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DOI 10.1002/pmic.201400525

RESEARCH ARTICLE

Cryopreservation-induced alterations in protein composition of rainbow trout semen 2 ¨ Joanna Nynca1 , Georg J. Arnold2 , Thomas Frohlich and Andrzej Ciereszko1 1

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima, Olsztyn, Poland 2 Laboratory for Functional Genome Analysis LAFUGA, Gene Center and Department of Biochemistry, ¨ Munich, Germany Ludwig-Maximilians-Universitat,

The aim of this study was to detect cryopreservation-induced alterations in the protein composition of rainbow trout semen using two independent methods 1DE SDS-PAGE prefractionation combined with LC-MS/MS and 2D difference gel electrophoresis followed by MALDI-TOF/TOF identification. Here, we show the first comprehensive dataset of changes in rainbow trout semen proteome after cryopreservation, with a total of 73 identified proteins released from sperm to extracellular fluid, including mitochondrial, cytoskeletal, nuclear, and cytosolic proteins. Our study provides new information about proteins released from sperm, their relation to sperm structure and function, and changes of metabolism of sperm cells as a result of cryopreservation. The identified proteins represent potential markers of cryoinjures of sperm structures and markers of the disturbances of particular sperm metabolic pathways. Further studies will allow to decipher the precise function of the proteins altered during rainbow trout cryopreservation and are useful for the development of extensive diagnostic tests of sperm cryoinjures and for the successful improvement of sperm cryopreservation of this economically important species.

Received: November 6, 2014 Revised: January 30, 2015 Accepted: March 13, 2015

Keywords: Animal proteomics / DIGE / Fish / Freezing-thawing / Semen / Shotgun proteomics

 1

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Correspondence: Dr. Nynca Joanna, Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland Fax: +48-89-524-01-24 E-mail: [email protected]

has still deleterious effects on the sperm compartments and their functions making an optimization of fish cryopreservation protocols indispensable. During cryopreservation procedures, cryoinjuries of the sperm are caused by the physical and chemical factors, including rapid change in temperature, intracellular ice formation, oxidative stress, and osmotic stress [2]. These events result in disturbances of DNA integrity [3], changes in sperm protein profiles [4, 5], and membrane damages. The membrane disruption leads to leakage of intracellular spermatozoa proteins, and subsequently to a reduced post-thawing motility, fertilizing ability [6], and larval survival [7]. In order to better understand the mechanism of cryodamages and to find markers for sperm damage after cryopreservation, there is a need to monitor sperm cryoinjures. A promising approach to identify key proteins affected by cryopreservation is to investigate the semen proteome before and after freezing-thawing process, which can extent the

Abbreviations: CK, creatine kinase; EFC, extracellular fluid of cryopreserved semen; EFF, extracellular fluid of fresh semen; FDR, false discovery rate

Colour Online: See the article online to view Figs. 1, 2, and 3 in colour.

Sperm cryopreservation continues to be one of the most frequently employed techniques for the use in the modern animal production, but in fish breeding this method is still not implemented on a commercial level. The creation of a cryopreserved sperm bank can be an effective strategy to protect the biological biodiversity of the fish population and provides the opportunity to preserve the sperm samples of the most valuable males, which can be used in fish hatcheries [1]. However, even with the most up to date procedures, cryopreservation

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existing knowledge regarding the nature of cryoinjures. So far only a few proteomic studies have been reported, in which Li et al. [5, 8] and Zilli et al. [4, 9] described the qualitative changes of common carp and sea bass sperm protein profiles in response to cryopreservation. However, only limited numbers of proteins (9 sperm proteins for sea bass and 12 proteins for common carp) were identified in these studies [4, 5, 8, 9]. Moreover, there is no information regarding the effect of cryopreservation on the protein composition of rainbow trout semen, relevant for an improvement of the cryopreservation protocols of this economically important species. The objective of this study was to investigate the changes in rainbow trout semen protein composition after cryopreservation in order to identify molecular markers of sperm cryoinjures. We applied two strategies for the identification of proteins enriched in extracellular fluid of cryopreserved semen (EFC): (i) the combination of protein fractionation by 1DE and HPLC-ESI-MS/MS (LC-MS/MS) and (ii) 2D DIGE followed by MALDI-TOF/TOF identification.

2

Materials and methods

2.1 Sample collection The experiments were carried out on sexually matured spring spawning rainbow trout (3+ year old) maintained in the Rutki Salmonid Research Laboratory at the Institute of Inland Fisheries in Olsztyn, Poland. Prior to milt collection, fish (n = 4) ˙ were anesthetized using Propiscin (1 ppm IFI, Zabieniec, Poland). Milt from rainbow trout males was obtained by gentle abdominal massage, with special care to avoid blood, urine, or feces contamination. Aliquots of semen from the same individual to be used fresh were centrifuged at 3000 × g for 20 min at 4⬚C followed by a further centrifugation of the supernatant at 10 000 × g for 10 min at 4⬚C, while semen aliquots to be cryopreserved were frozen (see below). Seminal plasma obtained after centrifugation of fresh semen was denoted as extracellular fluid of fresh semen (EFF). Supernatant obtained by the centrifugation of cryopreserved semen (consisting of seminal plasma and extender) was indicated as EFC. Approval of the Animal Experiments Committee in Olsztyn (no. 114/2011), Poland, was gained before starting experiments. 2.2 Semen analysis The motility parameters of fresh and cryopreserved sperm were examined with Computer-Assisted Sperm Analysis using the Hobson Sperm Cell Tracker as described by Dietrich et al. [10]. Sperm was activated at a dilution ratio of 1:300 with 1 mM CaCl2 , 20 mM Tris, 30 mM glycine, 125 mM NaCl, pH 9.0 supplemented with 0.5% bovine albumin. The sperm motility parameters, percentage of motile sperm (MOT), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN), and ampli C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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tude of lateral head displacement (ALH), were measured over a 12-s period, between 5 and 17 s postactivation time. Video recordings (two replicates per sample) were made using a microscope Olympus BX40 (Olympus Optical, Tokyo, Japan) with a 10× negative phase objective and a Sony CCD black and white camera (SPT-M108CE). Sperm concentration was measured using the spectrophotometric method [11]. Mean sperm concentration was 9.98 ± 1.53 × 109 spermatozoa per milliliter. 2.3 Cryopreservation The cryopreservation followed the procedure previously described [12] with the use of 0.3 M glucose and 10% methanol as extender. The extender did not contain any protein. EFC was obtained by centrifugation of thawed semen at 3000 × g for 20 min at 4⬚C followed by a further centrifugation of the supernatant at 10 000 × g for 10 min at 4⬚C. The extracellular fluid was concentrated using Amicon Ultra concentrators (Merc, Darmstadt, Germany) followed by the measurement of the protein concentration by performing a Bradford Protein Assay (Coomassie Plus Assay Reagent, Thermo Fisher Scientific, Rockford, IL, USA). 2.4 LC-MS/MS analysis 2.4.1 SDS-PAGE electrophoresis prefractionation and tryptic digestion of gel slices The SDS-PAGE of three biological replicates of EFF and EFC (40 ␮g/per well) was performed using a 4% stacking gel and a 12% separation gel on a mini-Protean II system (Bio-Rad, Hercules, CA, USA). After electrophoresis, gels were stained overnight by CBB R-250 and destained in 5% methanol with 7% acetic acid. Gels were washed twice in water and gel lanes were cut into 11 sections per lane for trypsin digestion and subsequent LC-MS/MS analysis. 2.4.2 LC-MS/MS protein identification Nano-flow LC-MS/MS (nano-LC-MS/MS) was performed with a nano-LC ultra-chromatographic device (Eksigent, Dublin, CA, USA) coupled to an LTQ mass spectrometer (Thermo Scientific, San Jose, CA, USA). Peptides were injected onto a C18 trap column (LC Packings Dionex Sunnyvale, CA, USA) and subsequently separated by RP chromatography using a nano-LC column (Dr. Maisch, AmmerbuchEntringen, Germany) at a flow rate of 280 nL/min with a 150-min linear gradient of 84% ACN and 0.1% formic acid. 2.4.3 Database searching and data analysis MS RAW data were processed using MASCOT Daemon and MASCOT Server version 2.1.03 (Matrix Science, London, UK). MS/MS data were searched against the NCBIr www.proteomics-journal.com

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Fish databases and the following parameters: (i) enzyme: trypsin; (ii) fixed modification: carbamidomethyl (C); (iii) variable modifications: oxidation (M), (iv) peptide mass tolerance: 2 Da, (v) MS/MS mass tolerance: 0.8 Da; (vi) peptide charges: 1+, 2+, and 3+; (vii) instrument: ESI trap; and (viii) allow up to one missed cleavage. The occurrence of false positives (FDR) was estimated by running searches using the same parameters against decoy databases. Scaffold version_2_04_00 (Proteome Software, Inc., Portland, OR, USA) was used to obtain a list of protein identifications with a false discovery rate (FDR) ࣘ 1%, requiring at least two individual peptides per protein with a minimum peptide probability of 95%. Normalized spectral counts, in Scaffold denoted as “quantitative value,” were exported to Microsoft Excel and used for the quantitative comparison (protein abundance) between EFF and EFC data. Protein abundance difference between EFF and EFC was performed exclusively for the proteins present in all the biological replicates. Corresponding proteins of EFF and EFC were considered to be more abundant in EFC if they fulfilled the following enrichment criteria: (i) normalized spectral counts need to be at least 2.5 times higher in EFC compared to corresponding EFF data and (ii) at least ten spectra per protein in at least one sample need to be acquired. A similar strategy has been applied by Piersma et al. [13] and Flenkenthaler et al. [14]. The statistical analysis of protein abundance was also performed (Section 2.7). 2.5 Two-dimensional difference gel electrophoresis analysis 2.5.1 Protein labeling with CyDye DIGE fluors and 2DE For 2D DIGE analysis, four biological replicates (individual fish) were used, including the three same biological replicates as used in SDS-PAGE LC-MS/MS analysis. For each of the two groups (EFF and EFC), an internal standard consisting of 50 ␮g protein aliquots from each sample of all four biological replicates was generated and labeled with fluorescent dyes following the 2D DIGE minimal labeling procedure. A dye swap (Cy3/Cy5) was performed between EFF and EFC samples (2/2 aliquots). Briefly, for each biological replicate, 50 ␮g protein aliquots of each sample type (fresh, cryopreserved, and internal standard) were labeled with 400 pmol of Cy 2, Cy3, or Cy5 (CyDye DIGE fluors, GE Healthcare, Uppsala, Sweden) by incubation on ice for 30 min in the dark. Reactions were quenched by incubation with 1␮L of 10 mM lysine/50mg protein (Sigma–Aldrich, St. Louis, MO, USA). Samples labeled with Cy3 (50 ␮g) were mixed with samples labeled with Cy5 (50 ␮g) and 50␮g of Cy2-labeled internal standard, and rehydration solution was added (7M urea, 2M thiourea, 2% CHAPS, DDT, 2% pharmalyte pH 3– 10, and 130 mM DTT) to a final volume of 450 ␮L. Mixed samples were loaded onto 24 cm immobilized pH gradient strips (pH 3–10, GE Healthcare). IEF was performed using an IPGPhore system (GE Healthcare) with the current limited  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

to 50 ␮A/strip and the following voltage program: 500 V for 7 h, 1000 V linear ramp for 1 h, and linear ramp to 8000 V for 3 h, then 8000 V constant for 5.5 h. The IPG strips were then equilibrated by being soaked first for 15 min in 50 mM Tris– HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 1% DTT, and a trace of bromophenol blue, and then for 15 min in the same solution but containing 2.5% iodoacetamide instead of DTT. Equilibrated gel strips were fixed on top of the gels using 0.5% agarose dissolved in SDS running buffer (25 mM Tris, 192 mM glycine, 0.2% SDS). As second dimension, a 12.5% SDS-PAGE was run using the ETTAN Dalt six electrophoresis unit (GE Healthcare) at 25 mA/gel until the tracking dye had run off the gel.

2.5.2 Gel imaging and data analysis Gels were scanned immediately after electrophoresis using a Typhoon 9400 fluorescence scanner (GE Healthcare) with parameters recommended for 2D DIGE experiments by the manufacturer. Image analysis and relative quantification were performed using DeCyder Differential Analysis Software (Version 5.02, GE Healthcare). The number of spots was estimated to 10 000 and a spot volume of 30 000 was used as a cut-off filter. The number of spots 10 000 is a parameter set in DeCyder, which ensures that all detectable spots are taken. Depending on gel or image quality, artifacts can be specifically filtered out by discrete parametric values such as volume filtering. This reflects a more adaptive and flexible strategy to eliminate noise and potentially questionable spots close to the threshold of detection. Differential in-gel analysis was used to calculate protein abundance alterations between samples on the same gel. The resulting spot maps for each biological replicate were then analyzed through biological variation analysis to provide statistical data on the differential protein expression that existed between extracellular fluid of fresh and cryopreserved semen. Abundance alterations of proteins between EFF and EFC were considered as relevant if (i) the corresponding spots were detected in all gels, (ii) the Student’s t-test reached levels of significance with p ࣘ 0.05 (including FDR-correction), and (iii) the intensity ratio of spots exceeded a factor of 2 and ࣘ−2, respectively. After DIGE analysis, gels were stained with CBB R-250 (Bio-Rad). Clearly visible spots with significant intensity differences between EFF and EFC, which were well separated from adjacent spots, were digested and identified by MALDI-TOF/TOF as described below.

2.5.3 MALDI-TOF/TOF protein identification Spots of interests were manually excised and subjected to tryptic digestion as described above. The peptide mixtures were desalted using ZipTip C-18 RP tips (Millipore, Billerica, MA, USA) and spotted in a matrix of 0.5% cyano-4hydroxy cinnamic acid on a 96-spot MALDI target. Peptide www.proteomics-journal.com

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Table 1. Sperm characteristics of fresh and cryopreserved rainbow trout semen (n = 4)

Parameter

Fresh semen

Cryopreserved semen

Motility (%) VCL (␮m/s) VSL (␮m/s) ALH (␮m) LIN (%) VAP (␮m)

87.3 ± 2.9 178.4 ± 29.6 52.9 ± 7.9 20.6 ± 5.9 30.3 ± 3.3 114.8 ± 14.4

33.3 ± 5.7** 101.9 ± 2.9* 45.6 ± 9.6 11.7 ± 4.4 38.8 ± 5.7 68.4 ± 15.7*

Significantly different from fresh sperm. *p < 0.05, **p < 0.01.

samples were analyzed on a TOF Autoflex-ToF/ToF mass spectrometer (Bruker Daltonics, Bremen, Germany). MS peptide mass fingerprint and fragment spectra from each individual spot were combined and used to search against NCBIr fish database (released on April 10, 2014) using the MASCOT search engine (version 2.2, Matrix Science Ltd., London, UK) in consideration of the following settings: (i) enzyme trypsin; (ii) fixed modifications carbamidomethyl; (iii) variable modifications methionine oxidation; (iv) allowed to one missed cleavages; (v) peptide tolerance 0.5 Da, MS/MS tolerance. 0.5 Da; (vi) peptide charge 1+; (viii) instrument MALDI-TOF-TOF. Using these settings, a combined MASCOT score of ࣙ100 was taken as significant (p < 0.05) with at least two peptides per protein with MASCOT ions scores ࣙ 30. 2.6 GO annotation UniProtKB database (www.uniprot.org) was used to annotate proteins enriched in EFC. Two independent sets of ontology were used in the annotation: the “biological processes” in which the proteins participate and their “cellular component.” 2.7 Statistical analysis The CASA results are presented as mean ± SD. For statistical procedures, data percentages were transformed by arcsin square root transformation. The parameters of fresh and cryopreserved sperm were compared using paired t-test. Normalized spectral counts, in Scaffold denoted as “quantitative value” of corresponding protein in EFF and ECF, were logarithmized and analyzed using paired t-test. All analyses were performed at a significance level of 0.05 using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

3

Results

3.1 Semen analysis The percentage of sperm motility of cryopreserved semen was above 33%, which was 62% lower in comparison to fresh semen (87%; Table 1). VCL and VAP declined simultaneously  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. One-dimensional representative SDS gel (12% polyacrylamide) stained by CBB R-250. (1) EFF (40 ␮g) and (2) EFC (40 ␮g). Boxes in 1 and 2 indicate fractions individually analyzed by LC-MS/MS. (M) Molecular weight marker ranging from 10 to 250 kDa.

in cryopreserved semen by 43 and 40%, respectively, but VSL, LIN, and ALH values were not affected by cryopreservation.

3.2 LC-MS/MS identification of proteins enriched in EFC SDS-PAGE profiles of EFF and EFC are shown in Fig. 1. The corresponding gel lanes EFF and EFC samples of the same individual were cut into corresponding 11 slices as outlined in Fig. 1. To get samples suitable for LC-MS/MS, each gel slice was individually subjected to the in-gel digestion procedure described in Section 2. Protein abundance between EFF and EFC obtained from three individuals was quantitatively compared applying a spectral counting approach as described in Section 2. The criteria applied to the SDS-PAGE LC-MS/MS dataset yielding a list of 26 proteins enriched in EFC (with normalized spectral counts > 2.5 higher than corresponding protein in the EFF) and 37 proteins found exclusively in the EFC (Table 2; Supporting Information Table 1).

3.3 2D DIGE analysis of extracellular fluid of fresh and cryopreserved semen proteomes The total number of protein identifications obtained after 1DE prefractionation combined with LC-MS/MS analysis was 189, whereas the total number of spots detected by 2D DIGE was 949. 2D DIGE analysis revealed alterations in protein abundance between EFF and EFC of four individual males. Applying DeCyder software criteria (the corresponding spots were detected in all gels, p < 0.05 after FDR correction, and a spot intensity ratio > 2), 75 protein spots with different intensity in the comparison between EFF and EFC were detected (Fig. 2A and B). The overlay of channel images is shown in Fig. 2C. www.proteomics-journal.com

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Proteomics 2015, 15, 2643–2654 Table 2. Proteins enriched in EFC identified by LC-MS/MS

Protein name

Accession number

Creatine kinase, testis isozyme (O. mykiss) FK506-binding protein 2 precursor (O. mykiss) Heat shock 90 kDa protein 1 beta isoform a (O. mykiss) Tubulin alpha chain, testis-specific (O. mykiss) S-phase kinase-associated protein 1A (O. mykiss) FK506-binding protein 1A (O. mykiss) Nucleoside diphosphate kinase (O. mykiss) L-lactate dehydrogenase B chain (Salmo salar) Ubiquitin-conjugating enzyme E2 variant 1 (O. mykiss) Nucleoside diphosphate kinase B (O. mykiss) Triosephosphate isomerase (O. mykiss) Cytosolic malate dehydrogenase (O. mykiss) Calmodulin (O. mykiss) Ubiquitin (O. mykiss) Actin beta (O. mykiss) Cofilin-2 (O. mykiss) 14-3-3A1 protein (O. mykiss) 14-3-3 Protein gamma-1 (O. mykiss) Proteasome subunit beta type 1-A (O. mykiss) 2-Amino-3-ketobutyrate coenzyme A ligase, mitochondrial precursor (O. mykiss) 14-3-3E1 protein (O. mykiss) Superoxide dismutase (O. mykiss) 14-3-3C1 protein (O. mykiss) 14-3-3 Protein beta/alpha-2 (O. mykiss) 14-3-3 Protein beta/alpha-1 (O. mykiss) 14-3-3C2 protein (O. mykiss) Acetyl-CoA acetyltransferase, mitochondrial precursor (Salmo salar) Acyl-coenzyme A dehydrogenase C-4 to C-12 straight chain (Salmo salar) ADP-ribosylation factor 1 (O. mykiss) ADP-ribosylation factor 4 (O. mykiss) Alpha-enolase (Salmo salar) S-adenosylhomocysteine hydrolase (Salmo salar) Calreticulin (Salmo salar) Carbamoyl-phosphate synthetase III (O. mykiss) Dipeptidyl-peptidase 3 (Salmo salar) Disulfide-isomerase A3 precursor (Salmo salar) Dynein light chain 1, cytoplasmic (O. mykiss) Electron transfer flavoprotein subunit alpha, mitochondrial precursor (O. mykiss) Glucose-regulated protein 78 kDa (O. mykiss) 94 kD glucose-regulated protein (O. mykiss) Glutamate dehydrogenase 1 (O. mykiss) Glutamate dehydrogenase 3 (O. mykiss) Glyceraldehyde-3-phosphate dehydrogenase (O. mykiss) GTP-binding nuclear protein Ran (O. mykiss) 60 kDa HSP, mitochondrial precursor (Salmo salar) Heat shock cognate 70 kDa protein (O. mykiss) Inositol monophosphatase (Salmo salar) Isocitrate dehydrogenase 2-1 (NADP+), mitochondrial (Salmo salar) Isocitrate dehydrogenase 2-2 (NADP+), mitochondrial (Salmo salar) Isocitrate dehydrogenase 3 (NAD+) alpha (Danio rerio) Malate dehydrogenase 2-1, NAD (mitochondrial) (Salmo salar)

gi|125314 (+2) gi|225705942 (+1) gi|185132161 (+1) gi|135427 (+2) gi|225703580 (+1) gi|225704200 (+2) gi|185135416 (+1) gi|223648586 gi|225703674 gi|225705930 (+1) gi|34221914 gi|24421241 gi|225703528 (+5) gi|225703194 gi|185132289 (+2) gi|225703646 gi|185134456 (+3) gi|185134404 (+2) gi|225704042 gi|225705272

43 15 83 50 19 12 17 36 17 17 22 12 17 15 42 19 28 28 26 40

gi|185134340 (+1) gi|185132317 (+1) gi|185134260 (+1) gi|185134239 (+2) gi|185134229 (+2) gi|185134281 (+1) gi|209155134

29 16 28 27 28 28 56

3.0 3.0 2.8 2.6 2.5 2.5 n.d.

gi|197631959 (+2)

46

n.d.

gi|225705508 (+1) gi|225703934 (+1) gi|223646978 (+1) gi|197632181 (+4) gi|224613524 gi|1518088 (+1) gi|209155668 (+1) gi|209153384 (+1) gi|225703558 (+5) gi|225703308

21 20 47 48 45 167 81 55 10 35

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

gi|60223019 gi|302353531 (+1) gi|21666610 (+1) gi|21666614 gi|185132746 (+2)

70 91 60 60 36

n.d. n.d. n.d. n.d n.d

gi|225703290 gi|209153200 gi|218931112 (+3) gi|223649210 (+1) gi|197632445 (+3)

24 61 71 30 51

n.d. n.d. n.d. n.d. n.d.

gi|197632447 (+1)

51

n.d

gi|29124437 (+2) gi|197632449

40 35

n.d n.d.

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Molecular mass (kDa)

Ratio EFC/EFFa)

t-Test p-value

23.9 11.3 7.0 6.7 6.6 5.2 5.7 5.0 5.0 4.9 4.2 4.2 4.0 3.8 3.4 3.3 3.3 3.3 3.2 3.2

0.0003 0.0009 0.0013 0.0092 0.0041 0.004 0.0037 0.0051 0.0046 0.0073 0.0019 0.0027 0.0097 0.0060 0.0070 0.0065 0.0022 0.0011 0.0096 0.012 0.012 0.0149 0.0021 0.0080 0.0075 0.0040

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Table 2. Continued

Protein name

Accession number

Molecular mass (kDa)

Ratio EFC/EFFa)

Nuclear transport factor 2 (O. mykiss) Ornithine carbamoyltransferase, mitochondrial precursor (Salmo salar) Ran-specific GTPase-activating protein (O. mykiss) Ras-related protein Rab-11B (O. mykiss) Succinate-CoA ligase GDP-forming alpha subunit (O. mykiss) Valosin-containing protein (O. mykiss) Testis catalytic subunit of cyclic adenosine 3 , 5 -monophosphate dependent protein kinase (O. mykiss) Thioredoxin domain-containing protein 12 precursor (Salmo salar) Transketolase (Salmo salar) Transitional ER ATPase (O. mykiss) Tubulin beta-2C chain (Oreochromis niloticus) V-type ATPase B subunit (O. mykiss)

gi|225705646 (+1) gi|209738442

15 40

n.d. n.d.

gi|225704164 gi|225705282 gi|223049433 (+1)

24 17 34

n.d. n.d. n.d.

gi|185132242 (+1) gi|185135984 (+2)

83 41

n.d. n.d

gi|221219350 (+1)

19

n.d.

gi|209156132 (+1) gi|225703688 (+1) gi|348515923 gi|185132186 (+1)

68 36 50 56

n.d. n.d n.d. n.d.

t-Test p-value

a) Ratio EFC/EFF (extracellular fluid of cryopreserved semen/fresh semen): ratio according to spectral counting. n.d.: the protein is only detected in extracellular fluid of cryopreserved semen and not in fresh semen.

3.4 Identification of enriched proteins in EFC From 75 differentially abundant protein spots, we were able to unambiguously identify 71 spots using MALDI-TOF-TOF (Table 3; Supporting Information Table 2). Most of identified proteins corresponded to those identified by LC-MS/MS spectral count approach, except ten proteins (aconite hydratase, NAD-dependent malic enzyme, aldehyde dehydrogenase family 7 member A1, T-complex protein 1, chaperonincontaining TCP1, S-formylgluthatione hydrolase, aspartate aminotransferase, 14-3-3- protein theta, ubiquitin carboxylterminal hydrolase isoenzyme L3, disulfide-isomerase precursor). Nine proteins were identified in more than one protein spot, obviously representing protein isoforms with different molecular weight and/or isoelectric point, for example creatine kinase (CK) (identified in 23 spots), isocitrate dehydrogenase (identified in 3 spots), aconitate hydratase (identified in 3 spots), or aspartate aminotransferase (identified in 2 spots). CK (spot no. 28 and no. 29) was identified with the highest fold-change in EFC (a fold change > 40). We did not record any differences in the protein isoforms. All isoforms identified for a specific protein responded in the same manner.

3.5 GO analysis of proteins enriched in cryopreserved semen Among 73 identified proteins enriched in EFC (63 proteins identified after LC-MS/MS and 10 additional proteins after DIGE), 62 proteins were classified into “biological process” and 60 proteins into “cellular component” of GO database  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

(Fig. 3). The classification of proteins according to “biological process” revealed the majority of proteins to be associated with metabolic process (53%), and response to stimulus (25%). Using the classification “cellular component,” most proteins were classified as mitochondrion (33%) and cytosol (32%) proteins (Fig. 3).

4

Discussion

This is the first comprehensive description of the protein abundance differences in rainbow trout semen proteome after cryopreservation. The application of 1D SDS-PAGE prefractionation combined with LC-MS/MS and the “Spectral counting” algorithm for quantification facilitated the identification of 63 proteins enriched in rainbow trout EFC. To confirm the validity of this dataset, extracellular fluids of fresh and cryopreserved semen were additionally analyzed by the 2D DIGE proteomic approach. Moreover, the application of 2D DIGE led to the identification of ten additional proteins affected by cryopreservation. The analysis of cryopreservationinduced alteration in the quantitative protein composition of rainbow trout EFC using two independent methods (LCMS/MS and 2D DIGE) ensures a high confidence of our results. Fish spermatozoa are characterized by simplified structure and physiology. They do not have acrosome and the duration of the sperm movement is very brief. The percentage of sperm motility is considered to be the most important semen parameter reflecting the usefulness of semen for fertilization [15, 16]. We confirmed this relationship for cryopreserved semen for salmonid fish indicating high www.proteomics-journal.com

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Figure 2. 2D DIGE representative gel for the comparison of EFF and EFC proteins. Numbered protein spots (1–75) correspond to differentially abundant proteins in EFC. (A) Single channel image (Cy 3) EFF proteins; (B) single channel image (Cy 5) EFC proteins; (C) 2D DIGE gel (overlay images): more abundant proteins in EFC are displayed in red (Cy5); (D) examples for the 3D charts and normalized DIGE intensity ratios, serving as indicators for changes in protein abundance in EFF and EFC (94 kD glucose-regulated protein, creatine kinase, isocitrate dehydrogenase).  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Table 3. Proteins enriched in EFC; identified by MALDI-TOF-TOF protein spots with changes in abundance of at least > 2- fold (p < 0.05)

Fold change

t-Test pValue

3 5 5 2 2 2 2 2 4 2 2 2 2 2 2

11.4 3.7 3.6 3.5 9.2 7.1 2.3 5.8 7.5 2.7 5.8 7.0 3.7 8.4 2.3

0.010 0.016 0.042 0.042 0.022 0.047 0.02 0.04 0.0045 0.02 0.015 0.012 0.012 0.010 0.01

149 118 108 112 120

3 2 2 2 2

4.1 4.5 10.9 5.1 7.2

0.0065 0.0073 0.0044 0.022 0.0073

gi|350543526 gi|125314 gi|125314 gi|125314 gi|125314 gi|125314 gi|125314 gi|125314 gi|125314 gi|209731104 gi|213514332

151 129 395 513 108 569 199 709 515 414 161

2 2 4 5 2 5 2 6 4 4 3

3.1 12.3 6.4 10.0 6.5 23.1 16.3 33.8 19.4 23.7 13.9

0.0097 0.0082 0.0069 0.0065 0.0078 0.009 0.0067 0.0097 0.015 0.0079 0.00930

gi|223672929 gi|225707654

216 143

2 2

9.8 14.7

0.0075 0.013

gi|22505994

133

2

9.0

0.0082

gi|46358344

239

3

8.3

0.0078

gi|198285519

235

3

7.9

0.0069

gi|1213511138 gi|223673113 gi|125314 gi|209154700 gi|259089502 gi|125314 gi|209738442

178 177 206 119 129 200 350

2 2 3 2 2 3 4

4.2 12.7 12.9 3.8 6.6 27.3 4.1

0.018 0.0037 0.0069 0.04 0.0035 0.0065 0.022

gi|209738442

276

3

7.7

0.009

gi|317119971 gi|197632449 gi|213512098 gi|125314

187 302 298 291

3 4 4 3

5.8 10.0 16.2 41.7

0.0097 0.032 0.0062 0.0035

Spot number in Fig. 2B

Identified protein

Accession number

MASCOT score

Number of peptides (ion score ࣙ 30)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

94 kD glucose-regulated protein Valosin-containing protein Valosin-containing protein Aconitate hydratase, mitochondrial Aconitate hydratase, mitochondrial Aconitate hydratase, mitochondrial Dipeptidyl peptidase 3 Glucose-regulated protein 78 kDa Glucose-regulated protein 78 kDa Transketolase Calreticulin precursor Disulfide-isomerase precursor Disulfide-isomerase precursor Alpha enolase NAD-dependent malic enzyme, mitochondrial-like Aldehyde dehydrogenase family 7 member A1 T-complex protein 1 subunit zeta Chaperonin-containing TCP1, subunit 7 Adenosylhomocysteinase B Succinyl-CoA ligase (ADP-forming) subunit beta; mitochondrial-like Beta-actin Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Isocitrate dehydrogenase 2-2 (NADP+), mitochondrial Creatine kinase Isocitrate dehydrogenase, mitochondrial precursor Isocitrate dehydrogenase, mitochondrial precursor Isocitrate dehydrogenase (NAD) subunit alpha, mitochondrial Electron transfer-flavoprotein alpha polypeptide S-formylglutathione hydrolase Glyceraldehyde-3-phosphate dehydrogenase Creatine kinase Malate dehydrogenase, cytoplasmic Creatine kinase Creatine kinase Ornithine carbamyoltransferase, mitochondrial precursor Ornithine carbamyoltransferase, mitochondrial precursor Malate dehydrogenase, cytoplasmic Malate dehydrogenase 2-1 NAD, mitochondrial Malate dehydrogenase 2-1 NAD, mitochondrial Creatine kinase

gi|303324549 gi|185132242 gi|185132242 gi|317419173 gi|38707983 gi|38707983 gi|213513776 gi|60223019 gi|60223019 gi|209156132 gi|209154212 gi|224613274 gi|224613274 gi|223672841 gi|348517316

146 459 361 103 110 102 164 129 380 94 103 108 106 187 168

gi|213514574 gi|41152046 gi|190340066 gi|213513453 gi|348524076

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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Proteomics 2015, 15, 2643–2654 Table 3. Continued

Spot number in Fig. 2B

Identified protein

Accession number

MASCOT score

Number of peptides (ion score ࣙ 30)

49 50 51

Creatine kinase Creatine kinase Testis catalytic subunit of cyclic adenosine 3 ,5 -monophosphate dependent protein kinase Aspartate aminotransferase, mitochondrial precursor Aspartate aminotransferase, mitochondrial precursor 14-3-3 Protein beta/alpha-1 14-3-3 Protein beta/alpha-2 14-3-3 Protein theta 14-3-3 Protein gamma-1 Ubiquitin carboxyl-terminal hydrolase isozyme L3 Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Creatine kinase Thioredoxin domain containing protein 12 Creatine kinase Creatine kinase Nuclear transport factor 2 FK506-binding protein 1B Dynein light chain 1, cytoplasmic Nucleoside diphosphate kinase

gi|213512882 gi|125314 gi|185135984

195 318 198

gi|209155580

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71

fertilizing ability of spermatozoa characterized by high percentage of motility [10, 17, 18]. Sperm motility parameters obtained in the present study were within the range established for trout semen cryopreserved with the application of 0.3 M glucose in 10% methanol as an extender, that is we previously observed 20 ± 5.4% sperm motility of cryopreserved rainbow trout semen [17]. It can be assumed that

Fold change

t-Test pValue

3 4 2

36.9 13.8 6.4

0.0035 0.011 0.018

175

2

12.1

0.0062

gi|209150416

118

2

10.0

0.0083

gi|82203881 gi|213512804 gi|41393183 gi|49227280 gi|226443186

262 407 105 101 157

2 3 2 2 2

2.8 3.5 2.1 3.3 2.5

0.042 0.044 0.046 0.046 0.031

gi|125314 gi|125314 gi|125314 gi|125314 gi|125314 gi|213512882 gi|226443348 gi|125314 gi|225704742 gi|209731592 gi|304434637 gi|225704956 gi|194500331

118 240 303 434 378 132 183 302 103 104 190 137 208

2 2 3 4 3 2 4 3 2 2 2 2 3

14.2 38.5 24.3 19.9 13.0 21.1 9.0 21.5 26.5 2.5 6.3 3.1 7.7

0.0069 0.0069 0.0035 0.0041 0.0035 0.0057 0.0065 0.0065 0.004 0.0078 0.016 0.044 0.016

all sperm samples used in our experiment were successfully cryopreserved. The obtained data are complementary to our previous results identifying 358 proteins of rainbow trout semen [19,20]. Most of the 73 proteins we found to be altered in abundance after the freezing-thawing procedure correspond to sperm proteins previously identified [20]. Furthermore, our

Figure 3. GO analysis of proteins enriched in EFC.

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study demonstrates the presence of eight additional proteins, formerly not detected in rainbow trout sperm. These proteins are mostly enzymes: ornithine carbamyoltransferase, adenosylhomocysteinase, aconite hydratase, NADdependent malic enzyme, aldehyde dehydrogenase family 7, S-formylglutathione hydrolase, aspartate aminotransferase, and 14-3-3 protein theta. The number of identified proteins in this study released after cryopreservation was higher compared to the previous studies addressing effects of cryopreservation on semen proteome of other fish species [4, 5, 8, 9]. Summing up, our results broadened the knowledge about the protein composition of rainbow trout semen, provided new important information about salmonid fish reproduction, and of cryoinjures of rainbow trout spermatozoa. Moreover, the results are relevant for the selection of biomarkers useful for the evaluation of cryopreservation technology. GO analysis for “cellular component” revealed that most of proteins (65%) enriched in extracellular fluid were of mitochondrial and cytosolic origin. Mitochondrial and cytosolic proteins were in equal proportion, which agrees with the results of O’Connell et al. [21], who found that the plasma and mitochondrial membranes of human sperm have similar susceptibility to cryopreservation. The presence of mitochondrial sperm proteins in extracellular fluid likely reflects the injury of the sperm mitochondria structures and membranes by cryopreservation and may result from leakage of proteins from spermatozoa. The identification of matrix proteins could indicate that damages to mitochondria were very severe. So far, mitochondria damages were examined with the application of fluorescence-based methods [10, 22]. Our study supplements these studies by providing a list of sperm proteins released from the mitochondrium and can be used in the future for the detailed evaluation of cryoinjures to particular mitochondria structures. The abundance alteration of cytosolic proteins during freezing-thawing procedures was demonstrated previously for higher vertebrates sperm [23] and also for common carp sperm [5]. In this study, we provided an extended list of cytosolic proteins as compared to published data [4, 5, 8], which appear in the extracellular fluid after the freezing-thawing process. CK was identified with the highest fold change (41.7) after cryopreservation, which indicates that this enzyme is rather loosely bound to cytoplasmic membranes, as opposed to possibly tightly bound 14-3-3 protein theta with the lowest fold change (2.1). We believe that our findings are the first step for targeted and detailed analysis of cryoinjures to spermatozoa, for example for the localization of these proteins, studies addressing the attachment of these proteins to particular sperm cytoplasmic membranes, and for experiments monitoring their concentration within sperm cells. The sperm cytoskeleton is organized in a dynamic intracellular network important for the maintenance of normal cell shape and coordinated cell movement, as well as for the appropriate cell volume regulation [24]. Since cytoskeleton proteins are osmosensitive and thermosensitive, a direct connection between cell volume regulation, flagellar morphol C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2015, 15, 2643–2654

ogy, and the actin cytoskeleton in the sublethal damage that occurs due to osmotic stress during cryopreservation is postulated [24]. In this study, the cryopreservation led to the significant release of cytoskeletal proteins, such as cofilin, dynein, actin, and tubulin. In summary, our results indicate that besides the destabilization of the spermatozoa movement apparatus, cryopreservation resulted in the alterations of cytoskeleton organization. Cryopreservation can cause DNA damage of spermatozoa in fish species [3]. However, the effect on nuclear proteins has not been examined. In this study, we found that rainbow trout EFC is enriched with nuclear proteins participating in signal transduction, such as members of the Ras family, but no structural nuclear proteins, such as protamines and histones, were found. Summing up, it can be postulated that cryopreservation causes disturbances to nuclear regulatory proteins of rainbow trout sperm, but does not affect structural proteins. Future research should be focused on the examination of the effect of the release of nuclear regulatory proteins on the progeny survival and development. Such research is important for evaluating the possible effect of cryopreservation on next generation of fish. Apart from lethal cryoinjuries through intracellular ice formation, there may be sublethal effects on spermatozoa at a molecular level, affecting sperm functions, mainly sperm motility and fertilizing ability. Our data suggest that these sublethal damages are related with the loss of numerous proteins. The GO analysis for “biological process” of protein released after cryopreservation revealed that most of them are involved in metabolic processes, such as glycolysis, tricarboxylic acid cycle and amino acids, carbohydrate, and fatty acids metabolism. The leakage of these proteins may explain the mechanism responsible for the decrease in sperm metabolic activity and in consequence the disturbances in ATP production and ATP regeneration resulting in the decline in sperm motility. This coincides with lower percentages of motile sperm, changed parameters of cryopreserved sperm movement trajectory, and its duration in comparison to fresh sperm. In summary, our results strongly suggest that cryoinjures did not only damage sperm structures, but also lead to the impairment of sperm metabolism. The latter can be important for semen quality during post-thaw storage, since it can lead to the deterioration of sperm physiology, especially quality and duration of sperm movement. Besides the proteins involved in sperm metabolism, we found the release of proteins engaged in diverse functions, such as response to stimulus, antioxidant protection, protein folding, and ubiqutination. The production of ROS during cryopreservation and a higher susceptibility of frozen–thawed spermatozoa to ROS ongoing with a loss of antioxidant and physical stress defenses were described [25]. For this reason, the release of sperm proteins participating in antioxidant protection, such as superoxide dismutase, may additionally strengthen the damages caused by ROS. HSPs are involved in cell protection by refolding denatured protein, removing damaged proteins by degradation and by blocking apoptosis [26]. www.proteomics-journal.com

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The ubiquitin–proteasome system is the major pathway for intracellular protein degradation, and ubiquitin-dependent proteolysis plays a role in sperm cell differentiation and cell cycle control throughout spermatogenesis and fertilization of fish [27]. Proteasomes, in turn, have also been identified as a regulator of sperm motility of salmonid fish through modulation of dynein cAMP-dependent phosphorylation [28]. Lower levels of HSPs and proteins related to ubiqutination after cryopreservation can potentially disturb protein protection and regulation of protein degradation during post-thaw semen storage. Summing up, the freezing–thawing of rainbow trout semen diminishes the antioxidant capacity and probably leads to disturbances in the ubiquitin–proteasome system in rainbow trout spermatozoa, which likely influences the motility. The activities of the intracellular enzymes are frequently used to evaluate the spermatozoan plasma membrane integrity, since the extracellular fluid enzyme concentrations increase after thawing as a result of damage to sperm membranes [29, 30]. So far, sperm enzymes are believed to be most applicable for the evaluation of spermatozoa damage. With our study we provided an extended list of diverse proteins, which can be used for a more comprehensive monitoring of sperm cryoinjures. We demonstrated the release of mitochondrial, cytoskeletal, nuclear, and cytosolic proteins, which could be new predictors of damages after freezingthawing procedure for particular sperm structures. Further studies should be conducted to select among these proteins the most suitable markers of sperm structure cryoinjures. Additional studies are needed to select the proteins that can be applied to the indication of the disturbances of particular sperm metabolic pathways. Recently, we have established the proteomic characterization of rainbow trout sperm [20]. Nine proteins identified in this study, such as CK, tubulin alpha chain, 14-3-3 C1 protein, 14-3-3 C2 protein, 14-3-3 protein beta/alpha-1, 143-3 protein beta/alpha-1, glucose-regulated protein 78 kDa, valosin-containing protein, and beta actin, were previously found to be the most prominent proteins in rainbow trout semen [20]. The presence of the major sperm proteins in the EFC might be attributed to membrane damage and consequently extracellular leaking. On the other hand, outer dense fiber of sperm tail protein 3 (gi|185132120) identified also as a major sperm protein was not released into EFC. This suggests some selectivity of cryodamage to sperm proteins. Further studies focused on the damages induced by cryopreservation without cryoprotectant are necessary in order to identify potential specific damages to spermatozoa during cryopreservation. In summary, we have applied for the first time in any fish species a combination of two different proteomic techniques to study the influence of cryopreservation process on rainbow trout semen. We identified spermatozoal proteins in extracellular fluid, which reflect the cryopreservation-induced alterations of the rainbow trout semen proteome. Our study provides new information about proteins released from sperm, their relation to sperm structure and function, and changes  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of metabolism of sperm cells as a result of cryopreservation. Furthermore, our data are the basis for studies deciphering the precise function of the proteins altered during rainbow trout cryopreservation and are useful for the development of extensive diagnostic tests of sperm cryoinjures and for the successful improvement of sperm cryopreservation of this economically important species. ´ We thank Mariola Słowinska for support in 2D DIGE analysis and Grzegorz Dietrich for technical assistance in semen cryopreservation and CASA analysis. This work was supported by Iuventus grant IP2011 0390 71 from Polish Ministry of Higher Education and funds appropriated to Institute of Animal Reproduction and Food Research. The authors have declared no conflict of interest.

5

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