Molecular Cloning of an Estrogen Receptor β Subtype from the Goldfish,Carassius auratus

Abstract

The brain of many teleost fish species, including the goldfishCarassius auratus, expresses exceptionally high levels of cytochrome P450 aromatase (estrogen synthetase). To begin investigating the molecular and cellular targets of estrogen action in goldfish brain, a polymerase chain reaction (PCR) cloning strategy was used to isolate an estrogen receptor (ER) complementary DNA (cDNA). The 2283-bp cDNA isolated from goldfish liver encoded a protein of 568 amino acids (aa) with an estimated molecular weight of 63,539. The goldfish ER had high overall sequence identity when compared to other vertebrate ER sequences: eel (64%), human β subtype (59%), human α subtype (46%), medaka (46%), and rainbow trout (47%). The highest degree of conservation was seen in the DNA-binding (94–100%) and ligand-binding (67–79%) domains. Phylogenetic analysis of the ER gene family indicated that the goldfish and eel ER are most closely related to mammalian ERβ subtypes, whereas previously identified fish, amphibian, and avian ER forms cluster separately with mammalian ERα subtypes. Using the goldfish ER cDNA (here designated gfERβ), multiple mRNA species (3.1– 8.6 kb) were detected by Northern blot analysis in goldfish liver and ovary but expression was below detection in brain. Using reverse transcription-PCR analysis, gfERβ mRNA was detected in forebrain, mid/hindbrain, pituitary, retina, liver, ovary, and testis. Further studies are required to determine whether an additional ERα subtype is present in the goldfish and whether ERα or ERβ forms have evolutionary precedence in vertebrates.