Transcriptomic analysis of the Non-Obstructive Azoospermia (NOA) to address gene expression regulation in human testis

There is a need to understand the molecular basis of testes under Non-Obstructive Azoospermia (NOA), a state of failed spermatogenesis. There has been a lack of attention to the transcriptome at the level of alternatively spliced mRNAs (iso-mRNAs) and the mechanism of gene expression regulation. Hence, we aimed to establish a reliable iso-mRNA profile of NOA-testes, and explore molecular mechanisms – especially those related to gene expression regulation. We sequenced mRNAs from testicular samples of donors with complete spermatogenesis (control samples) and a failure of spermatogenesis (NOA samples). We identified differentially expressed genes and their iso-mRNAs <i>via</i> standard NGS data analyses. We then listed these iso-mRNAs hierarchically based on the extent of consistency of differential quantities across samples and groups, and validated the lists <i>via</i> RT-qPCRs (for 80 iso-mRNAs). In addition, we performed extensive bioinformatic analysis of the splicing features, domains, interactions, and functions of differentially expressed genes and iso-mRNAs. Many top-ranking down-regulated genes and iso-mRNAs, i.e., those down-regulated more consistently across the NOA samples, are associated with mitosis, replication, meiosis, cilium, RNA regulation, and post-translational modifications such as ubiquitination and phosphorylation. Most down-regulated iso-mRNAs correspond to full-length proteins that include all expected domains. The predominance of alternative promoters and termination sites in these iso-mRNAs indicate their gene expression regulation <i>via</i> promoters and UTRs. We compiled a new, comprehensive list of human transcription factors (TFs) and used it to identify TF-'TF gene’ interactions with potential significance in down-regulating genes under the NOA condition. The results indicate that RAD51 suppression by HSF4 prevents SP1-activation, and SP1, in turn, could regulate multiple TF genes. This potential regulatory axis and other TF interactions identified in this study could explain the down-regulation of multiple genes in NOA-testes. Such molecular interactions may also have key regulatory roles during normal human spermatogenesis.

目前学界对于非阻塞性无精子症（Non-Obstructive Azoospermia, NOA，即精子发生障碍状态）患者的睾丸分子机制仍有待深入解析，且针对可变剪接mRNA（iso-mRNAs）层面的转录组研究及基因表达调控机制的关注尚且不足。为此，本研究旨在构建可靠的NOA患者睾丸可变剪接mRNA谱，并探索其分子调控机制，尤其是与基因表达调控相关的通路。我们对具备完整精子发生过程的供体睾丸样本（对照组）及精子发生障碍的NOA患者睾丸样本中的mRNA进行了测序，通过标准下一代测序（NGS）数据分析流程鉴定了差异表达基因及其对应的可变剪接mRNA。随后我们依据样本与分组间差异表达量的一致性程度，对这些可变剪接mRNA进行了层级排序，并通过实时定量聚合酶链反应（RT-qPCR）对其中80条可变剪接mRNA进行了验证。此外，我们针对差异表达基因及其可变剪接mRNA的剪接特征、蛋白结构域、分子相互作用及功能开展了全面的生物信息学分析。在NOA样本中一致性下调程度更高的顶级排名下调基因及可变剪接mRNA，多与有丝分裂、DNA复制、减数分裂、纤毛功能、RNA调控及泛素化、磷酸化等翻译后修饰过程密切相关。绝大多数下调的可变剪接mRNA对应包含所有预期结构域的全长蛋白。此类可变剪接mRNA中可变启动子与终止位点占比占优，提示其基因表达调控可通过启动子及非翻译区（UTRs）实现。我们整理了一套全新的人类转录因子（TFs）综合列表，并借此鉴定出在NOA状态下可能参与基因下调调控的转录因子-转录因子基因相互作用。研究结果显示，HSF4对RAD51的抑制可阻断SP1的激活，而SP1后续可调控多个转录因子基因。这一潜在调控轴及本研究中鉴定出的其他转录因子相互作用，可解释NOA患者睾丸中多个基因的下调现象，此类分子相互作用在正常人类精子发生过程中或许也发挥关键调控作用。