Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice [PRM1]

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2), are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing we generated Prm1-deficient mice and demonstrate, that Prm1+/- mice are subfertile while Prm1-/- are infertile. Prm1-/- and Prm2-/- sperm show high levels of reactive oxygen species (ROS)-mediated DNA damages and increased histone retention. In contrast, Prm1+/- sperm display only moderate DNA damages. The majority of Prm1+/- sperm were CMA3 positive indicating protamine-free DNA. This is not due to increased histone retention in Prm1+/- sperm. Additionally, we found that sperm from Prm1+/- and Prm1-/- mice contain high levels of incompletely processed PRM2. Further, the PRM1:PRM2 ratio is skewed from 1:2 in WT to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce the mature PRM2 which, together with PRM1 is able to hypercondense DNA. Hence, the species specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility. Comparative testis gene expression profiling of PRM1 deficient mice compared to WT精子形成（spermiogenesis）过程中的核心事件之一，是通过以鱼精蛋白替换绝大多数组蛋白，实现染色质的高度浓缩。在人类与小鼠中，鱼精蛋白1（PRM1/Prm1）与鱼精蛋白2（PRM2/Prm2）的表达呈现物种特异性比例。本研究采用CRISPR-Cas9介导的基因编辑技术构建了Prm1基因缺陷型小鼠，实验证实Prm1+/-小鼠表现为亚不育，而Prm1-/-小鼠完全不育。Prm1-/-与Prm2-/-小鼠的精子可检测到高水平的活性氧（ROS）介导的DNA损伤，且组蛋白滞留现象显著增加；与之相对，Prm1+/-小鼠的精子仅存在轻度DNA损伤。多数Prm1+/-小鼠的精子呈CMA3阳性，提示其DNA缺乏鱼精蛋白包裹，但该现象并非源于Prm1+/-精子中组蛋白滞留水平升高。此外，我们发现Prm1+/-与Prm1-/-小鼠的精子中存在大量未完全加工的PRM2。进一步研究显示，Prm1+/-小鼠体内的PRM1:PRM2比例从野生型（Wild Type, WT）的1:2偏移至1:5。本研究结果表明，PRM1对于PRM2的正常加工以生成成熟PRM2至关重要；成熟PRM2可与PRM1协同实现染色质高度浓缩。因此，必须精准调控物种特异性的PRM1:PRM2比例，方能维持完整的生育能力。本研究对PRM1缺陷型小鼠与野生型小鼠开展了睾丸基因表达谱对比分析。