Preliminary Study On Proteomics Before And After Buffalo Sperm Capacitation

Swamp buffalo(Bubalus Bubalis)is the most important germplasm resource,with multi-purpose animals in subtropical regions of southern China,providing a high quality of milk and meat.However,buffalo has a low reproductive efficiency and the obstacles of sperm fertilization process are vital factor of low fertilization rate.Unlike somatic cells,spermatozoa are highly specific differential cells.The transcription and translation process are completely silence in matured spermatozoa.Sperm capacitation is the prerequisite for successful fertilization and mainly relies on protein transport and post-translational modification.Proteomics has been applied to the study of mammalian sperm development and maturation.Series of specific proteins and tyrosine phosphorylated proteins involved in sperm capacitation were identified.However,proteomic study has been seldom reported in buffalo reproductive biology.In this study,the dynamic protein changes in sperm capacitation were discovered by proteomics technology in order to better understand the potential mechanism.The affective factors of sperm capacitation were analyzed from protein expression and post-translation modification.This study providing a new perspective for buffalo reproductive mechanism.The results were shown as follows:??Quantitative proteomics analysis before and after buffalo sperm capacitation.Using TMT(Tandem Mass Tag)label combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)to analysis the protein expression before and after sperm capacitation.A total of 1441 proteins were identified successfully,the control group was the noncapacitated spermatozoa group,and the experimental group was the capacitated spermatozoa group.93 proteins were identified as differentially expressed proteins(DEPs,change fold>1.2),of which 52 proteins were up-regulated and 41 proteins were down-regulated.Through the bioinformatics analysis of DEPs,most of the DEPs involved in the biological processes were shown by GO analysis,including transport,cytoskeleton organization,sexual reproduction and spermatogenesis.KEGG analysis showed that the signaling pathways involved in DEPs were metabolic pathway,PPAR signaling pathway and SNARE interactions in vesicular transport.STRING analysis revealed complex interactions between the identified proteins,including transports glycolysis and phosphorylation.Western Blot confirmed that the expression levels of VAMP4 and APOC3 proteins were consistent with quantitative proteomic analysis.??Phosphoproteomic analysis of buffalo sperm before and after capacitation.The proteins were extracted from before and after buffalo sperm capacitation,and the phosphorylated peptides were enriched for LC-MS/MS identification and bioinformatics analysis.The results showed that 260 proteins were identified in the noncapacitated spermatozoa group,including 495 phosphorylated peptides and 848 phosphorylated modification sites.A total of 284 proteins containing 627 phosphorylated peptides and 853 phosphorylated modification sites were identified in the capacitated spermatozoa group.Venn analysis showed that there were 113 phosphorylated proteins between the two groups,of which 147 unique to the noncapacitated spermatozoa group,and 171 unique to the capacitated spermatozoa group.GO analysis of the two groups of phosphorylated proteins revealed that the capacitated spermatozoa group had more items than the noncapacitated spermatozoa group,regardless of cellular components,biological processes or molecular functions,and were related to the process of sperm capacitation.The capacitated spermatozoa group participated in more signaling pathways.Tyrosine phosphorylated proteins were 28 out of 113 phosphorylated proteins.The expression levels of SYNE2 and TAT1 proteins were changed,the tyrosine phosphorylation sites of proteins ODF1 and FISP2 were changed,which may be the markers of sperm capacitation.In summary,this study constructed the profiles of differential expression proteins and phosphorylated proteins before and after sperm capacitation of buffalo,which will be contribute to understand the regulatory role of proteins expression changes and post-translational modification:in sperm capacitation,and made a theoretical foundation for improving the fertilization ability of buffalo.