Deletion of androgen receptor in the smooth muscle of the seminal vesicles impairs secretory function and alters its responsiveness to exogenous testosterone and estradiol

Abstract

The seminal vesicles (SVs), like much of the male reproductive tract, depend on androgen-driven stromal-epithelial interactions for normal development, structure, and function. The primary function of the SVs is to synthesize proteins that contribute to the seminal plasma and this is androgen dependent. However, the cell-specific role for androgen action in adult SVs remains unclear. This study analyzed the SV in mice with targeted ablation of androgen receptors specifically in smooth muscle cells (PTM-ARKO) to determine in vivo whether it is androgen action in a subset of the SV stroma, the smooth muscle cells, that drives epithelial function and identity. These mice have significantly smaller SVs in adulthood with less smooth muscle and reduced epithelial cell height. Less epithelial cell proliferation was observed in adult PTM-ARKO SVs, compared with controls, and production of seminal proteins was reduced, indicating global impairment of epithelial cell function in PTM-ARKO SVs. None of these changes could be explained by altered serum testosterone or estradiol concentrations. We also demonstrate altered SV responsiveness to exogenous testosterone and estradiol in PTM-ARKO mice, indicating that smooth muscle androgen receptors may limit the SV epithelial proliferative response to exogenous estrogens. These results therefore demonstrate that the smooth muscle cells play a vital role in androgen-driven stromal-epithelial interactions in the SV, determining epithelial cell structure and function as well as limiting the SV epithelial proliferative response to exogenous estrogens.

Figure 1

A, Characterization of Cre recombinase expression in adult PTM-ARKO seminal vesicles. Approximately 50% of male PTM-ARKOs were Cre positive, identified by the presence of a band at 100 bp. Control littermates were negative for Cre but were positive for the internal control gene. B, Deletion of AR in the adult SV was determined using RT-PCR spanning exon 2. Only the larger 765-bp WT band was seen in control SVs, whereas the smaller 613-bp KO band was seen in ARKO testes. Both bands were identified in PTM-ARKO SVs, showing deletion of AR in a proportion of cells. C, Immunohistochemistry for Cre recombinase (green) and SMA (blue) showed that Cre recombinase was expressed selectively (arrow) in the smooth muscle cells of the SVs but not in any other SV cell types or smooth muscle cells in control SVs (*). Note that the smooth muscle layer appears narrower in the PTM-ARKO than controls. D, GFP [expression of which is also driven by the MH promoter in this mouse (47)] was expressed in the smooth muscle cells in PTM-ARKO SVs at d 100 (*) but not in the outer stromal cell layer (arrow); GFP was not detected in any cells in control SVs. Scale bars, 50 μm. E, In contrast, AR was expressed in the smooth muscle cells of control SV (arrowhead) but not PTM-ARKO SV smooth muscle cells (*); AR continued to be expressed in the epithelial and outer stromal cells (arrow) in the PTM-ARKO SV. Scale bars, 25 μm.

Figure 2

Gross morphology of PTM-ARKO seminal vesicles. A, Quantification of SV weight in PTM-ARKO mice at d 21–100 showing that there is a significant reduction at d 35 onward but not at d 21. B, Quantification of empty SV minus secretion and SV secretion weight as well as secretion weight relative to SV weight in PTM-ARKO mice at d 100. Values are means ± sem (n = 6–14 mice). *, P < 0.05, **, P < 0.01, ***, P < 0.001, compared with age-matched controls.

Figure 3

Histological analysis of PTM-ARKO seminal vesicles. A, Hematoxylin and eosin staining of d 100 PTM-ARKO and control SVs composed of epithelia surrounded by a stromal compartment (arrow); this epithelium appeared more folded in PTM-ARKO SVs than control (arrowhead). Note the dense eosinophilic seminal secretions in the lumen of both the PTM-ARKO and control SV (*). Scale bars, 400 and 50 μm, respectively. B, Immunohistochemistry for cytokeratin (brown), highlighting the epithelial cells in d 100 PTM-ARKO and control SVs. C, Note that epithelial cell height is significantly reduced in PTM-ARKOs at d 100 but not d 12, compared with age-matched controls. Scale bars, 50 μm. D, SMA immunostaining (brown) identifying the smooth muscle cell layer (arrow) directly surrounding the epithelium in d 100 PTM-ARKO and control SVs; note the presence of an outer SMA-negative layer (arrowhead). E, The smooth muscle layer is narrower in the PTM-ARKO than the control, as confirmed quantitatively. Values are means ± sem (n = 3 mice). *, P < 0.05, **, P < 0.01, compared with age-matched controls. Scale bars, 50 μm.

Figure 4

Cell mitosis in PTM-ARKO seminal vesicles. Quantification of epithelial cell mitotic index in d 12 and 100 PTM-ARKO and control SVs. Values are means ± sem (n = 3 mice). *, P < 0.05, compared with age-matched control littermates.

Figure 5

SV secretion production in PTM-ARKO mice. A, Seminal secretion protein expression in PTM-ARKO and control adult mice. There are four major proteins observed in the seminal secretions in both KO and control mice with no obvious differences in expression. B, Relative gene expression in SVs from d 100 PTM-ARKO and control mice. Note that the only gene that significantly changes is Cox-1, which is significantly increased in PTM-ARKO mice, compared with controls. Values are means ± sem (n = 3 mice). *, P < 0.05, compared with control littermates.

Figure 6

ER expression in adult PTM-ARKO SVs. Immunoexpression of ERα (green) in adult PTM-ARKO and control SVs; nuclei are counterstained red.

Figure 7

Gross morphology and histology of adult PTM-ARKO mice exposed to exogenous T and E2. A, Quantification of SV weight in adult PTM-ARKO mice exposed to T and E2 for 13 wk. Note the increase in SV weight in control but not KO mice after exposure to T+E2. B, Hematoxylin and eosin staining of adult PTM-ARKO and control SVs exposed to T+E2, with dense esinophilic seminal secretion in the lumen of both the PTM-ARKO and control SV (*). Note the densely packed cell nuclei in both the stroma and epithelia in control and PTM-ARKO SVs exposed to T+E2; this is more pronounced in KOs than controls, with desquamation of epithelial cells obvious in some KO SVs (arrow). Values are means ± sem (n = 3 mice). ***, P < 0.001, compared with control littermates; **, P < 0.01, compared with untreated control mice.

Figure 8

Quantification of histological changes in adult PTM-ARKO seminal vesicles after exposure to T and E2. A, Epithelial cell height (immunostained for cytokeratin, brown) is significantly reduced in PTM-ARKO SVs treated with T+E2, compared with T+E2-treated controls (as reported in untreated mice). Exposure to T+E2 does not result in any change in epithelial cell height, compared with untreated PTM-ARKOs and controls, respectively. B, The depth of the smooth muscle cell layer (immunostained for SMA, brown, *) is significantly smaller in PTM-ARKO SVs treated with T+E2, compared with treated controls. Note that exposure to T+E2 results in a significant increase in this smooth muscle layer in PTM-ARKO and control SVs, compared with untreated PTM-ARKOs and controls, respectively. C, Quantification of epithelial cell mitosis revealed a significant increase in epithelial mitosis in control SVs treated with T+E2, compared with untreated controls. Note that there is significantly more epithelial cell mitosis in PTM-ARKO SVs than controls but that treatment with T+E2 does not significantly alter epithelial cell mitosis in PTM-ARKO mice. Values are means ± sem (n = 3 mice). *, P < 0.05, compared with control littermates or untreated mice; **, P < 0.01, compared with control littermates.