Proteomic Analysis Of Dezhou Donkey Sperm Before And After Cryopreservation And Identification Of Protein Biomarkers Related To Freezability

During cryopreservation the abundance of functionally relevant proteins such as internal membrane permeability and energy metabolism in sperm is changed and the loss of function of these proteins causes damage to the plasma membrane system and organelles,leading to a reduction in sperm thawing viability and fertilization capacity.Comparison of the changes in the abundance of sperm proteins before and after cryopreservation would provide a reference for the study of the mechanism of sperm cryoinjuries.Additionally,sperm differ in terms of freezability in individuals of the same species.Comparing the difference in protein abundance between of sperm from good freezability ejaculates(GFEs)and poor freezability ejaculates(PFEs)provides a possibility to screen for freeze resistant marker proteins and freeze resistant marker genes.However,studies on the mechanism of damage to donkey sperm cryopreservation and screening for anti-freeze marker proteins have not yet been reported.In this study,we compared the motility characteristics of fresh spermatozoa(F group)and frozen spermatozoa(FT group),as well as the spermatozoa in good freezability ejaculates(GFE group)and poor freezability ejaculates(PFE group)of donkeys,and applied TMT labeling combined with high performance liquid chromatography tandem mass spectrometry(HPLC-MS)to identify the different abundance proteins(DAPs)in the F/FT and GFE/PFE groups.According to the results of the differences in motility characteristics of spermatozoa and different abundance proteins analyses in each group we obtained the following conclusions.1.The sperm motility,plasma membrane integrity rate,acrosome integrity rate and high mitochondrial membrane potential rate of the frozen sperm group were significantly lower than those of the fresh sperm group(P < 0.05);The sperm motility,plasma membrane integrity,acrosomal integrity and high mitochondrial membrane potential rate of the sperm group with good freezability were higher than those of the sperm group with poor freezability,and the differences were significant(P < 0.05).2.2,682 proteins were quantitatively identified by tandem mass spectrometry(TMT)polypeptide labeling technique.Setting the threshold of difference change to 1.5-fold revealed that among the 175 differential proteins in the FT/F group,the abundance of 28 proteins was significantly increased,and the abundance of 147 proteins was significantly decreased.Setting the threshold of difference change to 1.3-fold revealed that among the 74 differential proteins in the GFE/PFE groups,the abundance of 58 proteins was significantly increased,and the abundance of 16 proteins was significantly decreased.3.Molecular bioinformatics analysis of the differential proteins revealed that DAPs from F/FT groups sperm samples include SERPINE2,MMP2,PRDX2,and CAT,which are mainly related to proteasome and cell vesicle composition,have peptidase and metal ion binding enzyme activities,and are associated with protein hydrolysis and phosphorylation biological processes.KEGG pathway enrichment analysis showed that DAPs are mainly involved in proteasomal and lysosomal regulatory pathways.DAPs in GFE group sperm samples and PFE group samples mainly include HSP90β1,HYOU1,HSPA5,DDOST,S100A12 and ANXA1.A variety of these DAPs are involved in biological processes such as copper and calcium binding,regulation of RNA biosynthesis process,positive regulation of innate immune response and negative regulation of programmed cell death.KEGG pathway enrichment analysis showed that the GF group upregulated proteins are mainly involved in N-glycan biosynthesis and protein processing in the endoplasmic reticulum.Analysis of the above results led us to the following conclusions:1.The loss of proteins related to acrosome integrity and oxidation-reduction ability such as SERPINE2,MMP2,PRDX2,and CAT after cryopreservation may cause the decline of acrosome integrity and antioxidant capacity of thawed sperm,and thus lead to the decline of various physiological indicators after sperm thawing.2.Proteins with calcium regulation and cell repair functions such as HSP90β1,HYOU1,HSPA5,DDOST,S100A12 and ANXA1 are in higher abundance in sperm with good freezability and can be considered as biomarkers of freezability in donkey sperm.