Hiroshi Urushitani

National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan

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Publications (30)84.19 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The organotin compounds have a high affinity for the retinoid X receptor (RXR), which is a transcriptional factor activated by retinoids that induce imposex in gastropods. However, the molecular mechanisms underlying the regulation of RXR and its related genes in gastropods remain unclear. We isolated a retinoic acid receptor (RAR)-like cDNA (TcRAR) in the rock shell, Thais clavigera, and examined the transcriptional activity of the TcRAR protein by using all-trans retinoic acid (ATRA). However, we did not observe any ligand-dependent transactivation by this protein. We also examined the transcriptional activity of the TcRAR-ligand binding domain fused with the GAL4-DNA binding domain by using retinoic acids, retinol, and organotins and again saw no noteworthy transcriptional induction by these chemicals. Use of a mammalian two-hybrid assay to assess the interaction of the TcRAR protein with the TcRXR isoforms suggested that TcRAR might form a heterodimer with the RXR isoforms. The transcriptional activity of domain-swapped TcRAR chimeric proteins (the A/B domain of TcRAR combined with the D-F domain of human RARα) was also examined and found to be ATRA-dependent. These results suggest that TcRAR is not activated by retinoic acids, but can form a heterodimer with TcRXR isoforms. These data contribute to our understanding of the mechanism by which RXR functions in gastropods.
    Aquatic toxicology (Amsterdam, Netherlands) 09/2013; 142-143C:403-413. DOI:10.1016/j.aquatox.2013.09.008 · 3.45 Impact Factor
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    ABSTRACT: We histologically examined normal differentiation and development of genital tract and gonad in the ivory shell Babylonia japonica (Buccinidae) to determine whether the formation of male-type genitalia in imposex-exhibiting females mimics the normal development of male genitalia in prosobranch gastropods. We used a wild-caught 2-year-old specimen and laboratory-reared juveniles aged 0-24 months. Gonad differentiation was unclear before age 14 months, but progressed after 16 months. Both sexes had complete genital tract and mature gonad at 20 months. However, differentiation and development occurred earlier in females than in males. Development of genital tract preceded gonad differentiation. Vas deferens morphogenesis in males resembled that in imposex-exhibiting females. These findings help to understand the morphogenesis of genital tract and gonad in prosobranch gastropods and will contribute to more in-depth studies of the mode of action of organotin compounds such as TBT and TPhT in imposex development in female prosobranch gastropods.
    Marine environmental research 07/2013; 93. DOI:10.1016/j.marenvres.2013.07.003 · 2.76 Impact Factor
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    ABSTRACT: Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ER, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ER, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% ( vs β1), 49% ( vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cER. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor–environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution. Copyright © 2011 John Wiley & Sons, Ltd.
    Journal of Applied Toxicology 01/2013; 33:41-49. DOI:10.1002/jat.1707 · 2.98 Impact Factor
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    ABSTRACT: Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones.
    Molecular and Cellular Endocrinology 11/2012; 365(2). DOI:10.1016/j.mce.2012.10.014 · 4.41 Impact Factor
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    ABSTRACT: We conducted histological observation of male germ cells and reproductive organs of the starspotted smooth-hound Mustelus manazo in Tokyo Bay to reveal any abnormality in male reproductive traits, as part of a study to elucidate the factors causing recent fluctuation in abundance of the population. Spermatogenesis proceeded in spermatocysts from the germinal zone in the ventral part of the testis to the degenerative zone in the dorsal part, where the spermatozoa were conveyed into the ciliated lumina of the attached terminal branches of the intratesticular ducts. The intratesticular ducts were classified from their terminal ends into branch, stem, and collecting tubules. The ducts formed in the germinal zone and grew as the spermatocysts developed. An opening formed through the wall of each of the most mature spermatocysts into a branch tubule; bundles of spermatozoa were evacuated through this opening into the branch and then the stem tubule and subsequently into the collecting tubules in the rete testis and the efferent duct connected to the epididymis. Spermatocysts that were unable to emit sperm because of failure of adhesion to the branch tubules were disorganized in situ, as were their spermatozoa. The collapsed spermatocysts seem to be cleared by hemophagocytosis with lymphocytes and leukocytes, which may have been recruited from the epigonal organ. There were no specific abnormalities in the spermatogenesis or the morphological structure of testes, which suggested that an abnormality of male reproductive traits was not the major cause of the recent fluctuation in the population abundance of this species. Details of the intratesticular duct system for sperm emission to the epididymis are the first findings in elasmobranchs worldwide.
    Fisheries Science 03/2012; 79(2). DOI:10.1007/s12562-012-0581-6 · 0.88 Impact Factor
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    ABSTRACT: Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.
    Aquatic toxicology (Amsterdam, Netherlands) 03/2012; 109:250-8. DOI:10.1016/j.aquatox.2011.09.004 · 3.45 Impact Factor
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    ABSTRACT: The characteristics of the development of male genitalia (penis and vas deferens) in imposex-exhibiting female rock shells, Thais clavigera, were histologically examined using specimens from a wild population and tributyltin (TBT)-exposed females in the laboratory. A variety of vas deferens morphogenesis patterns were observed in wild female T. clavigera, and the characteristics were summarized. The immature vas deferens at an initial stage, however, was only observed beneath or behind the penis, and no vas deferens was observed close to the vaginal opening (i.e., vulva) of the capsule gland in TBT-exposed females, which was different from the characteristics of vas deferens formation observed in wild females. Taking into consideration both the observed results from wild female specimens and from TBT-exposed females in the laboratory, the vas deferens sequence (VDS) index for T. clavigera was proposed as VDS 1-6.
    Marine environmental research 10/2011; 76:71-9. DOI:10.1016/j.marenvres.2011.10.001 · 2.76 Impact Factor
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    ABSTRACT: Anti-Müllerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Müllerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad-adrenal-mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59-53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator.
    Molecular and Cellular Endocrinology 02/2011; 333(2):190-9. DOI:10.1016/j.mce.2010.12.025 · 4.41 Impact Factor
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    ABSTRACT: The organotin compounds tributyltin (TBT) and triphenyltin (TPT) belong to a diverse group of widely distributed environmental pollutants that induce imposex in gastropods. These organotins have high affinity for retinoid X receptor (RXR), which is a transcription factor activated by retinoids, such as 9-cis retinoic acid (9cRA), in vertebrates. However, the molecular mechanisms underlying the regulation of RXR by retinoids and organotins have not been clarified in gastropods. We isolated two isoforms of RXR cDNAs, RXR isoform 1 (TcRXR-1) and RXR isoform 2 (TcRXR-2), in the rock shell Thais clavigera. The deduced amino acid sequences of TcRXR-1 and TcRXR-2 are highly homologous with those of other gastropods. These TcRXR isoforms displayed 9cRA-dependent activation of transcription in a reporter gene assay using COS-1 cells. The transcriptional activity of TcRXR-2, the encoded protein of which has five additional amino acids in the T-box of the C domain, was significantly lower than that of TcRXR-1. Decreases of the transcriptional activity by TcRXR-1 were observed when more than equal amount of TcRXR-2 fused expression vector was existed in a co-transfection assay. Immunoblot analysis showed several shifted bands for TcRXR isoforms resulting from phosphorylation. Mutation of potential phosphorylation sites from serine to alanine in the A/B domain of TcRXR-1 showed that, in the S89A/S103A mutant, there was a band shift and significantly higher transcriptional activity than in the controls when stimulated with 9cRA. Our findings could contribute to a better understanding of the role of interactions between RXR and retinoids and organotins, not only in the induction mechanism of imposex in gastropods but also in the endocrinology of mollusks.
    Aquatic toxicology (Amsterdam, Netherlands) 02/2011; 103(1-2):101-11. DOI:10.1016/j.aquatox.2011.02.012 · 3.45 Impact Factor
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    ABSTRACT: Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.
    General and Comparative Endocrinology 09/2010; 168(3):496-504. DOI:10.1016/j.ygcen.2010.06.010 · 2.47 Impact Factor
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    ABSTRACT: Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.
    General and Comparative Endocrinology 09/2010; 168(2):220-30. DOI:10.1016/j.ygcen.2010.01.002 · 2.47 Impact Factor
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    ABSTRACT: In the chain of study to further elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod mollusks, we established a polyclonal antibody against RXR of the rock shell Thais clavigera. Immunoblotting demonstrated that this antibody could recognize T. clavigera RXR. In males and imposex-exhibiting females, immunohistochemical staining with the antibody revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial and epidermal cells of the penis. These results suggest that the polyclonal antibody against T. clavigera RXR can specifically recognize RXR protein in tissues of T. clavigera and therefore is useful for evaluating RXR protein localization. Furthermore, RXR may be involved in the induction of male-type genitalia (penis and vas deferens) in normal male and organotin-exposed female rock shells.
    Ecotoxicology 12/2009; 19(3):571-6. DOI:10.1007/s10646-009-0447-6 · 2.71 Impact Factor
  • Yoshinao Katsu · Kaoru Kubokawa · Hiroshi Urushitani · Taisen Iguchi ·
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    ABSTRACT: Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog. Reporter gene assays revealed that the SR ortholog has molecular functions similar to those of the vertebrate ER. Surprisingly, the ER ortholog is an estrogen-insensitive repressor of SR-mediated transcription. Furthermore, we found that the SR ortholog can bind to both estrogen-responsive elements (EREs) and androgen-responsive elements (AREs) and mediates transcriptional activation by estrogens through both types of elements. Our findings suggest that the ancestral SR, but not ER, could bind estrone and induce the ERE- and ARE-dependent transactivation and that it gained the ability to be regulated by ketosteroid and recognize ARE specifically before jawless vertebrates split. These results highlight the importance of comparative experimental approaches for the evolutionary study of endocrine systems.
    Endocrinology 12/2009; 151(2):639-48. DOI:10.1210/en.2009-0766 · 4.50 Impact Factor
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    S Kohno · Y Katsu · H Urushitani · Y Ohta · T Iguchi · L.J. Jr. Guillette ·
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    ABSTRACT: Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.
    Sexual Development 11/2009; 4(1-2):73-87. DOI:10.1159/000260374 · 2.29 Impact Factor
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    ABSTRACT: Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six equine estrogens. In vitro estrogen receptor (ER) activation assays were applied in humans and fish to compare their potencies, followed by in vivo exposures of fish to equine estrogens and evaluation of bioaccumulation, estrogenic responses, and ER gene expression. The equine estrogen equilenin (Eqn), and its metabolite 17beta-dihydroequilenin (17beta-Eqn), were detected by tandem GC-MSMS in all STW influent samples and 83% of STW effluent samples analyzed, respectively, at low concentrations (0.07-2.6 ng/L) and were taken-up into effluent-exposed fish. As occurs in humans, these estrogens bound to and activated the fish ERs, with potencies at ERalpha 2.4-3490% of thatfor 17beta-estradiol. Exposure of fish for 21 days to Eqn and 17beta-Eqn induced estrogenic responses including hepatic growth and vitellogenin production at concentrations as low as 0.6-4.2 ng/L. Associated with these effects were inductions of hepatic ERalpha and ERbeta1 gene expression, suggesting ER-mediated mechanism(s) of action. These data provide evidence for the discharge of equine estrogens from HRT into the aquatic environment and highlight a strong likelihood that these compounds contribute to feminization in exposed wildlife.
    Environmental Science and Technology 06/2009; 43(10):3897-904. DOI:10.1021/es803135q · 5.33 Impact Factor
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    ABSTRACT: Globally, feminization responses in wild male freshwater fish are caused by exposure to estrogenic chemicals, including natural and synthetic estrogens, contained in effluentsfromwastewater treatment works. In U.K. rivers, feminization responses, including intersex, are widespread in wild roach (Rutilus rutilus) populations, and severely affected fish have a reduced reproductive success. We exposed roach to environmentally relevant concentrations of the contraceptive estrogen 17alpha-ethinylestradiol (EE2) for up to 2 years, including intermittent and repeated exposures,to determine effects on sexual development and subsequent responsiveness to estrogen. Exposure of roach to EE2 (at 4 ng/L) for 2 years resulted in sex reversal in males, leading to an all-female population with two cohorts in terms of their stages of ovarian development one paralleling the control females and one at a significantly less advanced stage, which we propose were sex-reversed males. Differing developmental and maturing rates of the putative sex-reversed males compared with control females would question their functional capability as females in the wild. Early-life exposure to environmentally relevant concentrations of EE2 sensitized females to estrogen, as determined by the measurement of the responses of estrogen-sensitive genes in a further EE2 challenge 398 days after the original exposure. In the wild, exposure to environmentally relevant concentrations of EE2 during early life has significantly wider implications for the sexual physiology in fish than has thus far been determined.
    Environmental Science and Technology 03/2009; 43(4):1219-25. DOI:10.1021/es802661p · 5.33 Impact Factor
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    ABSTRACT: Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.
    Molecular and Cellular Endocrinology 10/2007; 276(1-2):10-7. DOI:10.1016/j.mce.2007.06.004 · 4.41 Impact Factor
  • Taisen Iguchi · Yoshinao Katsu · Hiroshi Urushitani · Anke Lange · Charles R. Tyler ·
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    ABSTRACT: Pharmaceutical androgens and estrogens discharged into the aquatic environment are now known to induce adverse effects in fish and are a health concern for wildlife. Mosquitofish ( Gambusia affinis) exposed to a pharmaceutical androgen, trenbolone, used to enhance cattle growth and found to pollute waters below feedlots in the USA, has been shown to alter the development of the anal fin of the females and disrupt gonopodium development in fry. In our work, we showed that altered gonopodium development was associated with disruption in the normal patterns of expression of 2 cloned androgen receptors. Furthermore, exposure to trenbolone at 1 ∝g/L induced spermatocytes in the ovary of sexually mature females. Roach (Rutilus rutilus) living in UK rivers are exposed to estro- genic chemicals in effluents derived from sewage treatment works and this causes feminizing effects, including the development of oocytes in the testis of males. The contraceptive estrogen ethinylestr- adiol (EE2) is believed to contribute to these feminized responses. Our lab-based studies showed that gonadal feminization of roach could be induced by exposure to EE2 at 4 ng/L and the phenotypic responses were associated with altered patterns of expression of 2 cloned estrogen receptors (ERs) and aromatase genes. EE 2 was shown to induce similar feminized responses in the medaka ( Oryzias latipes). We established a reporter gene assay system for roach and medaka ERs and showed that specific environmental estrogens differentially activated the two fish ER subtypes. We also found evidence for an enhanced sensitivity for some estrogens to activate the medaka ERs compared with the roach ERs. EFFECTS OF TRENBOLONE ON MOSQUITOFISH
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    ABSTRACT: Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.
    Journal of Veterinary Medical Science 08/2007; 69(7):725-31. DOI:10.1292/jvms.69.725 · 0.78 Impact Factor
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    ABSTRACT: Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.
    Environmental Science and Technology 06/2007; 41(9):3368-74. DOI:10.1021/es062797l · 5.33 Impact Factor

Publication Stats

586 Citations
84.19 Total Impact Points


  • 2009-2013
    • National Institute for Environmental Studies
      • Center for Material Cycles and Waste Management Research
      Tsukuba, Ibaraki, Japan
  • 2005-2013
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan
  • 2007-2012
    • National Institutes Of Natural Sciences
      Edo, Tōkyō, Japan
  • 2003-2009
    • The Graduate University for Advanced Studies
      • School of Life Science
      Миура, Kanagawa, Japan
  • 2002
    • Yokohama City University
      Yokohama, Kanagawa, Japan