Expression Patterns of the chgH:rfp Transgene in Response to 17α-Ethinylestradiol (EE2) Exposure in Marine Medaka Oryzias dancena

Abstract

The functional utility of a transgenic marine medaka Oryzias dancena strain carrying the red fluorescent protein (RFP) gene driven by an endogenous choriogenin H (chgH) promoter was evaluated for its ability to detect waterborne 17α-ethinylestradiol (EE2), a synthetic estrogen derivative. The chgH:rfp transgenic marine medaka larvae showed an age-dependent tendency in the efficiency of EE2-mediated transgene expression, in which transgenic larvae older than 6 days post-hatching displayed a more effective response in their transgene expression to EE2 than did younger hatchlings. During experimental exposures to high concentrations of EE2 (200 to 1,000 ng/L), the transgenic responses in the hatchlings were broadly dose-and duration-dependent. With exposures using lower doses of EE2 (25, 50 and 100 ng/L), EE2-induced transgenic RFP was also observed in the transgenic larvae, although the lower doses required exposure of longer duration. Under the EE2 exposure and microscope assay conditions used in our study, transgenic marine medaka larvae exhibited a similar degree of EE2-mediated RFP phenotype expression at various salinity levels (0, 15 and 30 ppt).

Transgene chgH-rfp expression at developmental stages and reproductive status in marine medaka (Oryzias dancena)

“…However, in the present study, none of transgenic females showed naturally occurring RFP signals, suggesting that the regulation of transgenic chgH would not be robust yet in sexually differentiated, but immature, transgenic fish, which is generally in accordance with the anticipated role of the choriogenin as the main constitute of egg envelop (Hong et al 2009). However, these young immature individuals have been proven to respond readily to exogenously administered estrogens as reported previously (Cho et al 2013;Nam et al 2015).…”

“…Of various estrogen-responsive transgenic lines generated in marine medaka, the transgenic strain used in this study was the stable line showing the strongest and the most sensitive response to both natural and synthetic versions of the estrogen compounds. Characteristics of this transgenic line [labeled TG#038 in Cho et al (2013)] in details could be referred to our previous reports (Cho et al 2013;Nam et al 2015).…”

“…Because we aimed to examine primarily whether the estrogen-mediated RFP signal would be induced exclusively in the main target organ (i.e., liver) or ubiquitously also in other non-liver organs, we exposed transgenic males to high concentrations of estrogens [1.0 μg L −1 (for E2) and 0.5 μg L −1 (for EE2)]. These exposure conditions had been known to induce robustly endogenous chgH transcripts in male adults or RFP signals in transgenic larvae of this medaka species (Cho et al 2013;Nam et al 2015;Lee et al 2012). We did not consider the evaluation of the lowest-observed-effect concentrations (LOECs) of E2 and EE2 here.…”

“…The extremely high osmoregulatory capability of this species could facilitate the practical application of estrogenresponsive transgenic strains to a wide range of environmental salinity, as evidenced in the established transgenic strains carrying the red fluorescent protein (RFP) gene under the control of endogenous choriogenin H promoter (chgH-rfp transgenics) (Cho et al 2013;Nam et al 2015). Although the potential usefulness of chgHrfp transgenic marine medaka has been successfully demonstrated (Cho et al 2013), performance data on their estrogen-responsive function have been limited only to hatchling and larval stages.…”