The complete nuclear estrogen receptor family in the rainbow trout: Discovery of the novel ERα2 and both ERβ isoforms

Abstract

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERα (NR3A1) and ERβ (NR3A2). The ERβ subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERα have not been demonstrated in any fish species. In the rainbow trout ( Oncorhynchus mykiss), the only ER described is an isoform of the ERα subtype (i.e. ERα1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERα isoform, ERα2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERα1. Both the rainbow trout ERβ subtypes, ERβ1 [NR3A2a] and ERβ2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERαs (∼ 40% similarity). ERβ1 and ERβ2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERα subtype clade, with the closest relative to rainbow trout ERα2 being rainbow trout ERα1, suggesting a recent, unique duplication event to create these two isoforms. For the ERβ subtype there are two distinct subclades, one represented by the ERβ1 isoform and the other by the ERβ2 isoform. The rainbow trout ERβ1 and ERβ2 are not closely associated with each other, but instead fall into their respective ERβ subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms ( ERα1, ERα2, ERβ1, and ERβ2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.