Wave 1 follicles are remodeled prior to peri-puberty.

(A) Schematic diagrams of fetal and postnatal ovaries at the indicated stages (E = embryonic day; P = postnatal day). Wave 1 follicles, located in the ovarian medulla (below the dashed line), retain bipotential pre-granulosa cells (BPGs, green) and develop without delay. In contrast, cortical follicles, which become quiescent primordial follicles (Wave 2), undergo replacement of BPGs by epithelial pre-granulosa cells (EPGs, yellow). This replacement is completed by P5. By P21 (3 wk), wave 1 follicles undergo atresia, characterized by oocyte loss. Germ cells or oocytes are shown in blue. Developmental stages are indicated in the diagram. Dashed line: Medulla-cortex boundary. (B) Postnatal follicular developmental timeline: Wave 1 antral follicles emerge by 3 weeks (Figure S1A-C); Juvenile sexual development (peri-puberty) occurs at 4-5 weeks; Puberty and ovulation take place around 7-8 weeks. (C) Annotation of total follicles in P5 and P10 ovaries using Imaris software (gray balls). Each follicles with DDX4-postive oocyte was manually counted and labeled using the Imaris spot model. Scale bars: left, 50 µm; right, 70 µm. (D) Total follicle counts in P5 and P10 ovaries (mean ± SD; N = 3-4). ns, not significant. (E) Annotation of wave 1 follicles in P5 and P10 ovaries using Imaris software (gray balls). Follicles with oocytes larger than 20 µm in diameter, primarily located in the medulla, are classified as wave 1 follicles at the indicated times. Each gray ball represents one follicle. Scale bars: left, 50 µm; right, 70 µm. (F) Wave 1 follicle counts in P5 and P10 ovaries (mean ± SD; N = 3-4). ns, not significant. (G) 3D projection of the ovary at P14, reconstructed using the Imaris surface model. Scale bars = 50 µm. (H) Oocyte volume increase in wave 1 follicles over time (mean ± SD; N = 10). (Note: Nuclear volume is excluded, as DDX4 is a cytoplasmic protein.). ****p < 0.0001. (I) Representative images of ovary at 4 wk and 5 wk. Left (4 wk): arrowheads indicate an atretic follicle with a distorted oocyte and disorganized granulosa cells; arrow points to a developing healthy secondary follicle. Right (5 wk): arrowheads indicate degrading follicles at various stages; arrow points to a developing healthy secondary follicle. Scale bars = 100 µm. (J) Wave 1 follicle remodeling, indicated by oocyte loss over time (mean ± SD; N = 3-4). Oocyte loss begins around 3 wk and continues through 5 wk, accompanied by granulosa cell disorganization and cell death. ns, not significant; ** p < 0.01; ****p < 0.0001.

Wave 1 follicles lose granulosa cells during remodeling.

(A) Schematic diagram illustrating the activation of Foxl2-driven EYFP expression following TAM injection at E16.5. TAM administration at E16.5 induces the excision of a stop cassette, leading to EYFP expression in granulosa cells of follicles (see Methods). (B) Efficient EYFP labeling of granulosa cells in wave 1 medullary follicles of a 2-week ovary following TAM injection at E16.5, as shown in (A), magnified in the right. Scale bars: left, 100 µm; right, 30 µm. (C) EYFP-labeled follicles that have reached the primary/secondary follicle stage by 2 weeks are quantified using Imaris software, with annotation performed using the spot model. Left: A section showing annotated labeled follicles (white circles). Right: 3D projection using Imaris software, where labeled follicles are represented by gray spheres. Scale bars: left, 100 µm; right, 80 µm. (D) By 5 wk, EYFP-labeled follicles that have reached the preantral/antral follicle stage are quantified using Imaris software and annotated with the spot function. Left: A section showing annotated labeled follicles (white circles). Right: 3D projection using Imaris software, with labeled follicles represented by gray spheres. Scale bars = 200 µm. (E) Over 90% of wave 1 follicles contained EYFP-positive granulosa cells at 2 wk. At 5 wk, approximately 70% of normal antral follicles contained EYFP-positive granulosa cells. (F) Quantification of wave 1 follicle numbers (representative images shown in C-D) shows a decline from 2 to 5 weeks. ****p < 0.0001. Each dot represents one sample. (G) Representative section from a 5-week ovary reveals ongoing remodeling of wave 1 follicles. Follicles near the medullary core appear small and without oocytes (arrowheads), with few remaining EYFP-labeled granulosa cells, as seen in the follicle boxed in “1” and enlarged in G’. In contrast, a normal antral follicle in rectangle “2” enlarged in G’’, contains numerous EYFP-labeled granulosa cells. Right: The average number of EYFP-labeled granulosa cells in medullary follicles before remodeling (3 weeks) is compared to remodeled medullary follicles at 5 weeks. Each point represents one analyzed follicle. ****p < 0.0001. Scale bars: left, 200 µm; right, 50 µm. (H) About 80% of remodeled follicles still retain some EYFP-labeled granulosa cells at 5 wk, consistent with their wave 1 origin. Dashed line: follicle boundary; arrowheads: EYFP-positive granulosa cells. Each point represents one analyzed follicle. (I) Remnant granulosa cells of remodeled wave 1 follicles continue to express Foxl2 at 5 weeks. TAM was injected into Foxl2-CreERT2; R26R-LSL-tdTomato mice at 5 weeks, and ovaries were collected one week later. Arrowheads indicate remodeled follicles, with one remodeled follicle (outlined in the rectangle) enlarged on the right. Scale bars: left, 100 µm; right, 20 µm.

The labeling and quantification of boundary follicles

(A) At 2 wk, one or more granulosa cells in some primordial follicles located at the medulla-cortex boundary are labeled with EYFP (enlarged in “1”, “2”, “3”) following TAM injection, as shown in the schematic. These follicles are referred to as boundary follicles or, in accordance with the wave classification, wave 1.5 follicles. Dash line, medulla-cortex boundary. Scale bars: left, 100 µm; right, 20 µm. (B) Quantification shows that 7% (421 ± 74) of the total 5,843 ± 1,256 primordial follicles contained EYFP-labeled granulosa cells in the labeled experiment shown in (A). Each point represents one analyzed sample. Labeled PFs: Labeled primordial follicles. (C) Representative images of Foxl2-CreERT2; R26R-LSL-EYFP mouse ovary injected with TAM at E14.5. Boundary follicles labeled with EYFP are enlarged on the right. Dash line indicates the medulla-cortex boundary. Scale bars: left, 100 µm; right, 20 µm. (D) Approximately 70% of secondary follicles in 5 wk ovaries contain EYFP-labeled granulosa cells, suggesting they originate from labeled primordial follicles (wave 1.5) at 2 wk. TAM was injected at E16.5. Each point represents one analyzed sample. Scale bar = 50 µm.

Follicles remodel to form an interconnected stromal network rich in thecal secretory cells.

(A) Schematic diagram illustrating lineage labeling of Cyp17a1-expressing theca cells, interstitial gland cells, and potentially other cell types using Cyp17a1-iCre; R26R-LSL-tdTomato mouse model. (B) 2 wk ovary showing tdTomato-labeled theca cells sheathing follicles (arrowhead) and putative interstitial gland cells between follicles (rectangle). Scale bars: left, 20 µm; right, 5 µm. (C) 3D reconstruction of follicles with surrounding labeled theca cells and interconnected interstitial gland cells in a 2 wk ovary. Left: 3D projection using Imaris software. Right: Pseudo-color annotation using the Imaris surface model. Green: Oocytes. Red: Cyp17a1-expressing cells, including theca and interstitial gland cells. Scale bars = 20 µm. (D-E) Representative images of 2wk (D) and 5 wk (E) ovaries showing a dramatic increase in labeled theca, interstitial, and possibly other Cyp17a1-lineage cells in the ovarian medulla. Scale bars = 100 µm. (F-G) Oil Red O staining (red) reveals a significant increase in lipid droplet storage in ovarian medullary cells from 2 wk (F) to 5 wk (G) ovaries. Scale bars = 100 µm. (H) Immunofluorescence staining of HSD3B1 and PLIN1 at 5 wk shows that genes required for steroidogenesis and lipid droplet storage are highly expressed in medullary cells. Scale bars = 50 µm. (I) Electron microscopy of a remodeled follicle reveals cells with abundant lipid droplets (LD) and characteristic mitochondria (mt) (boxed region magnified in right panel). Scale bar = 10 µm. (J) 3D reconstruction of a 5 wk ovary labeled by tdTomato expression activated by Cyp17a1-iCre. Left: 3D projection using Imaris software. Right: Pseudo-color annotation using the Imaris surface model. Red: Thecal/interstitial network structure. Balls: Oocyte locations. Scale bars = 100 µm.

Single-cell RNA sequencing of 2 wk-6 wk ovaries

(A) UMAP plot of ovaries from 2 wk to 6 wk, showing 31 initial clusters (c0-c30) represented in different colors. Sequencing data from different ages were merged before summit to Seurat. (detailed information in the methods section). (B) The ovarian cells from 2 wk to 6 wk contribute to almost every cluster in the UMAP plot. O2w: scRNA sequencing data from 2 wk ovary. O3w: 3 wk ovary. O4w: 4 wk ovary. O5w: 5 wk ovary. O6w: 6 wk ovary. (C) The cell type groups are indicated by colored regions, with their relationship to the numbered initial clusters as shown in A. Granulosa cells: c1, c2, c3, c5, c10, c12, c20, c21, c24 and c30; Stromal cells: c0, c4, c7, c8, c9, 13, c17 and c19; Theca cells: c16; Hematopoietic cells: c23; Endothelial cells: c22 and c27; Pericytes: c18; Smooth muscle cells: c26; Epithelial cells: c25 and c28; Low UMI: c6, c11, c15 and c29; TBD (To be determined): c14. (D) Multiviolin plot of selected marker gene expression across different cell types. Y-axis: Gene names with expression levels normalized for display. X-axis: Cell types.

Further re-cluster analysis of sequencing data.

(A) UMAP plot of re-clustered granulosa cells from Figure 5A, showing 18 clusters (gc0-gc17). (B) The deduced ovarian follicle developmental stages are labeled and indicated by colored regions with their relationship to the numbered clusters shown in A. Primordial: gc11; Primary: gc3 and gc8; Secondary: gc1, gc4 and gc9; Antral: gc2, gc7, gc10 and gc13; Remodeling: gc0 and gc6; Mitotic: gc5, gc12, gc14, gc15, gc16 and gc17. (C) Dot plot of marker gene expression in granulosa cells across different follicle stages. Y-axis: Cell types. X-axis: Gene names with expression levels normalized for display. (D) Pseudotime trajectory analysis using Monocle3 reveals two pathway in secondary follicles: Path 1 leads to further development into antral follicles, while Path 2 results in remodeling follicles. (E) UMAP plot of re-clustered theca cell and stromal cells Figure 5A, showing 21 clusters (g0-g20). (F) The deduced mesodermal cell subgroups are labeled and indicated by colored regions and their relationship to the numbered clusters shown in panel E. Hoxd9+: g0; Tagln+: g1, g8, g14 and g17; Early TCs: g2 and g11; Late TCs: g9; Cxcl12+: g3, g5 and g20; Cd24a+: g4; Col11a1+: g6 and g7; Col14a1+: g10 and g19; Mitotic: g12, g13, g15 and g18; TBD (To be determined): g16. (G) Diagram of steroidogenic gene expression in theca and granulosa cells and their role in androgen and estrogen synthesis. Top panel: The diagram illustrates the synthesize of androgen in theca cells and estrogen in granulosa cells from cholesterol. Bottom panel: The estimated numbers of steroidogenic theca cells expressing three key steroidogenesis genes in the sequencing data— Cyp11a1, Hsd3b1, and Cyp17a1—are shown, along with those expressing all four essential genes (Cyp11a1, Hsd3b1, Cyp17a1, and Star). The total cell number are shown in the first row. From 2 wk to 5 wk, the theca cell population increases, followed by a decline at 6 weeks. Meanwhile, the number and percentage of theca cells expressing all four essential genes progressively increase. In contrast, the estimated numbers of granulosa cells expressing Hsd3b1, Hsd17b1, and Cyp19a1 follow a fluctuating pattern, decreasing from 2wk to 4 wk before rising again at 5 wk. Notably, 2 wk corresponds to the time of minipuberty. (H) Estimated numbers of granulosa cells in follicles across developmental subclasses: primordial, primary, secondary, antral, remodeling, and mitotic cycling.

Model of wave 1 follicle development and function.

Wave 1 follicles undergo remodeling during the peri-pubertal period to expand the population of androgen-producing theca and interstitial cells. Gradients between the medulla and cortex facilitate the sequential activation of primordial follicles, a process that continues into early adulthood. We refer to these sequentially activated follicles as boundary follicles (Wave 1.5). These follicles, whose granulosa cells are derived from bipotential pre-granulosa cells (BPGs), remain dormant in the medulla and cortex until recruiting signals—either positive or negative— become available. Boundary follicles are typically recruited around 2 weeks of age and constitute the majority of developing follicles by 5 weeks. In parallel, wave 1 follicles remodel to support androgen production through their remaining theca cells and associated interstitial glands.

Summary of whole mount C57BL/6J ovary analyses

Wave 1 follicle development in the early juvenile ovary.

(A-D) Representative images of wave 1 follicles stained for DDX4 (green) at P5, P7, P14 (2 wk), and P21 (3 wk). A: Early primary follicle with theca cells (arrow); B: Late primary follicle; C: Secondary follicle; D: Antral follicle. White arrowhead: non-surrounded nucleus; Red arrowhead: Pyknotic nuclei of granulosa cells. Scale bars in, A: 20 µm; others: 50 µm.

Wave 1 follicles begin to remodel by 3 weeks.

(A) Electron microscopy of follicles at 5 wk ovary. Left: Normal follicle. Middle: intermediate follicle, in which the oocyte cytoplasm shrinks and begins degrade, indicated by the space between the oocyte and ZP, the absence of microvilli, and folding of the ZP (red arrow). Right: Remodeled follicle, where the oocyte has disappeared but the ZP remains. Surrounding theca and interstitial gland cells further accumulate lipid droplets. The boxed region is magnified in the lower panel. Abbreviations: Oo = oocyte; ZP = zona pellucida; LD = lipid droplet; mt = mitochondria; mv = microvilli. Scale bars = 20 µm. (B) DAPI-stained DBA/2J ovaries showing remodeling wave 1 follicles (arrowheads) and normally developing follicles (arrows). Left (3 wk): Early remodeling follicles are observed in the central medulla (arrowheads). Right (7 wk): Wave 1 follicle derivatives retain an empty shape (arrowheads), remaining visible alongside normal follicles (arrows) and corpora lutea (asterisks). Ovaries were stained and imaged as whole mounts; only a single section is shown here. Scale bars = 200 µm.

Lineage labeling using Foxl2-CreERT2 and R26R-LSL-tdTomato

(A) Schematic diagram of the experiment. The Foxl2-CreERT2 mouse line was generated with EGFP inserted into the Foxl2 locus, resulting in weak EGFP signal detectable in the cytoplasm using an EGFP antibody. To rule out the possibility that the observed signal originated from the Foxl2 locus rather than ROSA26-EYFP, we repeated the experiment using the Foxl2-CreERT2; R26R-LSL-tdTomato mouse line. (B) At 2 wk, wave 1 follicle are labeled. By 5 wk, ome have turned over, as indicated by cavities left by disappeared oocytes and remaining tdTomato signals (arrowheads), while others have developed into antral follicles (arrow). One developing early primary follicle in the boxed region is shown in panel C. Scale bars are included in the images. (C) The EGFP channel shows a weak cytoplasmic signal from the Foxl2 locus, distinct from the strong, evenly distributed expression of EYFP from the ROSA-EYFP reporter shown in Figure 3/4. Scale bar, 20 µm. (D) Boundary follicles (enlarged on the right) were observed in the 2 wk ovary of Foxl2-CreERT2; R26R-LSL-tdTomato mouse line injected with TAM at E16.5. Scale bars: left, 50 µm; right: 10 µm.

Expansion of theca cells and its related cells - interstitial gland population.

(A) Immunofluorescence staining of CYP17A1 protein in the ovary shows a significant increase of theca cells and interstitial gland within the stroma from 3 wk to 5 wwk. Scale bars = 50 µm. (B) Immunofluorescence staining of Cyp17a1 lineage-traced ovary at 2 wk shows initial expression in theca cells and associated cells resembling interstitial gland cells (arrowheads). Additionally, a few granulosa cells were labeled (arrows), as previously reported (Bridges et al., 2008). Scale bar = 50 µm. (C) Structurally, steroidogenic theca cells closely resemble interstitial gland cells. In the electron microscopy images, red lines delineate distinct zones enriched in interstitial gland cells (1), theca cells (2), and granulosa cells (3), with each region magnified below. Zone 1 (rectangle labeled “1”) represents interstitial gland cells. Zone 2 (rectangle labeled “2”) contains steroidogenic theca cells. Zone 3 (rectangle labeled “3”) corresponds to granulosa cells. Abbreviations: nu – nucleus; LD – lipid droplet; mt – mitochondria. Scale bars are included in the images. (D) Cyp17a1 lineage-traced ovary at 4 wk and 7 wk shows a marked increase in the number of labeled theca cells and interstitial gland cells by 7 weeks. Extensive signal is also observed in the rete ovarii (arrowhead), indicating Cyp17a1 expression, as well as in the ovarian epithelium (arrow). Scale bars = 200 µm.

Analysis of single-cell RNA sequencing

(A) nFeature_RNA of the scRNA-seq data shows that clusers 6, 11, 15, 29 have low UMI. (B) Highlighting cluster 14 reveals its dispersed distribution. (C) The distribution of samples across clusters shows that cells from each sample contribute to all clusters (orig.ident). (D) Feature plot of marker gene expression from the reanalysis of mesenchymal cells, including late theca cells (Cyp11a1), early theca cells (Hhip), Tagln+ fibroblasts (Tagln), Hox9+ fibroblasts (Hox9), Col11a1+ fibroblasts (Col11a1), Cxcl12+ fibroblast (Cxcl12), Col14a1+ fibroblasts (Col14a1), Cd24a+ fibroblasts (Cd24a) and mitotic cells (Mki67).

Analysis of other cell types

(A) Expression plot of representative genes highly expressed in cluster 22, identified as endothelial cells in blood vessels, including Esam, Flt1, and Cd93. Red boxes in the top panel are enlarged in the bottom panel. cluster 27: Endothelial cells from lymphatic vessels. (B) Expression plot of representative genes highly expressed in cluster 27, identified as endothelial cells in lymphatic vessels, including Ccl21a, Lyve1, and Prox1. Red boxes in the top panel are enlarged in the bottom panel. (C) Expression plot of representative genes shared between pericytes and endothelial cells, including Ebf1, Emid1, and Apold1. Red boxes in the top panel are enlarged in the bottom panel. (D) Ridge plot of representative gene expression in epithelial cells from the ovarian surface epithelium (cluster 25) and the rete ovarii (cluster 28). (E) Further analysis of hematopoietic cells using re-clustering in Seurat identified 9 clusters across five samples. (F) The deduced hematopoietic cell subgroups are labeled and indicated by colored regions, including macrophages (clusters 0, 4, and 7), dendritic cells (cluster 3), neutrophils (cluster 5), B cells (cluster 1), T cells (cluster 2), NK cells (cluster 6), and mitotic cells (cluster 8). (G) Dot plot showing marker gene expression across different hematopoietic cell types.

Hormone receptor expression and XO mouse line.

(A) Expression levels of hormone receptors (Y-axis) across different cell clusters (X-axis). Androgen receptor (Ar) is expressed in nearly all cell types except hematopoietic cells. (Esr1) is primarily expressed in theca cells, epithelial cells, and smooth muscle cells, whereas estrogen receptor 2 (Esr2) is expressed in granulosa cells as expected. No expression of follicle-stimulating hormone receptor (Fshr) is detected in any cell type, while luteinizing hormone/choriogonadotropin receptor (Lhcgr) is specifically expressed in theca cells. Progesterone receptor (Pgr) is present in smooth muscle cells but absent in granulosa cells. (B) The total follicle count in XO mice at 1 week is significantly lower compared to control XX mice. Follicle counts were performed using Imaris software on whole-mount stained ovaries. N = 3–5. ** p < 0.01. (C) By 2 weeks, statistical difference between XO and XX mice were observed. Follicle counts were performed using Imaris software on whole-mount stained ovaries. N = 4–5. ** p < 0.01; ns, not significant.