Stem cell defects in ATM-deficient undifferentiated spermatogonia through DNA damage-induced cell-cycle arrest.
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 p19(Arf)-p53-p21(Cip1/Waf1) pathway were observed in Atm(-/-) undifferentiated spermatogonia. Moreover, suppression of p21(Cip1/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.
Takubo
Keiyo
K
Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. keiyot@gmail.com
Ohmura
Masako
M
Azuma
Masaki
M
Nagamatsu
Go
G
Yamada
Wakako
W
Arai
Fumio
F
Hirao
Atsushi
A
Suda
Toshio
T
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000235:genetics; D005434:Flow Cytometry; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000172:deficiency* / Q000235:genetics
D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases