Comparative Biochemistry and Physiology, Part D 12 (2014) 10–15
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In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa Mariola A. Dietrich a,⁎, Georg J. Arnold b, Thomas Fröhlich b, Andrzej Ciereszko a a b
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Department of Gamete and Embryo Biology, Olsztyn, Poland Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität, Munich, Germany
a r t i c l e
i n f o
Article history: Received 8 August 2014 Received in revised form 16 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: Carp Spermatozoa Proteome LC-MS/MS Fish
a b s t r a c t Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography-electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of proteins from carp spermatozoa identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalog of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability. The mass spectrometry data are available at ProteomeXchange with the dataset identifier PXD000877 (DOI: http://dx.doi.org/10.6019/PXD000877). © 2014 Elsevier Inc. All rights reserved.
1. Introduction Spermatozoa are highly specialized cells with distinct morphological and compositional differences compared to other somatic and germ cells, which reflect important roles in fertilization, embryo development and heredity. Spermatozoa of teleost fish exhibit special characteristics distinct from those of higher vertebrates including a lack of acrosome structure and interaction of sperm and egg at the micropyle level. In contrast to mammals, in most fish species fertilization occurs externally with spermatozoa being immotile on ejaculation. After release into the water spermatozoa become motile and metabolically active briefly up to 2 min (Billard et al., 1995a). However, the knowledge concerning protein components that determine the specific properties of fish semen, its quality and the fertilizing potential is very limited. To date, proteomic studies of sperm cells have been performed mainly in model organisms with fully sequenced genome i.e. in mammals (human, mouse, rat, cow, boar) and invertebrates (fruit fly, honeybee and nematode Caenorhabdilis elegans and ascidian Ciona intestinalis, Martinez-Heredia et al., 2006; Wang et al., 2013; Peddinti et al., 2008; Baker et al., 2008; Dorus et al., 2006; Collins et al., 2006; Byrne et al., 2012). In contrast, limited information is available on the proteomes of fish sperm. So far, two dimensional electrophoresis ⁎ Corresponding author. Tel.: +48 89 5393135. E-mail address: [email protected] (M.A. Dietrich).
http://dx.doi.org/10.1016/j.cbd.2014.09.003 1744-117X/© 2014 Elsevier Inc. All rights reserved.
(2DE) and mass spectrometry have been applied to study the differences in sperm protein profiles among sturgeon species (Li et al., 2010a, 2011), molecular mechanism determining the initiation of sea bream sperm motility (Zilli et al., 2008), the effect of domestication on semen quality (Forne et al., 2009) and the effect of cryopreservation on carp sperm injuries (Li et al., 2010b, 2013). However, in these studies only a few protein spots of spermatozoa were identified. Only, recently, Nynca et al. (2014) identified 206 proteins in spermatozoa of rainbow trout, a cold-water fish, which is a member of the salmonid family. However comprehensive information concerning protein composition of sperm carp has been not available yet. Common carp (Cyprinus carpio L.), a warm-water fish is a member of cyprinid family and is one of the most cultured fish all over the world, especially in Asia and Central and Eastern Europe (FAO, 2013). Besides being of commercial interest, it is also a research model organism within Teleostei. Many farmed fish species including carp do not spawn readily in captivity and hormonal treatments are necessary to either induce ovulation/spermiation or to synchronize gamete release of the two sexes at a time convenient for the fish farm (Zohar and Mylonas, 2001). Hormonal stimulation in common carp is a routine practice to obtain matured milt from majority of the males. Without hormonal stimulation, carp males either do not spawn at all or the collected milt is characterized by low and variable quality in terms of volume of milt, stage of spermatozoa maturity, sperm motility, and pH of seminal plasma (Saad and Billard, 1987; Drori et al., 1994).
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Male reproduction of cyprinid is distinct from salmonid in case of sperm structure, component of nuclear proteins, metabolism, mechanism of sperm activation as well as parameters of sperm movement (Billard et al., 1995b; Jamieson, 1991). The differences in sperm biology suggest different sperm protein patterns of fish species. Recently we have obtained information concerning proteome of carp seminal plasma (Dietrich et al., 2014). Identification of proteins from carp spermatozoa is necessary to obtain complete information regarding carp semen (consisting of seminal plasma and sperm cells) and to evaluate relationship between seminal plasma and spermatozoa. The knowledge concerning proteome of carp spermatozoa is also necessary for understanding carp sperm physiology and for identification of proteins important for sperm quality. Furthermore, this knowledge is important for optimization of artificial carp reproduction and for the development of cryopreservation techniques. The aim of the present study was to create an inventory of the most prominent carp sperm proteins with the use of one-dimensional electrophoresis (1-DE) prefractionation combined with high performance nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). 2. Material and methods 2.1. Semen collection Milt was obtained from common carp maintained at the Institute of Ichthyobiology and Aquaculture of the Polish Academy of Sciences in Gołysz, Poland. Twenty-four hours before the collection of semen, the male carp were injected intradorsaly with Ovopel (one pellet containing of 18–20 μg of GnRH analog and 8–10 mg of metoclopramide per one kg of fish body weight; Interfish Ltd, Hungary). The milt was obtained at the middle of spawning season (July 2nd) from 5 to 7 year old carp (n = 4) by gentle abdominal massage, with care not to pollute it with blood, feces or urine. Samples with spermatozoa content N10 × 109 ml−1 and N80% motile spermatozoa were selected for proteomic analysis. To obtain sperm, milt was centrifuged at 3000 ×g for 30 min (4 °C). After the removal of the supernatant, the pellets (containing the spermatozoa) were washed twice in a sperm immobilizing solution (20 mM Tris, 200 mM KCl, pH 8.0) by centrifugation at 1000 ×g at 4 °C for 30 min. The pellets were suspended in the protein extraction buffer (immobilization solution with 0.1% Triton), sonicated (5 s × 3), kept on ice for 1 h and centrifuged for 10 min at 14,000 ×g at 4 °C. Protein lysates were stored at − 80 °C until analysis. Approval by the Animal Experiments Committee in Olsztyn, Poland was gained before starting any experiments. 2.2. 1D-SDS-PAGE prefractionation and in-gel trypsin digestion Aliquots of 35 μg of proteins of carp sperm extract (n = 4) were mixed with sample loading buffer (65 mM Tris–HCl, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) mercaptoethanol, 1% bromophenol blue), heated (95 °C, 4 min), loaded onto polyacrylamide gel (4% stacking gel and 12% separating gel; 8.6 × 6.8 × 0.1 cm) and run in a Mini Protean II Cell (BioRad, Hercules, CA, USA) at 80 V for 15 min and 120 V for 1.5 h. After the electrophoresis gels were stained overnight using Coomassie Brillant Blue R250, destained at 5% (v/v) methanol with 7% (v/v) acetic acid and washed in water. The SDS-PAGE protein profile of carp sperm is shown in Fig. 1. After electrophoresis the line corresponded to each individual was cut into 12 slices and each slice was individually subjected to in gel trypsin digestion followed by LC-MS/MS. Each slice was destained by incubation in 40% acetonitrile (ACN) in 45 mM NH4HCO3 for 45 min at 37 °C, equilibrated with 50 mM NH4HCO3 for 10 min and then incubated in a solution of 5 mM DTT in 25 mM NH4HCO3 (volume sufficient to cover the gel) at 65 °C for 30 min for the reduction of cysteine residues. For the alkylation of proteins, the gel was incubated in a solution of 55 mM iodoacetamide in 25 mM NH4HCO3 at room
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temperature for 30 min, and the gel pieces were dehydrated in 100% ACN and dried. The gel pieces were treated in 10 μl 25 mM NH4HCO3 containing 1 μg modified sequencing grade trypsin (Promega, Madison, WI, USA) and incubated overnight at 37 °C. The supernatant was collected and preserved. The tryptic peptide mixture was extracted from the gel with 50 μl 50 mM NH4HCO3, followed by an extraction with 50 μl of 80% ACN. The ACN supernatant and the NH4HCO3 fractions were combined and dried using the SpeedVac concentrator (Vacuum Concentrator, Bachofer, Germany). 2.3. LC-MS/MS analysis LC-MS/MS was performed on an Eksigent Ultra nano-liquid chromatography device (Eksigent, Dublin, CA, USA) coupled to a linear ion trap mass spectrometer (LTQ, Thermo Electron, San Jose, CA, USA). Tryptic peptide solutions were reconstituted in 0.1% formic acid, injected onto a C18 reverse phase (RP) trap column (C18 PepMap100, 5 μm particle size, 100 Å, 300 μm × 5 mm column size; LC Packings Dionex, Sunnyvale, CA, USA) at a flow rate of 6 μl/min and subsequently separated by analytical RP chromatography using a nano-LC column (ReproSil-Pur C18 AQ, 2.4 μm; 150 mm × 75 μm, Dr. Maisch, Ammerbuch-Entringen, Germany) at a flow-rate of 280 nL/min. As mobile phase A water and as mobile phase B 84% ACN were used, both containing 0.1% formic acid. Eluted peptides were analyzed by mass spectrometry performed on a linear IT mass spectrometer. The ion spray voltage was set to 1.4 kV. MS and MS/MS measurements were performed using cycles of one MS scan (mass range 300–1600 m/z) and three subsequent data dependent MS/MS scans (collision energy 35%). 2.4. Database search and data analysis Tandem mass spectra were extracted using Xcalibur (Thermo Fisher, v 2.0), and uploaded to a Mascot version 2.1.03 (Matrix Science, London, UK,) server to search against a NCBInr Cyprinidae database (the database contained 99973 proteins and was generated on 2013.02.22) with a fragment ion mass tolerance of 0.8 Da and parent ion mass tolerance of 2 Da. The following parameters were used for MASCOT searches: one missed cleavage allowed, fixed carbamidomethylation of cysteines and oxidation of methionines as variable modification. The occurrence of false positives (FDR) was determined through running searches using the same parameters against a decoy database (sequence-reversed NCBInr Cyprinidae database). The Scaffold 2_04_00 (Proteome Software, Portland, Oregon, v. 2.0) was used to validate and quantify MS/MS-based peptide and protein identifications with the following criteria: 99.0% minimum protein ID probability, minimum number of two unique peptides per protein and a minimum peptide ID probability of 95%. Relative protein abundance was determined by Scaffold software using the terms “quantitative value” and “number of unique peptides”. Scaffold program calculates MWs but not pI values of identified proteins. Therefore to calculate pI values, FASTA file was generated after Scaffold analysis. Then, the generated FASTA file was exported and analyzed with JVirGel software (Java virtual 2D-Gel) which calculates molecular masses and pI values. GI numbers of identified proteins were matched to the UniProtKB database (www.uniprot.org) to obtain Gene Ontology annotations (GO) using the categories “molecular functions” and “biological process”. 2.5. Analysis of sperm The sperm motility was determined with Computer Assisted Sperm Analysis (CASA), as described by Wojtczak et al. (2007). The two-step method for motility measurement described by Rurangwa et al. (2002) was used. As a first step, carp semen was diluted 100-fold in an immobilizing buffer (94 mM NaCl, 27 mM KCl, 50 mM glycine,
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15 mM Tris–HCl, pH 7.5) in a small polyethylene tube. Next, to activate sperm motility, 1 μl of this solution was mixed with 19 μl of distilled water containing 0.5% albumin (final dilution 1:2000). Immediately after sperm activation, 0.7 μl of sperm subsamples was placed into a well of a 12-well multitest glass slide and covered with a coverslip. Percentage of motile sperm was analyzed for a 15–30 s post-activation time using Hobson Sperm Tracker (Hobson Vision Ltd, Baslow UK) with the setting as described by Wojtczak et al. (2007). Each sample was analyzed in duplicate. The sperm concentration was measured using a spectrophotometric method (Ciereszko and Dabrowski, 1993). 3. Results 3.1. 1D-SDS-PAGE prefractionation SDS-PAGE profiles of proteins from carp spermatozoa are shown in Fig. 1. The corresponding gel lanes of spermatozoa extract from four individual males were cut into ten slices and each slice was individually subjected to LC-MS/MS. 3.2. LC-MS/MS analysis of carp spermatozoa proteins From a total of 946,569 MS/MS spectra, 348 proteins were identified from spermatozoa of all four carp males (FDR 1%). Proteins identified with a protein ID probability N 99% (calculated probability of correct protein identification) with two different peptides at a minimum peptide ID probability of 95% were considered as identified. Protein abundance was estimated by the two Scaffold parameters: “quantitative value” and “number of unique peptides”. Table 1 shows a list of the ten most abundant proteins in carp spermatozoa in our dataset. A detailed list of all carp spermatozoa proteins identified in this study is provided in Supplementary data Table S1 together with their accession number, molecular mass, pI value, sequence coverage, and number of unique peptides. The amino acid sequences of identified peptides assigned to each protein are present as Supplementary data Table S2. 3.3. Gene ontology analysis Proteins identified after 1 DE and LC-MS/MS were categorized into gene ontology (GO) classes. Among the identified proteins, approximately 83% were assigned to a predicted function and classified into 6 categories according to their molecular function (Fig. 2). The
kDa 250 150 100 70 50 40 30 20 15
1
2
3
4
10
Fig. 1. Separation of proteins of common carp sperm extract (35 μg, n = 4) by one-dimensional SDS-PAGE (12% acrylamide). The rectangles (1–10) indicate the 10 gel slices individually subjected to LC-MS/MS. M—molecular mass marker (10–250 kDa). Proteins were stained with Coomassie Brillant Blue R-250.
majority of these proteins were classified as proteins with a catalytic activity (214 proteins). Among these proteins, 78 were classified as hydrolases, 61 as oxidoreductases, 44 as transferases, 20 as ligases, 13 as isomerases and 10 as lyases. Other prominent groups of spermatozoa proteins were assigned to binding and transporter proteins. Among the binding proteins, 110 were associated with nucleotide binding (ATP, GTP, NAD, NADP), 27 with cation binding (calcium, magnesium, potassium ions), 13 with DNA binding, six with protein binding and three with lipid binding. The remaining proteins were classified as proteins with enzyme regulator activity, electron carrier activity and antioxidant activity. Using the biological process database, 310 out of 347 proteins could be classified (Fig. 3), the majority of which are related to metabolic processes (254 proteins) as well as transport (44 proteins), response to stimulus (44 proteins), developmental process (26 proteins) and signal transduction (24 proteins). The remaining proteins are involved in cell motility (8 proteins) and spermatogenesis (4 proteins). To gain information about the biological relevance of carp spermatozoa proteins, we classified them into eight functional categories proposed for human and bull sperm proteome (Fig. 2; Martinez-Heredia et al., 2006; Byrne et al., 2012; Amaral et al., 2013). According this classification, the identified proteins were associated with: metabolism and energy production (32%); transcription, transport and protein turnover (30%); cell cycle, apoptosis and oxidative stress (10%); signal transduction (8%); cytoskeleton, flagella and cell movement (7%); nucleotide, ion and lipid binding and transport (6%); reproduction (2%) and with other, mostly unknown functions (Supplementary data Table S2). 4. Discussion The application of 1D-SDS-PAGE prefractionation combined with LC-MS/MS led to the identification of 348 proteins for carp spermatozoa. This is the highest number of proteins from fish spermatozoa identified by a proteomic approach to date. Among the 348 proteins of carp spermatozoa, 124 matched those previously described for the sperm proteome of rainbow trout out of 206 proteins (Nynca et al., 2014) indicating differences in protein composition among these species. Creatine kinase, tubulin, valosin-containing protein and glucose regulated protein were found as common abundant proteins within carp and rainbow trout spermatozoa. Other major proteins of carp spermatozoa such as S-adenosylhomocysteinase and NKEF-B were not identified in rainbow trout. The difference in protein composition and protein abundance between members of distinct fish families likely reflects specificity of carp and rainbow trout sperm biology. The analysis of gene ontology annotations for biological process revealed that, similar to rainbow trout and mammalian spermatozoa (Martinez-Heredia et al., 2006; Wang et al., 2013; Peddinti et al., 2008; Baker et al., 2008; Nynca et al., 2014), the majority of proteins of carp spermatozoa (62%) were involved in metabolic processes. Within this group we identified enzymes of the tricarboxylic acid cycle (19) and fatty acid oxidation (16) as well as respiration (14), glycolysis, gluconeogenesis (13) and amino acid metabolism (11). Almost all of these enzymes act within energy production pathways and ATP production, both representing a prerequisite for sperm movement. The domination of proteins involved in metabolism is consistent with the high energy demand for fish sperm motility. The major proteins of carp spermatozoa were functionally related to sperm motility. Two metabolic enzymes contributing to ATP generation such as mitochondrial ATP synthase and brain-type creatine kinase (CK) as well as dynein and tubulin were identified as major proteins of carp spermatozoa. Motility of carp spermatozoa depends mainly on sperm ATP synthetized by mitochondrial respiration and stored before activation rather than newly synthetized ATP (Perchec et al., 1995). Our study confirmed the presence of a high amount of mitochondrial ATP synthase, which together with creatine kinase is likely involved in
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Table 1 Summary table listing carp sperm protein abundance based on quantitative value and number of unique peptides provided by Scaffold. Identified proteins
Accession no
Organism
Quantitative value (max)
Number of unique peptides (max)
% coverage
Tubulin beta 2 (zgc:55461) Tubulin alpha 6 Dynein heavy chain 5, axonemal Brain creatine kinase (Zgc:154095) Heat shock protein HSP 90-alpha Valosin containing protein NKEF-B Natural killer cell enhancing factor S-adenosylhomocysteine hydrolase 60 kDa heat shock protein, mitochondrial ATP synthase subunit beta, mitochondrial Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)
gi|123232717 (+1) gi|37595424 (+1)
Danio rerio Danio rerio
313 204
36 34
73 58
gi|326679792 gi|115313427 (+2) gi|113681112 (+2) gi|122891315 (+5) gi|209977950 (+2) gi|182890144 (+3) gi|31044489 gi|47605558 (+1) gi|39645428 (+1)
Danio rerio Danio rerio Danio rerio Danio rerio Cyprinus carpio Danio rerio Danio rerio Cyprinus carpio Danio rerio
220 144 133 112 86 94 85 106 91
54 20 33 35 22 17 17 25 23
11 37 34 45 74 31 35 52 33
the generation and maintenance of an appropriate ATP level required for ATP hydrolysis by dynein ATPase localized within the flagellar motile apparatus, the axeneme (Inaba et al., 1998; Cosson et al., 2007). Besides tubulin and dynein, we identified other structural proteins (septin, actin, cofilin, sarcolemma associated protein, sperm associated antigen 6) which are involved in the maintenance of the structural integrity of mature sperm and in the regulation of flagella motility (Baltz et al., 1990; Inaba et al., 1999). The abundance of proteins involved in sperm motility and its energetics may reflect the physiology of spermatozoa which in case of freshwater fish spermatozoa is characterized by very vigorous but short (2 min) motility with high 50–90 Hz initial frequency of flagella beating (Perchec et al., 1995). S-adenosylhomocysteinase (Ahcy) was also identified as a major protein of carp spermatozoa. This enzyme is involved in two major
pathways: the synthesis of creatine and methylation. The synthesis of creatine in the mammalian reproductive tract was previously described by Lee et al. (1998). This energetic metabolic pathway leads to the production of phosphocreatine which plays an essential role in carp sperm motility. The involvement of Ahcy in carp sperm motility was suggested by Li et al. (2010b). Moreover, the presence of creatine kinase as major protein of carp sperm (see above) suggests that creatine synthesis takes place and related metabolic pathways are active in carp spermatozoa. In addition, it is further well known that Ahcy plays a key role in the maintenance of methylation homeostasis via regulation of the intracellular concentration of adenosylhomocysteine. The presence of a pathway involved in the methylation process in carp sperm cells was also supported by the identification of methionine adenosyltransferase in our study, which catalyzes the formation of
Fig. 2. Ontology analysis of identified carp sperm proteins. The classification of the protein set was performed according to the gene ontology terms: “Molecular function” (290 proteins) and “Biological process” (310 proteins).
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Fig. 3. Functional distribution of the identified proteins from carp sperm according to categories proposed for human and bull sperm proteome.
S-adenosylmethionine. The presence of Ahcy in carp sperm suggests its involvement in both energy production for sperm motility and epigenetic modification of sperm DNA. Another prominent group of proteins identified in our study were chaperones such as HSP90, HSP60, HSP70 and VCP (transitional endoplasmic reticulum ATPase). Stress-induced HSPs protect cells by refolding denatured protein, removing damaged proteins and by blocking apoptosis (Beere, 2004). Similarly, VCP acts as a chaperone to unfold proteins and transport them to specific cellular compartments or to the proteasome. HSPs and VCP were previously identified in mammalian mature sperm and were suggested to play a role in spermatogenesis, post-testicular maturation, membrane fusion and in antigen binding/presentation (Naaby-Hansen and Herr, 2010). We have recently identified HSP90 as a major protein of carp seminal plasma (Dietrich et al., 2014). This suggests that carp spermatozoa are well protected by chaperons within both sperm cells and sperm external environment, because chaperones can interact with the sperm plasma membrane (Hiyama et al., 2013). Our results revealed the presence of proteins involved in the protection of carp sperm against oxidative stress. NKEF-B, a homolog of mammalian peroxiredoxin, was identified as more abundant protein of carp spermatozoa within our database. Besides NKEF, other antioxidative enzymes such as glutathione S-transferase, glutathione peroxidase (GPx), superoxide dismutase (SOD), thioredoxin, glutaredoxin together with transferrin were identified. So far only SOD, GPx and glutathione reductase were identified by enzymatic measurements in carp spermatozoa (Li et al., 2010c). The later assumed that GPx is mainly involved in antioxidant response of carp spermatozoa to counterpart ROS stress after cryopreservation. Our results suggest that carp spermatozoa are well equipped with antioxidant proteins which are especially important for the protection of paternal genetic information to the next generation. Interestingly, we identified proteins involved in transcription, protein synthesis and turnover as well as protein transport and folding (30%). Although spermatozoa are transcriptionally and translationally inactive, we identified 19 proteins involved in these processes. The presence of these proteins may be a leftover of spermatogenesis or may reflect mitochondrial gene expression or protein synthesis with mitochondrial ribosomes (Gur and Breitbart, 2006); however this possibility is considered to be controversial (Amaral et al., 2013). Identification of spermatozoa proteins complemented our previous study regarding carp seminal plasma proteins (Dietrich et al., 2014) and led to obtain collection of carp semen (seminal plasma and spermatozoa) proteome dataset consisting of 485 proteins. A comparison of our dataset of proteins of carp spermatozoa with recently identified seminal plasma proteins (137 proteins, Dietrich et al., 2014)
revealed that about 15% of the identified proteins of carp spermatozoa were also detected in seminal plasma (Supplementary data Table S3). These proteins with the exception of HSP90 were not classified as abundant proteins in carp seminal plasma (Dietrich et al., 2014). The presence of sperm proteins like cytoskeletal proteins, creatine kinase, adenosylhomocysteinase, and LDH in seminal plasma could be the result of leakage from damaged spermatozoa, so their appearance in seminal plasma may well indicate spermatozoa damage. Moreover, some proteins like chaperones identified in both spermatozoa and seminal plasma could be sperm surface membrane proteins which are either incorporated into the membrane during membrane protein remodeling or adsorbed on the sperm surface from seminal plasma (Belleannee et al., 2011). The role of sperm membrane proteins is related to preservation of plasma membrane integrity or remodeling of the plasma membrane during maturation (Byrne et al., 2012). Further studies are necessary to elucidate the role of adsorbed proteins and commonly expressed proteins and to test if protein leakage from spermatozoa can be used as sperm quality indication. In conclusion, this study provides the first in-depth analysis of proteome of carp spermatozoa, with a total of 348 proteins identified by LC-MS/MS analysis. We found that major proteins of carp spermatozoa were functionally related to sperm motility and energy production as well as to the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. The availability of a catalog of carp spermatozoa proteins provides now the basis for subsequent fundamental research in male reproduction of fish. Generating a catalog of spermatozoa proteins is an important first step toward the understanding the mechanism underlying male reproduction of carp. Moreover, identification of high number of carp spermatozoa proteins provides new opportunities to develop novel biomarkers for carp sperm quality. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2014.09.003.
Acknowledgment We thank Florian Flenkenthaler, Daniela Deutsch, Miwako Kösters and Kathrin A. Otte for their excellent technical assistance. This work was supported by a Project 2011/01/D/NZ9/00628 from the National Science Centre (Identification and characterization of specific carp seminal plasma proteins—proteomics and classical approach), funds appropriated to the Institute of Animal Reproduction and Food Research and also supported by FP7 Project “REFRESH” 264103.
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Jamieson, B.G.M., 1991. Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne and Sydney (319 pp.). Lee, H., Kim, J.H., Chae, Y.-J., Ogawa, H., Lee, M.-H., Gerton, G.L., 1998. Creatine synthesis and transport systems in the male rat reproductive tract. Biol. Reprod. 58, 1437–1444. Li, P., Hulak, M., Rodina, M., Sulc, M., Li, Z.H., Linhart, O., 2010a. Comparative protein profiles: potential molecular markers from spermatozoa of Acipenseriformes (Chondrostei, Pisces). Comp. Biochem. Physiol. D 5, 302–307. Li, P., Hulak, M., Koubek, P., Sulc, M., Dzyuba, B., Boryshpolets, S., Rodina, M., Gela, D., Manaskova-Postlerova, P., Peknicova, J., Linhart, O., 2010b. Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L. Theriogenology 74, 413–423. Li, P., Li, Z.-H., Dzyuba, B., Hulak, M., Rodina, M., Linhart, O., 2010c. Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation techniques. Biol. Reprod. 83, 852–858. Li, P., Rodina, M., Hulak, M., Gela, D., Psenicka, M., Li, Z.H., Linhart, O., 2011. Physicochemical properties and protein profiles of sperm from three freshwater chondrostean species: a comparative study among Siberian sturgeon (Acipenser baerii), sterlet (Acipenser ruthenus) and paddlefish (Polyodon spathula). J. Appl. Ichthyol. 27, 673–677. Li, P., Hulak, M., Li, Z.H., Sulc, M., Psenicka, M., Rodina, M., Gela, D., Linhart, O., 2013. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues. Theriogenology 80, 84–89. Martinez-Heredia, J., Estanyol, J.M., Ballescà, J.L., Oliva, R., 2006. Proteomic identification of human sperm proteins. Proteomics 6, 4356–4369. Naaby-Hansen, S., Herr, J.C., 2010. Heat shock proteins on the surface of human sperm. J. Reprod. Immunol. 84, 32–40. Nynca, J., Arnold, G.J., Fröhlich, T., Otte, K., Ciereszko, A., 2014. Proteomic identification of rainbow trout sperm proteins. Proteomics 14, 1569–1573. Peddinti, D., Nanduri, B., Kaya, A., Feugang, J.M., Burgess, S.C., Memili, E., 2008. Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Syst. Biol. 2, 19. Perchec, G., Jeulin, C., Cosson, J., Andre, F., Billard, R., 1995. Relationship between sperm ATP content and motility of carp spermatozoa. J. Cell Sci. 108, 747–753. Rurangwa, E., Biegniewska, A., Slominska, E., Skorkowski, E.F., Ollevier, F., 2002. Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility. Comp. Biochem. Physiol. 131, 335–344. Saad, A., Billard, R., 1987. Spermatozoa production and volume of semen collected after hormonal stimulation in the carp, Cyprinus carpio. Aquaculture 65, 67–77. Statistical Yearbook, F.A.O., 2013. World food and agriculture. Food and Agriculture Organization of the United Nations; 2013. Wang, G., Guo, Y., Zhou, T., Shi, X., Yu, J., Yang, Y., Wu, Y., Wang, J., Liu, M., Chen, X., Tu, W., Zeng, Y., Jiang, M., Li, S., Zhang, P., Zhou, Q., Zheng, B., Yu, C., Zhou, Z., Guo, X., Sha, J., 2013. In-depth proteomic analysis of the human sperm reveals complex protein compositions. J. Proteomics 79, 114–122. Wojtczak, M., Dietrich, G.J., Irnazarow, I., Jurecka, P., Słowińska, M., Ciereszko, A., 2007. Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics. Comp. Biochem. Physiol. B 148, 426–431. Zilli, L., Schiavone, R., Storelli, C., Vilella, S., 2008. Molecular mechanisms determining sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus). Biol. Reprod. 79, 356–366. Zohar, Y., Mylonas, C.C., 2001. Endocrinemanipulations of spawning in cultured fish: from hormones to genes. Aquaculture 197, 99–136.
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Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd
In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa Mariola A. Dietrich a,⁎, Georg J. Arnold b, Thomas Fröhlich b, Andrzej Ciereszko a a b
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Department of Gamete and Embryo Biology, Olsztyn, Poland Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität, Munich, Germany
a r t i c l e
i n f o
Article history: Received 8 August 2014 Received in revised form 16 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: Carp Spermatozoa Proteome LC-MS/MS Fish
a b s t r a c t Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography-electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of proteins from carp spermatozoa identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalog of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability. The mass spectrometry data are available at ProteomeXchange with the dataset identifier PXD000877 (DOI: http://dx.doi.org/10.6019/PXD000877). © 2014 Elsevier Inc. All rights reserved.
1. Introduction Spermatozoa are highly specialized cells with distinct morphological and compositional differences compared to other somatic and germ cells, which reflect important roles in fertilization, embryo development and heredity. Spermatozoa of teleost fish exhibit special characteristics distinct from those of higher vertebrates including a lack of acrosome structure and interaction of sperm and egg at the micropyle level. In contrast to mammals, in most fish species fertilization occurs externally with spermatozoa being immotile on ejaculation. After release into the water spermatozoa become motile and metabolically active briefly up to 2 min (Billard et al., 1995a). However, the knowledge concerning protein components that determine the specific properties of fish semen, its quality and the fertilizing potential is very limited. To date, proteomic studies of sperm cells have been performed mainly in model organisms with fully sequenced genome i.e. in mammals (human, mouse, rat, cow, boar) and invertebrates (fruit fly, honeybee and nematode Caenorhabdilis elegans and ascidian Ciona intestinalis, Martinez-Heredia et al., 2006; Wang et al., 2013; Peddinti et al., 2008; Baker et al., 2008; Dorus et al., 2006; Collins et al., 2006; Byrne et al., 2012). In contrast, limited information is available on the proteomes of fish sperm. So far, two dimensional electrophoresis ⁎ Corresponding author. Tel.: +48 89 5393135. E-mail address: [email protected] (M.A. Dietrich).
http://dx.doi.org/10.1016/j.cbd.2014.09.003 1744-117X/© 2014 Elsevier Inc. All rights reserved.
(2DE) and mass spectrometry have been applied to study the differences in sperm protein profiles among sturgeon species (Li et al., 2010a, 2011), molecular mechanism determining the initiation of sea bream sperm motility (Zilli et al., 2008), the effect of domestication on semen quality (Forne et al., 2009) and the effect of cryopreservation on carp sperm injuries (Li et al., 2010b, 2013). However, in these studies only a few protein spots of spermatozoa were identified. Only, recently, Nynca et al. (2014) identified 206 proteins in spermatozoa of rainbow trout, a cold-water fish, which is a member of the salmonid family. However comprehensive information concerning protein composition of sperm carp has been not available yet. Common carp (Cyprinus carpio L.), a warm-water fish is a member of cyprinid family and is one of the most cultured fish all over the world, especially in Asia and Central and Eastern Europe (FAO, 2013). Besides being of commercial interest, it is also a research model organism within Teleostei. Many farmed fish species including carp do not spawn readily in captivity and hormonal treatments are necessary to either induce ovulation/spermiation or to synchronize gamete release of the two sexes at a time convenient for the fish farm (Zohar and Mylonas, 2001). Hormonal stimulation in common carp is a routine practice to obtain matured milt from majority of the males. Without hormonal stimulation, carp males either do not spawn at all or the collected milt is characterized by low and variable quality in terms of volume of milt, stage of spermatozoa maturity, sperm motility, and pH of seminal plasma (Saad and Billard, 1987; Drori et al., 1994).
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Male reproduction of cyprinid is distinct from salmonid in case of sperm structure, component of nuclear proteins, metabolism, mechanism of sperm activation as well as parameters of sperm movement (Billard et al., 1995b; Jamieson, 1991). The differences in sperm biology suggest different sperm protein patterns of fish species. Recently we have obtained information concerning proteome of carp seminal plasma (Dietrich et al., 2014). Identification of proteins from carp spermatozoa is necessary to obtain complete information regarding carp semen (consisting of seminal plasma and sperm cells) and to evaluate relationship between seminal plasma and spermatozoa. The knowledge concerning proteome of carp spermatozoa is also necessary for understanding carp sperm physiology and for identification of proteins important for sperm quality. Furthermore, this knowledge is important for optimization of artificial carp reproduction and for the development of cryopreservation techniques. The aim of the present study was to create an inventory of the most prominent carp sperm proteins with the use of one-dimensional electrophoresis (1-DE) prefractionation combined with high performance nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). 2. Material and methods 2.1. Semen collection Milt was obtained from common carp maintained at the Institute of Ichthyobiology and Aquaculture of the Polish Academy of Sciences in Gołysz, Poland. Twenty-four hours before the collection of semen, the male carp were injected intradorsaly with Ovopel (one pellet containing of 18–20 μg of GnRH analog and 8–10 mg of metoclopramide per one kg of fish body weight; Interfish Ltd, Hungary). The milt was obtained at the middle of spawning season (July 2nd) from 5 to 7 year old carp (n = 4) by gentle abdominal massage, with care not to pollute it with blood, feces or urine. Samples with spermatozoa content N10 × 109 ml−1 and N80% motile spermatozoa were selected for proteomic analysis. To obtain sperm, milt was centrifuged at 3000 ×g for 30 min (4 °C). After the removal of the supernatant, the pellets (containing the spermatozoa) were washed twice in a sperm immobilizing solution (20 mM Tris, 200 mM KCl, pH 8.0) by centrifugation at 1000 ×g at 4 °C for 30 min. The pellets were suspended in the protein extraction buffer (immobilization solution with 0.1% Triton), sonicated (5 s × 3), kept on ice for 1 h and centrifuged for 10 min at 14,000 ×g at 4 °C. Protein lysates were stored at − 80 °C until analysis. Approval by the Animal Experiments Committee in Olsztyn, Poland was gained before starting any experiments. 2.2. 1D-SDS-PAGE prefractionation and in-gel trypsin digestion Aliquots of 35 μg of proteins of carp sperm extract (n = 4) were mixed with sample loading buffer (65 mM Tris–HCl, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) mercaptoethanol, 1% bromophenol blue), heated (95 °C, 4 min), loaded onto polyacrylamide gel (4% stacking gel and 12% separating gel; 8.6 × 6.8 × 0.1 cm) and run in a Mini Protean II Cell (BioRad, Hercules, CA, USA) at 80 V for 15 min and 120 V for 1.5 h. After the electrophoresis gels were stained overnight using Coomassie Brillant Blue R250, destained at 5% (v/v) methanol with 7% (v/v) acetic acid and washed in water. The SDS-PAGE protein profile of carp sperm is shown in Fig. 1. After electrophoresis the line corresponded to each individual was cut into 12 slices and each slice was individually subjected to in gel trypsin digestion followed by LC-MS/MS. Each slice was destained by incubation in 40% acetonitrile (ACN) in 45 mM NH4HCO3 for 45 min at 37 °C, equilibrated with 50 mM NH4HCO3 for 10 min and then incubated in a solution of 5 mM DTT in 25 mM NH4HCO3 (volume sufficient to cover the gel) at 65 °C for 30 min for the reduction of cysteine residues. For the alkylation of proteins, the gel was incubated in a solution of 55 mM iodoacetamide in 25 mM NH4HCO3 at room
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temperature for 30 min, and the gel pieces were dehydrated in 100% ACN and dried. The gel pieces were treated in 10 μl 25 mM NH4HCO3 containing 1 μg modified sequencing grade trypsin (Promega, Madison, WI, USA) and incubated overnight at 37 °C. The supernatant was collected and preserved. The tryptic peptide mixture was extracted from the gel with 50 μl 50 mM NH4HCO3, followed by an extraction with 50 μl of 80% ACN. The ACN supernatant and the NH4HCO3 fractions were combined and dried using the SpeedVac concentrator (Vacuum Concentrator, Bachofer, Germany). 2.3. LC-MS/MS analysis LC-MS/MS was performed on an Eksigent Ultra nano-liquid chromatography device (Eksigent, Dublin, CA, USA) coupled to a linear ion trap mass spectrometer (LTQ, Thermo Electron, San Jose, CA, USA). Tryptic peptide solutions were reconstituted in 0.1% formic acid, injected onto a C18 reverse phase (RP) trap column (C18 PepMap100, 5 μm particle size, 100 Å, 300 μm × 5 mm column size; LC Packings Dionex, Sunnyvale, CA, USA) at a flow rate of 6 μl/min and subsequently separated by analytical RP chromatography using a nano-LC column (ReproSil-Pur C18 AQ, 2.4 μm; 150 mm × 75 μm, Dr. Maisch, Ammerbuch-Entringen, Germany) at a flow-rate of 280 nL/min. As mobile phase A water and as mobile phase B 84% ACN were used, both containing 0.1% formic acid. Eluted peptides were analyzed by mass spectrometry performed on a linear IT mass spectrometer. The ion spray voltage was set to 1.4 kV. MS and MS/MS measurements were performed using cycles of one MS scan (mass range 300–1600 m/z) and three subsequent data dependent MS/MS scans (collision energy 35%). 2.4. Database search and data analysis Tandem mass spectra were extracted using Xcalibur (Thermo Fisher, v 2.0), and uploaded to a Mascot version 2.1.03 (Matrix Science, London, UK,) server to search against a NCBInr Cyprinidae database (the database contained 99973 proteins and was generated on 2013.02.22) with a fragment ion mass tolerance of 0.8 Da and parent ion mass tolerance of 2 Da. The following parameters were used for MASCOT searches: one missed cleavage allowed, fixed carbamidomethylation of cysteines and oxidation of methionines as variable modification. The occurrence of false positives (FDR) was determined through running searches using the same parameters against a decoy database (sequence-reversed NCBInr Cyprinidae database). The Scaffold 2_04_00 (Proteome Software, Portland, Oregon, v. 2.0) was used to validate and quantify MS/MS-based peptide and protein identifications with the following criteria: 99.0% minimum protein ID probability, minimum number of two unique peptides per protein and a minimum peptide ID probability of 95%. Relative protein abundance was determined by Scaffold software using the terms “quantitative value” and “number of unique peptides”. Scaffold program calculates MWs but not pI values of identified proteins. Therefore to calculate pI values, FASTA file was generated after Scaffold analysis. Then, the generated FASTA file was exported and analyzed with JVirGel software (Java virtual 2D-Gel) which calculates molecular masses and pI values. GI numbers of identified proteins were matched to the UniProtKB database (www.uniprot.org) to obtain Gene Ontology annotations (GO) using the categories “molecular functions” and “biological process”. 2.5. Analysis of sperm The sperm motility was determined with Computer Assisted Sperm Analysis (CASA), as described by Wojtczak et al. (2007). The two-step method for motility measurement described by Rurangwa et al. (2002) was used. As a first step, carp semen was diluted 100-fold in an immobilizing buffer (94 mM NaCl, 27 mM KCl, 50 mM glycine,
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15 mM Tris–HCl, pH 7.5) in a small polyethylene tube. Next, to activate sperm motility, 1 μl of this solution was mixed with 19 μl of distilled water containing 0.5% albumin (final dilution 1:2000). Immediately after sperm activation, 0.7 μl of sperm subsamples was placed into a well of a 12-well multitest glass slide and covered with a coverslip. Percentage of motile sperm was analyzed for a 15–30 s post-activation time using Hobson Sperm Tracker (Hobson Vision Ltd, Baslow UK) with the setting as described by Wojtczak et al. (2007). Each sample was analyzed in duplicate. The sperm concentration was measured using a spectrophotometric method (Ciereszko and Dabrowski, 1993). 3. Results 3.1. 1D-SDS-PAGE prefractionation SDS-PAGE profiles of proteins from carp spermatozoa are shown in Fig. 1. The corresponding gel lanes of spermatozoa extract from four individual males were cut into ten slices and each slice was individually subjected to LC-MS/MS. 3.2. LC-MS/MS analysis of carp spermatozoa proteins From a total of 946,569 MS/MS spectra, 348 proteins were identified from spermatozoa of all four carp males (FDR 1%). Proteins identified with a protein ID probability N 99% (calculated probability of correct protein identification) with two different peptides at a minimum peptide ID probability of 95% were considered as identified. Protein abundance was estimated by the two Scaffold parameters: “quantitative value” and “number of unique peptides”. Table 1 shows a list of the ten most abundant proteins in carp spermatozoa in our dataset. A detailed list of all carp spermatozoa proteins identified in this study is provided in Supplementary data Table S1 together with their accession number, molecular mass, pI value, sequence coverage, and number of unique peptides. The amino acid sequences of identified peptides assigned to each protein are present as Supplementary data Table S2. 3.3. Gene ontology analysis Proteins identified after 1 DE and LC-MS/MS were categorized into gene ontology (GO) classes. Among the identified proteins, approximately 83% were assigned to a predicted function and classified into 6 categories according to their molecular function (Fig. 2). The
kDa 250 150 100 70 50 40 30 20 15
1
2
3
4
10
Fig. 1. Separation of proteins of common carp sperm extract (35 μg, n = 4) by one-dimensional SDS-PAGE (12% acrylamide). The rectangles (1–10) indicate the 10 gel slices individually subjected to LC-MS/MS. M—molecular mass marker (10–250 kDa). Proteins were stained with Coomassie Brillant Blue R-250.
majority of these proteins were classified as proteins with a catalytic activity (214 proteins). Among these proteins, 78 were classified as hydrolases, 61 as oxidoreductases, 44 as transferases, 20 as ligases, 13 as isomerases and 10 as lyases. Other prominent groups of spermatozoa proteins were assigned to binding and transporter proteins. Among the binding proteins, 110 were associated with nucleotide binding (ATP, GTP, NAD, NADP), 27 with cation binding (calcium, magnesium, potassium ions), 13 with DNA binding, six with protein binding and three with lipid binding. The remaining proteins were classified as proteins with enzyme regulator activity, electron carrier activity and antioxidant activity. Using the biological process database, 310 out of 347 proteins could be classified (Fig. 3), the majority of which are related to metabolic processes (254 proteins) as well as transport (44 proteins), response to stimulus (44 proteins), developmental process (26 proteins) and signal transduction (24 proteins). The remaining proteins are involved in cell motility (8 proteins) and spermatogenesis (4 proteins). To gain information about the biological relevance of carp spermatozoa proteins, we classified them into eight functional categories proposed for human and bull sperm proteome (Fig. 2; Martinez-Heredia et al., 2006; Byrne et al., 2012; Amaral et al., 2013). According this classification, the identified proteins were associated with: metabolism and energy production (32%); transcription, transport and protein turnover (30%); cell cycle, apoptosis and oxidative stress (10%); signal transduction (8%); cytoskeleton, flagella and cell movement (7%); nucleotide, ion and lipid binding and transport (6%); reproduction (2%) and with other, mostly unknown functions (Supplementary data Table S2). 4. Discussion The application of 1D-SDS-PAGE prefractionation combined with LC-MS/MS led to the identification of 348 proteins for carp spermatozoa. This is the highest number of proteins from fish spermatozoa identified by a proteomic approach to date. Among the 348 proteins of carp spermatozoa, 124 matched those previously described for the sperm proteome of rainbow trout out of 206 proteins (Nynca et al., 2014) indicating differences in protein composition among these species. Creatine kinase, tubulin, valosin-containing protein and glucose regulated protein were found as common abundant proteins within carp and rainbow trout spermatozoa. Other major proteins of carp spermatozoa such as S-adenosylhomocysteinase and NKEF-B were not identified in rainbow trout. The difference in protein composition and protein abundance between members of distinct fish families likely reflects specificity of carp and rainbow trout sperm biology. The analysis of gene ontology annotations for biological process revealed that, similar to rainbow trout and mammalian spermatozoa (Martinez-Heredia et al., 2006; Wang et al., 2013; Peddinti et al., 2008; Baker et al., 2008; Nynca et al., 2014), the majority of proteins of carp spermatozoa (62%) were involved in metabolic processes. Within this group we identified enzymes of the tricarboxylic acid cycle (19) and fatty acid oxidation (16) as well as respiration (14), glycolysis, gluconeogenesis (13) and amino acid metabolism (11). Almost all of these enzymes act within energy production pathways and ATP production, both representing a prerequisite for sperm movement. The domination of proteins involved in metabolism is consistent with the high energy demand for fish sperm motility. The major proteins of carp spermatozoa were functionally related to sperm motility. Two metabolic enzymes contributing to ATP generation such as mitochondrial ATP synthase and brain-type creatine kinase (CK) as well as dynein and tubulin were identified as major proteins of carp spermatozoa. Motility of carp spermatozoa depends mainly on sperm ATP synthetized by mitochondrial respiration and stored before activation rather than newly synthetized ATP (Perchec et al., 1995). Our study confirmed the presence of a high amount of mitochondrial ATP synthase, which together with creatine kinase is likely involved in
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Table 1 Summary table listing carp sperm protein abundance based on quantitative value and number of unique peptides provided by Scaffold. Identified proteins
Accession no
Organism
Quantitative value (max)
Number of unique peptides (max)
% coverage
Tubulin beta 2 (zgc:55461) Tubulin alpha 6 Dynein heavy chain 5, axonemal Brain creatine kinase (Zgc:154095) Heat shock protein HSP 90-alpha Valosin containing protein NKEF-B Natural killer cell enhancing factor S-adenosylhomocysteine hydrolase 60 kDa heat shock protein, mitochondrial ATP synthase subunit beta, mitochondrial Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)
gi|123232717 (+1) gi|37595424 (+1)
Danio rerio Danio rerio
313 204
36 34
73 58
gi|326679792 gi|115313427 (+2) gi|113681112 (+2) gi|122891315 (+5) gi|209977950 (+2) gi|182890144 (+3) gi|31044489 gi|47605558 (+1) gi|39645428 (+1)
Danio rerio Danio rerio Danio rerio Danio rerio Cyprinus carpio Danio rerio Danio rerio Cyprinus carpio Danio rerio
220 144 133 112 86 94 85 106 91
54 20 33 35 22 17 17 25 23
11 37 34 45 74 31 35 52 33
the generation and maintenance of an appropriate ATP level required for ATP hydrolysis by dynein ATPase localized within the flagellar motile apparatus, the axeneme (Inaba et al., 1998; Cosson et al., 2007). Besides tubulin and dynein, we identified other structural proteins (septin, actin, cofilin, sarcolemma associated protein, sperm associated antigen 6) which are involved in the maintenance of the structural integrity of mature sperm and in the regulation of flagella motility (Baltz et al., 1990; Inaba et al., 1999). The abundance of proteins involved in sperm motility and its energetics may reflect the physiology of spermatozoa which in case of freshwater fish spermatozoa is characterized by very vigorous but short (2 min) motility with high 50–90 Hz initial frequency of flagella beating (Perchec et al., 1995). S-adenosylhomocysteinase (Ahcy) was also identified as a major protein of carp spermatozoa. This enzyme is involved in two major
pathways: the synthesis of creatine and methylation. The synthesis of creatine in the mammalian reproductive tract was previously described by Lee et al. (1998). This energetic metabolic pathway leads to the production of phosphocreatine which plays an essential role in carp sperm motility. The involvement of Ahcy in carp sperm motility was suggested by Li et al. (2010b). Moreover, the presence of creatine kinase as major protein of carp sperm (see above) suggests that creatine synthesis takes place and related metabolic pathways are active in carp spermatozoa. In addition, it is further well known that Ahcy plays a key role in the maintenance of methylation homeostasis via regulation of the intracellular concentration of adenosylhomocysteine. The presence of a pathway involved in the methylation process in carp sperm cells was also supported by the identification of methionine adenosyltransferase in our study, which catalyzes the formation of
Fig. 2. Ontology analysis of identified carp sperm proteins. The classification of the protein set was performed according to the gene ontology terms: “Molecular function” (290 proteins) and “Biological process” (310 proteins).
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Fig. 3. Functional distribution of the identified proteins from carp sperm according to categories proposed for human and bull sperm proteome.
S-adenosylmethionine. The presence of Ahcy in carp sperm suggests its involvement in both energy production for sperm motility and epigenetic modification of sperm DNA. Another prominent group of proteins identified in our study were chaperones such as HSP90, HSP60, HSP70 and VCP (transitional endoplasmic reticulum ATPase). Stress-induced HSPs protect cells by refolding denatured protein, removing damaged proteins and by blocking apoptosis (Beere, 2004). Similarly, VCP acts as a chaperone to unfold proteins and transport them to specific cellular compartments or to the proteasome. HSPs and VCP were previously identified in mammalian mature sperm and were suggested to play a role in spermatogenesis, post-testicular maturation, membrane fusion and in antigen binding/presentation (Naaby-Hansen and Herr, 2010). We have recently identified HSP90 as a major protein of carp seminal plasma (Dietrich et al., 2014). This suggests that carp spermatozoa are well protected by chaperons within both sperm cells and sperm external environment, because chaperones can interact with the sperm plasma membrane (Hiyama et al., 2013). Our results revealed the presence of proteins involved in the protection of carp sperm against oxidative stress. NKEF-B, a homolog of mammalian peroxiredoxin, was identified as more abundant protein of carp spermatozoa within our database. Besides NKEF, other antioxidative enzymes such as glutathione S-transferase, glutathione peroxidase (GPx), superoxide dismutase (SOD), thioredoxin, glutaredoxin together with transferrin were identified. So far only SOD, GPx and glutathione reductase were identified by enzymatic measurements in carp spermatozoa (Li et al., 2010c). The later assumed that GPx is mainly involved in antioxidant response of carp spermatozoa to counterpart ROS stress after cryopreservation. Our results suggest that carp spermatozoa are well equipped with antioxidant proteins which are especially important for the protection of paternal genetic information to the next generation. Interestingly, we identified proteins involved in transcription, protein synthesis and turnover as well as protein transport and folding (30%). Although spermatozoa are transcriptionally and translationally inactive, we identified 19 proteins involved in these processes. The presence of these proteins may be a leftover of spermatogenesis or may reflect mitochondrial gene expression or protein synthesis with mitochondrial ribosomes (Gur and Breitbart, 2006); however this possibility is considered to be controversial (Amaral et al., 2013). Identification of spermatozoa proteins complemented our previous study regarding carp seminal plasma proteins (Dietrich et al., 2014) and led to obtain collection of carp semen (seminal plasma and spermatozoa) proteome dataset consisting of 485 proteins. A comparison of our dataset of proteins of carp spermatozoa with recently identified seminal plasma proteins (137 proteins, Dietrich et al., 2014)
revealed that about 15% of the identified proteins of carp spermatozoa were also detected in seminal plasma (Supplementary data Table S3). These proteins with the exception of HSP90 were not classified as abundant proteins in carp seminal plasma (Dietrich et al., 2014). The presence of sperm proteins like cytoskeletal proteins, creatine kinase, adenosylhomocysteinase, and LDH in seminal plasma could be the result of leakage from damaged spermatozoa, so their appearance in seminal plasma may well indicate spermatozoa damage. Moreover, some proteins like chaperones identified in both spermatozoa and seminal plasma could be sperm surface membrane proteins which are either incorporated into the membrane during membrane protein remodeling or adsorbed on the sperm surface from seminal plasma (Belleannee et al., 2011). The role of sperm membrane proteins is related to preservation of plasma membrane integrity or remodeling of the plasma membrane during maturation (Byrne et al., 2012). Further studies are necessary to elucidate the role of adsorbed proteins and commonly expressed proteins and to test if protein leakage from spermatozoa can be used as sperm quality indication. In conclusion, this study provides the first in-depth analysis of proteome of carp spermatozoa, with a total of 348 proteins identified by LC-MS/MS analysis. We found that major proteins of carp spermatozoa were functionally related to sperm motility and energy production as well as to the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. The availability of a catalog of carp spermatozoa proteins provides now the basis for subsequent fundamental research in male reproduction of fish. Generating a catalog of spermatozoa proteins is an important first step toward the understanding the mechanism underlying male reproduction of carp. Moreover, identification of high number of carp spermatozoa proteins provides new opportunities to develop novel biomarkers for carp sperm quality. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2014.09.003.
Acknowledgment We thank Florian Flenkenthaler, Daniela Deutsch, Miwako Kösters and Kathrin A. Otte for their excellent technical assistance. This work was supported by a Project 2011/01/D/NZ9/00628 from the National Science Centre (Identification and characterization of specific carp seminal plasma proteins—proteomics and classical approach), funds appropriated to the Institute of Animal Reproduction and Food Research and also supported by FP7 Project “REFRESH” 264103.
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