Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscape and underlying molecular signals during human spermatogenesis remain unknown. Here, we examined human spermatogenesis using transcriptome-wide single-cell RNA sequencing of 2,854 individual testicular cells from donors with normal spermatogenesis (donors with normal fertility or OA) and 174 testicular cells from one donor diagnosed as having nonobstructive azoospermia (NOA). Systematic bioinformatics analyses enabled us to delineate the full developmental trajectories of human spermatogenesis. A hierarchical model was established, which was characterized by the sequential and step-wise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Furthermore, we recapitulated key hallmarks of human spermatogenesis at the single-cell level, including spermatogonial development, mitosis-to-meiosis transition, meiotic recombination, meiotic sex chromosome inactivation, and histone-to-protamine exchange. Remarkably, BMPR1B and FGFR3 were identified as novel markers of human spermatogonial stem cells, indicating the potentially critical role of BMP and FGF signaling pathways for SSC maintenance. Further analysis identified several novel marker genes of specific cell type such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112, the expression patterns of which were confirmed via RNA FISH or immunostaining. We also demonstrated that scRNA-seq provided a platform to find differentially expressed genes correlated with one type of nonobstructive azoospermia (NOA) at the genome-wide transcriptional profiling level, which might pave way for further diagnosis and dissecting the underlying mechanisms of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders. Here we performed scRNA sequencing for 2,854 individual human testicular cells including spermatogonia, spermatocytes, spermatids, and testicular somatic cells (soma) from donors with normal fertility and obstructive azoospermia.Besides, another 174 human testicular cells from one NOA male were included as an example of infertilty. Some samples were sorted by FACS based on preliminary ploidy. These samples include "1N", "2N", or "4N" in the sample title. This information is also available in the sample characteristics.

精子发生（spermatogenesis）可产生成熟雄性配子，对于遗传信息在代际间的精准传递至关重要。然而，人类精子发生过程中的发育全景及潜在分子调控信号仍未被完全阐明。本研究针对人类精子发生开展分析：我们对2854个来自精子发生正常（生育能力正常或梗阻性无精子症（obstructive azoospermia, OA））供者的睾丸单细胞，以及1名确诊为非梗阻性无精子症（nonobstructive azoospermia, NOA）供者的174个睾丸细胞，进行了全转录组范围的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。通过系统性生物信息学分析，我们完整描绘了人类精子发生的全发育轨迹。本研究构建了一套层级化模型，该模型以三类精原细胞亚型、七类精母细胞亚型及四类精子细胞亚型的序贯逐步发育为核心特征。此外，我们在单细胞层面复现了人类精子发生的关键标志性事件，包括精原细胞发育、有丝分裂向减数分裂的转换、减数分裂重组、减数分裂性染色体失活以及组蛋白-鱼精蛋白替换。值得注意的是，本研究鉴定出BMPR1B与FGFR3可作为人类精原干细胞（spermatogonial stem cells, SSC）的新型标志物，提示BMP与FGF信号通路在精原干细胞维持中可能发挥关键调控作用。进一步分析还鉴定出多个特定细胞类型的新型标志物基因，包括HMGA1、PIWIL4、TEX29、SCML1及CCDC112，其表达模式已通过RNA荧光原位杂交（RNA fluorescence in situ hybridization, RNA FISH）或免疫染色得以验证。本研究同时证实，单细胞RNA测序可作为全基因组转录组层面筛选与非梗阻性无精子症相关差异表达基因的技术平台，这为男性不育的后续诊断及潜在发病机制解析奠定了基础。本研究成功重构了人类精子发生过程中细胞命运序贯转变所固有的转录调控程序，为解析男性相关生殖系统疾病提供了重要依据。本研究共对2854个人类睾丸细胞进行了单细胞RNA测序，这些细胞来自生育能力正常及梗阻性无精子症供者，涵盖精原细胞、精母细胞、精子细胞及睾丸体细胞（soma）。此外，本研究还纳入了1名非梗阻性无精子症男性的174个睾丸细胞，作为不育症研究的示例。部分样本基于初步倍性通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）进行了分选富集，此类样本的标题中包含"1N""2N"或"4N"字样，相关信息也可在样本特征中查阅。