This paper describes three distinct estrogen receptor (ER) subtypes: ER␣, ER␤, and a unique type, ER␥, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ER␥ arose through gene duplication from ER␤ early in the teleost lineage and indicates that ER␥ is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ER␥ shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ER␥s. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ER␤ and ER␥ fusion proteins can each bind estradiol-17␤ with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

Three forms of estrogen receptor: ERalpha, ERbeta (ERbetab), and a second ERbeta, ERbetaa (formerly ERgamma) are present in teleost fish. All ERbetaas share amino acid changes in the ligand binding domain that may influence ligand specificity and receptor function. We compared binding specificities of the three ERs of the teleost fish, Atlantic croaker Micropogonias undulatus. Bacterially expressed Atlantic croaker (ac) ERalpha, -betab, and -betaa fusion proteins showed specific, high affinity binding to 17beta-[(3)H]estradiol, with K(d) values of 0.61 +/- 0.013, 0.40 +/- 0.006, and 0.38 +/- 0.059 nm, respectively. Rank orders of binding were: diethylstilbestrol >> ICI182780 > 4-hydroxytamoxifen > ICI164384 > estradiol >/= zearalenone > moxestrol > tamoxifen > estrone >/= 17alpha-estradiol > estriol > 2-hydroxyestrone = genistein >> RU486 for acERalpha; ICI182780 > diethylstilbestrol > 4-hydroxytamoxifen > estradiol > ICI164384 > genistein > moxestrol > tamoxifen > zearalenone = estrone > estriol = 17alpha-estradiol > 2-hydroxyestrone >> RU486 for acERbetab; and estradiol >/= diethylstilbestrol > 4-hydroxytamoxifen > ICI182780 > ICI 164384 > estriol >/= genistein > moxestrol > zearalenone > estrone > 17alpha-estradiol > RU486 >/= tamoxifen > 2-hydroxyestrone for acERbetaa. acERbetaa showed higher relative binding affinities for estradiol, estriol, and RU486 and lower relative binding affinities for synthetic estrogens and antiestrogens than previously characterized ERs. Mutation of the conserved teleost substitutions (acERbetaaPhe(396)) to the ERalpha or ERbetab counterpart shifted diethylstilbestrol and tamoxifen affinities toward those of wild-type acERalpha and acERbetab, supporting the hypothesis that the positions with conserved residue changes in teleost ERs are important to ER structure and function.

The diversity and success of teleost fishes (Actinopterygii) has been attributed to three successive rounds of whole-genome duplication (WGD). WGDs provide a source of raw genetic material for evolutionary forces to act upon, resulting in the divergence of genes with altered or novel functions. The retention of multiple gene pairs (paralogs) in teleosts provides a unique opportunity to study how genes diversify and evolve after a WGD. This study examines the hypothesis that vitamin D receptor (VDR) paralogs (VDRα and VDRβ) from two distantly related teleost orders have undergone functional divergence subsequent to the teleost-specific WGD. VDRα and VDRβ paralogs were cloned from the Japanese medaka (Beloniformes) and the zebrafish (Cypriniformes). Initial transactivation studies using 1α, 25-dihydroxyvitamin D3 revealed that although VDRα and VDRβ maintain similar ligand potency, the maximum efficacy of VDRβ was significantly attenuated compared with VDRα in both species. Subsequent analyses revealed that VDRα and VDRβ maintain highly similar ligand affinities; however, VDRα demonstrated preferential DNA binding compared with VDRβ. Protein-protein interactions between the VDR paralogs and essential nuclear receptor coactivators were investigated using transactivation and mammalian two-hybrid assays. Our results imply that functional differences between VDRα and VDRβ occurred early in teleost evolution because they are conserved between distantly related species. Our results further suggest that the observed differences may be associated with differential protein-protein interactions between the VDR paralogs and coactivators. We speculate that the observed functional differences are due to subtle ligand-induced conformational differences between the two paralogs, leading to divergent downstream functions.

Sex steroid hormones regulate various neural functions that regulate vertebrate sociosexual behavior. A number of sex steroids can be synthesized de novo in the brain, including estrogens by the enzyme aromatase. Aromatase, the neuropeptides arginine vasotocin/vasopressin, and the monoamine neurotransmitter dopamine have all been implicated in the control of male sexual and aggressive behavior in a variety of vertebrates. This study examined the expression of brain aromatase in a teleost fish, the bluehead wrasse (Thalassoma bifasciatum), a teleost fish that exhibits sociallycontrolled behavioral and gonadal sex change. We used immunocytochemistry (ICC) to characterize distributions of aromatase-immunoreactive (ir) cells, and to examine their relationship with AVT-ir neurons, and tyrosine hydroxylase-ir (TH-ir) neurons in the key sensory and integrative areas of the brain of this species. Aromatase-ir appeared to be in glial cell populations, and was found in the dorsal and ventral telencephalon, the preoptic area of the hypothalamus, and the lateral recess of the third ventricle, among other brain areas. Aromatase-ir fibers are closely associated with AVT-ir neurons throughout the preoptic area, indicating the potential for functional interactions. Aromataseir cell bodies and fibers were also co-regionalized with TH-ir neurons, suggesting possible interaction between the dopaminergic system and neural estrogen production. The presence of aromatase in brain regions important in the regulation of sexual and aggressive behavior suggests local estrogen synthesis could regulate sex change through effects on signaling systems that subserve reproductive behavior and function.

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.