Peoteomics Analyses Of Capacitation And Acrosome Reaction In Frozen-Thawed Yak Spermatozoa

The yaks(Bos grunniens)are distinctive domestic in Qinghai-Tibetan Plateau,it provides meat and milk for Tibetans and other nomadic people in the area,but it has a reproductive rate lower than other domestic animals.Therefore,it is critical to boost yak reproductive efficiency using IVF and AI.The frozen semen plays a key role to achieve of these technologies,but the cryopreservation process results in changes in sperm motility,proteome composition,and tyrosine phosphorylation,and how these changes affect sperm capacitation and acrosome response,as well as the ability to achieve maximum fertilization potential,has not been determined.As highly specialized and differentiated cells,spermatozoa are largely devoid of transcriptional and translational functions.Protein phosphorylation,especially tyrosine phosphorylation,is particularly important for capacitation and capacitation-associated hyperactivated motility and acrosome reaction.In this study,the proteome and phosphorylated proteome of frozen-thawed spermatozoa,capacitated spermatozoa,and acrosome-reacted spermatozoa were systematically profiled in an in vitro capacitation system of yak frozen-thawed spermatozoa.Methods and techniques adopted in this research include LC-MS-MS,TMT,IMAC and other bioinformatics methods.This study aimed to identify the key signaling pathways,proteins,and phosphorylated proteins and their loci that regulate the frozen-thawed spermatozoa and acrosome reaction in yaks,and to provide a theoretical and practical basis for further research on the maturation mechanism of yak spermatozoa.The main findings of the study are as follows:1.Spermatozoa were incubated for different times with varying concentrations of heparin sodium in the in vitro capacitation process of yak frozen-thawed spermatozoa,acrosome reaction rate and tyrosine phosphorylation abundance were used to comprehensively evaluate the capacitation efficiency.It was found that the percentage of acrosome-reacted spermatozoa and the level of tyrosine-phosphorylated protein were significantly increased(P<0.05)after the addition of heparin sodium and that the optimal capacitation was reached with the concentration of 50 ug/m L and incubation time of 30 min.On this basis,different concentrations of caffeine and ouabain were added and the results showed that after the addition of caffeine,the percentage of acrosome-reacted spermatozoa and the expression level of tyrosine-phosphorylated protein both improved,and the addition of 5m M caffeine resulted in the best effect(P<0.05).However,the percentage of acrosome-reacted spermatozoa and the expression level of tyrosine phosphoprotein did not change after the addition of ouabain(P>0.05).Therefore,the preliminary judgment is that heparin sodium and caffeine played a synergistic role in the process of capacitation.2.Based on the above research,the optimal capacitation condition(50 ug/m L heparin and 5m M Caffeine incubation time of 30 min)for quantitative proteomics analysis of capacitation and acrosome reaction sperm.A total of 3,280 proteins were identified in yak frozen-thawed high-motility spermatozoa,capacitated spermatozoa,and acrosome-reacted spermatozoa,of which 3074 were quantified.Among them,68(3 up-regulated and 65 down-regulated)and 32(9 up-regulated and 23 down-regulated)proteins were differentially expressed during capacitation and acrosome reaction.The differentially expressed proteins were mainly involved in biological processes such as metabolism,redox reaction,and cell differentiation,and they were significantly enriched in metabolic and PPAR signaling pathways.Western blot experiments confirmed that LCS-1(an inhibitor that specifically inhibits SOD1 activity)inhibited the level of tyrosine phosphorylation during capacitation(P<0.05),suggesting that SOD1 may be a key protein in regulating tyrosine phosphorylation during capacitation.The differentially expressed proteins during the acrosome reaction were mainly involved in the biological processes of metabolism,fertilization,actin cytoskeleton reorganization,and sperm-egg recognition,and they were significantly enriched in the AMPK signaling pathway and insulin signaling pathways,among others.FITC-PSA staining experiments confirmed that 17-AAG(an inhibitor of both HSP90AA1 and HSP90AB1activity)inhibited the acrosome reaction(P<0.05)while NVP-BEP800(an inhibitor that specifically inhibits HSP90AB1)exerted no effect on the acrosome reaction(P>0.05).This finding indirectly suggested that HSP90AA1 might be a key protein regulating the acrosome reaction.3.Based on the above research,the optimal capacitation condition(50 ug/m L heparin and 5m M Caffeine incubation time of 30 min)for quantitative phosphoproteomics analysis of capacitation and acrosome reaction sperm.IMAC for the enrichment analysis of phosphorylated peptides was used to identify and analyze the phosphoproteome of yak frozen-thawed high-motility spermatozoa,capacitated spermatozoa,and acrosome-reacted spermatozoa.A total of 5,509 modified phosphorylation sites were identified on 1,377 proteins in yak spermatozoa,of which 3,938 sites on 853 proteins were quantified.This was the first study on the phosphoproteome of bovine spermatozoa.There were 148(55up-regulated,93 down-regulated)proteins and 259(89 up-regulated,170 down-regulated)phosphorylation sites differentially expressed in yak spermatozoa during capacitation;while in the acrosome reaction,227(60 up-regulated,167 down-regulated)proteins and 259(96up-regulated,483 down-regulated)phosphorylation sites were differentially expressed.Phosphorylated proteins differentially expressed during capacitation were significantly enriched in the metabolic and proteasomal signaling pathways,in which elevated levels of tyrosine phosphorylation at site 67 of SPA17,serine phosphorylation at site 14 of PSMD11,serine phosphorylation at site 735 of AKAP4,and threonine phosphorylation at sites 189 and 212 of AKAP3(P<0.05)enhanced the activity of PKA to regulate sperm capacitation.The differentially expressed phosphorylated proteins during the acrosome reaction were significantly enriched in the metabolic,AMPK signaling,and c GMP-PKG signaling pathways,where dephosphorylation on CABYR serine/threonine residues indicated that CABYR abolished binding to calcium during the acrosome reaction.In conclusion,the optimal condition for yak frozen-thawed spermatozoa capacitation is 30 min incubation in heparin at a concentration of 50 ug/ml supplemented with 5 m M caffeine.The analysis of the yak spermatozoa proteome and phosphorylated proteome provided a new understanding on the molecular basis of spermatozoa capacitation and acrosome reaction in yaks.The key signaling pathways and proteins regulating capacitation and acrosome reaction were found to provide a theoretical and practical basis for yak spermatozoa development and fertility.