Preliminary study of the homologous recombination repair pathway in mouse spermatogonial stem cells

Summary The present study was designed to detect DNA repair response through the homologous recombination pathway in mouse spermatogonial stem cells. Mouse spermatogonial stem cells ( mSSC s) were obtained from the adult DBA /2 mouse testes by MACS sorting. mSSC s and mice animals were divided into four groups (30 min, 2, 24 h, control) and treated with ionizing irradiation while the control group received pseudo‐irradiation. Proteins involved in the homologous recombination pathway (γH2 AX , ATM , RAD 51, Ct IP , and RPA 2) were assessed in mSSC s both in vitro and in vivo. Moreover, the non‐homologous end‐joining or homologous recombination ( NHEJ / HR ) reporter plasmids were transfected into mSSC s to assess NHEJ / HR pathway activity after DNA double‐strand break ( DSB ). γH2 AX , a classical DNA DSB marker, was absent in mSSC s both in vivo and in vitro after DSB repair, but was highly expressed in other tissue stem cells. In addition, ATM and phosphorylated ATM (p‐ ATM ) were involved in DNA damage response ( DDR ) in mSSC s. p‐ ATM foci were overexpressed immediately after irradiation (30 min and 2 h), but gradually decreased over the repair time. The HR pathway‐related proteins, Ct IP and RPA 2 were negatively regulated after treatment in Western blot ( WB ). NHEJ / HR reporter plasmid transfection indicated that the HR pathway played a minor role in mSSC s during DDR , consistent with the WB findings. This study demonstrates that mSSC s may have a unique response to DNA damage since crucial proteins involved in HR pathway were negatively regulated after DSB . In addition, the expression level of p‐ ATM , but not γH2 AX , was increased after DSB , suggesting that DNA damage repair in mSSC s might be a γH2 AX ‐independent response. Furthermore, the HR pathway may play a minor role during DDR in mSSC s.