Short-term storage-induced changes in the proteome of carp (Cyprinus carpio L.) spermatozoa

Abstract

Short-term storage of fish semen is a useful tool in aquaculture that enables the synchronization of male and female gamete availability. Unfortunately, at the same time, short-term storage causes oxidative stress and a significant decrease in sperm quality, including protein quality. However, comprehensive information regarding proteomic changes in fish semen upon short-term storage is lacking. We identified carp sperm proteins altered in abundance and carbonylation level after semen storage for 48 h and 120 h by analysis of extracellular medium (EM) and spermatozoa using proteomic approaches (two-dimensional difference gel electrophoresis (2D-DIGE) and 2D-oxyblot). Moreover, we assessed the motility, motion kinematics, oxidative stress and viability of spermatozoa using computer-assisted sperm analysis and flow cytometry.Short-term storage reduced viability, motility, curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and sperm concentration, whereas the number of ROS-positive cells increased at the same time (P < .05). Moreover, an increase in protein concentration and osmolality and a decrease of pH were observed in EM. We identified 52 sperm proteins enriched in EM, 54 proteins changed in abundance in spermatozoa and 19 sperm proteins increased in carbonylation level (P < .05) following storage. The analysis of EM and spermatozoa allowed for monitoring distinct disturbance to sperm structures during storage. EM and spermatozoa extract primarily contained cytosolic (60%) and flagellar (32%) proteins, respectively. Other sperm structures, such as nucleus, mitochondria and sperm membranes, were also affected by storage. The altered sperm proteins were primarily involved in energy metabolism, flagella movement, response to stress, and posttranslational modification and protein folding; the remaining proteins were associated with signal transduction and DNA repair.The simultaneous analysis of EM and spermatozoa following storage provides new insight into the complex mechanism of sperm damage in particular structures and cellular pathways. The availability of a catalogue of sperm proteins altered by storage provides a crucial tool for the future development of novel potential protein markers of sperm injury and for the improvement of short-term sperm preservation procedures commonly applied in aquaculture.