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Toxicotranscriptomics of single cell RNA-Seq and conventional RNA-Seq in TCDD-exposed testicular tissue

ABSTRACT: Spermatogenesis is a dynamic, tightly conserved process that is susceptible to environmental contaminants. Exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is believed to be a cause of idiopathic male infertility. To understand TCDD-induced disruption of spermatogenesis at the single cell level, we profiled adult zebrafish testes with and without early-life exposure to TCDD. Secondly, to investigate the potential for technical artifacts in scRNA-seq, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples, and included an intact control fish to account for dissociation-induced artifacts. We found scRNA-seq and bulk-seq of dissociated testes produced markedly different profiles both toxicologically and transcriptomically. Through scRNA-seq, we found TCDD exposure induces mid-late germ cell apoptosis while increasing the population of spermatogonial stem cells. Bulk-seq on the same cells returned results of immune dysregulation. The transcriptome was unequally detected by scRNA-seq and bulk-seq, with scRNA-seq biased towards detecting longer and more abundant transcripts, but this was not mediated by TCDD exposure.

ORGANISM(S): Danio rerio

PROVIDER: GSE193758 | GEO | 2022/05/28

REPOSITORIES: GEO

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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

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Toxicotranscriptomics of single cell RNA-Seq and conventional RNA-Seq in TCDD-exposed testicular tissue

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