Visual Detection Of Environmental Estrogens Transgenic Zebrafish Department To Establish And Applied Research

Environmental estrogens(EEs) are synthetic chemical compounds and plant-derived substance that are capable of imitating the functions of natural in vivo estrogen hormones.It has been known that estrogenic endocrine disrupters,which can cause intersexuality and reproduction decline in animals and human,can also induce synthesis of female-specific proteins such as vitellogenin(VTG) in male and juvenile fish.To establish a novel in vivo test system for rapidly and visibly detection of environmental estrogens,a transgenic zebrafish line was developed.First,the expression pattern of zvtg1 was demonstrated by RT-PCR and whole mount in situ hybridization in developing zebrafish exposed to synthetic estrogen 17αethinylestradiol(EE₂).Second,two DNA fragments upstream zvtg1 gene was cloned and the 1720bp fragment was identified as effective promoter in vitro by driving reporter green fluorescent protein(gfp) gene expression in MCF-7 cell line.Third,the zvtg1:gfp fusion DNA was microinjected into 1～2-cell stage embryos and faint green fluorescence in the liver was observed in larvae exposed to EE₂.Thus the promoter activity of 1720bp DNA fragment was identified in vivo.Fourth,in order to increasing GFP expression in transgenic zebrafish,a 39 base pairs DNA sequences contained the consensus estrogen response element(ere,GGTCAnnnTGACC) was synthesized according to the 5'end region of Xenopus vitellogenin A₂ gene,and was added upstream of the zvtg1 promoter.Then the fusion gene ere-zvtg1:gfp was microinjected into 1-2 cell stage embryos.Injected embryos were treated with 10ng/L EE₂ for 6 days and screened by examining fluorescence in the liver by using a fluorescence microscope.Founders were raised till sexually mature and individually mated with wild-type fish,and the offspring were examined fluorescence after being treated with 10ng/L EE₂ for 6 days.Of the 119 founders one germline-transmitted transgenic fish was identified.F1 fish fry obtained from the positive founder were screened by observing fluorescence and the positive F1 fish were raised.Two positive male F1 fish were survived and mated with wide type females to generate F2 fish.F2 males were mated with F2 females to breed F3 generation which was intercrossed with wide type zebrafish to generate F4 fish.Finally,the ere-zvtg1:gfp transgenic line was confirmed by 100%fluorescence induction in the F4 generation.In this transgenic line,GFP was exclusively expressed in the liver of the mature adult female.Male and larval transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17α-ethynylestradiol(EE₂). Concurrent of zvtg1 and gfp expression in embryos and larvae after EE₂ exposure was observed,which indicated that the expression of gfp transgene was driven by the zvtg1 promoter.Green fluorescence was first observed in the liver at 53,74,100 or 131 hours post fertilization(hpf) after exposure to 100,10,1 or 0.1 ng/L EE₂ respectively from 1-2 cell stage.As for mature male transgenic zebrafish,green fluorescence was observed after exposure to 100,10,1 or 0.1 ng/L EE₂ for 2,3,4 or 7 days respectively; as for mature female,fluorescence enhanced after exposure to relatively high concentrations of EE₂(10 and 100 ng/L).Green fluorescence in the liver was increased with prolonging of exposure time and was repeatedly induced after removal and re-addition of EE₂.We also demonstrated that GFP expression could be induced by other estrogenic compounds,includingβ-estradiol(E₂,0.1μg/L),Cadmium chloride(CdCl₂,10μg/L),zearalenone(50μg/L),estriol(E₃,1μg/L ), diethylstilbesterol(DES,50 ng/L) bisphenol A(BPA,1 mg/L)but not by week estrogenic compounds such as nonylphenol(NP,up to 10 mg/L),or non-estrogenic steroid hormone such as progesterone(up to 100 mg/L) and 17hydroxysteroid(up to 50 mg/L).These data suggest the transgenic zebrafish is sensitive and specific for detection of estrogenic compounds.Because the observed-effective concentrations are as low as those of environment and the observed-effective exposure times are very short,this transgenic fish is hopeful for monitoring environmental estrogens directly. rapidly and easily.Using this transgenic line,we investigated the molecular mechanism involved in EE₂ caused feminination and abnormal gonads in zebrafish.The results suggested estrogen receptorβwas needed for EE₂ induced zvtg1 expression in larval zebrafish. We also demonstrated high concentration of EE₂(500ng/L) disturbed the primordial germ cells(PGCs) migration in embryonic zebrafish,and this effect seemed having no relationship with estrogen receptors.