An atlas of cell types in the mammalian epididymis and vas deferens [single-cell RNA-Seq]

The epididymis is comprised of multiple segments/compartments that, in addition to concentrating sperm and preventing their premature activation, play key roles in remodeling the protein, lipid, and RNA composition of maturing sperm. In order to understand the complex roles for the epididymis in reproductive biology, we generated a single cell atlas of gene expression from the murine epididymis and vas deferens using droplet-based (10X Chromium) single cell RNA-Seq. Following single-cell RNA-Seq, quality control, and removal of reads attributable to contaminating cell-free RNA we were able to profiled the expression of 16878 genes in 8,880 individual cells with an average of 3110 (median = 1965) unique molecular identifiers (UMIs) per cell from across the epididymis and the vas deferens. We recovered all the key cell types of the epididymal epithelium, including principal cells, clear cells, and basal cells, along with its associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Our data confirm and extend prior studies of segment-restricted gene expression programs, and reveal that these region-specific programs are largely driven by principal cells. In addition to the extensive region-specific specialization of principal cells, we find evidence for functionally-specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Furthermore, we find several novel cell subpopulations, most notably including an Amylin-positive clear cell subtype, and predict intercellular signaling networks between the various cell types. Together, these data provide an atlas of cell composition across the epididymis, and a wealth of molecular hypotheses for future efforts in reproductive physiology. Sperm-depleted single cell suspensions were obtained from eight epididymides from four 10-12 weeks old FVB mice. The organ was cut into four segments that roughly corresponded to caput, corpus, cauda, and vas deferens. Single-cell RNA sequencing libraries were prepared using Chromium Single Cell 3' Reagent Kit V2 (10X Genomics).附睾由多个节段/分区构成，其不仅能够浓缩精子并防止其提前激活，还在重塑成熟精子的蛋白质、脂质与RNA组成过程中发挥关键作用。为解析附睾在生殖生物学中的复杂功能，我们基于液滴法（10X Chromium）单细胞RNA测序技术，构建了小鼠附睾与输精管的基因表达单细胞图谱。完成单细胞RNA测序、质量控制并去除游离RNA污染的测序读段后，我们对附睾与输精管的8880个单个细胞中的16878个基因表达情况进行了分析，每个细胞平均包含3110个（中位数为1965个）唯一分子标识符（UMI）。我们鉴定到附睾上皮的所有关键细胞类型，包括主细胞、亮细胞与基底细胞，同时也获取了相关支持细胞，即成纤维细胞、平滑肌细胞、巨噬细胞及其他免疫细胞。本研究数据验证并拓展了此前关于节段特异性基因表达程序的研究，同时揭示这些区域特异性程序主要由主细胞驱动。除主细胞存在广泛的区域特异性特化外，我们还发现基质细胞存在功能特化的亚群，尤为关键的是，本次分析鉴定出两种不同的亮细胞亚群。此外，我们还发现了数个全新的细胞亚群，其中最具代表性的为胰岛淀粉样多肽（Amylin）阳性的亮细胞亚型，并预测了不同细胞类型间的细胞间信号传导网络。综上，本研究数据构建了附睾整体细胞组成图谱，同时为生殖生理学领域的后续研究提供了丰富的分子假说。我们从4只10-12周龄的FVB小鼠的8个附睾中获取了去除精子的单细胞悬液。将附睾组织切割为四个节段，大致对应附睾头、附睾体、附睾尾与输精管。采用Chromium单细胞3'端试剂试剂盒V2（10X Genomics）构建单细胞RNA测序文库。