Fig 1.
Localization of nestin-GFP in the vasculature of the murine epididymis.
A: 3D-morphological analyses (depth 150μm): Nestin-GFP-positive cells are visible in the vasculature of the nestin-GFP mouse epididymis. B: Unlike blood vessels (v), epithelial cells (ep) and surrounding smooth muscle cells (SMCs) of the epididymal duct (d) are nestin-negative, DAPI (blue) labels the nuclei. C (B in a higher magnification): nestin-positive cells (asterisks) in the vasculature of the caput epididymidis.
Fig 2.
No colocalization of nestin-GFP and the endothelial cell marker CD31 in blood vessels.
A-C,D-F,G-I: In vessels of the caput epididymidis., nestin-GFP is not colocalized with CD31 (red). DAPI (blue) labels the nuclei. A,D,G: CD31-positive endothelial cells (e). B,E,H: Nestin-GFP-positive cells (asterisks) in the vascular walls of the nestin-GFP mouse epididymis. C,F,I: Merged images.
Fig 3.
Colocalization of nestin-GFP and the SMC marker SMA in blood vessels.
A-F, G-I: Different vessels (v) of the nestin-GFP mouse. The epididymis (caput) shows nestin-GFP-positive/SMA-positive SMCs. SMCs are indicated. DAPI (blue) labels the nuclei. A,C,D,F,G,I: SMA-positive SMCs of the vessels (v) and of the epididymal duct (d). D-F: Higher magnification of the vessels shown in A-C. C,F,I: Nestin-GFP-positive (asterisks) SMCs in vessels (v).
Fig 4.
Colocalization of nestin-GFP and the proliferation marker PCNA.
A-C, D-F: Different vessels (v) of the nestin-GFP mouse epididymis (caput) with nestin-GFP-positive / PCNA-positive cells. DAPI (blue) labels the nuclei. A,D: PCNA-positive cells (red) in the vascular wall (arrows). B,E: Nestin-GFP-positive cells (asterisks). C,F: Merged images.
Fig 5.
Nestin expression during postnatal development of the epididymis.
A-B: qPCR analysis of nestin mRNA expression in the epididymis during postnatal development of mouse (A) and rat (B) (n = 3, each). A: relative nestin mRNA expression in the epididymis of the postnatal mouse decreases between d1-d25 as shown by log-linear regression (p≤0.05). Regression line is shown in blue. Housekeeping gene: RPS18. B: relative nestin mRNA expression in the epididymis of the postnatal rat (d5, w12). The error bars indicate SEM. *P<0.01 indicates significant differences. Housekeeping gene: β-actin. C: Western Blot analysis shows increased protein expression of nestin in the anatomical regions of postnatal epididymis, caput (Ca), corpus (Co) and cauda (Cd) in the 5 day-old compared with the 12w-old rat (20μg protein per sample). Vinculin was used as loading control. D-F: Immunofluorescence staining of the vasculature in the caput epididymidis of postnatal rats (d5) using antibodies against nestin (red) and SMA FITC (green). D: Nestin-positive cells in the vascular wall E: SMA FITC-positive vascular cells. F: Merged images. G,H: PCNA staining of paraffin embedded tissue. G: PCNA is highly expressed in the vasculature of the epididymis (arrows) on postnatal d5. H: No PCNA-positive cells are visible in the vasculature of the epididymis of the 12w-old rat.
Fig 6.
Epididymis after depletion of Leydig cells by EDS treatment.
A: Western Blot analysis after EDS (ethane dimethane sulphonate) treatment in rats; on d7 and d14 after EDS a distinct increase of nestin expression is visible. On d28 and d48 nestin expression is lower again. The androgen receptor (AR) was barely detectable on d7 and d14. β-actin was used as loading control. B,C: Immunohistochemical staining of the rat epididymis (cauda) with the proliferation marker PCNA. B: PCNA-positive epithelial cells (ep) of the epididymal duct serve as an internal positive control. In a section of an untreated rat PCNA is only visible in epithelial cells (ep) but not in the vasculature (v). C: PCNA-positive cells are visible in a vessel (v) and in epithelial cells on d2 after EDS.
Fig 7.
Nestin expression in vessels of the human epididymis.
A,B: Paraffin sections (cauda) with nestin-positive cells in the blood vessels (v), but not in SMCs and epithelial cells (ep) of the epididymal duct (d). C-E, F-H: Immunofluorescence staining of two different vessels with antibodies against nestin (red) and SMA FITC (green). E,H: Merged image. It indicates the localization of nestin in vascular SMCs.