Single-cell RNAseq analysis of testicular germ and somatic cell development during the perinatal period

Pro-spermatogonia (SG) serve as the gateway to spermatogenesis. Using single-cell RNA sequencing (RNAseq), we studied the development of ProSG, their SG descendants, and testicular somatic cells, during the perinatal period in mice. We identified both gene and protein markers for 3 temporally distinct ProSG cell subsets, including a migratory cell population with a distinct transcriptome from the previously defined T1- and T2-ProSG stages. This intermediate (I)-ProSG subset translocates from the center of seminiferous tubules to the spermatogonial stem cell (SSC) "niche" in its periphery soon after birth. We identified 3 undifferentiated SG subsets at postnatal day 7, each of which express distinct genes, including transcription factor and signaling genes. Two of these subsets have the characteristics of newly emergent SSCs. We also molecularly defined the development of Sertoli, Leydig, and peritubular myoid cells during the perinatal period, allowing us to identify candidate signaling pathways acting between somatic and germ cells in a stage-specific manner during the perinatal period. Our study provides a rich resource for those investigating testicular germ and somatic cell developmental during the perinatal period. Single cell sequencing (10X genomics) from unfractionated perinatal testicular cells (embryonic day [E] 18.5, postnatal day [P] 2, and P7; with two biological replicates for each timepoint) were performed for this study.前精原细胞（Pro-spermatogonia）是精子发生的门户。本研究借助单细胞RNA测序（RNAseq），解析了小鼠围产期内前精原细胞、其分化产生的精原细胞（SG）后代以及睾丸体细胞的发育进程。我们鉴定出3个时间特异性的前精原细胞亚群的基因与蛋白标志物，其中包含一类转录组特征不同于已有定义的T1、T2前精原细胞阶段的迁移细胞群。该中间型（I-）前精原细胞亚群在出生后不久即从生精小管中央迁移至外周的精原干细胞（SSC）微环境中。我们在出生后第7天鉴定出3个未分化精原细胞亚群，每个亚群均表达独特的基因，包括转录因子及信号通路相关基因；其中两个亚群具备新生精原干细胞的特征。本研究还从分子层面解析了围产期内支持细胞（Sertoli细胞）、莱迪希细胞（Leydig细胞）与管周肌样细胞的发育过程，据此鉴定出围产期内以阶段特异性方式作用于体细胞与生殖细胞间的候选信号通路。本研究为关注围产期睾丸生殖细胞与体细胞发育的研究者提供了丰富的研究资源。本研究对未分选的围产期睾丸细胞（胚胎第18.5天[E18.5]、出生后第2天[P2]及第7天[P7]，每个时间点设置两个生物学重复）开展了10×基因组学（10X Genomics）单细胞测序。