Single-Cell RNA Sequencing Reveals an Atlas of Meihua Pig Testis Cells

Abstract

Mammalian spermatogenesis is a complex biological process that is regulated by multiple types of cells. The heterogeneity of these cells poses a challenge for analyzing different cell types at different developmental stages. To characterize the transcriptomic landscape of porcine spermatogenesis and identify potential marker genes for spermatogonia, an unbiased transcriptomic study of spermatogenesis in neonatal and sexually mature six-month-old Meihua pigs was performed using 10× Genomics single-cell RNA sequencing (scRNA-seq). Through the collection of scRNA-seq data from 13,839 cells from Meihua pig testes, three germ cells (spermatogonia, spermatocytes and spermatids) and eight somatic cells (Sertoli cells, Leydig cells, myoid/stromal cells, endothelial cells, T cells/macrophages and erythroblasts) were identified. Pseudo-timing analysis showed that myoid cells and stromal cells originated from common progenitors in Meihua pigs. Functional enrichment analysis revealed that the differentially expressed genes (DEGs) in testicular somatic cells were enriched in the pathways of Ribosome, Oxidative phosphorylation, Protein processing in endoplasmic reticulum, Retrograde endocannabinoid signaling, Cellular senescence and Insulin signaling. Meanwhile, in the three different germ cells, except for pathways which were the same as the first three pathways for somatic cells, DEGs were also enriched in the Spliceosome, Cell cycle, Autophagy and Mitophagy pathways. Furthermore, the candidate marker gene TKTL1 in spermatogonia was identified using immunohistochemistry and immunofluorescence. In conclusion, we collected transcription datasets and constructed single-cell developmental maps of germ cells and somatic cells during the testicular development of Meihua pigs, which provided new insights into the spermatogenesis of Meihua pigs and the development of various types of cells in their testes.

Keywords: Meihua pig; TKTL1; single-cell transcriptome sequencing; testis.

Figure 1

Histological observation of Meihua pig testicular tissue sections. (A) 1 week old. (B) 6 months old. SPG: spermatogonia; SPC: spermatocyte; SPT: spermatid; SC: Sertoli cell; LC: Leydig cell. Left bar = 200 µm; right bar = 50 µm.

Figure 2

Classification of single-cell subpopulations and their visualization. (A) UMAP clustering analysis of all testicular cells. (B) Cell clusters in UMAP according to groups, with cells colored based on age. (C) The expression pattern of the selected marker genes on the UMAP map. (D) UMAP plots of testicular cell clusters defined by scRNA-seq analysis; different colors represent different cell types. (E) The percentage of cells in each of the 11 clusters in two samples (Y-axis). Clusters are distinguished by color.

Figure 3

Pseudo-time analysis of myoid cells and stromal cells. (A) Marker genes of different cells with UMAP plot. (B) Violin plots of different cell type-specific gene expression patterns across different clusters. (C,D) The pseudo-time trajectory of the myoid cells and stromal cells, colored according to their predicted cluster (C) and pseudo-time. Cells with a darker color are in the front position of the pseudo-time, and cells with a lighter color are in the back position of the pseudo-time (D). The numbers in the figure represent the nodes of the pseudo-time trajectory.

Figure 4

Functional enrichment analysis of different types of testicular somatic cells. (A) GO analysis of DEGs in Sertoli cells. (B) KEGG analysis of DEGs in Sertoli cells. (C) GO analysis of DEGs in myoid/stromal cells. (D) KEGG analysis of DEGs in myoid/stromal cells.

Figure 5

Functional enrichment analysis of testicular germ cell types. (A) GO analysis of DEGs in germ cell types. (B) KEGG analysis of DEGs in germ cell types.

Figure 6

Expression and distribution of potential marker gene. (A,B) Re-clustering of spermatogonia, colored by assignment to six sub-clusters (A) and two samples (B). (C) Expression of UCHL1 and TKTL1 in Meihua pig spermatogonia. (D) Violin plots of UCHL1 and TKTL1 expression across six sub-clusters. (E) Immunohistochemistry analysis of location of TKTL1 in Meihua pig testis. The red arrows indicated the TKTL1-positive cells. Bar = 50 µm. (F) Immunofluorescence analysis of location of TKTL1 and UCHL1 in Meihua pig testis. Bar = 20 µm.