Epididymal segment-specific miRNA and mRNA regulatory network at the single cell level

Spermatozoa released from the testis cannot fertilize an egg before becoming mature and motile in the epididymis. Based on three bulk and one single-cell RNA-seq (scRNA-seq) data series, we compared mRNA or miRNA expression between epididymal segment-specific samples and the other samples. Hereby, we identified 570 differentially expressed mRNAs (DE-mRNAs) and 23 differentially expressed miRNAs (DE-miRNAs) in the caput, 175 DE-mRNAs and 15 DE-miRNAs in the corpus, 946 DE-mRNAs and 12 DE-miRNAs in the cauda. In accordance with respective DE-miRNAs, we predicted upstream transcription factors (TFs) and downstream target genes. Subsequently, we intersected target genes of respective DE-miRNAs with corresponding DE-mRNAs, thereby obtaining 127 upregulated genes in the caput and 92 upregulated genes in cauda. Enriched upregulated pathways included cell motility-related pathways for the caput, smooth muscle-related pathways for the corpus, and immune-associated pathways for the cauda. Protein-protein interaction (PPI) network was constructed to extract key module for the caput and cauda, followed by identifying hub genes through cytohubba. Epididymis tissues from six mice were applied to validate hub genes expression using qRT-PCR, and 7 of the 10 genes displayed identical expression trends in mice caput/cauda. These hub genes were found to be predominantly distributed in spermatozoa using scRNA-seq data. In addition, target genes of DE-miRNAs were intersected with genes in the PPI network for each segment. Subsequently, the miRNA and mRNA regulatory networks for the caput and cauda were constructed. Conclusively, we uncover segment-specific miRNA-mRNA regulatory network, upstream TFs, and downstream pathways of the human epididymis, warranting further investigation into epididymal segment-specific functions.