The complete nuclear estrogen receptor family in the rainbow trout: Discovery of the novel ER alpha 2 and both ER beta isoforms

• Published in: Gene Vol. 392; no. 1-2; pp. 164 - 173
• Main Authors: Nagler, J J, Cavileer, T, Sullivan, J, Cyr, D G, Rexroad, C
• Format: Journal Article
• Language: English
• Published: 01.05.2007
• Subjects: Oncorhynchus mykiss
• ISSN: 0378-1119
• DOI: 10.1016/j.gene.2006.12.030

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ER alpha (NR3A1) and ER beta (NR3A2). The ER beta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ER alpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ER alpha subtype (i.e. ER alpha 1, NR3A1a). The purpose of this study was to determine whether the gene for the other ER alpha isoform, ER alpha 2 (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 ER alpha 1. Both the rainbow trout ER beta subtypes, ER beta 1 [NR3A2a] and ER beta 2, [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 ER alpha s (~40% similarity). ER beta 1 and ER beta 2 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 ER alpha subtype clade, with the closest relative to rainbow trout ER alpha 2 being rainbow trout ER alpha 1, suggesting a recent, unique duplication event to create these two isoforms. For the ER beta subtype there are two distinct subclades, one represented by the ER beta 1 isoform and the other by the ER beta 2 isoform. The rainbow trout ER beta 1 and ER beta 2 are not closely associated with each other, but instead fall into their respective ER beta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ER alpha 1, ER alpha 2, ER beta 1, and ER beta 2) 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.