Cryopreservation Induces Acetylation of Metabolism-Related Proteins in Boar Sperm

Abstract

Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography-mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters.

Keywords: boar; energy metabolism; protein acetylation; sperm cryopreservation.

Figure 1

Identified proteins, peptides, sites and their correlation. Peptide length shows the number of amino acids present in different peptides (A); heat map is drawn using Pearson’s correlation coefficient (PCC): positive (close to +1), negative (close to −1) and no-correlation (close to 0) (B); boxplot shows the relative standard deviation between the fresh (Fs) and frozen (Fts) sperm: the smaller the overall relative standard deviation (RSD), the better the quantitative repeatability (C); volcano plot of the modified sites: orange and green dots represent the upregulated and downregulated modified proteins and sites, while grey indicated no change in expression (D); hierarchical diagram of the modified sites: the X-axis represents different fresh and frozen sperm groups and Y-axis represents the upregulated or downregulated proteins (E).

Figure 2

GO term analysis and Log2 fold change comparison between fresh and frozen–thawed groups. Gene ontology (GO) of proteins participating in biological functions (A); KEGG pathways regulated by modified proteins and sites (B); pie diagram showing subcellular localization of modified proteins and sites (C). All data are shown as Log2 fold change enrichment of the proteins for relative expression. The size of the circle in B indicates the number of proteins involved in a particular process along the Y-axis, while color intensity (strong red for higher and light blue for lesser roles) shows participation in different processes.

Figure 3

Cluster analysis showing the degree of enrichment of differentially expressed proteins in fresh and frozen sperm groups. Biological processes (A); molecular functions (B). The intense red color indicates the highest enrichment and blue indicates the lowest enrichment. Different groups (Q1, Q2, Q3 and Q4) and functions are given horizontally and vertically, respectively.

Figure 4

Protein–protein interactions of differentially acetylated proteins between fresh and post-thawed boar sperm. The green color indicates downregulated proteins, while red indicates upregulated proteins.

Figure 5

Relative mRNA and protein expressions of acetylated enzymes (IDH2, LDHC, MDH2 and SIRT5) in fresh and post-thawed boar sperm. (A) WB analysis indicates protein expression between fresh and frozen (post-thawed) sperms; (B) mRNA expression of IDH2, LDHC, MDH2 and SIRT5 between fresh and post-thawed sperms; (C) relative protein expressions of IDH2, LDHC, MDH2 and SIRT5 between fresh and post-thawed sperms; (D) WB analysis of SIRT5 in fresh, frozen, siSIRT5 and NC groups; (E) relative gene expression of SIRT5 in fresh, siSIRT5 and NC groups; (F) relative protein expression of SIRT5 in fresh, frozen, siSIRT5 and NC groups. All the data were subjected to statistical analysis and considered significant at (* p < 0.05) and highly significant at (** p < 0.01 and *** p < 0.001). Isocitrate dehydrogenase (IDH2); malate dehydrogenase (MDH2); sirtuin5 (SIRT5); lactate dehydrogenase (LDHC).

Figure 6

Knockdown of SIRT5 (siSIRT5) affects total and progressive sperm motilities. (A) Total and progressive motilities were significantly higher in the fresh group compared to the frozen (post-thawed) and siSIRT5-treated groups. Total motility was not significantly different among the fresh, siSIRT5 and NC groups before 6 h of transfection, but significant differences were observed at and after 6 h. (B) Progressive motility started to differ significantly at 3 h of transfection. (C) Total and progressive motilities were significantly higher in untreated frozen groups compared to siSIRT5-treated frozen groups. ** p < 0.01; *** p < 0.00.1. siSIRT5 6 h, siSIRT5 12 h X and siSIRT5 24 h X indicate transfection of sperm for 6 h, 12 h and 24 h, respectively, before freezing.

Figure 7

Knockdown of SIRT5 (siSIRT5) affects acrosomal integrity in boar sperm. (A) siSIRT5 sperms were subjected to PI and FITC-PNA stains: as shown in the image titled Merge, the red sperms (marked with a yellow arrow) are dead (PI⁺/FITC-PNA⁻); sperms with intact green caps have intact acrosome and membranes (blue arrow) (PI⁺/FITC-PNA⁺); sperms with damaged green caps are reacted ones (green arrow) (PI⁺/FITC-PNA⁺). (B) Acrosomal status was subjected to statistical analysis and evaluated after 24 h. The results are representative of at least three independent experiments (mean ± SEM). * p < 0.05; ** p < 0.01. Propidium iodide (PI); fluorescein isothiocyanate (FITC); peanut agglutinin (PNA); negative control (NC).

Figure 8

Mitochondrial membrane potential (MMP). Analysis of MMP by flow cytometry: blue color shows cells with higher MMP while purple indicates cells with lower MMP; control (without JC−1 dye), CCCP (positive control (PC)), cells with highly disrupted MMP), fresh (control), NC, frozen (post-thawed) and siSIRT5 treated groups are shown as a–f, respectively (A). The ratio of JC-1 red to green was sorted out to determine the statistical significance between the groups (B). * p < 0.05; ** p < 0.01; *** p < 0.001. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP); negative control (NC).