Abstract
Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo–distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal–distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.
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Acknowledgements
This work was supported by National Institutes of Health Grants RO1ES001332, U54DK104310, T32ES007015, U01DK110807, and U01DK110807-S1.
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Supplementary Fig. 1 CGRP+, VAChT+, and TH+ axons are denser during prostate branching morphogenesis and sexual maturity than during prostatic bud formation. E17.5, P9, and P50 mouse prostate tissue sections were immunostained with antibodies against TH+, VAChT+, and CGRP+ (green) and e-cadherin (CDH1, to visualize prostatic and urethral epithelium, red). Axon pixel densities were quantified in the 10 μM periductal spaces radiating outward from the basilar surface of prostatic epithelium and results are shown pictorially on the right. Yellow arrowheads identify varicose axons. Results are mean ± SE of seven mice per group and three non-adjacent tissue sections per mouse. Asterisks indicate significant differences (*p < 0.05, and **p < 0.01) between regions. Scale bar is 50 µm (TIFF 23808 kb)
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Supplementary Fig. 2 CGRP+ axon density in P50 mouse ventral prostate is greater in the periurethral and proximal region than the distal region while VAChT+ and TH+ axon density does not significantly differ between proximal–distal regions. P50 mouse prostate tissue sections were immunostained with antibodies against TH+, VAChT+, or CGRP+ (green) and e-cadherin (CDH1, to visualize prostatic and urethral epithelium, red). Regions selected for analysis are schematized at the top of the figure and representative images are shown at the bottom left. Axon pixel densities were quantified in the 10 μM periductal spaces radiating outward from the basal surface of prostatic epithelium and results are shown pictorially at the right. Results are mean ± SE of ten mice per group and three non-adjacent tissue sections per mouse. Asterisks indicate significant differences (**p < 0.01) between regions. Scale bar is 50 µm (TIFF 27271 kb)
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Supplementary Fig. 3 Graphs showing CGRP+ axon density in P50 mouse dorsal prostate is greater in the periurethral and proximal region than the distal region while VAChT+ and TH+ axon density does not significantly differ between proximal–distal regions. Axon densities in P50 mouse prostate tissue sections were determined using the procedure described in Methods and Fig. 1 legend. Axon pixel densities were quantified in the 10 μm periductal spaces radiating outward from the basal surface of prostatic epithelium and results graphed. (a) CGRP+ axon density in the periurethral and proximal region is significantly greater than in the distal region. (b) VAChT+ axon density is uniform across the periurethral, proximal, and distal regions. (c) TH+ axon density is uniform across the periurethral, proximal, and distal regions. Results are mean ± SE of ten mice per group and three non-adjacent tissue sections per mouse. Asterisks indicate significant differences (**p < 0.01) between regions. RS identifies the rhabdosphincter. Scale bar is 50 µm (TIFF 4497 kb)
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Supplementary Fig. 4 Graphs showing CGRP+ axon density in P50 mouse ventral prostate is greater in the periurethral and proximal region than the distal region while VAChT+ and TH+ axon density does not significantly differ between proximal–distal regions. Axon densities in P50 mouse prostate tissue sections were determined using the procedure described in Methods and Fig. 1 legend. Axon pixel densities were quantified in the 10 μm periductal spaces radiating outward from the basal surface of prostatic epithelium and results graphed. (a) The CGRP+ axon density in the periurethral and proximal region is significantly greater than in the distal region. (b) VAChT+ axon density is uniform across the periurethral, proximal and distal regions. (c) TH+ axon density is uniform across the periurethral, proximal and distal regions. Results are mean ± SE of ten mice per group and three non-adjacent tissue sections per mouse. Asterisks indicate significant differences (*p < 0.05, and **p < 0.01) between regions (TIFF 2233 kb)
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Supplementary Table 1 List of primary antibodies used in this study. The antibody target, vendor and catelog number, RRID number, host species, dilution, and proof of specificity is listed. (DOCX 31 kb)
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Turco, A.E., Cadena, M.T., Zhang, H.L. et al. A temporal and spatial map of axons in developing mouse prostate. Histochem Cell Biol 152, 35–45 (2019). https://doi.org/10.1007/s00418-019-01784-6
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DOI: https://doi.org/10.1007/s00418-019-01784-6