A) Rat corpus epididymidis stained for COX1 (green). Higher magnification is shown in inset. Arrows indicate basal cells that extend towards the lumen. Bars: 50 μm, 5 μm (inset). B) 3D-reconstruction of cauda epididymidis labeled for COX1 (green) showing two basal cells reaching towards the lumen (arrows). See Movie S1. Bar: 8μm. C) Oblique section of cauda epididymidis stained for Cldn1 (green). Basal cell body projections infiltrating between epithelial cells are seen as small dots (arrows). The inset shows two basal cells with intracellular COX1 (red) and membrane-bound Cldn1 (green). Bars: 20 μm, 5 μm (inset). D) 3D-reconstruction of corpus epididymidis double-stained for COX1 (red) and Cldn1 (green) showing several basal cell extensions reaching out to the lumen. See Movie S2. Bar: 8 μm. E) Quantification of the total number of basal cells in different regions of the epididymis and the proximal vas deferens. Data are represented as mean ± SEM. No significant differences were detected between the regions. p-IS: proximal initial segment, d-IS: distal initial segment, Inter-zone: intermediate zone, pCPT: proximal caput, dCPT: distal caput, pCPS: proximal corpus, mCPS: middle corpus, dCPS: distal corpus, pCD: proximal cauda, mCD: middle cauda, dCD: distal cauda, VD: proximal vas deferens. F) Open bars: percentage of basal cells detected with their body projection reaching the apical pole of the epithelium. Data are represented as mean ± SEM. Number of cells reaching the apical pole / total number of basal cells are indicated above the bars. Solid bars: percentage of basal cells detected at the apical border. G) Rat trachea stained for COX1 (red) and tubulin (green). Arrow shows a COX1-positive basal cell that extends towards the lumen (arrow). The cilia of adjacent ciliated cells are labeled for tubulin. Some unidentified COX1-positive cells are also detected. Bar = 15 μm. H) 3D-reconstruction of a trachea section double-stained for COX1 (red) and ZO1 (green) showing a basal cell reaching the apical border of the epithelium (arrow). Unidentified COX1-positive cells are also detected. See Movie S3. Bar: 5 μm. I) Rat coagulating gland stained for COX1 (green). Several basal cells extend towards the lumen (arrows). The inset shows a COX1-positive basal cell (green) visualized by DIC (arrows). Bars: 15 μm, 5 μm (inset). J) Human epididymis 5 μm section stained for COX1 (green). Numerous basal cells are seen in the basal region of the epithelium. Some basal cells are also detected, even on this thinner section, with their body projections reaching the apical region of the epithelium (arrows).
Lu: lumen, IT: interstitium. In some panels, nuclei and spermatozoa are stained in blue with DAPI.

A′, A″, A‴) Three examples of AGTR2 (green) and V-ATPase (red) labeling in cauda epididymidis. Arrows indicate AGTR2-labelled basal cells that send projections towards the lumen. Arrowheads show nearby V-ATPase-labeled clear cells. Nuclei are visualized with DAPI (blue). Bars: 5 μm. B) Epididymis stained using anti-AGTR2 antibody with (+ peptide) and without (AGTR2) pre-incubation with the immunizing peptide. Bar: 20 μm. C) Western blot detection of AGTR2. 180 μg of epididymal homogenates were loaded onto the gel. Two bands at around 44 and 88 kDa were detected (arrows). D) 3D-reconstruction showing AGTR2-positive basal cells (green; arrows). One basal cell sends a projection between principal cells. Two clear cells, stained apically for the V-ATPase (red), are visible (arrowheads). See also Movie S10. Bar: 5 μm. E) RT-PCR analysis of Agtr2 mRNA expression in clear cells, isolated by FACS from B1-EGFP mouse epididymides (GFP+), and in all other epididymal cell types (GFP-). While a positive signal was detected in the GFP negative cell population, no Agtr2 mRNA expression was observed in GFP-positive clear cells.
Lu: lumen. SMC: smooth muscle cells.

A,B,C) Confocal images showing clear cells perfused for 20 min with 1 μM ANGII (A), or pre-incubated for 10 min with PD123319 (1 μM; B) or losartan (1 μM; C), before addition of ANGII, still in the presence of antagonists. PD123319, but not losartan, prevented the ANGII-induced microvilli elongation. Arrows show the frontier between the base of apical microvilli and the cytoplasm of the cell. D) Mean effects of PD123319 and losartan on ANGII-mediated microvilli elongation. PD123319 inhibited the effect of ANGII at both 0.1 and 1 μM concentrations. Values were obtained from at least 10 cells per epididymis. Data are represented as mean ± SEM and “n” is the number of rat epididymis examined. *P<0.001 vs control; ns: no significant difference vs control.

Basal cells extend narrow projections between epithelial cells to reach the lumen. A new TJ is formed between the basal cell and adjacent epithelial cells. Basal cells express AGTR2 and luminal ANGII triggers the production of NO in these cells. NO then acts locally on clear cells to produce cGMP via activation of the sGC, which is enriched in these cells. cGMP induces the accumulation of V-ATPase in well developed apical microvilli in clear cells, which results in the increase of proton secretion.

A′, A″, A‴) Three different rotations of a 3D reconstruction of an epididymis section stained for Cldn1 (red) and ZO1 (green). Basal cells reach the TJs at the intersection between three epithelial cells (arrows; see Movie S4). Bar: 10 μm B) Conventional microscopy image of one basal cell (stained for Cldn1 in red) forming a tight junction (stained for ZO1 in green) with adjacent principal cells (arrow). A clear cell expressing apical V-ATPase (blue) is seen (arrowhead). The nuclei are also detected in blue (DAPI). Bar: 5 μm. C, D, E, F) Body projections of basal cells showing different patterns of interaction with TJs. C: no co-localization between Cldn1 and ZO1 (arrow; Movie S5); D: partial co-localization of Cldn1 with ZO1 (arrows; Movie S6); E: basal cell that penetrates the TJ (arrow; Movie S7); E: basal cell showing ZO1-stained TJ (green) with adjacent principal cells (arrows; Movie S8). G′, G″, G‴) Rotations of a 3D reconstruction of epididymis stained for Cldn1 (green) and F-actin (red). A Cldn1-positive basal cell reaches the luminal side (arrow) between F-actin-labelled principal cells (see also Movie S9). Bar = 5 μm. H′, H″) Enface view visualized by DIC and immunofluorescence Cldn1 labeling (green). The dotted lines in H″ indicate the junctions between epithelial cells. Arrows show the tricellular corners between epithelial cells. One corner is occupied by a Cldn1-positive basal cell.

(A,B,C) Confocal microscopy images of V-ATPase-labeled clear cells (green) luminally perfused in vivo under control conditions (A) or in the presence of 0.1 μM (B) or 1 μM ANGII (C) for 20 min. The arrows show the border between the base of the apical microvilli and the apical pole of the cell. Bars: 5 μm. D) Quantitative analysis of the dose-dependent effect of ANGII on the elongation of V-ATPase-labeled microvilli normalized for the apical width of the cell. Values are mean ± SEM obtained from at least 10 cells per epididymis from “n” number of epididymis per group. **P<0.001 vs control. E,F) V-ATPase immunogold labeling of the apical pole of a clear cell perfused under control conditions (E) or in the presence of luminal ANGII (1 μM) (F). Under control conditions, the V-ATPase is located mainly in the sub-apical pole, and a few short V-ATPase-labeled microvilli are detected. In the presence of ANGII, longer and more numerous V-ATPase-labeled microvilli are detected. Bars: 500 nm. G) Apical accumulation of V-ATPase by luminal ANGII (1 μM) in clear cells. The left axis shows the density of V-ATPase-associated gold particles in the apical membrane including microvilli (Gold / μm apical membrane). The right axis shows the total number of gold particles located in the apical membrane of clear cells normalized for the width of the cell (Gold / μm cell width). 28 cells were analyzed in each group. Data are expressed as means ± SEM. *P<0.0005. H) Effect of ANGII (1 μM) on proton secretion in cut-open proximal VD using a proton-selective electrode. After an initial spike due to disturbance of the proton gradient, a sustained increase in proton secretion (expressed as μV) was induced by ANGII. A marked inhibition was then observed following addition of concanamycin A (1 μM). I) Mean effect of ANGII (1 μM) on concanamycin-sensitive proton secretion (mean ± SEM, n=7) measured 30 min after addition of ANGII. *P<0.05.

A) Confocal images of V-ATPase-labeled clear cells (green) perfused under control conditions (control), or in the presence of 1 mM p-cpt-cGMP, or 1 mM SNP for 20 min. A marked elongation of V-ATPase-labelled microvilli is observed in the presence of p-cpt-cGMP and SNP, compared to control. The arrows indicate the border between the base of apical microvilli and the cytoplasm. Bars: 5 μm. B) Effect of ODQ or L-NAME on the ANGII-induced response. Pre-treatment for 10 min with ODQ (3 μM) or L-NAME (100 μM), followed by ANGII still in the presence of inhibitors for 20 min, abolished the V-ATPase apical accumulation and microvilli elongation induced by ANGII alone. Bars: 5 μm. C) Mean microvilli elongation in clear cells. Values were obtained from at least 10 cells per epididymis. Data are represented as mean ± SEM, and “n” is the number of epididymides examined. *P<0.01 vs control (CTL), **P<0.001 vs CTL, and #P<0.001 vs ANGII. D) Localization of β₁-sGC (green) and V-ATPase (red) in epididymis. V-ATPase-positive clear cells (arrows) show abundant β₁-sGC staining in their basolateral membrane and apical pole. Weaker and more uniform staining is also detected in principal, basal, and smooth muscle cells. Nuclei are visualized with DAPI (blue) in the merged panel. Bar: 10 μm. E) Western blot detection of β₁-sGC in rat (RE) and mouse epididymis (ME) (120 μg/lane). In both samples, a band at about 70 kDa was detected corresponding to the molecular weight of β₁-sGC. Additional bands at around 35kDa and 50kDa were also detected in rat and mouse epididymis, respectively, possibly indicating degradation products in these tissues. All bands were absent after pre-incubation of the antibody with the immunizing peptide. F) Inhibition of immunofluorescence staining using the antibody pre-incubated with the immunizing peptide (+ peptide). Bars: 50 μm.