Proteomic identification of cryostress in epididymal spermatozoa

Abstract

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.

Keywords: Cryopreservation; Cryostress; Protein; Spermatozoa.

Fig. 1

Effect of cryopreservation on sperm. a Viability b Motility before and after cryopreservation. Data are presented as mean ± SEM. (*P < 0.05, n = 9)

Fig. 2

Effect of cryopreservation on sperm motion kinematics. a Curvilinear velocity b Straight-line velocity c Average path velocity d Straightness e Wobble f Amplitude of lateral head g Beat-cross frequency h Linearity before and after cryopreservation. Data are presented as mean ± SEM. (*P < 0.05, n = 9)

Fig. 3

Effect of cryopreservation on live sperm capacitation status. a AR pattern. b B pattern. c F pattern assessed by combined CTC/H33258 staining. Data are presented as mean ± SEM. (*P < 0.05, n = 9)

Fig. 4

2-DE Separation of proteins by 2-DE. 2-DE gels were stained with silver nitrate and analyzed using PDQuest 8.0 software. Protein spots from (a) before cryopreservation and after cryopreservation. b The expression of seven proteins increased significantly after cryopreservation. c The expression of two proteins decreased significantly after cryopreservation. Differentially expressed (>3-fold) proteins were determined by comparing samples before and after cryopreservation (P < 0.05, n = 9). The line indicates the landmark of equal levels for before cryopreservation

Fig. 5

Expression of SOD and NDUFV2 before and after cryopreservation. a Ratio of SOD2 to α-tubulin expression before and after cryopreservation. b Ratio of NDUFV2 to α-tubulin expression before and after cryopreservation. c Expression of SOD, NDUFV2 and before and α-tubulin after cryopreservation. Data are presented as mean ± SEM. (*P < 0.05, n = 9)

Fig. 6

Signaling pathways associated with differentially expressed proteins as identified by Pathway Studio. a ROS metabolism is associated with SOD2. b Guanylate cyclase pathway is associated with CAPZB, NDPK, and ODF2. c EphrinR-actin signaling pathway is associated with CAPZB and ODF2. d Actin cytoskeleton regulation associated with CAPZB and ODF2. e Actin cytoskeleton assembly is associated with CAPZB