Figure 2. High-resolution three-dimensional (3D) imaging of the blood and lymphatic vasculature of the mouse epididymis after organ clearing.

(A) shows a schematic representation of the caput (red) and cauda (green) regionalization and segmentation commonly used to describe the mouse epididymis. (B) Representative multiplex immunostaining of the caput (left) and cauda (right) of the epididymis using five markers recognizing blood and lymphatic vessels is shown. An anti-MECA32 antibody (red) reveals PLVAP^pos blood vessels, particularly fenestrated vessels. An anti-GFP antibody revealing the VEGFR3:YFP transgene (green) and an anti-LYVE1 antibody (cyan). Anti-LYVE1 (cyan), anti-PROX1 (magenta), and anti-PDPN (white) antibodies were used as additional lymphatic markers. After organ clearance, high-resolution 3D imaging was performed with a light-sheet ultramicroscope (LaVision BioTec). The two extreme segments, IS (initial segment or S1) and S10, are delineated by a large dashed line, whereas the caput (left) and cauda (right) regions are delineated by a small dashed line. The scale bar is 500 µm. The PLVAP image corresponds to the contro-lateral epididymis used for the four lymphatic markers since it is not possible to use all five markers on the same organ.

Figure 2—figure supplement 1. Positive and negative immunohistochemical controls.

Panel A shows confocal views of multiplex labeling with the anti-GFP antibody (green) and the anti-mouse VEGFR3 antibody (red)on paraffin sections of an adult mouse epididymis, attesting that the two antibodies recognize the same structures. The scale bar is 20 µm. Panel B shows the compatibility of the four markers/dyes chosen for imaging blood and lymphatic vessels. Multiplex paraffin immunodetection of PLVAP/Alexa A555 (red), VEGFR3:YFP/GFP/Alexa A488 (green), LYVE1/Chromeo 494 (cyan), and PDPN/Alexa A647 (white) at the junction between the initial segment (IS)/S1 and the epididymal capsule. We noted the presence of fenestrated PLVAP^pos blood vessels at the periphery of the epididymal tubule of the IS (white arrowheads). These are also positive for VEGFR3:YFP/GFP but negative for LYVE1 and PDPN, demonstrating that there is no cross-detection between the labeling revealed with Chromeo 494 and those revealed by Alexa 488, Alexa 555, and Alexa 647. The peripheral lymphatic vessels of the organ located in the capsule (LV) are negative for PLVAP and positive for the three lymphatic markers (VEGFR3:YFP/GFP, LYVE1, and PDPN). We noted the presence of intertubular blood vessels (*), which present heterogeneous labeling. The upper one is only PLVAP^pos while the lower one is PLVAP^pos and VEGFR3:YFP^pos. In addition, note that the latter shows peripheral labeling for PDPN, while that is not the case for the upper one. A=artery. Panel C shows three-dimensional (3D) imaging obtained with a light-sheet ultramicroscope (LaVision Biotech) of the clarified epididymis caput and cauda after whole-mount incubation with the different dyes used to reveal the blood and lymphatic markers (Alexa 488 [green], Alexa 555 [red] Chromeo 494 [cyan], and Alexa 647 [white]).

Figure 2—figure supplement 2. Three-dimensional (3D) view of blood and lymphatic vascularization of whole epididymis after 3DISCO clarification.

Panel A shows representative 3D views of an epididymis of an adult VEGFR3:YFP transgenic mouse obtained by light-sheet ultramicroscopy (LaVision Biotech, 2× resolution) after immunodetection using the blood marker Meca32/PLVAP (red, left panel) or the lymphatic VEGFR3:YFP transgene, revealed using an anti-GFP antibody (green, middle panel). The right panel presents a merged view. Panel B presents a comparison of blood and lymphatic vessel density between the caput and cauda as described in Figure 2 legend. The Mann–Whitney test was used to determine statistical significance (**p<0.001, *p<0.01).

Figure 2—figure supplement 3. Cross-sectional views of the clarified caput and cauda epididymides.

Panel A presents median slices of the caput (left) and cauda (right) shown in Figure 2 for the PLVAP blood marker (red) and the lymphatic markers (VEGFR3:YFP, LYVE1, PROX1, and PDPN in green, cyan, magenta, and white, respectively). The epididymis used for PLVAP detection is the contralateral organ of the one used for lymphatic markers. The white-squared regions in each photograph (left in caput and cauda) are enlarged in the right photographs. PLVAP+ micro-vascularization (red) is visible at the peritubular epithelium level of the initial segment (IS)/S1 and its expression markedly decreases in the other segments. At higher magnification, a network of blood capillaries surrounds each tubule mainly at the level of the IS/S1 while intertubular vessels can be observed in segment 3 (S3). In the cauda (S10), PLVAP^pos vasculature is less dense and is mostly located in the intertubular space. The VEGFR3:YFP transgene (green), revealed by an anti-GFP antibody, is abundant in the caput and in particular in the IS/S1. Enlargements show punctiform labeling at the peritubular level and in the intertubular zone. On can note the presence of very large lymphatics at the intertubular level (white star). In the cauda (S10), reactivity is mainly interstitial, stringy, and compatible with flattened lymphatic vessels trapped in the extracellular matrix. We also noted at the S9/S10 boundaries (delimited by the dotted line) the presence of large lymphatic vessels (white asterisk), suggesting that important drainage takes place at this location. The lymphatic marker LYVE1 in the caput is mainly interstitial and clearly stronger in the IS/S1. We also noticed that the lymphatics detected with the transgene (VEGFR3:YFP) and with LYVE1 present differences (white arrows), in agreement with previous studies reporting heterogeneity for these markers at the level of the lymphatics (Pawlak and Caron, 2020; Ulvmar and Mäkinen, 2016). In the cauda, LYVE1 appears less present compared with VEGFR3:YFP. We also noticed differences in localization and intensity between these two lymphatic markers (white arrows). PROX1 (magenta) shows a profile comparable to those obtained for the transgene product and/or LYVE1 in both the caput and cauda. The labeling is punctiform (compatible with a nuclear localization) and filamentous at the level of the IS/S1. The stringy appearance can be explained by the use of a PROX1-biotinylated antibody that generates a fuzzier/coarser signal. PDPN (white) appears abundant in the caput and cauda at the peritubular level of the epididymal epithelium and in the interstitial space. Stereocilia in the initial segment but not in other segments of the caput also show some reactivity with PDPN. Because of the fuzzy PDPN pattern, it is difficult at this resolution to assert that PDPN strictly co-localizes with VEGFR3:YFP, LYVE1, and PROX1. Panel B shows light-sheet confocal (12×) resolution of the caput/IS-S2 region of a VEGFR3:YFP transgenic epididymis (green) or after PLVAP immunodetection (red). The superposition of the two channels (merge) suggests both co-localization but also specific territories. The far right picture shows the superposition of the surface rendering of the two markers, which allows distinguishing the lymphatic vasculature that expresses only the transgene (in dark green) from the PLVAP+/VEGFR3:YFP+ vessels (in light green).

Figure 2—video 1. Three-dimensional (3D) imaging of multiplex labeling of blood and lymphatic networks in the adult mouse caput epididymis.

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The video shows the overlay of the PLVAP^pos blood networks (red) and those obtained for the lymphatic markers VEGFR3:YFP (green), LYVE1 (cyan), and PDPN (white). 3D reconstruction from the light-sheet ultramicroscopy and video data were performed with IMARIS software.

Figure 2—video 2. Three-dimensional (3D) imaging of multiplex labeling of blood and lymphatic networks in the adult mouse corpus epididymis.

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The video shows the 3D reconstruction, using light-sheet ultramicroscopy data, of the PLVAP^pos blood vessel network and VEGFR3:YFP⁺ lymphatic vasculature in the corpus epididymis of the adult mouse. The PDPN^pos grid is shown to highlight the structure of the epididymis. The surface rendering of these two networks shows a difference in distribution. The blood network is evenly distributed in the corpus while the lymphatics show a greater density on the anterior edge (edge adjacent to the testis) with some large external collectors surrounding the organ. A few large lymphatic collectors cross the corpus from side to side at the septa (magenta arrow). Co-CL=corpus lymphatic collector.

Figure 2—video 3. Three-dimensional (3D) imaging of multiplex labeling of blood and lymphatic networks in the adult mouse cauda epididymis.

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The video shows the overlay of the PLVAP^pos blood networks (red) and those obtained for the lymphatic markers VEGFR3:YFP (green), LYVE1 (cyan), and PDPN (white). 3D reconstruction from the light-sheet ultramicroscopy data and video was performed with IMARIS software. The cross-sectional view shows the internal distribution of the different networks and the zoom shows the importance of the lymphatic network at the terminal.