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. Overall design: 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.