Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen.

During freezing and thawing, spermatozoa are exposed to physical and chemical stressors that result in adverse changes in sperm structures and physiological functions. The present study provides, for the first time, a comprehensive description of protein changes in the extracellular medium of cryopreserved semen. Using 2D-DIGE and a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, 183 proteins released from sperm to an extracellular medium were identified. The majority of released proteins were involved in metabolism and energy production. Moreover, proteins associated with a response to stress, apoptosis, small GTPase mediated signal transduction, transcription, translation, protein folding and turnover, reproduction and DNA repair were identified. The dominant group of released proteins was related to cytoplasm. Moreover, specific proteins associated with the membrane, mitochondria and nucleus were identified. The identification of a high number of proteins released from sperm provides new insight into the mechanism of cryodamage to the particular sperm structure and to specific metabolic pathways, which were affected by cryopreservation. The availability of a catalog of carp sperm proteins altered by cryopreservation provides a crucial tool for the development of novel potential biomarkers of cryoinjuries and for the improvement of a long-term sperm preservation procedure.