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Comparative proteomics analysis of male and female Persian sturgeon (Acipenser persicus) gonads
Animal Reproduction Science 111 (2009) 361–368
Short communication
Comparative proteomics analysis of male and female Persian sturgeon (Acipenser persicus) gonads Saeed Keyvanshokooh a , Mohammad Reza Kalbassi a,∗∗ , Saman Hosseinkhani b , Behrouz Vaziri c,∗ a
c
Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, 46414-356 Noor, Mazandaran, Iran b Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran Protein Chemistry Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, 69 Pasteur Street, 13164 Tehran, Iran
Received 24 September 2007; received in revised form 21 January 2008; accepted 13 March 2008 Available online 20 March 2008
Abstract In sturgeon aquaculture, the fish are sexed by an invasive surgical examination of the gonads. Development of a non-invasive procedure for sexing fish based on a molecular method is of special interest. In the present study a proteomics approach has been utilized to analyze a differential protein expression between mature male and female Persian sturgeon (Acipenser persicus) gonads. When comparing protein patterns on the 2-DE gels of the testis and ovary, 48 unique spots were distinguished in testis while only two spots were matchless in ovary. The spots were identified by MALDI-TOF/TOF analysis. The identified proteins are involved in metabolism and energy production, cell structure, transcription and translation, cell defense, signal transduction, transport, cell division, and none were directly linked to a sex-determining gene. The provided proteomics data could be considered as a starting base for subsequent studies focusing on the identification of proteins involved in sex determination and differentiation at different stages of gonadal maturation. © 2008 Elsevier B.V. All rights reserved. Keywords: Proteomics; Sex; Ovary; Testis; Acipenser persicus
∗ ∗∗
Corresponding author. Tel.: +98 21 66480780; fax: +98 21 66480780. Corresponding author. Tel.: +98 122 6253101; fax: +98 122 6253499. E-mail addresses: Kalbassi [email protected] (M.R. Kalbassi), [email protected] (B. Vaziri).
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.03.005
362
S. Keyvanshokooh et al. / Animal Reproduction Science 111 (2009) 361–368
1. Introduction In sturgeon aquaculture, where the main purpose is caviar production, a reliable method is needed to separate the fish according to gender. Males are destined to the meat market while females remain in culture for many more years under conditions of optimal growth and development. The availability of monosex populations of caviar-producing females would significantly enhance the economic viability of domestic caviar production systems (Logan et al., 1995). However, it is not possible to distinguish male fish from females by morphological characters at larval, juvenile and even adult stages. Currently, growers wait 3–4 years before fish are sexed by an invasive surgical examination of the gonads (Doroshov et al., 1997) and the development of a non-invasive procedure based on a molecular method is of special interest. Although it is known that all sturgeons are gonochoristic and evidence for female heterogametic genetic sex determination has been presented for the white sturgeon (Acipenser transmontanus) (Van Eenennaam et al., 1999), bester (Huso huso female × Acipenser ruthenus male) (Omoto et al., 2005) and shortnose sturgeon (Acipenser brevirostrum) (Flynn et al., 2006), PCR-based techniques have failed to identify sex-specific genes in sturgeon species (Hett and Ludwig, 2005; Wuertz et al., 2006; Keyvanshokooh et al., 2007) and alternative approaches are required (Wuertz et al., 2006). In this study, we have used a proteomics approach to analyze differential protein expression between mature male and female Persian sturgeon (Acipenser persicus) gonads. The objective of the present work was to search for proteins associated with sex determination. 2. Materials and methods 2.1. Fish sampling and protein extraction Wild fish were captured from the southern Caspian Sea during their spawning run and kept at Shahid Rajaee Sturgeon Propagation Center, Sari, Iran. Sex was determined by inspection of testes and ovaries of necropsied fish. Both gonads from each fish were examined for evidence of hermaphroditism. Sampling was performed from male and female fish in stage 5 of gonadal development. Gonad tissue samples from 15 Persian sturgeon of each sex stored at −70 ◦ C until protein extraction. The gonad tissues of each five fish were mixed before protein extraction and three pools were prepared for each sex. The tissue samples were homogenized in the presence of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 50 mM Tris, 0.2% carrier ampholyte, 1 mM PMSF, 0.25% RNase and 1% DNase). Each sample was maintained for 1 h at room temperature for protein release before centrifugation at 12,000 × g for 10 min at 20 ◦ C. Protein concentration was determined by Bradford assay using BSA standards (Bradford, 1976). 2.2. Two-dimensional gel electrophoresis Isoelectric focusing (IEF) was performed on immobilized pH gradients (IPG; pH 3–10, 17 cm) with Protean IEF Cell (BioRad, USA). A total of 150 g of proteins was used for analytical runs, and 1500 g of proteins was used for preparative runs to a total volume of 300 l of re-hydrating buffer (8 M urea, 4% CHAPS, 50 mM DTT, 0.2% ampholyte and a trace of bromophenol blue). Focusing was performed at 60,000 Vh. Once the IEF was completed, the strips were equilibrated in buffer containing 6 M urea, 50 mM Tris–HCl, pH 8.8, 20% glycerol, 2% SDS and 2% DTT for 20 min, followed by the same buffer without DTT and supplemented with 2.5% idoacetamide for 20 min. Separation of the second dimension was performed according to Keyvanshokooh and
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Vaziri (2007). Silver staining protocol was used to visualize protein spots in analytical gels. The preparative gels were stained using a modified colloidal Coomassie G-250 procedure. 2.3. Analysis of 2D gels and MALDI-TOF/TOF MS The gels were scanned using a GS-800 (BioRad). Spot detection and matching were performed using the PG200 software (Nonlinear Dynamics, UK). Each set of gel replicates for both testis and ovary samples were combined into average gels, which represented spots that were reproducibly present on each set of the triplicate gels. The spots that were present only in the protein map of ovary or testis were then selected for further MS analysis. In-gel digestion of samples and MALDI-TOF/TOF analysis was carried out according to Keyvanshokooh and Vaziri (2007). 3. Results and discussion Fig. 1 shows the 2DE map of the sturgeon testis and ovary proteins. When comparing protein patterns on the average gels of the testis and ovary, 48 unique spots were distinguished in testis while only two spots were matchless in ovary. These 50 spots were excised from 2D gels and identified by a combination of MALDI-TOF/TOF MS analysis and a database search. Number of peptides matched to the candidate protein sequence ranged from 3 to 33 and the MASCOT score was between 46 and 442 (Table 1). The 50 spots corresponding to 46 different proteins were characterized. Since the genome sequence of the sturgeon is still not complete, none of the identified proteins could be matched to sturgeon proteins. Only 10 spots (20.0%) matched to proteins in other fish species such as Danio rerio, Tetraodon nigroviridis, Paralichthys olivaceus, Oplegnathus fasciatus, and Ictalurus punctatus. As expected, the quality of the matches to fish proteins (average MASCOT score = 192.6) was better than to proteins from other organisms (average MASCOT score = 102.6). Some pro-
Fig. 1. 2-DE maps of the Persian sturgeon (Acipenser persicus) testis (A) and ovary (B) proteins. The first dimension was performed by IEF on pH 3–10 NL IPG strips, the second dimension on 8–12% gradient SDS-PAGE gels and the proteins were visualized by CBB G-250. The unique protein spots are indicated by arrow. For complete information see Table 1.
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Table 1 Proteins identified in Persian sturgeon ovary and testis using MALDI-TOF/TOF MS Spot
Protein name
Ovary proteins Cell division 1 DNA topoisomerase III 2
GA14055-PA
Testis proteins Metabolism and energy production 3 Glutamate dehydrogenase 4
5
PREDICTED: similar to 5-aminoimidazole-4-carboxamide-1beta-d-ribonucleotide transformylase/inosin Glutamine synthetase
6
MGC82998 protein
7 8
Enolase 2 Pyruvate kinase, muscle
9
Hypothetical protein RSP 0472
10 11
Acetyl-coenzyme A acetyltransferase 2 CoA-transferase family III
12
Pyruvate kinase
13 14
15 16
Enolase 2, gamma neuronal Mitochondrial glyceraldehyde-3-phosphate dehydrogenase Alpha enolase-1 Unnamed protein product
17
Isocitrate dehydrogenase (NADP+)
18 19
Enolase 2 Pyrophosphatase (inorganic) 1
Cytoskeletal and cell structure 20 Actin A12 21 22 23
PREDICTED: tubulin, alpha, ubiquitous isoform 5 Actin type 5, cytosolic Beta tubulin
Accession number/species
MASCOT score/number of peptides matched
gi|89902714/Rhodoferax ferrireducens T118 gi|125808971/Drosophila pseudoobscura
58/31
gi|51863477/Cercopithecus sabaeus gi|114583143/Pan troglodytes
278/24
gi|126648112/Algoriphagus sp. PR1 gi|49256179/Xenopus laevis gi|51467931/Danio rerio gi|45382651/Gallus gallus gi|77464041/Rhodobacter sphaeroides 2.4.1 gi|5174389/Homo sapiens
55/19
62/33
110/23
123/10 84/10 70/11 54/14 217/12
gi|70607455/Sulfolobus acidocaldarius DSM 639 gi|113207856/Crassostrea gigas gi|7305027/Mus musculus gi|52547708/Phaeodactylum tricornutum
56/15
gi|11999247/Amia calva gi|47211348/Tetraodon nigroviridis gi|4218518/Piromyces sp. E2 gi|51467931/Danio rerio gi|62955639/Danio rerio
206/11 188/9
gi|113233/Dictyostelium discoideum gi|109096506/Macaca mulatta gi|86169/Gallus gallus gi|10242162/Notothenia coriiceps
117/9
64/11 87/14 89/10
97/9 174/10 70/7
76/14 397/15 204/15
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Table 1(Continued ) Spot
Protein name
Accession number/species
MASCOT score/number of peptides matched
24
PREDICTED: hypothetical protein
70/18
25
Actin
26
TPM1 protein variant
gi|126305451/Monodelphis domestica gi|26522784/Galaxea fascicularis gi|62087662/Homo sapiens gi|82466662/Paralichthys olivaceus gi|422699/Gallus gallus
310/25
gi|78187764/Pelodictyon luteolum DSM 273 gi|45360923/Xenopus tropicalis gi|47218700/Tetraodon nigroviridis gi|71724948/Pseudosciaena crocea gi|443110/Homo sapiens
62/24
Cell defense and chaperones 27 Heat shock protein 60 kDa 28
30
Cell adhesion protein retina cognin—chicken (fragment) Peptidyl-prolyl cis–trans isomerase, PpiC-type Synuclein, beta
31
Unnamed protein product
32
Heat shock protein 60
33
Chain, lysozyme (E.C.3.2.1.17) mutant with Cys 77 replaced by Ala (C77a) Natural killer cell enhancing factor
29
34
gi|52219464/Ictalurus punctatus Translational and transcriptional regulation or DNA/RNA binding related 35 Hypothetical protein CBG14983 gi|39598303/Caenorhabditis briggsae 36 Osmolarity two component response gi|115423843/Bordetella regulator avium 197N 37 DEAD/DEAH box helicase gi|83942539/Sulfitobacter sp. EE-36 38 CDC48 like AAA ATpase gi|66357178/Cryptosporidium parvum Iowa II 39 PREDICTED: aspartyl-tRNA gi|114581044/Pan synthetase isoform 5 troglodytes 40 PREDICTED: similar to YB2 gi|126340161/Monodelphis domestica 41 Cold shock protein gi|53804855/Methylococcus capsulatus str. Bath 42 Response regulator receiver, gi|90418271/Aurantimonas transcriptional sp. SI85-9A1 43 Predicted protein gi|126135368/Pichia stipitis CBS 6054 Transport 44 Quinohemoprotein amine gi|56476668/Azoarcus sp. dehydrogenase, 60 kDa subunit EbN1 45 Putative FoF1 ATP synthase, subunit gi|78696953/Bradyrhizobium B sp. BTAi1
224/13 79/11
60/11
277/4 442/24 60/15 95/5
401/10
48/17 54/13 50/16 53/27 47/16 98/3 46/8 75/6 64/10
53/18 56/12
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Table 1(Continued ) Spot 46
Protein name
Accession number/species
MASCOT score/number of peptides matched
Catalase
gi|52354832/Oplegnathus fasciatus
106/10
gi|47223957/Tetraodon nigroviridis gi|15218697/Arabidopsis thaliana gi|55251215/Danio rerio
56/6
gi|28627817/Streptomyces roseochromogenes subsp. oscitans
64/25
Cell signaling 47 Unnamed protein product 48
Binding
49 Synaptosome-associated protein 25a Cell division 50 Topoisomerase II subunit B
51/22 95/14
tein spots were matched to the proteins of quite distant species such as cold shock protein of Methylococcus capsulatus. These highly conserved proteins might have an important function in the phylogenetically distant species. However, there is still the probability of a random match. Among the testis proteins, three proteins are represented by multiple spots. These proteins are pyruvate kinase and heat shock protein 60 kDa (HSP60), both represented by two spots, and enolase 2, represented by three spots. The multiple spots may result from phosphorylation, glycosylation, or other post-translational modifications (PTM). However, the biological significance of these heterogeneities remains uncertain and further research is necessary to determine the importance of PTM in reproduction biology. To further characterize the identified sturgeon gonad proteins, the proteins were classified with regard to their main known function, which was found in the SWISS-PROT and NCBI databases. Proteins with multiple isoforms were considered one protein. The largest group of sturgeon testis proteins (31.8%) was related to metabolism and energy production. Proteins related to translational and transcriptional regulation or DNA- and RNA-binding proteins, such as aspartyl-tRNA synthetase, account for 20.4% of identified sturgeon testis proteins. A large number of testis proteins were identified as chaperones, heat shock proteins, and oxidative stress defense enzymes (16%). Three protein spots identified as HSP were likely derived from two genes: hsp60 (spots 27 and 32) and hsp70 (spot 31). These proteins have been previously reported in the testis of pig (Huang et al., 2005) and mouse (Allen et al., 1988). HSPs have been demonstrated to be crucial for spermatogenesis (Sarge and Cullen, 1997; Neuer et al., 2000). In addition to their chaperoning function, the expression of these proteins in our study might reflect this role of HSPs. The cell structure protein class (16%) was composed of cytoskeletal proteins such as tubulin and actin. The remaining 15.8% of the identified testis proteins are implicated in diverse functions such as signal transduction (6.8%), transport (6.8%), and cell division (2.2%). Of the 50 unique protein spots of testis and ovary, none were directly linked to a sex-determining gene. Cytogenetic studies have not revealed the presence of heteromorphic sex chromosomes in either sex of any sturgeon species (Fontana and Colombo, 1974; Van Eenennaam et al., 1998a,b), although genetic evidence suggests that sturgeon species may have a genetic sex determination system with female heterogamety (WZ female, ZZ male) (Van Eenennaam et al., 1999; Omoto et
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al., 2005; Flynn et al., 2006). Based on this proved assumption, the female sturgeon should carry sex-specific sequences. Regarding the lack of proteins linked to a sex-determining sequence, our results support the idea that the homologous chromosomes, on which the sex-determining factor is found, are not extensively differentiated in sturgeon species. Searching the genome of sturgeon for sex markers, which failed to find any sex-linked sequences (Hett and Ludwig, 2005; Wuertz et al., 2006; Keyvanshokooh et al., 2007) seems to further strengthen this possibility. Although the above possibility cannot be excluded, it is also probable that the sex gene might function only in the early stages of sex differentiation. The probability of the presence of low abundant sex-linked gene products, which might have been beyond the sensitivity of the methods utilized, should also be considered. The reported proteomics data in the present work provides the basis for subsequent studies focusing on the identification of proteins involved in sex determination and differentiation at different stages of gonadal maturation. Acknowledgements This work was financially supported by Tarbiat Modares University as a Ph.D. research program. The authors would like to thank the Pasteur Institute of Iran for technical support of this research. We also wish to thank Dr. Richard Burchmore (The Sir Henry Wellcome Functional Genomics Facility, University of Glasgow) for mass spectrometry analysis. References Allen, R.L., Obrien, D.A., Eddy, E.M., 1988. A novel Hsp70-like protein (P70) is present in mouse spermatogenic cells. Mol. Cell. Biol. 8, 828–832. Bradford, M.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal. Biochem. 72, 248–254. Doroshov, S.I., Moberg, G.P., VanEenennaam, J.P., 1997. Observations on the reproductive cycle of cultured white sturgeon, Acipenser transmontanus. Environ. Biol. Fish 48, 265–278. Flynn, S.R., Matsuoka, M., Reith, M., Martin-Robichaud, D.J., Benfey, T.J., 2006. Gynogenesis and sex determination in shortnose sturgeon, Acipenser brevirostrum Lesuere. Aquaculture 253, 721–727. Fontana, F., Colombo, G., 1974. Chromosomes of Italian sturgeons. Experientia 30, 739–742. Hett, A.K., Ludwig, A., 2005. SRY-related (Sox) genes in the genorne of European Atlantic sturgeon (Acipenser sturio). Genome 48, 181–186. Huang, S.Y., Lin, J.H., Chen, Y.H., Chuang, C.K., Lin, E.C., Huang, M.C., Sun, H.F.S., Lee, W.C., 2005. A reference map and identification of porcine testis proteins using 2-DE and MS. Proteomics 5, 4205–4212. Keyvanshokooh, S., Vaziri, B., 2007. Proteome analysis of Persian sturgeon (Acipenser persicus) ova. Anim. Reprod. Sci., doi:10.1016/j.anireprosci.2007.10.008. Keyvanshokooh, S., Pourkazemi, M., Kalbassi, M.R., 2007. The RAPD technique failed to identify sex-specific sequences in beluga (Huso huso). J. Appl. Ichthyol. 23, 1–2. Logan, S.H., Johnston, W.E., Doroshov, S.I., 1995. Economics of joint production of sturgeon (Acipenser transmontanus Richardson) and Roe for Caviar. Aquaculture 130, 299–316. Neuer, A., Spandorfer, S.D., Giraldo, P., Dieterle, S., Rosenwaks, Z., Witkin, S.S., 2000. The role of heat shock proteins in reproduction. Hum. Reprod. Update 6, 149–159. Omoto, N., Maebayashi, M., Adachi, S., Arai, K., Yamauchi, K., 2005. Sex ratios of tniploids and gynogenetic diploids induced in the hybrid sturgeon, the bester (Huso huso female × Acipenser ruthenus male). Aquaculture 245, 39–47. Sarge, K.D., Cullen, K.E., 1997. Regulation of hsp expression during rodent spermatogenesis. Cell. Mol. Life Sci. 53, 191–197. Van Eenennaam, A.L., Murray, J.D., Medrano, J.F., 1998a. Mitotic analysis of the North American white sturgeon, Acipenser transmontanus Richardson (Pisces, Acipenseridae), a fish with a very high chromosome number. Genome 41, 266–271.
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Van Eenennaam, A.L., Murray, J.D., Medrano, J.F., 1998b. Synaptonemal complex analysis in spermatocytes of white sturgeon, Acipenser transmontanus Richardson (Pisces, Acipenseridae), a fish with a very high chromosome number. Genome 41, 51–61. Van Eenennaam, A.L., Van Eenennaam, J.P., Medrano, J.F., Doroshov, S.I., 1999. Evidence of female heterogametic genetic sex determination in white sturgeon. J. Hered. 90, 231–233. Wuertz, S., Gaillard, S., Barbisan, F., Carle, S., Congiu, L., Forlani, A., Aubert, J., Kirschbaum, F., Tosi, E., Zane, L., Grillasca, J.P., 2006. Extensive screening of sturgeon genomes by techniques revealed no sex-specific random screening marker. Aquaculture 258, 685–688.
Short communication
Comparative proteomics analysis of male and female Persian sturgeon (Acipenser persicus) gonads Saeed Keyvanshokooh a , Mohammad Reza Kalbassi a,∗∗ , Saman Hosseinkhani b , Behrouz Vaziri c,∗ a
c
Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, 46414-356 Noor, Mazandaran, Iran b Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran Protein Chemistry Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, 69 Pasteur Street, 13164 Tehran, Iran
Received 24 September 2007; received in revised form 21 January 2008; accepted 13 March 2008 Available online 20 March 2008
Abstract In sturgeon aquaculture, the fish are sexed by an invasive surgical examination of the gonads. Development of a non-invasive procedure for sexing fish based on a molecular method is of special interest. In the present study a proteomics approach has been utilized to analyze a differential protein expression between mature male and female Persian sturgeon (Acipenser persicus) gonads. When comparing protein patterns on the 2-DE gels of the testis and ovary, 48 unique spots were distinguished in testis while only two spots were matchless in ovary. The spots were identified by MALDI-TOF/TOF analysis. The identified proteins are involved in metabolism and energy production, cell structure, transcription and translation, cell defense, signal transduction, transport, cell division, and none were directly linked to a sex-determining gene. The provided proteomics data could be considered as a starting base for subsequent studies focusing on the identification of proteins involved in sex determination and differentiation at different stages of gonadal maturation. © 2008 Elsevier B.V. All rights reserved. Keywords: Proteomics; Sex; Ovary; Testis; Acipenser persicus
∗ ∗∗
Corresponding author. Tel.: +98 21 66480780; fax: +98 21 66480780. Corresponding author. Tel.: +98 122 6253101; fax: +98 122 6253499. E-mail addresses: Kalbassi [email protected] (M.R. Kalbassi), [email protected] (B. Vaziri).
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.03.005
362
S. Keyvanshokooh et al. / Animal Reproduction Science 111 (2009) 361–368
1. Introduction In sturgeon aquaculture, where the main purpose is caviar production, a reliable method is needed to separate the fish according to gender. Males are destined to the meat market while females remain in culture for many more years under conditions of optimal growth and development. The availability of monosex populations of caviar-producing females would significantly enhance the economic viability of domestic caviar production systems (Logan et al., 1995). However, it is not possible to distinguish male fish from females by morphological characters at larval, juvenile and even adult stages. Currently, growers wait 3–4 years before fish are sexed by an invasive surgical examination of the gonads (Doroshov et al., 1997) and the development of a non-invasive procedure based on a molecular method is of special interest. Although it is known that all sturgeons are gonochoristic and evidence for female heterogametic genetic sex determination has been presented for the white sturgeon (Acipenser transmontanus) (Van Eenennaam et al., 1999), bester (Huso huso female × Acipenser ruthenus male) (Omoto et al., 2005) and shortnose sturgeon (Acipenser brevirostrum) (Flynn et al., 2006), PCR-based techniques have failed to identify sex-specific genes in sturgeon species (Hett and Ludwig, 2005; Wuertz et al., 2006; Keyvanshokooh et al., 2007) and alternative approaches are required (Wuertz et al., 2006). In this study, we have used a proteomics approach to analyze differential protein expression between mature male and female Persian sturgeon (Acipenser persicus) gonads. The objective of the present work was to search for proteins associated with sex determination. 2. Materials and methods 2.1. Fish sampling and protein extraction Wild fish were captured from the southern Caspian Sea during their spawning run and kept at Shahid Rajaee Sturgeon Propagation Center, Sari, Iran. Sex was determined by inspection of testes and ovaries of necropsied fish. Both gonads from each fish were examined for evidence of hermaphroditism. Sampling was performed from male and female fish in stage 5 of gonadal development. Gonad tissue samples from 15 Persian sturgeon of each sex stored at −70 ◦ C until protein extraction. The gonad tissues of each five fish were mixed before protein extraction and three pools were prepared for each sex. The tissue samples were homogenized in the presence of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 50 mM Tris, 0.2% carrier ampholyte, 1 mM PMSF, 0.25% RNase and 1% DNase). Each sample was maintained for 1 h at room temperature for protein release before centrifugation at 12,000 × g for 10 min at 20 ◦ C. Protein concentration was determined by Bradford assay using BSA standards (Bradford, 1976). 2.2. Two-dimensional gel electrophoresis Isoelectric focusing (IEF) was performed on immobilized pH gradients (IPG; pH 3–10, 17 cm) with Protean IEF Cell (BioRad, USA). A total of 150 g of proteins was used for analytical runs, and 1500 g of proteins was used for preparative runs to a total volume of 300 l of re-hydrating buffer (8 M urea, 4% CHAPS, 50 mM DTT, 0.2% ampholyte and a trace of bromophenol blue). Focusing was performed at 60,000 Vh. Once the IEF was completed, the strips were equilibrated in buffer containing 6 M urea, 50 mM Tris–HCl, pH 8.8, 20% glycerol, 2% SDS and 2% DTT for 20 min, followed by the same buffer without DTT and supplemented with 2.5% idoacetamide for 20 min. Separation of the second dimension was performed according to Keyvanshokooh and
S. Keyvanshokooh et al. / Animal Reproduction Science 111 (2009) 361–368
363
Vaziri (2007). Silver staining protocol was used to visualize protein spots in analytical gels. The preparative gels were stained using a modified colloidal Coomassie G-250 procedure. 2.3. Analysis of 2D gels and MALDI-TOF/TOF MS The gels were scanned using a GS-800 (BioRad). Spot detection and matching were performed using the PG200 software (Nonlinear Dynamics, UK). Each set of gel replicates for both testis and ovary samples were combined into average gels, which represented spots that were reproducibly present on each set of the triplicate gels. The spots that were present only in the protein map of ovary or testis were then selected for further MS analysis. In-gel digestion of samples and MALDI-TOF/TOF analysis was carried out according to Keyvanshokooh and Vaziri (2007). 3. Results and discussion Fig. 1 shows the 2DE map of the sturgeon testis and ovary proteins. When comparing protein patterns on the average gels of the testis and ovary, 48 unique spots were distinguished in testis while only two spots were matchless in ovary. These 50 spots were excised from 2D gels and identified by a combination of MALDI-TOF/TOF MS analysis and a database search. Number of peptides matched to the candidate protein sequence ranged from 3 to 33 and the MASCOT score was between 46 and 442 (Table 1). The 50 spots corresponding to 46 different proteins were characterized. Since the genome sequence of the sturgeon is still not complete, none of the identified proteins could be matched to sturgeon proteins. Only 10 spots (20.0%) matched to proteins in other fish species such as Danio rerio, Tetraodon nigroviridis, Paralichthys olivaceus, Oplegnathus fasciatus, and Ictalurus punctatus. As expected, the quality of the matches to fish proteins (average MASCOT score = 192.6) was better than to proteins from other organisms (average MASCOT score = 102.6). Some pro-
Fig. 1. 2-DE maps of the Persian sturgeon (Acipenser persicus) testis (A) and ovary (B) proteins. The first dimension was performed by IEF on pH 3–10 NL IPG strips, the second dimension on 8–12% gradient SDS-PAGE gels and the proteins were visualized by CBB G-250. The unique protein spots are indicated by arrow. For complete information see Table 1.
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Table 1 Proteins identified in Persian sturgeon ovary and testis using MALDI-TOF/TOF MS Spot
Protein name
Ovary proteins Cell division 1 DNA topoisomerase III 2
GA14055-PA
Testis proteins Metabolism and energy production 3 Glutamate dehydrogenase 4
5
PREDICTED: similar to 5-aminoimidazole-4-carboxamide-1beta-d-ribonucleotide transformylase/inosin Glutamine synthetase
6
MGC82998 protein
7 8
Enolase 2 Pyruvate kinase, muscle
9
Hypothetical protein RSP 0472
10 11
Acetyl-coenzyme A acetyltransferase 2 CoA-transferase family III
12
Pyruvate kinase
13 14
15 16
Enolase 2, gamma neuronal Mitochondrial glyceraldehyde-3-phosphate dehydrogenase Alpha enolase-1 Unnamed protein product
17
Isocitrate dehydrogenase (NADP+)
18 19
Enolase 2 Pyrophosphatase (inorganic) 1
Cytoskeletal and cell structure 20 Actin A12 21 22 23
PREDICTED: tubulin, alpha, ubiquitous isoform 5 Actin type 5, cytosolic Beta tubulin
Accession number/species
MASCOT score/number of peptides matched
gi|89902714/Rhodoferax ferrireducens T118 gi|125808971/Drosophila pseudoobscura
58/31
gi|51863477/Cercopithecus sabaeus gi|114583143/Pan troglodytes
278/24
gi|126648112/Algoriphagus sp. PR1 gi|49256179/Xenopus laevis gi|51467931/Danio rerio gi|45382651/Gallus gallus gi|77464041/Rhodobacter sphaeroides 2.4.1 gi|5174389/Homo sapiens
55/19
62/33
110/23
123/10 84/10 70/11 54/14 217/12
gi|70607455/Sulfolobus acidocaldarius DSM 639 gi|113207856/Crassostrea gigas gi|7305027/Mus musculus gi|52547708/Phaeodactylum tricornutum
56/15
gi|11999247/Amia calva gi|47211348/Tetraodon nigroviridis gi|4218518/Piromyces sp. E2 gi|51467931/Danio rerio gi|62955639/Danio rerio
206/11 188/9
gi|113233/Dictyostelium discoideum gi|109096506/Macaca mulatta gi|86169/Gallus gallus gi|10242162/Notothenia coriiceps
117/9
64/11 87/14 89/10
97/9 174/10 70/7
76/14 397/15 204/15
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Table 1(Continued ) Spot
Protein name
Accession number/species
MASCOT score/number of peptides matched
24
PREDICTED: hypothetical protein
70/18
25
Actin
26
TPM1 protein variant
gi|126305451/Monodelphis domestica gi|26522784/Galaxea fascicularis gi|62087662/Homo sapiens gi|82466662/Paralichthys olivaceus gi|422699/Gallus gallus
310/25
gi|78187764/Pelodictyon luteolum DSM 273 gi|45360923/Xenopus tropicalis gi|47218700/Tetraodon nigroviridis gi|71724948/Pseudosciaena crocea gi|443110/Homo sapiens
62/24
Cell defense and chaperones 27 Heat shock protein 60 kDa 28
30
Cell adhesion protein retina cognin—chicken (fragment) Peptidyl-prolyl cis–trans isomerase, PpiC-type Synuclein, beta
31
Unnamed protein product
32
Heat shock protein 60
33
Chain, lysozyme (E.C.3.2.1.17) mutant with Cys 77 replaced by Ala (C77a) Natural killer cell enhancing factor
29
34
gi|52219464/Ictalurus punctatus Translational and transcriptional regulation or DNA/RNA binding related 35 Hypothetical protein CBG14983 gi|39598303/Caenorhabditis briggsae 36 Osmolarity two component response gi|115423843/Bordetella regulator avium 197N 37 DEAD/DEAH box helicase gi|83942539/Sulfitobacter sp. EE-36 38 CDC48 like AAA ATpase gi|66357178/Cryptosporidium parvum Iowa II 39 PREDICTED: aspartyl-tRNA gi|114581044/Pan synthetase isoform 5 troglodytes 40 PREDICTED: similar to YB2 gi|126340161/Monodelphis domestica 41 Cold shock protein gi|53804855/Methylococcus capsulatus str. Bath 42 Response regulator receiver, gi|90418271/Aurantimonas transcriptional sp. SI85-9A1 43 Predicted protein gi|126135368/Pichia stipitis CBS 6054 Transport 44 Quinohemoprotein amine gi|56476668/Azoarcus sp. dehydrogenase, 60 kDa subunit EbN1 45 Putative FoF1 ATP synthase, subunit gi|78696953/Bradyrhizobium B sp. BTAi1
224/13 79/11
60/11
277/4 442/24 60/15 95/5
401/10
48/17 54/13 50/16 53/27 47/16 98/3 46/8 75/6 64/10
53/18 56/12
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Table 1(Continued ) Spot 46
Protein name
Accession number/species
MASCOT score/number of peptides matched
Catalase
gi|52354832/Oplegnathus fasciatus
106/10
gi|47223957/Tetraodon nigroviridis gi|15218697/Arabidopsis thaliana gi|55251215/Danio rerio
56/6
gi|28627817/Streptomyces roseochromogenes subsp. oscitans
64/25
Cell signaling 47 Unnamed protein product 48
Binding
49 Synaptosome-associated protein 25a Cell division 50 Topoisomerase II subunit B
51/22 95/14
tein spots were matched to the proteins of quite distant species such as cold shock protein of Methylococcus capsulatus. These highly conserved proteins might have an important function in the phylogenetically distant species. However, there is still the probability of a random match. Among the testis proteins, three proteins are represented by multiple spots. These proteins are pyruvate kinase and heat shock protein 60 kDa (HSP60), both represented by two spots, and enolase 2, represented by three spots. The multiple spots may result from phosphorylation, glycosylation, or other post-translational modifications (PTM). However, the biological significance of these heterogeneities remains uncertain and further research is necessary to determine the importance of PTM in reproduction biology. To further characterize the identified sturgeon gonad proteins, the proteins were classified with regard to their main known function, which was found in the SWISS-PROT and NCBI databases. Proteins with multiple isoforms were considered one protein. The largest group of sturgeon testis proteins (31.8%) was related to metabolism and energy production. Proteins related to translational and transcriptional regulation or DNA- and RNA-binding proteins, such as aspartyl-tRNA synthetase, account for 20.4% of identified sturgeon testis proteins. A large number of testis proteins were identified as chaperones, heat shock proteins, and oxidative stress defense enzymes (16%). Three protein spots identified as HSP were likely derived from two genes: hsp60 (spots 27 and 32) and hsp70 (spot 31). These proteins have been previously reported in the testis of pig (Huang et al., 2005) and mouse (Allen et al., 1988). HSPs have been demonstrated to be crucial for spermatogenesis (Sarge and Cullen, 1997; Neuer et al., 2000). In addition to their chaperoning function, the expression of these proteins in our study might reflect this role of HSPs. The cell structure protein class (16%) was composed of cytoskeletal proteins such as tubulin and actin. The remaining 15.8% of the identified testis proteins are implicated in diverse functions such as signal transduction (6.8%), transport (6.8%), and cell division (2.2%). Of the 50 unique protein spots of testis and ovary, none were directly linked to a sex-determining gene. Cytogenetic studies have not revealed the presence of heteromorphic sex chromosomes in either sex of any sturgeon species (Fontana and Colombo, 1974; Van Eenennaam et al., 1998a,b), although genetic evidence suggests that sturgeon species may have a genetic sex determination system with female heterogamety (WZ female, ZZ male) (Van Eenennaam et al., 1999; Omoto et
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al., 2005; Flynn et al., 2006). Based on this proved assumption, the female sturgeon should carry sex-specific sequences. Regarding the lack of proteins linked to a sex-determining sequence, our results support the idea that the homologous chromosomes, on which the sex-determining factor is found, are not extensively differentiated in sturgeon species. Searching the genome of sturgeon for sex markers, which failed to find any sex-linked sequences (Hett and Ludwig, 2005; Wuertz et al., 2006; Keyvanshokooh et al., 2007) seems to further strengthen this possibility. Although the above possibility cannot be excluded, it is also probable that the sex gene might function only in the early stages of sex differentiation. The probability of the presence of low abundant sex-linked gene products, which might have been beyond the sensitivity of the methods utilized, should also be considered. The reported proteomics data in the present work provides the basis for subsequent studies focusing on the identification of proteins involved in sex determination and differentiation at different stages of gonadal maturation. Acknowledgements This work was financially supported by Tarbiat Modares University as a Ph.D. research program. The authors would like to thank the Pasteur Institute of Iran for technical support of this research. We also wish to thank Dr. Richard Burchmore (The Sir Henry Wellcome Functional Genomics Facility, University of Glasgow) for mass spectrometry analysis. References Allen, R.L., Obrien, D.A., Eddy, E.M., 1988. A novel Hsp70-like protein (P70) is present in mouse spermatogenic cells. Mol. Cell. Biol. 8, 828–832. Bradford, M.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal. Biochem. 72, 248–254. Doroshov, S.I., Moberg, G.P., VanEenennaam, J.P., 1997. Observations on the reproductive cycle of cultured white sturgeon, Acipenser transmontanus. Environ. Biol. Fish 48, 265–278. Flynn, S.R., Matsuoka, M., Reith, M., Martin-Robichaud, D.J., Benfey, T.J., 2006. Gynogenesis and sex determination in shortnose sturgeon, Acipenser brevirostrum Lesuere. Aquaculture 253, 721–727. Fontana, F., Colombo, G., 1974. Chromosomes of Italian sturgeons. Experientia 30, 739–742. Hett, A.K., Ludwig, A., 2005. SRY-related (Sox) genes in the genorne of European Atlantic sturgeon (Acipenser sturio). Genome 48, 181–186. Huang, S.Y., Lin, J.H., Chen, Y.H., Chuang, C.K., Lin, E.C., Huang, M.C., Sun, H.F.S., Lee, W.C., 2005. A reference map and identification of porcine testis proteins using 2-DE and MS. Proteomics 5, 4205–4212. Keyvanshokooh, S., Vaziri, B., 2007. Proteome analysis of Persian sturgeon (Acipenser persicus) ova. Anim. Reprod. Sci., doi:10.1016/j.anireprosci.2007.10.008. Keyvanshokooh, S., Pourkazemi, M., Kalbassi, M.R., 2007. The RAPD technique failed to identify sex-specific sequences in beluga (Huso huso). J. Appl. Ichthyol. 23, 1–2. Logan, S.H., Johnston, W.E., Doroshov, S.I., 1995. Economics of joint production of sturgeon (Acipenser transmontanus Richardson) and Roe for Caviar. Aquaculture 130, 299–316. Neuer, A., Spandorfer, S.D., Giraldo, P., Dieterle, S., Rosenwaks, Z., Witkin, S.S., 2000. The role of heat shock proteins in reproduction. Hum. Reprod. Update 6, 149–159. Omoto, N., Maebayashi, M., Adachi, S., Arai, K., Yamauchi, K., 2005. Sex ratios of tniploids and gynogenetic diploids induced in the hybrid sturgeon, the bester (Huso huso female × Acipenser ruthenus male). Aquaculture 245, 39–47. Sarge, K.D., Cullen, K.E., 1997. Regulation of hsp expression during rodent spermatogenesis. Cell. Mol. Life Sci. 53, 191–197. Van Eenennaam, A.L., Murray, J.D., Medrano, J.F., 1998a. Mitotic analysis of the North American white sturgeon, Acipenser transmontanus Richardson (Pisces, Acipenseridae), a fish with a very high chromosome number. Genome 41, 266–271.
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Van Eenennaam, A.L., Murray, J.D., Medrano, J.F., 1998b. Synaptonemal complex analysis in spermatocytes of white sturgeon, Acipenser transmontanus Richardson (Pisces, Acipenseridae), a fish with a very high chromosome number. Genome 41, 51–61. Van Eenennaam, A.L., Van Eenennaam, J.P., Medrano, J.F., Doroshov, S.I., 1999. Evidence of female heterogametic genetic sex determination in white sturgeon. J. Hered. 90, 231–233. Wuertz, S., Gaillard, S., Barbisan, F., Carle, S., Congiu, L., Forlani, A., Aubert, J., Kirschbaum, F., Tosi, E., Zane, L., Grillasca, J.P., 2006. Extensive screening of sturgeon genomes by techniques revealed no sex-specific random screening marker. Aquaculture 258, 685–688.