FIGURE 2.

Characterization of germ cell proliferative state and validation of the receptor–ligand analysis method. (A) Immunofluorescence of MAGEA3, POU5F1, and DDX4 in fetal testis (18 WD), revealing three germ cell types: POU5F1⁺/DDX4^low/MAGEA4− primordial germ cells (PGCs), POU5F1−/DDX4^high/MAGEA4⁺ SGON, and POU5F1−/DDX4^low/MAGEA4− transitory germ cells (TGCs). (B) UMAP visualization of isolated male (left panels) and female (right panels) germ cell (GC) clusters, showing cluster identification, estimated cell cycle (CC) phase, and MKI67 expression. Both male and female PGC show variable CC state and expression of MKI67, reflecting proliferation. However, gonia (GONs) differ between male and female, with MKI67 being expressed in oogonia (OGONs), but not in prospermatogonia (SGONs). (C) Immunofluorescence of MKI67 in fetal testis (18 WD) and ovary (19 WD). A subset of PGCs (POU5F1+) and TGCs (POU5F1−/DDX4^low) shows positive MKI67 staining, whereas SGONs (left panel: POU5F1−/DDX4^Hi, middle panel: MAGEA3⁺/DDX4^Hi) were never MKI67 positive. In fetal ovary, both PGC and premeiotic OGONs MKI67⁺ subsets are present. (D) Overview of identified germ cell subtypes, the markers used for their identification, and their proliferative state. (E) UMAP plots of male and female sets showing expression levels of KIT (in red) and KITLG (in blue). KIT is expressed in PGC clusters and premeiotic OGONs, while KITLG is expressed by GRA1–2 and Sertoli cells (SER). (F) CellphoneDB analysis of KIT–KITLG interaction between different clusters in male (top) and female (bottom) fetal gonads. Depicted are −log₁₀ p-values (circle size) and log₂ means (circle color) for the KIT–KITLG interacting pair, for selected pairwise cluster combinations. In the ovary, a significant KIT–KITLG interaction is observed from GRA to PGC and OGON clusters, while in males, a significant interaction is observed from SER to PGC clusters only. (G) Immunofluorescence of KIT with POU5F1 and DDX4, in PGCs (POU5F1⁺) and GON (POU5F1-) in fetal testis (20 WD) and ovary (18 WD). Consistent with scRNA-seq data, KIT is expressed by PGCs and OGONs, but not SGONs. Yellow boxes indicate zoom-in of a PGC, and cyan boxes indicate zoom-in of a GON. Scale bar is 10 μm.