/08/$15.00/0 Endocrinology 149(12): Copyright 2008 by The Endocrine Society doi: /en

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1 /08/$15.00/0 Endocrinology 149(12): Printed in U.S.A. Copyright 2008 by The Endocrine Society doi: /en Molecular Cloning, Characterization, and Evolutionary Analysis of Estrogen Receptors from Phylogenetically Ancient Fish Yoshinao Katsu, Satomi Kohno, Susumu Hyodo, Shigeho Ijiri, Shinji Adachi, Akihiko Hara, Louis J. Guillette, Jr., and Taisen Iguchi Okazaki Institute for Integrative Bioscience (Y.K., T.I.), National Institute for Basic Biology, National Institutes of Natural Sciences, and Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Okazaki , Japan; Department of Zoology (S.K., L.J.G.), University of Florida, Gainesville, Florida 32611; Laboratory of Physiology (S.H.), Ocean Research Institute, University of Tokyo, Nakano, Tokyo , Japan; and Faculty of Fisheries Sciences (S.I., S.A., A.H.), Hokkaido University, Hakodate, Hokkaido , Japan Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates, and they promote the growth and differentiation of the adult female reproductive system. To understand the evolution of vertebrate estrogen receptors (ESRs) and to evaluate estrogen receptor-ligand interactions in phylogenetically ancient fish, we used PCR techniques to isolate the cdna encoding ESRs from lungfish, sturgeon, and gar. Sequence analyses indicate that these fishes have two ESRs, ESR1 (ER ) and ESR2 (ER ), as previously reported for other vertebrate species, but a second type of ESR2 (ER 2) was not found as has been reported in a number of teleost fishes. Phylogenetic analysis of the ESR sequences indicated that the lungfish ESRs are classified to the tetrapod ESR group, not with the teleost fish ESRs as are the ESRs from gar and sturgeon. Using transient transfection First Published Online July 17, 2008 Abbreviations: DDE, Dichloro-diphenyl-ethylene; DDT, dichloro-diphenyl-trichloroethane; DES, diethylstilbestrol; DMSO, dimethylsulfoxide; E1, estrone; E2, 17 -estradiol; E3, estriol; EE2, ethynylestradiol; ERE, estrogen response element; ESR, estrogen receptor; RACE, rapid amplification cdna ends. Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. assays of mammalian cells, ESR proteins from these three ancient fishes displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. We also examined the estrogenic potential of o,p -dichloro-diphenyl-trichloroethane (o,p -DDT) and p,p - DDT as well as one of its common metabolites, p,p -dichlorodiphenyl-ethylene (p,p -DDE) on the ESRs from these fishes. Lungfish ESR1 was less sensitive to DDT/DDE than the ESR1 from the other two fishes. The response of lungfish ESR1 to these pesticides is similar to the pattern obtained from salamander ESR1. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine-disrupting mechanisms in three species of phylogenetically ancient fish and also expands our knowledge of ESR evolution. (Endocrinology 149: , 2008) STEROID HORMONES PLAY important roles in the reproductive biology of vertebrates. Many of the currently reported actions of steroid hormones, including estrogens, androgens, and progestogens, are mediated by specific receptors that are localized in the nucleus of target cells. Nuclear steroid hormone receptors form a superfamily of transcription factors that include progestogens, androgens, glucocorticoids, mineralocorticoids, vitamin D, and the retinoic acid receptors (1). Three distinct types of estrogen receptor (ESR) have been isolated to date in vertebrates. Teleost fish express ESR1 (ER ), ESR2a (ER ), and ESR2b (ER ), but the teleost ER form appears to be closely related to the teleost ESR2a, suggesting that it reflects the gene duplication event that occurred within the teleosts (2). Thornton (3) proposed that the ancestral condition for the jawed vertebrates (Gnathostomata) is the presence of two forms of ESR, corresponding to ESR1 and ESR2. Both forms of ESR have been found in fish, amphibians, reptiles, birds, and mammals. To date, cdnas encoding full-length ESR have not been cloned from any of the fishes designated as phylogenetically ancient (basal) fishes, yet these receptors appear to hold a basal location in the evolution of vertebrate steroid receptors. 17 -Estradiol (E2) is the principle estrogen in circulation and appears essential for normal ovarian development in many vertebrate species (4). In fishes, E2 is thought to be the main vitellogenic estrogen that stimulates hepatic production of vitellogenin, the precursor of egg yolk proteins (5). A number of studies strongly suggest that endogenous E2 acts as a natural inducer of ovarian differentiation in nonmammalian vertebrates (6 8). However, the molecular mechanisms of estrogen action on ovarian differentiation in nonmammalian vertebrates, especially phylogenetically ancient fish, remain poorly studied. In these fishes, the presence of an ESR has been reported using steroid-binding assays in sturgeon (9) and a partial sequence of cdna encoding an ESR from spotted gar, Lepisosteus oculatus (GenBank accession no. AY547320) has been reported. To further understand the molecular endocrinology of phylogenetically ancient fish as well as provide additional data on the evolution of vertebrate steroid hormone receptors, we isolated cdna clones encoding homologs of ESRs from lungfish, Protopterus dolloi and Protopterus annectens, Amur sturgeon, Acipenser schrenckii, and tropical gar, Atrac- 6300

2 Katsu et al. ESR of Phylogenetically Ancient Fish Endocrinology, December 2008, 149(12): tosteus tropicus. The resulting sequence data were analyzed to determine their phylogenic relationship with other known vertebrate ESRs. Furthermore, the transactivation function of the ESRs from these basal fish was determined by expressing the receptors in transiently transfected cultured cells using a general reporter gene assay. Materials and Methods Animals and chemical reagents African lungfish (Protopterus dolloi and Protopterus annectens) were purchased from a local commercial supplier. Lungfish were anesthetized in freshwater containing 0.02% ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich Corp., St. Louis, MO), and tissue samples were quickly dissected and frozen in liquid nitrogen. Amur sturgeon (A. schrenckii) purchased from a local supplier (Fujikin, Tsukuba, Japan) were reared at Nanae Fish Culture Experimental Station, Field Science Center for Northern Biosphere, Hokkaido University. The liver and vitellogenic ovary were collected from the female sturgeon, which was approximately 10 yr old. Samples of gonadal and liver tissue from the tropical gar, A. tropicus, were obtained from the Universidad Juárez Autónoma de Tabasco, Villahermosa, Tabasco, México. We obtained chemicals from Sigma-Aldrich E2, estrone (E1), estriol (E3), ethynylestradiol (EE2), and diethylstilbestrol (DES) or Chem Service (West Chester, PA) p,p -dichloro-diphenyl-trichloroethane (p,p - DDT), o,p -DDT, and p,p -dichloro-diphenyl-ethylene (p,p -DDE). All chemicals were dissolved in dimethylsulfoxide (DMSO). The concentration of DMSO in the culture medium did not exceed 0.1%. Molecular cloning of estrogen receptor For lungfish, sturgeon, and gar ESRs, two conserved amino acid regions in the DNA-binding domain (GYHYGVW) and the ligand-binding domain (NKGM/IEHL) of vertebrate ESR1 were selected, and degenerate oligonucleotides were used as primers for PCR. As a template for PCR, the first-strand cdna was synthesized from 2 g total RNA isolated from the liver (lungfish and gar) or the ovary (sturgeon). After amplification, an additional primer set corresponding to two amino acid sequences, CEGCKAF and NKGM/IEHL, was used for the second PCR. The amplified DNA fragments were subcloned with TA-cloning plasmid pcr2.1 vector (Invitrogen, Carlsbad, CA), sequenced using a Big- Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA) with T7 and M13 reverse primers and analyzed on the ABI PRISM 377 automatic sequencer (PE Applied Biosystems). The 5 - and 3 -ends of the ESRs (except for lungfish, P. annectens) were amplified by rapid amplification of the cdna end (RACE) using a SMART RACE cdna amplification kit (BD Biosciences Clontech, Palo Alto, CA). A single full-length transcript of the open reading frame was then amplified using the primer set at the 5 -untranslated region and 3 -untranslated region. Before cloning of the full-length ESR cdna of the lungfish, P. annectens, we found that the ESR sequences of P. dolloi and P. annectens were very similar. Thus, two primer sets at the 5 -untranslated region and 3 -untranslated region of P. dolloi s ESRs were used for the amplification of P. annectens ESRs. For ESR1, primer A (5 -TAGCAGAGACGCACAATGCCGCTGACC- 3 ) and primer B (5 -GTGAGACACTAGTGCTCAACAGAATCC-3 ) were used, and for ESR2, primer C (5 -TGCTTCAAGATGTCTGCACCTG- CACTG-3 and primer D (5 -GGAGCATAGACAGTAGCCAAGTCA- CAG-3 ) were used for PCR using KOD-plus DNA polymerase (TOYOBO Biochemicals, Osaka, Japan), respectively. The amplified DNA fragments were subcloned with plasmids pcr-blunt II vector (Invitrogen). Construction of plasmid vectors The full-coding regions of lungfish, P. dolloi ESRs, sturgeon ESRs, and gar ESRs obtained from this study and the ESRs from Japanese giant salamander ESR1 (AB252211), zebrafish ESR1 (AB037185) and medaka ESR1 (AB033491) were amplified by PCR with KOD DNA polymerase (TOYOBO Biochemicals). The PCR product was gel-purified and subcloned into pcdna3.1 vector (Invitrogen). An estrogen-regulated reporter vector containing four estrogen response elements (4xERE), named pgl3 4xERE was constructed as described previously (10). Transactivation assays Transactivation assay was performed as previously reported (11, 12). To examine the ligand interactions with the cloned ESRs, HEK293 cells were seeded in 24-well plates at cells per well in phenol-red-free DMEM (Sigma-Aldrich) supplemented with 10% charcoal/dextrantreated fetal bovine serum (Hyclone, South Logan, UT). After 24 h, the cells were transfected with 400 ng pgl3 4xERE, 100 ng prl-tk (as an internal control to normalize the variation in transfection efficiency; contains the Renilla reniformis luciferase gene with the herpes simplex virus thymidine kinase promoter; Promega), and 400 ng pcdna3.1-esr using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer s instructions. After 1 h of incubation, various steroid hormones or the pesticides (DDT and DDE) were applied to the medium at various concentrations. After 44 h, the cells were collected, and the luciferase activity of the cells was measured by a chemiluminescence assay with the Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured using a Turner Designs luminometer TD-20/20 (Promega). Promoter activity was calculated as firefly (Photinus pyralis)-luciferase activity/sea pansy (R. reniformis)-luciferase activity. All transfections were performed at least three times, employing triplicate sample points in each experiment. The values shown are mean se (sem) from three separate experiments, and doseresponse data and EC 50 were analyzed using GraphPad Prism (Graph Pad Software, Inc., San Diego, CA). Statistical methods Results are presented as mean se (sem). All multigroup comparisons were performed using one-way ANOVA followed by Bonferroni. Software used was GraphPad Prism version 5.0a. Phylogenic analysis Predicted amino acid sequences of the ESRs (ESR1 and -2) were aligned by Clustal X (13). The GenBank accession numbers of the sequences are provided in supplemental Table 1 (published as supplemental data on The Endocrine Society s Journals Online web site at The regions encompassing the DNAbinding (C) domain to ligand-binding (E) domain were used for the phylogenetic analyses. Any gaps were removed from the sequence under the MEGA program (14). Aligned sequences were analyzed by the PHYLIP package of programs for inferring phylogenies (15). The sequences were translated into 1000 bootstrapped data sets by the Seqboot program the PHYLIP. Phylogenic trees were generated using the 1000 data sets by the Protdist program using maximum likelihood with the Jones-Taylor-Thornton model in the PHYLIP. The consensus tree was generated from these 1000 trees by Consensus program with the majority rule in the PHYLIP. Results Molecular cloning and characterization of lungfish ESRs Using standard PCR techniques described above, partial DNA fragments were amplified from lungfish (P. dolloi) liver RNA. Two types of DNA fragment were obtained, and sequence analysis showed that the fragments had similarity to ESR1 and ESR2 (data not shown). Using the RACE technique, we were able to clone full-length lungfish ESR1 and ESR2 cdnas (GenBank accession no. AB for ESR1 and AB for ESR2). The cdna for ESR1 is composed of a predicted 591 amino acids with a calculated molecular mass of 66.2 kda, and ESR2 is composed of a predicted 561 amino acids with a calculated molecular mass of 63.3 kda. Using the nomenclature of Krust et al. (16), the lungfish ESR sequence can be divided into five domains based on its sequence identity to other species ESRs (Fig. 1). Amino acid sequences

3 6302 Endocrinology, December 2008, 149(12): Katsu et al. ESR of Phylogenetically Ancient Fish FIG. 1. Comparison of African lungfish (P. dolloi) ESR proteins with ESRs of several other species. A, Comparison of the structures between lungfish ESR1 and ESR2. The functional A/B to F domains are schematically represented with the numbers of amino acid residues indicated. The percentage of amino acid identity is depicted. B, Comparison of lungfish ESR1 with that of several other species (Gen- Bank accession numbers are human ESR1, M12674; chicken ESR1, X03805; gecko ESR1, AB240528; Xenopus ESR1, AB244211; and zebrafish ESR1, AB037185). C, Comparison of lungfish ESR2 with that of several other species (GenBank accession numbers are human ESR2, AB006590; chicken ESR2, AB036415; gecko ESR2, AB240529; Xenopus ESR2, AB244212; zebrafish ESR2a, AJ414566; and zebrafish ESR2b, AJ414567). A Lungfish ESR1 Lungfish ESR2 B Lungfish ESR1 Human ESR1 Chicken ESR1 Gecko ESR1 Xenopus ESR1 Zebrafish ESR1 C Lungfish ESR2 Human ESR2 Chicken ESR2 Gecko ESR2 Xenopus ESR A/B C D E F A/B C D E F A/B C D E F Zebrafish ESR2a Zebrafish ESR2b of lungfish ESR1 and ESR2 show an overall identity of 47%. The two lungfish ESRs share 30% identity in the A/B domain, 96% in the C domain (the DNA-binding domain), 29% in the D domain, 60% in the E domain (the ligand-binding domain), and 26% in the F domain (Fig. 1A). If our lungfish sequences are compared with five other species (human, chicken, gecko, Xenopus, and zebrafish), lungfish ESR1 shared 54-35, , 58-47, 69-62, and 35-25% identities in the A/B, C, D, E, and F domains, respectively (Fig. 1B). In contrast, lungfish ESR2 shared 41-33, , 51-33, 75-67, and 30-23% identities in the A/B, C, D, E, and F domains, respectively (Fig. 1C). Thus, domains C (DNA-binding domain) and E (ligand-binding domain) are highly conserved among all vertebrate ESRs studied to date. The overall identities of lungfish ESR1 with ESR1 in human, chicken, gecko, Xenopus, or zebrafish were 59, 62, 57, 62, and 52%, respectively, whereas the overall identities of lungfish ESR2 with ESR2 from the same species were 59, 59, 59 61, 52 (zebrafish ESR2a), and 55% (zebrafish ESR2b), respec-

4 Katsu et al. ESR of Phylogenetically Ancient Fish Endocrinology, December 2008, 149(12): FIG. 2. Transcriptional activities of African lungfish ESRs exposed to endogenous and pharmaceutical estrogens. Concentration-response profile for lungfish ESR1 (A) and lungfish ESR2 (B) activation by E1, E2, E3, and EE2. Results are expressed as means SEM; n 3. The y-axis indicates fold induction compared with the activity of vehicle (DMSO) treatment alone. TABLE 1. Gene transcriptional activities of estrogens mediated by African lungfish ESR1 and ESR2 EC 50 (M) 95% CI (M) RP (%) ESR1 E to E to E to EE to ESR2 E to E to E to EE to Shown are 95% confidence intervals (CI) of EC 50. Relative potency (RP) (EC 50 E2/EC 50 chemical X) 100. tively. These results indicate that lungfish ESRs are more similar to tetrapod forms than to teleost ESRs. Furthermore, we could not detect the second type of ESR2 (ESR2b) in lungfish, suggesting that lungfish ESR genes likely belong to the tetrapod but not the teleost clade. Vertebrate ESRs recognize a response element named ERE consisting of a palindromic repeat of the GGTCAnnnTGACC site (17) and induce a transcript of its downstream region (18). We examined the transcriptional activity of lungfish ESRs using a reporter gene assay and found that estrogens were effective in inducing luciferase activity. We detected that the estrogens E1, E2, E3, and EE2 stimulated luciferase activity from both lungfish ESR1 and ESR2 in a dose-dependent manner (Fig. 2). The EC 50 of lungfish ESR1 was m for E1, m for E2, m for E3, and m for EE2. The activity of lungfish ESR1 was slightly more sensitive to E2 than EE2, but it was more sensitive to E2 than to E1 and E3 in our assay system (Table 1). The EC 50 of lungfish ESR2 was m for E1, m for E2, m for E3, and m for EE2. Like lungfish ESR1, lungfish ESR2 was more responsive to E2 than to E1, E2, E3, and EE2 (Table 1). Intriguingly, E1 stimulated transcriptional activity with both lungfish ESR1 and ESR2; however, the fold activation induced by E1 on ESR1 was half that reached after exposure of ESR1 to the other steroids, suggesting that the conformational change induced by E1 intreraction may be not enough for full activation of lungfish ESR1. Molecular cloning and characterization of sturgeon ESRs Sturgeons belong to a very ancient fish group, existing since Late Cretaceous (19) and believed to have diverged from an ancient, pre-jurassic teleost lineage being approximately 200 million yr ago (20). Full-length clones of Amur sturgeon ESRs were obtained (supplemental Figs. S1 and S2, published as supplemental data on The Endocrine Society s Journals Online web site at We found two distinct clones that are similar to ESR1, and a third is similar to ESR2. The cdna for ESR1a (clone AF4) is composed of a predicted 523 amino acids with a calculated molecular mass of 59.5 kda, whereas ESR1b (clone AF9) is composed of a predicted 533 amino acids with a calculated molecular mass of 60.2 kda. The third clone, ESR2 (clone BF4), is composed of a predicted 553 amino acids with a calculated molecular mass of 62.4 kda (GenBank accession no. AB for ESR1a, clone AF4; no. AB for ESR1b, clone AF9; and no. AB for ESR2, clone BF4). The two sturgeon ESR1s showed high similarity when compared with each other in the A/B (83%), C (95%), D (78%), and E (83%) domains but less so in the F (46%) domain. Both sturgeon ESR1s showed low sequence similarity with sturgeon ESR2 (clone BF4) in the A/B (30-28%), D (32-28%), E (56-55%), and F (29%) domains because only the C domain, the DNAbinding domain, appeared conserved (95-93%) when the forms are compared. During PCR-based cloning, we found four variant types for sturgeon ESR1a (clone AF4, AF5, AF7, and AF8), suggesting that sturgeon ESR1a could possess no fewer than three distinct transcriptional starting points. Furthermore, we detected an insertion in the A/B domain of ESR1a (supplemental Fig. S1B). Although we have identified various ESR1a variants, at this time, we know nothing relative to the expression pattern of these forms or their tissue distribution or potential function. Thus, further studies are needed to clarify these points. Three distinct types of ESR2 (clone BF4, BF5, and BF9) also were obtained (supplemental Fig. S2). Sequences of these clones were very similar when compared (BF4 vs. BF5, 97%; BF4 vs. BF8, 98%; BF5 vs. BF8, 95%). The large number of forms in this species could be caused by polyploidy (21). So, we could not isolate the second type of sturgeon ESR2. Using the sturgeon ESRs obtained and a reporter gene assay, we found that E2 was effective in inducing luciferase

5 6304 Endocrinology, December 2008, 149(12): Katsu et al. ESR of Phylogenetically Ancient Fish FIG. 3. Transcriptional activities of Amur sturgeon ESRs exposed to E2. A, Transcriptional activities of sturgeon ESR1 splice variant clones exposed to E2. Concentration-response profiles are shown for sturgeon ESR1 clone AF4, AF5, AF7, and AF8. Results are expressed as means SEM; n 3. The y-axis indicates the fold induction compared with the activity of vehicle (DMSO) treatment alone. EC 50 values of E2-induced transactivation for each variant are M for AF4, M for AF5, M for AF7, and M for AF8. B, Transcriptional activities of sturgeon ESR2 clones exposed to E2. Concentration-response profiles are shown for sturgeon ESR2 clone BF4, BF5, and BF8. Results are expressed as means SEM;n 3. The y-axis indicates the fold induction compared with the activity of vehicle (DMSO) treatment alone. EC 50 values of E2-induced transactivation for each variant are M for BF4, M for BF5, and M for BF8. activity via all ESR1a clones (Fig. 3A). EC 50 values and relative potency were calculated and indicated that clone AF4 is the most sensitive for E2 when compared with the other three clones. The relative potency at the EC 50 of clone AF4 was 10-fold higher than that of the other clones (Fig. 3A). Furthermore, we found that E2 stimulated the transcriptional activities of all ESR2 clones (Fig. 3B). EC 50 values and relative potency were calculated and indicated that clone BF8 is the most sensitive for E2 among the other two clones. The relative potency of EC 50 of clone BF4 was 2-fold higher than that of clone BF5, whereas the other clone, BF8, was 4-fold higher than BF4 (Fig. 3B). Sturgeon ESR2, clone BF8, has a deletion region in the E domain (ligand-binding domain). This deletion of seven amino acids could influence the transcriptional activity and/or ligand-binding activity of this receptor isoform. The ability of natural and pharmaceutical estrogens to induce ER-dependent transcriptional activity of sturgeon ESR1a (clone AF4), ESR1b, and ESR2 (clone BF4) was also examined. After exposure to estrogens, all sturgeon ESRs FIG. 4. Transcriptional activities of Amur sturgeon ESRs exposed to endogenous and pharmaceutical estrogens. Concentration-response profiles are shown for sturgeon ESR1a (clone AF4) (A), ESR1b (B), and ESR2 (clone BF4) (C) activation by E1, E2, E3, EE2, and DES. Results are expressed as means SEM; n 3. The y-axis indicates fold induction compared with the activity of vehicle (DMSO) treatment alone. activated the expression of the luciferase reporter gene in a dose-dependent manner (Fig. 4). As shown in Fig. 4A, sturgeon ESR1a exhibited significant transcriptional activity at a concentration of 10 9 m E2. Both E1 and E3 activated sturgeon ESR1a transcription but were less effective (less potent) compared with E2. Both EE2 and DES also activated sturgeon ESR1a transcription, and DES exhibited higher potency when compared with E2. EC 50 values and relative potency indicated that sturgeon ESR1a was more sensitive to DES as compared with other estrogens. The relative potency, as indicated by the EC 50, of DES was 2-fold higher than that of E2

6 Katsu et al. ESR of Phylogenetically Ancient Fish Endocrinology, December 2008, 149(12): TABLE 2. Gene transcriptional activities of estrogens mediated by Amur sturgeon ESR1a, ESR1b, and ESR2 EC 50 (M) 95% CI (M) RP (%) ESR1a E to E to E to EE to DES to ESR1b E to E to E to EE to DES to ESR2 E to E to E to EE to DES to Shown are 95% confidence intervals (CI) of EC 50. Relative potency (RP) (EC 50 E2/EC 50 chemical X) 100. (Table 2). Like sturgeon ESR1a, sturgeon ESR1b also exhibited significant transcriptional activity, with a concentration of 10 9 m E2 inducing gene expression (Fig. 4B). Unlike ESR1a, sturgeon ESR1b activity was more sensitive to E2, and the transcriptional activity by E2 was 10-fold higher than that of DES (Table 2). Interestingly, sturgeon ESR2 was more A Gar ESR1 Gar ESR2 sensitive to estrogen stimulation when compared with either form of ESR1 (Table 2). Sturgeon ESR2 showed clear transcriptional activity at m E2. At this concentration, neither ESR1a nor ESR1b were stimulated (Fig. 4). Molecular cloning and characterization of gar ESRs The gar (family Lepisosteidae) is a group of predatory basal actinopterygians characterized by an elongate snout and a jaw joint anterior to the orbit (22). Gar is nonteleost, bony fishes that together with the bowfin, Amia calva, belong to the order Semionotiformes (23) and are sometimes grouped together as holosteans. Like lungfish and sturgeon, full-length clones of gar ESRs were obtained (GenBank accession no. AB for ESR1 and AB for ESR2). The cdna for ESR1 represents a predicted 581-amino-acid protein with a calculated molecular mass of 65.4 kda, whereas ESR2 is predicted to be 571 amino acids with a calculated molecular mass of 64.4 kda. A comparison of the amino acid sequences of gar ESR1 and ESR2 show an overall identity of 50% with 32% identity in the A/B domain, 96% in the C domain, 31% in the D domain, 63% in the E domain, and 26% in the F domain (Fig. 5A). If gar ESR1 protein sequences are compared with lungfish ESR1 and sturgeon ESR1a sequences, gar ESR1 shared 52-49% identity in the A/B domain, 98% identity in the C domain, 55-36% identity in the D domain, 81-64% identity in the E domain, and 25-17% identity in the F domain (Fig. 5B). When ESR2 sequence for A/B C D E F FIG. 5. Comparison of tropical gar ESR proteins with ESRs of the African lungfish (P. dolloi) and Amur sturgeon. A, Comparison of the structures between gar ESR1 and ESR2. The functional A/B to F domains are schematically represented with the numbers of amino acid residues indicated. The percentage of amino acid identity is depicted. B, Comparison of gar ESR1 with lungfish and sturgeon ESR1s (clone AF4). C, Comparison of gar ESR2 with lungfish and sturgeon ESR2s (clone BF4). B Gar ESR1 Lungfish ESR1 Sturgeon ESR A/B C D E F C Gar ESR2 A/B C D E F Lungfish ESR Sturgeon ESR

7 6306 Endocrinology, December 2008, 149(12): Katsu et al. ESR of Phylogenetically Ancient Fish gar is compared with lungfish ESR2 and sturgeon ESR2 (clone BE4), we observed that gar ESR2 shared sequence identity of 57-42% in the A/B domain, 98-96% in the C domain, 61-40% in the D domain, 73-71% in the E domain, and 33-27% in the F domain (Fig. 5C). The overall identities of gar ESR1 with lungfish and sturgeon ESR1s were 57 and 67%, respectively. The overall identities of gar ESR2 with lungfish and sturgeon ESR2s were 59 and 65%, respectively, whereas a similar comparison for gar ESR2 with lungfish and sturgeon ESR2s resulted in 59 and 65%, respectively. Transcriptional assays demonstrated that estrogens were effective in inducing luciferase activity by gar ESRs. We observed that E1, E2, E3, and EE2 stimulated luciferase activity of both gar ESR1 and ESR2 in a dose-dependent manner (Fig. 6). EC 50 of gar ESR1 was m for E1, m for E2, m for E3, and m for EE2. The gar ESR1 protein has similar sensitivities to E2 and EE2 (Table 3). EC 50 for lungfish ESR2 was m for E1, m for E2, m for E3, and m for EE2. Unlike gar ESR1, gar ESR2 was more sensitive to E2 than for the other estrogens tested (Table 3). Transcriptional activity of representative estrogenic pesticides Given the global contamination of wetlands with various persistent chemicals, such as organochlorines (e.g. DDT and TABLE 3. Gene transcriptional activities of estrogens mediated by tropical gar ESR1 and ESR2 EC 50 (M) 95% CI (M) RP (%) ESR1 E to E to E to EE to ESR2 E to E to E to EE to Shown are 95% confidence intervals (CI) of EC 50. Relative potency (RP) (EC 50 E2/EC 50 chemical X) 100. its metabolites such as DDE) and the fact that a number of these pesticides have been shown to bind to the ESRs of various vertebrates, we tested whether a similar phenomenon could be documented for the ESRs of the basal fish studied here. We compared the ligand-dependent induction of gene expression using ESR1 from lungfish, sturgeon, gar, an amphibian (Japanese giant salamander), and two teleost fish (zebrafish and medaka) using p,p -DDT, o,p -DDT and p,p -DDE. All ESR1s investigated were sensitive to o,p -DDT, which gave greater induction than p,p -DDT or p,p -DDE (Fig. 7). The activities of teleost fish, gar, and sturgeon ESR1s after exposure to 10 5 m o,p -DDT achieved maximal levels similar to that after E2-induced transactivities occurring after exposure to much lower doses (10 9 m). However, in salamander ESR1, the o,p -DDT-induced activity levels are half the levels of E2-induced ones. Moreover, p,p -DDT and p,p -DDE hardly increased the transactivity of salamander ESR1 even at 10 5 m concentration. Furthermore, we found that lungfish ESR1 showed an activity pattern similar to that of the salamander ESR1 (Fig. 7). Phylogenetic analysis of ancient fish ESRs To analyze the relationship among the teleost fish, phylogenetically ancient fish, and tetrapod ESRs and molecular evolution of ESR, we tried to isolate ESR cdna from another lungfish, P. annectens. Based on PCR technique, we isolated the cdna clones of ESR1 and ESR2 from lungfish, P. annectens (GenBank accession no. AB for ESR1 and AB for ESR2). Phylogenetic analysis indicates that lungfish ESRs are closer in sequence to the tetrapod ESRs than to teleost fish ESRs (Fig. 8) as suggested by our reporter gene assay data. Furthermore, sturgeon and gar ESR1 and ESR2 sequences cluster together and lie outside of teleost fish ESRs clade. FIG. 6. Transcriptional activities of tropical gar ESRs exposed to endogenous and pharmaceutical estrogens. Concentration-response profile for gar ESR1 (A) and gar ESR2 (B) activation by E1, E2, E3, and EE2. Results are expressed as means SEM; n 3. The y-axis indicates the fold-induction compared with the activity of vehicle (DMSO) treatment alone. Discussion Estrogens are implicated in a wide array of reproductive activities in vertebrates, such as gonadal differentiation, maturation of the female reproductive tract, and reproductive behaviors (24 26). In vertebrates, estrogens appear to induce both genomic and nongenomic cellular actions via nuclear and possibly G-coupled membrane receptors (25, 27). In 1990, a full sequence for the nuclear ESR was reported for a fish, the rainbow trout (28). Since then, many additional

8 Katsu et al. ESR of Phylogenetically Ancient Fish Endocrinology, December 2008, 149(12): FIG. 7. Ligand dependency of fish and amphibian ESR1s. Concentration-response profiles are shown for African lungfish ESR1 (A), Amur sturgeon ESR1a (clone AF4) (B), tropical gar ESR1 (C), zebrafish ESR1 (D), medaka ESR1 (E), and giant salamander ESR1 (F) activation by E2, p,p -DDT, o,p -DDT, and p,p -DDE. EC 50 values of E2-induced transactivation are presented for each species in each graph. Results are expressed as means SEM; n 3. The y-axis indicates the fold induction compared with the activity of vehicle (DMSO) treatment alone. sequences have been reported for teleost fishes, and three distinct types of ESR have been isolated to date in teleost (2). We report here the sequence for both ESR1 and ESR2 in phylogenetically ancient fishes, lungfish, sturgeon, and gar. Although a growing literature exists on sequence and evolutionary phylogeny for various vertebrate steroid receptors (29 31), few studies have examined hormone-induced transcriptional activity of these receptors with various known steroids so that cross-species comparison can be made. Our study adds important information to this database. We cloned and sequenced the lungfish ESRs, which have sequence similarity more closely related to the ESR genes reported from tetrapod than for those reported to date for teleost. Phylogenetic analysis also supported our conclusion that the lungfish ESRs belong to the tetrapod ESR clade. Recent phylogenetic studies, examining various mitochondrial and nuclear genes, support the hypothesis that lungfishes are more closely related to living relatives of the land vertebrates, the tetrapods, than to the fishes (32 36). Most increases in gene numbers occur through many independent tandem duplication events, yet rarely do entire genome duplications appear to have played a major role during the evolution of genomic and possibly phenotypic complexity (37). For many gene families, two paralogous copies are found in teleost such as zebrafish and pufferfish, for example, whereas only one ortholog is present in tetrapods (38). Furthermore, using three nuclear genes (fzd8, sox11, and tyrosinase), individual gene trees and a concatenated data set support the hypothesis that the fish-specific genome duplication occurred after the split of the Acipenseriformes (sturgeons) and the Semionotiformes (gars) from the lineage leading to teleost fish but before the divergence of Osteoglossiformes (39). We have isolated a single type of ESR2 DNA fragment from lungfish, sturgeon, and gar. No ESR2b sequences were amplified from lungfish, sturgeon, or gar, even after the PCR were repeated under various conditions; over 100 subcloned DNA fragments were sequenced without the detection of an ESR2b form. Our data support the hypothesis that the fish-specific genome duplication event occurred between the split of the Semionotiformes from the fish stem lineage and the origin of the Osteoglossomorpha ( million yr ago) (39). Thus, we propose the hypothesis that the presence of ESR2b reflects the gene duplication event that occurred within the teleost before the divergence of Osteoglossiformis (2). We examined the transactivation of ancient fish ESRs using reporter gene assay systems. All of the ESRs tested in our

9 6308 Endocrinology, December 2008, 149(12): Katsu et al. ESR of Phylogenetically Ancient Fish Petromyzon marinus (sea lamprey) Acipenser schrenckii (Amur sturgeon) ESR1a Acipenser schrenckii (Amur sturgeon) ESR1b Atractosteus tropicus (tropical gar) Teleostei (teleost fishes) Pleurodeles waltl (Iberian ribbed newt) Petromyzontiformes Chondrostei Neopterygii Protopterus dolloi (Slender African lungfish)) Dipnoi (lungfishes) Protopterus annectens (West African lungfish) Andrias japonicus (Japanese giant salamander) Anura (frogs and toads) Amniota (amniotes) Squalus acanthias (spiny dogfish) Acipenser schrenckii (Amur sturgeon) Atractosteus tropicus (tropical gar) Teleostei (teleost fishes) ESR2a Teleostei (teleost fishes) ESR2b Protopterus dolloi (Slender African lungfish) Protopterus annectens (West African lungfish) Anura (frogs and toads) Amniota (amniotes) Caudata Chondrostei Neopterygii Dipnoi (lungfishes) Actinopterygii (ray-finned fishes) Amphibia (amphibians) Chondrichthyes (cartilaginous fishes) Actinopterygii (ray-finned fishes) Amphibia (amphibians) ESR1 ESR2 0.1 EAASS Support: > 95% 90-95% 70-95% < 70% FIG. 8. Evolutionary relationships among ESR sequences for selected vertebrate species. The length of branches reflects the estimated number of substitutions along each branch and were estimated by maximum likelihood using the Jones-Taylor-Thornton model. The scale bar shows 0.1 expected amino acid substitutions per site. The thickness of blanches indicate the bootstrap values (percent). For complete tree and accessions, see supplemental Fig. S3 and Table S1. system had similar E2-induced transcriptional responses. Relatively high concentrations of E1 and E3 were necessary for transactivation of the ESRs from phylogenetically ancient fish when compared with the induction by E2. This phenomenon is similar to our previous work with transcriptional activity of other vertebrate ESRs (11, 40). The transactivation assay system that we have used is suitable for the analysis of ESR-induced transactivation from a wide array of species. Moreover, although we have used mammalian cultured cells, cells obtained from other species could also be used for this assay. Thus, future studies are needed to examine the basic regulation of ESR in detail in a wide array of species. Furthermore, additional studies that focus on ontogenic and sexually dimorphic responses in ancient fish could provide insight into the function of these steroids in critical life history attributes of an ancient fish species. In addition to an examination of the interaction of ESRs with endogenous steroids, there is a need to examine the possible role of environmental contaminants on the activation of steroid receptors. A significant number of contaminants have been shown to interact with the ESRs of mammals and some other vertebrates (41). Furthermore, a number of these contaminants are persistent global contaminants that could potentially affect the developmental and reproductive biology of fishes from numerous ecosystems, including the species studied here. Given the continuous use of DDT as an insecticide in tropical regions of Africa, Asia, and South America with malaria threats, the fact that this chemical and its metabolites are persistent, bioaccumulated, and biomagnified contaminants in all food webs studied to date, and that several previous studies have shown that DDT can induce estrogenic actions, it is worth further study. Concentrations of DDTs (DDT and its metabolites) were predominant in the sturgeon samples (42). We examined possible interactions between this pesticide and its metabolite p,p -DDE and the ESRs from a number of vertebrate species in our transactivation system. We observed that o,p -DDT had the potential to induce activation of the ESRs of all three species of phylogenetically ancient fish as well as a number of other species, although the induction of the lungfish and Japanese giant salamander ESR1 was significantly lower. Although induction was possible, much more DDT than E2 was required to induce gene expression in our assay. However, it should be noted that the o,p -DDT concentrations required for ESR1 induction were of the same magnitude as that described for E3. Also of interest is the differential response of the lungfish and salamander ESR1s when compared with those of the other fish because our phylogenetic and sequence analyses support the differential response. These data strongly support the hypothesis that risk of endocrine disruption cannot be predicted for all wildlife studies by simply examining binding and receptor activation for a few mammalian or fish species. Thus, studies should examine the molecular interactions between steroid receptors from a specific spe-

10 Katsu et al. ESR of Phylogenetically Ancient Fish Endocrinology, December 2008, 149(12): cies and native ligands as well as common contaminants in that species environment. Furthermore, future studies are needed to examine the basic regulation of ESR in detail as well as potential effects of environmental chemicals on the sex determination and growth of larvae or neonates in a wide array of species. In summary, we cloned and sequenced ESR1 and ESR2 from three phylogenetically ancient fish, two lungfishes, a sturgeon, and a gar. This is the first report of the full-sequence information and characterization of in vitro transcriptional activity by ligands of ESRs from such basal fishes. These data provide a useful approach for future studies examining the basic endocrinology of nonmammalian steroid hormone receptors. We have demonstrated that transactivation assays, using ESRs from nontraditional research species, such as these ancient fish, provide important initial insights into potential risks from contamination and confirm that such an approach could provide important data for conservation of species and ecological risk assessments. Furthermore, these data provide basic molecular data useful in examining the role of ESR in future studies, such as those examining gonadal development, reproductive biology, and evolutionary endocrinology. Acknowledgments We thank colleagues in our laboratories. We also thank Dr. Arlette Hernández-Franyutti of the División de Ciencias Básicas, Biología, Universidad Juárez Autónoma de Tabasco, Villahermosa, Tabasco, México, and Dr. Mari Carmen Uribe of the Laboratorio de Biología de la Reproducción Animal, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, for assistance in obtaining samples from the gar. Received May 7, Accepted July 9, Address all correspondence and requests for reprints to: Prof. Taisen Iguchi, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi , Japan. taisen@ nibb.ac.jp. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (Y. K. and T. I.), grants from the Ministry of Environment, Japan (Y. K. and T. I.), and a grant from the National Institute of Environmental Health Sciences R21 ES (L.J.G). Disclosure Statement: The authors have nothing to disclose. References 1. Blumberg B, Evans RM 1998 Orphan nuclear receptors: new ligands and new possibilities. Genes Dev 12: Hawkins MB, Thornton JW, Crews D, Skipper JK, Dotte A, Thomas P 2000 Identification of a third estrogen receptor and reclassification of estrogen receptors in teleosts. 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