Characterization of evolutionary trend in squamate estrogen receptor sensitivity

Abstract

Highlights• Estrogen receptors from three squamates were functionally characterized.• Comparisons of ligand sensitivities suggest an evolutionary trend in vertebrate ERα.• Squamates have high ligand sensitivity, while urodeles have low sensitivity.• Homology models also suggest similar trend in molecular ligand binding affinity. AbstractSteroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results 2 from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

Functional distinctions associated with the diversity of sex steroid hormone receptors ESR and AR

“…Cross-species comparative analyses from various vertebrates has revealed species differences in ESR sensitivity in response to endogenous estrogens, notably via the use of luciferase reporter gene assays [132]. For example, teleost Esr1s do not show much difference in responsiveness to E2, whereas species differences are more pronounced in tetrapods [133,134]. Amphibian Esrs appear to be less sensitive to E2 generally [135,136].…”

“…Amphibian Esrs appear to be less sensitive to E2 generally [135,136]. From vertebrates studies to date, the ESR1 in snakes -the Okinawa habu (Protobothrops flavoviridis, Viperidae) and Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae), have the highest estrogen sensitivity, followed by other reptilian and avian species [133,137]. ESRs from high sensitive animals may respond more quickly and have a lower demand for the amount of hormone required to trigger hormone activity compared with low sensitive animals.…”

Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus

“…Although genomic (receptor-mediated) and non-genomic signalling is recognized for sex steroid actions in other vertebrates, the second has been scantly studied in Squamata (Contrò et al, 2015;Yatsu et al, 2016;Yaşar et al, 2017). In genomic signal transduction, sex steroids exert their effects by activating nuclear receptors that bind to their promoter regions and modulate gene expression (Sato et al, 2016;Garg et al, 2017).…”

Homology models of mouse and rat estrogen receptor-α ligand-binding domain created by in silico mutagenesis of a human template: Molecular docking with 17β-estradiol, diethylstilbestrol, and paraben analogs

“…Indeed, in recent years, homology models in general have gained wide acceptance as a means of producing high-quality protein structures for research investigations and as docking targets [74, 81, 82]. With respect to estrogen receptors, homology models of ER receptors based on X-ray crystal structures of human ERα have been created for human (ERβ) [83], lizard (3 species) [84], medaka (3 varieties) [85], rainbow trout [86], rotifer [87], and zebrafish [88]. These estrogen receptor homology models have been used in evolutionary and physiological research and as docking targets with applications in ecological toxicology.…”

“…The best overall results were obtained with the simplest procedure: in silico mutagenesis and energy minimization with YASARA-Structure. This technique could be employed in future studies to gain insight into species differences in ligand binding [76], evolutionary or physiological investigations [84, 87, 88], studies of ERα-LBD mutations in breast cancer or other diseases [68–70, 89, 90, 92], ecological toxicology [85, 86, 124], and genetic engineering of mutant ERα-LBDs for use in bio-detection of ligands in assays or biosensors [93]. …”