The Preliminary Proteomics Study Of Seminal Exosomes On Male Infertility Caused By Varicocele

Objective:Varicocele is one of the main reasons for male infertility,but the mechanism of male infertility is still unkown.To establish a normal human seminal exosomes profile as a reference for the comparative proteomic study of seminal exosomes from infertility patients with and without varicocele,to depict the potential molecular function and to develop potential biomarkers of seminal exosomes in male infertility patients with varicocele,and to further construct experimental rat varicocele model for studying of the molecular mechanism of male infertility underlying varicocele associated seminal exosomes proteins.Methods:1.Exosomes were extracted from semen specimens from 18 healthy volunteers with step-by-step centrifugations and ultracentrifugation(12 cases)and 8%PEG6000 enrichment(6 cases),respectively.These pellets were characterized by nanoparticle tracking analysis,western blotting,and transmission electron microscopy;2.These 12 normal seminal exosomes were extracted by ultracentrifugation method,then were performed by comprehensive proteomic analysis,some selectively proteins identified were validated by western blottiong.3.The differentially expressed proteins between seminal exosomes from 6 case of male infertility and 6 normal fertile volunteers were obtained,which were analysed by bioinformatics(GO analysis,including cellular component,biological process,molecular function).4.The model of experimental rat varicocele was induced by partially occluding left renal vein and completely ligating the branches of the LSV,the feasibility of clip surgically inducing rat varicocele was evaluated compared to the conventional method.Results:1.The pellets was successfully obtained from these two methods,they positively expressed exosomal protein markers(CD63,ALIX and TSG101).Nanoparticle tracking analysis demonstrated that these pellets were 30 to 150 nm in diameter,transmission electron microscopy showed round or elliptic membrane vesicles.2.A total of 1474 proteins were identified in all seminal exosomes samples,which were mainly involved in the biological processes including protein metabolism,energy pathway,protein metabolism,cell growth/maintenance and transport.Some selectively proteins such as PHGDH,LGALS3BP,SEMG1,ACTB,ALIX,GAPDH were validated by western blotting,and the results were consistent with the proteomics data;.3.In all,1457 proteins were identified in the comparative proteomics analysis of seminal exosomes from varicocele,and 1331 proteins was quantitative.Compared with the normal fertility group,a number of 175 proteins were differentially expressed between seminal exosomes from men with and without varicocele(up-regulaed 76 proteins and down-regulated 99 proteins),GO analysis showed that these proteins were mainly involved in cell growth and maintainace,energy metabolism,transport biological process disorders;4.Compared with sham operation,the criterion for successful experimental varicocele is that the diameter of the left spermatic vein was much than in the conventional and clip group,and no pathological changes in the left kidney were observed.In the conventional group,3 rats failed to surgically induce varicocele including left renal vein tear bleeding and left kidney hydration and other complications,while silver clip group 1 rat silver clip slipping off left renal vein,and the success rate of varicocele were 75%and 87.5%,respectively.Conclusions:1.Extraction and identification of seminal exosomes based on PEG6000 method are successfully established.2.Comprehensive proteomic profiles of seminal exosomes from healthy fertile man were established,which facilitates comparative proteomic studies of seminal exosomes as a reference in male infertility and other diseases.3.The initial description of proteomic profiles of seminal exosomes from infertile men with varicocele and fertile men was performed,the differentially expressed proteins were mainly involved in cell growth/maintenance,energy pathways,metabolism,transport biological process disorders,which may play a vital role in the mechanism underlying varicocele associated male infertility,and these proteins are potential biomarkers in male infertility.4.With a new clip technique,surgically inducing varicocele rat model becomes convenient and safe,which lays foundation for study of the molecular mechanism of male infertility underlying varicocele associated seminal exosomes proteins..