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. 2019 Mar 14;9:4452. doi: 10.1038/s41598-019-41051-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Reduced expression of SOX9 and loss of proliferation of Sertoli cells in SC-SF-1^−/− mice. Immunostaining of SOX9 and BrdU in developing testes of control and SC-SF-1^−/− mice at E15.5 (A,B), E16.5 (C,D), E17.5 (E,F) and E18.5 (G,H). SC-SF-1^−/− testes demonstrated a marked decline in SOX9 positive cells (B,D,F,H) compared to control testes (scale bar = 200 μm). Pregnant mice were injected with BrdU (50 mg/kg b.wt.) 4 h prior to sacrifice. Reduced BrdU incorporation was observed in SC-SF-1^−/− testes (B,D,F,H) compared to controls (A,C,E,G). Proliferating Sertoli cells in the seminiferous tubules were identified with SOX9 and BrdU double positive staining (orange; merge 40X). Reduced proliferating Sertoli cells were observed in SC-SF-1^−/− testes through E15.5 to E18.5 (I–L) compared to their respective controls. (M) SOX9 protein levels were analyzed by western blotting in whole tissue extracts of control and SC-SF-1^−/− mice at E15.5. Histone H3 served as a protein loading control. (N) Quantification of western blots demonstrated marked decline in SOX9 expression in SC-SF-1^−/− testes compared to control testes. *p < 0.001 by two-tailed Student’s unpaired t test. CON = control.