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doi: 10.1242/10.1242/dev.00392

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Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production

¹ Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
² Department of Biochemistry, Akita University School of Medicine, Akita 010-8543, Japan
³ Department of Laboratory Sciences for Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
⁴ Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada
⁵ Amgen Research Institute, Ontario Cancer Institute, and University of Toronto, Toronto, Ontario M5G 2C1, Canada

View larger version (70K): [in a new window]   Fig. 1. Deletion of PTEN in PGCs. Immunohistochemical staining for PTEN was carried out on E15.5 control (A, Ptenflox/–: TNAP/Cre–) and mutant (B, Ptenflox/–: TNAP/Cre+) testes. In control mice, strong cytoplasmic staining was observed in germ cells but the expression level was low in somatic cells. In mutant mice, only background level of expression was observed both in germ and somatic cells. Scale bar: 20 µm.

View larger version (72K): [in a new window]   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.

View larger version (108K): [in a new window]   Fig. 4. Expression of the germ cell markers (Mvh and TRA98) and the immature cell marker (4C9) in the testes of control (A) and Pten mutant (B) mice during embryogenesis. Sections of testes at E11.5, E16.5 and P0 were double-stained with TRA98 (green) and anti-Mvh (red) antibodies. Adjacent or neighboring sections of the same samples were stained with 4C9 (brown) antibody. Similarly, sections of E13.5 gonads were double-stained with anti-Mvh (green) and 4C9 (red) antibodies, and adjacent sections were stained with TRA98 (green) antibody. At E13.5, all 4C9-positive PGCs expressed the germ-lineage-specific marker Mvh. In E16.5 mutant gonads, TRA98-positive teratoma cells, which were Mvh negative or weakly positive for Mvh (arrowheads), and outgrowth of these cells (arrow) were observed. In P0, large teratomas that were Mvh negative, weakly positive for TRA98 and 4C9 positive were observed. Scale bars: 50 µm. At each developmental stage, at least three mice were examined and essentially similar results were obtained.

View larger version (106K): [in a new window]   Fig. 3. Apoptosis in Pten mutant mice. (A) Sections of embryo testes from E13.5, E14.5 and E15.5 control and mutant mice were analyzed by the TUNEL assay. Scale bars: 20 µm. (B) The percentages of apoptotic PGCs at E13.5, E14.5 and E16.5. The percentage of apoptotic cells in Mvh-positive germ lineage cells (mean±s.d.) was calculated and analyzed using Student's t-test (P=0.22 at E13.5, *P<0.01 at E14.5 and P<0.001 at E16.5). Three to six mice were examined at each stage.

View larger version (28K): [in a new window]   Fig. 5. Extra-gonadal PGCs in Pten mutant mice. (A) Route of PGC migration. Transverse section of E12.5 embryos stained for alkaline phosphatase activity. Alkaline phosphatase-positive cells were stained red. The normal migration pathway is indicated by arrows. PGCs become incorporated into the wall of the developing hindgut (gut; E8.5), migrate actively into the dorsal mesentery (dm; E9.5) and then to the genital ridges (E10.5-12.5). The path of `out of route' PGCs is shown by a broken arrow. dm, dorsal mesentery; cw, coelomic wall; gnd, gonad; mes, mesonephros. (B) The percentage and the number of extra-gonadal PGCs at E12.5. The serial transverse sections of whole embryos were stained with alkaline phosphatase substrate as described in A. The number of PGCs in three sections was counted in every case. Ectopic PGCs were also stained with 4C9 antibody for confirmation. The results were analyzed by Student's t-test (*P<0.0025, P<0.01). (C) The distribution of PGCs at E12.5. The values for individual mice are plotted (Student's t-test; *P<0.05, P<0.01, and P<0.005).

View larger version (24K): [in a new window]   Fig. 6. In vitro culture of PGCs from Pten mutant mice. (A) Increased proliferation of E11.5 PGCs from Pten mutant mice. Cell suspensions of E11.5 gonads were seeded on Sl/Sl4-m220 feeder cells that expressed the membrane-bound form of SCF, and cultured in the presence or absence of LIF and bFGF. Changes in PGC number (%) were calculated by dividing the number of PGCs at day 3 by the number of seeded PGCs. Data are mean±s.d. (*P<0.005, P<0.05). (B) EG cell colonies formed in the presence of LIF and bFGF at day 6 after seeding. EG cells were visualized by alkaline phosphatase staining. (C) Increased EG cell production in E11.5 PGCs from Pten mutant mice. Multi-layered colonies that contained more than 20 cells were counted as EG cell colonies, as described previously (Koshimizu et al., 1996). The EG colony-forming efficiency (%) was calculated by dividing the number of EG cell colonies at day 6 by the number of seeded PGCs. The values for individual mice are plotted (P<0.01).

View larger version (64K): [in a new window]   Fig. 7. Differentiation capacity of Pten mutant-derived EG cells. (A) Embryoid bodies with various cell types formed when differentiation was induced in methylcellulose cultures for 8 days. Scale bars: 50 µm. (B) RNA from embryoid bodies was subjected to semi-quantitative RT-PCR analysis to examine the expression of ectodermal (Wnt1), endodermal (collagen IV) and mesodermal (T) markers. ß-Actin was used as a loading control. Expression of all three markers was induced both in control and in mutant EG cell-derived embryoid bodies. (C) Teratomas with differentiated cells that formed when EG cells were transplanted into nude mice. Scale bars:50 µm.

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