The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta 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, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (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 ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [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 ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 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 ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) 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.

Fig. 1

Alignment comparison of the deduced amino acid sequences for rainbow trout ERα1 (GenBank accession no. AJ242741), ERα2 (GenBank accession no. DQ177438), ERβ1 (GenBank accession no. DQ177439), and ERβ2 (GenBank accession no. DQ248229). The ‘*’ indicates positions which have a single, fully conserved residue. The different domains of the proteins, A–F, are indicated beginning at the N-terminus (top). The different domains are demarcated by the C- and E-domains, which are shaded in gray. Conserved residues within the transactivation factors AF-1 (A/B-domain) and AF-2 (F-domain) are boxed. The zinc-finger motif cysteines are indicated in bold within the C-domain.

Fig. 2

Maximum-likelihood estimate of the gene-family phylogeny for all currently documented fish ERs. This estimate was derived following an iterative search strategy, under the model selected using decision theory. Taxon labels in green are sequences of Acanthopterygii, in blue are Ostariophysi, in red are Protacanthopterygii (i.e., salmonids), and the others in black. The rainbow trout sequences reported in this study are in bold red. Branches denoted by two asterisks are supported by Bayesian posterior probabilities of 100%, those with a single asterisk have probabilities >95%. Nodes with support between 50% and 95% are indicated. Parsimony bootstraps are indicated below the branches.

Fig. 3

Histograms showing the relative ER mRNA expression, measured by real-time RT-PCR, for each ER isoform (A—ERα1: α 1; B—ERα2: α 2; C—ERβ1: β 1; D—ERβ2: β 2) in various rainbow trout tissues (tissue legend, from top to bottom, corresponds with left to right on the x-axis). The data is presented as a mean+/−standard deviation (n=three fish). Note: all ER mRNAs were detected in all tissues studied, however due to low ER mRNA levels in some cases the relative expression is not observable due to the y-axis scale used.