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.
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’’’).