The rete testis harbors Sertoli-like cells capable of expressing DMRT1

in Reproduction
Correspondence should be addressed to E A Malolina; Email: kate.ma85@gmail.com
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Sertoli cells (SCs) are supporting cells in the mammalian testis that proliferate throughout fetal and postnatal development but exit the cell cycle and differentiate at puberty. In our previous study, we isolated a population of highly proliferative Sertoli-like cells (SLCs) from the region of the adult mouse testis containing the rete testis and adjacent seminiferous tubules. Here RNA-seq of the adult SLC culture as well as qPCR analysis and immunofluorescence of the adult and immature (6 dpp) SLC cultures were performed that allowed us to identify SLC-specific genes, including Pax8, Cdh1, and Krt8. Using these, we found that SLCs are mostly localized in the rete testis epithelium; however, some contribution of transitional zones of seminiferous tubules could not be excluded. The main feature of SLCs indicating their relationship to SCs is DMRT1 expression. More than 40% of both adult and immature SLCs expressed DMRT1 at different levels in culture. Only rare DMRT1+ cells were detected in the adult rete testis, whereas more than 40% of cells were positively stained for DMRT1 in the immature rete testis. One more SC protein, AMH, was found in some rete cells of the immature testis. It was also demonstrated that SLCs expressed such SC genes as Nr5a1, Dhh, Gdnf, and Kitl and interacted with germ cells in 3D co-culture with immature testicular cells. All these similarities between SLCs and rete cells on one the hand and SCs on the other, suggest that rete cells could share a common origin with SCs.

Downloadable materials

  • Supplementary table 1. qRT‐PCR primers
  • Supplementary table 2. Antibodies
  • Supplementary table 3
  • Supplementary figure 1. LCM procedure on cell cultures live stained with SYBR Green I. Adult (A) and immature (B) SLCs were recognized as dense compact colonies (arrows). SCs were captured from ST cultures. To increase SC density, adult ST cultures were obtained by plating not fully digested tubule fragments. Adult SCs (C) were identified by their characteristic nuclear morphology, an inset shows a higher magnification of boxed SCs. Immature SCs (D), as shown by Wt1 staining (red), grew as clusters surrounded by Wt1- cells exhibiting a more dispersed pattern. Such clusters (E) were also visible after SYBR Green staining. Adult SLCs and SCs were grown for 9 and 5 days before LCM respectively; immature SLCs and SCs were captured on day 3 of culture. For SLC and immature ST cultures, from 7000 to 9000 cells were captured per sample; for adult ST culture, the number of cells was from 300 to 500 per sample. Scale bars: 200 μm, 50 μm (inset).
  • Supplementary figure 2. Negative control images for key antibodies used. Tissue sections (B,D,F,H,J,L,N) and cell cultures (P,Q,S,T,W,X) known not to express a target antigen were used to check for a non-specific signal; positively stained testis sections (A,C,E,G,I,K,M) with the rete testis (RT) and seminiferous tubules (ST) as well as SLC cultures (O,R,U,V) were added for comparison. Nuclei were counterstained with DRAQ5 or DAPI. MEF: mouse embryonic fibroblasts. Scale bars: 50 μm (A-N), 100 μm (O-X).
  • Supplementary figure 3. qRT-PCR of the culture samples used for RNA-seq and interstitial cell culture; Oil Red O staining of these cultures. (A) Hsd3b6 and Hsd17b3, markers of adult Leydig cells, were downregulated in the adult SLC and ST cultures; P<0.05. The data are represented as a fold change (log2) relative to RNA levels in the interstitial cell culture that was established from adult testes and grown for 24 hours before harvest. (B) Leydig cells labeled by Oil Red O staining (arrows) were detected in the interstitial cell culture (1) and ST culture (2) but not in the SLC culture (3). Nuclei were counterstained with hematoxylin. (C) Validation of RNA-seq analysis. RNA expression in the adult SLC culture is represented as a fold change (log2) relative to RNA expression in the ST culture. P<0.05 for all genes examined. All PCR data are presented as the mean ± SEM from 3 biological replicates. Scale bars: 100 μm.
  • Supplementary figure 4. ACTA2 expression in the adult SLC cultures. YAC supplementation prevented the expression of a mesenchymal marker ACTA2 in cultured SLCs, as shown by immunostaining (A,B) and qRT-PCR (C). Peritubular myoid cells (Wt1- cells) remained ACTA2 expression in culture with YAC. Nuclei were counterstained with DAPI. In (C) LCM samples were used (mean ± SEM, 3 biological replicates). *P<0.05. Scale bars: 250 μm.
  • Supplementary figure 5. Immunolocalization of SOX9, WT1, and DMRT1 in the adult mouse testis. (A, B) SOX9 (red, in A) and WT1 (red, in B) antibodies labeled SCs from seminiferous tubules (ST) and transitional zones (TZ) with high intensity and epithelial cells of the rete testis (RT) with low intensity. A DMRT1 (green) antibody in (B) labeled SCs from seminiferous tubules (ST) as well as spermatogonia (asterisks) with high intensity, SCs from transitional zones (TZ) with lower intensity, and rete testis epithelium was mostly unstained. Nuclei were counterstained with DRAQ5 (blue). (A’-A’’’,B’-B’’’) Higher magnifications of boxed cells in (A,B). Scale bars: 50 μm (A-B); 30 μm (A’-A’’’,B’-B’’’).

 

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    Morphological appearance of SLC colonies. Phase-contrast (A, C and E) and bright-field (B, D and F) images of SLC colonies from adult cultures without YAC (A and B) and with YAC (C and D), and from immature culture (E and F). Cells were stained with hematoxylin in (B, D and F). Arrows point to SLC colonies. Scale bars: 200 µm (A, C); 50 µm (B, D and F); 100 µm (E).

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    Characterization of adult and immature SLCs. (A and C) Representative colonies of adult (A) and immature (C) SLCs positively stained for WT1 (red) and having heterogeneous DMRT1 expression (green). (A’–A’’’) Higher magnifications of boxed cells with different intensities of a DMRT1 signal, (A’) shows an adult SC outside the colony. (1, 2) Higher magnifications of boxed immature SLCs (1) and SCs (2). Nuclei were counterstained with DAPI (blue). (B) The percent of SLCs with different levels of DMRT1 in the adult cultures with and without YAC, and in the immature culture at day 9 and day 3, respectively (mean ± s.e.m., 3–6 biological replicates). *P < 0.001. (D and E) Expression of SLC markers revealed by RNA-seq analysis in adult (D) and immature (E) SLC colonies obtained by LCM (mean ± s.e.m., three biological replicates). *P < 0.05. Scale bars: 250 µm (A), 200 µm (C), 40 µm (A’–A’’’, 1, 2).

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    Immunofluorescence detection of some genes revealed by RNA-seq in the adult and immature SLC cultures. (A and C) SLCs (SOX9+ colonies, red) were labeled with a PAX8 antibody (green) in the adult (A) and immature cultures (C). (B) The percent of PAX8+ SLCs in the adult culture (mean ± s.e.m., three biological replicates). (D and E) SLCs (WT1+ colonies, red) were positively stained for CDH1 (green) in the adult (D) and immature cultures (E). (F and G) SLCs (WT1+ colonies, red) were labeled with a KRT8 antibody (yellow) in the adult (F) and immature cultures (G); some of them co-expressed DMRT1 (green, indicated by arrows). Arrowheads point to adult SCs. Scale bars: 200 µm (A, C, D, E, F and G).

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    Immunofluorescence examination of adult SLCs at 1 day of culture. (A) Clusters of SLCs, small WT1+ cells (red), were labeled with KRT8 (yellow) and CDH1 (green) antibodies. Asterisks indicate CDH1−KRT8− SLCs. (B) Some KRT8+ SLCs co-expressed DMRT1 (green, indicated by dots). Compare to a SC (arrowhead, inset) that was KRT8 negative and had characteristic nuclear morphology. (C) The percent of CDH1+ and KRT8+ cells among SLCs, and the percent of KRT8+ cells among DMRT1+ SLCs (mean ± s.e.m., three biological replicates). (D) Staining of the culture with an ace-TUB antibody; a WT1+KRT8− cell with a high ace-TUB signal (green) is indicated by an arrow. Nuclei in (A, B and D) were counterstained with DAPI (blue). Scale bars: 50 µm (A, B and D); 25 µm (inset in B).

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    SLC markers revealed by RNA-seq analysis were detected in the epithelium of the adult rete testis by immunofluorescent staining. (A) SOX9+ (red) epithelial cells of the rete testis (RT) were labeled with a PAX8 antibody (green). ST, seminiferous tubules; TZ, SCs from transitional zones. (B) A stitched image of the rete testis epithelium positively stained for KRT8 (green) and CDH1 (red). (B’) shows a higher magnification of a boxed area with CDH1+KRT8+ cells (dots) and CDH1+KRT8− cells (arrows). (C) SCs from transitional zones were brightly stained for ace-TUB (green) but were KRT8 (red) negative. Nuclei were counterstained with DRAQ5 (blue). Scale bars: 50 µm (A, B, C, D and E); 25 µm (B’).

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    SLC markers were detected in the immature rete testis by immunofluorescent staining. (A) SOX9+ (red) epithelium of the rete testis (RT) was positively stained for PAX8 (green). An arrowhead points to a seminiferous tubule with SOX9+PAX8− SCs. (B) Cells of the rete testis expressed CDH1 (green) and KRT8 (red). (B’) shows a higher magnification of a boxed area, arrows point to a KRT8 signal in seminiferous tubules (ST). (C) An ace-TUB signal (green) was detected in the rete testis marked by CDH1 staining (yellow) and in seminiferous tubules; a WT1 antibody (red) labeled both rete cells and SCs. Nuclei were counterstained with DRAQ5 (blue). Images in (A and B) were stitched together from individual images. Scale bars: 50 µm (A, B’ and C); 100 µm (B).

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    Cells in the adult and immature rete testis epithelium expressed DMRT1. (A) A stitched image of the immature rete testis (RT) marked by co-expression of CDH1 (red) and SOX9 (blue) with many DMRT1+ (green) rete cells. ST, seminiferous tubules. Asterisks indicate CDH1+DMRT1+ germ cells in the rete epithelium that were SOX9-negative. (B) Some DMRT1+ (green) rete cells expressed KRT8 (red) in the immature testis (indicated by arrows). (C and D) Rare rete cells expressed DMRT1 (green) in the adult testis. TZ, transitional zones. (C’, C’’ and D’) show higher magnifications of boxed areas with DMRT1+ rete cells positive (C’, C’’, asterisks) or negative (D’, an arrowhead) for KRT8 (red). Nuclei were counterstained with DRAQ5 (blue). Scale bars: 50 µm (A, C and D); 25 µm (B, inset).

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    Cells in the immature rete testis and SLCs in culture expressed SC genes. (A and A’) AMH+ (yellow) cells in the rete testis (RT) positively stained for CDH1 (green) are indicated by asterisks, SOX9 (red) labels SCs and rete cells. Arrows point to AMH+ cells not expressing CDH1 (i.e., SCs). (B and B’) AMH+ (yellow) cells in the rete positively stained for PAX8 (green) are indicated by arrowheads. ST, seminiferous tubules. (A’ and B’) Higher magnifications of boxed areas represent maximum projections of serial confocal optical sections; orthogonal projections of areas denoted by yellow lines are shown in the right and bottom panels. (C) A stitched image of the immature (6 dpp) rete testis stained for AMH (blue), CDH1 (red), and DMRT1 (green). Dots point to AMH+ rete cells. A dotted line outlines part of the rete without DMRT1+ and AMH+ cells. ED, efferent duct. Nuclei were counterstained with DRAQ5 (blue). (D and E) Expression of the selected genes in the adult (D) and immature (E) SLC colonies obtained by LCM. Dashed lines indicate the gene expression levels in SCs. The data are presented as the mean ± s.e.m. from three biological replicates. *P < 0.05. Scale bars: 100 µm (A, B and C); 20 µm (A’ and B’).

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    Adult SLCs contacted with germ cells in the 3D co-culture with immature testicular cells. (A and B) The most cells from the SLC culture identified by GFP staining (green) were SLCs (SOX9+ cells, red, A) with different levels of DMRT1 expression (yellow), and not peritubular myoid cells (ACTA2+ cells, red, B). Asterisks indicate SLCs. A dot points to a GFP+SOX9− cell. (C, D and E) Some germ cells (arrowheads) positively stained for DDX4 (red, C), a pre-meiotic marker STRA8 (red, D), and a meiotic marker SCP3 (red, E) were closely associated with GFP+ cells. Dotted lines in (B and E) outline tubule-like structures. (C, D and E) represent maximum projections of serial confocal optical sections; orthogonal projections of areas denoted by yellow lines are shown in the right and bottom panels. (E’) represents a higher magnification of one of the optical sections in (E, arrow) and shows a spermatocyte in metaphase with an adjacent GFP+ cell. (F and G) Single apoptotic cells labeled by anti-cCASP3 antibody (red, F) and germ cells labeled by anti-DDX4 antibody (red, G) were located at different sites, at the center and in the periphery of 3D-culture samples respectively. An inset in (F) shows a higher magnification of a boxed area. Nuclei were counterstained with DRAQ5 (blue). Scale bars: 20 µm (A, B, C, D and E); 50 µm (F and G); 5 µm (E’); 25 µm (inset).

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