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. 2016 Jun 21;5:e15635. doi: 10.7554/eLife.15635

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© 2016, Barrionuevo et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 4. — (A) Double immunofluorescence for SOX9 and FOXL2 in TX-treated control (Sox9^f/f;Sox8^-/-) testis (a), Sox9/8 SC-DKO mutant testes analyzed at P105 (45 datx) and P150 (90 datx) (b,c) and a control ovary (d) (b' is a higher magnification of the area marked in b). Colocalization of SOX9 and FOXL2 was rare and the few observed cells showed weak fluorescence for both proteins (arrowheads in b'). (B) Triple immunofluorescence for SOX9, DMRT1 and PCNA (germ cell marker) in P150 (90 datx) TX-treated control (Sox9^f/f;Sox8^-/-) testes (a) and Sox9/8 SC-DKO mutant testes at P90 (30 datx) (b–d) and P120 (60 datx) (e-g). Different cell types can be identified: SS: Sertoli cells with strong staining for both DMRT1 and SOX9 (SOX9⁺ DMRT1⁺ PCNA^-; strong yellow); WS: Sertoli cells with weak staining for both DMRT1 and SOX9 (SOX9⁺ DMRT1⁺ PCNA^-; pale yellow); SP: spermatocytes (SOX9^- DMRT1^- PCNA⁺; blue); SG: proliferating spermatogonia (SOX9^- DMRT1⁺ PCNA⁺; purple), arrow (SOX9^- DMRT1⁺ PCNA^-; red). Non-proliferating spermatogonia could be confused in Sox9/8 SC-DKO mice with recombined DMRT1⁺ SOX9^- Sertoli cells in which SOX9 already disappeared, but the number of the former cell type is so low that they can be ignored. (C) Double immunofluorescence for DMRT1 and WT1 in P90 (30 datx) TX-treated control (Sox9^f/f;Sox8^-/-) (a) and mutant testes (b). (D) Double immunofluorescence for DMRT1 and FOXL2 in P150 (90 datx) TX-treated control (Sox9^f/f;Sox8^-/-) testis (a), Sox9/8 SC-DKO mutant testes (b–c) and control ovary (d) (b' is a higher magnification of the area marked in b). Colocalization of both proteins was rare (arrowheads in b'). Scale bar in Dd represent 50 µm in A and D; scale bar in Bg represents 25 µm in B; scale bar in Cb represents 50 µm in C.

DOI: http://dx.doi.org/10.7554/eLife.15635.019

Figure 4—figure supplement 1. — (A) Double immunofluorescence for SOX9 and FOXL2 in TX-treated control (Sox9^f/f;Sox8^-/-) testis (a), Sox9/8 SC-DKO mutant testes analyzed at P105 (45 datx) and P150 (90 datx) (b,c) and a control ovary (d) (b' is a higher magnification of the area marked in b). Colocalization of SOX9 and FOXL2 was rare and the few observed cells showed weak fluorescence for both proteins (arrowheads in b'). (B) Triple immunofluorescence for SOX9, DMRT1 and PCNA (germ cell marker) in P150 (90 datx) TX-treated control (Sox9^f/f;Sox8^-/-) testes (a) and Sox9/8 SC-DKO mutant testes at P90 (30 datx) (b–d) and P120 (60 datx) (e-g). Different cell types can be identified: SS: Sertoli cells with strong staining for both DMRT1 and SOX9 (SOX9⁺ DMRT1⁺ PCNA^-; strong yellow); WS: Sertoli cells with weak staining for both DMRT1 and SOX9 (SOX9⁺ DMRT1⁺ PCNA^-; pale yellow); SP: spermatocytes (SOX9^- DMRT1^- PCNA⁺; blue); SG: proliferating spermatogonia (SOX9^- DMRT1⁺ PCNA⁺; purple), arrow (SOX9^- DMRT1⁺ PCNA^-; red). Non-proliferating spermatogonia could be confused in Sox9/8 SC-DKO mice with recombined DMRT1⁺ SOX9^- Sertoli cells in which SOX9 already disappeared, but the number of the former cell type is so low that they can be ignored. (C) Double immunofluorescence for DMRT1 and WT1 in P90 (30 datx) TX-treated control (Sox9^f/f;Sox8^-/-) (a) and mutant testes (b). (D) Double immunofluorescence for DMRT1 and FOXL2 in P150 (90 datx) TX-treated control (Sox9^f/f;Sox8^-/-) testis (a), Sox9/8 SC-DKO mutant testes (b–c) and control ovary (d) (b' is a higher magnification of the area marked in b). Colocalization of both proteins was rare (arrowheads in b'). Scale bar in Dd represent 50 µm in A and D; scale bar in Bg represents 25 µm in B; scale bar in Cb represents 50 µm in C.

DOI: http://dx.doi.org/10.7554/eLife.15635.019