Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor α gene P.I.S. Pinto a, ⁎, R. Teodósio a , S. Socorro b , D.M. Power a , A.V.M. Canário a a Centro de Ciências do Mar (CCMAR), CIMAR-Laboratório Associado, University of Algarve, Campus de Gambelas, 8005–139 Faro, Portugal b CICS-UBI - Health Sciences Research Center, University of Beira Interior, Av. Infante D. Henrique, 6200–506 Covilhã, Portugal abstract article info Article history: Accepted 26 April 2012 Available online 3 May 2012 Keywords: Alternative splicing Estrogen receptor Estrogen responsiveness Genomic organization Multiple transcripts Teleost fish Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Estrogens play important roles in the regulation of a wide range of physiological processes in female and male vertebrates. Estrogen functions are mainly mediated by specific estrogen receptors (ERs), which regulate the transcription of target genes by binding as homo or hetero dimers to their promoters (classical mechanism) or by interacting with other transcription factors, and may also mediate rapid non-genomic actions through activation of intracellular signalling cascades (Bjornstrom and Sjoberg, 2005). Two ER subtypes encoded by different genes (ERα and ERβ) have been characterized in mammals and show different but complementary functional properties, tissue expression and biological roles (Matthews and Gustafsson, 2003). In several teleost fish species, duplicated ERβ (βa and βb) genes have been identified which appear to have different functional properties, tissue distribution and expression patterns during development (e.g. Menuet et al., 2002; Tingaud-Sequeira et al., 2004; Pinto et al., 2006a). In mammals, variant ER transcripts are co-expressed with the wild-type (wt) ERs in normal and tumor tissues (e.g. Perlman et al., 2005; Nott et al., 2008; Springwald et al., 2010). These transcripts may result from multiple mechanisms, the most common being alternative promoter usage and alternative mRNA splicing with deletion of one or more exons (Tiffoche et al., 2001; Hirata et al., 2003; Perlman et al., 2005; Ishunina and Swaab, 2008; Nott et al., 2008). While some variant mRNAs only differ from the wild type in their stability or translation efficiency (Kos et al., 2002), others produce variant proteins with different functional properties that may stimulate or inhibit the transcriptional activities of the wt-ERα and/or ERβ proteins, or may mediate non-classical estrogen actions (e.g. Bollig and Miksicek, 2000; Flouriot et al., 2000; Lin et al., 2003; Peng et al., 2003). Some of the variant proteins were detected in vivo in mammalian tissues (Park et al., 1996; Flouriot et al., 2000; Poola et al., 2005; Ishunina and Swaab, 2008) and their potential to modulate the in vivo estrogenic actions during normal physiology or in disease is starting to be recognized (Ishunina and Swaab, 2008; Nott et al., 2008; Weickert et al., 2008; Ishunina and Swaab, 2010). In fish, however, information about variant ER transcripts and their functional significance is limited. cDNAs encoding N-terminal truncations of ERα/ERβa have been reported in rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus puntactus) and sole (Solea solea)(Pakdel et al., 2000; Patiño et al., 2000; Caviola et al., 2007); cDNAs encoding internal truncations or extensions and one antisense ERα mRNA in channel catfish (Patiño et al., 2000), and cDNAs with different 5′ or 3′ untranslated regions (UTR) have been reported for tilapia (Oreochromis aureus) and Gene 503 (2012) 18–24 Abbreviations: 18S, 18S ribosomal RNA; bp, base pairs; E 2 , estradiol-17β; ER, estrogen receptor; ERE, estrogen-response element; EST, expressed sequence tags; ICI, ICI 182,780; RT-PCR, reverse transcriptase-polymerase chain reaction; qPCR, quantitative real-time RT-PCR; UTR, un-translated region; wt, wild-type. ⁎ Corresponding author. Tel.: + 351 289800100x7336; fax: + 351 289800069. E-mail addresses: ppinto@ualg.pt (PIS. Pinto), rteodosio@ualg.pt (R. Teodósio), ssocorro@fcsaude.ubi.pt (S. Socorro), dpower@ualg.pt (DM. Power), acanario@ualg.pt (AVM. Canário). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.081 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene