Molecular Cloning, Characterization, and Evolutionary Analysis of Estrogen Receptors from Phylogenetically Ancient Fish

Abstract

Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates, and they promote the growth and differentiation of the adult female reproductive system. To understand the evolution of vertebrate estrogen receptors (ESRs) and to evaluate estrogen receptor-ligand interactions in phylogenetically ancient fish, we used PCR techniques to isolate the cDNA encoding ESRs from lungfish, sturgeon, and gar. Sequence analyses indicate that these fishes have two ESRs, ESR1 (ERα) and ESR2 (ERβ), as previously reported for other vertebrate species, but a second type of ESR2 (ERβ2) was not found as has been reported in a number of teleost fishes. Phylogenetic analysis of the ESR sequences indicated that the lungfish ESRs are classified to the tetrapod ESR group, not with the teleost fish ESRs as are the ESRs from gar and sturgeon. Using transient transfection assays of mammalian cells, ESR proteins from these three ancient fishes displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. We also examined the estrogenic potential of o,p′-dichloro-diphenyl-trichloroethane (o,p′-DDT) and p,p′-DDT as well as one of its common metabolites, p,p′-dichloro-diphenyl-ethylene (p,p′-DDE) on the ESRs from these fishes. Lungfish ESR1 was less sensitive to DDT/DDE than the ESR1 from the other two fishes. The response of lungfish ESR1 to these pesticides is similar to the pattern obtained from salamander ESR1. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine-disrupting mechanisms in three species of phylogenetically ancient fish and also expands our knowledge of ESR evolution.