Published online Dec 26, 2022. doi: 10.4252/wjsc.v14.i12.822
Peer-review started: August 4, 2022
First decision: September 29, 2022
Revised: October 7, 2022
Accepted: November 30, 2022
Article in press: November 30, 2022
Published online: December 26, 2022
Processing time: 138 Days and 20.1 Hours
Spermatogonial stem cells (SSCs) are the origin of spermatogenesis, which continuously generates spermatozoa through self-renewal and differentiation. Although we have identified many molecules and pathways that regulate SSC function in mice, the mechanisms regulating human SSCs are not yet fully revealed.
To explore the regulatory mechanisms of human SSCs, we analyzed human testis single-cell RNA sequencing (scRNA-seq) data from the GSE149512 and GSE112013 datasets. We found that SPOC domain-containing protein 1 (SPOCD1) is differentially expressed in human SSCs. This study explored the role of SPOCD1 in human proliferation and apoptosis, which will help to expand the understanding of SSC regulation.
To investigate the functions and mechanisms of SPOCD1 in human proliferation and apoptosis, and to explore the potential effects on spermatogenesis.
In this study, scRNA-seq was used to detect differentially expressed genes in human SSCs, in which the SPOCD1 gene is highly expressed in human SSCs. Immunohistochemistry was used to investigate the expression pattern of SPOCD1 in human testicular tissue. Subsequently, we used small interfering RNA to knockdown SPOCD1 in human SSC lines and dissected the role of SPOCD1 in human SSCs by Cell Counting Kit-8, Western blot analysis, 5-ethynyl-2’-deoxyuridine, fluorescence-activated cell sorting, and terminal deoxynucleotidyl transferase dUTP nick end labeling. RNA-seq was used to explore gene expression alterations induced by SPOCD1 downregulation. Finally, we identified the functional target genes of SPOCD1 by rescue experiments.
The scRNA-seq and immunohistochemical results showed that SPOCD1 was predominantly localized to human SSCs. Knockdown of SPOCD1 in human SSC lines resulted in a significant decrease in cell proliferation and induced apoptosis. RNA-seq results showed that SPOCD1 knockdown caused the significant downregulation of genes such as adenylate kinase 4 (AK4) and affected pathways such as tumor necrosis factor and cyclic AMP. Overexpression of AK4 in SPOCD1 knockdown cells significantly responded to the changes in cell proliferation and apoptosis caused by SPOCD1 inhibition.
We demonstrated that SPOCD1 was predominantly localized to human SSCs and regulated its proliferation and apoptosis through AK4. Our study provides new insights into regulating human SSCs and potential novel targets for treating male infertility.
Future studies will explore the correlation between SPOCD1 and abnormal human spermatogenesis in large samples. These include screening for potentially curative mutations of SPOCD1 in azoospermia patients and exploring the association between abnormal SPOCD1 expression and azoospermia in large samples using deep learning.