Fig 2: Prkar2b and Marcks were two representative markers for indicating the imbalance of GCs composition in obese mice(A) qPCR assay of GCs isolated from RD or HFD mouse growing follicles for representative up-regulated genes identified in Figure 2A. Expression levels were normalized to Rps24. Two technical duplicates were performed in individual qPCR experiments, and the representative result of three independent biological replicates is shown. Error bars indicate the mean ± SEM.(B) Immunofluorescence staining for Prkar2b and Marcks in ovarian tissues from RD and HFD mice. Tissues were counterstained with DAPI and Gdf9 or Ddx4.(C) Boxplots showing expression levels of Cyp17a1 for ETCs, and Ar for GCs, in RD and the obese mice. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. denotes not significant (Wilcoxon rank-sum test).(D) Trend lines showing relative expression levels for Ar in two branches, State 1–2 and State 1–3, identified by using Monocle2.(E) Boxplots showing expression levels of Ar for Ldhb+ GCs, Htra1+ GCs, and Inhbb+ GCs, in RD and the obese mice. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. denotes not significant (Wilcoxon rank-sum test).

Fig 3: Alternations in subgroup composition of ovarian GCs from the obese mice(A) Venn diagram showing the up-regulated genes in GCs overlapping between Lep KO, Lepr KO, and HFD mice compared with RD mice.(B) Boxplots showing expression levels of Inhbb, Prkar2b and Marcks in GCs from RD, Lep KO, Lepr KO and HFD mice, respectively. The asterisks above indicate statistical significance. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001 (Wilcoxon rank-sum test).(C) Dot plot depicting the average expression levels of 22 up-regulated genes identified in Figure 2A in Inhbb+ GCs.(D) Pseudotime trajectory of granulosa cells in Figure 1B. Trajectory was inferred by Monocle 2. Cells are color coded by State inferred in the pseudotime analysis.(E) Bar plot showing the percentage of GCs in State 3 from RD and the obese mice, respectively. The Chi-squared test was performed.(F) Heatmap depicting genes with pseudotemporal expression patterns in GCs from RD and the obese mice, which were identified using Monocle2. Gene expression levels were scaled using Monocle2.(G) Heatmap depicting representative pathways enriched in GCs from different states based on the results of GSVA (gene set variation analysis). Enrichment scores are scaled and color coded. Group and State for GCs are as indicated.See also Tables S2 and S4.

Fig 4: Snf2h regulates transcription by altering chromatin accessibility. (A) Metaplot showing the ATAC-seq signals at transcription start sites (TSSs) in WT and CKO growing oocytes (GO1) and fully grown GV oocytes (FGO). (B) Heat map showing the ATAC peaks classified according to their changes in CKO versus WT oocytes. Each row represents a locus (ATAC-seq peak center ± 5 kb), and the green gradient color indicates the ATAC-seq signal intensity. (C) Metaplot showing the ATAC-seq signals at TSS of down-regulated genes in CKO FGO. (D) Genome browser views showing the ATAC-seq and RNA-seq signals at Prkar2b, Ccnb2, and Ndc80 loci. The pink boxes indicate the ATAC peaks at the promoter regions, and the green boxes indicate the RNA-seq signals.

Fig 5: Androgens induced expression of Prkar2b and Marcks in vitro in granulosa cells isolated from RD mice(A) Schematic representation of the androgen stimulation experiments on GCs isolated from 12- to 14-day-old RD mice. DHT, dihydrotestosterone; T, testosterone.(B) qPCR assay of GCs under different treatments for Inhbb, Marcks, and Prkar2b. Expression levels were normalized to Rps24. Two technical duplicates were performed in individual qPCR experiments. Error bars indicate the mean ± SEM. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. denotes not significant (Wilcoxon rank-sum test).(C and D) Immunofluorescence staining for Prkar2b (C) and Marcks (D) in cultured GCs with T or DHT treatment. Tissues were counterstained with DAPI. Scale bars are as indicated. Density plot shows immunofluorescence signal intensity in cells for Prkar2b (C) and Marcks (D) under different treatments. The signal intensity was measured with Zeiss ZEN. The mean signal intensity of specific antibody staining is indicated as a dashed line. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001 and n.s. denotes not significant. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. denotes not significant (Wilcoxon rank-sum test).