Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis

Abstract

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

Identification of the co-differentially expressed hub genes involved in the endogenous protective mechanism against ventilator-induced diaphragm dysfunction

“…B The other network with Tmprss13 and Cst3 as core genes, showing that there is a certain degree of crosstalk between apoptosis and autophagy (See figure on next page.) protein could negatively regulate autophagy in SSCs [34]. An extrapolation of this function would suggest that increased CST3 expression, such as that observed in this study, could suppress autophagy, which is known to contribute to VIDD.…”

Germline FOXJ2 overexpression causes male infertility via aberrant autophagy activation by LAMP2A upregulation

“…Moreover, the level of autophagy increased in high-fat diet mice with disrupted spermatogenesis and male fertility, and autophagy was also overactivated in sperm samples from obese subfertile male patients [ 41 , 42 ]. Recent studies have found that LAMP2 was highly expressed in human spermatogonia, and some autophagy-related genes were dysregulated in the testicular germ cells of patients with non-obstructive azoospermia (NOA), suggesting that autophagy is related to male infertility [ 43 ].…”

Construction and external validation of a 5-gene random forest model to diagnose non-obstructive azoospermia based on the single-cell RNA sequencing of testicular tissue

“…Nowadays, the widespread applications of gene sequencing, especially scRNA-seq, have deepened the knowledge of NOA [ 25 , 26 ]. For instance, Wang et al had disclosed the unique role of autophagy homeostasis in the spermatogenesis of NOA cases through scRNA-seq analysis [ 27 ]. Liu et al utilized scRNA-seq to detect the genetic change of ACE2 in testicular cells of normal and NOA patients, uncovering the possible mechanisms of how SARS-CoV-2 affected testicular cells [ 28 ].…”