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.

DNA损伤是人类精子的普遍特征，与卵母细胞受精能力受损和后代突变负荷增加有关。但是，这种损害的病因学仍然不清楚。在这项研究中，我们证明了通过暴露于外源无细胞DNA（cfDNA）引发哺乳动物精子中DNA损伤的主要途径。体外将人和小鼠的精子暴露于cfDNA（小牛胸腺，小鼠的肝和鲑鱼的睾丸）会引起精子DNA损伤的剂量依赖性增加，这可以通过同时存在DNase来有效地抑制。这种损伤的诱导并没有伴随着精子活动力或活力的任何变化，并且与氧化应激的诱导没有直接关系。在体内，外源DNA的注射再次促使精子DNA片段化的增加，这可以通过事先施用DNase来逆转。类似地，短暂性单侧睾丸局部缺血的诱导会导致DNA片段化的增加，这种增加在24小时内是明显的，并且至少持续14天。预先给予DNase可以完全抑制的机制。我们得出结论，外源cfDNA激活人类精子中的防御反应，与核酸酶介导的DNA片段化相关，可能涉及TLR9和CD4的参与。这些新颖的见解对我们对雄性种系中DNA片段化的理解具有重要意义，并为补救这种状况开辟了新途径。