Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality

Abstract

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein-protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

Keywords: Sertoli cell; data-independent acquisition; germ cell; sheep; spermatogenesis; targeted proteomics; testis.

Figure 1

Differential protein expression profiles. (A) Volcano plot showing protein expression profiles between 3M and 1Y groups, 1Y and 3Y groups, and 3M and 3Y groups. (B) Venn diagram showing the shared and unique proteins. (C) Cluster analysis of proteins showing similar pattern of expression during testis development. 3M, 1Y, and 3Y correspond to 3-month-old, 1-year-old, and 3-year-old, respectively.

Figure 2

The GO annotation of DAPs in the four clusters. (A–D) represent the bubble plots of the significant GO terms for DAPs in cluster 1, cluster 2, cluster 3, and cluster 4, respectively. BP, biological process; CC, cellular component; MF, molecular function.

Figure 3

Representative significantly enriched KEGG pathway categories of DAPs in the four clusters.

Figure 4

Verification of 18 proteins with differential abundance identified by DIA analysis by PRM (n = 4 per group). Data represent the mean ± SD. Significance levels: two asterisks, p, 0.01; one asterisk, p, 0.05; ns, not significant. 3M, 1Y, and 3Y correspond to 3-month-old, 1-year-old, and 3-year-old, respectively.

Figure 5

Verification of 18 differentially abundant proteins at the mRNA level (n = 8 for each group). (A) Relative mRNA expression monitored by qPCR. (B) Heatmap of mRNA expression derived from our RNA-seq data (NCBI SRA accessions: SRR11348536–SRR11348547). Significance levels: two asterisks, p, 0.01; ns, not significant. 3M, 1Y, and 3Y correspond to 3-month-old, 1-year-old, and 3-year-old, respectively.

Figure 6

The PPI network and expression heat map of DAPs enriched at different stages of spermatogenesis. Node size indicates surrounding gene numbers (degrees) in the network. Spg, spermatogonia; pSpc, primary spermatocyte; sSpc, secondary spermatocyte; Std, spermatid; Spz, spermatozoa. 3M, 1Y, and 3Y correspond to 3-month-old, 1-year-old, and 3-year-old, respectively.

Figure 7

Identification of testicular microenvironment-relevant proteins. (A) The PPI network of protein-coding gene sets enriched on testicular microenvironment-related pathways. Network nodes represent proteins, and network edges represent protein-protein interactions. Node size indicates surrounding protein numbers (degrees) in the network. The proteins correlated with cell junctions are shown in red, and proteins correlated with glycolysis/gluconeogenesis are shown in blue. (B) The gene-pathway network according to cell junctions and glycolysis-related genes and their enriched pathways.