MOLECULAR CLONING OF ESTROGEN RECEPTORS FROM FATHEAD MINNOW (PIMEPHALES PROMELAS) AND BLUEGILL (LEPOMIS MACROCHIRUS) FISH: LIMITED PISCINE VARIATION IN ESTROGEN RECEPTOR-MEDIATED REPORTER GENE TRANSACTIVATION BY XENOESTROGENS

摘要

Estrogen receptors (ERs) play essential roles in estrogen function. Because they may be targets of environmental chemicals, concern exists that exposure to some anthropogenic agents may result in disruption of endocrine systems in human and wildlife populations. Various kinds of ERs have been reported, and a number of in vitro assays have been established to test hormone-like activity of chemicals. In the present study, cDNAs encoding ERs from fathead minnow (Pimephales promelas) and bluegill (Lepomis macrochirus) fish, which are useful for environmental monitoring, were cloned and sequenced. Four complete, full-length ERα and ERβ cDNA sequences could be derived, with an ATG start site and a TGA termination signal included in each. Fathead minnow ERa has 602 amino acid residues, bluegill ERα1 has 582, bluegill ERα2 has 506, and bluegill ERβ has 554. When the cDNAs were inserted into the pRc/RSV vector and transfected into HeLa cells (derived from cervical cancer cells) with a reporter plasmid, the encoding proteins were confirmed to be functional through interaction of the receptor-ligand complex with the estrogen-responsive element (ERE). Here, we report the analysis of piscine differences in ER-dependent transactivation with some chemicals using reporter gene assays. In agonist assays, fold-induction of luciferase activity significantly varied among ERs, but responsiveness to each chemical demonstrated certain similarities. These reporter gene assays appear to be very useful for estimating adverse effects of chemicals, including hormone-like or antihormone-like activity, in fish.