Molecular Architecture of Meiotic Chromosomes

减数分裂对有性生殖至关重要，因为它使单倍体细胞的产生成为可能。它由两个连续的细胞分裂组成，没有介入DNA复制。在第一次减数分裂前期，同源染色体对，突触和重组。这些过程对于确保第一次减数分裂过程中正确的染色体分离是必不可少的。在第二次分裂中，姐妹染色单体分离，形成单倍体配子。由于同源染色体在减数分裂I期间的重组和它们的独立分类，生殖细胞在遗传上不同于原始母细胞（精原细胞或卵原细胞）。减数分裂的高度生物学相关性是毋庸置疑的：它为有性生殖提供了基础，并代表了遗传变异的最大自然来源。减数分裂染色体突触和减数分裂重组依赖于减数分裂特异性结构的形成：突触复合体、内聚蛋白轴和重组结节。值得注意的是，这些结构的形成揭示了发生在减数分裂染色体轴上的相互依赖的过程。因此，确定减数分裂染色体轴的分子组织是理解生殖系基因组稳定性的一个关键方面。利用免疫荧光显微镜研究了减数分裂染色体轴向结构的蛋白质定位，但其分辨率仅为200 nm左右。一些轴向染色体成分的定位也被应用于免疫金电镜。在这里，蛋白质可以在nm分辨率下定位，而定位精度主要受一抗和二抗以及金颗粒大小的限制。由于金颗粒含量低，样品制备通常耗时且定量分析繁琐。由于可实现的结构分辨率不仅取决于定位精度，还取决于Nyquist-Shannon采样定理所描述的信号密度，因此利用免疫金EM构建不同蛋白质的蛋白质定位图仍然具有挑战性。为了克服这些障碍，我们建议通过提供定量的蛋白质分布图来研究减数分裂染色体轴向结构的分子结构和相互作用以及与核膜的相互作用。为此，超分辨率成像（dSTORM）结合单色和双色单定位显微镜和纳米定位平均位置测定将应用于野生型和选定的敲除小鼠，并结合电子显微镜断层扫描以及分子和生化方法。由于不孕症和非整倍体（即唐氏综合症）通常是减数分裂缺陷的结果，因此这些研究将与动物和人类生殖健康领域相关。

Meiosis is essential for sexual reproduction in that it enables the generation of haploid cells. It consists of two successive cellular divisions without intervening DNA replication. During the first meiotic prophase homologous chromosomes pair, synapse and recombine. These processes are essential to ensure correct chromosome segregation during the reductional first meiotic division. During the second division, sister chromatids are separated leading to the formation of haploid gametes. As a result of recombination between the homologous chromosomes and their independent assortment during meiosis I, germ cells are genetically different from the original mother cell (spermatogonia or oogonia). The high biological relevance of meiosis is beyond question: it provides the basis for sexual reproduction and represents the largest natural source of genetic variability. Meiotic chromosome synapsis and meiotic recombination depend on the formation of meiosis-specific structures: synaptonemal complexes, cohesin axes and recombination nodules. Remarkably, formation of these structures revealed to be interdependent processes that take place in the meiotic chromosome axis. Thus, defining the molecular organization of the meiotic chromosome axis is a key aspect for the understanding of genome stability in the germ line. Protein localization of meiotic chromosome axial structures has been approached by immunofluorescence microscopy, but the resolution achieved is only about 200 nm. The localization of some of the axial chromosome components has also been approached by immunogold EM. Here, proteins can be localized with nm resolution, whereas the localization precision is mainly limited by the size of the primary and secondary antibody and the size of the gold particle. Sample preparation is usually time consuming and quantitative analysis tedious, because of the low amount of gold particles. Since the structural resolution achievable is determined not only by the localization precision but also by the signal density as described by the Nyquist-Shannon sampling theorem, the construction of proteins localization maps of different proteins by immunogold EM remains challenging. To circumvent these obstacles we propose to investigate the molecular architecture and interactions of meiotic chromosome axial structures to each other and with the nuclear envelope by providing quantitative protein distribution maps. To this end, super-resolution imaging (dSTORM) in combination with one- and dual-color single localization microscopy and average position determination with nanometer position will be applied to wild-type and selected knockout mice, together with electron microscope tomography as well as molecular and biochemical approaches. As infertility and aneuploidies (i.e. Down syndrome) are often the consequence of a defective meiosis, these investigations would be of relevance for the field of animal and human reproductive health.