Spatio-temporal landscape of mouse epididymal cells and specific mitochondria-rich segments defined by large-scale single-cell RNA-seq

In total, 40,623 cells were detected and further clustered into 8 identified cell populations. Focused analyses revealed the subpopulations of principal cells, basal cells, clear/narrow cells, and halo/T cells. Notably, two subtypes of principal cells, the Prc7 and Prc8 subpopulations were enriched as stereocilia-like cells according to GO analysis. Further analysis demonstrated the spatially specific pattern of the DEGs in each cell cluster. Unexpectedly, the abundance of mitochondria and mitochondrial transcription (MT) was found to be higher in the corpus and cauda epididymis than in the caput epididymis by scRNA-seq, immunostaining, and qPCR validation. In addition, the spatio-temporal profile of the DEGs from the P42 and P56 epididymis, including transiting spermatozoa, was depicted. Five 42 days (P42) and ten 56 days (P56) old of wild-type C57Bl/6J mice were sacrified, then the epididymis were dissected and divided into three segments (caput, corpus, and cauda). Ten epididymis from five mice were cut into the different segment and immediately transferred into the GEXSCOPETM Tissue Preservation Solution (Singleron Biotechnologies) in an Eppendorf tube labeled as EP (caput), EO (corpus) and ED (cauda) on ice. For P56 epididymis, samples were two replicates. The procedure was repeated in a mice until all segments were collected in each tube. The pooled segments were further to the procedure of tissue dissociation and single cell preparation.本研究共检测到40623个细胞，并进一步聚类为8个经明确鉴定的细胞群。针对性分析揭示了主细胞、基底细胞、亮细胞/窄细胞以及晕细胞/T细胞的亚群特征。值得注意的是，经基因本体（Gene Ontology, GO）分析发现，两类主细胞亚群Prc7与Prc8可富集为静纤毛样细胞（stereocilia-like cells）。进一步分析揭示了各细胞群中差异表达基因（Differentially Expressed Genes, DEGs）的空间特异性表达模式。出乎意料的是，通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）、免疫染色与实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）验证，发现附睾体与附睾尾中的线粒体丰度及线粒体转录（MT）水平均高于附睾头。此外，本研究还刻画了P42与P56龄小鼠附睾（包括迁移中的精子）中差异表达基因的时空表达谱。选取5只42日龄（P42）及10只56日龄（P56）的野生型C57Bl/6J小鼠，处死后剥离其附睾，并将附睾分为头、体、尾三段。将五只小鼠的十个附睾分别切割为对应节段，随后立即转移至装有GEXSCOPETM组织保存液（Singleron Biotechnologies）的Eppendorf离心管中，冰上分别标记为EP（附睾头）、EO（附睾体）与ED（附睾尾）。对于P56龄小鼠的附睾样本，设置两个生物学重复。该操作逐小鼠重复进行，直至每支离心管中收集完对应节段的全部样本。合并后的节段样本将进一步开展组织解离与单细胞制备流程。