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代表精子质量控制检查点的染色质超浓缩，因为不完全的组蛋白替换和/或异常的鱼精蛋白-1（PRM 1）与鱼精蛋白-2（PRM 2）的比率与男性生育力低下有关。近年来，精子染色质已成为生殖医学的一个新兴领域。我们能够证明PRM 2缺陷型雄性小鼠由于精子DNA片段水平高度升高和精子完全不动而不育（Schneider et al. 2016）。这一观察结果连同以下事实：（1）已经产生了缺乏PRM 1的第二小鼠系，以及（2）已知来自生育力低下男性的精子表现出异常的PRM 1与PRM 2比率，代表了所提出的DFG-GACR项目的基础，其中两个申请人结合他们的专业知识并汇集他们的实验室资源，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，（生育力低下的男性，杂合子小鼠）或缺失（纯合子小鼠）鱼精蛋白表达之后是不完全的染色质凝聚，最终导致LINC功能障碍（核骨架和细胞骨架的接头）网络系统，由于异常的组蛋白翻译后修饰（PTM），阻止染色质与INM的结合，和/或或LINC复合物组分的异常表达阻碍核骨架和细胞骨架之间的信息交换。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和核膜之间的相互作用，而Hortova小组将专注于核膜和精子细胞骨架之间的相互作用。首先，将鉴定野生型和鱼精蛋白缺陷小鼠之间选定的核（鱼精蛋白，修饰的组蛋白）和细胞质（肌动蛋白丝，微管）蛋白与LINC复合物蛋白（LBR，SUN，KASH）的定位和相互作用的差异。其次，将在小鼠中获得的数据与（亚）生育力男性进行比较。这种复杂的方法将增加我们对人类生育力低下的病理生理学中涉及的分子因素之间的串扰的知识，与不正确的组蛋白鱼精蛋白交换有关。申请人有共同的科学兴趣，但表现出不同的技术专长，完美互补。拟议的项目将嵌入一个高度刺激的科学环境中，Steger与Schorle教授（波恩，D）保持两个DFG资助的项目，Schorle教授应用CRISPR/Cas9技术产生了鱼精蛋白缺陷小鼠品系，Hortova拥有一个GACR资助的研究配子中蛋白质-蛋白质相互作用的项目。蛋白质-蛋白质相互作用参与的核质通讯的预期结果将有助于我们了解染色质完整性和精子活力的分子调控机制的新方面。

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.