The Preliminary Study Of Motility And Mechanisms Of Action Underlying The Decrease Of Pseudomonas Aeruginosa On Boar Sperm

Pseudomonas aeruginosa is the dominant strain in boar semen,which can reduce the sperm motility,pregnancy rate and litter size of sow by damaging the sperm plasma membrane,acrosome and ultrastructure of mitochondria.At present,the specific mechanism of action is still unclear.In particular,there are few reports about the effects of Pseudomonas aeruginosa on sperm energy metabolism and protein post-translational modification levels.Therefore,in this study,the reduction of sperm motility as the starting point,in assessing the role of Pseudomonas aeruginosa on sperm motility and morphological changes,mainly for sperm energy metabolism pathways and protein post-translational modification levels changes,in-depth investigate the effects of Pseudomonas aeruginosa on glycolysis,mitochondrial oxidative phosphorylation,phosphorylation and acylation levels,in order to elucidate the effects of Pseudomonas aeruginosa on duroc sperm motility and mechanism of action,providing theoretical basis and reference for the development of boar semen quality standards.This study mainly includes the following four parts:1.The effects of different concentrations Pseudomonas aeruginosa on boar sperm were analyzed specific mechanism of action of Pseudomonas aeruginosa on sperm in this experiment.With the increase of Pseudomonas aeruginosa concentrations,the results showed that the sperm motility was significantly decreased(P<0.05),plasma membrane,acrosome integrity and mitochondrial membrane potential were also significantly decreased(P<0.05).The electron microscopy showed that the sperm plasma membrane,acrosomal and mitochondrial ultrastructure were severely damaged,confirmed that Pseudomonas aeruginosa mainly through the nanotube channels and the release of OMVs to reduce sperm motility.2.The maintenance of sperm motility is mainly derived from glycolysis and mitochondrial oxidative phosphorylation energy metabolism pathways.At the same time,it is closely link with protein posttranslational modification levels.Does Pseudomonas aeruginosa reduce sperm motility by regulating boar sperm energy metabolism and protein post-translational modification levels? With the increase of Pseudomonas aeruginosa concentrations,the results showed that the activity of GAPDH,the content of NADH in the electron transport chain and the level of intracellular ATP in the process of glycolysis were significantly decreased(P<0.05).Meanwhile,deacetylation,desuccinylation modification levels were increased,the reduction of c AMP level can promote protein serine/threonine and tyrosine dephosphorylation levels,following the c AMP-PKA signaling pathway to reduce sperm motility.3.For the first time,we found that OMVs of Pseudomonas aeruginosa played an important role in reducing boar sperm motility in this study.To further explore the ways of OMVs affect sperm energy metabolism and posttranslational modification of protein,sperm were treated with different concentrations of OMVs in this experiment.With the increase of OMVs concentrations,the results showed that the activity of GAPDH,a key enzyme in the process of glycolysis,was decreased,but there was no significant difference(P>0.05).The content of NADH and intracellular ATP in the electron transport chain were significantly decreased(P<0.05).At the same time,the levels of deacetylation and desuccinylation were increased,but there were no significant difference(P>0.05).The decrease of c AMP level can promote protein serine/threonine and tyrosine dephosphorylation levels,following the c AMP-PKA signaling pathway to reduce boar sperm motility.The results suggested that OMVs as the mainly mechanism of action of Pseudomonas aeruginosa,but it was different from that Pseudomonas aeruginosa affected sperm motility and mechanism of action,mainly through the effects on NADH and ATP levels,following c AMP-PKA signaling pathway affected sperm protein phosphorylation modification levels and reduce sperm motility.4.In order to screen the specific proteins,which can reduce sperm motility in OMVs,the types and quantities of OMVs proteins were investaged by mass spectrometry.A total of 152 proteins were identified in OMVs.Candidate proteins were identified as Pa AP,Opr F,Opr E,Opr D,Opr H,Opr C,Hap 2 and Hcp and so on.The results of KEGG pathway enrichment showed that it mainly involved in the process of aminoacyl t RNA biosynthesis,arginine biosynthesis,β-lactam resistance,C5-branched dibasic acid metabolism and cationic antimicrobial peptide resistance.The results of GO enrichment showed that the difference expression of genes were related to the metabolic processes of organonitrogen compounds,the types of cytoplasmic part components and the function of the bounding protein molecules.Taken together,Pseudomonas aeruginosa mainly through the adhesion of sperm to form nanotube channels and OMVs and other mechanisms of action,disrupt the morphological structure of sperm,inhibit the key enzyme GAPDH activity in glycolysis,and reduce the mitochondrial oxidative electron transport chain NADH content and intracellular ATP level.At the same time,promote protein deacetylation and desuccinylation,reduce c AMP level,thereby promoting protein serine/threonine dephosphorylation,tyrosine dephosphorylation levels,phosphorylation changes follow c AMP-PKA signaling pathway further reduces sperm motility.