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仍然很大程度上是未知的。出乎意料的是，我们在这些燕麦睾丸的未分化生殖细胞(精原细胞)中发现了上突变，这在成熟的、活动的精子中没有检测到。这表明精原亚群和质量控制检查点的存在，阻止了具有异常表观基因组的精子的产生。此外，对这些男性的睾丸组织进行的单细胞RNA测序(scRNA-Seq)显示，精原细胞室的动态发生了变化，精原干细胞的生态位也发生了变化。整合甲基组和转录组数据表明，在燕麦样本中，精原细胞的差异甲基化区域和生殖细胞衰竭阶段的差异表达基因之间存在关联。除了这些常见的变化外，我们还发现了患者特有的变化，需要扩大研究队列。这项应用的总体目标是探索分子特征(在转录和DNA甲基化水平上)，区分那些有助于成熟精子形成的精原细胞亚群和那些未能进展的精原细胞亚群，并评估干细胞生态位对这些过程的影响。我们将利用这一独特的途径获得具有严格特征的人类睾丸活检组织，以进行以下研究工作。与正常对照组相比，将对患有严重燕麦不育男性的睾丸组织进行scRNA-Seq(10倍基因组学)，以产生反映生殖细胞失效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，将进行超高分辨率的靶向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被睾丸组织中的多光谱蛋白质成像所证实。通过在表观遗传学、转录和蛋白质水平上的分析，我们将对人类精子发生中指导生殖细胞分化和质量控制的机制有一个无与伦比的、完整的看法，这可能导致对燕麦综合征的更好的亚型。

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.