Comparative Analysis of Epididymis Cauda of Yak before and after Sexual Maturity

Abstract

Epididymis development is the basis of male reproduction and is a crucial site where sperm maturation occurs. In order to further understand the epididymal development of yak and how to regulate sperm maturation, we conducted a multi-omics analysis. We detected 2274 differential genes, 222 differential proteins and 117 co-expression genes in the cauda epididymis of yak before and after sexual maturity by RNA-seq and proteomics techniques, which included TGFBI, COL1A1, COL1A2, COL3A1, COL12A1, SULT2B1, KRT19, and NPC2. These high abundance genes are mainly related to cell growth, differentiation, adhesion and sperm maturation, and are mainly enriched via extracellular matrix receptor interaction, protein differentiation and absorption, and lysosome and estrogen signaling pathways. The abnormal expression of these genes may lead to the retardation of epididymal cauda development and abnormal sperm function in yak. In conclusion, through single and combined analysis, we provided a theoretical basis for the development of the yak epididymal cauda, sperm maturation, and screening of key genes involved in the regulation of male yak reproduction.

Keywords: RNA-seq; epididymis; proteomics; sperm maturation; yak.

Figure 1

Histological observation of epididymis and testis. (A) Epididymis caput. (B) Epididymis corpus. (C) Epididymis cauda. (D) Testicular tissue. The blue box shows a partial enlarged (40×) view of the organization.

Figure 2

RNA-seq data summary. (A) The expression distribution of FPKM in each sample. (B) FPKM violin diagram. (C) PCA diagram. (D) Correlation heatmap and cluster analysis among samples. (E) Gene volcano map of the difference between 6m and 30m groups. (F) Cluster analysis of different gene groups. Y6m: 6-month-old group. Y30m: 30-month-old group.

Figure 3

GO, KEGG, and GSEA analyses for DEGs. GO enrichment analysis of DEGs showed that (A) was upregulated and (B) was downregulated. KEGG enrichment analysis of DEGs showed that (C) was upregulated and (D) was downregulated. GSEA enrichment results revealed upregulation of (E) and downregulation of (F). GO enrichment analysis ranked 10 entries from large to small according to the respective −log10 p value. KEGG analysis exhibited some significant enrichment results. GSEA analysis selected the top KEGG for enrichment analysis verification.

Figure 4

Overview of proteome sequencing data. (A) PCA diagram of two sample groups. (B) Differential protein volcano map. (C) Cluster analysis of differential proteins.

Figure 5

GO and KEGG enrichment analyses. (A) GO enrichment of upregulated DEPs. (B) KEGG enrichment analysis of upregulated DEPs. (C,D) GO and KEGG enrichment analyses of downregulated DEPs.

Figure 6

Network diagram of differential protein interaction in epididymal cauda. The protein–protein interaction network diagram was drawn using STRING. Cluster 1 and Cluster 2 represented upregulated DEP aggregation. Cluster 3 and Cluster 4 were formed when DEPs were downregulated.

Figure 7

Integration analysis. The differential genes and differential proteins were analyzed together. (A) presents the correlation between DEGs and DEPs. (B) presents the nine-quadrant diagram.

Figure 8

Analysis of GO and KEGG genes with the same expression trend. (A,B) present the GO and KEGG enrichment of co-up-regulated genes. (C,D) present GO and KEGG enrichment analyses with downregulated expression of proteins and mRNA.

Figure 9

Differential genes and differential proteins were verified through qRT-PCR and 4D-PRM. (A) Differential genes were verified through qRT-PCR, and the results were presented as Log₂FC. (B) The selected differential proteins were validated through 4D-PRM.