Identification of the Regulatory Network of Autophagy and Novel Autophagic Genes in Normal Spermatogenesis and Male Infertility via Single-Cell Transcriptome Analysis

Autophagy has been implicated to be involved in spermatogenesis and dysfunction of autophagy can lead to male infertility. However, sophisticated transcriptional signatures of autophagic genes in spermatogenesis and its significance remain uncharacterized. Here, we explore the transcriptional profiling of autophagy-related genes across human spermatogenesis in donors with normal fertility and nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Core component gene sets in each step of autophagy exhibit stage-specific expression pattern in human spermatogenesis, which is further confirmed by immunostaining; in this context, we further uncover a unique stage-specific enrichment of a cohort autophagy-related genes. Human-to-mouse comparison analysis reveals that this stage-specific expression pattern of autophagy-related genes is highly conserved in mammals and dysregulation of this pattern is associated with human male infertility. Moreover, we discover several stage-specific autophagic genes, which have not been identified to regulate spermatogenesis before. Among these candidate genes, Cst3, an actively expressed autophagic gene in spermatogonia and early spermatocytes, is involved in the regulation of SSC maintenance and subsequent male germ cell development. Downregulation of Cst3 increases the autophagy activity in mouse SSCs, which exerts its roles by regulating the expression of SSC core factors such as Oct4, Id1 and Nanos3. Collectively, our study provides novel insights into the global transcriptional signatures of autophagy-related genes and confirms the role of proper autophagy activity in SSC maintenance and normal spermatogenesis, opening new avenues for further investigation of the correlation between autophagy and spermatogenesis as well as male infertility. Here we performed scRNA sequencing for 432 individual human testicular cells including spermatogonia, spermatocytes, spermatids, and testicular somatic cells (soma) from donors with nonobstructive azoospermia Note from submitter: Due to the Regulations of the People's Republic of China on the Management of Human Genetic Resources, raw data related to humans needs to be uploaded to the National Genomics Data Center, China National Center for Bioinformation (CNCB-NGDC), so we cannot upload raw data to NCBI. The GSA number accession is HRA000317.