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

doi:10.1016/j.theriogenology.2013.03.021

Theriogenology

Volume 80, Issue 2, 15 July 2013, Pages 84-89

https://doi.org/10.1016/j.theriogenology.2013.03.021 Get rights and content

Abstract

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.

Introduction

Cryopreservation of fish sperm is considered a valuable technique for artificial reproduction and genetic improvement. However, the quality of cryopreserved sperm is usually lower than that of fresh sperm, because the freeze–thaw procedure and cryoprotectant negatively affects DNA and protein integrity, membrane lipids, spermatozoa motility, fertilization, and larva survival [1], [2].

A significant change in spermatozoa motility parameters after the freeze–thaw procedure has been reported in several fish species [3]. Zilli et al. [1] reported that cryopreservation had a strong effect on the phosphorylation state of proteins in sea bass (Dicentrarchus labrax) sperm. Studies of mammalian sperm demonstrated that freezing and thawing led to an increase in protein phosphorylation [4], [5]. Phosphorylation and dephosphorylation of structural and regulatory proteins are involved in the regulation of most cellular processes in eukaryotes, particularly in signal transduction, and play an essential role in the coordination and regulation of intracellular pathways [6], [7].

Currently, limited information is available concerning the effects of the freeze–thaw procedure and cryoprotectants on the phosphorylation of sperm proteins in fish. In our previous studies [8], [9], we reported a negative effect of cryopreservation on common carp (Cyprinus carpio L.) spermatozoa with respect to protein composition and of the potential for cryopreservation to induce oxidative stress. Systematic immunoblot screening coupled with matrix-associated laser desorption/ionization time-of-flight mass spectrometry was used to investigate the presence of phosphorylated/dephosphorylated proteins in fresh and frozen/thawed common carp spermatozoa and to explain their primary role in the spermatozoa.

Section snippets

Broodstock handling and collection of gametes

Common carp were reared at the experimental station of the Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, Vodnany, Czech Republic. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by the Ethical Committee of the University of South Bohemia. Before experimentation, six 3-year-old male common carp were individually marked with passive inductive transponder tags and stocked in 4-m³ hatchery

Effect of cryopreservation on spermatozoa motility and velocity

Motility characteristics of spermatozoa obtained from common carp were evaluated in fresh and frozen/thawed sperm using computer-assisted analysis. After cryopreservation the percent motility and spermatozoa velocity were significantly reduced compared with fresh sperm (91.30% and 115.61 μm/s). The percent motility in the DMSO group (47.26%) was significantly higher than that in the EG group (29.87%), and spermatozoa velocity in the DMSO group (63.85 μm/s) was significantly lower than that in

Discussion

The effect of the freeze/thaw procedure and cryoprotectants on the phosphorylation state of proteins in common carp sperm was evaluated. Cryopreservation with DMSO and EG extenders was shown to significantly alter the phosphorylation state of sperm proteins, the dephosphorylation of tyrosine residues (Band-Tyr1, Fig. 1A), and the phosphorylation of threonine residues (Band-Thr1, Fig. 1B). Detailed comparison of cryopreservation procedures revealed two additional protein bands, Band-Tyr2 and

Conclusions

Studies performed on mammalian sperm, such as cattle [22], sheep [4], and horse [5] demonstrated that the freeze/thaw procedure led to an increase of phosphorylation of tyrosine residues. This phosphorylation pattern was similar to that observed during capacitation. It has also been suggested that the increase in protein phosphorylation detected in mammalian frozen/thawed spermatozoa might be because of membrane modifications that determine conformational changes of these proteins [4], or

Acknowledgments

This work was supported by the Ministry of Education, Youth and Sport CENAQUA CZ.1.05/2.1.00/01.0024, Grant agency of University of South Bohemia GAJU 114/2013/Z, and the institutional research concept of the Institute of Microbiology RVO61388971.

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Citation Excerpt :
Flow cytometry is used to obtain reliable data about cell membrane or mitochondrial status, antioxidant status is evaluated using different methodologies, and chromatin integrity is also considered as an important checkpoint (reviewed by Cabrita et al., 2014). Moreover, proteome analysis contributes to deeper understanding in sperm physiology, revealing changes in proteins related to membrane traffic and organization, metabolism or signal transduction (Li et al., 2010, 2013; Nynca et al., 2015a), providing valuable information on the nature of cryodamage (Fig. 2). In addition, beyond fertilization rate, different studies have evaluated the long-term development of the progeny (Pérez-Cerezales et al., 2011; Viveiros et al., 2012).
This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved.
This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.

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Theriogenology

Abstract

Introduction

Section snippets

Broodstock handling and collection of gametes

Effect of cryopreservation on spermatozoa motility and velocity

Discussion

Conclusions

Acknowledgments

References (22)

Effect of cryopreservation on phosphorylation state of proteins involved in sperm motility initiation in sea bream

Cryobiology

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus)

Theriogenology

The coordinated action of protein tyrosine phosphatases and kinases in cell signaling

Trends Biochem Sci

The age of crosstalk: phosphorylation, ubiquitination, and beyond

Mol Cell

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L

Theriogenology

Cryopreservation of carp sperm

Aquaculture

Cryopreservation of tench, Tinca tinca, sperm: sperm motility and hatching success of embryos

Theriogenology

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.)

Cryobiology

Autophos-phorylation of GRP 94 (endoplasmin)

J Biol Chem

Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors

Biochem Biophys Acta

GRP94 resides within cardiac sarcoplasmic reticulum vesicles and is phosphorylated by casein kinase

J Biol Chem

Cited by (20)

Proteomics in fish health and aquaculture productivity management: Status and future perspectives

Spermatology and sperm ultrastructure in farmed coho salmon (Oncorhynchus kisutch)

Cryopreservation of coho salmon sperm (Oncorhynchus kisutch): Effect on sperm function, oxidative stress and fertilizing capacity

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility

Protein profile of Dabry's sturgeon (Acipenser dabryanus) spermatozoa and relationship to sperm quality

Cryobanking of aquatic species

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

Abstract

Introduction

Section snippets

Broodstock handling and collection of gametes

Effect of cryopreservation on spermatozoa motility and velocity

Discussion

Conclusions

Acknowledgments

Cryobiology

Theriogenology

Trends Biochem Sci

Mol Cell

Theriogenology

Aquaculture

Theriogenology

Cryobiology

J Biol Chem

Biochem Biophys Acta

J Biol Chem

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