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©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4
Dai Zhou, Fang Zhu, Zeng-Hui Huang, Huan Zhang, Li-Qing Fan, Jing-Yu Fan
Dai Zhou, Fang Zhu, Zeng-Hui Huang, Huan Zhang, Li-Qing Fan, Jing-Yu Fan, Institute of Reproduction and Stem Cell Engineering, School of Basic Medicine Science, Central South University, Changsha 410000, Hunan Province, China
Dai Zhou, Zeng-Hui Huang, Huan Zhang, Li-Qing Fan, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha 410000, Hunan Province, China
Dai Zhou, College of Life Sciences, Hunan Normal University, Changsha 410000, Hunan Province, China
Dai Zhou, Zeng-Hui Huang, Huan Zhang, Li-Qing Fan, Clinical Research Center for Reproduction and Genetics in Hunan Province, Changsha 410000, Hunan Province, China
Jing-Yu Fan, Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, United States
Author contributions: Fan JY designed the study and supervised the laboratory experiments; Zhou D conducted the experiments and drafted the manuscript; Zhu F assisted in bioinformatics analysis; Huang ZH and Zhang H assisted with the experiments and sample collection; Fan LQ contributed new reagents and analytic tools; all authors read and approved the final manuscript.
Supported by the National Natural Science Foundation for Young Scholars of China, No. 82201771; National Natural Science Foundation of China, No. 32270912; Natural Science Foundation of Changsha, No. kq2202491; Research Grant of CITIC-Xiangya, No. YNXM202109 and No. YNXM202115; and Hunan Provincial Grant for Innovative Province Construction, No. 2019SK4012.
Institutional review board statement: The study was reviewed and approved by the Reproductive & Genetic Hospital of CITIC-Xiangya, Basic Medical Science School, Central South University Institutional Review Board (Approval No. LL-SC-2021-025).
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: The authors have no conflicts of interest to declare.
Data sharing statement: All data are available from the corresponding author upon reasonable request jingyu@email.sc.edu.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Jing-Yu Fan, MD, PhD, Researcher, Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter St, GSRC 109, Columbia, SC 29208, United States. jingyu@email.sc.edu
Received: August 4, 2022
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
ARTICLE HIGHLIGHTS
Research background
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.
Research motivation
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.
Research objectives
To investigate the functions and mechanisms of SPOCD1 in human proliferation and apoptosis, and to explore the potential effects on spermatogenesis.
Research methods
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.
Research results
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.
Research conclusions
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.
Research perspectives
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.
