Defining the cell and molecular origins of the primate ovarian reserve

Abstract

The primate ovarian reserve is established during late fetal development and consists of quiescent primordial follicles in the ovarian cortex, each composed of granulosa cells surrounding an oocyte in dictate. As late stages of fetal development are not routinely accessible for study with human tissue, we exploited the evolutionary proximity of the rhesus macaque to investigate primate follicle formation. Similar to human prenatal ovaries, the rhesus also develops multiple types of pre-granulosa (PG) cells, with the majority of primordial follicles derived from PG2 with small variable contributions from PG1. We observed that activated medullary follicles recruit fetal theca cells to establish a two-cell system for sex-steroid hormone production prior to birth, providing a cell-based explanation for mini puberty.

Fig. 1.. Dynamic changes to the ovary precede follicle formation.

(A) Illustration of key events and morphological transitions during human and rhesus macaque ovarian development. Cortex (c), medulla (m), anterior (a) and posterior (p) labels show section image orientation – all subsequent images are positioned according to these axes. Roman numerals refer to key events: i – definitive ovary formation from gonadal ridge; ii – sex determination; iii – germline cells enter meiosis (leptotene oocytes observed); iv – ovigerous cord formation; v – diplotene oocytes observed; vi – primordial follicles emerge; vii – early activated follicles observed. (B) Immunofluorescence analysis in W5 (D34), W6 (D41), W8 (D50), W15 (D100), W19 (D130) and 6 months postnatal rhesus ovaries for primordial germ cell (SOX17, green; PRDM1, magenta; TFAP2C, yellow); late-stage germ cell (VASA, green) and granulosa (KRT19, yellow; FOXL2, magenta) markers (bottom panels at higher magnification in the developing cortex). Arrows show rare FOXL2+ cells at W5. Nuclei counterstained with DAPI (grey). All scale bars 50 μM.

Fig. 2.. Pregranulosa (PG) cells emerge from mesenchymal-like progenitors at W5–6.

(A, B) Spatial distribution of genes of interest in Visium CytAssist analysis of W5 and W6 ovaries mapped onto a black and white H&E image. Ovary (white), mesonephros (orange) and adrenal (yellow) regions are outlined; expression level scaled from blue (min) to yellow (max). (C) CosMx spatial molecular imager (SMI) analysis of W6_3 ovaries; left panels, distribution of annotated cell clusters in the selected fields of view (FOVs); right panels, zoom in on ovary region with cell segmentation overlay showing germline, pre-granulosa and stromal cells (additional clusters marked “Others”). White dashed line indicates ovary region. (D) Immunofluorescence analysis for known genes (WT1, blue; GATA4, orange; FOXL2, yellow; VASA, green; NR2F2, magenta) and genes identified in the DEG analysis (NR5A1, red; GATM, cyan) in W5 and W6 ovaries. Scale bars 50 μM.

Figure 3.. PG cells undergo a major transcriptional shift between embryonic and fetal stages.

(A) UMAP plot of single cells collected from rhesus fetal ovaries at W8, W15 and W19 colored according to Seurat clusters (n=3 biological replicates per timepoint).(B) Bar graph of annotated cell type proportions at each time point.(C) UMAP plot of W8 cells colored by cell annotation from the analysis in Fig. 3A. (D) Expression of ovary-enriched genes identified in the Visium CytAssist spatial transcriptomics analysis at W5 and W6 cast on the UMAP plot from Fig.3C. Normalized expression plotted on a high-to-low scale (indigo-yellow).(E) UMAP plot of granulosa subset (clusters a1, a3 and a4 from 3A) re-clustered and colored according to Seurat clusters. (F) Bar graph of granulosa sub-cluster proportions at each time point. (G) Dot plot of known granulosa-associated genes, or genes identified as highly enriched in each cluster (rectangles). Expression plotted on a high-to-low scale (indigo-yellow); dot size reflects percentage of cells expressing given gene. *KRT18 = ENSMMUG00000031911), *COL9A3 = ENSMMUG00000016859.

Figure 4.. PG1.5 and PG2 contribute to both quiescent and activated follicles.

(A) VASA (green), NR2F2 (magenta), FOXL2 (yellow), NR5A1 (red), GATM (cyan) expression and DAPI (grey) at W8, W15 and W19. Scale bars 50 μM. (B) VASA (yellow), KRT19 (cyan), FOXL2 (magenta), expression and DAPI (grey) in primordial follicles at W19. Scale bars 50 μM. (C) Spatial distribution of germline bins (E) and granulosa PG1 subcluster bins identified in Visium CytAssist HD analysis of W19_3 mapped onto a black and white H&E image. See Methods for details.

Figure 5.. Early activated follicles recruit fetal theca cells and produce hormones.

(A) Visium CytAssist HD spatial distribution of granulosa cluster g9 and theca clusters t0 and t2 bins identified in W19_3 mapped onto a black and white H&E image. (B) VASA (green), CYP17A1 (yellow) expression and DAPI (grey) at W19 and 6 MPN. (C) NR5A1 (yellow), FOXL2 (magenta), PAPP-A (red), CYP19A1 (cyan) expression and DAPI (grey) in fetal activated follicles at W19. Magnified area (dashed) is shown in the inset. (D) VASA (green), CYP17A1 (yellow), FOXL2 (magenta), WT1 (blue) expression and DAPI (grey) at W19 and 6MPN. Magnified area (dashed) is shown in the inset. All scale bars 50 μM.