Spatio-temporal landscape of mouse epididymal cells and specific mitochondria-rich segments defined by large-scale single-cell RNA-seq

Abstract

Spermatozoa acquire their fertilizing ability and forward motility during epididymal transit, suggesting the importance of the epididymis. Although the cell atlas of the epididymis was reported recently, the heterogeneity of the cells and the gene expression profile in the epididymal tube are still largely unknown. Considering single-cell RNA sequencing results, we thoroughly studied the cell composition, spatio-temporal differences in differentially expressed genes (DEGs) in epididymal segments and mitochondria throughout the epididymis with sufficient cell numbers. In total, 40,623 cells were detected and further clustered into 8 identified cell populations. Focused analyses revealed the subpopulations of principal cells, basal cells, clear/narrow cells, and halo/T cells. Notably, two subtypes of principal cells, the Prc7 and Prc8 subpopulations were enriched as stereocilia-like cells according to GO analysis. Further analysis demonstrated the spatially specific pattern of the DEGs in each cell cluster. Unexpectedly, the abundance of mitochondria and mitochondrial transcription (MT) was found to be higher in the corpus and cauda epididymis than in the caput epididymis by scRNA-seq, immunostaining, and qPCR validation. In addition, the spatio-temporal profile of the DEGs from the P42 and P56 epididymis, including transiting spermatozoa, was depicted. Overall, our study presented the single-cell transcriptome atlas of the mouse epididymis and revealed the novel distribution pattern of mitochondria and key genes that may be linked to sperm functionalities in the first wave and subsequent wave of sperm, providing a roadmap to be emulated in efforts to achieve sperm maturation regulation in the epididymis.

Fig. 1. Overview of scRNA sequencing of mouse epididymis regions.

a Schematic of mouse epididymis collection and analysis. b UMAP visualization of the eightcell clusters identified in the epididymis after erythrocyte filtration. c UMAP visualization of the three regions of the mouse epididymis in P42 and P56 mice. Each epididymal region of the P56 epididymis has two biological replicates.

Fig. 2. Features of principal cell subpopulations.

a UMAP representation of the subpopulations of principal cells aligned in the caput, corpus, and cauda. Eight subclusters of principal cells were identified. b Heatmap showing the top 10 marker genes for each subpopulation (log1pRPM scaled by row). c GO enrichment analysis for the principal subpopulation. d Violin plot of actin (Actb) in the principal subpopulation (y-axis log1pRPM). The expression level of Actb was the highest in the Prc7 subcluster. e Violin plot showing representative marker genes for the principal subpopulation Prc7 (y-axis log1pRPM).

Fig. 3. Segmental DEGs of the mouse epididymis.

a The spatial proportion of each cell cluster is illustrated. b The distribution of the number of segmental DEGs in each cell cluster. c Heatmaps of DEG comparisons in three epididymal regions from the epididymal epithelial cell clusters (log1pRPM scaled by row). d Representative spatially specific DEGs of principal cells in three epididymal segments (y-axis log1pRPM).

Fig. 4. Characterization of the mitochondrial distribution in epididymal regions.

a The segmental percentage of mitochondrial transcripts (MTs) in epididymal epithelial cell clusters. b Stitched image of immunostaining of the mitochondrial marker cytochrome c in the mouse epididymis. Scale bar = 1 mm. c Representative images of cytochrome c staining in the caput, corpus and cauda epididymis. Scale bar = 50 µm. Red: cytochrome c (cyto C), Blue: DAPI for nuclear count staining. d Corresponding statistics of cytochrome c staining in the segmental epididymis. **P < 0.01; ***P < 0.001. e UMAP plots of mitochondrial transcript genes (mt-cytb and mt-Nd1) in the indicated cell clusters (color values were log1pRPM). f Representative mitochondrial transcript genes (mt-cytb and mt-Nd1) in sperm-depleted epididymal segments by qPCR analysis. The statistical significance was corpus or cauda vs. caput. ****P < 0.00001.

Fig. 5. Segmental DEG comparison of first-wave sperm (P42) production and adult sperm (P56).

a Heatmaps of DEGs for P42 and P56 spermatozoa in the caput, corpus, and cauda epididymis (log1pRPM scaled by row). b Violin plots showing segmental expression of spermatozoa DEG genes at different stages. NA: There were no representative DEGs that were highly expressed in P56 sperm compared to P42 sperm in the caput epididymis.