Animal Reproduction Science 152 (2015) 99–107

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Differential protein expression in chicken spermatozoa before and after freezing–thawing treatment Chuen-Yu Cheng a,1 , Pin-Rong Chen a,1 , Chao-Jung Chen b,c , Shin-Han Wang a , Chih-Feng Chen a,d,e , Yen-Pai Lee a , San-Yuan Huang a,d,e,f,∗ a

Department of Animal Science, National Chung Hsing University, Taichung 40227, Taiwan Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan Graduate Institute of Integrated Medicine, China Medical University, Taichung 40402, Taiwan d Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan e Center for the Integrative and Evolutionary Galliformes Genomics, iEGG Center, National Chung Hsing University, Taichung, 40227, Taiwan f Center of Nanoscience and Nanotechnology, National Chung Hsing University, Taichung, 40227, Taiwan b c

a r t i c l e

i n f o

Article history: Received 10 May 2014 Received in revised form 21 October 2014 Accepted 9 November 2014 Available online 2 December 2014 Keywords: Chicken Sperm Cryopreservation Proteome

a b s t r a c t The biological characteristics of rooster sperm that has undergone freezing treatment remain elusive. This study analyzed the change in sperm proteins after freezing–thawing treatment by using a proteomic approach. Semen from three 36-wk-old L2 strain Taiwan country chickens were used. A qualifying ejaculate containing more than 80% motility and volume 200 ␮L was used for cryopreservation. The proteomic analysis explored 55 protein spots that differed significantly before and after freezing–thawing treatment (P < 0.05). Among the 55 protein spots, expression levels of 19 proteins decreased after treatment. Forty-five differentially expressed protein spots were identified and belong to 33 proteins. Results of gene ontology analysis revealed that most differentially expressed proteins were involved in molecular function of the cellular metabolism process (28%) and cellular carbohydrate metabolism process (15%), and were associated with molecular function of oxidoreductase activity (19%) and protein binding (18%). The differentially expressed proteins before and after freezing–thawing treatment, including fructose-bisphosphate aldolase C, triosephosphate isomerase, aconitate hydratase, tubulin and outer dense-fiber protein, are associated with sperm energy metabolism and flagellum structure. In conclusion, freezing–thawing treatment significantly affects the expression of proteins related to sperm metabolism and structure in chicken spermatozoa. The differing levels of these proteins could be valuable for further enhancing the fertility of frozen–thawed chicken spermatozoa. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan. Tel.: +886 4 22870613x245. E-mail address: [email protected] (S.-Y. Huang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anireprosci.2014.11.011 0378-4320/© 2014 Elsevier B.V. All rights reserved.

The Food and Agriculture Organization of the United Nations announced that, in 2000, 9% of the global farm animal population was in critical condition and 39% was threatened with extinction (Sawicka et al., 2011). Consequently, preserving gametes to prevent species extinction and maintain gene diversity is becoming increasingly

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urgent. Avian semen cryopreservation was first conducted in 1942 but failed to achieve hatched chicks until 1949 (Polge, 1951; Polge et al., 1949; Shaffner, 1942). The quality of spermatozoa decreased after being frozen and thawed due to high osmolality, mechanical harm, ice crystal formation, and pH changes (Benson et al., 2012; Glander and Schaller, 1999; Long, 2006; Purdy et al., 2009). The biological changes of spermatozoa after cryopreservation include the disturbance of adenosine triphosphate (ATP) production, causing a decrease in the motility of spermatozoa (Long, 2006). Sperm are highly differentiated cells without organelles such as the endoplasmic reticulum, Golgi complex, peroxisomes, lysosomes, and ribosomes. Therefore, gene expression (transcription and translation) does not occur in mature spermatozoa (Boerke et al., 2007). However, the structure and quantity of semen proteins are still changeable after ejaculation and affect capacitation, acrosome reaction, sperm membrane protection, prevent oxidative stress, and antimicrobial activity (Manaskova et al., 2003; Moura et al., 2007). Proteomic analysis has been applied to characterize changes in sperm proteins under various conditions (Labas et al., 2014; Park et al., 2013; Sharma et al., 2013; Partyka et al., 2013). Studies in boar spermatozoa revealed that cyopreservation of boar spermatozoa caused differential expression of 41 proteins which were related to sperm capacitation, adhesion, energy supply and sperm-oocyte binding and fusion (Chen et al., 2014). A high hydrostatic pressure treatment of boar spermatozoa before cryopreservation increased the expression of proteins related to sperm-zona pellucida reactions while decreased the expression of proteins associated with sperm motility (Huang et al., 2009). In poultry, Froman et al. (2011) analyzed the differential expression of proteins in sperm exhibiting high and low motility in New Hampshire roosters. Furthermore, Labas et al. (2014) identified 1165 proteins involved in oxidoreduction mechanisms, energy processes, proteolysis and protein localizations that affected the fertility of rooster sperm. However, the proteomic change of rooster spermatozoa after cryopreservation is still elusive. The purpose of this study was to investigate the change of protein expression in chicken sperm after freezing–thawing treatment. 2. Materials and methods 2.1. Animal care, semen collection, and evaluation of semen quality Three 36-week-old roosters of L2 strain Taiwan country chicken (TCC) were used in this study. All roosters were kept in mechanically controlled chambers at 25 ◦ C, a relative humidity of 55%, and a 14L:10D photoperiod. The roosters were fed commercial feed (breeder feed, DaChan, Tainan, Taiwan) and water ad libitum. Semen was collected by abdominal massage (Burrows and Quinn, 1937). Sperm motility was assessed using a phase contrast microscope (BX51, Olympus, Japan), and represented as a percentage (modified from Blesbois et al., 2008). Sperm viability was assessed using a Live/Dead sperm viability kit (Invitrogen, CA, USA) (Chalah et al., 1999). Semen with sperm motility

higher than 80% and semen volume higher than 200 ␮L was used for cryopreservation. The roosters were healthy and with good semen quality when compared to their contemporaries. 2.2. Preparation of the semen and freezing and thawing of rooster sperm The freezing and thawing protocols were based on previously established methods (Tselutin et al., 1999). The Lake extender (composed of 19.2 g of sodium glutamate, 8 g of fructose, 5 g of potassium acetate, 0.7 g of magnesium acetate, and 3 g of polyvinylpyrrolidone dissolved in 1 L of distilled water; Lake, 1968) was used for cryopreservation and diluted with 1:1. The total volume of semen was divided into fresh and freezing treatment groups. Semen for cryopreservation was cooled at 5 ◦ C for 20 min, added to 6% dimethylacetamide (DMA), and then maintained at 0 ◦ C for 1 min. The semen was then dropped directly into liquid nitrogen by using a pipette and stored at −196 ◦ C for more than a week. Frozen semen was thawed at 60 ◦ C for 2 min with an equal volume of extender. Sperm motility and viability were evaluated as described in the previous section. 2.3. Analysis of proteins using two-dimensional gel electrophoresis For protein analysis, semen was centrifuged twice at 300 × g for 10 min to remove the semen plasma, extender, and cryoprotectant. The sperm was lysed using a lysis buffer [9.5 M urea, 2% NP-40, 2% v/v Ampholyte 3-10, and 65 mM dithiothritol (DTT; USB Corporation, Cleveland, OH, USA)] and quantified using a 2D Quant Kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Approximately 500 ␮g of sperm protein was used for two-dimensional gel electrophoresis (2-DE) analysis (Huang et al., 2009). In brief, isoelectric focusing (IEF) was conducted using 18-cm IPG DryStrips (pH 3–10 linear) and the IPGphor 3 system (GE Healthcare Bio-Sciences AB). Sperm proteins were mixed with an equal volume of rehydration buffer [8 M urea, 2% 3-[(3-cholamidopropyl) dimethylamonio]-1propanesulfonate (CHAPS; Sigma–Aldrich, Inc., Saint Louis, MO, USA), and 0.5% v/v Pharmalyte 3–10 NL], loaded onto a strip holder, and then covered with a DryStrip and cover fluid. The DryStrips were rehydrated at 30 V for 12 h with the sample and focused for 64,000 volthours (V h) for 7–9 h. After focusing, the strips were equilibrated in a sodium dodecyl sulfate (SDS) equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 0.002% bromophenol blue) containing 100 mM DTT for 15 min, followed by equilibration in an SDS equilibration buffer containing 250 mM iodoacetamide for 15 min. The equilibrated DryStrips were layered on top of a vertical 12.5% SDS–polyacrylamide gel for second-dimension separation using the Daltsix Vertical electrophoresis system (GE Healthcare Bio-Sciences AB). The separation was conducted under a condition of 2.5 W/gel for 25 min at 15 ◦ C, followed by 8 W/gel until the dye front reached the bottom of the gel. The PageRuler Unstained Protein Ladder (Fermentas International Inc., Vilnius, Lithuania) which contains synthetic peptides of

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molecular weights ranging from 10 to 200 kDa was used as molecular weight standards. 2.4. Staining and imaging of the 2-DE gels After electrophoresis, the gels were stained with colloidal Coomassie blue (SERVA blue G, SERVA Electrophoresis GmbH, Heidelberg, Germany) for at least 14 h (Neuhoff et al., 1988). Following staining, the gels were neutralized using 0.1 M Tris/phosphoric acid (pH 6.5) for 3 min and destained using 25% methanol until the background was clear. The gels were then scanned using an Image Scanner III (GE Healthcare Bio-Sciences AB) and saved as TIFF images for analysis. 2.5. Analysis of protein expression levels before and after freezing–thawing treatment Protein spots on the 2-DE gels were detected and analyzed using the Melanie 7 software package (GeneBio, Geneva, Switzerland). To determine the levels of protein expression in spermatozoa, protein spots on all triplicate 2-DE gels from three roosters before and after freezing were quantified. The ratio of the volume of each spot to the total volume (RVol) of all quantified spots was generated by the software (Huang et al., 2009). The RVol was used to determine the expression level of each protein spot. The expression ratio of differentially expressed spots was represented by the ratio of the RVol of the high-value protein spot to that with the low value. 2.6. Protein identification by using mass spectrometry 2.6.1. In-gel digestion Differentially expressed protein spots were cut from the gels for identification. In-gel trypsin digestion was conducted according to Havlis et al. (2003), with minor modifications (Huang et al., 2009). Gel plugs were washed twice with double-distilled water, followed by 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate and then with pure ACN. Gel plugs were dried in a SpeedVac evaporator (Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and subjected to in-gel digestion or stored at −20 ◦ C. For in-gel digestion, gel plugs were rehydrated using 20 ng/␮L of trypsin (Promega Corporation, Madison, WI, USA) in 25 mM ammonium bicarbonate at 4 ◦ C for 30 min. Digestion was allowed to perform at 37 ◦ C overnight. After digestion, peptide products were recovered using 40% ACN and 1% trifluoracetic acid (TFA; Riedel-deHaen, Seelze, German). 2.6.2. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis The digested samples were spotted directly onto a 600-␮m/384-well AnchorChipTM sample target (Bruker Daltonics, Bremen, Germany). An equal volume of a 1 mg/mL solution of CHCA in 0.1% TFA/50% ACN was then added. The MALDI mass spectra were obtained using a Bruker autoflex TOF mass spectrometer equipped with a 384-sample scout source (Bruker Daltonics). An external peptide calibration standard containing angiotensin II

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([M+H]+ 1046.54), angiotensin I ([M+H]+ 1296.68), substance P ([M+H]+ 1347.74), bombesin ([M+H]+ 1619.82), ACTH clip 1–17 ([M+H]+ 2093.09), ACTH clip 18–39 ([M+H]+ 2465.20), and somatostatin 28 ([M+H]+ 3147.47) was used to calibrate the instrument. Spectra were acquired in the reflectron mode. Peptide masses were examined against a comprehensive nonredundant protein sequence database (NCBInr 20130524 version with 25805290 sequences and 8915431356 residues; SwissProt 2013 05 version with 540052 and 191770152 residues) by using the Mascot program (Perkins et al., 1999) for protein identification. The search criteria were taxonomy for Mammalia, fixed carbamidomethyl modification, variable oxidation modification, a mass accuracy of 50–300 ppm, and a maximum of one missed cleavage site. Positive identification was achieved when a minimum of five peptides were matched and the mass accuracy and modification of the score were matched the protein or mixture of proteins in the database with a significant probability of P < 0.05. Protein identification was further confirmed using MALDITOF/TOF MS/MS analysis. 2.6.3. MALDI-TOF/TOF MS analysis The AnchorChip target was subjected to MALDITOF/TOF analysis by using a Bruker UltraFlex III MALDITOF/TOF MS (Bruker Daltonics) equipped with a delayed extraction ion source. We analyzed metastable ions generated by laser-induced decomposition in the laser-induced forward transfer (LIFT) mode (Bruker Daltonics). The precursor ion and its corresponding fragment ions were selected in a time gate, followed by further acceleration in the LIFT cell at 19.0 kV. The fragment ions were accelerated into the second field-free region and separated in the twostage gridless reflectron. The reflectron voltage was set at 27.4 kV. Masses were processed using the FlexAnalysisTM 2.4 software (Bruker Daltonics). We identified the proteins by searching for MS/MS spectra in the NCBInr database (NCBInr 20130524 version and SwissProt 2013 05 version) by using BioTools 3.0 software (Bruker Daltonics) combined with the MASCOT program. The search criteria were as follows: taxonomy for bony vertebrates, fixed modification of carbamidomethyl modification, variable modifications of oxidation modification, 100 ppm for the precursor ion, and 0.5 Da for fragment ions. Positive identification was achieved when scores exceeded the minimal significance individual ion score (P < 0.05). 2.7. Gene ontology (GO) annotation of differentially expressed proteins The official gene symbols of the differentially expressed proteins were used to investigate and categorize the GO annotations. The original GO annotations (cellular components, molecular functions, and biological processes) were downloaded from the NCBI Entrez Gene database and analyzed further. 2.8. Statistical analysis The semen quality traits and RVols of the protein spots on all 2-DE gels from the three roosters before and after

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freezing were subjected to ANOVA analysis using the general linear model procedure in the Statistical Analysis System software (SAS, 2010). The comparison of means between treatments was conducted by the least-squares means method in the software. The difference was considered significant at P < 0.05. 3. Results 3.1. Motility and viability of chicken spermatozoa before and after freezing–thawing treatment The data of fresh and the post-thaw chicken spermatozoa showed that the freezing–thawing treatment significantly decreased the motility by 20.0% (90.0 ± 3.5% vs. 70.0 ± 3.5%; P < 0.05) and viability by 64.3% (90.3 ± 3.7% vs. 26.0 ± 3.7%; P < 0.01). 3.2. Quantitative changes in sperm protein levels after freezing–thawing treatment The protein profiles of fresh and frozen–thawed chicken sperm are shown in Fig. 1. The patterns of the protein profiles were similar between the two groups and around 500 protein spots were detected on each gel. The expression levels of 495 protein spots on each 2-DE gel were quantified. Fifty-five protein spots showed a significant difference between the fresh and frozen–thawed spermatozoa (Table 1; P < 0.05). The expression levels of 36 protein spots increased and 19 spots decreased in the frozen–thawed spermatozoa. 3.3. Identification of proteins with different levels between fresh and frozen–thawed sperm The purported identities of the differentially expressed proteins are listed in Supplementary Table 1. In total, 45 spots were identified and belong to 33 proteins. The remaining 10 spots could not be identified, suggesting that these proteins may be unknown. The expression levels of tubulin ␤-3, dynein intermediate chain, dynein, axonemal, light intermediate chain 1, and tektin 2, hosphatidylethanolamine-binding protein 1, protien kinase C inhibitor, homer 1, superoxide dismutase, dihydropyrimidinase, fructose-bisphosphate aldolase C, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, l-lactate dehydrogenase, NADH dehydrogenase 1 alpha subcomplex subunit, creatine kinase, and cytochrome Bc1 complex decreased while the level of tubulin ␣-3, outer dense-fiber protein, and tektin 5 increased aconitate hydratase, voltage-dependent anion-selective channel protein 2, heat shock protein 70 increased in the frozen–thawed sperm (Table 1). 3.4. Bioinformatic analysis of differentially expressed proteins between fresh and frozen–thawed sperm The result on the GO annotations of the 33 differentially expressed proteins is shown in Fig. 2. Regarding cellular component, most of the proteins were located in mitochondria (31%), microtubules (18%), cytosol (18%), and

cytoplasm (15%). Regarding biological process, the majority of the proteins were involved in cell metabolic process (28%), cellular carbohydrate metabolic process (15%), signal transduction (9%), cellular component biogenesis (6%), and sperm motility (6%). Regarding molecular function, 19% of the proteins participated in oxidoreductase activity, 18% in protein binding, 12% in hydrolase activity, and 9% in lyase activity. 4. Discussion The semen characteristics of the three L2 strain TCC roosters used in this study were comparable to our previous observations (semen volume 333.3 ± 82.3 ␮L vs. 253.2 ± 61.2 ␮L, motility 90.0 ± 3.5% vs. 62.9 ± 11.4%, sperm concentration 5.67 ± 1.34 × 109 /mL vs. 4.55 ± 1.33 × 109 /mL, and viability 90.3 ± 3.7% vs. 72.28 ± 8.25%; Cheng et al., unpublished data). Thus the three roosters can be considered to be healthy and with normal semen quality for further freezing–thawing treatment and representative for proteomic analysis. Proteomic and bioinfomatic analysis showed that 45 of the 55 differentially expressed protein spots were identified and belong to 33 proteins. The functions of these differentially expressed proteins with known identities are listed in Table 2. 4.1. Freezing–thawing treatment changes the levels of proteins related to sperm flagellum structures Proteomic analysis showed that the expression levels of tubulin ␣-3, outer dense-fiber protein, and tektin 5 increased, and those of tubulin ␤-3, dynein intermediate chain, dynein, axonemal, light intermediate chain 1, and tektin 2 decreased after the freezing and thawing of chicken sperm (Tables 1 and 2). Tubulin is associated with the mobility of sperm flagellum (O’Donnell and O’Bryan, 2014). Tektins could stabilize the binding of dyneins, nexin, and radial spokes to tubulin (Nojima et al., 1995). Furthermore, Pixton et al. (2004) reported that the level of outer dense-fiber protein 2 was significantly higher in sperm samples that failed to fertilize during in vitro fertilization. Increase of outer dense-fiber protein 2 may imply that the structure of the sperm flagellum is unstable (Bedford and Calvin, 1974) and thus decrease the motility of spermatozoa by disturbing the normal functions of the flagellum. In the present study, the changes of sperm flagellum structure related proteins may infer that the structure of sperm flagellum was damaged and then caused a decrease of sperm motility in rooster after freezing–thawing treatment. 4.2. Proteins relate to energy metabolism were affected by freezing–thawing treatment Freezing–thawing treatment decreased energy metabolism related enzymes like fructose-bisphosphate aldolase C, triosephosphate isomerase, glyceraldehyde3-phosphate dehydrogenase, l-lactate dehydrogenase, NADH dehydrogenase 1 alpha subcomplex subunit, creatine kinase (CK), and cytochrome Bc1 complex and increased aconitate hydratase in chicken sperm

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Fig. 1. The protein profiles and differentially expressed spots of chicken spermatozoa before (A) and after (B) the freezing and thawing process. Fivehundred micrograms of soluble proteins were separated using IEF with an 18-cm IPG DryStrip of pH 3–10 NL, followed by 12.5% SDS–PAGE. Proteins were visualized using colloidal Coomassie blue staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2. The annotation of cellular component (A), biological process (B), and molecular function (C) of differentially expressed proteins in chicken spermatozoa before and after freezing–thawing treatment. The original GO annotations were downloaded from the NCBI Entrez Gene database and analyzed further. The percentages are the total hits divided by the number of annotated proteins for each GO category.

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Table 1 The differentially expressed rooster sperm proteins before and after freezing–thawing treatment# . Spot no.*

Protein identity

Fresh

Frozen–thawed

P value

Expression ratio$

8 9 30 43 55 61 95 106 112 124 135 146

Creatine kinase (EC 2.7.3.2) chain Bb Creatine kinase (EC 2.7.3.2) chain Bb Tubulin beta-3 chain 2310014H01Rik protein Similar to dynein intermediate chain Similar to dynein, axonemal, light intermediate chain 1 Aconitate hydratase, mitochondrial Unknown l-lactate dehydrogenase B chain Chain F, Cytochrome Bc1 Complex NADH dehydrogenase 1 alpha subcomplex subunit 7 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (EC 1.2.1.12) Voltage-dependent anion-selective channel protein 2 Unknown Phosphatidylethanolamine-binding protein 1 Similar to outer dense fiber protein Proacrosin protein Dermokine-delta 3 Similar to dihydropyrimidinase Hypothetical protein (tubulin polymerization-promoting protein family member 2) Unknown NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 Unknown Superoxide dismutase [Cu-Zn] Protein kinase C inhibitor Unknown Tubulin beta-3 chain Similar to testis specific gene A2 Unknown Similar to tubulin alpha-3/alpha-7 chain Unknown Unknown Similar to dihydropyrimidinase Heat shock protein 70 Unknown Similar to Tektin 2 (testicular) Unknown Hypothetical protein (NDUFB10-NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10) Homer protein homolog 1-like Peptidylprolyl isomerase A (cyclophilin A) Peptidylprolyl isomerase A (cyclophilin A) Tubulin alpha-3 chain Triosephosphate isomerase Fructose-bisphosphate aldolase C B-creatine kinase Hypothetical protein (tektin-5) Similar to Tektin 2 (testicular) Proacrosin protein G-2 and S-phase expressed 1 Tubulin beta-3 chain Similar to dihydropyrimidinase Creatine kinase (EC 2.7.3.2) chain Bb Alpha-enolase Similar to haloacid dehalogenase-like hydrolase domain containing 1A Alpha tubulin

1.224 ± 0.213 0.526 ± 0.019 0.174 ± 0.037 0.319 ± 0.089 0.115 ± 0.0255 0.077 ± 0.021 0.045 ± 0.038 0.031 ± 0.004 0.802 ± 0.124 0.156 ± 0.022 0.036 ± 0.001 0.195 ± 0.019

0.649 ± 0.079 0.278 ± 0.012 0.303 ± 0.018 0.119 ± 0.018 0.053 ± 0.003 0.023 ± 0.008 0.221 ± 0.158 0.016 ± 0.007 0.387 ± 0.065 0.039 ± 0.034 0.014 ± 0.001 0.111 ± 0.008

0.012 0.011 0.006 0.0192 0.013 0.015 0.029 0.037 0.006 0.015

Differential protein expression in chicken spermatozoa before and after freezing-thawing treatment.

The biological characteristics of rooster sperm that has undergone freezing treatment remain elusive. This study analyzed the change in sperm proteins...
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