Figure 1.
Developmental characteristics of 129T2/SvJ mouse gonads.
A. H&E stained cross section of a teratoma collected from an adult 129T2/SvJ mouse testis. RTC: residual testis cords. B. Bright field images showing hindlimb morphology of typical E12.5 129T2/SvJ and CD1 embryos (Left images). Immunofluorescent staining of E12.5 fetal testis sections from 129T2/SvJ and CD1 mice. AMH is shown in green, while MVH is shown in red. DAPI (blue) marks the cell nuclei. Scale bars; 10 um. C. Immunofluorescence showing the typical extent of the germ cell populations marked by MVH staining (red) enclosed by testis cords in E15.5 129T2/SvJ, C57Bl/6 and CD1 fetal testes (a further four independent individuals are shown in Figure S1). DAPI (blue) marks the cell nuclei. Scale bars; 200 um. D. Quantification of the gonad area, total cord number, average cord area, number of germ cells per cord area, the number of germ cells per cord and the number of germ cells per section for whole testis sections taken from E15.5 129T2/SvJ, C57Bl/6 and CD1 embryos (Data represents +/−SEM, eight-ten sections for at least 4 testes). Statistical differences compared to 129T2/SvJ were calculated: * indicates p<0.05, ** indicates p<0.01.
Figure 2.
Fluorescence activated flow cytometric analysis of S-phase progression and cell cycle state in teratoma susceptible and non-susceptible fetal mouse germ cells.
(A) (i) Typical example of E12.5-E15.5 129T2/SvJ germ cells analysed for EdU incorporation after 2 hours of in-vivo exposure to EdU. MVH positive cells were isolated against an MVH stained limb control (MVH negative) sample ([14], not shown) while the EdU gate was set against an EdU negative control. (ii) ModFit cell cycle analysis based on DNA content assessed by propidium iodide staining in the same germ cell populations as shown in (i). (B) Germ cell proliferation based on EdU incorporation in E12.5-E15.5 129T2/SvJ, C57Bl/6 and CD1 fetal gonads. Data is represented by 3-6 biological replicates for each time point and strain analysed. Error bars represent standard deviation. (C–F) ModFit analysis of germ cell cycle state, based on propidium iodide staining for DNA content in E12.5-E15.5 129T2/SvJ, C57Bl/6 and CD1 fetal gonads. C: percentage cells in G0/G1 for the 129T2/SvJ, C57Bl/6 and CD1 strains, D–F: percentage of cells in each cell cycle stage for 129T2/SvJ, CD1 and C57Bl/6. Data is represented by 3–6 biological replicates +/− standard deviation for each time point and strain analysed.
Figure 3.
Immunofluorescent staining of cell cycle regulators, KI-67 and retinoblastoma (RB) in E13.5–15.5 fetal testis sections from 129T2/SvJ mice.
(A) KI-67 (green) (B) Total RB (green, E13.5 only shown) (C) phosphorylated RB (green, E13.5 and E14.5 shown), (D) p15INK4b, (E) p16INK4a and (F) p27KIP1 (G) an example of the few cords showing retained expression of phosphorylated RB and KI67 at E14.5. MVH (red) marks the germ cells and DAPI (blue) marks the cell nuclei. Scale bars; 10 um for A–F, 50 um for G.
Figure 4.
Male fetal germ cell differentiation markers are activated as 129T2/SvJ germ cells enter mitotic arrest.
Immunofluorescent staining of (A) NANOS2 (green), (B) DNMT3L (green), and (C) MILI (red) in E13.5–15.5 fetal testis sections from 129T2/SvJ mice. The high power image (HP) (A, far right) shows enrichment of NANOS2 protein to a cytoplasmic body that is largely MVH negative and has a NANOS2 negative core. MVH (red) marks the germ cells in A and B while GFP marks the germ cells in C. DAPI (blue) marks the cell nuclei. Scale bars; 10 um.
Figure 5.
The core pluripotency proteins OCT4 and SOX2 are down-regulated as 129T2/SvJ germ cells enter mitotic arrest.
Immunofluorescent staining of (A) OCT4 and (B) SOX2 in E13.5–15.5 fetal testis sections from 129T2/SvJ mice. MVH (red) marks the germ cells and DAPI (blue) marks the cell nuclei. Scale bars; 10 um.
Figure 6.
DPPA2 is depleted, while DPPA4 and FGFR3 are enriched in the nuclei of differentiating male germ cells.
Immunofluorescent staining of (A) DPPA2, (B) DPPA4 and (C) FGFR3 in E13.5–15.5 fetal testis sections from 129T2/SvJ mice. MVH (red) marks the germ cells and DAPI (blue) marks the cell nuclei. Scale bars; 10 um.
Figure 7.
Schematic representation of early male fetal germ cell development.
Only mice of the 129T2/SvJ strain form germ cell derived teratomas. Testis determination is initiated by SRY, which activates SOX9 and AMH. Testis cords are formed by E12.5. Germ cells are committed to male development at around E12.5 and enter mitotic arrest between E12.5 and E14.5. This is moderately strain dependent, with germ cells of the outbred CD1 strain entering mitotic arrest slightly earlier than those for the inbred 129T2/SvJ and C57Bl/6 strains. During mitotic arrest the G1 cyclins (particularly Cyclin E1 and Cyclin E2) are repressed, the G1-S cell cycle inhibitors p27KIP1, p15INK4b, and p16INK4a are upregulated and pRB is activated. Expression of male germ cell differentiation markers including NANOS2, MILI, DNMT3L and FGFR3 increases between E12.5 and E14.5, while the core pluripotency markers OCT4, SOX2 and NANOG are repressed. DPPA2 and DPPA4 exhibit reciprocal expression patterns in E12.5-E15.5 129T2/SvJ male germ cells. This scheme summarises data from the present study, and references 10, 26 and 27.
Table 1.
Primary Antibodies.