- Title
- Investigation of the molecular mechanisms underlying estrogen-mediated induction of vitellogenin gene expression in the Sydney rock oyster, Saccostrea glomerata
- Creator
- Tran, Thi Kim Anh
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Environmental estrogens are known to interfere with normal development and reproduction in a wide range of marine invertebrates. Previous studies have developed an oyster biomonitor to indicate the presence of estrogenic compounds in marine environments through exploiting the induction of the egg yolk protein precursor vitellogenin (Vtg). Despite this advance, the mechanism through which estrogens exert their action on Vtg gene expression, in particular the functional role of the estrogen receptor (ER), is currently unknown. In an attempt to fill this knowledge gap, the present research aims to isolate the genes encoding Vtg (sgVtg) and ER (sgER) from the Sydney rock oyster Saccostrea glomerata and investigate different potential mechanisms contributing to estrogen mediated induction of sgVtg. Our results indicated that the deduced protein of sgVtg is substantially longer than those of the other oyster Vtgs reported so far and contains all the conserved domains as found in other marine molluscs. The sgVtg promoter contains multiple putative half-EREs which are closely spaced, implying that they may function as an estrogen response unit (ERU) to interact with ER. In line with the potential involvement of ER in sgVtg regulation, the induction of sgVtg mRNA expression in ovarian explants was shown to be abolished by the ER antagonist ICI 182, 780. Considering that the vertebrate-like ER so far reported in molluscs lacks estrogen-binding ability, this finding supports the requirement of a novel estrogen-binding receptor for gene activation. The sgER cDNA is predicted to encode a 477-amino acid protein, which contains a DNA binding domain (DBD) and a ligand binding domain (LBD) conserved among vertebrate and invertebrate ERs. Comparison of the sgER LBD sequence with those of other ligand-dependent ERs indicated that the sgER LBD is degenerate at several conserved residues critical for ligand binding and receptor activation. Its inability to bind estrogens was then confirmed by a ligand binding assay using fluorescent-labelled E2 and purified sgER protein. The 5′-flanking region of sgER contains three putative ½EREs and several other putative elements for ER-interacting transcription factors, suggesting potential autoregulation of sgER expression. sgER mRNA is ubiquitously expressed in various tissues, with the highest expression level observed in the ovary where sgVtg is highly expressed. Functional analyses, including luciferase gene reporter assays, point mutation of ½ EREs and an electrophoretic mobility shift assay (EMSA), confirmed that sgER binds and activates the sgVtg promoter through ½EREs. In addition, sgER mRNA was significantly upregulated following in vitro and in vivo exposure to E2 and the enhancing effect of E2 on sgER expression was abolished by co-treatment with the specific ER antagonist ICI 182, 780 in vitro. These findings support the presence of a novel estrogen-binding receptor in S. glomerata. To elucidate whether estrogens modulate sgVtg and sgER expression at the epigenetic level, we assessed the DNA methylation levels of a 5’ intragenic CpG island in sgVtg and a promoter CpG island in sgER in ovaries after E2 exposure in vivo. Bisulfite sequencing revealed that both of these CpG islands are hypomethylated in both control and E2-treated oysters. Neither significant differential DNA methylation nor correlation between DNA methylation and mRNA levels was observed for either sgVtg or sgER. Overall, the findings from this research provides new molecular insights into how environmental estrogens regulate Vtg expression in marine molluscs and lays the foundation for further research into the mechanism of action of estrogenic compounds on molluscan vitellogenesis.
- Subject
- vitellogenin; estrogen receptor; gene expression; estrogen responsive elements; Sydney rock oyster; molluscs; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1343169
- Identifier
- uon:29100
- Rights
- Copyright 2017 Thi Kim Anh Tran
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT01 | Thesis | 13 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 476 KB | Adobe Acrobat PDF | View Details Download |