Difference Analysis Of Fresh And Capacitated Boar Sperm Proteins And Functional Verification Of PFK

When the sperm is released from the male reproductive tract,it does not have the ability to fertilize the ovum.The sperm needs to move to the female uterus or fallopian tube for a period of time before it obtains the ability to combine with the oocyte to form a fertilized egg.The sperm modification process that leads to the functional transformation is called"capacitation".The main energy sources of capacitation are mitochondrial aerobic oxidation and glycolysis,but the specific mechanism of these two pathways has not been clear.In this study,we first used the principle of proteomics and i TRAQ technology to analyze the protein difference of fresh and capacitated boar sperm.Then,the key proteins that affect sperm capacitation were obtained by bioinformatics technology,such as functional annotation,expression level cluster analysis,KEGG signal pathway analysis and so on.Finally,the biological information of the key proteins was analyzed,and the effects of the key proteins on the antioxidation and glycometabolism of capacitated sperm were verified.The results will provide theoretical basis for the study of sperm capacitation mechanism.The specific methods and results are as follows:Experiment 1,the protein difference analysis,based on i TRAQ technology,of fresh and capacitated boar sperm.The sperm of epididymis head of sexually mature boar incubated with capacitated solution was used as the experimental group,and the non capacitated group without adding bicarbonate and Ca²⁺was used as the control group.Firstly,the two groups of sperm proteins were labeled and sequenced by i TRAQ technology,then the differential protein data were searched in the GO database,and finally the differential genes were enriched by KEGG signal pathway.475 differential proteins were detected in fresh and capacited spermatozoa,of which 303 proteins were up-regulated and 172 proteins were down-regulated.These differential proteins were the main ones of cell components,such as cell membrane（13.71%）,organelle（10.66%）,cytoplasm（9.40%）,which were mainly involved in the biological processes（nitrogen metabolism,redox,biosynthesis,metabolism of organic cycle compound and metabolism of cell aromatic compound）and the molecular functions（oxidoreductase activity,transport activity,substrate specific transporter activity,nuclear acid binding,ionic transmembrane transport activity and cofactor binding）.Oxidative phosphorylation,cholesterol metabolism,ketone synthesis and degradation,aminoacyl t RNA biosynthesis,sphingolipid signaling pathway and lysine degradation are the main signaling pathways of sperm capacitation.Experiment 2,cloning and bioinformatics analysis of boar sperm PFK（phosphofructokinase,PFK）gene.PFK gene of boar sperm was cloned by puc57 vector and product bioinformatics was analyzed.The results showed that PFK contained 782 amino acids,the theoretical size of the protein was 83.819 Ku,the theoretical isoelectric point was6.96,and it was an acidic hydrophilic protein,the molecular formula was C₃₆₇₄H₅₈₉₇N₁₀₅₁O₁₁₀₉S₄₀.In addition,PFK gene contained 2 N-glycosylation sites,14 O-glycosylation sites and 35 phosphorylation sites,which can promote sperm surface modification;there were binding sites of PKC and PKA specific protein kinase,which can participate in hexose metabolism and monosaccharide metabolism.Experiment 3,the effect of PFK on the antioxidation of capacitated sperm.After PFK was applied to capacitated sperm for 2 h,the content of antioxidants was detected,and SDHA was used as the internal reference gene to analyze the expression of SOD,CAT,TR1 and GPx4m RNA.When the concentration of PFK was 0.5μmol/L,the activity of SOD and T-AOC was significantly higher than that of the control group（P<0.01）.5μmol/L PFK significantly increased CAT content（P<0.01）,and 1μmol/L PFK was the optimal concentration to inhibit MDA production（P<0.01）.When the concentration of PFK was more than 0.5μmol/L,the m RNA expression of SOD and TR1 gene decreased gradually（P<0.01）.0.5-5μmol/L PFK didn’t significantly affected the m RNA expression of CAT（P>0.05）,but 0.125μmol/L PFK significantly up-regulated the parameter（P<0.01）.The expression level of GPx4 gene showed a concentration dependence,gradually increasing with the increase of PFK concentration（P<0.01）,and reaching the maximum value at 1μmol/L.Experiment 4,the effect of PFK on the glycometabolism related substances and gene expression of capacitated sperm.After incubation of capacitated spermatozoa with PFK,the content of glycometabolism related substances was detected,andβ-actin was used as the internal reference gene to analyze the expression of glycometabolism related genes.When the concentration of PFK was above 0.5μmol/L,the content of F1,6P2 increased significantly（P<0.05）,while the content of F1,6P2 was not significantly affected by 0.125-0.5μmol/L PFK（P>0.05）.With the increase of PFK concentration,the activity of HK was significantly inhibited（P<0.01）,and the total cholesterol content gradually increased（P<0.05）.0.125μmol/L PFK significantly increased the activity of tyrosinase（P<0.05）.When the concentration was more than 0.25μmol/L,the activity of tyrosinase significantly increased（P<0.01）,and reached the optimal level at 5μmol/L.The expression of PFK,SLC2A3 and MCT was significantly increased with the increase of PFK concentration（P<0.01）.When the concentration of PFK was 0.5μmol/L,the expression of HK-1 was significantly inhibited（P<0.01）.In conclusion,it was firstly made by screening the differential proteins of fresh and capacitated boar sperm in this study.Then,the physical/chemical properties and biological characteristics of PFK were studied by bioinformatics simulation.Finally,by analyzing the effect of PFK on the antioxidation and glycometabolism of capacitated sperm,the author verified that PFK can significantly enhance the antioxidation and glucometabolism of capacitated sperm,which laid a molecular foundation for the study of capacitation mechanism of boar sperm.