The androgen receptor depends on ligand‐binding domain dimerization for transcriptional activation
2021; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês
10.15252/embr.202152764
ISSN1469-3178
AutoresSarah El Kharraz, Vanessa Dubois, Martin E. van Royen, Adriaan B. Houtsmuller, Ekaterina Pavlova, Nina Atanassova, Nguyen T. Tien, Arnout Voet, Roy Eerlings, Florian Handle, Stefan Prekovic, Elien Smeets, Lisa Moris, Wout Devlies, Claes Ohlsson, Matti Poutanen, Kevin J. Verstrepen, Geert Carmeliet, Kaisa‐Mari Launonen, Laura Helminen, Jorma J. Palvimo, Claude Libert, Dirk Vanderschueren, Christine Helsen, Frank Claessens,
Tópico(s)Hormonal and reproductive studies
ResumoArticle18 October 2021free access Source DataTransparent process The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation Sarah El Kharraz Sarah El Kharraz orcid.org/0000-0003-1444-2531 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Vanessa Dubois Vanessa Dubois orcid.org/0000-0001-8894-2980 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Martin E van Royen Martin E van Royen orcid.org/0000-0002-6814-0996 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Adriaan B Houtsmuller Adriaan B Houtsmuller orcid.org/0000-0003-0967-0740 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Ekatarina Pavlova Ekatarina Pavlova orcid.org/0000-0002-8821-653X Institute of Experimental Morphology Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Search for more papers by this author Nina Atanassova Nina Atanassova orcid.org/0000-0002-9032-3588 Institute of Experimental Morphology Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Search for more papers by this author Tien Nguyen Tien Nguyen orcid.org/0000-0002-6300-0302 Department of Chemistry, KU Leuven, Leuven, Belgium Search for more papers by this author Arnout Voet Arnout Voet orcid.org/0000-0002-3329-2703 Department of Chemistry, KU Leuven, Leuven, Belgium Search for more papers by this author Roy Eerlings Roy Eerlings orcid.org/0000-0001-9661-8312 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Florian Handle Florian Handle orcid.org/0000-0002-7558-5635 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Stefan Prekovic Stefan Prekovic orcid.org/0000-0002-7051-9321 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Elien Smeets Elien Smeets orcid.org/0000-0002-0975-5253 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Lisa Moris Lisa Moris Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Wout Devlies Wout Devlies orcid.org/0000-0003-0725-6300 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Claes Ohlsson Claes Ohlsson orcid.org/0000-0002-9633-2805 Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Matti Poutanen Matti Poutanen orcid.org/0000-0002-8953-1734 Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden Research Centre for Integrative Physiology and Pharmacology, Turku Center for Disease Modeling, University of Turku, Turku, Finland Search for more papers by this author Kevin J Verstrepen Kevin J Verstrepen orcid.org/0000-0002-3077-6219 VIB Laboratory for Systems Biology and KU Leuven Laboratory for Genetics and Genomics, VIB - KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Geert Carmeliet Geert Carmeliet orcid.org/0000-0001-8324-4462 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Kaisa-Mari Launonen Kaisa-Mari Launonen orcid.org/0000-0003-2770-0464 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Laura Helminen Laura Helminen orcid.org/0000-0002-0870-9319 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Jorma J Palvimo Jorma J Palvimo orcid.org/0000-0003-2373-0578 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Claude Libert Claude Libert orcid.org/0000-0001-6408-036X VIB Center for Inflammation Research, VIB, Ghent, Belgium Department for Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Dirk Vanderschueren Dirk Vanderschueren orcid.org/0000-0003-1395-0104 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Christine Helsen Christine Helsen orcid.org/0000-0003-1048-2798 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Frank Claessens Corresponding Author Frank Claessens [email protected] orcid.org/0000-0002-8676-7709 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Sarah El Kharraz Sarah El Kharraz orcid.org/0000-0003-1444-2531 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Vanessa Dubois Vanessa Dubois orcid.org/0000-0001-8894-2980 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Martin E van Royen Martin E van Royen orcid.org/0000-0002-6814-0996 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Adriaan B Houtsmuller Adriaan B Houtsmuller orcid.org/0000-0003-0967-0740 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Ekatarina Pavlova Ekatarina Pavlova orcid.org/0000-0002-8821-653X Institute of Experimental Morphology Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Search for more papers by this author Nina Atanassova Nina Atanassova orcid.org/0000-0002-9032-3588 Institute of Experimental Morphology Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Search for more papers by this author Tien Nguyen Tien Nguyen orcid.org/0000-0002-6300-0302 Department of Chemistry, KU Leuven, Leuven, Belgium Search for more papers by this author Arnout Voet Arnout Voet orcid.org/0000-0002-3329-2703 Department of Chemistry, KU Leuven, Leuven, Belgium Search for more papers by this author Roy Eerlings Roy Eerlings orcid.org/0000-0001-9661-8312 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Florian Handle Florian Handle orcid.org/0000-0002-7558-5635 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Stefan Prekovic Stefan Prekovic orcid.org/0000-0002-7051-9321 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Elien Smeets Elien Smeets orcid.org/0000-0002-0975-5253 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Lisa Moris Lisa Moris Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Wout Devlies Wout Devlies orcid.org/0000-0003-0725-6300 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Claes Ohlsson Claes Ohlsson orcid.org/0000-0002-9633-2805 Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Matti Poutanen Matti Poutanen orcid.org/0000-0002-8953-1734 Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden Research Centre for Integrative Physiology and Pharmacology, Turku Center for Disease Modeling, University of Turku, Turku, Finland Search for more papers by this author Kevin J Verstrepen Kevin J Verstrepen orcid.org/0000-0002-3077-6219 VIB Laboratory for Systems Biology and KU Leuven Laboratory for Genetics and Genomics, VIB - KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Geert Carmeliet Geert Carmeliet orcid.org/0000-0001-8324-4462 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Kaisa-Mari Launonen Kaisa-Mari Launonen orcid.org/0000-0003-2770-0464 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Laura Helminen Laura Helminen orcid.org/0000-0002-0870-9319 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Jorma J Palvimo Jorma J Palvimo orcid.org/0000-0003-2373-0578 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Claude Libert Claude Libert orcid.org/0000-0001-6408-036X VIB Center for Inflammation Research, VIB, Ghent, Belgium Department for Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Dirk Vanderschueren Dirk Vanderschueren orcid.org/0000-0003-1395-0104 Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium Search for more papers by this author Christine Helsen Christine Helsen orcid.org/0000-0003-1048-2798 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Frank Claessens Corresponding Author Frank Claessens [email protected] orcid.org/0000-0002-8676-7709 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Sarah El Kharraz1, Vanessa Dubois2, Martin E Royen3, Adriaan B Houtsmuller3, Ekatarina Pavlova4, Nina Atanassova4, Tien Nguyen5, Arnout Voet5, Roy Eerlings1, Florian Handle1, Stefan Prekovic1,6, Elien Smeets1, Lisa Moris1, Wout Devlies1, Claes Ohlsson7, Matti Poutanen7,8, Kevin J Verstrepen9, Geert Carmeliet2, Kaisa-Mari Launonen10, Laura Helminen10, Jorma J Palvimo10, Claude Libert11,12, Dirk Vanderschueren2, Christine Helsen1,† and Frank Claessens *,1,† 1Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium 2Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, Belgium 3Department of Pathology, Erasmus MC, Rotterdam, The Netherlands 4Institute of Experimental Morphology Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria 5Department of Chemistry, KU Leuven, Leuven, Belgium 6Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands 7Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden 8Research Centre for Integrative Physiology and Pharmacology, Turku Center for Disease Modeling, University of Turku, Turku, Finland 9VIB Laboratory for Systems Biology and KU Leuven Laboratory for Genetics and Genomics, VIB - KU Leuven Center for Microbiology, Leuven, Belgium 10Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland 11VIB Center for Inflammation Research, VIB, Ghent, Belgium 12Department for Biomedical Molecular Biology, Ghent University, Ghent, Belgium † These authors contributed equally to this work as senior authors *Corresponding author. Tel: +32 16 330253; E-mail: [email protected] EMBO Reports (2021)22:e52764https://doi.org/10.15252/embr.202152764 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition. Synopsis This study reveals the contribution of ligand-binding domain (LBD) dimerization to androgen receptor (AR) activity. Disrupting LBD dimerization affects multiple receptor functions, proposing this interface as new therapeutic target. Disrupting LBD dimerization in vitro slightly reduces ligand and DNA binding, interactions with a subset of coregulators as well as transactivation. In vivo, it leads to androgen insensitivity with absence of accessory sex glands, despite high circulating LH, testosterone and androstenedione levels. In vivo, the mutation leads to loss of binding to AR binding sites in chromatin, despite nuclear translocation and chromatin binding. In the testis, the AR regulates expression of HSD17B3, the enzyme which converts androstenedione into testosterone. Introduction The androgen receptor (AR) is a nuclear receptor that belongs to the subfamily of steroid receptors. After binding of its cognate ligand, the AR translocates to the nucleus, binds DNA, and recruits co-regulators and RNA polymerase II to initiate transcription (Aranda & Pascual, 2001). All steroid receptors have a conserved DNA-binding domain (DBD), a ligand-binding domain (LBD), and a variable N-terminal domain (NTD) (Escriva et al, 2004). Steroid receptors assemble as homodimers to perform their classical role as transcription factors. For the AR and for other steroid receptors, dimerization via the DBD has been well characterized and is crucial for the recognition of the bipartite, inverted DNA repeats that act as androgen response elements (AREs) (Shaffer et al, 2004; Sahu et al, 2014). A second receptor dimerization mechanism is AR-specific and is named the N/C interaction. It occurs through binding of the 23FQNLF27 motif in the NTD of the AR to the coactivator-binding groove on the surface of the AR LBD (He et al, 2000, 2004; He & Wilson, 2002; Li et al, 2006; van Royen et al, 2007, 2012). The third dimerization mechanism, which happens via the LBDs, has been more controversial for the AR. The only evidence for AR LBD dimerization comes back from two-hybrid protein–protein interaction assays (Doesburg et al, 1997). No further evidence became available, until Nadal et al (2017) published a unique crystal structure of AR LBD dimers. Although the dimerization surface differs from the one described for other nuclear receptors (Brzozowski et al, 1997; Tanenbaum et al, 1998; Williams & Sigler, 1998; Bledsoe et al, 2002; Nadal et al, 2017), biophysical data confirmed this dimerization interface (Nadal et al, 2017). However, the exact in vivo role of LBD dimerization in receptor functioning remains unknown and has not been studied for any steroid receptor. Mutations in the AR LBD have been described in patients with androgen insensitivity syndrome (AIS). These patients have an intersex phenotype ranging from mild (e.g., normal male phenotype with infertility) to complete (female phenotype with undescended testes and absence of Wolffian and Müllerian ducts) AIS (Batista et al, 2018). Most AR LBD mutations can be correlated with reduced ligand and/or co-regulator binding. However, some of these mutations are located within the LBD dimerization interface. Hence, the associated AIS phenotype might be explained by the fact that these mutations disrupt LBD dimerization. The W752R mutation, discovered in two siblings suffering from AIS (Boehmer et al, 2001), was predicted to disrupt the LBD dimer interface without strongly affecting other AR functions (Nadal et al, 2017). To study the physiological relevance of LBD dimerization for AR activity, we generated a mouse model (ARLmon for monomeric LBD of the AR) bearing the corresponding murine W731R point mutation. Results The W752R mutation disrupts AR LBD dimerization In the center of the human AR (hAR) LBD dimer interface, Trp 752 is part of an important stabilizing π-stack (Nadal et al, 2017). In the wild-type (WT) LBD dimer, the two opposing tryptophans stabilize the interface via hydrogen bonds (Fig 1A). Molecular dynamics (MD) simulations were used to investigate the effect of W752 replacement by R or A on AR LBD dimer stability. Based on the model, tryptophan replacement by arginine (W752R) retains the possibility of hydrogen bond formation but induces an electrostatic repulsion of the positive charges and a steric clash due to the larger arginine side group (Fig 1A). Replacing the tryptophan by alanine (W752A) would disrupt the stabilizing π-stack without adding electrostatic interactions. Since arginine could still form a hydrogen bond, it investigated whether it disrupts dimerization. For both W752R and W752A, MD simulations indicated that the interface was disrupted, with one protein bending away from the other (Fig EV1A–C). This is reflected in the calculated free energy of binding for the dimer interface shifting from −34.24 kJ/mol for the WT receptor to −19.38 kJ/mol for the W752R mutant and to −14.01 kJ/mol for the W752A mutant (Table EV1A and Fig EV1D and E). To study the influence of the mutations on the stability of the monomer–ligand interactions, we calculated the binding free energy of the AR LBD bound to dihydrotestosterone (DHT) in the presence of the AF2 crystal peptide. No differences in free energy were observed in both mutants, showing no major effect of the replacement on ligand-induced receptor stabilization (Table EV1B). The W752R mutation was selected for further study. To confirm the effect of the W752R mutation on AR LBD dimerization in a cellular context, we performed acceptor-bleaching fluorescence resonance energy transfer microscopy (abFRET) (van Royen et al, 2009). In the presence of 10 nM DHT, a clear abFRET signal was detected between two WT LBDs, whereas no abFRET signal occurred between two W752R LBDs (Fig 1B). A yeast two-hybrid experiment confirmed the interactions between two WT LBDs, but not between two W752R LBDs (Fig EV1F). Figure 1. Disrupting AR LBD dimerization A. Crystal structure of the human AR LBD core dimer based on PDB 5JJM (1 monomer in gray and 1 monomer in pink). W752, located at the interface of the AR LBD dimer, is involved in hydrogen bond formation with T756 of the neighboring LBD and thereby stabilizes the interface. R752 disrupt this stabilization by steric hindrance and charge repulsion. The side chains of both W and R are shown at position 752. A close-up of the LBD-LBD interface is given. The ligand (DHT) is depicted as spheres. B. Acceptor photobleaching FRET after transfection of Hep3B cells with labeled WT LBD or labeled W752R-mutated LBD. Representative confocal images of Hep3B cells transiently expressing WT or W752R AR in the presence of 10 nM DHT are shown below the bars. Scale bar = 10 µm. The bar graphs show means ± SEM (biological replicates, n = 54 (WT LBD) and n = 65 (W752R LBD), unpaired two-tailed Student’s t-test, ***P < 0.001). C. Representative pictures of the AGD of 13-week-old WT male (upper left), WT female (lower left), ARLmon/Y (upper right), and AR−/Y (lower right) mice. D. Upper panel: a representative picture of the urogenital tract of a WT male and an ARLmon/Y mouse. Lower panel: a representative picture of the testis of a WT male and an ARLmon/Y mouse. Scale bar = 1 cm. E, F. Serum levels of T (E) and LH (F) in WT males, and ARLmon/Y and AR−/Y mice at the age of 13 weeks. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, ***P < 0.001, ns = not significant). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Molecular dynamics of the AR LBD dimerization interface and yeast two-hybrid assay A. Molecular dynamics simulations based on the WT AR LBD dimer crystal structure. B. Molecular dynamics simulations based on the W752R AR LBD dimer model. C. Superposed model of WT LBD dimer (pink) and W752R LBD dimer (white). D. Molecular dynamics simulations based on the W752A AR LBD dimer model. E. Superposed model of WT LBD dimer (pink) and W752A LBD dimer (purple). F. Yeast two-hybrid assay on human WT LBD and human W752R LBD. RFP signal normalized to 0 nM DHT is shown. The bar graphs show means ± SEM (biological replicates, n = 3, unpaired two-tailed Student’s t-test, ***P < 0.001, ns = not significant). Download figure Download PowerPoint The ARLmon/Y mice have a feminized phenotype The corresponding murine LBD dimer disrupting mutation, W731R, was introduced in the AR gene of C57BL/6J mice by CRISPR/Cas9 to generate ARLmon/Y males (Appendix Fig S1A and B). To estimate the importance of LBD dimerization in normal AR functioning, we compared the ARLmon/Y males with WT littermates and global AR knockout mice (AR−/Y), in which the AR is no longer expressed (De Gendt et al, 2004). Longitudinal follow-up of the anogenital distance (AGD), an external marker for sexual differentiation in mice (Schwartz et al, 2019), showed a notable difference between WT males and ARLmon/Y mice (Figs 1C and EV2A). The AGD of ARLmon/Y mice was up to three times reduced compared with WT males and comparable to that of WT females and AR−/Y. Nipple development was clearly visible in ARLmon/Y and AR−/Y mice, while it was absent in WT males. Body weight and composition of ARLmon/Y were comparable to those of WT females and AR−/Y (Fig EV2B and C). The testes of ARLmon/Y mice were cryptorchid and had an intermediate weight between testes of WT males and AR−/Y mice (Fig EV2D). Dissection of the urogenital tract revealed the absence of male reproductive organs including seminal vesicles, vas deferens, prostate, and epididymis in ARLmon/Y (Fig 1D). Kidneys of both ARLmon/Y and AR−/Y mice weighed 16% less when compared to WT (Fig EV2E). Serum testosterone (T) and luteinizing hormone (LH) were increased by sixfold and 45-fold in ARLmon/Y mice compared with WT males, respectively (Fig 1E and F). No significant increase was observed for follicle-stimulating hormone (FSH) in ARLmon/Y mice (Fig EV2F). Click here to expand this figure. Figure EV2. Detailed evaluation of the ARLmon/Y phenotype A. Evolution of the anogenital distance (AGD) over time. Average is shown, and shaded areas represent SEM (biological replicates, n ≥ 10). B. Body weight followed over time. Average is shown, and shaded areas represent SEM (biological replicates, n ≥ 10). C. Total amount of fat at 12 weeks of age determined by EchoMRI normalized to body weight. The bar graphs show means ± SEM (biological replicates, n ≥ 10, one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, ns = not significant). D. Testes weight normalized to body weight of 13-week-old WT males, and ARLmon/Y and AR−/Y mice. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, ***P < 0.001). E. Kidney weight normalized to body weight of 13-week-old WT males, and ARLmon/Y and AR−/Y mice. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, ns = not significant). F. Serum levels of FSH in WT males, and ARLmon/Y and AR−/Y mice at the age of 13 weeks. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, ns = not significant). G. Upper panel: H&E staining on testis of a WT male, and ARLmon/Y or AR−/Y mouse. Lower panel: immunofluorescence staining of the AR (green). Nuclei are shown in blue. Orange, blue, and white arrows indicate LC, SC, and peritubular myoid cells, respectively. Scale bar = 50 µm. H–I. Relative contribution of seminiferous epithelium (H) and interstitium (I) in testis from 13-week-old mice. Proportions are expressed relative to total testis volume. The bar graphs show means ± SEM (biological replicates, n = 5, one-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001, ns = not significant). Download figure Download PowerPoint The testicular function is strongly affected in ARLmon/Y mice The AR regulates cellular composition and histological appearance of the testis (Wang et al, 2009). To get further insight into the remaining activity of the ARLmon, we performed testicular analysis. Histological analysis of the testes of ARLmon/Y mice showed that the diameter of the seminiferous tubules was smaller compared with that of WT males, with fewer spermatogenic cells (Fig EV2G, upper panel). Nevertheless, in comparison with AR−/Y mice, seminiferous tubules of ARLmon/Y mice had a larger cross-sectional area with further developed spermatogenic cells (Fig EV2G, upper panel). Seminiferous epithelium was decreased in ARLmon/Y and AR−/Y mice (Fig EV2H). In contrast, the interstitium in the testes of ARLmon/Y and AR−/Y mice was significantly increased compared with WT testes (Fig EV2I). Immunofluorescence staining showed nuclear AR expression in both WT and ARLmon/Y testes, whereas AR was not detected in testis from AR−/Y mice (Fig EV2G, lower panel). AR-positive Leydig cells (LC), Sertoli cells (SC), and peritubular myoid cells were present in both WT and ARLmon/Y testes, with striking hyperplasia of the androgen-producing LC in the ARLmon/Y (Table 1; Fig EV2G, lower panel). A higher percentage of spermatogenesis-supporting SC was observed in the ARLmon/Y and AR−/Y testes, although the absolute nuclear volume of SC per testis was reduced in both genotypes compared with WT (Table 1). Further examination of the histology of ARLmon/Y testes uncovered the patchy presence of differentiated elongated spermatids (ESd), albeit at very low numbers as illustrated in Table 1. Quantification of the spermatogenic cells confirmed that the relative numbers of spermatogonia (Sg) were increased in ARLmon/Y testes, while the percentage of the specific spermatogenic cell types decreased the further they develop toward the elongated stage (Table 1). Measurement of intratesticular T concentrations showed a significant increase in androstenedione (A-dione), T, and DHT in ARLmon/Y mice compared with WT (Appendix Fig S2A–C). To gain further insight into their testicular phenotype and the expression of AR-regulated genes, we performed RNA-sequencing (RNA-seq) analysis on whole testis of five mice per genotype: WT, ARLmon/Y, and AR−/Y. Comparing gene expression between ARLmon/Y or AR−/Y mice and WT males confirmed the more severe phenotype of AR−/Y testes. Indeed, compared with WT, more genes were up- and downregulated in AR−/Y mice and with a higher magnitude than in ARLmon/Y testes (Fig 2A and B). A large part of the differentially expressed genes is specific to the spermatogenic lineage. The comparison of SC- and LC-specific transcripts extracted from the bulk RNA-seq data showed clear relative differences between WT, ARLmon/Y, and AR−/Y (Fig 2C). In general, relative expression levels of SC- and LC-specific genes tended to be higher in ARLmon/Y mice compared with WT, corresponding to the higher percentage of SC and LC and fewer germ cells in ARLmon/Y testes. However, several well-known AR-regulated genes, such as the SC-specific Rhox5 and the LC-specific Insl3, were almost completely silenced in ARLmon/Y. This was confirmed by qPCR (Fig 2D). Furthermore, steroidogenic enzyme expression was dysregulated in ARLmon/Y testes. Indeed, the expression of genes encoding enzymes involved in the uptake of cholesterol (Star), the conversion of cholesterol into pregnenolone (Cyp11a1), and the hydroxylation of pregnenolone (Cyp17a1) were higher compared with WT (Fig 3A, confirmed by qPCR in Appendix Fig S2D). Hsd3b1, which is responsible for the formation of A-dione, was also significantly higher expressed in testes of ARLmon/Y (Fig 3A), which correlated with the higher LH and A-dione serum levels (Figs 1F and 3B). Malfunctioning of the AR in the hypothalamic–pituitary–gonadal (HPG) axis also led to increased expression levels of the LH receptor (LHR) in testes of both ARLmon/Y and AR−/Y mice (Appendix Fig S2E). Surprisingly, the expression level of Hsd17b3, which is responsible for the final conversion of A-dione into T, was found to be fivefold lower in ARLmon/Y than in WT (Fig 3C, confirmed by qPCR in Appendix Fig S2F) and even 13-fold lower in AR−/Y mice. Because the AR is a transcription factor, we performed co-transfections of AR with a luciferase reporter under the control of the Hsd17b3 promoter. We selected the region ranging from −230 bp to +1 bp because it is most conserved between mammalians. The reporter construct was androgen-responsive when co-transfected with a WT mouse AR (mAR), but not in the presence of the W731R mAR (Fig 3D). Similarly, the Insl3 promoter (–132 to +1 bp) also conferred an
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