Proteomic identification of cryostress in epididymal spermatozoa

Background: 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.