Proteomics Identification And Protein Pathway Analysis Of Yanbian Yellow Cattle Sperm

Sperm capacitation is a process prior to fertilization that is essential for producing high-quality living embryos.Although considerable progress has been made in understanding the signaling pathways involved in sperm capacitation,the molecular underpinnings of these processes remain to be elucidated.Many processes prior to fertilization,such as sperm capacitation,are protein-dependent,in this way,post-translational modification of proteins plays a key role in the enabling mechanism.It has also been shown that during capacitation,sperm can replace degraded proteins or synthesize new ones,which are essential for both capacitation and fertilization.Proteomics has been applied to sperm capacitation in mammals.and its molecular mechanisms have been elucidated in species such as humans and bulls.Mass spectrometry is an integrated technique used in proteomics to elucidate the level and composition of proteins in cells.Therefore,in this study,the semen of yanbian yellow cattle was used to compare the capacitated and non-capacitated in vitro,and the expression differences of the two groups of proteins were analyzed by proteomics.In this experiment,3 healthy and disease-free yanbian yellow cattle aged 3-5 years were selected from 20 cattle,and their semen was collected,numbered as A,B and C,respectively,and two treatment groups were set up:capacited and non-capacited sperm.The experiment was mainly divided into two parts:The first part mainly studied the physiological and biochemical characteristics of the capacited sperm of yanbian yellow cattle.Specifically,biological characteristics of bovine sperm were detected through the microbial dynamic(static)image detection system(CASA);The integrity of sperm plasma membrane and acrosome membrane was determined by coomassie staining and hypotonic swelling.Sperm proteins from the two treatment groups were extracted and SDS PAGE and Western Blot were used for phosphorylation analysis;The changes of mitochondrial membrane potential before and after sperm capacitation were detected by JC-1 staining;Flow cytometry(BDaccuri C6)was used to detect sperm cell apoptosis and plasma membrane lipid disorder.In vitro fertilization and development of attuned sperm and bovine oocytes were also observed.The second part is mainly about the identification and the analysis of protein pathway of the capacited and non-capacited sperm proteome.Specifically,SDS PAGE and Western Blot were performed to extract sperm proteins from the capacited and non-capacited groups for mass spectrometry analysis,Liquid chromatography-mass spectrometry(lc-ms)was used in this study.Biological information was analyzed through GO enrichment,KEGG Pathway enrichment and protein-protein interaction(PPI).The experimental results are as follows:1.After detection by the microbial dynamic(static)image detection system(CASA),the viability,integrity of plasma membrane and acrosome membrane,and mitochondrial membrane potential of the capaccited spermatozoa of yanbian yellow cattle were all lower than that of the non-capacited treatment group;2.By using the microbial dynamic(static)image detection system(CASA)and Western Blot,the Curvilinear velocity and protein tyrosine phosphorylation level of the capacited sperm of yanbian yellow cattle were higher than that of the non-capacited sperm;3.According to the flow cytometry(BDaccuri C6),the apoptosis and membrane lipid disorder of the capacitated sperm of yanbian yellow cattle were greater than that of the non-capacitated sperm;4.After capacited,spermatozoa of yanbian yellow cattle were cultured in vitro with oocytes,the cleavage rate could reach above 46%;5.Compared with non-capacitated sperm,89 protein expressions in capacitated sperm were up-regulated and 509 protein expressions were down-regulated;6.By Western Blot analysis,the expression levels of PSMD1 and HSPA5 in the capacitated sperm group(H)were significantly lower than those in the uncapacitated sperm group(F).In this study,proteomics was used to analyze the differences in protein expression between in vitro capacitated and non-capacitated sperm of yanbian yellow cattle.It was found that 89 proteins were up-regulated and 509 proteins were down-regulated in the process of sperm capacitation.Western blot was used to detect the expression levels of PSMD1 protein and HSPA5 protein,and the expression levels of both PSMD1 and HSPA5 were down-regulated after the capacitated protein was observed.The changes we found in these key proteins during sperm capacitation can help screen the key proteins that affect sperm capacitation,enhance our understanding of the molecular processes involved in yanbian yellow cattle sperm capacitation.It is valuable for further understanding of the regulation mechanism of spermatogenesis and provide a framework for further study of this process.