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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve <italic>Mytilus edulis</italic></title><author><name sortKey="Ciocan, Corina M" sort="Ciocan, Corina M" uniqKey="Ciocan C" first="Corina M." last="Ciocan">Corina M. Ciocan</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cubero Leon, Elena" sort="Cubero Leon, Elena" uniqKey="Cubero Leon E" first="Elena" last="Cubero-Leon">Elena Cubero-Leon</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Minier, Christophe" sort="Minier, Christophe" uniqKey="Minier C" first="Christophe" last="Minier">Christophe Minier</name><affiliation><nlm:aff id="aff2"><addr-line>Laboratoire d'Ecotoxicologie, Universite du Havre, Le Havre, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rotchell, Jeanette M" sort="Rotchell, Jeanette M" uniqKey="Rotchell J" first="Jeanette M." last="Rotchell">Jeanette M. Rotchell</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21818309</idno><idno type="pmc">3144882</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144882</idno><idno type="RBID">PMC:3144882</idno><idno type="doi">10.1371/journal.pone.0022326</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">000226</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve <italic>Mytilus edulis</italic></title><author><name sortKey="Ciocan, Corina M" sort="Ciocan, Corina M" uniqKey="Ciocan C" first="Corina M." last="Ciocan">Corina M. Ciocan</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cubero Leon, Elena" sort="Cubero Leon, Elena" uniqKey="Cubero Leon E" first="Elena" last="Cubero-Leon">Elena Cubero-Leon</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Minier, Christophe" sort="Minier, Christophe" uniqKey="Minier C" first="Christophe" last="Minier">Christophe Minier</name><affiliation><nlm:aff id="aff2"><addr-line>Laboratoire d'Ecotoxicologie, Universite du Havre, Le Havre, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rotchell, Jeanette M" sort="Rotchell, Jeanette M" uniqKey="Rotchell J" first="Jeanette M." last="Rotchell">Jeanette M. Rotchell</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline.</p></sec><sec><title>Methodology/Principal Findings</title><p>Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences.</p></sec><sec><title>Conclusions</title><p>The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Goldberg, Ed" uniqKey="Goldberg E">ED Goldberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Andral, B" uniqKey="Andral B">B Andral</name></author><author><name sortKey="Stanisiere, Jy" 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Ciocan</name></author><author><name sortKey="Cubero Leon, E" uniqKey="Cubero Leon E">E Cubero-Leon</name></author><author><name sortKey="Puinean, Am" uniqKey="Puinean A">AM Puinean</name></author><author><name sortKey="Hill, Em" uniqKey="Hill E">EM Hill</name></author><author><name sortKey="Minier, C" uniqKey="Minier C">C Minier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cubero Leon, E" uniqKey="Cubero Leon E">E Cubero-Leon</name></author><author><name sortKey="Ciocan, Cm" uniqKey="Ciocan C">CM Ciocan</name></author><author><name sortKey="Hill, Em" uniqKey="Hill E">EM Hill</name></author><author><name sortKey="Osada, M" uniqKey="Osada M">M Osada</name></author><author><name sortKey="Kishida, M" uniqKey="Kishida M">M Kishida</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gagne, F" uniqKey="Gagne F">F Gagné</name></author><author><name sortKey="Bouchard, B" uniqKey="Bouchard B">B 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Adessi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hines, Ga" uniqKey="Hines G">GA Hines</name></author><author><name sortKey="Bryan, Pj" uniqKey="Bryan P">PJ Bryan</name></author><author><name sortKey="Wasson, Km" uniqKey="Wasson K">KM Wasson</name></author><author><name sortKey="Mcclintock, Jb" uniqKey="Mcclintock J">JB McClintock</name></author><author><name sortKey="Watts, Sa" uniqKey="Watts S">SA Watts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, W" uniqKey="Zhu W">W Zhu</name></author><author><name sortKey="Mantione, K" uniqKey="Mantione K">K Mantione</name></author><author><name sortKey="Jones, D" uniqKey="Jones D">D Jones</name></author><author><name sortKey="Salamon, E" uniqKey="Salamon E">E Salamon</name></author><author><name sortKey="Cho, Jj" uniqKey="Cho J">JJ Cho</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Le Curieux Belfond, O" uniqKey="Le Curieux Belfond O">O Le Curieux-Belfond</name></author><author><name sortKey="Moslemi, S" uniqKey="Moslemi S">S Moslemi</name></author><author><name sortKey="Mathieu, M" uniqKey="Mathieu M">M Mathieu</name></author><author><name sortKey="Seralini, Ge" uniqKey="Seralini G">GE Seralini</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Janer, G" uniqKey="Janer G">G Janer</name></author><author><name sortKey="Porte, C" uniqKey="Porte C">C Porte</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Le Curieux Belfond, O" uniqKey="Le Curieux Belfond O">O Le Curieux-Belfond</name></author><author><name sortKey="Fievet, B" uniqKey="Fievet B">B Fievet</name></author><author><name sortKey="Seralini, Ge" uniqKey="Seralini G">GE Seralini</name></author><author><name sortKey="Mathieu, M" uniqKey="Mathieu M">M Mathieu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peck, M" uniqKey="Peck M">M 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sortKey="Matsuoka, M" uniqKey="Matsuoka M">M Matsuoka</name></author><author><name sortKey="Odo, S" uniqKey="Odo S">S Odo</name></author><author><name sortKey="Harayama, S" uniqKey="Harayama S">S Harayama</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vandesompele, J" uniqKey="Vandesompele J">J Vandesompele</name></author><author><name sortKey="De Preter, K" uniqKey="De Preter K">K De Preter</name></author><author><name sortKey="Pattyn, F" uniqKey="Pattyn F">F Pattyn</name></author><author><name sortKey="Poppe, B" uniqKey="Poppe B">B Poppe</name></author><author><name sortKey="Van Roy, N" uniqKey="Van Roy N">N Van Roy</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21818309</article-id><article-id pub-id-type="pmc">3144882</article-id><article-id pub-id-type="publisher-id">PONE-D-11-02229</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0022326</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology</subject><subj-group><subject>Anatomy and Physiology</subject><subj-group><subject>Endocrine System</subject><subj-group><subject>Endocrine Physiology</subject><subj-group><subject>Hormones</subject></subj-group></subj-group></subj-group><subj-group><subject>Reproductive System</subject><subj-group><subject>Sexual Reproduction</subject></subj-group></subj-group></subj-group><subj-group><subject>Biochemistry</subject><subj-group><subject>Hormones</subject></subj-group></subj-group><subj-group><subject>Computational Biology</subject><subj-group><subject>Genomics</subject><subj-group><subject>Genome Expression Analysis</subject></subj-group></subj-group></subj-group><subj-group><subject>Ecology</subject><subj-group><subject>Bioindicators</subject><subject>Ecophysiology</subject><subject>Marine Ecology</subject></subj-group></subj-group><subj-group><subject>Genetics</subject><subj-group><subject>Animal Genetics</subject><subject>Gene Expression</subject></subj-group></subj-group><subj-group><subject>Genomics</subject><subj-group><subject>Genome Expression Analysis</subject></subj-group></subj-group><subj-group><subject>Marine Biology</subject><subj-group><subject>Marine Ecology</subject></subj-group></subj-group><subj-group><subject>Molecular Cell Biology</subject><subj-group><subject>Signal Transduction</subject><subj-group><subject>Membrane Receptor Signaling</subject></subj-group></subj-group><subj-group><subject>Gene Expression</subject></subj-group></subj-group><subj-group><subject>Zoology</subject><subj-group><subject>Animal Physiology</subject><subject>Malacology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Earth Sciences</subject><subj-group><subject>Marine and Aquatic Sciences</subject><subj-group><subject>Marine Monitoring</subject><subject>Water Quality</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve <italic>Mytilus edulis</italic></article-title><alt-title alt-title-type="running-head">Molluscan Gametogenesis and Endocrine Disruption</alt-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Ciocan</surname><given-names>Corina M.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Cubero-Leon</surname><given-names>Elena</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Minier</surname><given-names>Christophe</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Rotchell</surname><given-names>Jeanette M.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref><xref ref-type="author-notes" rid="fn1"><sup>¤</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Biology and Environmental Science, University of Sussex, Brighton, United Kingdom</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Laboratoire d'Ecotoxicologie, Universite du Havre, Le Havre, France</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Laudet</surname><given-names>Vincent</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">Ecole Normale Supérieure de Lyon, France</aff><author-notes><corresp id="cor1">* E-mail: <email>j.rotchell@hull.ac.uk</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: JMR CMC EC-L CM. Performed the experiments: CMC EC-L. Analyzed the data: JMR CMC EC-L. Contributed reagents/materials/analysis tools: JMR CMC EC-L. Wrote the paper: JMR CMC EC-L.</p></fn><fn id="fn1" fn-type="current-aff"><label>¤</label><p>Current address: Department of Biological Sciences, University of Hull, Hull, United Kingdom</p></fn></author-notes><pub-date pub-type="collection"><year>2011</year></pub-date><pub-date pub-type="epub"><day>27</day><month>7</month><year>2011</year></pub-date><volume>6</volume><issue>7</issue><elocation-id>e22326</elocation-id><history><date date-type="received"><day>27</day><month>1</month><year>2011</year></date><date date-type="accepted"><day>26</day><month>6</month><year>2011</year></date></history><permissions><copyright-statement>Ciocan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</copyright-statement><copyright-year>2011</copyright-year></permissions><abstract><sec><title>Background</title><p>While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline.</p></sec><sec><title>Methodology/Principal Findings</title><p>Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences.</p></sec><sec><title>Conclusions</title><p>The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.</p></sec></abstract><counts><page-count count="8"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Bivalve molluscs, such as <italic>Mytilus sp.</italic>, are currently used as sentinel organisms to monitor exposure to a variety of chemical contaminants in international programmes such as “Mussel Watch” <xref ref-type="bibr" rid="pone.0022326-Goldberg1">[1]</xref>–<xref ref-type="bibr" rid="pone.0022326-Viarengo1">[3]</xref>. Their sessile nature, wide geographical distribution, large populations and large filtering rates make them excellent indicator species for environmental toxicology applications <xref ref-type="bibr" rid="pone.0022326-Rittschof1">[4]</xref>. It is apparent that molluscs take up and bioaccumulate potentially endocrine disrupting chemicals <xref ref-type="bibr" rid="pone.0022326-Morcillo1">[5]</xref>, <xref ref-type="bibr" rid="pone.0022326-Labadie1">[6]</xref> and they are sensitive to endocrine disruption at environmentally relevant concentrations <xref ref-type="bibr" rid="pone.0022326-Langston1">[7]</xref>–<xref ref-type="bibr" rid="pone.0022326-Gagn1">[10]</xref>.</p><p>In vertebrate reproductive endocrinology it is well recognised that the sex steroids, namely estrogens, androgens, progestins, mineralcorticoids, and glucocorticoids or glucorticoid hormones, play a key role via their binding to steroid receptors. Sex steroid hormones and their role in supporting molluscan reproduction are still unclear. It is established that vertebrate-like steroids, particularly estrogens (estradiol E2, estrone E1), androgens (testosterone), and progestins are present in various tissues of molluscs <xref ref-type="bibr" rid="pone.0022326-DeLongcamp1">[11]</xref>–<xref ref-type="bibr" rid="pone.0022326-Zhu1">[14]</xref>. A number of enzymatic activities and regulatory, including non-genomic, pathways in molluscs have also been characterised <xref ref-type="bibr" rid="pone.0022326-DeLongcamp1">[11]</xref>, <xref ref-type="bibr" rid="pone.0022326-LeCurieuxBelfond1">[15]</xref>–<xref ref-type="bibr" rid="pone.0022326-Janer1">[16]</xref>. The occurrence of sex steroids is therefore not in doubt, yet their source (endogenous or exogenous) <xref ref-type="bibr" rid="pone.0022326-LeCurieuxBelfond2">[17]</xref>, <xref ref-type="bibr" rid="pone.0022326-Peck1">[18]</xref>, and role in molluscs is less clear. It is also important to distinguish between the presence of estrogens and an endogenous role. For example, while there are reports of putative hydroxysteroid dehydrogenase and cholesterol esterase-like unpublished sequences for <italic>Haliotis diversicolor</italic> (ADV02385) and <italic>Biomphalaria glabrata</italic> (LIBEST_021038, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/">www.ncbi.nlm.nih.gov/</ext-link>), there is no report of side-chain cleavage activities in any bivalve mollusc in the literature, to our knowledge, and this reaction is the first step to make an estrogen in vertebrates.</p><p>Historically, the role of estrogens in the hormonal regulation of the reproduction in bivalves was suggested to be similar to that which they fulfil in the vertebrate endocrine system. Studies have shown that injection of E2 directly into the gonads of <italic>Crassostrea gigas</italic> causes a significant increase in oocyte diameter and egg yolk protein vitellin (Vn) content in the female oyster ovary <xref ref-type="bibr" rid="pone.0022326-Li1">[19]</xref>. Also, in scallop, <italic>Patinopecten yessoensis</italic> and <italic>P. magellanicus</italic>, direct injection of E2 into gonads resulted in an increase of Vn in the ovary and serotonin (5-HT)-induced gamete release <xref ref-type="bibr" rid="pone.0022326-Osada1">[20]</xref>, <xref ref-type="bibr" rid="pone.0022326-Wang1">[21]</xref>. Estrogens are thought to bring about the induction of the 5-HT receptor on the oocyte membrane and in turn trigger spawning <xref ref-type="bibr" rid="pone.0022326-Osada2">[22]</xref>. The levels of E2 in bivalves have also been shown to vary along the year; the profile is synchronised with variations of oocyte diameter and gonad index <xref ref-type="bibr" rid="pone.0022326-Osada3">[23]</xref>. Subsequently, E2 is considered to exhibit a seasonal change associated with the reproductive cycle and to be involved in the regulation of several reproductive processes in bivalves such as vitellogenesis <xref ref-type="bibr" rid="pone.0022326-Osada3">[23]</xref>.</p><p>The role and metabolism of E2 in bivalves is however debated. For instance, exposure of <italic>M. edulis</italic> and <italic>Anodonta cygnea</italic>, either waterborne or injected E2, failed to induce the production of Vn-like proteins in the hemolymph and gonads <xref ref-type="bibr" rid="pone.0022326-Riffeser1">[24]</xref>. Also, functional studies using the invertebrates <italic>Aplysia californica</italic>, <italic>Octopus vulgaris</italic>, <italic>Thais clavigera</italic>, <italic>Marisa cornuarietis</italic> and <italic>C. gigas</italic> have shown that the ER does not bind E2 or is unresponsive <xref ref-type="bibr" rid="pone.0022326-Thornton1">[25]</xref>–<xref ref-type="bibr" rid="pone.0022326-Matsumoto1">[28]</xref>. Puinean et al (2006) also reported an absence of <italic>ER</italic> mRNA induction in <italic>M. edulis</italic> (at the mature stage of gametogenesis) following E2 aqueous exposure <xref ref-type="bibr" rid="pone.0022326-Puinean1">[29]</xref>. Possible explanations for the lack of induction in bivalves have been suggested <xref ref-type="bibr" rid="pone.0022326-Labadie1">[6]</xref>, <xref ref-type="bibr" rid="pone.0022326-Thornton1">[25]</xref>, <xref ref-type="bibr" rid="pone.0022326-Bannister1">[27]</xref>, <xref ref-type="bibr" rid="pone.0022326-Puinean1">[29]</xref>. The role of estrogens and their functional mechanism of action in bivalves are therefore far from clear.</p><p>The bivalve response to exogenous estrogens has been the focus of recent research. Significant natural variation was observed in <italic>M. edulis ER</italic> mRNA expression, with significantly lower values during January, February and July compared with other times of the year <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. <italic>M. edulis</italic> exposed to E2 and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days also displayed a significant increase in <italic>ER</italic> mRNA expression <italic>provided</italic> mussels were exposed to estrogens at the early stage of gametogenesis <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. In contrast, mature mussels exposed to estrogens displayed no statistically significant change in <italic>ER</italic> mRNA expression <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>, <xref ref-type="bibr" rid="pone.0022326-Puinean1">[29]</xref>. Gonad <italic>VTG</italic> mRNA expression also showed up-regulation in estrogen exposed mussels at the early stages of development <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. In a parallel study, <italic>serotonin receptor</italic> and <italic>cyclooxygenase</italic> mRNA expression were also observed modulated by E2 exposure in <italic>M. edulis</italic> <xref ref-type="bibr" rid="pone.0022326-CuberoLeon1">[9]</xref>. Combined, these data suggest that estrogens may have an impact on reproduction processes in bivalves.</p><p>Building on these observations, the aim of this study was to adopt an exploratory approach to identify novel genes differentially expressed in the maturation process and the estrogen response in the marine bivalve, <italic>M. edulis</italic>.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>SSH Analysis</title><p>The 206 putative mRNA sequences were compared with sequences in the NCBI GenBank database using the blastx and blastn algorithms. Forty seven (22%) of the sequences, with a small number of duplicates, from the forward (up-regulated genes) and reverse (down-regulated genes) libraries could be matched to genes from different organisms, mainly invertebrate species (<xref ref-type="table" rid="pone-0022326-t001">Tables 1</xref> and <xref ref-type="table" rid="pone-0022326-t002">2</xref>). The remaining sequenced clones showed similarity to unidentified hypothetical or novel proteins or showed no similarity with the sequences deposited in the database. In some of these latter cases, there was high similarity to sequences identified in other mussel EST libraries: sixteen from the E2-exposed enriched library were highly similar (blastn <italic>E = </italic>3.0<sup>−115</sup> to 1.0<sup>−100</sup>) to sequences identified in SSH libraries that were constructed from mussels treated with an inactivated cocktail of <italic>Vibrio</italic> (AM880859) or exposed to a variety of environmental stressors (ES389965.1).</p><table-wrap id="pone-0022326-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.t001</object-id><label>Table 1</label><caption><title>Differentially expressed (subtracted) mRNAs identified in <italic>M. edulis</italic> testis at two stages of gonadal development.</title></caption><alternatives><graphic id="pone-0022326-t001-1" xlink:href="pone.0022326.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Clone accession no.</td><td align="left" rowspan="1" colspan="1">Category & gene identity</td><td align="left" rowspan="1" colspan="1">Length (bp)</td><td align="left" rowspan="1" colspan="1">Homolog species/Accession no.</td><td align="left" rowspan="1" colspan="1"><italic>E</italic>-value</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">HQ690234</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt101">a</xref>Senescence-associated protein</td><td align="left" rowspan="1" colspan="1">155</td><td align="left" rowspan="1" colspan="1"><italic>Brugia malayi</italic> XP_001900327.1</td><td align="left" rowspan="1" colspan="1">5.0<italic>E</italic><sup>−10</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AJ492924.1</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt101">a</xref>Histone 2A</td><td align="left" rowspan="1" colspan="1">301</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic> AJ492924.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−108</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AY484747.1</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt101">a</xref>16S ribosomal protein</td><td align="left" rowspan="1" colspan="1">931</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic> AY484747.1</td><td align="left" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">FM995162.1</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt101">a</xref>Vitelline coat lysin M7 precursor</td><td align="left" rowspan="1" colspan="1">635</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic> BAA03551.1</td><td align="left" rowspan="1" colspan="1">3.0<italic>E</italic><sup>−123</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678182</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt101">a</xref>Sialic acid binding lectin</td><td align="left" rowspan="1" colspan="1">397</td><td align="left" rowspan="1" colspan="1"><italic>Helix pomatia</italic> ABF00124.1</td><td align="left" rowspan="1" colspan="1">4.0<italic>E</italic><sup>−15</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678180</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Testis-specific serine/threonine kinase 1 (TSTK1)</td><td align="left" rowspan="1" colspan="1">815</td><td align="left" rowspan="1" colspan="1"><italic>Strongylocentrotus purpuratus</italic> XP_787865.1</td><td align="left" rowspan="1" colspan="1">4.0<italic>E</italic><sup>−70</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678181</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Testis-specific A-kinase-anchoring-protein</td><td align="left" rowspan="1" colspan="1">182</td><td align="left" rowspan="1" colspan="1"><italic>Gallus gallus</italic> XP_002162537.1</td><td align="left" rowspan="1" colspan="1">9.0<italic>E</italic><sup>−6</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ67816</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Histone H2A isoform 2</td><td align="left" rowspan="1" colspan="1">332</td><td align="left" rowspan="1" colspan="1"><italic>Haliotis discus discus</italic> ACJ12611.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−53</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678184</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Beta-tubulin</td><td align="left" rowspan="1" colspan="1">511</td><td align="left" rowspan="1" colspan="1"><italic>C. gigas</italic> AAU93877.1</td><td align="left" rowspan="1" colspan="1">9.0<italic>E</italic><sup>−81</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AB257133</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>ER</td><td align="left" rowspan="1" colspan="1">111</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic> BAF34366.2</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−12</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678183</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Bindin precursor 5 repeat variant (acrosomal protein)</td><td align="left" rowspan="1" colspan="1">392</td><td align="left" rowspan="1" colspan="1"><italic>C. gigas</italic> ABQ18234.1</td><td align="left" rowspan="1" colspan="1">7.0<italic>E</italic><sup>−14</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ678185</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Phosphodiesterase 1</td><td align="left" rowspan="1" colspan="1">533</td><td align="left" rowspan="1" colspan="1"><italic>S. purpuratus</italic> NP_001091918.1</td><td align="left" rowspan="1" colspan="1">5.0<italic>E</italic><sup>−62</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AY130198.1</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt102">b</xref>Cytochrome c oxidase subunit III</td><td align="left" rowspan="1" colspan="1">254</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic> AAV68300.1</td><td align="left" rowspan="1" colspan="1">2.0<italic>E</italic><sup>−31</sup></td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label>a</label><p>Down-regulated in early developing testes relative to mature testes.</p></fn><fn id="nt102"><label>b</label><p>Up-regulated in early developing testes relative to mature testes.</p></fn></table-wrap-foot></table-wrap><table-wrap id="pone-0022326-t002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.t002</object-id><label>Table 2</label><caption><title>Differentially expressed (subtracted) mRNAs identified in <italic>M. edulis</italic> testis following E2 exposure.</title></caption><alternatives><graphic id="pone-0022326-t002-2" xlink:href="pone.0022326.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Clone Accession No.</td><td align="left" rowspan="1" colspan="1">Category & gene identity (BlastX)</td><td align="left" rowspan="1" colspan="1">Length (bp)</td><td colspan="2" align="left" rowspan="1">Homolog species/Accession no.</td><td align="left" rowspan="1" colspan="1"><italic>E</italic>-value</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">HQ664951</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Complement C1q-like protein</td><td align="left" rowspan="1" colspan="1">111</td><td align="left" rowspan="1" colspan="1"><italic>Ailuropoda melanoleuca</italic></td><td align="left" rowspan="1" colspan="1">XP_002918680.1</td><td align="left" rowspan="1" colspan="1">7.0<italic>E</italic><sup>−5</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690237</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Alpha tubulin</td><td align="left" rowspan="1" colspan="1">297</td><td align="left" rowspan="1" colspan="1"><italic>C. gigas</italic></td><td align="left" rowspan="1" colspan="1">BAD80736.1</td><td align="left" rowspan="1" colspan="1">2.0<italic>E</italic><sup>−51</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690238</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Beta tubulin</td><td align="left" rowspan="1" colspan="1">501</td><td align="left" rowspan="1" colspan="1"><italic>Rattus norvegicus</italic></td><td align="left" rowspan="1" colspan="1">NP_954525.1</td><td align="left" rowspan="1" colspan="1">2.0<italic>E</italic><sup>−94</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690235</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Ribosomal protein L7</td><td align="left" rowspan="1" colspan="1">240</td><td align="left" rowspan="1" colspan="1"><italic>C. gigas</italic></td><td align="left" rowspan="1" colspan="1">AJ557884.1</td><td align="left" rowspan="1" colspan="1">3.0<italic>E</italic><sup>−30</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690239</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Bromodomain adjacent to zinc finger domain, 1A</td><td align="left" rowspan="1" colspan="1">369</td><td align="left" rowspan="1" colspan="1"><italic>G. gallus</italic></td><td align="left" rowspan="1" colspan="1">XP_426440.2</td><td align="left" rowspan="1" colspan="1">7.0<italic>E</italic><sup>−24</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690240</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Elongation factor 1 gamma</td><td align="left" rowspan="1" colspan="1">162</td><td align="left" rowspan="1" colspan="1"><italic>Saccoglossus kowalevskii</italic></td><td align="left" rowspan="1" colspan="1">NP_001171816.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−13</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ664949</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Vitelline envelope zona pellucida domain 9</td><td align="left" rowspan="1" colspan="1">900</td><td align="left" rowspan="1" colspan="1"><italic>Haliotis rufescens</italic></td><td align="left" rowspan="1" colspan="1">ABE72949.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−26</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ664950</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>C1q domain containing protein</td><td align="left" rowspan="1" colspan="1">141</td><td align="left" rowspan="1" colspan="1"><italic>Argopecten irradians</italic></td><td align="left" rowspan="1" colspan="1">ADD17343</td><td align="left" rowspan="1" colspan="1">7.0<italic>E</italic><sup>−5</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ664948</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>RWD domain containing protein 4A</td><td align="left" rowspan="1" colspan="1">240</td><td align="left" rowspan="1" colspan="1"><italic>Caligus rogercresseyi</italic></td><td align="left" rowspan="1" colspan="1">ACO11028.1</td><td align="left" rowspan="1" colspan="1">3.0<italic>E</italic><sup>−17</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ664952</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Hemaglutinin/amoebocyte aggregation factor precursor</td><td align="left" rowspan="1" colspan="1">240</td><td align="left" rowspan="1" colspan="1"><italic>Salmo salar</italic></td><td align="left" rowspan="1" colspan="1">ACI68653.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−10</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">YP_073337</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Cytochrome c oxidase subunit II</td><td align="left" rowspan="1" colspan="1">448</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic></td><td align="left" rowspan="1" colspan="1">YP_073337.1</td><td align="left" rowspan="1" colspan="1">4.0<italic>E</italic><sup>−56</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">YP_073338.1</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>NADH dehydrogenase subunit 1</td><td align="left" rowspan="1" colspan="1">647</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic></td><td align="left" rowspan="1" colspan="1">YP_073338.1</td><td align="left" rowspan="1" colspan="1">4.0<italic>E</italic><sup>−61</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690236</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt103">a</xref>Triosephosphate isomerase TIM</td><td align="left" rowspan="1" colspan="1">156</td><td align="left" rowspan="1" colspan="1"><italic>Metapenaeus ensis</italic></td><td align="left" rowspan="1" colspan="1">AAP79983.1</td><td align="left" rowspan="1" colspan="1">3.0<italic>E</italic><sup>−13</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AAV68423</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt104">b</xref>Cytochrome c oxidase subunit 1</td><td align="left" rowspan="1" colspan="1">534</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic></td><td align="left" rowspan="1" colspan="1">AAV68411.1</td><td align="left" rowspan="1" colspan="1">8.0<italic>E</italic><sup>−90</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">AAV68416</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt104">b</xref>Cytochrome b</td><td align="left" rowspan="1" colspan="1">302</td><td align="left" rowspan="1" colspan="1"><italic>M. edulis</italic></td><td align="left" rowspan="1" colspan="1">AAV68404.1</td><td align="left" rowspan="1" colspan="1">6.0<italic>E</italic><sup>−28</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690243</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt104">b</xref>Ferritin-like protein</td><td align="left" rowspan="1" colspan="1">492</td><td align="left" rowspan="1" colspan="1"><italic>Pinctada fucata</italic></td><td align="left" rowspan="1" colspan="1">AAQ12076.1</td><td align="left" rowspan="1" colspan="1">2.0<italic>E</italic><sup>−78</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690241</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt104">b</xref>Senescence-associated protein</td><td align="left" rowspan="1" colspan="1">318</td><td align="left" rowspan="1" colspan="1"><italic>Trichoplax adhaerens</italic></td><td align="left" rowspan="1" colspan="1">XP_002118266.1</td><td align="left" rowspan="1" colspan="1">6.0<italic>E</italic><sup>−49</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">HQ690244</td><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt104">b</xref>Spectrin beta chain</td><td align="left" rowspan="1" colspan="1">293</td><td align="left" rowspan="1" colspan="1"><italic>Harpegnathos saltator</italic></td><td align="left" rowspan="1" colspan="1">EFN75523.1</td><td align="left" rowspan="1" colspan="1">1.0<italic>E</italic><sup>−10</sup></td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt103"><label>a</label><p>Down-regulated in control testes relative to E2-exposed testes.</p></fn><fn id="nt104"><label>b</label><p>Up-regulated in control testes relative to E2-exposed testes.</p></fn></table-wrap-foot></table-wrap></sec><sec id="s2b"><title>Validation of Differential mRNA Expressions</title><p>Six <italic>target</italic> mRNAs were selected for qPCR validation of the SSH differential expression results (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1A–F</xref>). Both <italic>vitelline coat lysin precursor</italic> mRNA (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1A</xref>) and <italic>sialic acid binding lectin</italic> (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1B</xref>) were statistically significantly differentially regulated according to testis stage of maturity, up-regulated as the testis mature. Conversely, <italic>testis-specific serine/threonine kinase 1 (TSTK1)</italic>(<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1C</xref>) and <italic>ER</italic> (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1D</xref>) mRNA expressions measured using qPCR are statistically significantly down-regulated in mature testis. <italic>C1q domain containing protein</italic>, identified as down-regulated in control mussels compared with E2-exposed mussels by SSH, was confirmed as such using qPCR (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1E</xref>). <italic>Cytochrome b</italic> mRNA expression was statistically significantly down-regulated in E2-exposed mussels relative to control samples, again confirming the SSH result (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1F</xref>).</p><fig id="pone-0022326-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.g001</object-id><label>Figure 1</label><caption><title>Real-time quantitative RT-PCR validation of differential screening results of <italic>M. edulis</italic> developing gonad versus mature gonad samples (1A–1E) and <italic>M. edulis</italic> experimentally-exposed to E2 (1F–1H).</title><p>Data plotted as mean±SEM, n = 15 samples. * = <italic>p</italic><0.05; ** = <italic>p</italic><0.01.</p></caption><graphic xlink:href="pone.0022326.g001"></graphic></fig><p>Two further <italic>target</italic> mRNAs highlighted by SSH were employed in a reverse analysis using qPCR (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1G–H</xref>). <italic>RWD domain containing protein 4A</italic>, highlighted by SSH as down-regulated in control mussel testis samples relative to E2-exposed samples (<xref ref-type="table" rid="pone-0022326-t002">Table 2</xref>), was identified using qPCR as down-regulated in early developing testis samples relative to mature samples (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1G</xref>). <italic>TSTK1</italic> highlighted by SSH as up-regulated in early developing stages of mussel testis samples relative to mature samples (<xref ref-type="table" rid="pone-0022326-t001">Table 1</xref>), was identified using qPCR as down-regulated in E2-exposed testis samples relative to control samples (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1H</xref>).</p></sec></sec><sec id="s3"><title>Discussion</title><p>Using the SSH approach we generated libraries enriched for genes that vary between early developing and mature mussels, as well as control and E2 experimentally exposed individuals. These libraries were produced from mussel testis and, because of the limited genomic resources over three quarters of the sequences could not be identified, or could only be matched to other ESTs of unknown function. This success rate of identification (22%) is comparable to similar studies using molluscs (6–12% <xref ref-type="bibr" rid="pone.0022326-Boutet1">[30]</xref>, <xref ref-type="bibr" rid="pone.0022326-Craft1">[31]</xref>). The sequence and species with the highest identity using BLAST analysis are cited in the Tables, yet this can give arbitrary results and accordingly the GenBank accession numbers for each sequence isolated are also cited to facilitate further characterisation.</p><p>In the subtractions reported here four separate libraries were constructed using: a) cDNA from immature males as driver (reverse subtraction 1), b) cDNA from mature males as driver (forward subtraction 1), c) cDNA from untreated immature males as driver (reverse subtraction 2) and d) cDNA from E2-treated immature males as driver (forward subtraction 2). A number of transcripts of interest were selected for additional characterization by qPCR and are discussed below.</p><sec id="s3a"><title>mRNA Transcripts Differentially Regulated in Testis at Two Stages of Gametogenesis</title><p>In the developing testis tissue samples (<xref ref-type="fig" rid="pone-0022326-g002">Fig. 2B</xref>) sequences associated with sperm development, cell signalling, cell cycle and electron transport were isolated (<xref ref-type="table" rid="pone-0022326-t001">Table 1</xref>) and would be consistent with an early stage of gametogenesis in which there is supporting cell initiation tubule formation and cell proliferation occurring. Two sperm-associated kinases were identified that may have roles similar to testes-specific kinases reported in the scallop, <italic>A. purpuratus</italic> (ES469344, <xref ref-type="bibr" rid="pone.0022326-Boutet1">[30]</xref>). The sperm-associated kinases differ with scallop, however, in that the mussel homolog is up-regulated in immature/early developing gonad, yet the scallop homolog is down-regulated at this stage of gametogenesis. Interestingly, the testis-specific serine/threonine kinase 1 activity, naturally up-regulated in early developing mussels (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1C</xref>) was statistically significantly down-regulated following experimental exposure to E2 (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1H</xref>).</p><fig id="pone-0022326-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.g002</object-id><label>Figure 2</label><caption><title>Sections of testis at different stages of the mussel gametogenic cycle.</title><p>A, resting stage, characterized by the presence of connective tissue, narrow tubules, few residual germ cells undergoing cytolysis. B, early gametogenesis stage, characterized externally by flat and colourless shape, and microscopically by visible presence of connective tissue, some growing follicles (less than 30% of examined area), few spermatozoa in the centre of some follicles. C, mature stage, characterized externally by thick, milk-coloured aspect, and microscopically by 90–95% of examined area covered by follicles, some fully grown, packed with spermatozoa, very little surrounding connective tissue. Scale bars: 100 µm.</p></caption><graphic xlink:href="pone.0022326.g002"></graphic></fig><p>Histones, tubulin, and cytochrome c oxidase have also previously been isolated in bivalve testes though their maturation-specific levels were not reported. Up-regulation of <italic>ER</italic> in early stages of development is consistent with previous work using mussel <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. The observed up-regulation of acrosomal binding protein and putative vitelline coat lysin in the developing testis are likely related to one another. Bivalve acrosomal proteins, including lysin, are released upon binding of sperm to the egg vitelline envelope, lysin then creates a hole for sperm to pass via binding to a vitelline envelope glycoprotein vitelline envelope receptor for lysin (VERL) <xref ref-type="bibr" rid="pone.0022326-Aagaard1">[32]</xref>. In the developing testis the acrosomal binding protein and lysin appear up-regulated (<xref ref-type="table" rid="pone-0022326-t001">Table 1</xref>), yet the VERL is not up-regulated until we analyse the testis at mature stages of gametogenesis (<xref ref-type="table" rid="pone-0022326-t001">Table 1</xref>). In the testis tissues at the mature stage of gametogenesis, a VERL-like sequence was isolated and apparently up-regulated. VERL is currently used as a marker of female sex in mussels <xref ref-type="bibr" rid="pone.0022326-Sedik1">[33]</xref>, and its appearance in male samples observed herein may suggest that approach be reviewed.</p><p>The few sequences identified and up-regulated in the mature testis (<xref ref-type="fig" rid="pone-0022326-g002">Fig. 2C</xref>) were cell cycle and apoptosis related (<xref ref-type="table" rid="pone-0022326-t001">Table 1</xref>). For instance, senescence-associated protein is likely involved in blocking cell cycle, preventing initiation of maturation. Such sequences may be indicative that the testis are in preparation for a move towards the mature/spawning/spent stage of gametogenesis.</p></sec><sec id="s3b"><title>mRNA Transcripts Differentially Regulated in Testis Following E2 Exposure</title><p>Several interesting mRNA transcripts were identified and validated as differentially expressed in E2-exposed mussel testis samples (<xref ref-type="table" rid="pone-0022326-t002">Table 2</xref>; <xref ref-type="fig" rid="pone-0022326-g001">Fig. 1E–F and H</xref>). Here we limit the discussion to two: <italic>vitelline envelope zona pellucida domain</italic> protein and the <italic>RWD domain containing protein</italic>.</p><p><italic>Vitelline envelope zona pellucida domain</italic> mRNA expression was down-regulated in control compared with E2-exposed mussel testis (<xref ref-type="table" rid="pone-0022326-t002">Table 2</xref>). Vitelline envelope proteins in vertebrates share a common structural motif, the zona pellucida domain <xref ref-type="bibr" rid="pone.0022326-Aagaard1">[32]</xref>. The expression of such proteins has been proposed as a sensitive biomarkers of environmental estrogens in vertebrates for the following reasons: E2 induction has been observed in different teleost species <xref ref-type="bibr" rid="pone.0022326-Hyllner1">[34]</xref>; the observed induction of <italic>vitelline envelope</italic> mRNA isoforms precedes <italic>ER</italic> and <italic>VTG</italic> mRNA induction in E2 injected juvenile Arctic char; and the expression remains high (up to 36 days) <xref ref-type="bibr" rid="pone.0022326-Westerlund1">[35]</xref>. It is also argued that xenoestrogen-induced changes in vitelline envelope would have a higher potential for ecologically adverse effects because it involves critical population parameters in terms of fertilization and mechanical protection of the eggshell <xref ref-type="bibr" rid="pone.0022326-Arukwe1">[36]</xref>. Another advantage of adopting vitelline envelope proteins as a biomarker relates to a report of minimal seasonal variation in eelpout over a yearly cycle <xref ref-type="bibr" rid="pone.0022326-Larsson1">[37]</xref>. Here, we also observe increased <italic>vitelline envelope zona pellucida</italic> induction in E2-exposed testis relative to control mussel testis samples, and as such, this may represent a promising biomarker of estrogen pollution in bivalves.</p><p><italic>RWD domain containing protein 4</italic> was identified as differentially expressed in this study. A phylogenetic analysis of the sequence (using MEGA 5 software, maximum likelihood) was conducted to further investigate its' identity (<xref ref-type="fig" rid="pone-0022326-g003">Fig. 3</xref>). The isolated <italic>M. edulis</italic> partial RWD sequence clusters with an anemone RWD domain containing protein 4 sequence rather than the vertebrates.. However, Gir2, a related RWD superfamily protein, represents a different branch from all the other RWD sequences, including that isolated from mussel (<xref ref-type="fig" rid="pone-0022326-g002">Fig. 2</xref>). <italic>RWD domain containing protein 4</italic> was identified as down-regulated in control compared with E2-exposed mussel testis (<xref ref-type="table" rid="pone-0022326-t002">Table 2</xref>). In a parallel analysis using qPCR, <italic>the RWD domain containing protein 4</italic> mRNA expression was statistically significantly up-regulated in mature testis compared to early developing testis samples (<xref ref-type="fig" rid="pone-0022326-g001">Fig. 1G</xref>). The <italic>M. edulis RWD domain containing protein 4</italic> mRNA expression is thus up-regulated naturally as part of the maturation status, and apparently susceptible to exogenous induction following experimental exposure of early stage mussels to E2. Currently there is no information available in the literature regarding RWD domain proteins other than for RWDD1 isoform in rats <xref ref-type="bibr" rid="pone.0022326-Kang1">[38]</xref>. The RWD domain containing protein 1 counterpart in rat is referred to as small androgen receptor (AR) interactin protein (data from RDG-Rat Genome Database). RWDD1 enhances transactivation activity of AR in mice thymus, and as such, RWDD1 is considered an AR co-regulator <xref ref-type="bibr" rid="pone.0022326-Kang1">[38]</xref>. Further work is required to determine if the <italic>M. edulis</italic> RWD domain containing protein, differentially expressed in mature gonads, represents any such component of a non-genomic nuclear receptor pathway.</p><fig id="pone-0022326-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.g003</object-id><label>Figure 3</label><caption><title>Phylogenetic analysis of the <italic>M. edulis</italic> partial Rwd sequence with published RWD domain containing protein 4 sequences from different species: human (<italic>H. sapiens</italic> NP_057036), rat (<italic>Rattus norvegicus</italic> EDL78909), frog (<italic>Rana catesbeiana</italic> ACO52001), ant (<italic>C. floridanus</italic> EFN74794), fruit fly (<italic>Drosophila pseudoobscura</italic> XP_001356904), anemone (<italic>Nematostella vectensis</italic> XP_001639511), nematode (<italic>Ascaris suum</italic> ADY48558) as well as a related protein family member Gir2 (from <italic>M. anisopliae</italic> EFZ00157 and <italic>Saccharomyces.cerevisiae</italic> NP_010436), and an unrelated protein ras (from <italic>M. galloprovincialis</italic> ABC46896).</title></caption><graphic xlink:href="pone.0022326.g003"></graphic></fig><p>In conclusion, several differentially regulated genes, including testis-specific kinases, vitelline lysin and envelope sequences, have been isolated from mussel testis. The differentially expressed mRNAs, isolated from testis at two stages of maturation and following experimental estrogen exposure, provide evidence that mussels may be impacted by exogenous estrogen exposure.</p></sec></sec><sec sec-type="methods" id="s4"><title>Methods</title><sec id="s4a"><title>Sample Collection and Histological Analysis</title><p>For all the analyses, mussels were collected at low tide near Brighton Pier, U.K. (50°49′ longitude and 0°8′ latitude) on April 2007 and February 2008, kept in seawater and immediately brought to the laboratory. Mussels were dissected and a piece of gonad (approximately 0.5 cm<sup>2</sup>) was fixed in 4% formaldehyde prior to histology processing, a second and third piece from the same individual mussel was used for the molecular and chemical analyses using methods described previously <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>, <xref ref-type="bibr" rid="pone.0022326-Puinean1">[29]</xref>. Histological analysis of the gonads was performed as described previously <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. The gonads of male mussels synchronized at early gametogenesis stages (<xref ref-type="fig" rid="pone-0022326-g002">Fig. 2B</xref>) and mature stages (<xref ref-type="fig" rid="pone-0022326-g002">Fig. 2C</xref>) were kept in RNAlater™ (Qiagen Ltd., Crawley, U.K.) for further analysis using suppression subtractive hybridization (SSH).</p></sec><sec id="s4b"><title>Experimental E2 Exposure</title><p>Mussels collected in February 2008 (size 4.43±0.34 cm; synchronized at early gametogenesis) were placed in aquaria with 60 l of artificial seawater (InstantOcean, Sarrebourg, France) at a light regime of 12 hrs light/12 hrs dark. Following acclimatization (4 d), the mussels were exposed for 10 d to a nominal concentration of 50 ng/l of E2 under semistatic conditions or kept as a non-exposed control as described previously <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>, <xref ref-type="bibr" rid="pone.0022326-Boutet1">[30]</xref>. The E2 concentrations in the aquaria water (control and exposed) were analysed and are described previously <xref ref-type="bibr" rid="pone.0022326-Ciocan1">[8]</xref>. Male gonads were immersed in RNAlater™ (Qiagen Ltd., Crawley, U.K.) and selected for SSH analysis.</p></sec><sec id="s4c"><title>SSH</title><p>The SSH procedure was used to isolate and enrich for genes differentially-expressed between 1. mussels at the early stages of gonad development versus mature stage, as well as 2. mussels exposed experimentally to E2 while at the early stages of gonad development versus control mussels at the same stage of early gonad development. Total RNA was extracted from each mussel using Nucleospin RNA II (Macherey Nagel, U.K.) according to the manufacturer's protocol. For each group, equal amounts of RNA were pooled from each mussel (8 mussels in each group). cDNA was synthesised using SuperSMART PCR cDNA Synthesis reagents (Clontech, France). The forward- and reverse-subtracted libraries were produced using PCR-Select cDNA Subtraction reagents (Clontech, France) according to the manufacturer's protocol. The differential PCR products generated by SSH were inserted in a pCR<sup>R</sup>2.1 linearized vector and the constructs were transformed into competent TOP10 <italic>E.coli</italic> (Invitrogen). Sixty randomly selected colonies from each subtracted library were inoculated in LB broth and screened by PCR for inserts using vector-based primers. A total of 40 clones per library were randomly selected for sequencing (GATC Biotech U.K.) directly from the PCR product. Sequence identities were obtained by BLAST searches against the NCBI nucleic acid and protein databases. Sequence reads with <italic>E</italic>-value<10<sup>−5</sup> were filtered out.</p></sec><sec id="s4d"><title>Quantitative Real-Time PCR Analysis of <italic>Target</italic> mRNA Expression in <italic>M. edulis</italic> Testis</title><p>Six target mRNAs, identified using SSH, were selected for further investigation using real-time quantitative RT-PCR. In order to increase the statistical power of the analysis 15 individual samples (all males) were analyzed from each group. In brief, total RNA was isolated from gonadal tissue using RNeasy reagents (Qiagen, U.K.) and treated with RNA-free DNase I (Qiagen, U.K.) to remove genomic DNA. RNA concentrations were measured with the Quant-iT RNA assay kit (Invitrogen, U.K.) using a Qubit fluorometer (Invitrogen, U.K.). Reverse transcription of 1 µg of total RNA samples was carried out using Transcriptor First Strand cDNA synthesis reagents (Roche Applied Science, U.K.). Mussel species (<italic>M. edulis</italic>) was confirmed by PCR amplification of the <italic>Glu</italic> gene <xref ref-type="bibr" rid="pone.0022326-Inoue1">[39]</xref>. Real-time PCR reactions were performed in duplicate, in a final volume of 25 µl containing 12.5 µl of qPCR Fast Start SYBR Green Master Rox (Roche Applied Science, U.K.), 5 µl of diluted cDNA (1/60) and 3.75 µM primers (<xref ref-type="table" rid="pone-0022326-t003">Table 3</xref>). A control lacking cDNA template was included in qPCR analysis to determine the specificity of target cDNA amplification. Amplification was detected with a Mx3005P real time PCR system (Stratagene, U.K.). For each <italic>target</italic> mRNA, melting curve, gel picture and sequences were analysed in order to verify the specificity of the amplified products and the absence of primer dimers. The amplification efficiency of each primer pair was calculated using a dilution series of cDNA. A normalization factor, calculated using geNorm software <xref ref-type="bibr" rid="pone.0022326-Vandesompele1">[40]</xref> and based on the expression levels of the best performing reference transcripts in the gonadal samples, was used for accurate normalization of real-time RT-PCR data. The most stable reference mRNAs used for normalization in the developing and mature gonadal samples were <italic>18S rRNA</italic> (L33448), <italic>elongation factor 1-alpha</italic> (AF063420), and <italic>alpha-tubulin</italic> (DQ174100). For the E2-exposed samples the most stable reference transcripts used for normalization were <italic>18S rRNA</italic>, <italic>28S rRNA</italic> (Z29550) and <italic>elongation factor 1-alpha</italic>.</p><table-wrap id="pone-0022326-t003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0022326.t003</object-id><label>Table 3</label><caption><title>Primer sequences used for expression analysis of selected differentially expressed <italic>target</italic> mRNAs in mussel testis tissue and reference transcripts.</title></caption><alternatives><graphic id="pone-0022326-t003-3" xlink:href="pone.0022326.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Target/Reference mRNA</td><td align="left" rowspan="1" colspan="1">Forward primer 5′-3′</td><td align="left" rowspan="1" colspan="1">Reverse primer 5′-3′</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Vitelline coat lysin</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">TATCATCAAGACAGAAAGCAGACAG</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">CCATATGGTAAACTGCGTTTTAGTC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">Sialic acid binding lectin</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">AATATCAGTTCAACGAAGCCTACAC</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">AATCCTACCACTTTGCTTACTTGTG</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">Testis-specific serine kinase 1 (TSTK1)</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">ATAGACCGACTTACCGTGCTGT</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">CTGTGCTGAAAATCTTTTGGTG</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">ER</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">GGAACACAAAGAAAAGAAAGGAAG</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">ACAAATGTGTTCTGGATGGTG</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">C1q domain</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">GGAACCCTTGCTGTAAACACGC</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">AGGTCTAAAGCAGCCAAGCCAG</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">Cytochrome b</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">CCAGTGGAACCTTATATCR</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">TTTCAAATCTACAGGACGGC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">RWD domain containing protein 4</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">AAGAGCTGGAAGTCCCCCATTC</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">GTCCGCTGTCCATGAAATCTCC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">18S rRNA</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">GTGCTCTTGACTGAGTGTCTCG</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">CGAGGTCCTATTCCATTATTCC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">28S rRNA</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">AGCCACTGCTTGCAGTTCTC</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">ACTCGCGCACATGTTAGACTC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">Elongation factor 1-alpha</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">CACCACGAGTCTCTCCCAGA</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">GCTGTCACCACAGACCATTCC</named-content></td></tr><tr><td align="left" rowspan="1" colspan="1">Alpha-tubulin</td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">TTGCAACCATCAAGACCAAG</named-content></td><td align="left" rowspan="1" colspan="1"><named-content content-type="gene">TGCAGACGGGCTCTCTGT</named-content></td></tr></tbody></table></alternatives></table-wrap></sec><sec id="s4e"><title>Statistical Analysis</title><p>All statistical analyses were carried out using SPSS Inc. Chicago, U.S.A. (version 17.0). All data was tested for normality and homogeneity of variances. For data normally distributed, independent t-tests were performed to compare the means. For not normally distributed data non-parametric Mann-Whitney <italic>U</italic> comparison tests were performed to compare the means. Statistical significance was accepted at <italic>p</italic><0.05.</p></sec></sec></body><back><fn-group><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p><bold>Funding: </bold>This work was funded by the European Regional Development Fund in the framework of the INTERREG IV A France (Channel) - England programme (DIESE project number 4040). 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