Analyses of the consequences of a protamine-2-deficiency in mouse and man: molecular mechanisms, reactive oxygen species and an approach to therapy.

使用CRISPR/Cas9，我们在第一次授权期间建立并分析了鱼精蛋白-2缺陷小鼠系。该结果与早期的出版物相反，后者声称鱼精蛋白-2的一个等位基因的丢失会导致不育。因此，我们首先建立了小鼠品系，从而可以详细研究精蛋白-2在精子发生过程中对单倍体基因表达和染色质组织的基本调节中的作用。它代表了一个理想的模型系统，以更好地了解和最终治疗的后果，改变鱼精蛋白1/2比例在生育力低下的男子。最初的质谱分析显示，这种精子显示出控制活性氧（ROS）水平所需的蛋白质不平衡。例如，过氧化氢酶在Prm-2缺陷的精子中检测不到。因此，DNA、膜和蛋白质的进行性损伤可能是氧化应激的结果。这一假说的研究不仅与基础研究相关，而且可能为治疗不育男性开辟新的途径。在下一个资助期，我们建议建立一个睾丸-器官培养系统，可以详细分析ROS水平及其与精子完整性破坏的相关性。接下来，将用缓冲ROS水平的抗氧化剂处理培养物，以确定观察到的缺陷在多大程度上是由ROS引起的。我们预计，我们将能够减少DNA损伤，膜和蛋白质的缺陷。然后，我们将测试这种只有鱼精蛋白1的精子是否能够使卵子受精。这将通过IVF，ICSI或ROSI（圆形精子细胞注射）完成。这将是有趣的，看看鱼精蛋白到组蛋白的替代动力学，进一步，这些鸡蛋是否能够启动和完成胚胎发生。此外，我们有第一个数据表明，Prm-2缺陷精子显示组蛋白翻译后修饰模式的改变。这种修改被讨论参与表观遗传，并将在拟议的项目中进行更详细的分析。此后，将这些数据与生育力低下男性的情况进行比较。拟议的实验建立在第一个赠款期的试剂、工具和实验的基础上，第一个赠款期的申请和赠款仅为24个月。由于两个申请人之间的高效和富有成效的合作，仅在16个月后就可以发表Prm-2缺陷小鼠。

Using CRISPR/Cas9 we established and analyzed a Protamine-2-deficient mouse line during the first grant period. The results are in contrast to earlier publications, which claim that loss of one allele of Protamine-2 leads to infertility. Hence, we were first in establishing mouse lines, which allow studying in detail the role of Protamine-2 in basic regulation of haploid gene-expression and chromatin-organization during spermiogensis. It represents an ideal model system to better understand and eventually treat the consequences of an altered Protamine1/2 ratio in subfertile men. An initial Mass Spec analysis revealed, that such sperm displays an imbalance in the proteins necessary to control the level of reactive oxygen species (ROS). For example, Catalase is not detectable in Prm-2-deficent sperm. Thus, the progressive damage of DNA, membranes and proteins could be a consequence of oxidative stress. Research on this hypothesis is not only relevant for basic research, but could also open up new avenues to treat subfertile men, who show altered Protamine1/2 ratio.For the next grant period, we propose to set up a testes-organ culture system, which allows for a detailed analysis of ROS-levels and correlation to destruction of sperm integrity. Next, the culture will be treated with anti-oxidants buffering the ROS levels in order to determine, to which extent the defect observed is due to ROS. We anticipate, that we will be able to reduce the DNA damage, the defects of the membranes and the proteins. Then, we will test, whether such -Protamine-1-only- sperm is able to fertilize eggs. This will be done using IVF, ICSI or ROSI (round spermatid injection). It will be interesting to see the dynamics of Protamine-to-Histone replacement and, further, whether such eggs are able to initiate and complete embryogenesis. Further, we have first data indicating that Prm-2-deficient sperm shows alterations in the pattern of post-translational Histone modifications. Such modifications are discussed to be involved in epigenetic inheritance and will be analyzed in more detail in the proposed project. Thereafter, the data will be compared to the situation in subfertile men. The proposed experiments built on the reagents, tools and experiments from the first grant period, which was requested and granted for 24 months only. Due to the efficient and fruitful collaboration between the two applicants, publication of the Prm-2 deficient mouse was possible after 16 months only.