Proteomics 2015, 15, 4175–4179
4175
DOI 10.1002/pmic.201500066
DATASET BRIEF
Sperm proteome of Mytilus galloprovincialis: Insights into the evolution of fertilization proteins in marine mussels Yanjie Zhang1 , Huawei Mu1 , Stanley C. K. Lau2 , Zhifeng Zhang3∗ and Jian-Wen Qiu1 1
Department of Biology, Hong Kong Baptist University, Hong Kong, P. R. China Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, P. R. China 3 Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, P. R. China 2
Cataloging the sperm proteome of an animal can improve our understanding of its sperm-egg interaction and speciation, but such data are available for only a few free-spawning invertebrates. This study aimed to identify the sperm proteome of Mytilus galloprovincialis, a free-spawning marine mussel. We integrated public transcriptome datasets by de novo assembly, and applied SDS-PAGE coupled LC-MS/MS analysis to profile the sperm proteome, resulting in the identification of 550 proteins. Comparing the homologous sperm protein coding genes between M. galloprovincialis and its closely related species M. edulis revealed that fertilization proteins have the highest mean nonsynonymous substitution rate (Ka/Ks = 0.62) among 11 functional groups, consistent with previous reports of positive selection of several fertilization proteins in Mytilus. Moreover, 78 sperm proteins in different functional groups have Ka/Ks values > 0.5, indicating the presence of many candidate sperm proteins for further analysis of rapid interspecific divergence. The MS data are available in ProteomeXchange with the identifier PXD001665.
Received: February 10, 2015 Revised: April 15, 2015 Accepted: June 2, 2015
Keywords: Animal proteomics / Mussel / Mytilus galloprovincialis / Sperm / Sperm protein
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Sperm are equipped with many proteins that play various roles in the fertilization process, such as sperm locomotion, sperm-egg recognition, acrosomal reaction, and sperm penetration into the egg. Free-spawning marine invertebrates have been used as an excellent model to study how sperm proteins have contributed to reproductive isolation and speciation [1]. Several sperm acrosomal proteins (i.e. bindin of sea urchins [2], lysin of abalone [3], and M7 and M3 lysins of mussels [4, 5]) have been well studied with respect to their roles in sperm-egg recognition and fusion. Rapid evolution in these sperm proteins has been reported, and such rapid evolution
Correspondence: Dr. Jian-Wen Qiu, Department of Biology, Hong Kong Baptist University, 224 Waterloo Road, Kowloon, Hong Kong, P. R. China E-mail: [email protected] Fax: +852-34115995
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is considered an important evolutionary force of reproductive isolation [3, 6–8]. The blue mussels Mytilus spp. (Mollusca: Bivalvia: Mytilidae) are common marine bivalves widely distributed from the polar to the tropical regions. Several species of Mytilus, especially M. edulis, M. galloprovincialis, and M. trossulus, are important in aquaculture. Their distribution ranges overlap due to postglacial migration and human-mediated introduction, and they have established hybrid populations in their contact zones [9, 10], providing a system to study the role of fertilization in speciation. A previous proteomic study showed that the hybrids of M. edulis and M. galloprovincialis expressed more variable proteins than each of the two ∗ Additional corresponding author: Dr. Zhifeng Zhang, E-mail: [email protected] Colour Online: See the article online to view Fig. 1 in colour.
www.proteomics-journal.com
4176
Y. Zhang et al.
species, which might have contributed to their postzygotic isolation [11]. Mytilus species have an unusual system of mitochondrial DNA inheritance [12]. Their offspring inherit both maternal and paternal mtDNA, a phenomenon called doubly uniparental inheritance, providing an interesting model to study the heredity of the mitochondrial genome [13]. The rapid development of proteomic techniques has allowed scientists to identify large numbers of sperm proteins in two marine mollusks–975 in the abalone Haliotis rufescens [14], and 77 in the mussel M. edulis [15]. The present study aimed to the report sperm proteome of M. galloprovincialis using a 1D SDS-PAGE coupled LC-MS/MS approach, and compare sperm proteins between M. edulis and M. galloprovincialis to reveal the rates of sequence divergence in different protein functional groups. Mature specimens of M. galloprovincialis, originally collected from the aquaculture rafts off the western coast of Qingdao, were bought from a wet market in Qingdao. The adductor muscle of four individuals was sampled for DNA extraction after spawning. The COI, 28S, Glu-5’, and Glu3’ gene segments were amplified by PCR to confirm their species identity (Supporting Information Fig. 1). The mussels spawned soon after submersion into filtered seawater. Sperm released into the seawater were collected by pipetting. Sperm samples from three males, concentrated by centrifugation for 2 min at 8000 g, were treated as three biological replicates to account for potential intraspecific difference in sperm protein profile. Urea solution (8 M) with 50 mM Tris (pH = 7.5) was used to resuspend the sperm pellets and the samples were transported to Hong Kong Baptist University on dry ice, and stored at –80⬚C until use. Proteome analysis was conducted using an established method [16]. In brief, protein quantity was analyzed using a RC DC Protein Assay Kit (BIO-RAD, CA, USA) after sonication of the samples for 5 min, and centrifugation to remove the insoluble pellet. For each replicate, 100 g total protein extract was used in SDS-PAGE. The gel was then destained with 1% acetic acid, and cut into eight fractions with proteins of different molecular mass (Supporting Information Fig. 2). Following in-gel trypsin digestion and desalting using Sep-Pak C18 cartridges, the fractions were dried using a SpeedVac, and each fraction was reconstituted in 10 L of 0.1% formic acid and analyzed using a LTQ Velos Ion Trap Mass Spectrometer (Thermo Scientific, MA, USA) with a 90-min LC gradient. The MS data were searched against a M. galloprovincialis transcriptome database, which was de novo assembled using CLC Genomics Workbench version 7.5 with 30 public M. galloprovincialis transcriptome datasets downloaded from the NCBI SRA Database (Supporting Information Table 1). The contigs obtained were translated into protein sequences using the GETORF online server (http://imed.med.ucm.es/ cgi-bin/emboss.pl?_action=input&_app=getorf) with 200 bp as the minimum nucleotide number of ORF. Reverse concatenated sequences were added to form a decoy database. The MS data generated for each fraction of the 1DE gel were submitted to Mascot version 2.2 (Matrix Sciences Ltd., London, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 4175–4179
UK) with the following settings: 0.8 Da for peptide tolerance and MS/MS tolerance; fixed modification: carbamidomethyl (cysteine); variable modification: oxidation (M) and deamidation (NQ). Up to one missed trypsin cleavage was allowed. Protein identification was considered valid only when the following criteria were met: ions score for peptide assignment > 95% confidence level (individual ions score > 41); significance threshold p < 0.05; peptide length ࣙ 8; false discovery rate < 1%. Proteins identified by only one matched spectrum/peptide were also removed. All identified proteins from three replicates were pooled for annotation and classification by BLST2GO combined with WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). De novo assembly of the M. edulis transcriptome was also conducted, using 50 public M. edulis datasets (Supporting Information Table 2). Ka/Ks ratios were calculated using all orthologous sperm proteins of M. galloprovincialis and M. edulis. The bidirectional best hit method [17] was applied using BLASTP to identify orthologous genes with the threshold of 1e × 10−5 . ParaAT1.0 [18] was used to align the DNA sequence of each pair of orthologous genes. The output files were used to estimate Ka/Ks ratios using the MLWL method implemented in KaKs Calculator [19]. Sequence divergent rate of each specific functional category was compared with that of all homologous sperm genes using a two-tailed unpaired Student’s t-test [20]. A total of 550 proteins were identified from the M. galloprovincialis sperm, with 384, 398, and 382 from the three biological replicates, respectively (Supporting Information Fig. 3 and Table 3). The functions of the identified proteins were described based on the GO assignment, supplemented with manual annotation using the UniProt database. Among the 550 identified proteins, 108 (20%) were uncharacterized (Fig. 1). “Energy metabolism” was the largest group (30%), which includes proteins involved in electron transport chain, lipid metabolism, tricarboxylic acid cycle, glycolysis and gluconeogenesis, and energy homeostasis. Similar results have been reported in previous studies of sperm proteome [15, 21], which are consistent with the high energy demand required for sperm motility. “Protein synthesis and degradation” is the second largest group (14%). Among this group, proteins with degradation function dominated (34%), consistent with the presence of an effective ubiquitinproteasome system in sperm, which is required for the sperm to create a hole in the egg vitelline coat and the elimination of defective sperm [22]. The ubiquitin-proteasome system in mussel sperm has been suggested to be involved in tagging sperm mitochondria for destruction, thus may play a role in doubly uniparental inheritance [13]. “Spermatogenesis and sperm motility” includes 10% of the sperm proteins, among them a large proportion (81%) is related to sperm motility. “Cytoskeleton” includes 12% of the sperm proteins, which are structural proteins universal to cells. Proteins classified into “signal pathway” and “ion channel and transport” groups, which are required for sperm capacitation and motility [23], account for 5% and 3% of the identified proteins, respectively. www.proteomics-journal.com
4177
Proteomics 2015, 15, 4175–4179
Figure 1. Functional distribution of proteins identified from the sperm of M. galloprovincialis.
The “fertilization” group, although small (3%), contains several proteins that have been shown to play various roles in fertilization. Specifically, surface protein Sp17, which may mediate sperm-egg recognition, has also been detected in the M. edulis sperm proteome [15]. M7 and M3 lysins have been well-studied using the individual gene/protein approach for their roles in sperm-egg recognition and dissolution of the egg vitelline coat in Mytilus [4, 5]. In addition, bindin f-lectin has been considered as the critical motif of the oyster sperm bindin [24]. Sperm-associated antigen protein 6, reported in the M. edulis sperm proteome [15] and highly expressed at the mature oyster testis [25], has been shown to be critical in the sperm-egg interaction of many animals [26]. A total of 312 orthologous sperm genes out of the 550 identified by MS were found between M. galloprovincialis and M. edulis. These genes (nearly 212 kb in total, Supporting
Information Table 3) have a mean sequence coverage (i.e. ratio of the nucleotide sequence length used for Ka/Ks calculation over the protein coding region length) of 84%, slightly higher than that (82%) of fruit fly sperm protein coding genes [19]. The mean Ka/Ks values differ substantially among the different functional groups (Fig. 2). The “cytoskeleton” group has the lowest mean Ka/Ks value (0.18), indicating purifying selection in many genes in this group. The “fertilization” group containing nine proteins has the highest mean Ka/Ks value (0.62), which warrants more detailed analysis of positive selection. Among the 312 orthologues, 78 have a Ka/Ks value > 0.5 and are thus candidates for more detailed analysis of positive selection [27] (Supporting Information Fig. 4). This list includes M7 lysin (Ka/Ks = 0.70) and M3 lysin (Ka/ Ks = 0.56) that are known to have undergone positive selection, as well as proteins in many different functional groups.
Figure 2. Substitution rate analysis of orthologous genes between M. galloprovincialis and M. edulis. Genes are grouped by functions. Each bar represents the mean ± SE of all genes in a particular functional group. Number to the right of each bar indicates the number of genes in each group. Significant differences (p < 0.05) are indicated with an asterisk.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com
4178
Y. Zhang et al.
Nine sperm proteins show clear signature of adaptive evolution (Ka/Ks >1), including two involved in fertilization, one in sperm motility, three in energy metabolism, one in protein degradation, one carrier protein, and one uncharacterized protein. Although these orthologues have allowed us to compare the interspecific divergence of sperm proteins between the two species of Mytilus, it should be pointed out that there are limitations in orthologue comparison and Ka/Ks calculation in the nonmodel species: First, although we have integrated many transcriptome datasets, the proteome databases were still incomplete because they were translated from the assembled transcripts that covered only part of the genome, and many of the transcripts have not been annotated; Second, the copy number of a gene might not be identical between the two proteome databases, and without a sequenced genome, it was not possible to determine whether the ORF region was complete for every protein, although our results for most of the sperm genes should be reasonably robust as they have a full or nearly full ORF based on the predictions by TargetIdentifier [28]; Third, the Ka/Ks calculations do not have strong statistical power when only pairwise comparisons were made [27]. However, although multiple alignments are desirable for site model analysis, such sequence data are not available yet. In conclusion, we have identified 550 proteins from the M. galloprovincialis sperm, most of which are reported for the first time. It is seven times more than that of the number of M. edulis sperm proteins, identified using 2DE coupled nESIMS analysis [15]. The M. galloprovincialis sperm proteome can be used to enhance our understanding of fundamental processes of fertilization, heredity, reproductive isolation, and speciation in Mytilus. This work was supported by Hong Kong Baptist University (Grant number FRG2/13-14/009). The LC-MS/MS analysis was conducted at the Biosciences Central Research Facility, The Hong Kong University of Science & Technology. The authors have declared no conflict of interest.
References [1] Kosman, E. T., Levitan, D. R., Sperm competition and the evolution of gametic compatibility in externally fertilizing taxa. Mol. Hum. Reprod. 2014, 20, 1190–1197. [2] Vacquier, V. D., Moy, G. W., Isolation of bindin: the protein responsible for adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA 1977, 74, 2456–2460. [3] Swanson, W. J., Aquadro, C. F., Vacquier, V. D., Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg’s receptor for lysin (VERL) and positive Darwinian selection of sperm lysin. Mol. Biol. Evol. 2001, 18, 376–383. [4] Lima, T. G., McCartney, M. A., Adaptive evolution of M3 lysin—a candidate gamete recognition protein in the Mytilus edulis species complex. Mol. Biol. Evol. 2013, 30, 2688–2698.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 4175–4179 [5] Springer, S. A., Crespi, B. J., Adaptive gamete-recognition divergence in a hybridizing Mytilus population. Evolution 2007, 61, 772–783. [6] Pujolar, J. M., Pogson, G. H., Positive Darwinian selection in gamete recognition proteins of Strongylocentrotus sea urchins. Mol. Ecol. 2011, 20, 4968–4982. [7] Moy, G. W., Springer, S. A., Adams, S. L., Swanson, W. J. et al., Extraordinary intraspecific diversity in oyster sperm bindin. Proc. Natl. Acad. Sci. 2008, 105, 1993–1998. [8] Riginos, C., McDonald, J. H., Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus. Mol. Biol. Evol. 2003, 20, 200–207. [9] Koehn, R. K., The genetics and taxonomy of species in the genus Mytilus. Aquaculture 1991, 94, 125–145. [10] Fra¨ısse, C., Roux, C., Welch, J. J., Bierne, N., Gene flow in a mosaic hybrid zone: is local introgression adaptive? Genetics 2014, 197, 939–51. [11] Diz, A. P., Skibinski, D. O., Evolution of 2-DE protein patterns in a mussel hybrid zone. Proteomics 2007, 7, 2111–2120. [12] Skibinski, D. O., Gallagher, C., Beynon, C. M., Sex-limited mitochondrial DNA transmission in the marine mussel Mytilus edulis. Genetics 1994, 138, 801–809. [13] Zouros, E., Biparental inheritance through uniparental transmission: the doubly uniparental inheritance (DUI) of mitochondrial DNA. Evol. Biol. 2013, 40, 1–31. [14] Palmer, M. R., McDowall, M. H., Stewart, L., Quaddi, A. et al., Mass spectrometry and next-generation sequencing reveal an abundant and rapidly evolving abalone sperm protein. Mol. Reprod. Dev. 2013, 80, 460–465. [15] Diz, A. P., Dudley, E., Skibinski, D. O., Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis. Proteomics 2012, 12, 1949–1956. [16] Sun, J., Zhang, H., Wang, H., Heras, H. et al., First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. J. Proteome Res. 2012, 11, 4240– 4248. [17] Overbeek, R., Fonstein, M., D’Souza, M., Pusch, G. D. et al., The use of gene clusters to infer functional coupling. Proc. Natl. Acad. Sci. 1999, 96, 2896–2901. [18] Zhang, Z., Xiao, J., Wu, J., Zhang, H. et al., ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem. Bioph. Res. Co. 2012, 419, 779–781. [19] Zhang, Z., Li, J., Zhao, X. Q., Wong, G. K. et al., KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics 2006, 4, 259–263. [20] Dorus, S., Busby, S. A., Gerike, U., Shabanowitz, J. et al., Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. 2006, 38, 1440– 1445. ¨ [21] Nynca, J., Arnold, G. J., Frohlich, T., Otte, K., Ciereszko, A., Proteomic identification of rainbow trout sperm proteins. Proteomics 2014, 14, 1569–1573. [22] Sutovsky, P., Sperm proteasome and fertilization. Reproduction 2011, 142, 1–14.
www.proteomics-journal.com
Proteomics 2015, 15, 4175–4179 ´ C., Felix, R., Nishigaki, T., Trevino, ˜ C. L., [23] Darszon, A., Beltran, Ion transport in sperm signaling. Dev. Biol. 2001, 240, 1–14. [24] Wu, Q., Li, L., Zhang, G., Crassostrea angulata bindin gene and the divergence of fucose-binding lectin repeats among three species of Crassostrea. Mar. Biotechnol. 2011, 13, 327– 335. [25] Dheilly, N. M., Lelong, C., Huvet, A., Kellner, K. et al., Gametogenesis in the Pacific Oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes. PLoS One 2012, 7, e36353.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4179 [26] Suri, A., Family of sperm associated antigens: relevance in sperm-egg interaction and immunocontraception. Soc. Reprod. Fertil. Suppl. 2006, 63, 433–443. [27] Clark, N. L., Swanson, W. J., Pervasive adaptive evolution in primate seminal proteins. PLoS Genet. 2005, 1, e35. [28] Min, X. J., Butler, G., Storms, R., Tsang, A., TargetIdentifier: a webserver for identifying full-length cDNAs from EST sequences. Nucleic Acids Res. 2005, 33, 669– 672.
www.proteomics-journal.com
4175
DOI 10.1002/pmic.201500066
DATASET BRIEF
Sperm proteome of Mytilus galloprovincialis: Insights into the evolution of fertilization proteins in marine mussels Yanjie Zhang1 , Huawei Mu1 , Stanley C. K. Lau2 , Zhifeng Zhang3∗ and Jian-Wen Qiu1 1
Department of Biology, Hong Kong Baptist University, Hong Kong, P. R. China Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, P. R. China 3 Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, P. R. China 2
Cataloging the sperm proteome of an animal can improve our understanding of its sperm-egg interaction and speciation, but such data are available for only a few free-spawning invertebrates. This study aimed to identify the sperm proteome of Mytilus galloprovincialis, a free-spawning marine mussel. We integrated public transcriptome datasets by de novo assembly, and applied SDS-PAGE coupled LC-MS/MS analysis to profile the sperm proteome, resulting in the identification of 550 proteins. Comparing the homologous sperm protein coding genes between M. galloprovincialis and its closely related species M. edulis revealed that fertilization proteins have the highest mean nonsynonymous substitution rate (Ka/Ks = 0.62) among 11 functional groups, consistent with previous reports of positive selection of several fertilization proteins in Mytilus. Moreover, 78 sperm proteins in different functional groups have Ka/Ks values > 0.5, indicating the presence of many candidate sperm proteins for further analysis of rapid interspecific divergence. The MS data are available in ProteomeXchange with the identifier PXD001665.
Received: February 10, 2015 Revised: April 15, 2015 Accepted: June 2, 2015
Keywords: Animal proteomics / Mussel / Mytilus galloprovincialis / Sperm / Sperm protein
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Sperm are equipped with many proteins that play various roles in the fertilization process, such as sperm locomotion, sperm-egg recognition, acrosomal reaction, and sperm penetration into the egg. Free-spawning marine invertebrates have been used as an excellent model to study how sperm proteins have contributed to reproductive isolation and speciation [1]. Several sperm acrosomal proteins (i.e. bindin of sea urchins [2], lysin of abalone [3], and M7 and M3 lysins of mussels [4, 5]) have been well studied with respect to their roles in sperm-egg recognition and fusion. Rapid evolution in these sperm proteins has been reported, and such rapid evolution
Correspondence: Dr. Jian-Wen Qiu, Department of Biology, Hong Kong Baptist University, 224 Waterloo Road, Kowloon, Hong Kong, P. R. China E-mail: [email protected] Fax: +852-34115995
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is considered an important evolutionary force of reproductive isolation [3, 6–8]. The blue mussels Mytilus spp. (Mollusca: Bivalvia: Mytilidae) are common marine bivalves widely distributed from the polar to the tropical regions. Several species of Mytilus, especially M. edulis, M. galloprovincialis, and M. trossulus, are important in aquaculture. Their distribution ranges overlap due to postglacial migration and human-mediated introduction, and they have established hybrid populations in their contact zones [9, 10], providing a system to study the role of fertilization in speciation. A previous proteomic study showed that the hybrids of M. edulis and M. galloprovincialis expressed more variable proteins than each of the two ∗ Additional corresponding author: Dr. Zhifeng Zhang, E-mail: [email protected] Colour Online: See the article online to view Fig. 1 in colour.
www.proteomics-journal.com
4176
Y. Zhang et al.
species, which might have contributed to their postzygotic isolation [11]. Mytilus species have an unusual system of mitochondrial DNA inheritance [12]. Their offspring inherit both maternal and paternal mtDNA, a phenomenon called doubly uniparental inheritance, providing an interesting model to study the heredity of the mitochondrial genome [13]. The rapid development of proteomic techniques has allowed scientists to identify large numbers of sperm proteins in two marine mollusks–975 in the abalone Haliotis rufescens [14], and 77 in the mussel M. edulis [15]. The present study aimed to the report sperm proteome of M. galloprovincialis using a 1D SDS-PAGE coupled LC-MS/MS approach, and compare sperm proteins between M. edulis and M. galloprovincialis to reveal the rates of sequence divergence in different protein functional groups. Mature specimens of M. galloprovincialis, originally collected from the aquaculture rafts off the western coast of Qingdao, were bought from a wet market in Qingdao. The adductor muscle of four individuals was sampled for DNA extraction after spawning. The COI, 28S, Glu-5’, and Glu3’ gene segments were amplified by PCR to confirm their species identity (Supporting Information Fig. 1). The mussels spawned soon after submersion into filtered seawater. Sperm released into the seawater were collected by pipetting. Sperm samples from three males, concentrated by centrifugation for 2 min at 8000 g, were treated as three biological replicates to account for potential intraspecific difference in sperm protein profile. Urea solution (8 M) with 50 mM Tris (pH = 7.5) was used to resuspend the sperm pellets and the samples were transported to Hong Kong Baptist University on dry ice, and stored at –80⬚C until use. Proteome analysis was conducted using an established method [16]. In brief, protein quantity was analyzed using a RC DC Protein Assay Kit (BIO-RAD, CA, USA) after sonication of the samples for 5 min, and centrifugation to remove the insoluble pellet. For each replicate, 100 g total protein extract was used in SDS-PAGE. The gel was then destained with 1% acetic acid, and cut into eight fractions with proteins of different molecular mass (Supporting Information Fig. 2). Following in-gel trypsin digestion and desalting using Sep-Pak C18 cartridges, the fractions were dried using a SpeedVac, and each fraction was reconstituted in 10 L of 0.1% formic acid and analyzed using a LTQ Velos Ion Trap Mass Spectrometer (Thermo Scientific, MA, USA) with a 90-min LC gradient. The MS data were searched against a M. galloprovincialis transcriptome database, which was de novo assembled using CLC Genomics Workbench version 7.5 with 30 public M. galloprovincialis transcriptome datasets downloaded from the NCBI SRA Database (Supporting Information Table 1). The contigs obtained were translated into protein sequences using the GETORF online server (http://imed.med.ucm.es/ cgi-bin/emboss.pl?_action=input&_app=getorf) with 200 bp as the minimum nucleotide number of ORF. Reverse concatenated sequences were added to form a decoy database. The MS data generated for each fraction of the 1DE gel were submitted to Mascot version 2.2 (Matrix Sciences Ltd., London, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 4175–4179
UK) with the following settings: 0.8 Da for peptide tolerance and MS/MS tolerance; fixed modification: carbamidomethyl (cysteine); variable modification: oxidation (M) and deamidation (NQ). Up to one missed trypsin cleavage was allowed. Protein identification was considered valid only when the following criteria were met: ions score for peptide assignment > 95% confidence level (individual ions score > 41); significance threshold p < 0.05; peptide length ࣙ 8; false discovery rate < 1%. Proteins identified by only one matched spectrum/peptide were also removed. All identified proteins from three replicates were pooled for annotation and classification by BLST2GO combined with WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). De novo assembly of the M. edulis transcriptome was also conducted, using 50 public M. edulis datasets (Supporting Information Table 2). Ka/Ks ratios were calculated using all orthologous sperm proteins of M. galloprovincialis and M. edulis. The bidirectional best hit method [17] was applied using BLASTP to identify orthologous genes with the threshold of 1e × 10−5 . ParaAT1.0 [18] was used to align the DNA sequence of each pair of orthologous genes. The output files were used to estimate Ka/Ks ratios using the MLWL method implemented in KaKs Calculator [19]. Sequence divergent rate of each specific functional category was compared with that of all homologous sperm genes using a two-tailed unpaired Student’s t-test [20]. A total of 550 proteins were identified from the M. galloprovincialis sperm, with 384, 398, and 382 from the three biological replicates, respectively (Supporting Information Fig. 3 and Table 3). The functions of the identified proteins were described based on the GO assignment, supplemented with manual annotation using the UniProt database. Among the 550 identified proteins, 108 (20%) were uncharacterized (Fig. 1). “Energy metabolism” was the largest group (30%), which includes proteins involved in electron transport chain, lipid metabolism, tricarboxylic acid cycle, glycolysis and gluconeogenesis, and energy homeostasis. Similar results have been reported in previous studies of sperm proteome [15, 21], which are consistent with the high energy demand required for sperm motility. “Protein synthesis and degradation” is the second largest group (14%). Among this group, proteins with degradation function dominated (34%), consistent with the presence of an effective ubiquitinproteasome system in sperm, which is required for the sperm to create a hole in the egg vitelline coat and the elimination of defective sperm [22]. The ubiquitin-proteasome system in mussel sperm has been suggested to be involved in tagging sperm mitochondria for destruction, thus may play a role in doubly uniparental inheritance [13]. “Spermatogenesis and sperm motility” includes 10% of the sperm proteins, among them a large proportion (81%) is related to sperm motility. “Cytoskeleton” includes 12% of the sperm proteins, which are structural proteins universal to cells. Proteins classified into “signal pathway” and “ion channel and transport” groups, which are required for sperm capacitation and motility [23], account for 5% and 3% of the identified proteins, respectively. www.proteomics-journal.com
4177
Proteomics 2015, 15, 4175–4179
Figure 1. Functional distribution of proteins identified from the sperm of M. galloprovincialis.
The “fertilization” group, although small (3%), contains several proteins that have been shown to play various roles in fertilization. Specifically, surface protein Sp17, which may mediate sperm-egg recognition, has also been detected in the M. edulis sperm proteome [15]. M7 and M3 lysins have been well-studied using the individual gene/protein approach for their roles in sperm-egg recognition and dissolution of the egg vitelline coat in Mytilus [4, 5]. In addition, bindin f-lectin has been considered as the critical motif of the oyster sperm bindin [24]. Sperm-associated antigen protein 6, reported in the M. edulis sperm proteome [15] and highly expressed at the mature oyster testis [25], has been shown to be critical in the sperm-egg interaction of many animals [26]. A total of 312 orthologous sperm genes out of the 550 identified by MS were found between M. galloprovincialis and M. edulis. These genes (nearly 212 kb in total, Supporting
Information Table 3) have a mean sequence coverage (i.e. ratio of the nucleotide sequence length used for Ka/Ks calculation over the protein coding region length) of 84%, slightly higher than that (82%) of fruit fly sperm protein coding genes [19]. The mean Ka/Ks values differ substantially among the different functional groups (Fig. 2). The “cytoskeleton” group has the lowest mean Ka/Ks value (0.18), indicating purifying selection in many genes in this group. The “fertilization” group containing nine proteins has the highest mean Ka/Ks value (0.62), which warrants more detailed analysis of positive selection. Among the 312 orthologues, 78 have a Ka/Ks value > 0.5 and are thus candidates for more detailed analysis of positive selection [27] (Supporting Information Fig. 4). This list includes M7 lysin (Ka/Ks = 0.70) and M3 lysin (Ka/ Ks = 0.56) that are known to have undergone positive selection, as well as proteins in many different functional groups.
Figure 2. Substitution rate analysis of orthologous genes between M. galloprovincialis and M. edulis. Genes are grouped by functions. Each bar represents the mean ± SE of all genes in a particular functional group. Number to the right of each bar indicates the number of genes in each group. Significant differences (p < 0.05) are indicated with an asterisk.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com
4178
Y. Zhang et al.
Nine sperm proteins show clear signature of adaptive evolution (Ka/Ks >1), including two involved in fertilization, one in sperm motility, three in energy metabolism, one in protein degradation, one carrier protein, and one uncharacterized protein. Although these orthologues have allowed us to compare the interspecific divergence of sperm proteins between the two species of Mytilus, it should be pointed out that there are limitations in orthologue comparison and Ka/Ks calculation in the nonmodel species: First, although we have integrated many transcriptome datasets, the proteome databases were still incomplete because they were translated from the assembled transcripts that covered only part of the genome, and many of the transcripts have not been annotated; Second, the copy number of a gene might not be identical between the two proteome databases, and without a sequenced genome, it was not possible to determine whether the ORF region was complete for every protein, although our results for most of the sperm genes should be reasonably robust as they have a full or nearly full ORF based on the predictions by TargetIdentifier [28]; Third, the Ka/Ks calculations do not have strong statistical power when only pairwise comparisons were made [27]. However, although multiple alignments are desirable for site model analysis, such sequence data are not available yet. In conclusion, we have identified 550 proteins from the M. galloprovincialis sperm, most of which are reported for the first time. It is seven times more than that of the number of M. edulis sperm proteins, identified using 2DE coupled nESIMS analysis [15]. The M. galloprovincialis sperm proteome can be used to enhance our understanding of fundamental processes of fertilization, heredity, reproductive isolation, and speciation in Mytilus. This work was supported by Hong Kong Baptist University (Grant number FRG2/13-14/009). The LC-MS/MS analysis was conducted at the Biosciences Central Research Facility, The Hong Kong University of Science & Technology. The authors have declared no conflict of interest.
References [1] Kosman, E. T., Levitan, D. R., Sperm competition and the evolution of gametic compatibility in externally fertilizing taxa. Mol. Hum. Reprod. 2014, 20, 1190–1197. [2] Vacquier, V. D., Moy, G. W., Isolation of bindin: the protein responsible for adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA 1977, 74, 2456–2460. [3] Swanson, W. J., Aquadro, C. F., Vacquier, V. D., Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg’s receptor for lysin (VERL) and positive Darwinian selection of sperm lysin. Mol. Biol. Evol. 2001, 18, 376–383. [4] Lima, T. G., McCartney, M. A., Adaptive evolution of M3 lysin—a candidate gamete recognition protein in the Mytilus edulis species complex. Mol. Biol. Evol. 2013, 30, 2688–2698.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 4175–4179 [5] Springer, S. A., Crespi, B. J., Adaptive gamete-recognition divergence in a hybridizing Mytilus population. Evolution 2007, 61, 772–783. [6] Pujolar, J. M., Pogson, G. H., Positive Darwinian selection in gamete recognition proteins of Strongylocentrotus sea urchins. Mol. Ecol. 2011, 20, 4968–4982. [7] Moy, G. W., Springer, S. A., Adams, S. L., Swanson, W. J. et al., Extraordinary intraspecific diversity in oyster sperm bindin. Proc. Natl. Acad. Sci. 2008, 105, 1993–1998. [8] Riginos, C., McDonald, J. H., Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus. Mol. Biol. Evol. 2003, 20, 200–207. [9] Koehn, R. K., The genetics and taxonomy of species in the genus Mytilus. Aquaculture 1991, 94, 125–145. [10] Fra¨ısse, C., Roux, C., Welch, J. J., Bierne, N., Gene flow in a mosaic hybrid zone: is local introgression adaptive? Genetics 2014, 197, 939–51. [11] Diz, A. P., Skibinski, D. O., Evolution of 2-DE protein patterns in a mussel hybrid zone. Proteomics 2007, 7, 2111–2120. [12] Skibinski, D. O., Gallagher, C., Beynon, C. M., Sex-limited mitochondrial DNA transmission in the marine mussel Mytilus edulis. Genetics 1994, 138, 801–809. [13] Zouros, E., Biparental inheritance through uniparental transmission: the doubly uniparental inheritance (DUI) of mitochondrial DNA. Evol. Biol. 2013, 40, 1–31. [14] Palmer, M. R., McDowall, M. H., Stewart, L., Quaddi, A. et al., Mass spectrometry and next-generation sequencing reveal an abundant and rapidly evolving abalone sperm protein. Mol. Reprod. Dev. 2013, 80, 460–465. [15] Diz, A. P., Dudley, E., Skibinski, D. O., Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis. Proteomics 2012, 12, 1949–1956. [16] Sun, J., Zhang, H., Wang, H., Heras, H. et al., First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. J. Proteome Res. 2012, 11, 4240– 4248. [17] Overbeek, R., Fonstein, M., D’Souza, M., Pusch, G. D. et al., The use of gene clusters to infer functional coupling. Proc. Natl. Acad. Sci. 1999, 96, 2896–2901. [18] Zhang, Z., Xiao, J., Wu, J., Zhang, H. et al., ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem. Bioph. Res. Co. 2012, 419, 779–781. [19] Zhang, Z., Li, J., Zhao, X. Q., Wong, G. K. et al., KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics 2006, 4, 259–263. [20] Dorus, S., Busby, S. A., Gerike, U., Shabanowitz, J. et al., Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. 2006, 38, 1440– 1445. ¨ [21] Nynca, J., Arnold, G. J., Frohlich, T., Otte, K., Ciereszko, A., Proteomic identification of rainbow trout sperm proteins. Proteomics 2014, 14, 1569–1573. [22] Sutovsky, P., Sperm proteasome and fertilization. Reproduction 2011, 142, 1–14.
www.proteomics-journal.com
Proteomics 2015, 15, 4175–4179 ´ C., Felix, R., Nishigaki, T., Trevino, ˜ C. L., [23] Darszon, A., Beltran, Ion transport in sperm signaling. Dev. Biol. 2001, 240, 1–14. [24] Wu, Q., Li, L., Zhang, G., Crassostrea angulata bindin gene and the divergence of fucose-binding lectin repeats among three species of Crassostrea. Mar. Biotechnol. 2011, 13, 327– 335. [25] Dheilly, N. M., Lelong, C., Huvet, A., Kellner, K. et al., Gametogenesis in the Pacific Oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes. PLoS One 2012, 7, e36353.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4179 [26] Suri, A., Family of sperm associated antigens: relevance in sperm-egg interaction and immunocontraception. Soc. Reprod. Fertil. Suppl. 2006, 63, 433–443. [27] Clark, N. L., Swanson, W. J., Pervasive adaptive evolution in primate seminal proteins. PLoS Genet. 2005, 1, e35. [28] Min, X. J., Butler, G., Storms, R., Tsang, A., TargetIdentifier: a webserver for identifying full-length cDNAs from EST sequences. Nucleic Acids Res. 2005, 33, 669– 672.
www.proteomics-journal.com
