title: Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.
abstract: Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5'- and 3'-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5' CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0-3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs.
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Thi Kim Anh
authors.initials: TK
authors.lastname: Tran
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Geoff R
authors.initials: GR
authors.lastname: MacFarlane
authors.affiliation: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong Special Administrative Region, China.
authors.forename: Richard Yuen Chong
authors.initials: RY
authors.lastname: Kong
authors.affiliation: New South Wales Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach, NSW 2316, Australia.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: O'Connor
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia. Electronic address: Richard.Yu@newcastle.edu.au.
authors.forename: Richard Man Kit
authors.initials: RM
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004967:Estrogens / Q000633:toxicity*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D010125:Ostreidae / Q000187:drug effects* / Q000235:genetics; D010053:Ovary / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014818:Vitellogenesis / Q000187:drug effects; D014819:Vitellogenins / Q000737:chemistry / Q000235:genetics*; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D018076:DNA, Complementary; D004967:Estrogens; D014819:Vitellogenins; D014874:Water Pollutants, Chemical
keywords: Estrogen receptor; Estrogen responsive element; Gene expression; Molluscs; Sydney rock oyster; Vitellogenin