Analysis Of The Differential Proteomics Between Fresh And Frozen-thawed Dorper Sheep Spermatozoa Based On TMT Technique

The process of cryopreservation of sperm results in lethal or sublethal damage to spermatozoa,which affects the pregnancy rate of animals during artificial insemination,but the mechanism of cryopreservation damage of sheep sperm is not clear.In this study,the physiological indexes of fresh spermatozoa and frozen-thawed spermatozoa of Dorper sheep were analyzed.The differential proteins between fresh sperm group and frozen-thawed sperm group were identified by TMT(Tandem Mass Tag)labeling combined with high performance liquid chromatography-tandem mass spectrometry(HPLC-MS).The results are as follows:1、There were significant differences in sperm motility,sperm plasma membrane integrity,acrosome integrity,average velocity and vibration index between fresh sperm group(F group)and freeze-thawed sperm group(FT group).The linear velocity of fresh spermatozoa was not significantly different from that of frozen-thawed spermatozoa.2、A total of 2039 proteins were identified and 2024 proteins were quantified.1.5 times the difference change threshold,365 differential proteins were found,of which 63 were up-regulated and 302 were down-regulated.3、Through the molecular bioinformatics analysis of the differential proteins of Dorper sheep before and after freezing and thawing,it was found that:The main results are as follows:(1)GO annotation analysis is mainly involved in the biological process of GO analysis:cell process,metabolic process,single organism process and so on.The GO analysis of cell composition was mainly as follows:intracellular,organelle and membrane.Molecular function GO analysis is mainly as follows:binding function and catalytic function.(2)KEGG pathway analysis showed that differential proteins were mainly involved in five biological pathways:thermogenesis,oxidative phosphorylation,Parkinson’s disease,Huntington disease and Alzheimer’s disease.(3)Through the analysis of the interaction network of differentially expressed proteins,it was found that ubiquinone oxidoreductase subunit B6,proteasome 26S nonATP enzyme subunit 4 and TCP-1 complex were at the core position compared with other proteins.4、Fourteen differentially expressed proteins were selected for PRM verification.The results showed that the quantitative expression abundance of PRM was consistent with the change of protein abundance recorded in TMT analysis.To sum up,10 proteins such as heat shock protein 90abl(HSP90abl),TCP-1 complex(CCT8),calpain 11(CAPN11)and fibrin like protein 2(FGL2)play an important role in sperm cryopreservation.Proteasome 26S non-ATP subunit 4(PSMD4),TCP-1 complex(CCT8),matrix metalloproteinases 2(MMP2)and calpain 11(CAPN11)related proteins accounted for a large proportion of the significant proteins.Changes in these proteins may affect the quality of cryopreserved semen.The preliminary experimental results provide a reference for further improving the effect of low temperature storage of sheep spermatozoa.