Prm2 deficiency triggers a Reactive Oxygen Species (ROS)-mediated destruction cascade during epididymal sperm maturation in mice

Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Prm2-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm`s antioxidant capacity, indicated by downregulation of key proteins like SOD1 and PRDX5, ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination. Testicular mRNA of wildtype, Prm2-deficient and Prm2-heterozygous mice鱼精蛋白（Protamines）是父本精子基因组的保护屏障。在精子形成（spermiogenesis）过程中，鱼精蛋白会替换大部分组蛋白（histones），使DNA高度浓缩，从而保护基因组免受环境有害因子的侵害。多项研究已证实，鱼精蛋白替换异常与小鼠和人类的雄性不育密切相关。除DNA完整性受损外，鱼精蛋白缺失的人类及小鼠精子还会出现多种继发效应，包括运动能力下降与头部形态异常。本研究采用Prm2（Protamine 2）缺失型小鼠模型结合无标记定量蛋白质组学（label-free quantitative proteomics），解析上述效应的潜在分子机制。研究发现，精子抗氧化能力丧失，具体表现为超氧化物歧化酶1（SOD1）、过氧化物还原酶5（PRDX5）等关键蛋白的下调，最终会在附睾精子成熟阶段触发氧化应激介导的损伤级联反应；附睾精子中8-羟基脱氧鸟苷（8-OHdG）这一氧化应激介导DNA损伤的生物标志物水平升高，也验证了这一结论。Prm2缺失型睾丸精子未受上述影响，且在体外通过卵胞浆内单精子注射（intracytoplasmic sperm injection, ICSI）注入卵母细胞后，可正常发育至囊胚期植入前胚胎。本研究为阐明Prm2的功能及其下游分子对精子功能的调控机制提供了新视角，同时也为鱼精蛋白替换异常的不育男性探索新型治疗方案提供了重要参考。野生型、Prm2缺失型及Prm2杂合型小鼠的睾丸mRNA