Proteomic identification of cryostress in epididymal spermatozoa

摘要

Backgroune:Cryopreservation of epididymal spermatozoa is important in cases in which it is not possible to collect semen using normal methods,as the sudden death of an animal or a catastrophic injury.However,the freezing and thawing processes cause stress to spermatozoa,including cold shock,osmotic damage,and ice crystal formation,thereby reducing sperm quality,We assessed the motility (％),motion kinematics,capacitation status,and viability of spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining.Moreover,we identified proteins associated with cryostress using a proteomic approach and performed western blotting to validate two-dimensional electrophoresis (2-DE) results using two commercial antibodies.Results:Cryopreservation reduced viability (％),motility (％),straight-line velocity (VSL),average path velocity (VAP),amplitude of lateral head displacement (ALH),and capacitated spermatozoa,whereas straightness (STR) and the acrosome reaction increased after cryopreservation (P ＜ 0.05).Nine proteins were differentially expressed (two proteins decreased and seven increased) (＞3 fold,P ＜ 0.05) before and after cryopreservation.The proteins differentially expressed following cryopreservation are putatively related to several signaling pathways,including the ephrinR-actin pathway,the ROS metabolism pathway,actin cytoskeleton assembly,actin cytoskeleton regulation,and the guanylate cyclase pathway.Conclusion:The results of the current study provide information on epididymal sperm proteome dynamics and possible protein markers of cryo-stress during cryopreservation.This information will further the basic understanding of cryopreservation and aid future studies aiming to identify the mechanism of cryostress responses.