Fig 2: Immunophenotypic analysis of cell types within the theca/stroma layer.a Violin plots of transcripts differentially expressed across theca clusters. Genes that were used to distinguish discrete lineage-specific sub-populations are enclosed within dotted lines: red = sTC, green = pfTC and blue = aTC. Red lines show median expression levels for all cells in that cluster; black asterisks show levels that are significantly enriched (p value ≤ 1 × 10−40.) b Representative immunolabeling of antral follicles for THBD, ACTA2 and ANPEP; arrows are color-coded and indicate position of the layer where identified cells are located. c Immunolabeling of antral follicles for THBD, ACTA2 and the EC marker CD34; arrows show representative cells: green = uniquely expressing THBD, blue = ECs that stain for both CD34 and THBD, and red = cells that are positive for ACTA2. d Immunolabeling for CYP17A1 and ANPEP, blue arrows show representative cells that stain for ANPEP, while green cells show ANPEP+CYP17A1+ cells. e–f Immunolabeling for BGN and PDPN (e) or ACTA2 (f). g Antral follicle stained for the transcription factor TCF21, ACTA2 and ANPEP; arrows indicate representative ANPEP+ cells that show reduced TCF21. h Immunolabeling of an antral follicle for PTCH1, THBD and GLI1; white arrows indicate THBD+ cells showing decreased expression of PTCH1 and GLI1. i Labeling of TCF21 and THBD in antral follicles; white arrows show THBD cells that are also positive for TCF21. Scale bars in (b–i) – 100 μm. Nuclear counterstain (DAPI) is shown in white in (b–i); white boxes in (b–i) indicate areas magnified in adjacent panels; white dotted line in (b–f) and (h–i) shows the GC basement membrane.