A novel pathway for the induction of DNA damage in human spermatozoa involving extracellular cell-free DNA

DNA damage is a common feature of human spermatozoa associated with an impaired capacity to fertilize the oocyte and an increased mutational load in the offspring. However, the etiology of this damage remains poorly defined. In this study we demonstrate that a major pathway for the induction of DNA damage in mammalian spermatozoa is triggered by exposure to exogenous cell free DNA (cfDNA). Exposure of human and mouse spermatozoa to cfDNA (calf thymus, mouse liver and salmon testes) in vitro induced a dose-dependent increase in sperm DNA damage that could be effectively suppressed by the concomitant presence of DNase. The induction of such damage was not accompanied by any concomitant change in sperm motility or vitality and was not directly associated with the induction of oxidative stress. In vivo the injection of exogenous DNA again precipitated an increase in sperm DNA fragmentation that could be reversed by the prior administration of DNase. Similarly, the induction of a transient unilateral testicular ischemia induced an increase in DNA fragmentation that was evident within 24 h and sustained for at least 14 days via mechanisms that could be completely suppressed by the prior administration of DNase. We conclude that exogenous cfDNA activates a defensive response in human spermatozoa associated with the nuclease-mediated induction of DNA fragmentation, possibly involving the participation of TLR9 and CD4. These novel insights have significant implications for our understanding of DNA fragmentation in the male germ line and open up new pathways for the remediation of this condition.