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, 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.

Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change

“…Consistent with this hypothesis, the expression of estrogen receptors were found in the testis of teleosts, such as rainbow trout (Nagler et al 2007) and zebrafish (Menuet et al 2002). The overlapped expression of both CYP19 genes in the testis of the ricefield eel may offer a more refined regulation of testicular estrogen production, and thus male reproductive function.…”

Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle

“…In teleost fish, two subtypes of ERb (ERb1 and ERb2 -also named ERg) have been identified in distant taxa. In contrast, most fish species have only ERa, but two ERa isoforms (ERa1 and ERa2) have recently been reported in salmonids (rainbow trout; Nagler et al (2007)) and cyprinids (goldfish C. auratus and phoenix barb S. denticulatus; Zhu et al (2008)). Atlantic cod has a single ERa subtype, supporting the hypothesis that the two ERa subtypes (ERa1 and ERa2) reported in goldfish, rainbow trout, and phoenix barb are derived from the recent tetraploidization event that is thought to have occurred in the salmonid and cyprinid lineages.…”

Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17α-ethynylestradiol

“…Since this event salmonids have undergone Robertsonian rearrangements reducing their chromosome complement (66). Despite these rearrangements, genome duplication can be observed with additional alleles (beyond the normal two) having been identified for many genes (67)(68)(69). The presence of such additional alleles in rainbow trout may reduce detrimental effects of embryonic monosomy through some type of compensation, or partial compensation, which allows embryos to survive longer as a more equal level of transcript formation is maintained (i.e., 4:3 in trout vs. 2:1 in mammal monosomy).…”