@article{yost_pow_hawkins_kullman_2014, title={Bridging the Gap From Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes}, volume={48}, ISSN={["1520-5851"]}, DOI={10.1021/es404093n}, abstractNote={This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hERα. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hERα and hERβ, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ERβ subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ERβ subtypes are critically involved in the teleost estrogenic response, with the ERα:ERβ ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures.}, number={9}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, author={Yost, Erin E. and Pow, Crystal Lee and Hawkins, Mary Beth and Kullman, Seth W.}, year={2014}, month={May}, pages={5211–5219} }
 @article{kollitz_hawkins_whitfield_kullmam_2014, title={Functional diversification of vitamin D receptor paralogs in teleost fish after a whole genome duplication event}, volume={155}, DOI={10.1210/en.2014-1505}, abstractNote={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.}, number={12}, journal={Endocrinology}, author={Kollitz, E. M. and Hawkins, M. B. and Whitfield, G. K. and Kullmam, Seth}, year={2014}, pages={4641–4654} }
 @article{mccaffrey_hawkins_godwin_2011, title={Sexual Phenotype Differences in zic2 mRNA Abundance in the Preoptic Area of a Protogynous Teleost, Thalassoma bifasciatum}, volume={6}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0023213}, abstractNote={The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum) is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.}, number={8}, journal={PLOS ONE}, author={McCaffrey, Katherine and Hawkins, Mary Beth and Godwin, John}, year={2011}, month={Aug} }
 @article{marsh_creutz_hawkins_godwin_2006, title={Aromatase immunoreactivity in the bluehead wrasse brain, Thalassoma bifasciatum: Immunolocalization and co-regionalization with arginine vasotocin and tyrosine hydroxylase}, volume={1126}, ISSN={["1872-6240"]}, DOI={10.1016/j.brainres.2006.09.017}, abstractNote={Sex steroid hormones regulate various neural functions that control 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 the bluehead wrasse (Thalassoma bifasciatum), a teleost fish that exhibits socially controlled 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 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. Aromatase-ir 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 that local estrogen synthesis could regulate sex change through effects on signaling systems that subserve reproductive behavior and function.}, journal={BRAIN RESEARCH}, author={Marsh, K. Erica and Creutz, Lela M. and Hawkins, M. Beth and Godwin, John}, year={2006}, month={Dec}, pages={91–101} }
 @article{hawkins_godwin_crews_thomas_2005, title={The distributions of the duplicate oestrogen receptors ER-beta a and ER-beta b in the forebrain of the Atlantic croaker (Micropogonias undulatus): evidence for subfunctionalization after gene duplication}, volume={272}, ISSN={["1471-2954"]}, DOI={10.1098/rspb.2004.3008}, abstractNote={Teleost fishes have three distinct oestrogen receptor (ER) subtypes: ER-α, ER-βa (or ER-γ) and ER-βb. ER-βa and ER-βb arose from a duplication of an ancestralER-βgene early in the teleost lineage. Here, we describe the distribution of the three ER mRNAs in the hypothalamus and cerebellum of the Atlantic croaker to address two issues: the specific functions of multiple ERs in the neuroendocrine system and the evolution and fate of duplicated genes. ER-α was detected in nuclei of the preoptic area (POA) and hypothalamus previously shown to possess ER-αs in teleosts. AcER-βb, but not ER-βa, labelling was detected in the magnocellular neurons of the POA, nucleus posterior tuberis, the nucleus recessus posterior and cerebellum. By contrast, acER-βa, but not ER-βb, was detected in the dorsal anterior parvocellular POA and suprachiasmatic nucleus. Both ER-βs were found in posterior parvocellular and ventral anterior POA nuclei, the ventral hypothalamus, and periventricular dorsal hypothalamus. The differences we observed in ER subtype mRNA distribution within well-characterized brain nuclei suggest that ER-βa and ER-βb have distinct functions in the neuroendocrine control of reproduction and behaviour, and provide evidence that the teleostER-βparalogues have partitioned functions of the ancestralER-βgene they shared with tetrapods.}, number={1563}, journal={PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES}, author={Hawkins, MB and Godwin, J and Crews, D and Thomas, P}, year={2005}, month={Mar}, pages={633–641} }
 @article{hawkins_thomas_2004, title={The  unusual binding properties of the third distinct teleost estrogen receptor subtype ER beta a are accompanied by highly conserved amino acid changes in the ligand binding domain}, volume={145}, DOI={10.1210/en.2003.0806}, abstractNote={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.}, number={6}, journal={Endocrinology}, author={Hawkins, M. B. and Thomas, P.}, year={2004}, pages={2968–2977} }
 @article{hawkins_thornton_crews_skipper_dotte_thomas_2000, title={Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts}, volume={97}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.97.20.10751}, abstractNote={
            This paper describes three distinct estrogen receptor (ER)
 subtypes: ERα, ERβ, and a unique type, ERγ, 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 ERγ arose through gene
 duplication from ERβ early in the teleost lineage and indicates that
 ERγ is present in other teleosts, although it has not been recognized
 as such. The Atlantic croaker ERγ shows amino acid differences in
 regions important for ligand binding and receptor activation that are
 conserved in all other ERγs. The three ER subtypes are genetically
 distinct and have different distribution patterns in Atlantic croaker
 tissues. In addition, ERβ and ERγ fusion proteins can each bind
 estradiol-17β 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.
          }, number={20}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Hawkins, MB and Thornton, JW and Crews, D and Skipper, JK and Dotte, A and Thomas, P}, year={2000}, month={Sep}, pages={10751–10756} }