Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity, Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa

Fish sperm cryopreservation is considered as a valuable technique for artificial reproduction and genetic improvement (Chao & Liao, 2001; Kopeika et al., 2007; Rana, 1995; Suquet et al., 2000). Semen quality must be monitored when attempts are made to increase the efficiency of artificial fertilization, to cryopreserve only sperm of high quality, and to evaluate frozen-thawed sperm. Cryopreserved sperm usually shows, with respect to fresh sperm, a lower quality, since the freezing–thawing procedure affects DNA and protein integrity (Labbe et al., 2001; Zilli et al., 2003, 2005), membrane lipids (Maldjian et al., 2005; Müller et al., 2008), sperm motility (Linhart et al., 2000; Ritar, 1999; Rodina et al., 2007; Zilli et al., 2005), fertilization ability (Gwo & Arnold, 1992; Rana, 1995), and also larval survival (Suquet et al., 1998). Spermatozoa genome alteration due to cryopreservation may affect only late embryonic development and larval survival (Kopeika et al., 2003a, 2003b, 2004; Suquet et al., 1998), but not the early events in embryonic development, because these are controlled by maternally inherited information (Braude et al., 1988). On the contrary, defects in sperm proteins (degradation and/or change of the phosphorylation state) may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huang et al., 1999; Lessard et al., 2000). The most common parameters used to evaluate sperm quality are fertilization ability, motility (rate and duration) and cellular (chemical and/or biochemical) parameters. Fertilizing capacity is the most conclusive test of sperm quality but the use of this marker is laborious and requires the availability of eggs (McNiven et al., 1992). Motility is normally evaluated as percentage and duration, but some authors also use velocity, flagellum beat frequency, or other parameters measured by computer-assisted sperm analysis (Ciereszko et al., 1996; Cosson et al., 2000; Rurangwa et al., 2001). Cellular bio markers has been used to evaluate spermatozoa quality of different fish species such as Atlantic salmon (Aas et al., 1991; Hwang & Idler, 1969), rainbow trout (Ciereszko & Dabrowski, 1994; Lahnsteiner et al.,1996a, 1998) and sea bass (Zilli et al., 2004). All these parameters have been also used to evaluate the effect of cryopreservation on spermatozoa quality. Here we reviewed data obtained by our group, on the effect of freezing-thawing procedures on sea bass and sea bream sperm. In particular, data concerning the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state, are reported

Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

Palabras clave extraídas del título[EN] Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservationSIThis work was supported by Apulia Region (Italy) under the program between the Apulia Region and the University of Salento. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Studies of Sparus aurata sperm motility by computer-assisted sperm analysis (CASA).

Aim: Sperm of Sparus aurata, like those of other teleosts, become activated when spawned into the external medium. Several extracellular factors (ions, osmotic pressure, O2/CO2, sperm activating peptides) have been reported to control sperm motility activation. These factors act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane and determining the dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation. In the present study we have investigated the role of different proteins involved in this signal transduction cascade using cryopreserved sperm of Sparus aurata. Methods: Studies have been performed by using cryopreserved sperm (Fabbrocini et al., Cryobiology 40:46-53, 2000). To investigate the role played by different proteins involved in the signal transduction cascade we evaluated: (1) the effect of specific protein inhibitors on sperm motility (assessed by CASA), (2) quantitative and qualitative evaluation of phosphorylated proteins (tyrosine-phosphorylated, serine-phosphorylated and threonine-phosphorylated) by western blot analysis in activated and non-activated sperm. Results: Results obtained indicated that: (1) calcium and potassium were not involved in sperm motility activation; (2) osmolality values <1,000 mOsm/Kg inhibited the sperm motility activation; (3) inhibition of Adenylyl Cyclase by 500 μM MDL-12330A hydrochloride and Protein-Kinase A (PKA) by 50 μM U73122 prevented the sperm activation; (4) inhibition of Tyrosine-kinase (by 100 μM AG18) and CAM-Kinase (by 50 μM KN93) were ineffective on sperm activation, but determined changes in sperm motility parameters. Particularly, AG18 significantly decreased the Curvilinear Velocity (VCL), while KN93 significantly decreased both Curvilinear Velocity (VCL) and Straight Line Velocity (VSL). Conclusion: In Sparus aurata sperm, an important factor controlling sperm motility activation was the osmolality of the external medium. The osmotic shock induces sperm activation by cAMP/PKA signaling pathway. In addition also Tyrosine-kinases and CAMkinases seems to be involved in the control of sperm motility

Role of protein phosphorylation/dephosphorylation in fish sperm motility activation: State of the art and perspectives

In many fish species, the spermatozoa are immotile in the testes and seminal plasma, and motility is induced when they are released in the aqueous environment. Extracellular factors control the activation of the axoneme through signal transduction across the plasma membrane. The present review examines the existing literature concerning the axoneme activation in fish, with particular emphasis on the role played by the protein phosphorylation/ dephosphorylation process, since post-transciptional modifications are involved in the mechanisms of sperm motility activation in many animals with external fertilization (starfish, sea urchins, sea cucumber, fish). We report the current understanding of the role played by the changes of protein phosphorylation state in sperm motility activation of teleost fish and provide additional tools for evaluating the gamete quality before and after cryopreservation procedure and, therefore, improve fish farm management. Statement of relevance: The present review examines the existing literature concerning the signaling pathways involved in the axoneme activation in fish, in order to better understand the molecular mechanism regulating spermmotility initiation and to show how the proteins that change their phosphorylation status after spawning can be used as biomarkers for sperm quality and cryodamage

Molecular mechanism regulating axoneme activation in marine fish: a review

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Development of AFLP markers to determine genetic diversity in population of sea bass Dicentrarchus labrax in two areas of mediterranean sea

The use of new tools for the study of genetic of marine populations is important for the development and implementation of genetic improvement programs. The use of DNA markers in marine population has made little progress compared with their use in terrestrial species. DNA markers can be used to verify pedigrees as well as to screen wild populations to maximise diversity in founder animals. These knowledges can be applied to many aspects such as stock management, creation of reserves, etc. In particular they can be applied to commercially important species such as Dicentrarchus labrax that is an important farmed specie in Europe. The information available on population genetic structure of sea bass suggests that there are significant population subdivision in both Atlantic and Mediterranean parts of its range. Further work in this area will benefit from the availability of useful molecular markers. AFLP markers provide an efficient method for DNA fingerprinting in many species of plants and animals. In this work new AFLP markers were generated for sea bass (Dicentrarchus labrax). Two main factors affect the number of AFLP bands generated per individual: 1) the specific endonucleases used and 2) the number of selective bases added to the primers in the selective amplification step. The AFLP markers utilised in this study were generated by EcoRI and MseI restriction and by using primers with three selective bases. These AFLP markers were used to start an investigation on population structure of sea bass in southern Italy by comparing the animals captured in two different geographic areas in Puglia: Acquatina lagoon and Alimini lagoon

Proteome analysis of normal and transformed thyroid cells

In the present study we used the two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to compare the protein expression in two cell lines (PC-Cl3 and PC-E1A+raf). PC-Cl3 is a immortalized rat thyroid cell line which in vitro retain biochemical characteristics of differentiated thyroid cell. PC-E1A+raf originates by stable transfection with E1A and v-raf oncogenes of the PC-Cl3 cells, and displays a malignant phenotype. The protein profiles differed between the two cell lines as revealed by visual inspection and by image analysis software. We identified 414 ± 10 (n = 10) spots in PC-Cl3, among these 11 significantly decreased, eight were absent and six increased in 2-D gel obtained with PC-E1A+raf. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in the bioinformatics analysis (Profound, Mascot, ad MS-fit). In particular, one of the spot showed homology with a GAPDH (glyceraldehydes-3-phosphate dehydrogenase, one with H(+) transporting ATP-synthase and one with PEBP (phoshatidylethanolamine-binding protein). The present work shows that the transfection with E1A and v-raf oncogenes of the PC-Cl3 cells causes the change of protein expression of PC-Cl3. Two out of three of these identified spots (ATP-synthase and GAPDH) seems to be involved in the cell apoptosis