Yoshinao Katsu

Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

Estrogen Receptor 1 (ESR1; ERα), not ESR2 (ERβ), Modulates Estrogen-Induced Sex Reversal in the American Alligator, a Species With Temperature-Dependent Sex Determination

All crocodilians and many turtles exhibit temperature-dependent sex determination where the temperature of the incubated egg, during a thermo-sensitive period (TSP), determines the sex of the offspring. Estrogens play a critical role in sex determination in crocodilians and turtles, as it likely does in most nonmammalian vertebrates. Indeed, administration of estrogens during the TSP induces male to female sex reversal at a male-producing temperature (MPT). However, it is not clear how estrogens override the influence of temperature during sex determination in these species. Most vertebrates have 2 forms of nuclear estrogen receptor (ESR): ESR1 (ERα) and ESR2 (ERβ). However, there is no direct evidence concerning which ESR is involved in sex determination, because a specific agonist or antagonist for each ESR has not been tested in nonmammalian species. We identified specific pharmaceutical agonists for each ESR using an in vitro transactivation assay employing American alligator ESR1 and ESR2; these were 4,4,4-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), respectively. Alligator eggs were exposed to PPT or WAY 200070 at a MPT just before the TSP, and their sex was examined at the last stage of embryonic development. Estradiol-17β and PPT, but not WAY 200070, induced sex reversal at a MPT. PPT-exposed embryos exposed to the highest dose (5.0 μg/g egg weight) exhibited enlargement and advanced differentiation of the Müllerian duct. These results indicate that ESR1 is likely the principal ESR involved in sex reversal as well as embryonic Müllerian duct survival and growth in American alligators.

Molecular cloning and characterization of anti-Müllerian hormone (AMH) from the Japanese wrinkled frog, Rana rugosa

The role of anti-Müllerian hormone (AMH) during gonad development has been studied extensively in many species of mammal, bird, reptile, and fish but remains unresolved in amphibians. In male mammalian embryos, Sox9 activates AMH expression, which initiates regression of the Müllerian ducts. However, Sox9 (Sry-related HMG box 9) is unlikely to initiate AMH in chicken, because AMH precedes Sox9 expression in this species. To clarify whether AMH is involved in testicular differentiation in amphibians, we cloned the full-length AMH cDNA from the Japanese wrinkled frog, Rana rugosa. The AMH gene, which appears to be autosomal, is exclusively expressed in the testis of adult frog among 8 different tissues examined; Sertoli cells are probably responsible for its expression. AMH expression was found in the undifferentiated gonad of both male and female tadpoles, increasing in the differentiating testis. Moreover, we observed consensus binding sites for Sox9 in the 5-flanking region of the AMH gene. Sox9 stimulated statistically significant AMH expression in luciferase reporter assays when coexpressed in Xenopus kidney-derived A6 cells. However, Sox9 expression showed no sexual dimorphism when AMH expression was up-regulated in the developing testis. These results, taken together, suggest that AMH is probably involved in testicular differentiation in R. rugosa, although an additional, perhaps tissue-specific, transcription factor may be required for the regulation of AMH transcription.

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes.

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3 untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3 UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.

Allosteric role of the amino-terminal A/B domain on corticosteroid transactivation of gar and human glucocorticoid receptors.

We studied the role of the A/B domain at the amino terminus of gar (Atractosterus tropicus) and human glucocorticoid receptors (GRs) on transcriptional activation by various glucocorticoids. In transient transfection assays, dexamethasone [DEX] and cortisol had a lower half-maximal response (EC50) for transcriptional activation of full length gar GR than of human GR. Both GRs had similar responses to corticosterone, while 11-deoxycortisol had a lower EC50 for gar GR than for human GR. In contrast, constructs of gar GR and human GR consisting of their hinge (D domain), ligand binding domain (LBD) (E domain) fused to a GAL4 DNA-binding domain (DBD) had a higher EC50 (weaker response) for all glucocorticoids. To study the role of the A/B domain, which contains an intrinsically disordered region, we investigated steroid activation of chimeric gar GR and human GR, in which their A/B domains were exchanged. Replacement of human A/B domains with the gar A/B domains yielded a chimeric GR with a lower EC50 for DEX and cortisol, while the EC50 increased for these steroids for the human A/B-gar C/E chimera, indicating that gar A/B domains contributes to the lower EC50 of gar GR for glucocorticoids. Our data suggests that allosteric signaling between the A/B domains and LBD influences transcriptional activation of human and gar GR by different steroids, and this allosteric mechanism evolved over 400 million years before gar and mammals separated from a common ancestor.

Identification and Characterization of the Androgen Receptor From the American Alligator, Alligator mississippiensis

Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay. We showed that AmAR responded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly, we found a spliced form of the AR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.

Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator.

Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, binds to a variety of chemical compounds including various environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. This receptor regulates expression of target genes through dimerization with the AHR nuclear translocator (ARNT). Since AHR-ARNT signaling pathways differ among species, characterization of AHR and ARNT is important to assess the effects of environmental contamination and for understanding the molecular mechanism underlying the intrinsic function. In this study, we isolated the cDNAs encoding three types of AHR and two types of ARNT from a reptile, the American alligator (Alligator mississippiensis). In vitro reporter gene assays showed that all complexes of alligator AHR-ARNT were able to activate ligand-dependent transcription on a xenobiotic response element. We found that AHR-ARNT complexes had higher sensitivities to a ligand than AHR-ARNT2 complexes. Alligator AHR1B showed the highest sensitivity in transcriptional activation induced by indigo when compared with AHR1A and AHR2. Taken together, our data revealed that all three alligator AHRs and two ARNTs were functional in the AHR signaling pathway with ligand-dependent and isoform-specific transactivations in vitro.

Evolution of corticosteroid specificity for human, chicken, alligator and frog glucocorticoid receptors

We investigated the evolution of the response of human, chicken, alligator and frog glucocorticoid receptors (GRs) to dexamethasone, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol and aldosterone. We find significant differences among these vertebrates in the transcriptional activation of their full length GRs by these steroids, indicating that there were changes in the specificity of the GR for steroids during the evolution of terrestrial vertebrates. To begin to study the role of interactions between different domains on the GR in steroid sensitivity and specificity for terrestrial GRs, we investigated transcriptional activation of truncated GRs containing their hinge domain and ligand binding domain (LBD) fused to a GAL4 DNA binding domain (GAL4-DBD). Compared to corresponding full length GRs, transcriptional activation of GAL4-DBD_GR-hinge/LBD constructs required higher steroid concentrations and displayed altered steroid specificity, indicating that interactions between the hinge/LBD and other domains are important in glucocorticoid activation of these terrestrial GRs.

A second estrogen receptor from Japanese lamprey (Lethenteron japonicum) does not have activities for estrogen binding and transcription.

Estrogens regulate many physiological responses in vertebrates by binding to the estrogen receptor (ER), a ligand-activated transcription factor. To understand the evolution of vertebrate ERs and to investigate how estrogen acts in a jawless vertebrate, we used degenerate primer sets and PCR to isolate DNA fragments encoding two distinct ER subtypes, Esr1a and Esr1b from the Japanese lamprey, Lethenteron japonicum. Phylogenetic analysis indicates that these two ERs are the result of lineage-specific gene duplication within the jawless fishes, different from the previous duplication event of Esr1 (ERα) and Esr2 (ERβ) within the jawed vertebrates. Reporter gene assays show that lamprey Esr1a displays both constitutive and estrogen-dependent activation of gene transcription. Domain swapping experiments indicate that constitutive activity resides in the A/B domain of lamprey Esr1a. Unexpectedly, lamprey Esr1b does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esr1b suggests that although estradiol fits into the steroid binding site, some stabilizing contacts between the ligand and side chains that are found in human Esr1 and Esr2 are missing in lamprey Esr1b.

Corticosteroid and progesterone transactivation of mineralocorticoid receptors from Amur sturgeon and tropical gar

The response to a panel of steroids by the mineralocorticoid receptor (MR) from Amur sturgeon and tropical gar, two basal ray-finned fish, expressed in HEK293 cells was investigated. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone (Prog) were between 13 and 150u2005pM. For gar MR, EC50s were between 8 and 55u2005pM. Such low EC50s support physiological regulation by these steroids of the MR in sturgeon and gar. Companion studies with human and zebrafish MRs found higher EC50s compared with EC50s for sturgeon and gar MRs, with EC50s for zebrafish MR closer to gar and sturgeon MRs than was human MR. For zebrafish MR, EC50s were between 75 and 740u2005pM; for human MR, EC50s were between 65 pM and 2u2005nM. In addition to Prog, spironolactone (spiron) and 19nor-progesterone (19norP) were agonists for all three fish MRs, in contrast with their antagonist activity for human MR, which is hypothesized to involve serine-810 in human MR because all three steroids are agonists for a mutant human Ser810Leu-MR. Paradoxically, sturgeon, gar, and zebrafish MRs contain a serine corresponding to serine-810 in human MR. Our data suggest alternative mechanism(s) for Prog, spiron, and 19norP as MR agonists in these three ray-finned fishes and the need for caution in applying data for Prog signaling in zebrafish to human physiology.