TY - JOUR
T1 - Stem Cell Defects in ATM-Deficient Undifferentiated Spermatogonia through DNA Damage-Induced Cell-Cycle Arrest
AU - Takubo, Keiyo
AU - Ohmura, Masako
AU - Azuma, Masaki
AU - Nagamatsu, Go
AU - Yamada, Wakako
AU - Arai, Fumio
AU - Hirao, Atsushi
AU - Suda, Toshio
N1 - Funding Information:
The observation of accumulated DNA damage in Atm −/− undifferentiated spermatogonia led us to investigate involvement of the p19 Arf -p53 pathway, which along with the p16 Ink4a -pRb pathway ( Sherr, 2004 ) is a critical tumor suppressor cascade. The p19 Arf -p53 pathway is activated by various oncogenic cellular stresses, including Ras activation, to actuate a cell-cycle checkpoint and apoptosis. Recently, it was reported that loss of genomic integrity also activates p19 Arf -p53 signals ( Colombo et al., 2005 ). We previously examined p16 Ink4a activation in Atm −/− testis and found that it was not activated ( Takubo et al., 2006 ). Therefore, we used immunohistochemistry to analyze the p19 Arf -p53 pathway. In wild-type seminiferous tubules, p19 Arf protein was only observed in spermatogonia, particularly in Plzf-positive undifferentiated spermatogonia—shown as punctate nuclear staining ( Figure 5 C) localized at nucleoli ( Figures 5 F and 5G). In contrast, Plzf-positive Atm −/− undifferentiated spermatogonia showed intense p19 Arf staining throughout the nucleus ( Figures 5 C and 5E). Increased p19 Arf expression was supported by quantitative PCR of isolated undifferentiated spermatogonia ( Figure 5 D). Although p19 Arf protein typically localizes in the nucleolus, activation of p19 Arf changed its localization to regions outside the nucleolus ( Olson, 2004 ). In Atm −/− undifferentiated spermatogonia, p19 Arf protein distributed throughout the nucleus ( Figures 5 E and 5F), which gave additional evidence of the activation of p19 Arf . p19 Arf protein outside the nucleolus stabilizes p53 by suppressing mdm2, an E3 ubiquitin ligase facilitating p53 ubiquitination and degradation ( Sherr, 2004 ). Correlated with p19 Arf activation in undifferentiated spermatogonial nuclei, p53 protein was specifically evident in Plzf-positive undifferentiated spermatogonia, as revealed by flow cytometry ( Figure 5 H) and immunocytochemistry ( Figure 5 I). These data support activation of p19 Arf -p53 tumor suppressor mechanisms in undifferentiated spermatogonia and provide evidence for undifferentiated germ cell-specific degeneration mechanisms in Atm −/− mice. Our observation is unanticipated because ATM acts as an essential activator of p53 directly and indirectly to induce cell-cycle checkpoint responses ( Shiloh, 2003 ).
Funding Information:
We thank Drs. P. J. McKinnon, P. Leder, and M. Okabe for transgenic mice. We also thank Drs. H. Saya, T. Kitamura, and A. Iwama for fruitful discussions, T. Ogawa for critical reading, and Ms. A. Kumakubo, Ms. A. Ono, and Ms. T. Hirose for essential support. K.T. is a research fellow of the Japan Society for the Promotion of Science. A.H. was supported by grants-in-aid from the Stem Cell Research of the Ministry of Education, Science, Sports, and Culture, Japan, and a CREST grant by the JST. T.S. was supported by a grant-in-aid from the Specially Promoted Research of the Ministry of Education, Science, Sports, and Culture, Japan.
PY - 2008/2/7
Y1 - 2008/2/7
N2 - Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm-/- mice. Atm-/- testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm-/- undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19Arf-p53-p21Cip1/Waf1 pathway were observed in Atm-/- undifferentiated spermatogonia. Moreover, suppression of p21Cip1/Waf1 in an Atm-/- background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.
AB - Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm-/- mice. Atm-/- testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm-/- undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19Arf-p53-p21Cip1/Waf1 pathway were observed in Atm-/- undifferentiated spermatogonia. Moreover, suppression of p21Cip1/Waf1 in an Atm-/- background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.
KW - STEMCELL
UR - http://www.scopus.com/inward/record.url?scp=38649099103&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=38649099103&partnerID=8YFLogxK
U2 - 10.1016/j.stem.2007.10.023
DO - 10.1016/j.stem.2007.10.023
M3 - Article
C2 - 18371438
AN - SCOPUS:38649099103
VL - 2
SP - 170
EP - 182
JO - Cell Stem Cell
JF - Cell Stem Cell
SN - 1934-5909
IS - 2
ER -