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First published online 11 April 2007
doi: 10.1242/dev.003004

Development 134, 1853-1859 (2007)
Published by The Company of Biologists 2007
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Akt mediates self-renewal division of mouse spermatogonial stem cells

Jiyoung Lee1, Mito Kanatsu-Shinohara1,2, Kimiko Inoue3, Narumi Ogonuki3, Hiromi Miki3, Shinya Toyokuni4, Tohru Kimura5, Toru Nakano5, Atsuo Ogura3 and Takashi Shinohara1,*

1 Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
2 Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
3 The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki 305-0074, Japan.
4 Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
5 Department of Pathology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.


Figure 1
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  		Fig. 1. Effect of GDNF on GS cell proliferation. (A) Effect of GDNF on cell recovery after 6 days in culture on MEFs (left) or laminin (right) (mean±s.e.m.; n=5). (B) Appearance of wild-type (left and center) and myr-Akt-Mer (right) GS cells, 6 days after passage on laminin. (C) Apoptosis of GS cells in the absence of GDNF. Apoptotic cells were detected by TMR red staining. Apoptotic cells were observed when GDNF was removed from the culture medium. (D) Analysis of cell cycle distribution, 5 days after passage on laminin. Significantly more cells are in the S phase in the presence of GDNF or 4OHT. (E) Growth curve for myr-Akt-Mer GS cells that were maintained by 4OHT on laminin. (F) Effect of EGF and bFGF on cell recovery after 5 days of culture on laminin (mean±s.e.m.; n=5). Whereas the wild-type GS cells can grow in the presence of EGF, myr-Akt-Mer GS cells are unable to respond to EGF. Scale bars: in B,C, 100 µm.

 

Figure 2
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  		Fig. 2. Inhibition of GDNF-induced proliferation by LY294002. Cells were cultured in the presence of 33 µM LY294002 or 25 µM PD98059. (A) LY294002 decreases cell recovery after 6 days in culture on MEFs (mean±s.e.m.; n=5). (B) Analysis of cell cycle distributions, 5 days after passage on laminin in the presence of 33 µM LY294002 (left) or 25 µM PD98059 (right). Significantly more of the cells are in the G1 phase in the presence of LY294002. (C) Appearance of GS cells that were cultured on MEFs in the presence of 33 µM LY294002. Whereas control untreated cells form large colonies within 6 days (left), only small colonies are found in the presence of LY294002 (middle). However, these small colonies reinitiate growth after removal of LY294002 and form large colonies, 4 days after supplementation with fresh medium (right). (D) Quantification of spermatogonial stem cells in culture by germ cell transplantation. The same number of GS cells (2.5x105 cells per six-well plate) was cultured on MEFs for 6 days with or without 33 µM LY294002, and the total number of spermatogonial stem cells was quantified by transplanting 5x104 cells into the seminiferous tubules of infertile recipient testes. The total numbers of stem cells were estimated by multiplying the total cell recovery by the stem cell concentration in the injected cell suspension. Results from three independent experiments (mean±s.e.m.; n=9 for control, and n=13 for LY294002). Scale bar: in C, 100 µm.

 

Figure 3
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  		Fig. 3. GDNF signaling in wild-type and myr-Akt-Mer GS cells. (A) Phosphorylation of Akt and Gsk3ß in wild-type GS cells. GS cells on laminin were starved for 4 days, and then left untreated or treated with GDNF. Where indicated, cells were also incubated for 45 minutes with 33 µM LY294002 before the addition of GDNF. (B) Phosphorylation of Akt and Gsk3ß in myr-Akt-Mer GS cells. The myr-Akt-Mer GS cells on laminin were cultured with GDNF or indicated concentrations of 4OHT for 6 days. White and black arrowheads represent endogenous Akt and myr-Akt-Mer, respectively. (C) Phosphorylation of MAPK after GDNF or 1 µM 4OHT stimulation. (D) Expression of cyclin molecules after GDNF or 1 µM 4OHT stimulation.

 

Figure 4
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  		Fig. 4. Phenotypic analysis of GS cells. (A) Characterization of cell surface antigens by flow cytometry. Red shading, specific antibody; unshaded, unstained control. (B) RT-PCR analysis of GS cells. GS cells on laminin were cultured for 9 days in the indicated conditions. (C) Suppression of Neurog3 transcript expression by GDNF/4OHT identified by real-time PCR analysis (mean±s.e.m.; n=3). Expression of Neurog3 was enhanced in both wild-type and myr-Akt-Mer GS cells in the absence of GDNF.

 

Figure 5
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  		Fig. 5. COBRA of wild-type and myr-Akt-Mer GS cells (left) and offspring derived from myr-Akt-Mer GS cells (right). GS cells on laminin were cultured for 6 days in the indicated conditions. The PCR products of each DMR region were digested with the indicated restriction enzymes with recognition sequences containing CpG in the original unconverted DNA. Black and white arrows indicate the sizes of the unmethylated and methylated DNA fragments, respectively. Levels of percentage methylation, as estimated by the intensity of each band, are indicated below the gels. C, cleaved; U, uncleaved.

 

Figure 6
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  		Fig. 6. Germline transmission of myr-Akt-Mer GS cells. (A) A recipient testis with EGFP fluorescence in the GS cell-derived germ cell colonies. Arrows indicate the green fluorescence of donor cell colonization. (B) Histological appearance of a recipient testis that underwent germ cell transplantation showing round spermatids. (C) Offspring derived from the microinjection of oocytes with round spermatids, showing fluorescence. (D) PCR analysis of tail DNA samples from the offspring. The Neo transgene is detected in two of the four offspring. Scale bars: in A, 500 µm; in B, 50 µm.

 

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© The Company of Biologists Ltd 2007