Proteomic identification of cryostress in epididymal spermatozoa

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.

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.

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.