Analysis of estrogenic effects by quantification of green fluorescent protein in juvenile fish of a transgenic medaka

Abstract

TheChgH-GFP strain of the teleost medaka contains a regulatory region of the estrogen-responsive choriogenin H (chgH) gene fussed to the green fluorescent protein (GFP) gene. The strain was developed for the identification of environmental estrogens by noninvasive analysis of GFP fluorescence. In the present study, a quantification method for GFP by image analysis was establishedand applied to the analysis of time- and concentration-dependent GFP fluorescence in juvenile fish. Concentration-response analyses were performed with fish exposed for 14 d to 17beta-estradiol (0.37-367 pM), genistein (0.37-367 nM), or p-nonylphenol (0.367-1,835 nM). By means of image analysis, it was shown that ChgH-GFP was induced at 183.5 pM or greater 17beta-estradiol. Time-course and recovery experiments indicated a strong accumulation of GFP in the liver. Results of reverse transcriptase-polymerase chain reaction analysis of choriogenin H and vitellogenin demonstrated induction of gene expression for the same rangeof concentrations as that for GFP analysis. Neither expression of these genes nor GFP fluorescence was induced by genistein and p-nonylphenol. Although the ChgH-GFP strain failed to detect these weakly estrogenic compounds, the simplicity of the GFP quantification during early life stages of fish offers promising possibilities for further developments of transgenic strains using different target regulatory sequences.

The National BioResource Project Medaka (NBRP Medaka): An Integrated Bioresource for Biological and Biomedical Sciences

“…2G): Choriogenins are precursor proteins present in the egg envelope that are expressed in the mature female liver following induction with estrogen. In this transgenic line, the GFP gene was introduced into the transparent see-through line STII under the control of the choriogenin H gene regulatory region [21,40]. Estrogenic activity in the water can then be monitored as an expression of GFP in the liver of adult males or larvae.…”

Aquatic animal models of human disease: Selected papers and recommendations from the 4th Conference

“…As is seen with certain of the fish species reported herein advantages include: small size, rapid development and short generation time, and transparent, externally developing embryos that facilitate experiments in developmental toxicology and mixture evaluation. Transgenic species (Linney and Udvadia, 2004a; Kurauchi et al, 2005; Scholz et al, 2005; Chen et al, 2007) and gene knockdown (Nasevicius and Ekker, 2000) are well-developed approaches serving as powerful tools for mechanistic research. These and other advantages of small fish models have been described in detail elsewhere for zebrafish (Carvan et al, 2005; Hill et al, 2005; Linney et al, 2004b,) medaka (Oxendine et al, 2006), Atlantic killifish ( Fundulus heteroclitus ) (Burnett et al, 2007) and rainbow trout ( Oncorhynchus mykiss )Bailey et al, 1996).…”

Detection of environmental estrogenicity using transgenic medaka hatchlings (Oryzias latipes) expressing the GFP-tagged choriogenin L Gene

“…Using yolk-sac larvae of this strain, they also observed GFP induction in liver 24 h after the onset of aqueous exposure to 0.63 nM 17α-estradiol (E2). Further, Scholz et al [30] developed a method of quantifications of GFP using ChgH stain juvenile medaka. ChgL and ChgH show very weak sequence homology but induction of different sensitivity may be possible for these genes by E2.…”