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
]. 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 [4
]. 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
]. 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
], 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 [9
] and in vitro
] 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
]. The different ER subtypes are also widely expressed in body tissues in early life stages, from embryos to young larvae [15
], 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 [16
]. 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 [17
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 [18
]. 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
Use of tissue-specific promoters has become a powerful tool for studies on endogenous gene expression [22
] and to analyse the function of promoters [9
] 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 [26
]. 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
]. The Tol2
transposon system, originally identified in the medaka [29
], has recently been used to enhance the success rate of DNA integration into the zebrafish genome [30
]. This is illustrated in the work of [30
]) 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
]. 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
]. 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.