and AR(-) groups, and Log-rank method was used to determine the association between AR- positive expression and 5-year overall survival as well as disease free survival. Results Among 568 breast cancer patients, 174(30.6%) TNBC patients were identified, including 84 patiens with AR(-) expression and 90 patients with AR(+ ) expression.During 5 years' follow-up, reccurrence rate was 35.7%(30/84) in AR(-) group and 20.0%(18/90) in AR(+ ) group; 5-year overall survival rate was 73.8% in AR(-) group and 81.1% in AR(+ ) group.There was no significant association between AR(+ ) expression and 5-year death or recurrence rate, while after adjusting for age, menstruation, tumor size and lymph node metastasis, AR(+ ) expression was significantly associated with decreased 5-year death and recurrence rate, adjusted hazard ratio(HR) was 0.794, 95CI(0.430-1.021) and 0.722, 95CI(0.451-0.965) respectively. Conclusion AR(-) expression is associated with increased risk of death and recurrence and is a promising predictive marker for poor clinical outcomes. Key words: Triple negative breast cancer; Androgen receptor; Estrogen receptor; Progesterone receptor; Human epidermal growth factor receptor 2; Survival rate

The androgen receptor (AR) plays a critical role in sexual differentiation and in the virilization of the male reproductive system. A clear understanding of AR expression at the early stages of sexual development will help elucidate the sensitivity of perinatal animals to endocrine modulation by external agents, such as some environmental chemicals. Immunohistochemistry was used in this study to localize the AR in the differentiating testis and epididymis of Sprague-Dawley rats starting from gestation day 15 until postnatal day 21. Positive AR staining was found on gestation day 15 in the mesenchymal as well as in the epithelial cells in the mesonephros. Weak staining was also observed in a small number of interstitial cells in the primordial testis at this age. The fetal interstitial and peritubular myoid cells showed positive AR immunoreactivity early in development, but the Sertoli cells did not overtly express the receptors until postnatal day 5. The intensity of staining and number of AR-positive cells in the testis and epididymis increased over time. The epithelium in the mesonephros-derived tissues, including rete testis and epididymis, appeared to exhibit a higher capacity to express AR than the rest of the testicular tissue. The results demonstrate that AR expression in the primordial male reproductive system is highly specific to time and cell type and modify previous understanding on the timing of AR expression in the testicular tissue. Since AR-positive cells at various developmental stages may be potential sites of interaction with chemicals that adversely affect sexual differentiation, improved understanding of AR ontogeny will help in investigating the effects of AR-reactive agents, such as environmental antiandrogens, with respect to specific windows of sensitivity.

Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR as a target for radiosensitization. In addition, 70-95% of all estrogen receptor (ER) positive (ER+) breast cancers also have coexpression of AR, suggesting extended utility of AR inhibition in the radiosensitization of these AR+, ER+ tumors. Here we assessed the efficacy of AR inhibition in ER+, AR+ breast cancers to better understand the role of AR signaling across breast cancer models. Further, we also investigated the effect of ER inhibition on radiosensitization of ER+ breast cancer models. Methods: IC50 values were determined for MDV3100 (enzalutamide), ARN-509 (apalutamide), and ODM-201 (darolutamide) in TNBC cell lines (AR+ TNBC: MDA-MB-453, ACC-422, and SUM-185PE, and AR- TNBC: MDA-MB-231) and ER+ breast cancer cell lines (AR+, ER+: ZR-75-1, BT-474, CAMA-1, and AR-, ER+: MCF-7). IC50 values for tamoxifen were determined for ER+ breast cancer cell lines (MCF-7, T47D, ZR-75-1), and ER- (SUM-159) cells. Clonogenic survival assays were performed to assess radiosensitization with ER or AR inhibition with tamoxifen or second generation anti-androgens, respectively, in TNBC and ER+ breast cancer models. Results: AR inhibition with enzalutamide, apalutamide, and darolutamide showed limited single agent growth inhibition efficacy in AR+ TNBC and AR+, ER+ breast cancer cell lines (IC50 &amp;gt; 10 μM). AR inhibition with enzalutamide did not induce radiosensitivity in vitro. In AR+, ER+ CAMA-1 cells, AR blockade with enzalutamide had a radioprotective effect with enhancement ratios (enhR) of 0.76-0.83. No radiosensitization was observed in BT-474 (enhR: 0.92-1.01) or ZR-75-1 cells (enhR: 0.94-1.00). Radiosensitization was also assessed with anti-androgens apalutamide and darolutamide in AR+ breast cancer models. Inhibition of ER with tamoxifen, however, induced radiosensitization in MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells. No radiosensitization was observed with tamoxifen in ER- SUM-159 cells. Conclusion: Although AR is a mediator of radioresistance in AR+ TNBC, AR inhibition does not provide comparable radiosensitization in AR+, ER+ models and may actually confer a radioprotective effect. In contrast, our results demonstrate ER inhibition is an effective radiosensitizing strategy in ER+ breast cancers, independent of AR status. This work highlights the complexities of androgen and estrogen receptor signaling in AR+, ER+ breast tumors and underscores the necessity for understanding context dependent effects when translating into patients with AR+ breast cancer. Citation Format: Anna R. Michmerhuizen, Amanda Zhang, Rachel Schwartz, Andrea M. Pesch, Benjamin C. Chandler, Cassandra L. Ritter, Meilan Liu, Kari Wilder-Romans, Daniel E. Spratt, Daniel R. Wahl, Shyam Nyati, Lori J. Pierce, Corey Speers. Hormone receptor inhibition as a strategy for radiosensitization of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6271.

Objective To study reproductive functional lesions of Di (2-ethylhexyl) Phthalate(DEHP) on epididymis, and to find antagon which can repress the toxicity. Methods In vivo study,postnatal day 20 (PND20) and PND50 male KM mice were randomly divided into normal control, cornoil, DEHP and DEHP + Zinc gluconate group. In each stage and group, after been garaged for 10days, epididymal histopathologic changes and androgen receptor (A.R) and estrogen receptor (ER) inepididymis were detected. In vitro study, epididymal epithelial cells of PND20 and PNDS0 mice werecultured. Viability of epididymal epithelial ceils were measured using MTT assay, content of sialic acid(SA) and the activity of α-1,4-glucosidase and lactic acid dehydrogenase (LDH) were investigated. Re-sults DEHP induced epididymis atrophy and conspicuous histopathologic uhrastructure changes, up-regulated AR and down-regulated ER. No conspicuous variations were found in DEHP + Zinc gluco-hate group. Conclusions DEHP can induce lesions of epididymaI structure and function. Zinc is an an-tagon to DEHP toxicity. Key words: Dicthylhexyl phthalate; Reproduction; Epididymis; Zinc; Mice

Androgens acting via androgen receptor (AR) play essential roles in the prostate development, growth and pathogenesis of benign prostate hyperplasia (BPH) and prostate cancer. Over the last three decades, intensive studies have been carried out to elucidate the molecular basis of androgen action in the prostate. It has been realized that there are two natural potent androgens in the mammal including humans. Although testosterone is the major androgen secreted from the testes, dihydrotestosterone (DHT) is the main androgen in the prostate to mediate the androgen action via the AR. Only one AR has been identified up to date, a member of the steroid/nuclear receptor superfamily, which is a ligand-dependent nuclear transcription factor. When androgens bind to the AR, this results in a conformational change within the AR, leading to the recruitment of co-regulators and transcription factors which mediate androgen-target gene expression. Androgen actions in the prostate can also be modulated by other hormones such as estrogens via estrogen receptors (ER). There are two known isoforms of the ER, ERα and ERβ, which are both co-expressed with AR in the prostate and prostate tumor cells, which provides an anatomical basis for a direct interplay between AR and ER. Although it is well known that androgens are important for prostate development and for the pathogenesis of BPH and prostate cancer, the precise mechanisms as to how androgens control these processes are not yet fully understood. Furthermore, evidence for the direct modulation of androgenAR actions by other hormones within the prostate or prostate tumor cells is emerging, and the clinical implications of these hormones is being explored. In this article, we will assess the importance of androgens, and especially DHT, in prostate physiology and in the pathogenesis of BPH and prostate cancer. We will also review the molecular nature of the interactions between AR and ER and their respective ligands in prostate cells, as well their potential clinical implications.

Sexual Differentiation of the Nervous System: Where the Action Is

Genetic studies indicate that the actions of testosterone and dihydrotestosterone are mediated by a single androgen receptor (AR). The basis for the differential effects of these hormones during development is not known. To address this problem, we have turned to the rabbit, a species in which the endocrine and metabolic events in male sexual differentiation have been studied in detail. As a first step, we have cloned a partial cDNA encoding the rabbit prostate AR and have analyzed the expression of the cDNA in tissues of rabbit embryos at the time during which male phenotypic differentiation takes place. The coding sequence of the rabbit AR cDNA reveals a high degree of conservation with the sequences of the human, mouse, and rat ARs. By Northern analysis the principal transcript expressed in the mesonephros and the anlage of the external genitalia on gestation day 18 appeared to be identical to the mRNA expressed in adult prostate and epididymis.

We have studied the ability of the synthetic androgen methyltrienolone (R1881) to maintain testis and accessory organ weights, as compared to the effect of testosterone propionate (TP). In contrast to TP, R1881 is not metabolized and does not significantly bind to androgen-binding protein (ABP). Thirty-six rats were treated with ethane dimethane sulphonate (EDS) and GnRH antagonist (Org30267) to abolish all testicular androgen production, and recombinant human FSH (rec-hFSH, Org32489) was administered to ensure adequate FSH levels. Of these rats, five groups of four rats were treated daily with 0-, 50-, 100-, 200-, and 400-microgram TP, s.c., and four groups of four rats were treated daily with 150-, 300-, 600-, and 1200-microgram R1881, s.c. One control group of four rats received vehicle injections only. EDS treatment, followed by GnRH antagonist and rec-hFSH treatment for 17 days, significantly reduced testis, prostate, and seminal vesicle weights (P < 0.001, P < 0.01, P < 0.001, respectively). Simultaneous treatment with androgens prevented this organ weight decrease, in a dose-dependent manner. In all TP-treated animals, relative weights (% of control) of the acces, sory sex organs were significantly higher than the relative testis weights (P < 0.001). However, there was no difference in relative weights between testis and accessory sex organs in the R1881-treated animals. In another series of experiments, we investigated the effect of treatment with Finasteride, a 5 alpha-reductase inhibitor, on testis and accessory sex organ weights in rats treated with EDS and TP. Treatment with EDS, TP (300 micrograms/day) and Finasteride (40 mg/kg/day) did not alter testis weight as compared to the effect of treatment with EDS and TP alone. Prostate and seminal vesicle weights were, however, markedly reduced (significantly different from rats treated with EDS and TP alone; P < 0.01 and P < 0.05, respectively). Immunohistochemical analysis of androgen-receptor (AR) expression in the testis revealed that testicular AR immunoexpression is androgen dependent and that FSH alone is not able to maintain AR immunoexpression. Furthermore, the stage-dependent pattern of AR immunoexpression in Sertoli-cell nuclei, during the spermatogenic cycle, is identical in all TP- and R1881-treated rats. It is concluded that testes, prostate, and seminal vesicles are equally stimulated when the androgen receptor in these tissues is exposed to the same intracellular concentration of free androgen and that the low 5 alpha-reductase activity in the testis plays a critical role in the differential response of the testis and the accessory sex organs to T. Furthermore, stage-dependent AR immunoexpression in Sertoli cells does occur in the absence of testicular androgen production and is not due to androgen metabolism or local differences in androgen concentration.