Fig 2: TC17 validation in vivo. Treatment started at 8 weeks old mice. A After Dox treatment (4 weeks), CTRL and TC17 mice were sacrificed, and ovaries were collected (N = 4). RNAscope was performed for Cyp17 probe and Draq5 to stain DNA. Representative confocal micrographs of CTRL (upper panel) and TC17 transgenic mouse ovaries (lower panel). Panels show the effects of Dox treatment in the Cyp17 expression (in red). An antisense control probe was used as control from the vendor. B qPCR validation of Cyp17 expression in TC17 ovaries compared with CTRL ovaries (N = 6). After Dox treatment (8 weeks), ovaries were collected, and qPCR was performed Graphs show fold change means ± s.e.m relative expression to Cyp17 following normalization to the housekeeping gene. Data were analyzed using the two-tailed Mann–Whitney test (***p < 0.001)

Fig 3: Lipid droplet storage in Leydig cells and changes in testosterone synthase from gene expression and protein levels were examined. Cholesterol storage in Leydig cells between the two groups was visualized by BODIPY staining (A,B). The relative mRNA levels of the following indicators, Lhcgr, Srb1, Star, Cyp17a1, Cyp11a1, 3ß-hsd, and Cyp19a1, were detected by qRT-PCR (C). Also, the protein levels of cholesterol transporter SRB1 and testosterone synthase STAR, CYP11A1, and CYP17A1 were observed by Western Blot (D,E) and immunofluorescence staining experiments (F–I). * Significant differences compared to the control (p < 0.05).

Fig 4: PS-MPs treatment downregulated the expression of steroidogenic enzymes and StAR in testes. Mice were given drinking water comprising PS-MPs of various sizes for 180 sustained days. A, B The expression of 3ß-HSD, 17ß-HSD, P450scc, P450c17, and StAR in testes was measured by western blotting. The expression levels were quantified with ImageJ (n = 3). Data are expressed as means ± SD. *P < 0.05, **P < 0.01 vs. control. #P < 0.05, ##P < 0.01 vs. 100 µg/L group. C, D The expression of StAR in mouse testicular tissues of the control group and 1000 µg/L group was tested by immunofluorescence staining. Testicular tissues were stained with StAR (red) and DAPI (blue), arrows: positive expression (scale bar = 50 µm). The expression levels of StAR were quantified with ImageJ (n = 3). Data are expressed as means ± SD. **P < 0.01 vs. control. #P < 0.05, ***P < 0.001 vs. control

Fig 5: The transcriptomic effect of Cyp17 overexpression is the TC17 ovary. RNA sequencing reveals Cyp17 effect on ovary gene regulation. A Upon Cyp17 induction, 721 genes were upregulated, and 290 genes were downregulated (adj. p-value < 0.05). B Among these genes, well-known ovary morphogenesis transcriptional regulators such as Cyp11, Prlr were upregulated, while Ihh and Runx1 were downregulated. Volcano plot indicates the relative fold change and p-value of genes in B. C GO analysis of the 1011 differentially expressed genes included terms such as extracellular matrix and collagen-containing extracellular matrix (see Additional file 1: Figure S4 for full-term list). D–E The induction and repression of Lhcgr and Fshr respectively, as found by RNA-seq, were confirmed using qPCR