Overview of single‐cell RNA sequencing analysis and its application to spermatogenesis research

Abstract Background Single‐cell transcriptomics allows parallel analysis of multiple cell types in tissues. Because testes comprise somatic cells and germ cells at various stages of spermatogenesis, single‐cell RNA sequencing is a powerful tool for investigating the complex process of spermatogenesis. However, single‐cell RNA sequencing analysis needs extensive knowledge of experimental technologies and bioinformatics, making it difficult for many, particularly experimental biologists and clinicians, to use it. Methods Aiming to make single‐cell RNA sequencing analysis familiar, this review article presents an overview of experimental and computational methods for single‐cell RNA sequencing analysis with a history of transcriptomics. In addition, combining the PubMed search and manual curation, this review also provides a summary of recent novel insights into human and mouse spermatogenesis obtained using single‐cell RNA sequencing analyses. Main Findings Single‐cell RNA sequencing identified mesenchymal cells and type II innate lymphoid cells as novel testicular cell types in the adult mouse testes, as well as detailed subtypes of germ cells. This review outlines recent discoveries into germ cell development and subtypes, somatic cell development, and cell–cell interactions. Conclusion The findings on spermatogenesis obtained using single‐cell RNA sequencing may contribute to a deeper understanding of spermatogenesis and provide new directions for male fertility therapy.