Fig. 2. (A) Hematoxylin and Eosin (HE)-stained sections of P0 testes. All Pten mutant mice developed bilateral teratomas with multiple foci (n=9). ec, ectodermal vesicle; ed, endodermal vesicle; ud, undifferentiated cells. Scale bar: 20 µm. (B) Immunostaining with anti-phospho-Akt antibody (top) showed hyper-phosphorylation of Akt in germ cells and early teratoma foci in E16.5 mutant mice. The same sections were stained with propidium iodide (PI) (bottom) and the morphology of nuclei was examined by confocal microscopy at 0.8 µm optical sections (arrowheads in a-c). Some of the hyper-phosphorylated cells had pyknotic nuclei (a) and mitotic figures (b,c). Scale bars: 10 µm. (C) Sections of E14.5 testes were double-stained with anti-Mvh antibody and PI, and analyzed by confocal microscopy at 0.8 µm optical sections. Mitotic figures were observed in Pten mutant PGCs (arrowheads). Scale bars: 5 µm. (D) The percentage of mitotic PGCs at E13.5, E14.5 and E16.5 in control and mutant mice. The sections were stained with PI and anti-Mvh antibody. The percentage of mitotic cells in Mvh-positive cells (mean±s.d.) was calculated and analyzed by Student's t-test (P=0.48 at E13.5, *P<0.01 at E14.5 and P<0.002 at E16.5). Three to six mice were examined at each stage.