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Updated: Mar 13, 2026
Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye
Published on: January 14, 2021
Transcriptome analysis of the responses to methyl methanesulfonate treatment in mouse pachytene spermatocytes and
Hui Zhang1, Chuanchao Zhang1, Jinting Yan1
1CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
Abstract:
Spermatogenesis is threatened by DNA alkylating agents, one major category of DNA damaging agents. Currently, little is known about the alterations in transcriptome profiling of the mouse spermatogenic cells in response to DNA alkylation at distinct stages of spermatogenesis. In this study, RNA sequencing (RNA-seq) was performed in pachytene spermatocytes (PS) and round spermatids (RS) at 0 or 30min following Methyl Methanesulfonate (MMS) treatment and with untreated controls. A large number of differentially expressed genes (DEGs) were identified by comparison of the three groups in PS and RS, respectively. Functional analyses of all DEGs highlighted the protein ubiquitination pathway and DNA damage response (DDR) network being the two main biological processes in common in the two cell types. Further analyses of the DEGs with 2-fold or more changes between 30min repair and control group indicated that several cytokine signaling pathways were the most strongly affected in PS and DDR related pathways in RS, respectively. Gene ontology (GO) analyses directly showed differential biological process (BP) affected between PS and RS, with "regulation of transcription" being most overrepresented in PS and "cellular response to stress" in RS, respectively. Moreover, 374 DDR-related genes in PS and 158 in RS among all DEGs were filtered and clustered, which showed dynamic expression patterns in PS and RS. Our analyses provide a transcriptional landscape for male germ cells in response to MMS during spermatogenesis.
Insights
DNA alkylating agents like Methyl Methanesulfonate (MMS) impact spermatogenesis. This study reveals key transcriptional changes in mouse pachytene spermatocytes and round spermatids, highlighting DNA damage response and protein ubiquitination pathways.
Area of Science:
- Reproductive biology
- Molecular genetics
- Toxicology
Background:
- Spermatogenesis is vulnerable to DNA alkylating agents, which can cause DNA damage.
- The specific transcriptome alterations in mouse spermatogenic cells following DNA alkylation remain largely uncharacterized.
- Understanding these changes is crucial for assessing male reproductive health risks.
Purpose of the Study:
- To investigate the global transcriptome profiling of mouse spermatogenic cells (pachytene spermatocytes and round spermatids) after Methyl Methanesulfonate (MMS) exposure.
- To identify differentially expressed genes (DEGs) and key biological pathways affected by DNA alkylation at distinct spermatogenic stages.
- To provide a comprehensive transcriptional landscape of male germ cells responding to DNA damage.
Main Methods:
- RNA sequencing (RNA-seq) was performed on pachytene spermatocytes (PS) and round spermatids (RS) from mice treated with Methyl Methanesulfonate (MMS) or control.
- Samples were collected at 0 and 30 minutes post-MMS treatment to capture early transcriptional responses.
- Bioinformatic analyses, including DEG identification, functional enrichment (Gene Ontology), and pathway analysis, were employed.
Main Results:
- A substantial number of DEGs were identified in both PS and RS following MMS treatment.
- The protein ubiquitination pathway and DNA damage response (DDR) network were commonly affected in both cell types.
- Distinct pathways were modulated: cytokine signaling in PS and DDR-related pathways in RS at 30 minutes post-treatment.
- Gene Ontology analysis revealed "regulation of transcription" in PS and "cellular response to stress" in RS as prominent affected processes.
- Dynamic expression patterns were observed for 374 DDR-related genes in PS and 158 in RS.
Conclusions:
- MMS exposure induces significant and stage-specific transcriptional reprogramming in mouse spermatogenic cells.
- The study elucidates the early molecular events, including DDR and protein ubiquitination, critical for germ cell response to DNA alkylation.
- These findings establish a transcriptional landscape crucial for understanding the impact of DNA damaging agents on male fertility.
