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

摘要

The present study was designed to detect DNA repair response through the homologous recombination pathway in mouse spermatogonial stem cells. Mouse spermatogonial stem cells (mSSCs) were obtained from the adult DBA/2 mouse testes by MACS sorting. mSSCs and mice animals were divided into four groups (30min, 2, 24h, control) and treated with ionizing irradiation while the control group received pseudo-irradiation. Proteins involved in the homologous recombination pathway (H2AX, ATM, RAD51, CtIP, and RPA2) were assessed in mSSCs both invitro and invivo. Moreover, the non-homologous end-joining or homologous recombination (NHEJ/HR) reporter plasmids were transfected into mSSCs to assess NHEJ/HR pathway activity after DNA double-strand break (DSB). H2AX, a classical DNA DSB marker, was absent in mSSCs both invivo and invitro 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 mSSCs. p-ATM foci were overexpressed immediately after irradiation (30min and 2h), but gradually decreased over the repair time. The HR pathway-related proteins, CtIP and RPA2 were negatively regulated after treatment in Western blot (WB). NHEJ/HR reporter plasmid transfection indicated that the HR pathway played a minor role in mSSCs during DDR, consistent with the WB findings. This study demonstrates that mSSCs 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 H2AX, was increased after DSB, suggesting that DNA damage repair in mSSCs might be a H2AX-independent response. Furthermore, the HR pathway may play a minor role during DDR in mSSCs.