Identification And Analysis Of Differential Proteins In Sex-Sorting Sperm Of Goat And Sheep

With the intensive and large-scale development of mutton sheep industry in China,efficient breeding techniques have been gradually promoted and applied,such as semen preservation,synchronous estrus and artificial insemination,which can rapidly improve the population quality and production efficiency.High quality breeding ram has excellent genetic merits,can rapidly improve the production performance.Sexual sperm(X and Y sperm)of ram that been separated by the high speed flow cytometry based on live sperm DNA content difference,combined with the artificial insemination or sexual embryo transfer technology,can give full play to the reproductive potential of high quality breeding ram,expand the coverage rate of improved breed and promote the production of sheep.However,there are few studies on the molecular mechanism of sheep sexual sperm.The rise and update of proteomic methods provide an opportunity to reveal the molecular regulatory mechanism of sheep X and Y sperm at the protein level.This research focus on high quality sheep and goats,we carry out the comparative proteomic study of X and Y sperm from East friensian sheep and Boer goat using the high speed flow cytometry coupled with DIA based LC-MS/MS technique.In addition,we carry out the comparative proteomic study of seminal plasma between Hu sheep and Anhui white goat using the TMT based LC-MS/MS technique.The main results were obtained as follows:(1)Correlation between the biochemical indexes of seminal plasma and the conventional quality parameters of sperm,the conventional blood physiological and biochemical indexes were studied,X and Y sperm were sorted.The results showed that the sperm motility was more than 78%,the sperm density was more than 12×10^8/m L.There were proteins(such as ALB and GLB),enzymes(such as AST,LDH),non-enzymes(such as FUN,TG,TC),etc in seminal plasma,and these components were correlated with conventional sperm quality parameters,wherein some components were significantly different between sheep and goats.Blood indexs such as HCT,MCV,ALT,TG,ALB,AST,the values slightly deviated from the theoretical values,indicator values of all other tests were within the theoretical reference ranges.For sexual sperm sorted by high speed flow cytometry,the motility was ≥ 70%,the rate of normal morphology was ≥ 75%,the density was up to 10×10^8 /m L,and the sorting purity of X and Y sperm was ≥ 85%.(2)A total of 18,006 peptides and 1914 proteins were identified,of which 1897 proteins were quantified in the sorting sperm proteomic analysis based on the DIA LC-MS/MS technique,with 22 differentially expressed proteins(DEPs)were screened in the comparison group of X sperm and Y sperm from sheep,wherein 19 were up-regulated and 3 were downregulated.Chromatin DNA binding,RNA binding,ATP binding and transmembrane transport,protein transmembrane transport,cysteine endopeptidase activity activation,NADH dehydrogenase(ubiquitin)activity were significantly enriched and involved in spermic function regulation,among which the DEPs such as HNRPU,NUBP2,TCPQM,H13,RS5,CCT5,ASPH and NDUFB4 may play a regulatory role in the formation of chromatin structure during spermiogenesis,DNA repair,sperm excretion and motility maintenance,and sperm-egg fusion information communication.(3)A total of 15,226 peptides and 1961 proteins were identified,of which 1947 proteins were quantified in the sorting sperm proteomic analysis based on the DIA LC-MS/MS technique,with 24 DEPs were screened in the comparison group of X sperm and Y sperm from goats,wherein 9 were up-regulated and 15 were down-regulated.Spermatogenesis,ATPdependent RNA helicase activity,chromosome separation,chromatin DNA binding,spermzona pellucida binding,superoxide dismutase and other functional items were significantly enriched and involved in the regulation of goat sperm function,among which the DEPs such as CYLC2,DDX3 Y,CCDC112,ACTN4,ARSA,GRPL2 and SODC may play regulatory roles in sexual spermatogenesis,spermatogenesis chromatin structure,sex determination,metabolism,flagellum formation,sperm-egg binding.(4)A total of 10,137 peptides and 2416 proteins were identified,of which 2055 proteins were quantified in the seminal plasma proteomic analysis based on the TMT based LC-MS/MS technique,with 258 DEPs were screened in the comparison group of sheep and goats,wherein88 were up-regulated and 170 were down-regulated.Glucose homeostasis,cell motility,cell differentiation,embryonic placenta development,regulation of protein serine/threonine kinase activity,embryonic morphogenesis,sexual reproduction,spermatid development,sperm-egg recognition,fertilization,regulation of immune response,response to nutrient,and cell surface receptor signaling pathway were significantly enriched and involved in sperm reproductive process,among which the up-regulated DEPs such as MMP2,RNASE9,ZPBP,ROS1,and down-regulated DEPs such as IGFBP5,PLG,ADM,EGFR,SPINT2,ADAM2,ALPL,PDC-109 may play regulatory roles in spermiogenesis and energy supply for motility,sperm resistance to microenvironmental stimulation,and sperm delivery and survival.The differential proteins network interaction analysis showed that ENTPD6,ENTPD3,ENPP1,ENPP3,GMPR2 and NT5 E were clustered into a cluster,and CAT,PLG,TTR,FUCA2,EGFR,WFDC2 were the key nodes in the interaction network,which were the potential target proteins.In summary,this research established the differentially expressed protein profiles of sexsorted sperm and seminal plasma from sheep and goat,and identified several potential key proteins,which provided some theoretical reference for further study on the molecular regulation mechanism of sheep sexual sperm and improvement of the utilization efficiency of sheep semen.