Molecular mechanisms of spermatogenic failure at transcriptional and epigenetic level

在生殖医学和男科中心就诊的男性不育患者中，约有一半表现出严重的精子参数异常（称为少弱畸形精子症，OAT）。到目前为止，这种表型的潜在分子机制在很大程度上仍然未知。出乎意料的是，我们发现表突变存在于这些OAT睾丸的未分化生殖细胞（精原细胞）中，而在成熟的活动精子中未检测到。这表明存在精原细胞亚群和质量控制检查点，防止产生具有异常表观基因组的精子。此外，这些男性睾丸组织的单细胞RNA测序（scRNA-Seq）揭示了精原细胞室的动态变化和精原干细胞生态位的组成改变。甲基化组和转录组数据的整合表明精原细胞差异甲基化区域与OAT样本中生殖细胞衰竭阶段特异性差异表达基因之间存在关联。除了这些常见的改变，我们还发现了患者的具体变化，有必要扩大研究coherent.The总体目标的应用程序是探索的分子特征（在转录和DNA甲基化水平），区分那些精原细胞亚群，有助于形成成熟的精子，从那些未能取得进展，并评估这些过程中的干细胞生态位的影响。我们将利用这一独特的途径获得严格表征的人类睾丸活检，用于以下研究工作。与正常对照相比，将对患有重度OAT的不育男性的睾丸组织进行scRNA-Seq（10 x Genomics），以生成反映生殖细胞衰竭的转录机制的最大可用数据集。为了严格评估这些男性精原细胞中的差异甲基化区域，将进行超高分辨率靶向DNA甲基化分析。这些广泛的数据集在细胞水平上的相关性将通过睾丸组织中的多光谱蛋白质成像来证实。通过在表观遗传、转录和蛋白质水平上的应用分析，我们将获得指导人类精子发生中生殖细胞分化和质量控制的机制的无与伦比的综合观点，这可能导致OAT综合征的更好的亚分类。

About half of the patients with male infertility presenting at the Centre of Reproductive Medicine and Andrology display severely abnormal sperm parameters (termed oligoasthenoteratozoospermia, OAT). To date, the underlying molecular mechanisms for this phenotype remain largely unknown. Unexpectedly, we found epimutations present in the undifferentiated germ cells (spermatogonia) of these OAT testes, which were not detected in mature, motile sperm. This suggests the existence of spermatogonial subpopulations and quality control checkpoints, preventing sperm with an abnormal epigenome from being produced. Furthermore, single cell RNA sequencing (scRNA-Seq) of testicular tissues from these men revealed changed dynamics of the spermatogonial compartment and an altered composition of the spermatogonial stem cell niche. Integration of methylome and transcriptome data indicated an association between differentially methylated regions in spermatogonia and differentially expressed genes specific for the stages of germ cell failure in OAT samples. Apart from these common alterations, we also found patient-specific changes that necessitate expansion of the study cohort.The overall objective of this application is to explore the molecular features (at transcriptional and DNA methylation level) that distinguish those spermatogonial subpopulations that contribute to the formation of mature sperm from those which fail to progress, and to assess the impact of the stem cell niche on these processes. We will capitalize on the unique access to rigorously characterized human testicular biopsies for the following research endeavour. scRNA-Seq (10x Genomics) will be performed on testicular tissues of infertile men with severe OAT compared to normal controls to generate the largest available dataset reflecting the transcriptional mechanisms of germ cell failure. To rigorously assess the differentially methylated regions in the spermatogonia of these men, ultra-high resolution targeted DNA methylation analysis will be performed. The relevance of these extensive datasets on cellular level will be corroborated by multi-spectral protein imaging in testicular tissues. By applying analysis at the epigenetic, transcriptional, and protein level we will gain an unparalleled and integrated view of the mechanisms guiding germ cell differentiation and quality control in human spermatogenesis which could result in better subclassification of the OAT syndrome.