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Cited by 112Estrogen and related hormone effectsProstate Cancer Treatment and ResearchHormonal and reproductive studies

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Visualising Androgen Receptor Activity in Male and Female Mice

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Dart, D. Alwyn, Waxman, Jonathan, Aboagye, Eric O., Bevan, Charlotte L.·2013·PLoS ONE
DOI 10.1371/journal.pone.0071694PMC PMC3737126
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Abstract

Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic "ARE-Luc" mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.

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Figures

Figure 1 Analysis of the androgen receptor reporter construct in stably transfected cells. (A) Schematic representation of the ARE-tk-Luc androgen receptor reporter construct (hP = hPEST degradation signal). (B) Western blot analysis of steroid receptor expression in MCF-7, LNCaP and Du145 cells growing in full media. (C) Western blot analysis of PR expression in hormone starved MCF-7 cells -/+ E2 treatment for 24hr. (D), Luciferase activity from hormone treated LNCaP/Luc cells – treated for 24hours with 0-10nM mibolerone (Mib), estrogen (E2), progesterone (P4) and dexamethasone (Dex) (left hand side) and with 0-100nM of the androgens - (Mib), dihydrotestosterone (DHT), testosterone (Tes), androstenedione (A-dione) and dehydroepiandrosterone (DHEA) for 24 hours (right hand side). (E) Luciferase activity from hormone treated MCF-7/Luc cells – treated for 24hours with 0-10nM Mib, E2 or P4 or with additional 24hr pre-treatment with E2 (10nM) to induce PR expression. (F) Q-PCR quantification of relative expression of the steroid receptors AR, ER, and PR and luciferase transcripts in MCF-7/Luc cells, grown in full medium, transfected with siRNA against androgen receptor. (F) Luciferase activity from Du145/Luc cells treated with 0-100nM dex or mib (left hand side) or pre-transfected with an additional AR or empty vector expression constructs (right hand side). **P<0.01, *P<0.05 (t-test analysis).
Figure 1 Analysis of the androgen receptor reporter construct in stably transfected cells. (A) Schematic representation of the ARE-tk-Luc androgen receptor reporter construct (hP = hPEST degradation signal). (B) Western blot analysis of steroid receptor expression in MCF-7, LNCaP and Du145 cells growing in full media. (C) Western blot analysis of PR expression in hormone starved MCF-7 cells -/+ E2 treatment for 24hr. (D), Luciferase activity from hormone treated LNCaP/Luc cells – treated for 24hours with 0-10nM mibolerone (Mib), estrogen (E2), progesterone (P4) and dexamethasone (Dex) (left hand side) and with 0-100nM of the androgens - (Mib), dihydrotestosterone (DHT), testosterone (Tes), androstenedione (A-dione) and dehydroepiandrosterone (DHEA) for 24 hours (right hand side). (E) Luciferase activity from hormone treated MCF-7/Luc cells – treated for 24hours with 0-10nM Mib, E2 or P4 or with additional 24hr pre-treatment with E2 (10nM) to induce PR expression. (F) Q-PCR quantification of relative expression of the steroid receptors AR, ER, and PR and luciferase transcripts in MCF-7/Luc cells, grown in full medium, transfected with siRNA against androgen receptor. (F) Luciferase activity from Du145/Luc cells treated with 0-100nM dex or mib (left hand side) or pre-transfected with an additional AR or empty vector expression constructs (right hand side). **P<0.01, *P<0.05 (t-test analysis).CC BY · PubMed Central
Figure 2 Androgen receptor reporter activity in transgenic mice. (A), Bioluminescent imaging of male and female ARE-Luc and C57/Bl/6J wild type mice, injected with 150mg/kg luciferin substrate, and imaged with a CCCD camera after 10 minutes. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (B), Luciferase enzymatic assays from tissue homogenates taken from ARE-Luc mice, normalised to protein content (Bradford assay). Male mice upper panel, female mice lower panel.
Figure 2 Androgen receptor reporter activity in transgenic mice. (A), Bioluminescent imaging of male and female ARE-Luc and C57/Bl/6J wild type mice, injected with 150mg/kg luciferin substrate, and imaged with a CCCD camera after 10 minutes. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (B), Luciferase enzymatic assays from tissue homogenates taken from ARE-Luc mice, normalised to protein content (Bradford assay). Male mice upper panel, female mice lower panel.CC BY · PubMed Central
Figure 3 Bioluminescence analysis of ex vivo tissues. Ex vivo bioluminescence imaging of tissues taken from ARE-Luc mice injected with 150mg/kg luciferin substrate and killed and dissected after 10 minutes. (A), Images of male tissues (upper panel) and female mice (lower panel). (B), Images of coronal sections through the cerebral cortex of a male and female mouse brain. CC - Cerebral Cortex, Th - Thalamus, Hth - Hypothalamus. (c), Image of the underside of the male brain, showing pituitary and optic nerve bundles. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence. Images are representative of the pattern seen in several mice.
Figure 3 Bioluminescence analysis of ex vivo tissues. Ex vivo bioluminescence imaging of tissues taken from ARE-Luc mice injected with 150mg/kg luciferin substrate and killed and dissected after 10 minutes. (A), Images of male tissues (upper panel) and female mice (lower panel). (B), Images of coronal sections through the cerebral cortex of a male and female mouse brain. CC - Cerebral Cortex, Th - Thalamus, Hth - Hypothalamus. (c), Image of the underside of the male brain, showing pituitary and optic nerve bundles. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence. Images are representative of the pattern seen in several mice.CC BY · PubMed Central
Figure 4 Bicalutamide treatment reduces androgen receptor activity in male mice. Bioluminescent imaging of male mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. (A), Graph indicates measured bioluminescence signal from the gonadal region at the indicated timepoints; error bars represent the standard error from three mice in each group. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (C), panel showing ex vivo imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the testes and the prostate taken ex vivo, from male mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice. **P<0.01, *P<0.05 (t-test analysis).
Figure 4 Bicalutamide treatment reduces androgen receptor activity in male mice. Bioluminescent imaging of male mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. (A), Graph indicates measured bioluminescence signal from the gonadal region at the indicated timepoints; error bars represent the standard error from three mice in each group. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (C), panel showing ex vivo imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the testes and the prostate taken ex vivo, from male mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice. **P<0.01, *P<0.05 (t-test analysis).CC BY · PubMed Central
Figure 5 Bicalutamide treatment reduces androgen receptor activity in female mice. (A), Bioluminescent imaging of female mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. Graph indicates measured bioluminescence signal from the abdominal region at the indicated timepoints, error bars represent the standard error from three mice. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (C), panel showing ex vivo imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the ovaries and the intestines taken ex vivo, from female mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice, **P<0.01, *P<0.05 (t-test analysis).
Figure 5 Bicalutamide treatment reduces androgen receptor activity in female mice. (A), Bioluminescent imaging of female mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. Graph indicates measured bioluminescence signal from the abdominal region at the indicated timepoints, error bars represent the standard error from three mice. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm2. (C), panel showing ex vivo imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the ovaries and the intestines taken ex vivo, from female mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice, **P<0.01, *P<0.05 (t-test analysis).CC BY · PubMed Central
Figure 6 Analysis of AR and luciferase expression within a sample of mouse tissues. (A), Immunohistochemical co-localisation staining for AR (nuclear and cytoplasmic_ and luciferase (cytoplasmic) on consecutive formalin-fixed tissue sections taken from the ARE-Luc male mice. Upper panel represents image at 10x magnification, with box inset and lower panel showing 40x magnification. (B), Immunohistochemical staining for AR in a variety of female mouse tissues.
Figure 6 Analysis of AR and luciferase expression within a sample of mouse tissues. (A), Immunohistochemical co-localisation staining for AR (nuclear and cytoplasmic_ and luciferase (cytoplasmic) on consecutive formalin-fixed tissue sections taken from the ARE-Luc male mice. Upper panel represents image at 10x magnification, with box inset and lower panel showing 40x magnification. (B), Immunohistochemical staining for AR in a variety of female mouse tissues.CC BY · PubMed Central
Figure 7 Upper panel - Mouse tissues: A, prostate; B, testis; C, thymus; D, brain; E, skin and hair follicle; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, lacrimal gland; M, salivary gland; N, bone marrow; O, eye; P, heart. Lower panel - Human tissue: A, prostate; B, testis; C, thymus; D, brain; E, skin; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, breast; M, intestine and omentum; N, bone marrow; O, ovary and follicle; P, lung.
Figure 7 Upper panel - Mouse tissues: A, prostate; B, testis; C, thymus; D, brain; E, skin and hair follicle; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, lacrimal gland; M, salivary gland; N, bone marrow; O, eye; P, heart. Lower panel - Human tissue: A, prostate; B, testis; C, thymus; D, brain; E, skin; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, breast; M, intestine and omentum; N, bone marrow; O, ovary and follicle; P, lung.CC BY · PubMed Central

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  1. C Platell, 2000, The omentum, World J Gastroenterol, 6, 169

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Metadata

Authors
Dart, D. A., Waxman, J., Aboagye, E. O., & Bevan, C. L.
Affiliations
Imperial College London (GB)
Corresponding author
Bevan, Charlotte L.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-08-07
Volume
8
Issue
8
Pages
e71694
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0071694
PMC
PMC3737126
OpenAlex
W2045176719
Cited by
112
References
66
Citations by year
2026: 4 · 2025: 7 · 2024: 15 · 2023: 7 · 2022: 8 · 2021: 10 · 2020: 7 · 2019: 6 · 2018: 6 · 2017: 11 · 2016: 13 · 2015: 10 · 2014: 7 · 2013: 1
FWCI
6.84
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ORCA Online Research @Cardiff (Cardiff University) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Spiral (Imperial College London) (submittedVersion, other-oa)
Funders
  • Genesis Research Trust
  • Imperial College London
  • Medical Research Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Endocrinology › Estrogen and related hormone effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Prostate Cancer Treatment and Research
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
Keywords
androgen receptor, androgen signaling, transgenic mouse model, luciferase reporter, in vivo imaging, bicalutamide, androgen receptor antagonists, estrogen receptor, sex differences
MeSH terms
Androgen Antagonists, Anilides, Animals, Chlorocebus aethiops, Female, Humans, Male, Mice, Inbred C57BL, Mice, Transgenic, Molecular Sequence Data, Nitriles, Organ Specificity, Receptors, Androgen, Tosyl Compounds, Transcriptional Activation, Genes, Reporter, COS Cells, Luciferases, Firefly, Mice, Mice, 129 Strain, MCF-7 Cells

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