4.6 Article

Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

Journal

THERIOGENOLOGY
Volume 80, Issue 2, Pages 84-89

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/j.theriogenology.2013.03.021

Keywords

Cryopreservation; Sperm; Phosphorylation; Protein

Funding

  1. Ministry of Education, Youth and Sport CENAQUA [CZ.1.05/2.1.00/01.0024]
  2. Grant agency of University of South Bohemia GAJU [114/2013/Z]
  3. institutional research concept of the Institute of Microbiology [RVO61388971]

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The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage. (C) 2013 Elsevier Inc. All rights reserved.

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