Proteomics Study On The Role Of Melatonin In Frozen Bovine Sperm

The semen cryopreservation has an important and positive impact on improving breeds and preserve the excellent germplasm resources.Post-thaw fertility of sperm is dramatic decreased and differs significantly among the bulls,which is a common problem for cryopreservation of semen.Melatonin is a strong antioxidant and can remove excess active oxygen in sperm cells,to improve the quality of post-thaw sperm.Here the proteomics parallelled to phosphoproteomics of pre-freezing,post-freezing and adding melatonin post-freezing sperm samples were used to explore the molecular mechanism of sperm cryo-damage and protection role of melatonin on the semen cryopreservation.In this thesis,the effect of different sample preparation methods on bovine sperm proteomic was systematically studied.Using cryo-sperm of Angus cattle as the experimental material,5 different lysates were used to extract the sperm protein,and3 different enzymatic hydrolysis methods were used to obtain the peptide fragments.The LC-MS/MS system used the same method to obtain mass spectrometry data.The experimental results show that the protein extraction ability of bovine sperm BS is the strongest and BU is the weakest;BT,BR and BD are equivalent.But BS-FASP is not suitable for bovine sperm proteomics research.The proteins identified by in-gel enzymolysis are more than FASP method,but The process of enzymolysis in-gel is complicated and time-consuming.The BD-based solution digestion method identified the largest number of proteins,the membrane protein extraction and identification ability was strong,and the operation time was the shortest,which is an ideal method for sample preparation of cattle sperm.In this paper,proteomics and phosphoproteomics were conducted on sperm samples of Mongolian cattle and Luxi cattle pre-freezing,post-freezing and adding melatonin post-freezing.The experimental results show that the semen freezing-thawing process seriously affects the sperm mitochondrial oxidative phosphorylation process.The mitochondrial inner membrane respiratory chain complex?,?and?partial proteins,mitochondrial membrane pore protein VDAC2,PPIF,and Cytochrome C expression are increased.Excessive oxidative respiration increases the expression of Cytochrome C,and changes in mitochondrial membrane pores cause Cytochrome C to be released from the mitochondrial inner membrane to the cytoplasm,resulting in apoptosis.The addition of appropriate concentration of melatonin(10~-66 M)reduces the change of the above proteins and decrease the freezing damage to sperm,but high concentration of melatonin(10~-33 M)will damage sperm motility.In the study,it was found that the expression of heat shock protein HSP90B1 decreased in sperm after freeze-thawing,and the decrease degree was reduced after adding 10~-66 M melatonin,but HSP90B1 decreased significantly after adding 10~-33 M melatonin,which was associated with sperm vitality data,indicating that the protective effect of melatonin on frozen sperm may be related to HSP90B1 protein.The results of phosphoproteomics showed that the freeze-thawing of semen significantly increased the phosphorylation level of sperm protein.These phosphorylated proteins KEGG are mainly enriched in the glycolysis/gluconeogenesis energy pathway,and protein phosphorylation modification plays an important role in the regulation of sperm stress.The addition of appropriate concentration of melatonin can effectively inhibit the change of phosphorylation modification of sperm protein during the freeze-thaw process.The results of sperm proteomics research showed that Mongolian cattle identified more proteins than Luxi cattle,and the differential proteins were mainly about acrosomal vesicle-related proteins.The difference in the number of sperm proteins and phosphorylated peptides before and after freezing in Luxi cattle was significantly higher than that in Mongolia.From the molecular level,it was confirmed that the freezing damage rate of Mongolian cattle sperm was lower.The results of motif enrichment of differentially phosphorylated peptides show that there are mainly 4 motifs enriched for differentially phosphorylated peptides after freezing and before freezing in Luxi cattle.After melatonin was added,the amount of motifs enriched in the differentially phosphorylated peptides of sperm after freezing and before freezing was reduced to 1 and it was similar to the results of motif enrichment of Mongolian cattle after freezing and before freezing.Therefore,the mechanism of action of melatonin in sperm freezing may be to reduce the changes of protein phosphorylation modification during freezing by changing the activity of kinases,thereby reducing the cryo-damage of sperm.