Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 1

Cell & Tissue Research

The cell–cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions – a novel architectonic cell-cell

junction system

Lisa M. Domke (l.domke@dkfz.de) • Werner W. Franke (w.franke@dkfz.de) Helmholtz Group for Cell Biology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

Electronic Supplementary Material

Supplementary Figures S1 – S12

Fig. S1 (high-resolution image available for download from the authors' homepage)

Fig. S1 Double-label immunofluorescence microscopy of cryotomy cross-sections through seminiferous tubules (a-a’’) and excurrent duct epithelium (b-b’’) of frozen bovine testis after reactions with monoclonal mouse antibodies to the hemidesmosomal marker protein HD230/233 (a, a’’, b, b’’; red) and rabbit antibodies against the extracellular matrix (ECM) protein collagen IV (a’-a’’, b’-b’’; green). In a-a’’ note the totally negative reaction of HD antibodies, compared with the positive reaction of collagen IV in both the peritubular (brackets) and the perivascular walls (V, vessels) of the testis (a-a’’), whereas an intensely positive HD reaction is seen in distinct hemidesmosomal structures at the basal plasma membrane of the initial part of the excurrent duct epithelium (b-b’’). In contrast reactions of collagen IV of ECM material are seen in the specific layers of the peritubular and blood vessel (V) walls in the interstitium (I). Nuclei have been stained blue with DAPI (a’’, b’’). L, lumen. Bars 20 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 2

Fig. S2 (high-resolution image available for download from the authors' homepage)

Fig. S2 Double-label immunofluorescence microscopy of cryotomy cross-sections through frozen human testis after reactions with rabbit antibodies against the smooth muscle marker protein smoothelin (a, a’’, a’’’; red) and monoclonal mouse antibodies against smooth muscle α-actin (SMA; a’-a’’’; green). Colocalization of both smooth muscle marker proteins (yellow merge colour) is seen in extended regions of the peritubular wall cells around the seminiferous tubules but not in all regions (L, lumen; on optical phase contrast background in a’’’). I, interstitial space. Bar 50 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 3

Fig. S3 (high-resolution image available for download from the authors' homepage)

Fig. S3 Double-label immunofluorescence microscopy of cryotomy cross-sections through seminiferous tubules of frozen bull testis after reactions with monoclonal mouse antibodies against the protein talin (a, a’’; red) and rabbit antibodies to the smooth muscle marker protein SMA (a’-a’’; green). Note the positive staining in peritubular wall LSMCs (denoted by brackets). Reactions with rabbit antibodies against protein SM22-α (b, b’’, b’’’; red) and monoclonal mouse antibodies to desmin (b’-b’’’; green) also show positive reactions and frequent colocalizations in peritubular wall cells (denoted by brackets) surrounding the seminiferous tubules (L, lumen). Here SMCs of a blood vessel (V) in the interstitial region (I) appear only positive for SM22α. Bars 20 µm (a), 50 µm (b)

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 4

Fig. S4 (high-resolution image available for download from the authors' homepage)

Fig. S4 Double-label immunofluorescence microscopy of cryotomy cross-sections through seminiferous tubules of frozen bovine (a, b), human (c) and rat (d) testes after reactions with monoclonal mouse antibodies against the protein calponin (a, a’’, b, b’’, c, c’’, d, d’’; red) and rabbit antibodies to the smooth muscle marker protein SMA (a’-a’’, b’-b’’, c’-c’’, d’-d’’; green). Note the positive merger staining indicative of colocalization (yellow merger color) in peritubular wall LSMCs (denoted by brackets) surrounding the seminiferous tubules (L, lumen) and in blood vessel (V) walls in the interstitial region (I). Bars 50 nm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 5

Fig. S5 (high-resolution image available for download from the authors' homepage)

Fig. S5 Double-label immunofluorescence microscopy of cryotomy sections through seminiferous tubules (L, lumen) of rat testis. After reactions with monoclonal mouse antibodies against SMA (a, a’’, a’’’; red) and guinea pig antibodies to the intermediate-size filament protein vimentin (a’-a’’’; green) all Sertoli cells show positive reactions for vimentin filament bundles but are totally negative for SMA. In contrast, peritubular SMCs (brackets) show frequently overlapping localization of SMA and vimentin (a’’ and a’’’; yellow merge colour). Note the numerous spermatids in the lumina (a’’’). After reactions with monoclonal mouse antibodies against E-cadherin (b, b’’, c; red) and rabbit antibodies to N-cadherin (b’-b’’, c; green) Sertoli cells are positive for N-cadherin but negative for E-cadherin. Note, however, that a positive reaction for E-cadherin is seen in peritubular wall cells (brackets). I, interstitial space. Bars 20 μm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 6

Fig. S6 (high-resolution image available for download from the authors' homepage)

Fig. S6 Double-label immunofluorescence microscopy of a cryotomy cross-section through a seminiferous tubule (L, lumen) of bull testis, after reaction with antibodies against two different kinds of intermediate-sized filament proteins, desmin (a, a’’, a’’’; red; monoclonal mouse antibodies) and cytokeratins 8 and 18 (a’-a’’’; green; guinea pig antibodies). Sertoli cells are totally negative for both markers. Note, however, in the peritubular LSMC wall the occurrence of desmin IFs (red) but also cytokeratin-positive IFs (green) as well as hybrid-appearing fibril structures (yellow merge colour; some of the latter are denoted by white arrows). Bar 20 μm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 7

Fig. S7 (high-resolution image available for download from the authors' homepage)

Fig. S7 Double-label immunofluorescence microscopy of a cryotomy section through frozen boar testis tissue, showing seminiferous tubules (L, lumen) and interstitial (I) tissue after reactions with monoclonal mouse antibodies to SMA (a, a’’, a’’’; red) and guinea pig antibodies to the intermediate filament proteins cytokeratins 8 and 18 (a’-a’’’; green). Cells positive for SMA are seen as well as cell structures positive for cytokeratin only and also small regions with partly overlapping reactions pretending colocalization of both antibodies (yellow merge colour in a’’ and a’’’). Peritubular walls are denoted by brackets (phase contrast background in a’’’). By contrast, here the smooth muscle cells of the blood vessels (V) are only positive for SMA. Bar 20 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 8

Fig. S8 (high-resolution image available for download from the authors' homepage)

Fig. S8 Immunofluorescence microscopy of a cryotomy cross-section through frozen bull testis tissue after reactions with monoclonal mouse antibodies to protein myozap (a, a’’, a’’’; red) and guinea pig antibodies to cytokeratins 8 and 18 (a’-a’’’; green). Positive reaction and partial colocalization is seen in peritubular wall cells (brackets in a’’’) surrounding the seminiferous tubules (L, lumen). The SMCs of blood vessel (arrows) walls shown here in the interstitial region (I) are positive only for myozap. Nuclei have been stained blue with DAPI (a’’’). Bar 20 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 9

Fig. S9 (high-resolution image available for download from the authors' homepage)

Fig. S9 Tile scan survey figure of double-label immunofluorescence microscopy of a cryotomy cross-section through the epithelium of an anterior part of the excurrent duct system of bull testis after reactions with monoclonal mouse antibodies against smoothelin (a, a’’, a’’’; red) and rabbit antibodies to desmin (a’-a’’’; green). Note the intense positive staining of both SMC markers in cells of the peritubular walls encasing the epithelial ducts (colocalization as indicated by yellow merge colour) and total absence of any immunostaining reaction in most of the interstitial space (I) and the interior of the ductal lumina (L) which are rich in spermatozoa. Bar 200 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 10

Fig. S10 (high-resolution image available for download from the authors' homepage)

Fig. S10 Double-label immunofluorescence microscopy of a cryotomy cross-section through a part of the excurrent duct system of frozen bull testis (L, lumen), showing the relatively thick peri-epithelial SMC walls (denoted by bracket in a’’). The SMCs are positively immunostained for smooth muscle myosin (a-a’’; red; monoclonal mouse antibodies) and SMA (a’-a’’; green; rabbit antibodies). A very similar reaction is seen in the SMCs of the blood vessel (V) walls in the interstitial region (I). Bar 50 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 11

Fig. S11 (high-resolution image available for download from the authors' homepage)

Fig. S11 Tile scan survey figure of double-label immunofluorescence microscopy of a cryotomy cross-section through a more posterior (epididymal) part of the excurrent duct system of frozen boar testis after reactions with monoclonal mouse antibodies against smoothelin (a, a’; red) and guinea pig antibodies to protein myozap (a, a’; green). Note here the intense and extended positive reaction of smoothelin in the thick smooth muscle (M) tissue, separated from the epithelium (brackets) by a mesenchymal cell-dominated SMC-free peri-epithelial and interstitial (I) region and the specific myozap immunostaining of the zonula adhaerens of the excurrent duct epithelium. L, lumen. Bars 100 µm

Domke & Franke – CTR – Electronic Supplementary Material – Supplementary Figures – p. 12

Fig. S12 (high-resolution image available for download from the authors' homepage)

Fig. S12 Electron micrographs of ultrathin cross-sections through peritubular walls of seminiferous tubules of rat testes, showing a single LSMC layer (brackets) closely covered by ECM layers. Note the flattened, discoid nucleus in the LSMC layer (a-d). The peritubular wall is generally covered by an extremely thin non-SM cell (arrowheads) the nucleus of which protrudes into the interstitial space (e). SC, Sertoli cell. Bars 2 µm