Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice [cPRM2]

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete PRM2 incorporation, resulting in a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research. Comparative testis gene expression profiling of cleaved-PRM2 deficient mice compared to WT鱼精蛋白（Protamines）是一类独特的精子特异性蛋白，可在受精前包装并保护父本染色质。部分哺乳动物物种表达两种精蛋白：精蛋白1（PRM1）与精蛋白2（PRM2），而其余物种仅需PRM1即可完成精子染色质包装。PRM1与PRM2的物种特异性比例失衡与不育症密切相关。与PRM1不同，PRM2以前体蛋白形式生成，其包含一段高度保守的N端结构域，被称为切割型精蛋白2（cleaved PRM2，cP2），该结构域会在染色质浓缩过程中被逐步切除。其羧基末端部分称为成熟型精蛋白2（mature PRM2，mP2），可与DNA结合，并与PRM1共同介导染色质高度浓缩。学界普遍认为，切除cP2结构域对于正常染色质浓缩至关重要，但目前对cP2的具体功能仍尚不明确。我们构建了仅缺失cP2结构域、但仍可表达mP2的基因工程小鼠。研究结果表明，cP2结构域对于精子染色质完全鱼精蛋白化以及雄性小鼠的生育能力均不可或缺。缺失cP2的精子存在PRM2整合不全的问题，会导致鱼精蛋白比例严重失衡、过渡蛋白滞留，以及睾丸特异性组蛋白变体H2A.L.2的异常滞留。在附睾转运过程中，缺失cP2的精子似乎会经历活性氧（Reactive Oxygen Species，ROS）介导的降解，最终发生完全性DNA断裂。综上，cP2结构域似乎是精子染色质浓缩过程中，组蛋白、过渡蛋白与鱼精蛋白之间复杂串扰的关键调控节点之一。本研究为解析cP2结构域在父本染色质包装中的功能迈出了关键第一步，同时也为后续相关研究开辟了新方向。我们还开展了切割型PRM2缺陷小鼠与野生型（Wild Type，WT）小鼠的睾丸基因表达谱比较分析。