Selective androgen receptor modulators (SARMs) have specific impacts on the mouse uterus

Abstract

Selective androgen receptor modulators (SARMs) have been proposed as therapeutics for women suffering from breast cancer, muscle wasting or urinary incontinence. The androgen receptor (AR) is expressed in the uterus but the impact of SARMs on the function of this organ is unknown. We used a mouse model to compare the impact of SARMs (GTx-007/Andarine®, GTx-024/Enobosarm®), Danazol (a synthetic androstane steroid) and dihydrotestosterone (DHT) on tissue architecture, cell proliferation and gene expression. Ovariectomised mice were treated daily for 7 days with compound or vehicle control (VC). Uterine morphometric characteristics were quantified using high-throughput image analysis (StrataQuest; TissueGnostics), protein and gene expression were evaluated by immunohistochemistry and RT-qPCR, respectively. Treatment with GTx-024, Danazol or DHT induced significant increases in body weight, uterine weight and the surface area of the endometrial stromal and epithelial compartments compared to VC. Treatment with GTx-007 had no impact on these parameters. GTx-024, Danazol and DHT all significantly increased the percentage of Ki67-positive cells in the stroma, but only GTx-024 had an impact on epithelial cell proliferation. GTx-007 significantly increased uterine expression of Wnt4 and Wnt7a, whereas GTx-024 and Danazol decreased their expression. In summary, the impact of GTx-024 and Danazol on uterine cells mirrored that of DHT, whereas GTx-007 had minimal impact on the tested parameters. This study has identified endpoints that have revealed differences in the effects of SARMs on uterine tissue and provides a template for preclinical studies comparing the impact of compounds targeting the AR on endometrial function.

Keywords: Andarine; DHT; Danazol; Enobosarm; GTx-024; Ostarine; SARM; androgen; endometrium; uterus.

Figure 1

Compartment-specific changes in morphometric parameters induced by AR modulation in the mouse uterus. Female C57BL/6J mice were ovariectomised and treated with AR ligands as described in the ‘Materials and methods’ section. (A) Changes in total body weight of treated mice between the time of ovariectomy and tissue collection. (B) Changes in uterine weight normalised to animal weight following treatments. (C) Representative H&E uterine cross-sections of treated mice are shown, with increases in uterine size being accompanied by enlargement of individual cells. (D) Cross-section of a mouse uterus stained with H&E. Uterine cellular compartments are highlighted (myometrium, stroma, glands (G) and luminal epithelium (LE)). Analysis of surface area (in mm²) of the uterus (myometrium + endometrium), the stromal compartment, the epithelial compartment (glandular + luminal) and the myometrium following treatments. n = 10–14/treatment group. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons between treatment groups. Plain stars (*) indicate comparisons with VC, while stars above lines demonstrate comparisons between indicated treatment groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Scale bars: 500 μm. DHT, dihydrotestosterone; VC, vehicle control.

Figure 2

Treatment with the SARM GTx-024 altered AR protein levels in the uterus. (A) Uterine cross-sections of treated mice were stained with immunohistochemistry for AR (brown) and were counterstained with haematoxylin (blue). Uterine compartments are indicated (luminal epithelium (L), glandular epithelium (G) and myometrium (M)). Scale bars: 100 μm. (B) High-throughput image analysis of AR-stained sections using StrataQuest revealed a significant increase in the percentage of AR-positive cells in all uterine compartments following treatment with GTx-024 compared to VC. DHT upregulated AR in the stroma, glands and myometrium, while uterine sections of GTx-007- and Danazol-treated mice exhibited no change in the percentage of AR-positive cells compared to VC treatment. n = 8–14/treatment group. Kruskal–Wallis with Dunn’s multiple comparisons test was used for comparisons between treatment groups. Plain stars (*) indicate comparisons with VC, while stars above lines demonstrate comparisons between indicated treatment groups (*P < 0.05, **P < 0.01, ****P < 0.0001). DHT, dihydrotestosterone; VC, vehicle control.

Figure 3

Compartment-specific effects of SARM on uterine cellular proliferation. (A) Uterine cross-sections of treated mice were stained with immunohistochemistry for the proliferation marker Ki67 (brown) and were counterstained with haematoxylin (blue). Scale bars: 200 μm. (B) High-throughput image analysis of Ki67-stained sections using StrataQuest revealed a significant increase in the percentage of Ki67-positive cells in the stromal compartment following treatment with DHT, GTx-024 and Danazol. GTx-024 treatment induced a significant increase in the percentage of Ki67-positive cells in the glands and the myometrium, with DHT only partially mirroring this effect. n = 8–14/treatment group. Kruskal–Wallis with Dunn’s multiple comparisons test was used for comparisons between treatment groups. Plain stars (*) indicate comparisons with VC, while stars above lines demonstrate comparisons between indicated treatment groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). DHT, dihydrotestosterone; VC, vehicle control.

Figure 4

Uterine stimulation by DHT, GTx-024 and Danazol induces glandular expansion. (A) Uterine cross-sections of treated mice were stained with immunohistochemistry for the gland-specific transcription factor Foxa2 (brown) and were counterstained with haematoxylin (blue). Scale bars: 500 μm. (B) Quantification of endometrial glands identified significant increase in the absolute number of glands in the endometrium of mice treated with DHT, GTx-024 and Danazol. Normalisation to endometrial area (epithelial + stromal) revealed that the density of endometrial glands is unchanged following treatments. n = 9–14/treatment group. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons between treatment groups. Plain stars (*) indicate comparisons with VC, while stars above lines demonstrate comparisons between indicated treatment groups (*P < 0.05, **P < 0.01, ***P < 0.001). DHT, dihydrotestosterone; VC, vehicle control.

Figure 5

Whole-uterus gene expression changes of proteins involved in proliferation and stromal–epithelial cross-talk. Gene expression analysis by RT-qPCR of mRNAs extracted from whole-uterine homogenates of mice treated with AR ligands; individual genes are identified above the relevant results panels. n = 10–14/treatment group. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons between treatment groups. Plain stars (*) indicate comparisons with VC, while stars above lines demonstrate comparisons between indicated treatment groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). DHT, dihydrotestosterone; VC, vehicle control.