Fig 2: Localization of the regulatory elements involved in the cooperation between SOX8 and SF-1 to activate the Cx43 promoter. TM4 Sertoli cells were co-transfected with an empty expression vector (CTL) or expression vectors for SOX8 and/or SF-1 along with 5′ deletion constructs of the mouse Cx43 promoter. The number of experiments, each performed in triplicate, is indicated. Results are shown as fold activation over control (CTL) (±SEM). Statistical analyses were performed using a two-way ANOVA followed by a Tukey multiple comparisons test (different letters: P < 0.05).

Fig 3: NR5A1 heterozygous mutations detected in this study. c.15C > A; p.(Tyr5*) was identified in patient I1. c.124C > G; p.(Gln42Glu) was identified in patient I2. c.250C > T; p.(Arg84Cys) was identified in both patient I3 and patient I4. c.937C > T; p.(Arg313Cys) was identified in patient I5. c.1075_1089del; p.(Leu359_Leu363del) and c.1211_1212insTA; p.(Tyr404fs) were identified in patients I6 and I7, respectively. Patient I8 had an exon 6 duplication in NR5A1. Patient I9 had chromosome 9: 127,213,317-127570245_del (0.36 Mb del), containing NR5A1, NR6A1, MIR181A2HG, MIR181A2, MIR181B2 and OLFML2A

Fig 4: NR5A1 gene transcriptional activity assays and protein expression analysis. (A-C) NR5A1 gene transcriptional activity assays of WT NR5A1 and its variants. A luciferase reporter assay was used to assess the transcriptional activities of WT NR5A1 and its variants. Three promoter regions of the target genes (CYP11A1, CYP19A1 and AMH) of NR5A1 were included. (D) Western blot analysis of the protein expression levels of WT NR5A1 and its variants. Anti-GAPDH was used as a control. (E) Quantitative graph of the western blot analysis (D). One-way ANOVA was used. Data were represented as mean ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: Recruitment of SF-1 to the proximal region of the mouse Cx43 promoter. Chromatin immunoprecipitation (ChIP) assays for SF-1 recruitment on the Cx43 promoter were performed with TM4 Sertoli cells. Indicated Cx43 promoter regions were amplified following ChIP assays. Recruitment using a normal rabbit IgG was used as a negative control. Histone H3 as well as cJUN were used as the positive controls for protein-DNA complexes and recruitment to the proximal region of the Cx43 promoter, respectively. Amplification of a region of the second intron of Rpl30 was used as a negative control for recruitment of SF-1 to DNA (E). Results are presented as percentages of input (±SEM). Statistical analyses were performed using an ANOVA, followed by a Bonferroni multiple comparisons' test. Statistically significant differences from control (IgG) are indicated by an asterisk (*P < 0.05). The proximal region of the Cx43 promoter was investigated for SF-1 conserved regulatory elements using the CONTRAV3 platform [33] (F).