Fig 2: Proposed model of Theca differentiation.In early growing follicles, including primary and secondary stages, activated (ENG+, ACTA2+) cells are identifiable near the follicular basal membrane. These cells differentiate to the multiple cell lineages observed within the theca layer of antral follicles, which include sTCs (ACTA2+, PDPN+, TCF21+/neg), aTCs (ANPEP+CYP17A1+/neg), and pfTCs (ANPEPnegTHBD+) as well as Linneg TPCs (PTCH1+Linneg) with multilineage potential. Differentiation along this trajectory is driven by a reciprocal expression pattern of the transcription factors TCF21 and NR5A1, which govern specification of sTC and aTC identity, respectively.

Fig 3: Immunolabeling and RNA ISH reveals progressive emergence of theca lineage markers.a Immunostaining for ACTA2, ENG and ANPEP in primary (i), secondary (ii) and early antral (iii) follicles. Red, blue, and red/blue arrows point to cells that are positive for ENG, ACTA2 or both markers, respectively; in (iii) green arrows indicate ANPEP+ cell layer, while red arrows indicate an ENG+ area that is negative for both ANPEP and ACTA2. b–c Staining of early antral follicles for ANPEP and CYP17A1 (b), or ANPEP and THBD (c); red arrows indicate THBD+ cells and blue arrows show ANPEP+ cells. d Representative Immunolabeling for TCF21, ACTA2 and ANPEP in (i) primordial, (ii) transitioning primary, (iii) primary, (iv) secondary and (v) early antral follicles. Blue arrows indicate ACTA2+ cells and green arrows indicate TCF21+ cells; white boxes indicate areas magnified in bottom panels. e RNA ISH labeling for TCF21 RNA in a primary follicle. f Staining for ACTA2 and GLI1 in a secondary follicle; representative cells expressing both markers are indicated with red and blue arrow. g RNAScope labeling for PTCH1 RNA in primary and secondary follicles. Blue channel shows transmitted light in (e and g). Scale bars – 100 μm. a–e Nuclear counterstain (DAPI) is shown in white in (a–d and f). as = anti-sense RNA probe in (e and g).

Fig 4: 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.

Fig 5: Mitotic index of THBD+, ANPEP+ and CYP17A1+ reveals decreased proliferation with acquisition of androgenic fate.a Representative staining for Ki67, THBD and ANPEP in antral follicles; green arrows indicate Ki67+ cells. b Schematic of experimental approach for sequential labeling with thymidine analogues; red and green dots indicate times of EdU and CldU injections, respectively. c–e Xenografted antral follicles labeled to detect EdU incorporation (red) ki67 (green) and either CYP17A1, ANPEP, or THBD as indicated (blue). Cells within the CYP17A1/ANPEP/THBD compartment that stained positive for EdU (d) or Ki67 (e) were quantified. f Schematized representation of proliferation among sub-populations in antral follicles. g Representative images of ovarian xenograft tissue containing pre-antral follicle stages immunolabeled for EdU (red) and CldU (green). h Quantification of proliferative cells within the GC and theca compartment of early follicles (primordial, primary, secondary <5 GC layers), as measured by incorporation of EdU (red bar), CldU (green bar) or both (black bar). Nuclear counterstain (DAPI) is shown in white in (a and c) and in blue in (g). Scale bars: in (a) – 100 μm; in (c) large panels 1 mm, small panels 50 μm; in (g) left panel – 100 μm, Primary and Secondary panels – 50 μm;. p values *<0.05, **<0.005, ***<0.0005, ****<0.00005.