Fig 2: Collagen IV and laminin granules in human fetal germ cells. Collagen IV (A,C) and laminin (B,D) granules (in green) in the cytoplasm of germ cells (red) in both developing female first trimester (W10.5) and second trimester (W17) gonads. The same secondary antibody was used to detect OCT4 and VASA, both germ cell markers, because the two proteins localize to different cellular compartments (nucleus and cytoplasm, respectively) and their expression can therefore be clearly distinguished. 3D reconstructions from high magnification confocal z-projections from two different angles show that these granules are present throughout the cytoplasm of the germ cells. Scalebar is 2.5 µm.

Fig 3: Dynamics of oocyte reserve depletion and regeneration in adult female pStra8-HSVtk mice following GCV exposure and removal in vivo. (a) Number of primordial oocyte-containing follicles in ovaries of young adult pStra8-HSVtk mice following 21 days of treatment with vehicle (PBS) or GCV (10 mg kg-1; completed on postpartum day 69), or 21 days after cessation of GCV treatment (Post-GCV, completed on postpartum day 90). Data represent the mean ± s.e.m. (n = 4–6 mice per group; *P < 0.05). (b) Number of atretic (dying, dead) oocytes in ovaries of young adult pStra8-HSVtk mice treated with vehicle (PBS) or GCV (10 mg kg-1) for up to 21 days (mean ± s.e.m., n = 4–6 mice per group). (c) Stra8 mRNA levels (normalized against ß-actin mRNA levels) in ovaries contralateral to those used to derive the oocyte counts shown in panel a (mean ± s.e.m., n = 4–6 mice per group; *P < 0.05). (d) Yield of FACS-purified OSCs (percent of total viable cells sorted) from ovaries of young adult pStra8-HSVtk mice following 21 days of treatment with vehicle (PBS) or GCV (10 mg kg-1). Data represent the mean ± s.e.m., n = 3 mice per group. (e) Sycp3 expression in ovaries of young adult pStra8-HSVtk mice 21 days after ceasing treatment with vehicle (PBS) or GCV (10 mg kg-1). Data represent the mean ± s.e.m., n = 3–4 mice per group; *P < 0.05. (f) Number of Ddx4-positive germ cells with punctate nuclear localization of Sycp3 protein in ovaries contralateral to those used for panel e (mean ± s.e.m., n = 3 mice per group; *P < 0.05). (g) Immunofluorescence images of Ddx4-positive germ cells (blue) co-expressing Sycp3 (red) and Ser139-phospho-H2afx (green) in ovaries of young adult pStra8-HSVtk mice 21 days after ceasing a 3-week course of GCV treatment (10 mg kg-1). DAPI nuclear stain, white; scale bars, 10-µm.

Fig 4: Germ cells in second trimester human fetal adrenals. Histological sections of human adrenals of second trimester male (W22) and female (W20.5). (A,B) Germ cells were identified by expression of POU5F1 (red) and/or DDX4 (green). (C,D) DDX4+ (green) germ cells do not express H2AFX (red), however, H2AFX+ cells were present in the adrenals (white arrows). (E,F) DDX4+ (red) germ cells were not TUNEL-positive (green), however TUNEL+ cells were present in the adrenals (white arrows). Note the presence of autofluorescent red blood cells. Some germ cells are highlighted in dotted boxes. Magnified views of dotted boxes are shown as inserts and the insert number corresponds to its location. Scale bars are 100 µm in A-F and 20 µm in all inserts.

Fig 5: Germ cells in first trimester human fetal adrenals. Histological sections of human adrenals (and adjacent kidney) in first trimester male (W10) and female (W13). (A,B) Germ cells were identified by expression of POU5F1 (red) and/or DDX4 (green). (C,D) DDX4+ (green) germ cells do not express H2AFX (red), however, many H2AFX+ cells were present in the adrenals (white arrows). (E,F) DDX4+ (red) germ cells were not TUNEL-positive (green), however, many TUNEL+ cells were present in the adrenals (white arrows). Note the presence of autofluorescent red blood cells. Some germ cells are highlighted in dotted boxes. Magnified views of dotted boxes are shown as inserts and the insert number corresponds to its location. Scale bars are 200 µm in A,B,E,F, 100 µm in C,D and 20 µm in all inserts.