Figure 1

Estrogen enlarged the bi-potential gonad in zebrafish. (A) A schematic showing the procedure of the classification according to PGC area and gene expression analyses. (B) The bi-potential gonad area of the selected cyp17a1+/+ and cyp17a1−/− fish at 23 dpf. Dark green circles: top three bipotential gonad area. Brown circles: last three bi-potential gonad area. (C,D) Representative images of the selected cyp17a1+/+ and cyp17a1−/− fish from the PGC-rich group at 23 dpf. EGFP-labeled gonads were pointed by white arrows. Scale bar, 250 µm. (E,F) Representative images of the selected cyp17a1+/+ and cyp17a1−/− fish from the PGC-less group at 23 dpf. EGFP-labeled gonads were pointed by white arrows. Scale bar, 250 µm. (G) Gene expression heat map of cyp19a1a in cyp17a1+/+ zebrafish from PGC-rich and PGC-less groups at 23 dpf. (H) The bi-potential gonad area of the selected cyp17a1+/+ and cyp17a1−/− fish with estradiol treatment from 16 to 23 dpf. E2, 17 β-estradiol. n.s., no significance. * p < 0.05.
Figure 2

Gene expression heat map and gene set enrichment analysis. (A) DEGs from the comparison between cyp17a1+/+ and cyp17a1−/− fish of the PGC-rich group at 23 dpf. Volcano plot showing genes, including amh and gsdf, that were differentially expressed in cyp17a1+/+ and cyp17a1−/− fish of the PGC-rich group at 23 dpf. (B) Gene expression heat map of gonadal differentiation of cyp17a1+/+ and cyp17a1−/− fish of the PGC-rich group at 23 dpf. (C) DEGs from the comparison between cyp17a1+/+ and cyp17a1−/− fish of the PGC-less group at 23 dpf. Volcano plot showing genes, including amh and gsdf, that were differentially expressed in cyp17a1+/+ and cyp17a1−/− fish of the PGC-less group at 23 dpf. (D) Gene expression heat map of gonadal differentiation of cyp17a1+/+ and cyp17a1−/− fish of the PGC-less group at 23 dpf. (E) Veen analysis of DEGs. Up: upregulated. Down: downregulated. (F) Scatter plot of the intersected DEGs. Red dots: co-upregulated. Orange dots: co-downregulated.
Figure 3

The transcriptome results could be validated with qPCR, but downregulated amh and gsdf were observed in cyp17a1−/− fish after estradiol treatment from 16 to 23 dpf. (A,B) Relative expression of amh and gsdf in cyp17a1+/+ and cyp17a1−/− fish of PGC-rich and PGC-less groups at 23 dpf with qPCR. Upregulated expression of amh and gsdf was observed in cyp17a1−/− fish of PGC-rich and PGC-less groups at 23 dpf. (C,D) Relative expression of amh and gsdf in cyp17a1+/+ and cyp17a1−/− fish of PGC-rich and PGC-less groups treated with estradiol from 16 to 23 dpf. E2, 17 β-estradiol. Different letters in the bar charts represent significant differences.
Figure 4

Upregulated expression of amh and gsdf was observed in cyp19a1a−/− fish of PGC-rich and PGC-less groups at 23 dpf. (A) A schematic showing the procedure of the classification according to PGC area and gene expression analyses. (B) The bi-potential gonad area of the selected cyp19a1a+/+ and cyp19a1a−/− fish at 23 dpf. Dark green circles: top three bipotential gonad area. Brown circles: last three bi-potential gonad area. (C,D) Representative images of the selected cyp19a1a+/+ and cyp19a1a−/− fish from the PGC-rich group at 23 dpf. EGFP-labeled gonads were pointed by white arrows. Scale bar, 300 µm. (E,F) Representative images of the selected cyp19a1a+/+ and cyp19a1a−/− fish from the PGC-less group at 23 dpf. EGFP-labeled gonads were pointed by white arrows. Scale bar, 300 µm. (G,H) Relative expression of amh and gsdf in cyp19a1a+/+ and cyp19a1a−/− fish of the PGC-rich and PGC-less groups at 23 dpf. n.s., no significance. * p < 0.05. Different letters in the bar charts represent significant differences.
Figure 5

Upregulated expression of amh and gsdf was observed in WT fish of PGC-rich and PGC-less groups treated with tamoxifen from 16 to 23 dpf. (A) Upregulated expression of amh was observed in WT fish of PGC-rich and PGC-less groups treated with tamoxifen from 16 to 23 dpf. (B) Upregulated expression of gsdf was observed in WT fish of PGC-rich and PGC-less groups treated with tamoxifen from 16 to 23 dpf. Con, control. Treat, treated with TAM. TAM, tamoxifen. Different letters in the bar charts represent significant differences.
Acknowledgments
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