Tlapakova T et al. (2016), Identification and characterization of Xenopus ...

XB-ART-52308

Biol Open 2016 Sep 15;59:1275-82. doi: 10.1242/bio.019265.

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Identification and characterization of Xenopus tropicalis common progenitors of Sertoli and peritubular myoid cell lineages.

Tlapakova T , Nguyen TM , Vegrichtova M , Sidova M , Strnadova K , Blahova M , Krylov V .

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The origin of somatic cell lineages during testicular development is controversial in mammals. Employing basal amphibian tetrapod Xenopus tropicalis we established a cell culture derived from testes of juvenile male. Expression analysis showed transcription of some pluripotency genes and Sertoli cell, peritubular myoid cell and mesenchymal cell markers. Transcription of germline-specific genes was downregulated. Immunocytochemistry revealed that a majority of cells express vimentin and co-express Sox9 and smooth muscle α-actin (Sma), indicating the existence of a common progenitor of Sertoli and peritubular myoid cell lineages. Microinjection of transgenic, red fluorescent protein (RFP)-positive somatic testicular cells into the peritoneal cavity of X. tropicalis tadpoles resulted in cell deposits in heart, pronephros and intestine, and later in a strong proliferation and formation of cell-to-cell net growing through the tadpole body. Immunohistochemistry analysis of transplanted tadpoles showed a strong expression of vimentin in RFP-positive cells. No co-localization of Sox9 and Sma signals was observed during the first three weeks indicating their dedifferentiation to migratory-active mesenchymal cells recently described in human testicular biopsies.

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Species referenced: Xenopus tropicalis
Genes referenced: acta2 cd44 cyp11a1 cyp17a1 dazl ddx25 ddx4 itgb1 kitlg lif odc1 sox9 thy1 vim
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	Fig. 1. In vitro characterization of X. tropicalis cell culture. (A,B) Testicular somatic cell culture in morphology of adherent feeder layer (XtTSC) (A) and after long-term cultivation which enables the forming of colonies (XtTSCc) (B). (C) X. tropicalis transgenic XtTSC expressing Katushka RFP under CAG promotor (XtTSC-RFP). (D) Transgenic Katushka RFP expressing XtTSC in colonies (XtTSCc-RFP). (E,F) Structure of in vitro testicular cell colony visualized by TEM. In the colony the cells are placed in an extensive amount of extracellular matrix with two or three tight layers of XtTSCs surrounding the colony at the edge (E). Both XtTSC and XtTSCc are present in the centre of the colony (F). The XtTSCc are clearly several times smaller than the XtTSC. Red arrowheads, XtTSC; blue arrowheads, XtTSCc. (G) X. tropicalis cell culture proliferation during long-term cultivation in medium with or without recombinant mouse LIF. (H) Representative graph of FACS sorting after nucleofection. Only ∼15% of living transgenic cells with the highest intensity of fluorescent signal were sorted (blue area). Scale bars A,C:100 μm; scale bars B,D: 200 μm; scale bars E,F: 10 μm.
	Fig. 2. Expression analysis of X. tropicalis testicular culture. (A) RT-PCR analysis of X. tropicalis testis, XtTSC, XtTSCc, XtTSC-RFP and XtTSCc-RFP. (B) Hierarchical clustering presented as a qPCR heatmaps of germ cell markers and selected testis associated markers. A scale of colours indicates level of expression (the highest expression is shown by bright red, whereas the lowest expression is shown by bright green). Similarity between cell types/genes is indicated by the height at which the dendrograms are joined. The RNA spike represents a highly stable transcript across the cell types and odc1 represents housekeeping gene.
	Fig. 3. Immunocytochemistry of X. tropicalis testicular cell culture expressing Katushka RFP. Positive staining for Sma (marker of peritubular myoid cells, green) (A) and vimentin (marker of mesenchymal cells and peritubular myoid cells, green) (B). Nuclei were counterstained with DAPI (blue). (C-E) Double staining with Sox9 (marker of Sertoli cells, green) (C) and Sma (blue) (D) antibodies. (E) Merge of C and D. Yellow arrowhead, cell expressing both antigens; red arrowheads, cells expressing only Sox9 or only Sma. (F-H) Double staining with Sox9 (green) (F) and vimentin (blue) (G) antibodies. (H) Merge of F and G. Scale bars: 50 μm.
	Fig. 4. Immunohistochemistry of agarose embedded testicular sections from X. tropicalis and mouse adult males. (A,C) Double staining with Sox9 (red) and Sma (green) antibodies. White arrowheads indicate potential common precursor cells for Sertoli and PTM cell lineages expressing both antigens in X. tropicalis (A) and mouse (C) samples. Insets show a detailed view of structures marked with white arrowheads in underlying figures. (B,D) Staining with vimentin (green) antibody on X. tropicalis (B) and mouse (D) samples. Nuclei were counterstained with DAPI (blue). Scale bars: 40 μm.
	Fig. 5. Migration potential of X. tropicalis testicular somatic cells after allogeneic transplantation into peritoneal cavity of tadpoles in stage 41. (A) Scheme of XtTSCc-RFP preparation prior to transplantation employing isolation of cell colonies using 40 μm sieve and subsequent single cell dissociation with papain. 500 cells were microinjected dorsally into peritoneal cavity. (B-G) Observation of RFP-positive cells in transplanted tadpoles under stereo microscopy. (B) Cell deposit in pronephros and tail 1 day after microinjection. (C,D) Cell deposits in pronephros 13 days after microinjection. (E) Migration of RFP-positive cells into heart atrium 15 days after transplantation. (F,G) Cell-to-cell net growing through the tadpole body observable 30 days after microinjection. (H) Immunohistochemistry of agarose-embedded sections of transplanted tadpoles using antibodies against Sox9, Sma and vimentin (green) and Katushka RFP (red) 0, 1 and 30 days after transplantation. RFP-positive cells were stained with vimentin antibody but not Sox9 or Sma even 2 h after transplantation. 30 days after microinjection, few cells start to express Sox9 indicating potential redifferentiation into Sertoli cells or chondrocytes where Sox9 is considered as a specific marker. The first three figures in each line were taken under fluorescence microscopy. The figures on the right side were taken under fluorescence stereomicroscopy. Nuclei were counterstained with DAPI (blue). Scale bars B-G, H (0 and 1 day after transplantation), 300 μm; scale bars in H (30 days after transplantation, staining with Sox9 and Sma antibodies), 900 μm; scale bars in H (30 days after transplantation, staining with vimentin antibody), 200 μm.

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