Submitted to: Gene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2006
Publication Date: 5/1/2007
Citation: Nagler, J., Cavileer, T., Sullivan, J., Cyr, D., Rexroad III, C.E. 2007. The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel er.2 and both er. isoforms. Gene Vol 392(1-2)164-173. Interpretive Summary: Estrogens are hormones of great biological interest due to their multi-faceted and wide-ranging effects in vertebrate animals. For an estrogen to exert its biological effect it must interact with estrogen receptors, whose activities are directed either at the cell surface or the cell nucleus. In this report we describe our efforts to identify all of the nuclear estrogen receptors for the rainbow trout. Due to a whole-genome duplication event suspected to have occurred in ray-finned fishes, many genes found in single copies in the mammalian genome are often found to be duplicated in these fishes. We identified and characterized the novel DNA sequences of three nuclear estrogen receptors from the rainbow trout, completing the identification of this gene family for this species and for the first time in any fish.
Technical 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, ERa (NR3A1) and ERb (NR3A2). The ERb subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERa have not be demonstrated in any fishes. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERa subtype (i.e. ERa1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERa isoform, ERa2 (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 ERa1. Both the rainbow trout ERb subtypes, ERb1 [NR3A2a] and ERb2, [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 ERas (~40% similarity). ERb1 and ERb2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides 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 ERa subtype clade, with the closest relative to rainbow trout ERa2 being rainbow trout ERa1, suggesting a recent, unique duplication event to create these two isoforms. For the ERb subtype there are two distinct subclades, one represented by the ERb1 isoform and the other by the ERb2 isoform. The rainbow trout ERb1 and ERb2 are not closely associated with each other, but instead fall into their respective ERb subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERa1, ERa2, ERb1, and ERb2) 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.