CSIRO PUBLISHING
Reproduction, Fertility and Development, 2015, 27, 504–512 http://dx.doi.org/10.1071/RD13224
Shotgun proteomics of rainbow trout ovarian fluid Joanna Nynca A,C, Georg J. Arnold B, Thomas Fro¨hlich B and Andrzej Ciereszko A A
Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland. B Laboratory for Functional Genome Analysis (LAFUGA), Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universita¨t, Feodor Lynen Str. 25, 81377 Munich, Germany. C Corresponding author. Email: [email protected]
Abstract. In the present study we used a shotgun proteomic approach to identify 54 proteins of rainbow trout ovarian fluid. The study has unravelled the identity of several proteins not previously reported in fish ovarian fluid. The proteome of trout ovarian fluid consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. Most trout ovarian fluid proteins correspond to follicular fluid proteins of higher vertebrates, but 15% of the proteins were found to be different, such as those related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown functions (UPF0762 protein C6orf58 homologue). The present study could help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality. Additional keywords: immunity, mass spectrometry, ovarian fluid, rainbow trout, shotgun proteomics. Received 17 July 2013, accepted 18 December 2013, published online 30 January 2014
Introduction Mature rainbow trout oocytes are expelled at ovulation into the coelomic cavity, where they remain immersed in a semiviscous fluid known as ovarian or coelomic fluid. Ovarian fluid (OF) is formed by filtration from the blood plasma and the secretory activity of ovarian epithelia, and it may contain postovulatory or broken egg components (Matsubara et al. 1985). The composition of the OF is geared towards egg storage and extension of the fertilisation period during both natural spawning and artificial fertilisation (Lahnsteiner et al. 1995). The inorganic composition of the OF of fish is well described; in salmonids, this fluid has electrolyte concentrations similar to blood plasma (Rosengrave et al. 2009). However, knowledge regarding the protein composition of trout OF is limited to trout ovulatory proteins (Coffman et al. 2000), apolipoproteins, vitellogenins and lectins (Rime et al. 2004). The biochemical composition and pH of OF can reflect the quality of the eggs. The presence of components from broken eggs was found to decrease the pH of trout OF and consequently its ability to activate sperm motility (Dietrich et al. 2007). Using proteomic methods, Rime et al. (2004) reported potential markers of egg quality, such as vitellogenin fragments and apolipoproteins, in trout OF that may be associated with oocyte postovulatory aging. Studies on OF protein components may Journal compilation Ó CSIRO 2015
also contribute to a better understanding of the mechanisms underlying oocyte development and quality. In the present study we used the shotgun proteomic method to investigate rainbow trout OF proteins. We provided a brief overview of the biological processes and molecular functions of the proteins identified based on gene ontology (GO) analysis. Materials and methods Animals and gamete collection Rainbow trout spawners were maintained in an experimental fish hatchery at the Inland Fisheries Institute, Rutki, Poland. Eggs were collected from five female rainbow trout of the spring-spawning Rutki strain (4 years of age) and placed in a sieve. The OF was then poured off and collected. The fish were stocked in concrete ponds (150 m3) supplied with water from the river Radunia. The temperature of the water was 10–128C. OF samples were centrifuged at 3000g for 10 min at 48C to remove red blood cells and debris. The collected supernatants were stored at 808C until electrophoresis was conducted. The protein concentration was determined using the Coomassie Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and bovine serum albumin (BSA) as a calibration standard. www.publish.csiro.au/journals/rfd
Shotgun proteomics of rainbow trout ovarian fluid
kDa
M
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OF1
OF2
OF3
OF4
505
OF5
250 150 100 70 50 40 30 20 15
10
Fig. 1. One-dimensional gel electrophoresis separation of five replicates (OF1, OF2, OF3, OF4, OF5) of rainbow trout ovarian fluid proteins under reducing conditions. M, molecular mass marker (10–250 kDa). The boxes represent the 11 gel slices that were digested individually and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS).
All experiments described herein were approved by the Animal Experiments Committee of Olsztyn (no. 39/2011), Poland. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in-gel protein digestion OF proteins (40 mg) from five different individuals were analysed separately by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% stacking gel and a 12% separation gel with a mini ProteanTM II device (Bio-Rad, Hercules, CA, USA). Following electrophoresis, the gel was stained with Coomassie blue dye overnight (50% methanol, 10% acetic acid, 0.05% Coomassie brilliant blue R-250) and then destained with 5% methanol and 7% acetic acid (Fig. 1). The gel was then washed in water and each protein lane was cut into 11 slices. The slices were then washed twice in 50 mM NH4HCO3, reduced for 30 min at 558C with 45 mM dithioerythritol and then alkylated by 30 min incubation with 100 mM iodoacetamide. After extensive washing in 50 mM NH4HCO3, gel slices were minced and subjected to overnight digestion at 378C with 1 mg porcine trypsin (Promega, Madison, WI, USA) per slice. The resulting peptides were eluted from the gel pieces in two successive washes, first with 50 mM NH4HCO3 and then with 80% acetonitrile (ACN). The NH4HCO3 and the ACN supernatant were collected, pooled and concentrated using a SpeedVac concentrator (Bachofer, Vacuum Concentrator, Reutlingen, Germany). Peptide samples were reconstituted with 10 mL of 0.1% formic acid (FA) and applied to an Eksigent Ultra nano-liquid chromatography device (Eksigent, Dublin, CA, USA) coupled to a linear ion trap (IT) mass spectrometer (LTQ; Thermo Electron, San Jose, CA, USA). The procedures, which included cutting protein lanes, digestion with trypsin and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, were performed separately for five different OF samples.
LC-MS/MS analysis and database searches Samples were injected onto a C18 trap column (C18 Pep˚ , 300 mm 5 mm column Map100, 5 mm particle size, 100 A size; LC Packings Dionex, Sunnyvale, CA, USA) and subsequently separated by reverse-phase chromatography using a nano-LC column (ReproSil-Pur C18 AQ, 2.4 mm; 150 mm 75 mm; Dr Maisch, Ammerbuch-Entringen, Germany) at a flow rate of 280 nL min1. The mobile phases consisted of water (Mobile Phase A) and 84% ACN (Mobile Phase B), and both contained 0.1%FA. The ionspray voltage was set to 1.4 kV and the mass spectrometry (MS) and MS/MS analyses were performed using cycles of one MS scan (mass range m/z 300–1600) and three subsequent dependent MS/MS scans (collision energy 35%). MASCOT v. 2.4.0 (Matrix Science, Boston, MA, USA) was used to compare the MS/MS data against the proteins on the NCBIr Oncorhynchuss mykiss (www.ncbi.nlm.nih.gov/protein/ ?term=oncorhynchus+mykiss, accessed 1 July 2013) and NCBIr Fish (www.ncbi.nlm.nih.gov/protein/?term=fishes, accessed 1 July 2013) databases. The MS/MS ion searches were performed with the following settings: (1) trypsin chosen as a protein-digesting enzyme and up to one missed cleavage tolerated; (2) carbamidomethylation of cystein as a fixed modification; (3) oxidation of methionine as a variable modification; (4) peptide tolerance of 2 Da; (5) MS/MS tolerance of 0.8 Da; (6) peptide charge 1þ, 2þ and 3þ; (7) instrument electrospray ionization ion-trap (ESI-TRAP). The occurrence of false positives (false discovery rate; FDR) was estimated by running searches using the same parameters against decoy databases (sequence-reversed NCBIr Oncorhynchuss mykiss and Fish databases). Search results were merged using Scaffold v. 4.2.1 (Proteome Software, Portland, OR, USA). Peptide identifications were accepted if they could be established at .95%
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probability; protein identifications (FDR ,1%) were accepted if they could be established at .99% probability and contained at least two unique peptides. GO annotation The UniProtKB database (www.uniprot.org, accessed 1 July 2013) was used to annotate the proteins identified. Two independent sets of ontology were used in the annotation: (1) the biological process in which the proteins participate; and (2) their molecular function. Western blot analysis Polyclonal antibodies against rainbow trout seminal plasma transferrin, prostaglandin D synthase (PGDS) and a1-antiproteinase were used to confirm the identification of these proteins in trout OF. The polyclonal antibodies against transferrin, PGDS and a1-antiproteinase were developed by Nynca et al. (2011a, 2011b) and Mak et al. (2004). After SDS-PAGE of OF, proteins were transferred to a nitrocellulose membrane (0.45 mm; Sigma, St Louis, MO, USA) in a Mini Trans-Biol Cell (Bio-Rad) at 60 V for 90 min at 48C. Non-specific binding was blocked by placing the membrane in a 5% solution of non-fat dry milk at room temperature. The membrane was incubated overnight at 48C with polyclonal antibodies diluted with Trisbuffered saline Tween-20 (TBST) at a ratio of 1 : 40 000 (transferrin), 1 : 5000 (PGDS) and 1 : 1000 (a1-antiproteinase). After rinsing the membrane with TBST to remove unbound primary antibodies, the membrane was exposed to alkaline phosphatase-conjugated anti-rabbit antibodies (1 : 20 000) for 90 min at room temperature. Products were visualised by incubating the membrane in a solution of alkaline phosphate buffer (100 mM TRIS-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) with nitroblue tetrazolium (5% Nitro Blue Tetrazolium; Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (5% 5-bromo-4-chloro3-indolyl phosphate; Sigma) in the dark for 3 min. The membrane was then washed for 3 min with 2 mM EDTA and running water to stop the colour reaction. Results LC-MS/MS analysis Fifty-four proteins were identified in rainbow trout OF using an SDS-PAGE-based proteomic approach. A detailed list of all proteins identified in the present study is provided in Table 1, together with their accession number, molecular mass, sequence coverage and number of unique peptides assigned to each protein (see also Table S1, available as Supplementary Material to this paper). Gene ontology Of the 54 OF proteins, 28 were classified according to the term ‘biological process’ and 33 proteins were assigned to the term ‘molecular functions’. GO analysis for the proteins assigned to the ‘biological process’ group revealed that most of the OF proteins were involved in the metabolic process (43%), followed by transport (26%) and then response to stimulus (26%; Fig. 2a). GO analysis of the ‘molecular function’ group revealed that
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most of these proteins (49%) were implicated in binding (mostly ions (45%), lipids (23%) and carbohydrates (18%)), followed by catalytic functions (Fig. 2b). The proteins were also grouped into five functional categories based on the information generated by GO annotations and proposed for purple sea urchin (Dheilly et al. 2013) and sea star (Franco et al. 2011) coelomic fluid. Western blot analysis Polyclonal antibodies against rainbow trout seminal plasma transferrin, PGDS and a1-antiproteinase cross-reacted with trout OF proteins separated by SDS-PAGE (Fig. 3). The migration rates of the main bands reflected molecular masses of these proteins obtained by proteomic methods (see Table 1) and published results (Nynca et al. 2011a, 2011b). Additional bands of low migration rates presumably represent complexes of the a1-antiproteinase with target proteins (Mak et al. 2004; Wojtczak et al. 2007). Discussion In the present study we generated the first protein catalogue of rainbow trout OF using one-dimensional electrophoresis (1-DE) prefractionation combined with LC-MS/MS. For three proteins tested, we were able to validate proteins identified by shotgun proteomics using western blot analysis. We identified 54 proteins, mostly associated with lipid binding and metabolism. The lack of a fully sequenced rainbow trout genome hampers the success of sequence homology searching of databases. The National Center for Biotechnology Information (NCBI) Oncorhynchus mykiss database contains approximately 110fold fewer proteins (7065) than the NCBI Homo sapiens database (78 6331; http://www.ncbi.nlm.nih.gov/protein/?term= homo+sapiens, accessed 1 July 2013). Moreover, the large amounts and multiple forms of vitellogenin present in trout OF mask and hinder the successful identification of other less abundant proteins. In the present study, we confirmed the presence of putative markers of oocyte quality (different forms of vitellogenin, apolipoproteins A-I-1 and mannose-binding lectin) reported by previous studies of the proteome of rainbow trout OF (Bobe and Goetz 2001; Rime et al. 2004) and found additional proteins that had not been reported earlier for trout OF. The GO analysis of molecular function revealed that the predominant proteins of trout OF are associated with binding and catalytic activity. Our data show a similar pattern of molecular functions that have been reported for human follicular fluid and oocytes (Ambekar et al. 2013), as well as sea star coelomocytes (Franco et al. 2011). Moreover, most trout OF proteins (85%) can be found in the follicular fluid and eggs of higher vertebrates (Angelucci et al. 2006; Twigt et al. 2012; Ambekar et al. 2013). However, 15% of our proteins were found to differ from those found in higher vertebrates, such as proteins related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown function (UPF0762 protein C6orf58 homologue). A few proteins were also present in trout OF
44
gi|95931876 gi|185132366 (þ1)
gi|212420009
gi|3123011 (þ1)
gi|1066855 (þ1)
gi|3894096
gi|522209345
gi|522209364
Serum albumin Sex hormone-binding globulin precursor Sex hormone-binding globulin a
Vitellogenin, short (¼ VTG)
Vitellogenin, partial
Vitellogenin
Vitellogenin As
Vitellogenin C
Carbohydrate binding C-Type lectin receptor B C-Type mannose-binding lectin precursor Mannan-binding lectin H2 precursor
19 44
gi|295419235
Serum albumin 1 protein
27 21 26
gi|223049425 (þ1) gi|185132516 (þ1)
gi|159147213 (þ1)
143
183
34
50
183
31
16 31 19
gi|229890014 (þ1) gi|185133428 (þ1) gi|11095799
Apolipoprotein AII Apolipoprotein E precursor Prostaglandin D synthase
30
30
gi|185132822 (þ2)
gi|6686384 (þ2)
Binding Lipid binding and metabolism Apolipoprotein AI-1 precursor
Molecular mass (kDa)
Apolipoprotein AI-2 precursor
Accession no.
Protein
6
5 2
16
7
3
3
82
8
6 9
9
5 14 3
13
19
No. unique peptides
11
45 5
46
1667
456
313
2392
37
65 49
70
85 85 21
188
450
Quantitative value
Transport Primary spermatocyte growth Primary spermatocyte growth
Transport
Lipid transport Lipid metabolic process
Lipid transport, cholesterol metabolic process Lipid transport, cholesterol metabolic process
Biological process
Carbohydrate binding
Carbohydrate binding Carbohydrate binding
Lipid transporter activity, nutrient reservoir activity Lipid transporter activity Lipid transporter activity Lipid transporter activity Lipid transporter activity
Steroid binding
Steroid binding
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O. mykiss
O. mykiss O. mykiss
Oncorhynchus clarkii
O. clarkii
O. mykiss
O. mykiss
Oncorhynchus tshawytscha O. mykiss
O. mykiss O. mykiss
O. mykiss
O. mykiss O. mykiss O. mykiss
O. mykiss
Lipid binding
Lipid binding Transporter activity, small molecule binding Lipid and metal binding
Oncorhynchuss mykiss
Sequence source organism
Lipid binding
Molecular function
Table 1. Proteins identified in rainbow trout ovarian fluid by sodium dodecyl sulfate]polyacrylamide gel electrophoresis and liquid chromatography]tandem mass spectrometry ECM, extracellular matrix
Shotgun proteomics of rainbow trout ovarian fluid 507
46 25 50 13 55 75
gi|400364966
gi|225705018 (þ1) gi|1848139 gi|11095771 gi|134285833
gi|218931236 (þ1)
gi|1352103
Immune response Complement C3
106 94 67 64
65 65 25
gi|185133413 (þ1)
gi|185132432 (þ1)
gi|185133255 (þ1)
gi|116616
gi|3982895
gi|185132505 (þ1)
gi|58201845
gi|9256318 gi|345556 (þ1)
Complement factor Bf-2
Complement factor H precursor
Immunoglobulin mu heavy chain secretory form IgM heavy chain Ig light chain-fragment
93
85
193
gi|185135626 (þ1)
Complement component 4 precursor Complement component C6 precursor Complement component C7–2 precursor Complement component C9 precursor Complement component C9
180
gi|431608
Complement component C3, partial
182
26
Molecular mass (kDa)
gi|159147215 (þ1)
Accession no.
Mannan-binding lectin H3 precursor Ion transport Cobalamin-binding protein, partial Heme-binding protein 2 Hemopexin-like protein Hemopexin-like protein variant 1 Lymphocyte cytosolic protein 1 precursor Transferrin precursor
Protein
6 2
14 3
17
3
8
11
25
2
2
9
5
28
112
2 36 9 2
4
9
Quantitative value
Regulation of complement activation
Complement activation, classical pathway Complement activation
Immune response
Immune response
Immune response
Complement activation, inflammatory response Complement activation, inflammatory response
Iron ion transport and homeostasis
Biological process
Serine-type endopeptidase activity Heparin binding
Endopeptidase inhibitor activity
Endopeptidase inhibitor activity
Endopeptidase inhibitor activity
Ferric iron binding
Metal ion binding Metal ion binding Calcium ion binding
Cobalamin binding
Carbohydrate binding
Molecular function
O. mykiss O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss O. mykiss O. mykiss O. mykiss
O. mykiss
O. mykiss
Sequence source organism
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7
3
5
5
6
2
2
3
5
19
22
2 8 3 2
3
3
No. unique peptides
Table 1. (Continued)
508 J. Nynca et al.
51
gi|185134355 (þ1)
gi|195954322
Matrix metalloproteinase-2 precursor Myeloid cell lineage chitinase
Other UPF0762 protein C6orf58 homologue Triose-phosphate isomerase
74
gi|54300680
Cysteine proteinase inhibitor
40 22
gi|56310256 (þ2)
gi|34221914
15
71
gi|185135584 (þ1)
Coagulation factor II precursor
48
gi|185132174 (þ1)
43
58
gi|185132234 (þ1)
gi|185132376 (þ1)
1933 50
gi|528510293 gi|185134311 (þ1)
3922
Cathepsin D precursor
PREDICTED: titin isoform X7 Vitelline envelope protein gamma precursor Zona pellucida glycoprotein 2.3 precursor Proteolysis a1-Antiproteinase-like protein precursor
gi|528490869
48
gi|15028982 (þ1)
PREDICTED: low-quality protein: titin
42 170
gi|19309743 (þ2) gi|551527443
62
20
gi|185133875 (þ1)
gi|15028976 (þ1)
16
gi|266485 (þ4)
Keratin E1 type II
Precerebellin-like protein precursor Cell structure, shape b-Actin Collagen a-1(V) chain-like, partial Keratin S8 type I
Lysozyme C II
2
3
3
2
3
2
4
10
4
5 12
5
2
2
7 5
2
2
2
11
6
3
8
5
12
81
4
8 50
8
5
4
24 9
15
2
Gluconeogenesis
Chitinase activity
Proteolysis
Blood coagulation, proteolysis
Proteolysis
Proteolysis
Cell morphogenesis, regulation of Rho protein signal transduction; sarcomere organisation
Cell wall macromolecule catabolic process
Triose-phosphate isomerase activity
Serine-type endopeptidase inhibitor activity Aspartic-type endopeptidase activity Serine-type endopeptidase activity Cysteine-type peptidase activity Metalloendopeptidase activity Chitin catabolic process; carbohydrate metabolic process
ATP binding ECM structural constituent Structural molecule activity Structural molecule activity ATP binding, Rab GTPase activator activity, structural constituent of muscle
Lysozyme activity
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
Danio rerio O. mykiss
Danio rerio
O. mykiss
O. mykiss Xiphophorus maculatus O. mykiss
O. mykiss
O. mykiss
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(a) Developmental process
Response to stimulus
Transport
Metabolic process
0
(b)
5
10
15
20
25
30
35
40
45
50
Enzyme regulator activity Structural molecule
Transporter activity
Catalytic activity
Binding 0
5
10
15
20
25
30
35
40
45
50
55
Percentage Fig. 2. Bar graphs showing the rainbow trout ovarian fluid proteome in terms of (a) biological process and (b) molecular function.
(a)
(b) OF1 OF2 OF3
M
kDa
(c) OF1 OF2 OF3
202.403 114.802
kDa
73.058
202.403 114.802 78.058
47.891
47.891
34.111
34.111
27.046
17.014
M
OF1 OF2 OF3
27.046
17.014
6.026 6.026
Fig. 3. Cross-reactivity between polyclonal antibodies against (a) transferrin, (b) prostaglandin D synthase and (c) a1-antiproteinase and ovarian fluid samples (OF1, OF2, OF3). M, molecular mass marker (202.403–6.026 kDa).
in multiple forms compared with results from higher vertebrates (e.g. apolipoprotein AI (apoAI-2), hemopexin (hemopexin-like variant 1), serum albumin (serum albumin 1 protein) and forms of complement B (Bf-2) and complement component C7 (C7–2);
Ambekar et al. 2013). These proteins from trout OF could be related to the specific mode of fish reproduction. Most of the proteins identified seem to be evolutionarily conserved, because they are also observed in higher vertebrates.
Shotgun proteomics of rainbow trout ovarian fluid
Proteins involved in lipid binding and metabolism dominated within 54 trout OF proteins identified. Those proteins are likely involved in the formation and metabolism of egg yolk, which predominates in rainbow trout oocytes. The most prominent protein was vitellogenin, a member of the lipid transfer protein family, which also includes microsomal triglyceride transfer protein and apolipoproteins. Vitellogenin forms the major egg yolk proteins, providing the energy reserve for nourishment of the developing embryos of oviparous vertebrates and invertebrates. In addition, vitellogenin reduces oxidative stress by scavenging free radicals or via antimicrobial and antiviral activities (Seehuus et al. 2006; Garcia et al. 2010). Previously, Rime et al. (2004) indicated the presence of only Apo-AI in rainbow trout OF. In the present study, different types of apolipoproteins, including Apo-AI-1, Apo-AI-2, Apo-II and Apo-E, were identified in trout OF for the first time. Apolipoproteins participate in reverse cholesterol and fatty acid transport and steroidogenesis, as well as in other functions, such as having a regulatory role in the complement system (Magnado´ttir and Lange 2004) and antiviral and antimicrobial activity (Concha et al. 2003). Furthermore, the increased amount of vitellogenin breakdown products together with Apo-AI was found to be related to the postovulatory aging of trout oocytes (Rime et al. 2004). Summing up, the presence of vitellogenin and apolipoproteins indicates the importance of lipid uptake and storage during oocyte development, as well as the importance of protection against pathogens. Moreover, the presence of multiple forms of apolipoprotein may indicate a complex mechanism involved in lipid transport and metabolism in rainbow trout OF. We identified proteins involved in the binding of carbohydrates (including lectins) and ions, such as hemopexin, transferrin, ferritin and serum albumin. In addition to their transporting functions, these proteins are recognised as acute-phase proteins, the expression of which is induced by inflammation caused by infection, tissue injury, stress or immunological disorders (Gruys et al. 2005). Lectins in particular are crucial in the innate immune system, including cell agglutination and apoptosis, and act as opsonins to enhance phagocytosis (Kim et al. 2011). Furthermore, lectins have been suggested to have a variety of other functions, such as preventing polyspermy, participating in the formation of the egg envelope and signal transduction and regulating carbohydrate metabolism (Dong et al. 2004). Transferrin, hemopexin and ferritin are responsible for the iron shuttle in vertebrates and are involved in defence mechanisms against bacteria (Gomme and McCann et al. 2005; Dietrich et al. 2011). Moreover, other proteins identified in the present study, including complements, a1-antiproteinase-like protein, precerebellin-like protein and lysozyme C-II, have also been found to be key components of the innate immune system (Bayne and Gerwick 2001). Salmonids spawn in shallow streams with rocky bottoms and are therefore prone to injuries. The plethora of acute-phase proteins in rainbow trout OF strongly suggests that the defence mechanism is well developed in this fluid and may provide efficient protection of oocytes. Our results demonstrated the presence of OF proteins involved in proteolysis and cell shape and structure. Proteases participate in proteolysis accompanying maturation and ovulation processes. Cathepsin D has been determined as the enzyme
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responsible for the limited cleavage of vitellogenin to yield yolk proteins (Carnevali et al. 1999). In addition, follicular atresia is likely controlled by cathepsins and affects the potential fecundity of the organism (Carnevali et al. 2006). It is suggested that the presence of structural protein, such as actin, in the follicular fluid of higher vertebrates is related to changes in microfilaments and degradation of extracellular matrix during the preovulatory phase (Fahiminiya et al. 2011). Summing up, trout OF proteins seem to be involved in the ovulation of oocytes into the body cavity and the maturation of oocytes before spawning. However, at present, conclusions regarding the role of OF proteins are speculative and further studies are needed to prove the specific role of these proteins. In conclusion, the present study provides the first initial overview of the rainbow trout OF proteome. The study has unravelled the identity of several proteins not previously reported in fish OF. Most of the trout OF proteins correspond to follicular fluid proteins of higher vertebrates. The proteome of trout OF consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. This study can help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality. Acknowledgements This work was supported by a grant from the National Science Centre (2011/ 01/D/NZ9/00619), as well as by funding from the Operational Program Development of the Fisheries Sector and Coastal Areas 2007-2013 (OR-61724-OR1400001/10). This work was also supported by funds appropriated to the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn and by Project ‘‘REFRESH’’ 264103 (7th Framework Programme EU-Research Potential-CapacitiesFP7-REGPOT-2010-1-264103) from the European Commission.
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trutta f lacustris, Salvelinus alpinus and Hucho hucho. Reprod. Nutr. Dev. 35, 465–474. doi:10.1051/RND:19950501 Magnado´ttir, B., and Lange, S. (2004). Is apolipoprotein A-I a regulating protein for the complement system of cod (Gadus morhua L.)? Fish Shellfish Immunol. 16, 265–269. doi:10.1016/S1050-4648(03)00061-5 Mak, M., Mak, P., Olczak, M., Szalewicz, A., Głogowski, J., Dubin, A., Wtorek, W., and Ciereszko, A. (2004). Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma. Biochim. Biophys. Acta 1671, 93–105. doi:10.1016/J.BBAGEN.2004.02.001 Matsubara, T., Hara, A., and Takano, K. (1985). Immunochemical identification and purification of coelomic fluid-specific protein in chum salmon (Oncorhynchus keta). Comp. Biochem. Physiol. 81, 309–314. Nynca, J., Dietrich, M. A., Bilin´ska, B., Kotula-Balak, M., Kiełbasa, T., Karol, H., and Ciereszko, A. (2011a). Isolation of lipocalin-type protein from rainbow trout seminal plasma and its localization in the reproductive system. Reprod. Fertil. Dev. 23, 381–389. doi:10.1071/RD10118 Nynca, J., Słowin´ska, M., Dietrich, M. A., Bilin´ska, B., Kotula-Balak, M., and Ciereszko, A. (2011b). Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system. Comp. Biochem. Physiol. B 158, 106–116. doi:10.1016/J.CBPB.2010.10.002 Rime, H., Guitton, N., Pineau, C., Bonnet, E., Bobe, J., and Jalabert, B. (2004). Post-ovulatory ageing and egg quality: A proteomic analysis of rainbow trout coelomic fluid. Reprod. Biol. Endocrinol. 2, 26. doi:10.1186/1477-7827-2-26 Rosengrave, P., Taylor, H., Montgomerie, R., Metcalf, V., McBride, K., and Gemmell, N.J. (2009). Chemical composition of seminal and ovarian fluids of chinook salmon (Oncorhynchus tshawytscha) and their effects on sperm motility traits. Comp. Biochem. Physiol., Part A 152, 123–129. doi:10.1016/J.CBPA.2008.09.009 Seehuus, S. C., Norberg, K., Gimsa, U., Krekling, T., and Amdam, G. V. (2006). Reproductive protein protects functionally sterile honey bee workers from oxidative stress. Proc. Natl Acad. Sci. USA 103, 962–967. doi:10.1073/PNAS.0502681103 Twigt, J., Steegers-Theunissen, R. P., Bezstarosti, K., and Demmers, J. A. (2012). Proteomic analysis of the microenvironment of developing oocytes. Proteomics 12, 1463–1471. doi:10.1002/PMIC.201100240 Wojtczak, M., Całka, J., Głogowski, J., and Ciereszko, A. (2007). Isolation and characterization of a1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma. Comp. Biochem. Physiol. B 148, 264–276. doi:10.1016/J.CBPB.2007.06.004
www.publish.csiro.au/journals/rfd
Reproduction, Fertility and Development, 2015, 27, 504–512 http://dx.doi.org/10.1071/RD13224
Shotgun proteomics of rainbow trout ovarian fluid Joanna Nynca A,C, Georg J. Arnold B, Thomas Fro¨hlich B and Andrzej Ciereszko A A
Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland. B Laboratory for Functional Genome Analysis (LAFUGA), Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universita¨t, Feodor Lynen Str. 25, 81377 Munich, Germany. C Corresponding author. Email: [email protected]
Abstract. In the present study we used a shotgun proteomic approach to identify 54 proteins of rainbow trout ovarian fluid. The study has unravelled the identity of several proteins not previously reported in fish ovarian fluid. The proteome of trout ovarian fluid consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. Most trout ovarian fluid proteins correspond to follicular fluid proteins of higher vertebrates, but 15% of the proteins were found to be different, such as those related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown functions (UPF0762 protein C6orf58 homologue). The present study could help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality. Additional keywords: immunity, mass spectrometry, ovarian fluid, rainbow trout, shotgun proteomics. Received 17 July 2013, accepted 18 December 2013, published online 30 January 2014
Introduction Mature rainbow trout oocytes are expelled at ovulation into the coelomic cavity, where they remain immersed in a semiviscous fluid known as ovarian or coelomic fluid. Ovarian fluid (OF) is formed by filtration from the blood plasma and the secretory activity of ovarian epithelia, and it may contain postovulatory or broken egg components (Matsubara et al. 1985). The composition of the OF is geared towards egg storage and extension of the fertilisation period during both natural spawning and artificial fertilisation (Lahnsteiner et al. 1995). The inorganic composition of the OF of fish is well described; in salmonids, this fluid has electrolyte concentrations similar to blood plasma (Rosengrave et al. 2009). However, knowledge regarding the protein composition of trout OF is limited to trout ovulatory proteins (Coffman et al. 2000), apolipoproteins, vitellogenins and lectins (Rime et al. 2004). The biochemical composition and pH of OF can reflect the quality of the eggs. The presence of components from broken eggs was found to decrease the pH of trout OF and consequently its ability to activate sperm motility (Dietrich et al. 2007). Using proteomic methods, Rime et al. (2004) reported potential markers of egg quality, such as vitellogenin fragments and apolipoproteins, in trout OF that may be associated with oocyte postovulatory aging. Studies on OF protein components may Journal compilation Ó CSIRO 2015
also contribute to a better understanding of the mechanisms underlying oocyte development and quality. In the present study we used the shotgun proteomic method to investigate rainbow trout OF proteins. We provided a brief overview of the biological processes and molecular functions of the proteins identified based on gene ontology (GO) analysis. Materials and methods Animals and gamete collection Rainbow trout spawners were maintained in an experimental fish hatchery at the Inland Fisheries Institute, Rutki, Poland. Eggs were collected from five female rainbow trout of the spring-spawning Rutki strain (4 years of age) and placed in a sieve. The OF was then poured off and collected. The fish were stocked in concrete ponds (150 m3) supplied with water from the river Radunia. The temperature of the water was 10–128C. OF samples were centrifuged at 3000g for 10 min at 48C to remove red blood cells and debris. The collected supernatants were stored at 808C until electrophoresis was conducted. The protein concentration was determined using the Coomassie Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and bovine serum albumin (BSA) as a calibration standard. www.publish.csiro.au/journals/rfd
Shotgun proteomics of rainbow trout ovarian fluid
kDa
M
Reproduction, Fertility and Development
OF1
OF2
OF3
OF4
505
OF5
250 150 100 70 50 40 30 20 15
10
Fig. 1. One-dimensional gel electrophoresis separation of five replicates (OF1, OF2, OF3, OF4, OF5) of rainbow trout ovarian fluid proteins under reducing conditions. M, molecular mass marker (10–250 kDa). The boxes represent the 11 gel slices that were digested individually and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS).
All experiments described herein were approved by the Animal Experiments Committee of Olsztyn (no. 39/2011), Poland. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in-gel protein digestion OF proteins (40 mg) from five different individuals were analysed separately by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% stacking gel and a 12% separation gel with a mini ProteanTM II device (Bio-Rad, Hercules, CA, USA). Following electrophoresis, the gel was stained with Coomassie blue dye overnight (50% methanol, 10% acetic acid, 0.05% Coomassie brilliant blue R-250) and then destained with 5% methanol and 7% acetic acid (Fig. 1). The gel was then washed in water and each protein lane was cut into 11 slices. The slices were then washed twice in 50 mM NH4HCO3, reduced for 30 min at 558C with 45 mM dithioerythritol and then alkylated by 30 min incubation with 100 mM iodoacetamide. After extensive washing in 50 mM NH4HCO3, gel slices were minced and subjected to overnight digestion at 378C with 1 mg porcine trypsin (Promega, Madison, WI, USA) per slice. The resulting peptides were eluted from the gel pieces in two successive washes, first with 50 mM NH4HCO3 and then with 80% acetonitrile (ACN). The NH4HCO3 and the ACN supernatant were collected, pooled and concentrated using a SpeedVac concentrator (Bachofer, Vacuum Concentrator, Reutlingen, Germany). Peptide samples were reconstituted with 10 mL of 0.1% formic acid (FA) and applied to an Eksigent Ultra nano-liquid chromatography device (Eksigent, Dublin, CA, USA) coupled to a linear ion trap (IT) mass spectrometer (LTQ; Thermo Electron, San Jose, CA, USA). The procedures, which included cutting protein lanes, digestion with trypsin and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, were performed separately for five different OF samples.
LC-MS/MS analysis and database searches Samples were injected onto a C18 trap column (C18 Pep˚ , 300 mm 5 mm column Map100, 5 mm particle size, 100 A size; LC Packings Dionex, Sunnyvale, CA, USA) and subsequently separated by reverse-phase chromatography using a nano-LC column (ReproSil-Pur C18 AQ, 2.4 mm; 150 mm 75 mm; Dr Maisch, Ammerbuch-Entringen, Germany) at a flow rate of 280 nL min1. The mobile phases consisted of water (Mobile Phase A) and 84% ACN (Mobile Phase B), and both contained 0.1%FA. The ionspray voltage was set to 1.4 kV and the mass spectrometry (MS) and MS/MS analyses were performed using cycles of one MS scan (mass range m/z 300–1600) and three subsequent dependent MS/MS scans (collision energy 35%). MASCOT v. 2.4.0 (Matrix Science, Boston, MA, USA) was used to compare the MS/MS data against the proteins on the NCBIr Oncorhynchuss mykiss (www.ncbi.nlm.nih.gov/protein/ ?term=oncorhynchus+mykiss, accessed 1 July 2013) and NCBIr Fish (www.ncbi.nlm.nih.gov/protein/?term=fishes, accessed 1 July 2013) databases. The MS/MS ion searches were performed with the following settings: (1) trypsin chosen as a protein-digesting enzyme and up to one missed cleavage tolerated; (2) carbamidomethylation of cystein as a fixed modification; (3) oxidation of methionine as a variable modification; (4) peptide tolerance of 2 Da; (5) MS/MS tolerance of 0.8 Da; (6) peptide charge 1þ, 2þ and 3þ; (7) instrument electrospray ionization ion-trap (ESI-TRAP). The occurrence of false positives (false discovery rate; FDR) was estimated by running searches using the same parameters against decoy databases (sequence-reversed NCBIr Oncorhynchuss mykiss and Fish databases). Search results were merged using Scaffold v. 4.2.1 (Proteome Software, Portland, OR, USA). Peptide identifications were accepted if they could be established at .95%
506
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probability; protein identifications (FDR ,1%) were accepted if they could be established at .99% probability and contained at least two unique peptides. GO annotation The UniProtKB database (www.uniprot.org, accessed 1 July 2013) was used to annotate the proteins identified. Two independent sets of ontology were used in the annotation: (1) the biological process in which the proteins participate; and (2) their molecular function. Western blot analysis Polyclonal antibodies against rainbow trout seminal plasma transferrin, prostaglandin D synthase (PGDS) and a1-antiproteinase were used to confirm the identification of these proteins in trout OF. The polyclonal antibodies against transferrin, PGDS and a1-antiproteinase were developed by Nynca et al. (2011a, 2011b) and Mak et al. (2004). After SDS-PAGE of OF, proteins were transferred to a nitrocellulose membrane (0.45 mm; Sigma, St Louis, MO, USA) in a Mini Trans-Biol Cell (Bio-Rad) at 60 V for 90 min at 48C. Non-specific binding was blocked by placing the membrane in a 5% solution of non-fat dry milk at room temperature. The membrane was incubated overnight at 48C with polyclonal antibodies diluted with Trisbuffered saline Tween-20 (TBST) at a ratio of 1 : 40 000 (transferrin), 1 : 5000 (PGDS) and 1 : 1000 (a1-antiproteinase). After rinsing the membrane with TBST to remove unbound primary antibodies, the membrane was exposed to alkaline phosphatase-conjugated anti-rabbit antibodies (1 : 20 000) for 90 min at room temperature. Products were visualised by incubating the membrane in a solution of alkaline phosphate buffer (100 mM TRIS-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) with nitroblue tetrazolium (5% Nitro Blue Tetrazolium; Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (5% 5-bromo-4-chloro3-indolyl phosphate; Sigma) in the dark for 3 min. The membrane was then washed for 3 min with 2 mM EDTA and running water to stop the colour reaction. Results LC-MS/MS analysis Fifty-four proteins were identified in rainbow trout OF using an SDS-PAGE-based proteomic approach. A detailed list of all proteins identified in the present study is provided in Table 1, together with their accession number, molecular mass, sequence coverage and number of unique peptides assigned to each protein (see also Table S1, available as Supplementary Material to this paper). Gene ontology Of the 54 OF proteins, 28 were classified according to the term ‘biological process’ and 33 proteins were assigned to the term ‘molecular functions’. GO analysis for the proteins assigned to the ‘biological process’ group revealed that most of the OF proteins were involved in the metabolic process (43%), followed by transport (26%) and then response to stimulus (26%; Fig. 2a). GO analysis of the ‘molecular function’ group revealed that
J. Nynca et al.
most of these proteins (49%) were implicated in binding (mostly ions (45%), lipids (23%) and carbohydrates (18%)), followed by catalytic functions (Fig. 2b). The proteins were also grouped into five functional categories based on the information generated by GO annotations and proposed for purple sea urchin (Dheilly et al. 2013) and sea star (Franco et al. 2011) coelomic fluid. Western blot analysis Polyclonal antibodies against rainbow trout seminal plasma transferrin, PGDS and a1-antiproteinase cross-reacted with trout OF proteins separated by SDS-PAGE (Fig. 3). The migration rates of the main bands reflected molecular masses of these proteins obtained by proteomic methods (see Table 1) and published results (Nynca et al. 2011a, 2011b). Additional bands of low migration rates presumably represent complexes of the a1-antiproteinase with target proteins (Mak et al. 2004; Wojtczak et al. 2007). Discussion In the present study we generated the first protein catalogue of rainbow trout OF using one-dimensional electrophoresis (1-DE) prefractionation combined with LC-MS/MS. For three proteins tested, we were able to validate proteins identified by shotgun proteomics using western blot analysis. We identified 54 proteins, mostly associated with lipid binding and metabolism. The lack of a fully sequenced rainbow trout genome hampers the success of sequence homology searching of databases. The National Center for Biotechnology Information (NCBI) Oncorhynchus mykiss database contains approximately 110fold fewer proteins (7065) than the NCBI Homo sapiens database (78 6331; http://www.ncbi.nlm.nih.gov/protein/?term= homo+sapiens, accessed 1 July 2013). Moreover, the large amounts and multiple forms of vitellogenin present in trout OF mask and hinder the successful identification of other less abundant proteins. In the present study, we confirmed the presence of putative markers of oocyte quality (different forms of vitellogenin, apolipoproteins A-I-1 and mannose-binding lectin) reported by previous studies of the proteome of rainbow trout OF (Bobe and Goetz 2001; Rime et al. 2004) and found additional proteins that had not been reported earlier for trout OF. The GO analysis of molecular function revealed that the predominant proteins of trout OF are associated with binding and catalytic activity. Our data show a similar pattern of molecular functions that have been reported for human follicular fluid and oocytes (Ambekar et al. 2013), as well as sea star coelomocytes (Franco et al. 2011). Moreover, most trout OF proteins (85%) can be found in the follicular fluid and eggs of higher vertebrates (Angelucci et al. 2006; Twigt et al. 2012; Ambekar et al. 2013). However, 15% of our proteins were found to differ from those found in higher vertebrates, such as proteins related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown function (UPF0762 protein C6orf58 homologue). A few proteins were also present in trout OF
44
gi|95931876 gi|185132366 (þ1)
gi|212420009
gi|3123011 (þ1)
gi|1066855 (þ1)
gi|3894096
gi|522209345
gi|522209364
Serum albumin Sex hormone-binding globulin precursor Sex hormone-binding globulin a
Vitellogenin, short (¼ VTG)
Vitellogenin, partial
Vitellogenin
Vitellogenin As
Vitellogenin C
Carbohydrate binding C-Type lectin receptor B C-Type mannose-binding lectin precursor Mannan-binding lectin H2 precursor
19 44
gi|295419235
Serum albumin 1 protein
27 21 26
gi|223049425 (þ1) gi|185132516 (þ1)
gi|159147213 (þ1)
143
183
34
50
183
31
16 31 19
gi|229890014 (þ1) gi|185133428 (þ1) gi|11095799
Apolipoprotein AII Apolipoprotein E precursor Prostaglandin D synthase
30
30
gi|185132822 (þ2)
gi|6686384 (þ2)
Binding Lipid binding and metabolism Apolipoprotein AI-1 precursor
Molecular mass (kDa)
Apolipoprotein AI-2 precursor
Accession no.
Protein
6
5 2
16
7
3
3
82
8
6 9
9
5 14 3
13
19
No. unique peptides
11
45 5
46
1667
456
313
2392
37
65 49
70
85 85 21
188
450
Quantitative value
Transport Primary spermatocyte growth Primary spermatocyte growth
Transport
Lipid transport Lipid metabolic process
Lipid transport, cholesterol metabolic process Lipid transport, cholesterol metabolic process
Biological process
Carbohydrate binding
Carbohydrate binding Carbohydrate binding
Lipid transporter activity, nutrient reservoir activity Lipid transporter activity Lipid transporter activity Lipid transporter activity Lipid transporter activity
Steroid binding
Steroid binding
Reproduction, Fertility and Development (Continued )
O. mykiss
O. mykiss O. mykiss
Oncorhynchus clarkii
O. clarkii
O. mykiss
O. mykiss
Oncorhynchus tshawytscha O. mykiss
O. mykiss O. mykiss
O. mykiss
O. mykiss O. mykiss O. mykiss
O. mykiss
Lipid binding
Lipid binding Transporter activity, small molecule binding Lipid and metal binding
Oncorhynchuss mykiss
Sequence source organism
Lipid binding
Molecular function
Table 1. Proteins identified in rainbow trout ovarian fluid by sodium dodecyl sulfate]polyacrylamide gel electrophoresis and liquid chromatography]tandem mass spectrometry ECM, extracellular matrix
Shotgun proteomics of rainbow trout ovarian fluid 507
46 25 50 13 55 75
gi|400364966
gi|225705018 (þ1) gi|1848139 gi|11095771 gi|134285833
gi|218931236 (þ1)
gi|1352103
Immune response Complement C3
106 94 67 64
65 65 25
gi|185133413 (þ1)
gi|185132432 (þ1)
gi|185133255 (þ1)
gi|116616
gi|3982895
gi|185132505 (þ1)
gi|58201845
gi|9256318 gi|345556 (þ1)
Complement factor Bf-2
Complement factor H precursor
Immunoglobulin mu heavy chain secretory form IgM heavy chain Ig light chain-fragment
93
85
193
gi|185135626 (þ1)
Complement component 4 precursor Complement component C6 precursor Complement component C7–2 precursor Complement component C9 precursor Complement component C9
180
gi|431608
Complement component C3, partial
182
26
Molecular mass (kDa)
gi|159147215 (þ1)
Accession no.
Mannan-binding lectin H3 precursor Ion transport Cobalamin-binding protein, partial Heme-binding protein 2 Hemopexin-like protein Hemopexin-like protein variant 1 Lymphocyte cytosolic protein 1 precursor Transferrin precursor
Protein
6 2
14 3
17
3
8
11
25
2
2
9
5
28
112
2 36 9 2
4
9
Quantitative value
Regulation of complement activation
Complement activation, classical pathway Complement activation
Immune response
Immune response
Immune response
Complement activation, inflammatory response Complement activation, inflammatory response
Iron ion transport and homeostasis
Biological process
Serine-type endopeptidase activity Heparin binding
Endopeptidase inhibitor activity
Endopeptidase inhibitor activity
Endopeptidase inhibitor activity
Ferric iron binding
Metal ion binding Metal ion binding Calcium ion binding
Cobalamin binding
Carbohydrate binding
Molecular function
O. mykiss O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss O. mykiss O. mykiss O. mykiss
O. mykiss
O. mykiss
Sequence source organism
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7
3
5
5
6
2
2
3
5
19
22
2 8 3 2
3
3
No. unique peptides
Table 1. (Continued)
508 J. Nynca et al.
51
gi|185134355 (þ1)
gi|195954322
Matrix metalloproteinase-2 precursor Myeloid cell lineage chitinase
Other UPF0762 protein C6orf58 homologue Triose-phosphate isomerase
74
gi|54300680
Cysteine proteinase inhibitor
40 22
gi|56310256 (þ2)
gi|34221914
15
71
gi|185135584 (þ1)
Coagulation factor II precursor
48
gi|185132174 (þ1)
43
58
gi|185132234 (þ1)
gi|185132376 (þ1)
1933 50
gi|528510293 gi|185134311 (þ1)
3922
Cathepsin D precursor
PREDICTED: titin isoform X7 Vitelline envelope protein gamma precursor Zona pellucida glycoprotein 2.3 precursor Proteolysis a1-Antiproteinase-like protein precursor
gi|528490869
48
gi|15028982 (þ1)
PREDICTED: low-quality protein: titin
42 170
gi|19309743 (þ2) gi|551527443
62
20
gi|185133875 (þ1)
gi|15028976 (þ1)
16
gi|266485 (þ4)
Keratin E1 type II
Precerebellin-like protein precursor Cell structure, shape b-Actin Collagen a-1(V) chain-like, partial Keratin S8 type I
Lysozyme C II
2
3
3
2
3
2
4
10
4
5 12
5
2
2
7 5
2
2
2
11
6
3
8
5
12
81
4
8 50
8
5
4
24 9
15
2
Gluconeogenesis
Chitinase activity
Proteolysis
Blood coagulation, proteolysis
Proteolysis
Proteolysis
Cell morphogenesis, regulation of Rho protein signal transduction; sarcomere organisation
Cell wall macromolecule catabolic process
Triose-phosphate isomerase activity
Serine-type endopeptidase inhibitor activity Aspartic-type endopeptidase activity Serine-type endopeptidase activity Cysteine-type peptidase activity Metalloendopeptidase activity Chitin catabolic process; carbohydrate metabolic process
ATP binding ECM structural constituent Structural molecule activity Structural molecule activity ATP binding, Rab GTPase activator activity, structural constituent of muscle
Lysozyme activity
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
O. mykiss
Danio rerio O. mykiss
Danio rerio
O. mykiss
O. mykiss Xiphophorus maculatus O. mykiss
O. mykiss
O. mykiss
Shotgun proteomics of rainbow trout ovarian fluid Reproduction, Fertility and Development 509
510
Reproduction, Fertility and Development
J. Nynca et al.
(a) Developmental process
Response to stimulus
Transport
Metabolic process
0
(b)
5
10
15
20
25
30
35
40
45
50
Enzyme regulator activity Structural molecule
Transporter activity
Catalytic activity
Binding 0
5
10
15
20
25
30
35
40
45
50
55
Percentage Fig. 2. Bar graphs showing the rainbow trout ovarian fluid proteome in terms of (a) biological process and (b) molecular function.
(a)
(b) OF1 OF2 OF3
M
kDa
(c) OF1 OF2 OF3
202.403 114.802
kDa
73.058
202.403 114.802 78.058
47.891
47.891
34.111
34.111
27.046
17.014
M
OF1 OF2 OF3
27.046
17.014
6.026 6.026
Fig. 3. Cross-reactivity between polyclonal antibodies against (a) transferrin, (b) prostaglandin D synthase and (c) a1-antiproteinase and ovarian fluid samples (OF1, OF2, OF3). M, molecular mass marker (202.403–6.026 kDa).
in multiple forms compared with results from higher vertebrates (e.g. apolipoprotein AI (apoAI-2), hemopexin (hemopexin-like variant 1), serum albumin (serum albumin 1 protein) and forms of complement B (Bf-2) and complement component C7 (C7–2);
Ambekar et al. 2013). These proteins from trout OF could be related to the specific mode of fish reproduction. Most of the proteins identified seem to be evolutionarily conserved, because they are also observed in higher vertebrates.
Shotgun proteomics of rainbow trout ovarian fluid
Proteins involved in lipid binding and metabolism dominated within 54 trout OF proteins identified. Those proteins are likely involved in the formation and metabolism of egg yolk, which predominates in rainbow trout oocytes. The most prominent protein was vitellogenin, a member of the lipid transfer protein family, which also includes microsomal triglyceride transfer protein and apolipoproteins. Vitellogenin forms the major egg yolk proteins, providing the energy reserve for nourishment of the developing embryos of oviparous vertebrates and invertebrates. In addition, vitellogenin reduces oxidative stress by scavenging free radicals or via antimicrobial and antiviral activities (Seehuus et al. 2006; Garcia et al. 2010). Previously, Rime et al. (2004) indicated the presence of only Apo-AI in rainbow trout OF. In the present study, different types of apolipoproteins, including Apo-AI-1, Apo-AI-2, Apo-II and Apo-E, were identified in trout OF for the first time. Apolipoproteins participate in reverse cholesterol and fatty acid transport and steroidogenesis, as well as in other functions, such as having a regulatory role in the complement system (Magnado´ttir and Lange 2004) and antiviral and antimicrobial activity (Concha et al. 2003). Furthermore, the increased amount of vitellogenin breakdown products together with Apo-AI was found to be related to the postovulatory aging of trout oocytes (Rime et al. 2004). Summing up, the presence of vitellogenin and apolipoproteins indicates the importance of lipid uptake and storage during oocyte development, as well as the importance of protection against pathogens. Moreover, the presence of multiple forms of apolipoprotein may indicate a complex mechanism involved in lipid transport and metabolism in rainbow trout OF. We identified proteins involved in the binding of carbohydrates (including lectins) and ions, such as hemopexin, transferrin, ferritin and serum albumin. In addition to their transporting functions, these proteins are recognised as acute-phase proteins, the expression of which is induced by inflammation caused by infection, tissue injury, stress or immunological disorders (Gruys et al. 2005). Lectins in particular are crucial in the innate immune system, including cell agglutination and apoptosis, and act as opsonins to enhance phagocytosis (Kim et al. 2011). Furthermore, lectins have been suggested to have a variety of other functions, such as preventing polyspermy, participating in the formation of the egg envelope and signal transduction and regulating carbohydrate metabolism (Dong et al. 2004). Transferrin, hemopexin and ferritin are responsible for the iron shuttle in vertebrates and are involved in defence mechanisms against bacteria (Gomme and McCann et al. 2005; Dietrich et al. 2011). Moreover, other proteins identified in the present study, including complements, a1-antiproteinase-like protein, precerebellin-like protein and lysozyme C-II, have also been found to be key components of the innate immune system (Bayne and Gerwick 2001). Salmonids spawn in shallow streams with rocky bottoms and are therefore prone to injuries. The plethora of acute-phase proteins in rainbow trout OF strongly suggests that the defence mechanism is well developed in this fluid and may provide efficient protection of oocytes. Our results demonstrated the presence of OF proteins involved in proteolysis and cell shape and structure. Proteases participate in proteolysis accompanying maturation and ovulation processes. Cathepsin D has been determined as the enzyme
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responsible for the limited cleavage of vitellogenin to yield yolk proteins (Carnevali et al. 1999). In addition, follicular atresia is likely controlled by cathepsins and affects the potential fecundity of the organism (Carnevali et al. 2006). It is suggested that the presence of structural protein, such as actin, in the follicular fluid of higher vertebrates is related to changes in microfilaments and degradation of extracellular matrix during the preovulatory phase (Fahiminiya et al. 2011). Summing up, trout OF proteins seem to be involved in the ovulation of oocytes into the body cavity and the maturation of oocytes before spawning. However, at present, conclusions regarding the role of OF proteins are speculative and further studies are needed to prove the specific role of these proteins. In conclusion, the present study provides the first initial overview of the rainbow trout OF proteome. The study has unravelled the identity of several proteins not previously reported in fish OF. Most of the trout OF proteins correspond to follicular fluid proteins of higher vertebrates. The proteome of trout OF consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. This study can help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality. Acknowledgements This work was supported by a grant from the National Science Centre (2011/ 01/D/NZ9/00619), as well as by funding from the Operational Program Development of the Fisheries Sector and Coastal Areas 2007-2013 (OR-61724-OR1400001/10). This work was also supported by funds appropriated to the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn and by Project ‘‘REFRESH’’ 264103 (7th Framework Programme EU-Research Potential-CapacitiesFP7-REGPOT-2010-1-264103) from the European Commission.
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