The sperm LINC complex: The LINK between chromatin integrity, sperm motility and male fertility

在精子发生过程中，组蛋白-鱼精蛋白交换之后是染色质超凝集，这是精子质量控制的关卡，因为不完全的组蛋白替换和/或异常的鱼精蛋白-1(PRM1)/鱼精蛋白-2(PRM2)比例与男性不育有关。在过去的几年里，精子染色质发展成为生殖医学的一个新兴领域。我们能够证明PRM2缺陷的雄性小鼠是不育的，这是由于碎裂的精子DNA水平高度升高和完全静止的精子(Schneider等人。2016)。这一观察结果，加上以下事实：(1)已经产生了第二个缺乏PRM1的小鼠品系，(2)已知来自不育男性的精子显示出异常的PRM1和PRM2比率，这构成了拟议的DFG-GACR项目的基础，在该项目中，两位申请者都结合了他们的专业知识，并汇集了他们的实验室资源，以便更详细地研究从染色质到细胞骨架(运动性)的信号转导。申请人假设鱼精蛋白表达减少(不育男性、杂合子小鼠)或缺失(纯合子小鼠)，随后是染色质不完全凝聚，最终导致组蛋白翻译后修饰(PTM)阻止染色质与INM结合和/或LINC复杂成分的异常表达，从而阻碍核骨架和细胞骨架之间的信息交换，从而导致LINC(核骨架和细胞骨架连接物)网络系统功能失调。由于小组特定的专业知识和前期工作，Steger小组将专注于精子染色质与核膜之间的相互作用，而Hortova小组将专注于核膜与精子细胞骨架之间的相互作用。首先，在野生型和鱼精蛋白缺陷型小鼠之间，我们将识别所选择的核蛋白(鱼精蛋白、修饰组蛋白)和细胞质蛋白(肌动蛋白细丝、微管)与LINC复合体蛋白(LBR、SUN、KASH)的定位和相互作用的差异。其次，在老鼠身上获得的数据将与(亚)有生育能力的男性进行比较。这种复杂的方法将增加我们对参与人类不育症病理生理学的分子因素之间的串扰的了解，这些因素与不正确的组蛋白到鱼精蛋白的交换有关。申请者有共同的科学兴趣，但展示了不同的技术专长，相互完美互补。拟议的项目将被嵌入到一个高度刺激的科学环境中，因为Steger与Schorle教授(波恩，D)共同维持着两个由DFG资助的项目，Schorle教授应用CRISPR/Cas9技术创造了鱼精蛋白缺乏的小鼠系，而Hortova拥有一个由GACR资助的项目，研究配子中的蛋白质-蛋白质相互作用。关于核质通讯中蛋白质-蛋白质相互作用的预期结果将有助于我们从新的角度理解染色质完整性和精子活力的分子调控机制。

During spermiogenesis, histone-to-protamine exchange is followed by chromatin hypercondensation representing a sperm quality control checkpoint, as incomplete histone replacement and/or aberrant protamine-1 (PRM1) to protamine-2 (PRM2) ratios are associated with male subfertility. Within the last years, sperm chromatin developed into an emerging field of reproductive medicine. We were able to demonstrate that PRM2-deficient male mice are infertile due to highly elevated levels of fragmented sperm DNA and completely immotile sperm (Schneider et al. 2016). This observation together with the facts that (1) a second mouse line lacking PRM1 has been generated and (2) sperm from subfertile men are known to exhibit an aberrant PRM1 to PRM2 ratio, represent the basis for the proposed DFG-GACR project, where both applicants join their expertise and pool their lab resources in order to study the signal transduction from chromatin to cytoskeleton (motility) in more detail. Applicants hypothesize that decreased (subfertile men, heterozygous mice) or missing (homozygous mice) protamine expression is followed by incomplete chromatin condensation finally resulting in a dysfunctional LINC (Linker of Nucleoskeleton and Cytoskeleton) network system due to aberrant histone post-translational modifications (PTMs) that prevent binding of chromatin to the INM and/or aberrant expression of LINC complex components impeding exchange of information between nucleoskeleton and cytoskeleton. Due to group-specific expertise and pre-work, the Steger group will focus on interactions between sperm chromatin and nuclear membrane, whereas the Hortova group will concentrate on interactions between nuclear membrane and sperm cytoskeleton. Firstly, differences in both localization and interaction of selected nuclear (protamines, modified histones) and cytoplasmic (actin filaments, microtubules) proteins with LINC complex proteins (LBR, SUN, KASH) between wild-type and protamine-deficient mice will be identified. Secondly, data obtained in mice will be compared with (sub)fertile men. This complex approach will increase our knowledge on the crosstalk between molecular factors involved in the pathophysiology of human subfertility associated with incorrect histone to protamine exchange. Applicants share common scientific interest, but exhibit different technical expertise perfectly complementing each other. The proposed project will be embedded in a highly stimulating scientific environment, as Steger maintain two DFG-funded projects with Prof. Schorle (Bonn, D), who generated the protamine-deficient mouse lines applying CRISPR/Cas9 technology, and Hortova holds a GACR-funded project studying protein-protein interaction in gametes. Expected results on protein-protein interactions involved in the nucleus-cytoplasm communication will contribute to new aspects of our understanding of molecular regulation mechanisms of chromatin integrity and sperm motility.