Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos
© Lee et al.; licensee BioMed Central Ltd. 2012
Received: 13 February 2012
Accepted: 24 June 2012
Published: 24 June 2012
Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).
The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.
Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.
KeywordsOestrogenic endocrine disrupting chemicals Oestrogen response elements UAS-GAL4 Transient expression assay Zebrafish
Decades of research have shown that a number of natural and man-made chemicals interfere with the endocrine system and can result in adverse health effects in humans, mammals and fish [1–3]. Wildlife living in, or closely associated with the aquatic environment have been shown to be especially impacted by these so-called endocrine-disrupting chemicals (EDCs), because our freshwaters, estuaries and oceans act as sinks for chemical discharges . Oestrogenic EDCs in the environment are of particular concern. Endogenous oestrogens are a group of closely related steroid hormones essential in the development and functioning of the reproductive system. Extensive work has been conducted on natural (e.g. 17β-oestradiol) and synthetic oestrogens (e.g. 17α-ethinyloestradiol from the contraceptive pill) and their interactions in the vertebrate body, including their tissue distributions, mechanisms of action and pathways of elimination [5–7]. Furthermore, adverse effects of exposure to environmental oestrogens have received considerable research attention in a number of animal species, but this work is largely restricted to adults and juveniles [6, 8], and there has been little study of effects on embryos. Nothing is known regarding the relative sensitivities of the different cell types and tissues to oestrogenic EDCs, or other contaminants, during embryogenesis. Furthermore, in spite of the widespread concern for EDCs in the aquatic environment, there are very few bioassay systems that are sufficiently sensitive for accurate prediction of adverse biological effects. Moreover, conventional methods of EDCs detection such as tissue culture  and in vitro techniques [10, 11] are limited in their capacity to elucidate oestrogen signalling pathways and tissue specific physiological impacts.
Teleost fish have three oestrogen receptors (ER), ERalpha, ERbeta-1 and ERbeta-2, that show tissue specific patterns of expression and function in adults [12–14]. The different ER subtypes are also widely expressed in body tissues in early life stages, from embryos to young larvae , suggesting crucial roles of these signalling pathways in early development. Indeed, recently it was found that knockdown of ER-beta2 in the zebrafish suppressed normal development of the lateral line neuromast cells . Endogenous oestrogen receptors activated by oestrogenic chemicals bind to oestrogen response elements within regulatory regions of oestrogen-responsive genes. Response elements are recognized by nuclear transcription factors, including members of the steroid/nuclear receptor super family that then, together with various other regulatory factors mediate transcription of the associated downstream genes .
The adopted model in this work, the zebrafish (Danio rerio) has become one of the most commonly used animals for examining effects of aquatic pollutants . Furthermore, with the available genomic resources and suitability of this species for molecular manipulations, the zebrafish has been applied more widely for research in developmental biology and understanding of disease processes. The medaka (Oryzias latipes) is another model species widely used in ecotoxicology research and for the development of transgenic techniques. Many studies have shown that early life stages of fish have the greatest sensitivity to environmental contaminants and effects on development in the zebrafish and medaka are greatly facilitated by the fact that their embryos are transparent. [19–21].
Use of tissue-specific promoters has become a powerful tool for studies on endogenous gene expression  and to analyse the function of promoters [9, 23–25] and transgenic fish have become an established technique in developmental analyses. This generally includes using a specific promoter and a green fluorescent protein (GFP) or a luciferase reporter gene. To improve the efficiency of the sensitivity, tissue specificity and ease of generating transgenic fish, various manipulated gene systems have recently been introduced. One of these is the Gal4-UAS system. This is now used widely for the over expression of transgenes in various transgenic animals, including zebrafish . This system comprises a two-part expression system that utilises the yeast transcription activator protein Gal4 and its target sequence UAS (Upstream Activated Sequence), to which Gal4 binds to activate gene transcription [27, 28]. The Tol2 transposon system, originally identified in the medaka , has recently been used to enhance the success rate of DNA integration into the zebrafish genome [30, 31]. This is illustrated in the work of [30, 32]) where used of Tol2 increased the transgenesis rate of linear DNA from 5% to 50%.
Transgenic zebrafish have considerable potential for use in aquatic ecotoxicology to screen and test for hormone mimics and potentially to develop a more advanced system for assessing health impacts of chemicals. As a consequence there has been considerable activity in a number of laboratories to develop transgenic zebrafish as tools for screening and testing of chemicals [33–36]. Transgenic fish have the advantage that tissue specific effects of EDCs can be identified to allow for more directed and detailed studies to inform on health outcomes. However, it is time consuming, both to produce and maintain the stable transgenic lines. As a consequence, a number of studies have investigated the use of transient expression assays to examine the spatial and temporal expression of reporter genes that are fused to the regulatory regions of various genes in zebrafish embryos [37, 38]. To date, biosensor transgenic fish (TG fish) have only been generated in the zebrafish and medaka, and such technology has not been applied widely to other fish species. In theory, however, having developed the technology for these model species, it would be possible to develop a functional ‘transient assay’, by which vector DNA is transiently introduced into the fish embryo, in almost any fish species where the eggs produced can physically be injected, and thus examine the effect of chemical exposure specifically in those fish species.
To date, there have not been any reported transient expression assays for the detection of oestrogenic EDCs. The transient expression assay process normally involves the injection of fertilised embryos with a construct and followed by assaying of the response (e.g. GFP or Luciferase) once the embryos/larvae have reached the desired stage of development. A major advantage of the transient assay would be that the technique is applicable to a variety of fish species for examining tissue, stage and chemicals specific responses without the need to generate TG fish lines.
With a plan to develop a rapid and sensitive transient expression assay system to evaluate the oestrogenic activity of environmental chemicals in different fish embryos, we utilised a Gal4ff-UAS system that incorporates a two-step signal amplification process and a synthetic oestrogen responsive element with 3EREs. A major aim in this initial work was to make a construct that was highly responsive to oestrogenic EDCs. We then examined the responsiveness of the oestrogen response elements and tissue and stage specific responses in zebrafish using a transient expression assay. Finally, we tested our plasmid in another fish species, the medaka, to show that transient expression assay is suitable for observation of effects of oestrogenic compounds in other fish test species.
Results and discussion
Construction of reporter gene vectors with oestrogen responsive promoters
In the sequence of results reported, we first developed a novel transient assay system using a synthetic oestrogen responsive element, Tol2 and Gal4-UAS systems and the GFP reporter gene, responsive to environmental oestrogen. We then investigated the functional capability of this construct and response to a range of potent and weak environmental oestrogens using a transient expression assay using green fluorescent microscopy in zebrafish embryos/larvae. Finally, we proofed our vector system further in another fish species, the medaka.
In a mammalian cell culture system, Sathya et al. (1997)  showed that a construct with 3EREs produced an enhanced expression compared with 2EREs and 4EREs, especially in response to the natural oestrogen, oestrdaiol-17β (E2). We therefore similarly adopted three copies of ERE in our construct. For the transcriptional initiation, the TATA box sequence was inserted at 158 bp upstream of the Gal4ff start codon and the Tol2 transposon system was adopted to provide a more efficient transient reporter analyses. After the sequential synthesis of five successive constructs, we generated a novel plasmid vector system, p3ERE-TATA-Gal4ff containing, Tol2 and a poly A tail and employing two steps for amplification of the signal (Figure 1) where Gal4ff activates “UAS-GFP’. In the activation process, the first step, binding of oestrogens to ER and subsequent binding to ERE, induces the synthesis of GAL4ff protein and in the second step Gal4ff protein induces synthesis of GFP. This two-step process allows amplification of the resulting signal which, in theory, should give rise to a more “sensitive” transient expression assay system for detecting oestrogen chemicals than has been developed previously. In our approach, we used Gal4ff rather than the more commonly used Gal-VP16/UAS system, because GAL4ff is reported to be less toxic than GAL4-VP16 in zebrafish cells .
Induction of GFP in the zebrafish embryo transient expression assay
In the transient microinjection studies, expression of gene in zebrafish and medaka embryos was mosaic because the microinjected recombinant DNAs were not integrated to all cells. We minimised this problem by flanking the transgene with a Tol2 transposon and we were then able to identify target tissues for oestrogens in early life stages more effectively .
No differences in mortality rate were observed between both control and oestrogen chemical treated groups (> 95% survival), with the exception for 100 μg NP/L where the most embryos died by 24 hpf. The lack of toxicity effects in zebrafish embryos at the exposure concentrations adopted (with the exception of 100 ug NP/L) compared very favourably with previously reported findings (e.g. Scholz and Gutzeit (2000) for EE2 and Kishida et al. (2001) for E2).
GFP expression was seen at 24 h after exposure to EE2 (data not shown), albeit at low level, whereas there was no GFP expression detected in the controls at this time. There was stronger and more specific tissue GFP expression in EE2 treated embryos at 72 and 96 hpf, focused mainly in the liver, muscle and heart and this was therefore adopted as the time period for assessing tissue specific effects of oestrogens in the transient assay.
Percentage of plasmid injected zebrafish expressing GFP in different body tissues after exposure to oestrogens (at 72 hpf)
lateral line neuromast
To assess the consistency of transient expression of GFP, we counted the number of GFP-positive embryos for different tissues at 72 hpf. As shown in Table 1, for controls no GFP expression was observed in the liver, heart and forebrain, but weak GFP expression signals were detected in some embryos in somite muscles (13.3%) and the otic vesicle (16%). In contrast, in injected embryos (72 hpf), strong GFP expression occurred in the muscle and heart on exposure to both EE2 and E2. This was also the case for the liver in injected embryos exposed to both EE2 and E2: 74% for 1000 ng EE2/L, 62% for 100 ng EE2/L and 71% for 1000 ng E2/L. For the forebrain, the proportion of embryos expressing GFP were 33% and 26% for exposure to 1000 ng EE2/L and 1000 ng E2/L, respectively. For the NP exposure, the proportion of injected embryos showing GFP expression in muscle was 71% and 56% for the heart.
Assessments of the relative potencies of various environmental oestrogens in our transient assay were consistent with those reported in the literature for other assays systems, with EE2 being the most potent compound compared with E2, NP and BPA. Legler et al. (2002)  similarly reported that EE2 was the most potent (xeno) oestrogen compared with E2, NP and di(2-ethylhexyl)phthalate (DEHP) in their transgenic zebrafish assay. Relative potency estimates for these chemicals however, can vary depending on the assessment method. For example EE2 was shown to be 1.25-fold more potent than E2 in yeast-based in vitro assay  but had around a 30 times higher potency than E2 and E1 for VTG induction (measured as a protein in the plasma or VTG mRNA in the liver) in female zebrafish  in in vivo. Thorpe et al. (2003)  using a VTG induction assay in vivo, reported that EE2 was between 11 to 27 times more potent than E2. The responses to oestrogen in our zebrafish transient assay developed have shown it to be as sensitive as some other widely used oestrogen detection systems in fish (e.g. VTG induction), and as sensitive as available oestrogen responsive TG fish .
Induction of GFP in the medaka embryo transient expression assay
GFP expression observed in the skin and gallbladder in medaka was not observed in zebrafish, which might suggest that oestrogen receptor expression varies between fish species or oestrogen receptor binding affinities for different fish species might be different in the different embryos, at least for these organs. Differences between fish species have been shown for tissue distribution of ER subtypes as well as differences in ER affinities for environmental oestrogens ([59–61]). We do not yet know how the oestrogenic response pattern observed by GFP expression is related to the expression of ERs. The available literature suggests that ERs are expressed ubiquitously in the whole embryos both in zebrafish and medaka with an elevated expression in the neuromast cells in zebrafish [16, 62]. Given this, the tissue specific response of GFP to oestrogenes may be as a consequence of the co-factors associated with ER signalling pathway.
Despite the fact that transient expression analysis has been used in various areas of developmental biology, there has been no report using transient expression analysis for detection of oestrogen compounds. The vector system we have developed and applied in a transient assay effectively detects oestrogenic responses in body tissues. Furthermore, the transient assay system is as sensitive as many of the available TG fish for detecting oestrogens, in some cases considerably more so  There are however, exceptions to this, where the most sensitive TG fish developed have been shown to respond to oestrogens at 1 ng EE2/L (5 ng E2/L)  and 10 ng EE2/L (300 pM E2 (approximately 81 ng E2/L)) [33, 35]. Drawbacks of the transient expression assay over TG fish includes the fact that mosaicism can make it more difficult to detect GFP expression in physically small tissues such as lateral line neuromast.
Comparing our transient assay system with VTG induction, a well established biomarker of oestrogen exposure, the detection threshold for EE2 (as an example) in zebrafish in vivo is 2 ng EE2/L . Induction of VTG in medaka and common carp hepatocytes in culture is reported at 30 ng E2/L  and 0.01 μM E2, respectively. The EC50 concentration (half maximal effective concentration) for EE2 in the MCF-7 cell assay and yeast-oestrogen screen (YES) from Folmar et al. (2002)  is 0.017 nM (5 ng/L) and 0.29 nM (86 ng/L), respectively.
The vector system developed detected oestrogenic EDCs in both the zebrafish and medaka, indicating the likely wide suitability for application to other fish species. This system would be a particularly powerful technique for use in fish species with long generation times and/or where there are other difficulties for generating transgenic lines in those species. The transient expression assay further provides a novel in vivo system for investigating oestrogenic effects on embryo development in fish.
DNA construct for developing a transient assay
The construct, called pTol2_3ERE-TATA-Gal4ff was based on the sequence of EREc38 (EREc38 5’-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3’) published by Sathya et al. (1997) . The construct was generated by Polymerase Chain Reaction (PCR) using primers (upstream primer CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCA GCC AGGTCAGAGTG and downstream primer CTGAATGTGTTATTTTAGCTCAGG TCACTC TGACCTGGCTGAATGTGTTAT). The following conditions were used for 8 PCR cycles : denaturation at 96 °C for 1 min, annealing at 60 °C and extension at 72 °C for 1 min. Multiple tandem copies of EREc38 (nEREc38, where n = number of ERE copies) were obtained by PCR. EREc38 oligomers were prepared by restriction digestion of the 3ERE with SmaI. The reporter gene vector pKS(+) was digested with XhoI and XbaI. After obtaining the 3ERE fragment, a second fragment called TATA was constructed following the same protocol, using the following primers: (upstream primer GGCGTCGACTCTAGAGGGTATATAATAGAT CTGCGATCTAAGTAAGCTTGG downstream primer CGCGGGCCCGGCTTTACCAACAG TACCGGAATGCCAAGCTTACTTAGATCG). TATA was digested with ApaI and SalI. Gal4-ff (amplified by PCR using primers: upstream primer GCCGGGCCCGCCACCATGAAGCTA CTGCTGTC TTCT downstream primer CGCGGTACCGATTAGTTACCCGGGAGC) was inserted into the vector which already contained the 3ERE and TATA. The BamHI and NotI fragments of p3ERE-TATA-Gal4ffwere inserted into the BamHI and NotI sites of the plasmid pBR322 vector , the latter containing the Tol2 transposon. The positive clones were confirmed by sequencing. The resulting plasmid was designated as pTol2_3ERE-TATA-Gal4ff.
Fish maintenance and microinjection of embryos with DNA
Zebrafish were maintained at 28 ± 1 °C under a 14 h light/10 h dark photoperiod and were fed Tetramin dry tropical flake food (Tetramin; Tetraweke, Melle, Germany) twice a day. Fertilised zebrafish eggs were collected within 20 minutes post-fertilisation. Medaka were maintained at 28 °C on a 14:8 light:dark cycle. All adult medaka in this study were fed Artemia twice a day. The concentration of plasmid was 20 pg/nl and 0.05% phenol red was added to make the solution visible. Under a dissecting microscope, fertilised eggs were placed in injection plate and eggs were microinjected with a syringe into the centre of the cell over the yolk-cytoplasm boundary. Embryos were microinjected at the 1–2 cell stage with 2 nl DNA using a microinjector (INTRACEL, PICOSPRITZER®III). Some of the embryos were also exposed to various chemicals as detailed below.
17α-ethinyloestradiol (≥98% purity). 17β-oestradiol (98% purity), and 4-Nonylphenol (Acros Organics) were purchase from Sigma Chemical Co. Ltd. The stock solutions of EE2, E2 and NP were prepared in acetone and subsequently stored at 4 °C, until required for the exposures. The working solutions were prepared 3 days before use in the exposures. Prior to exposure, the solvent was evaporated under a stream of nitrogen and the working solutions made up with water and stirred vigorously for 1 day. The nominal concentration of treatment solutions were: 17α-ethinyloestradiol (EE2): 1000 ng/L, 100 ng/L and 10 ng/L; 17β-oestradiol (E2): 1000 ng/L, 100 ng/L and 10 ng/L; 4-Nonylphenol (NP): 100 μg/L and 10 μg/L Chemicals were dissolved in embryo culture water. Fifty embryos per chemical concentration were exposed for up to 72 hours at 28 °C. All experiments were run in duplicate and were repeated at least seven times.
The transient expression assay using fluorescent microscopy detection
The plasmid pTol2_ERE-TATA-Gal4ff was mixed in a 1:1 ratio with the plasmid pTol2_UAS-GFP and then mixed with transposase mRNA in a ratio of 1:2. The mixture was microinjected (20 pg/nl) into 1–2 cell stage embryos of zebrafish. Injected zebrafish embryos were cultured for up to 72 hours (zebrafish) with and without exposure to E2, EE2 and NP, and GFP expression examined by fluorescence microscopy (Leica DMI 4000 B). In the studies with medaka, the plasmid pTol2_ERE-TATA-Gal4ff with transposase mRNA was mixed in a 1:1 ratio with the plasmid pTol2_UAS-GFP and this was then injected into 1–2 cell stage medaka embryos (20 pg/nl). Injected medaka eggs were exposed to EE2 (100 ng/L) and embryo culture water for 11 days at 28 °C (±1 °C). GFP expression was observed under a fluorescent microscope (NIKON SMZ1500).
Synthesis of transposase mRNA
Transposase mRNA was synthesised in vitro using pCS-TP which carries Tol2 transposase cDNA. The plasmid pCS-TP was digested with NotI and purified by phenol: chloroform extraction and ethanol precipitation. RNA synthesis was performed using mMessage mMachine SP6 kit (Ambion). The synthesized mRNA was purified with Quick spin column for radiolabeled RNA purification (Roche), purified by phenol: chloroform extraction and by the subsequently ethanol precipitated. The RNA precipitated was suspended in distilled water and used for co-microinjection with pTol2_ERE-TATA-Gal4ff.
Western blot analysis
Western blot analysis was employed to quantify GFP expression. Embryos were transferred to a 1.5 ml tube filled with lysis buffer (2x LDS (Lithium dodecyl sulfate) sample buffer (Invitrogen), 2-Mercapto ethanol and distilled water), incubated at 95 °C for 5 minutes and the vessel tapped to get them to the bottom of tube. Samples were homogenised five times and centrifuged for 1 min at 1000 rpm. Protein samples were applied to 5% polyacrylamide-SDS gel and subjected to electrophoresis at 110 V for 2 hour and separated proteins were transferred to nitrocellulose membrane. The membranes were blocked for 1 hour in blocking solution (5% skimmed milk in 1x phosphate buffered saline (PBS) + 0.1% Tween (PBSTx)), and washed three times with distilled water (ddH2O). The membranes were incubated overnight at 4 °C with primary antibody, rabbit anti-GFP (ams Biotechonology), diluted 1:2500 in blocking solution. The membranes were washed for 3 x 15 minutes in PBSTx. The membranes were incubated with HRP-Goat Anti-Rabbit IgG (Invitrogen, Carlsbad, U.S.A) at 1:2000 in blocking solution for 2 hours and then washed 3 x 15 minutes in PBSTx again. For detection, Western blotting luminol reagent (Thermo Scientific) was used. The intensity of GFP was analysed using Image J (http://rsbweb.nih.gov/ij/), normalised to the intensity of alpha-tubulin band and indicated as fold increase in GFP over the level in control larvae.
All data are reported as mean ± SEM. Statistical evaluation were performed using Student’s t-test and the comparison between controls and each exposed groups. Statistical significance is indicated at the p <0.05 level.
We thank the fish facility for fish husbandry, Koichi Kawakami for the Tol2 system. This work was co-funded by the Natural Environmental Research Council (reference NE/I014470/1) and the University of Exeter (CRT/TK). OL was funded by a studentship from Gwanglim, Korea.
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