title: Differential proteome association study of freeze-thaw damage in ram sperm.
abstract: In this study proteomics analysis was performed to investigate damage caused to ram sperm by the freeze-thaw process. Sperm motility, viability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) content were measured to evaluate sperm quality. Compared with fresh groups, motility, viability and ATP content were all lower in freeze-thawed sperm (P < 0.001), and ROS content was higher (P < 0.001). Moreover, 25 differential protein spots were detected in two-dimensional gels using PDQuest 8.0 software and the corresponding proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) coupled with searching of the NCBI protein sequence database. Among these proteins, hexokinase1 (HXK1), the enzyme that catalyzes the first step of glycolysis in the sperm glycolytic pathway, is known to be associated with sperm motility. Casein kinase II subunit alpha (CSNK2A2), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. We used immunoblotting and immunofluorescence to analyze the expression and localization of these two proteins. HXK1 and CSNK2A2 expression levels in fresh sperm were significantly higher than that in freeze-thawed sperm (P < 0.001). HXK1 and CSNK2A2 were detected in the main part of the sperm flagellum, and the immunofluorescence signal from these proteins was weakened in the freeze-thawed group. Decreased expression of HXK1 and CSNK2A2 may be associated with decreased sperm motility and viability following freeze-thawing.
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: He
authors.affiliation: Animal Husbandry and Veterinary Research Institute of Gansu Province, Pingliang 744000, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China. Electronic address: zhaoxx@gsau.edu.cn.
authors.forename: Xingxu
authors.initials: X
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China.
authors.forename: Youji
authors.initials: Y
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Hu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D047390:Casein Kinase II / Q000096:biosynthesis*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D006593:Hexokinase / Q000096:biosynthesis*; D006801:Humans; D008297:Male; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods*; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism
chemical_list: D020543:Proteome; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; C472074:HK1 protein, human; D006593:Hexokinase; D047390:Casein Kinase II
keywords: Cryopreservation; Proteome; Ram sperm