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. 2011 May;141(5):653-63.
doi: 10.1530/REP-10-0493. Epub 2011 Feb 10.

A dense network of dendritic cells populates the murine epididymis

Nicolas Da Silva  1 , Virna Cortez-Retamozo, Hans-Christian Reinecker, Moritz Wildgruber, Eric Hill, Dennis Brown, Filip K Swirski, Mikael J Pittet, Sylvie Breton
Affiliations

A dense network of dendritic cells populates the murine epididymis

Nicolas Da Silva et al. Reproduction. 2011 May.

Abstract

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are 'foreign' to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. In this study, we report a previously unrecognized dense network of dendritic cells (DCs) located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal DCs (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.

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Figures

Figure 1
Figure 1
The epididymis is densely populated by CD11c+ and CX3CR1+ cells. (a and b) “mosaic” pictures of whole CD11c-EYFP and CX3CR1-GFP mouse epididymis sections, respectively. IS: initial segments, H: head (caput), B: body (corpus), T: tail (cauda), VD: vas deferens. (c and d) higher magnification pictures of the initial segments, showing numerous CD11c-EYFP+ cells located at the periphery of the epididymal tubule. Bars = 2 mm (a and b), 250 µm (c) and 50 µm (d). High-resolution pictures for panels a and b are available online (online material S1 and S2).
Figure 2
Figure 2
Epididymal CD11c+ and CX3CR1+ cells are peritubular and have stellate/dendriform morphology. (a-d) Visualization of CX3CR1-GFP+ and CD11c-EYFP+ eDCs using live cell confocal microscopy. Unfixed tissue slices were observed within 15 minutes after dissection. (a and b) and (c and d) represent 2 confocal planes of the same field in CD11c-EYFP and CX3CR1-GFP tissues, respectively. The lumen (L) is populated by spermatozoa that are invisible in these pictures; eDCs are located at the periphery of the epididymal tubule (e-g). Conventional fluorescence microscopy visualization of CX3CR1-GFP-positive eDCs (20, 40 and 60X objectives, respectively). Nuclei were stained (red) to better appreciate the organization of the tissue. (h and i) High magnification pictures of CD11c-EYFP+ cells in caput and cauda epididymidis, respectively, showing the distinctive morphology of eDCs in these regions. Cells are more ramified in the head compared to the tail. Bars = 100 (a-e), 50 (f) and 10 µm (g-i).
Figure 3
Figure 3
In the proximal epididymidis, eDCs project thin processes toward the epididymal lumen. (a) Cross section of the epididymal duct of a CD11c-EYFP mouse (initial segments) showing numerous intraepithelial dendrites converging toward the lumen (L). (b) Three-dimensional view of a CD11c-EYFP+ eDC, showing the bright cellular body, long lateral extensions (arrows) and slender transepithelial processes (arrowheads). (c and d) Two optical sections of epididymal CD11c-EYFP+ DCs taken at different depths. (c) The section runs through the cellular body of one DC (arrow), which has numerous lateral extensions. (d) The perpendicular intraepithelial processes appear as circular dots (arrowheads) in this section taken closer to the lumen. (e) Cross-section of the epididymal duct (initial segments) showing CX3CR1-GFP+ eDCs (green) projecting processes toward the tight junctions labeled with ZO-1 (red). In contrast, DC do not project visible intraepithelial dendrites in the epididymis tail (f). E: epithelium. Blue = DAPI. Bars = 10 µm (a-e) and 20 µm (f).
Figure 4
Figure 4
Flow cytometry analysis of cell surface markers in epididymal and splenic DCs isolated from C57BL/6 mice. Cell suspensions were prepared from C57BL/6 mouse whole epididymis and spleen and stained with monoclonal antibodies to 13 markers. (a) Recapitulation of the phenotype of 2 major CD11c+ eDC populations that express or do not express CD103 (gated from the histogram shown on the left). (b) Representative histograms of the phenotype of epididymal and splenic DCs. The percentage represents the mean of at least two independent experiments. Red histograms represent isotype controls
Figure 5
Figure 5
Expression of macrophage markers in the epididymis. F4/80 immunolabeling of CD11c-EYFP mouse epididymis revealed 3 subsets of cells that express CD11c (green), F4/80 (red), or both markers (yellow). CD11c-F4/80+ cells (a, c, e, arrowheads) tend to be more interstitial than CD11c+F4/80− cells. However, high magnification pictures d and e show that cells positive for both markers are also present in the peritubular region. (f-h): the macrophage marker CD206 (mannose receptor) is expressed exclusively by interstitial cells. Sections of CD11c-EYFP (green) mouse epididymis were immunolabeled with an anti-CD206 antibody (red). While CD11c+ cells are located mostly in the peritubular region, the CD206+ macrophages are exclusively interstitial. Higher magnification (h) reveals the non-stellate morphology of interstitial CD206+ cells, contrasting with eDCs (green). DNA staining (in a, blue) helps to appreciate the structure of the tissue, with rows of nuclei from epithelial cells (E) and luminal nuclei from spermatozoa. Bars = 50 (a-c, f), 25 (g) and 10 µm (d, e, h). L = lumen.
Figure 6
Figure 6
In vitro antigen presentation to T cells by epididymal and splenic DCs. CD11b+ CD11c+ DCs were isolated from C57BL/6 epididymis and spleen by FACS, as well as CD4+ OTII and CD8+ OT-I T cells. DCs and CFSE-labeled T cells were cocultured for 5 days in the presence (blue) or absence (red) of OVA protein. Proliferation of T cells was assessed by flow cytometry. The percentage of proliferating cells is indicated in blue. Histograms are representative of two independent experiments.
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