Cell-cell interactions during the formation of primordial follicles in humans

Abstract

Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC-syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell-cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility.

Figure S1.. Ovarian cords architecture in second trimester and postnatal human ovaries.

(A) Immunofluorescence for DDX4/POU5F1 in 12, 14, 18, 30, 38 WPF, and 2-yr-old ovaries. Dotted line separates inner cortex from outer cortex. (B) Immunofluorescence for SYCP3/TP63 in 30, 38 WPF, and 2 yr old ovaries. Dotted line separates the inner cortex from the outer cortex.

Figure 1.. Human fetal germ cell (hFGC)–syncytia in human ovarian cords.

(A) Representative transmission electron microscopy image of human ovarian cords. Cord boundaries between ovarian cords and stroma are outlined with dotted red lines. Pre-granulosa cells inside ovarian cords are marked with yellow asterisks. (B) Transmission electron microscopy images of hFGCsyncytia without intercellular bridges showing nuclei at interphase and pachytene. Cell membranes are highlighted with yellow dotted lines. Dashed box is shown magnified below. (C) Immunofluorescence for DDX4/CTNNB1 and DDX4/phosphorylated ezrinradixinmoesin (pERM) in 18 WPF ovary. CTNNB1 and pERM label the cell membrane; DDX4 label hFGCs in the ovarian cords. (D) Immunofluorescence for PDPN/ALPL in 17 WPF ovary. PDPN and ALPL label the cell membrane of mitotic hFGCs. (E) Percentage of mononucleated and multinucleated DDX4+ hFGCs in 14–17 and 18–20 WPF ovaries (mean ± SD). (F) Immunofluorescence for pERM/SYCP3 or TP63 in ovaries of different ages showing synchronized (left panel) and asynchronized (right panel) multinucleated germ cells. The single channel for SYCP3 or TP63 is shown at the bottom. SYCP3 marks meiotic hFGCs and TP63 marks oocytes.

Figure S2.. Nuclear structure of human fetal germ cells (hFGCs) in meiotic prophase I in the human fetal ovary.

(A, B, C, D) TEM images of hFGCs during prophase I from a 18WPF ovary showing hFGCs in leptotene (A), zygotene (B) and pachytene (C, D). Numbered dashed boxes are magnified at the bottom showing details of pairing chromosomes and their synaptonemal complex (green arrows), attachment of chromosome telomeres to the nuclear envelope (orange arrows) and mitochondria (pink arrows).

Figure S3.. Identity of cell types and establishment of cell–cell adhesion in the ovary.

(A) Transmission electron microscopy images of mouse fetal ovary at embryonic day (E)14.5. White asterisks mark germ cell nuclei in mFGCsyncytia. The yellow arrow points to an intercellular bridge that is no longer connected to the cell membrane. (B) Transmission electron microscopy images of mouse fetal ovary at E17.5. White asterisks mark germ-cell nuclei in mFGCsyncytia. Black arrows point to somatic cell protrusions that invaginate into the germ-cell syncytium. (C) Uniform manifold approximation and projections plot showing cell-cluster identity (CL_ID) of the main cell types present in 18–26 WPF human fetal ovaries. Dashed line depicts the gonadal somatic cells that are further sub-clustered (see Fig 3E). (D) Uniform manifold approximation and projections plots depicting expression levels of genes of interest. Dashed line depicts the gonadal somatic cells. (E) Immunofluorescence for KRT18/KRT19/FOXL2 (left panel) and KRT8/KRT19/FOXL2 (right panel) in a 17 WPF ovary. (F) Immunofluorescence for DDX4/NOTCH2 in an 18 WPF ovary.

Figure 2.. Human fetal germ cell (hFGC)–syncytia show intercellular bridges and constriction by pre-granulosa cells.

(A) Transmission electron microscopy (TEM) images of intercellular bridges between adjacent hFGCs (top) and hFGCsyncytium (bottom). Dashed boxes are shown magnified to the right. Red arrows point to the mitochondria. Blue arrows point to the intercellular bridge between adjacent hFGCs. (B) Quantification of the diameter of intercellular bridges in TEM images of 17 WPF_a, 18 WPF_a, 18 WPF_b ovaries. Median is shown as the black line. (C) Immunofluorescence for GM130/TJP1/DDX4 in 16, 18, and 20 WPF ovaries (top) and immunofluorescence for TOM20/TP63/DDX4 in 14, 17, and 20 WPF ovaries (bottom). GM130 marks the Golgi complex, TOM20 marks the mitochondria, and DDX4 marks hFGCs. (D) Immunofluorescence for DDX4/KIF23 in 18 WPF ovary (top panels) and adult testis (bottom panels). Dashed boxes are shown magnified to the right. Arrow points to KIF23+ DDX4+ germ cells. (E) Violin plot show the expression (in ln of transcripts per million + 1) of TEX14 in different germline and soma clusters. Each dot is a single cell. (F) RNA FISH for TEX14 combined with immunofluorescence for phosphorylated ezrinradixinmoesin in 18 and 20 WPF ovaries. Yellow dashed lines mark the border of ovarian cords. (G) TEM images displaying normal morphology of multinucleated hFGCs (top panels) and apoptotic cells (bottom panels). Dashed box is shown magnified to the right. Arrows point to the mitochondria. (H) Immunofluorescence for DDX4/CTNNB1/cCASP3 in an 18 WPF ovary. Dashed boxes are shown magnified at the bottom. Arrow points to cCASP3+ DDX4+ hFGC. cCASP3 marks apoptotic cells. (I) Percentage of mononucleated and multinucleated DDX4+ hFGCs in 14–17 and 18–20 WPF ovaries (mean ± SD) in apoptosis (cCASP3+) (mean + SD).

Figure 3.. Characterization of human pre-granulosa and granulosa cells subpopulations.

(A) Transmission electron microscopy images of pre-granulosa cells intercalated between meiotic human fetal germ cells (hFGCs) inside ovarian cords in a 17 WPF ovary. Pre-granulosa cells inside ovarian cords are marked with yellow asterisks. Dashed box is shown magnified at the bottom. (B) Transmission electron microscopy images of pre-granulosa cells constricting meiotic multinucleated hFGCs inside ovarian cords in a 17 WPF ovary. Pre-granulosa cells inside ovarian cords are marked with yellow asterisks. Dashed box is shown magnified at the bottom. Red arrows indicate protrusions of the pre-granulosa cells. (C) Immunofluorescence for FOXL2/VIM/CDH1 in 12 WPF (top panels) and 16 WPF ovaries (bottom panels). Dashed boxes are shown magnified to the right. Arrow points to triple-positive pre-granulosa cells intercalated between hFGCs inside ovarian cords. The stromal compartment and ovarian cords are separated by white dotted lines. FOXL2 marks (pre)granulosa cells. (D) Immunofluorescence for YAP/panKRT/FOXL2 in an 18 WPF ovary. Dashed box is shown magnified at the bottom. The stromal compartment and ovarian cords are separated by white dotted lines. panKRT marks (pre)granulosa cells. (E) Uniform manifold approximation and projections (UMAP) plot showing cluster identity (CL_ID) and age distribution (WPF) of the gonadal somatic cells. (F) UMAP plots depicting the expression levels of genes of interest. (G) Whole-mount immunofluorescence for KRT19/NOTCH3/DDX4 in a 17 WPF ovary. White asterisks mark primordial follicles. Ovarian cords are separated by yellow dotted lines. The merged and single channels are shown. (H) Immunofluorescence for panKRT/JAG1 in 20 WPF (left panel) and UMAP plot showing JAG1 expression in ovarian cells (right panel). Related to Fig S3D.

Figure 4.. Dynamics of cadherin expression during the formation of primordial follicles.

(A) Immunofluorescence for CDH1/CDH2 in 18 and 20 WPF ovaries. (B) Immunofluorescence for CDH1/TP63/DDX4 in a 20 WPF ovary (left panel) and immunofluorescence for CDH1/TP63 in a 2-yr-old ovary (right panel). (C) Quantification of CDH1 and CDH2 expression at the interface between human fetal germ-cells/oocytes and (pre)granulosa cells in 18 and 20 WPF ovaries. Representative images for CDH1/CDH2 in 18 and 20 WPF ovaries, with single fluorescence channels used for quantification (region used depicted as dashed line) (left panels); with associated plots of grey values for CDH1 and CDH2 per cell (middle panels); and plot depicting signal intensity (mean ± SD) (right panel). (***P < 0.001). (D) Confocal images of RNA FISH for CDH1 and CDH2 combined with immunofluorescence for pKRT in ovarian cords at 18 WPF and primordial follicles (PF) at 20 WPF.

Figure 5.. Cell–cell interactions during the formation of primordial follicles.

(A) Quantification of TJP1 expression at the interface between human fetal germ cells (hFGCs)/oocytes and (pre)granulosa cells in 16, 18, and 20 WPF ovaries. Representative images for TJP1/KRT19/DDX4 immunofluorescence used for quantification (region used is depicted by dashed lines) and white arrows point to TJP1+ tight junctions (TJ) (left top panels); with associated plots of grey values for TJP1+ per cell (left bottom panels); and plot depicting signal intensity (mean ± SD) (right panel). (***P < 0.001; ns, not significant). (B) Transmission electron microscopy images showing TJ between the granulosa cells and the oocyte in a primordial follicle at 18 WPF. Dashed box is shown magnified at the bottom. Arrows point to TJ. (C) Immunofluorescence for KRT19/GJA1/FOXL2 in an 18 WPF ovary. Dashed boxes are shown magnified to the bottom. FOXL2 marks (pre)granulosa cells. (D) Transmission electron microscopy images showing cellular interdigitations between the granulosa cells and the oocyte in a primordial follicle at 18 WPF. Dashed box is shown magnified at the right. Arrows point to the interdigitations. (E) Immunofluorescence for panKRT/COLIV/DDX4 in a 20 WPF ovary. DDX4 marks hFGCs and oocytes. (F) Cartoon depicting a model for the transition between ovarian cord and primordial follicle in humans from mononucleated hFGCs and hypothetical for multinucleated hFGCs.