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

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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.