The Association Of Protein Markers With Cryoinjury In Ram Spermatozoa

Freezing and thawing procedures are mostly harmful to spermatozoa morphology and function,since temperature and osmotically induced changes occur in the fluidity,permeability,and lipid composition of spermatozoa membranes.Cryopreservation could alter the quality of the spermatozoa's head,membranes,acrosome,tail and nucleus,since spermatozoa is highly sensitive to oxidative stress.These cryoinjuries could decrease spermatozoa motility and fertility,and affected fertilization and the early events after fertilization.These results are related to the absence or loss of spermatozoa proteins.At present,there is no report on the explicit mechanism of cryoinjury in ram spermatozoa,the rise and popularity of proteomics techniques provide an opportunity for revealing the mechanism of cryoinjury from the protein level in ram spermatozoa.In this study,the same batch of fresh and frozen-thawed spermatozoa were used,differential proteins were identified by two-dimensional electrophoresis(2-DE)combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Through construction of protein interaction network to screen the protein markers,and then the immunoblotting,immunofluorescence and Real-time PCR were used to verify the content of the proteins.Finally,the correlation of proteins and the quality of spermatozoa was analyzed.The aim of this study was to reveal the cryoinjury mechanism of ram by screening protein markers,and to provide candidate molecular targets for the study of cryoprotectants.The results showed:(1)A significant decrease in spermatozoa motility,viability,ATP content,plasma membrane integrity,acrosome integrity and mitochondrial function,and increase DNA damage and ROS content was observed in frozen–thawed ram spermatozoa.(2)25 differential protein spots were obtained by using the PDQuest8.0 software to analyze the 2-DE gel(fold Change was ?2),among these spots,17 spots were increased and 8 spots decreased in 2-DE gels of frozen-thawed spermatozoa.These spots were identified as 25 proteins using MALDI-TOF-TOF MS coupled with searching of the NCBI protein sequence database.The results of Gene ontology annotation(GO)and KEGG enrichment indicated that the freezing and thawing process caused change of the whole level of ram spermatozoa proteome,and the differential proteins involved a multiple biological processes.(3)Based on the protein interaction network,there are two protein markers of cryoinjury were screened.CK2(CK2? and CK2?')is involved in spermatozoa apoptosis.HXK1 is involved in glycolysis/gluconeogenesis and is hence associated with ATP generation.(4)Western blotting showed that the contents of CK2?'CK2?'and HK1 protein were decreased in frozen-thawed spermatozoa.Immunofluorescence localization showed that CK2? was mainly distributed in the midpiece of spermatozoa,CK2?'was mainly distributed in spermatozoa acrosome,and HK1 was mainly distributed in the head and midpiece of spermatozoa.(5)CK2?'protein content was increased and HK1 protein content has no significant change in the frozen-thawed seminal plasma,indicating that the freezing and thawing process could cause the CK2?'protein leakage into seminal plasma,and the HK1 protein was degraded within the spermatozoa.(6)CK2?'and HK1 m RNA level were decreased in the frozen-thawed spermatozoa,indicating that freeze-thaw process could cause the CK2?'and HK1 m RNA degradation.(7)Compared with fresh spermatozoa by SPSS correlation analysis,in frozen-thawed spermatozoa,the positive correlation between acrosome intergrity and CK2?' protein abundance was significant,the negative correlation between DNA damage and CK2?' protein abundance in was significant,the negative correlation between DNA damage and CK2?' protein abundance was significant,the negative correlation between apoptosis and CK2?' protein abundance was significant,the positive correlation between motility and HK1 protein abundance was significant and the positive correlation between ATP content and HK1 protein abundance was significant.These results indicated that CK2 and HK1 protein can be as protein markers of cryoinjury in ram spermatozoa.It was concluded that it is feasible to use a comparative proteomics to screen protein markers of cryoinjury in ram spermatozoa,and to obtain more comprehensive and reliable information of differential proteins.The protein markers of cryoinjury can be screened by analysis of protein interaction.Through comparing with the spermatozoa quality parameters of frozen-thawed spermatozoa,the correlation between protein markers and quality of frozen-thawed spermatozoa can be obtained by verifying the content and distribution of proteins.The study of potential protein markers is beneficial to reveal the intrinsic molecular mechanism of cryoinjury in ram spermatozoa,and to provide a theoretical basis for the study of ram spermatozoa cryoprotectant.