Preliminary Study On Proteome Of Seminal Plasma Exosomes In Buffalo

The buffalo(Bubalus bubalis)is an excellent large livestock with both milk and meat in the subtropical region of southern China.Buffalo has a wide range of industrial development value.However,in the middle-temperate zone,the reproductive efficienty of the buffalo is lower than other bovine species.Low sperm motility is one of the important influencing factors.This greatly limited the development of the milk buffalo industry and breed improvement of local buffalo.Sperm is a highly specialized and concentrated cell which can no longer grow and divide.The transcription and translation functions of the sperm genome are nearly silence,and sperm mainly depends on the internal environment of spermatoplasmic development to regulate its maturation process.Normal fertilization depends on not only internal factors in sperm development,but also external factors in the seminal plasma that affect sperm function.Seminal exosomes are extracellular vesicles derived from numerous adnexal gonads in the male reproductive tract,which play an important role in sperm maturation and fertilization.Proteomics is an important approach to study the maturation and fertilization of mammalian sperm,which can comprehensively search for proteins that affect sperm function and high-throughtput screen key genes or biomarkers.At present,there are few proteomic reports on seminal plasma exosomes proteomics study.Therefore,this study explored the relationship between buffalo seminal plasma exosomes and sperm function through proteomics techniques,and the key factors of exosomes were found to regulating sperm function.This study is helpful to explain the causes of low sperm motility in buffalo and can be applied to guide the breeding of new buffalo breeds.The results were shown as follows:1.Isolation and identification of buffalo seminal plasma exosomes.The improved differential centrifugation combined with sucrose desity gradient method was used to isolate and extract exosomes of high and low motility seminal plasma.The protein markers CD81,TSG101 and Alix of exosomes were validated by Western blot.The particle size of exosomes was analyzed by Nanoparticle Tracking Analyzer(NTA),and the results showed that the particle size was concentrated on 119.4 nm,accounting for 99.2%.The morphology of exosomes was observed by Transmission Electron Microscopy(TEM),and the results showed that the exosomes had an obvious saucer-like structure.2.The proteomic profiles of buffalo sperm,seminal plasma and seminal plasma exosomes were established and analysis.Using Liquid Chromatography tandem Mass Spectrometry(LC-MS/MS)proteomics strategy,a total of 1894,1247 and 1343 proteins were identified in spermatozoa,seminal plasma and seminal plasma exosomes,respectively.GO analysis showed that the proteins of spermatozoa,seminal plasma and exosome were mainly involved in REDOX process and protein transport process,with ATP and GTP binding ability,and were located on exosomes,cytoplasm and mitochondria.KEGG analysis showed that the metabolic pathways were the most abundant proteins in sperm,seminal plasma and seminal plasma exosomes.Spermatozoa has the largest number of proteins involved in pathways related to energy metabolism.In seminal plasma and seminal plasma exosomes,only a small amount of proteins are involved in amino acid metabolism and assimilation pathways.The seminal plasma stabilizes the internal environment with complement system and the proteasome pathway.Proteins in exosomes only participate in the proteasome pathway,indicating that proteasome is the main way for exosomes to exert their protective function.3.Quantitative proteomic analysis of high and low motility seminal plasma exosomes in buffalo.i TRAQ combined with Liquid Chromatography tandem Mass Spectrometry(LC-MS/MS)proteomics strategy was used to quantitatively analyze high and low motility seminal plasma exosomes.High motility buffalo seminal plasma exosomes was the control group,a total of 160 differentially expressed proteins(change folder >1.2),including 119 down-regulated proteins and 41 up-regulated proteins,and the changes of differential proteins showed a general downward trend.GO analysis of the differential proteins showed that most of the differential proteins were located in sperm and vesicles,with activities of hydrolase and metalloproteinase,and were involved in sperm-egg recognition,fertilization,single fertilization,sperm-zona pellucida binding,monobiotic reproduction,cell recognition and other processes.KEGG analysis results showed that differential proteins were mainly involved in the PPRP signaling pathway,calcium signaling pathway,c AMP signaling pathway,etc.,and the expression levels of 10 proteins such as IZUMO1 were significantly down-regulated in low-motility seminal plasma exosomes.In summary,the exosomes of buffalo seminal plasma were isolated and identified.The proteome of the buffalo spermatozoa,seminal plasma and exosomes were constructed.The differential expression proteins of exosomes of high and low motility seminal plasma were identified.A series of exosome proteins were identified.The relationship of exosome and buffalo sperm motility was further analyzed.This study provides a new perspective for the study of reproductive mechanism of buffalo.