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YoshinaoName
200901001689839406
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- Xiaozhi Lin, Wataru Takagi, Susumu Hyodo, Shigeho Ijiri, Yoshinao Katsu, Michael E BakerACS pharmacology & translational science 5 (2) 52 - 61 2022/02/11We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with the human PR provides insights into the evolution of steroid activation of the human PR. At 1 nM steroid, the elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20β-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone), and 11-deoxycortisol. The human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of the human PR. RU486, an important clinical antagonist of the human PR, did not inhibit progesterone activation of the elephant shark PR. Cys-528 in the elephant shark PR corresponds to Gly-722 in the human PR, which is essential for RU486 inhibition of the human PR. Confirming the importance of Cys-528 in the elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. To investigate the physiological relevance of Gly-722 in the human PR and Cys-528 in the elephant shark PR, we studied steroid activation of the Gly722Cys human PR and Cys528Gly elephant shark PR. Compared to the wild-type human PR, there was an increase in the activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to the human glycine-722 PR that first evolved in the platypus PR, a basal mammal.
- Yoshinao Katsu, Shin Oana, Xiaozhi Lin, Susumu Hyodo, Laurent Bianchetti, Michael E BakerPloS one 17 (8) e0272219 2022We wanted to clone the glucocorticoid receptor (GR) from slender African lungfish (Protopterus dolloi) for comparison to the P. dolloi mineralocorticoid receptor (MR), which we had cloned and were characterizing, as well as for comparison to the GRs from humans, elephant shark and zebrafish. However, although sequencing of the genome of the Australian lungfish (Neoceratodus forsteri), as well as, that of the West African lungfish (Protopterus annectens) were reported in the first three months of 2021, we could not retrieve a GR sequence with a BLAST search of GenBank, when we submitted our research for publication in July 2021. Moreover, we were unsuccessful in cloning the GR from slender African lungfish using a cDNA from the ovary of P. dolloi and PCR primers that had successfully cloned a GR from elephant shark, Xenopus and gar GRs. On October 21, 2021 the nucleotide sequence of West African lungfish (P. annectens) GR was deposited in GenBank. We used this GR sequence to construct PCR primers that successfully cloned the GR from the slender spotted lungfish. Here, we report the sequences of nine P. dolloi GR isoforms and explain the basis for the previous failure to clone a GR from slender African lungfish using PCR primers that cloned the GR from elephant shark, Xenopus and gar. Studies are underway to determine corticosteroid activation of these slender African lungfish GRs.
- Yoshinao Katsu, Shin Oana, Xiaozhi Lin, Susumu Hyodo, Michael E. BakerJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 215 0960-0760 2022/01Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.
- Kaori Oka, Hidemasa Bono, Asato Kuroiwa, Shusuke Fujioka, Atsushi Shimizu, Yoshinao Katsu, Kyoko MiuraJournal of Molecular Endocrinology 66 (4) 299 - 311 0952-5041 2021/04 [Refereed]
Naked mole-rats (Heterocephalus glaber) inhabit subterranean burrows in savannas and are thus unable to access free water. To identify their mechanism of osmoregulation in xeric environments, we molecularly cloned and analyzed the mineralocorticoid receptor (MR) gene, required for hormone-dependent regulation of genes contributing to body fluid homeostasis. Most vertebrates harbor a single MR homolog. In contrast, we discovered that MR is duplicated in naked mole-rats. The amino acid sequence of naked mole-rat MR1 is 90% identical to its mouse ortholog, and MR1 is abundantly expressed in the kidney and the nervous system. MR2 encodes a truncated protein lacking DNA- and ligand-binding domains of MR1 and is expressed in diverse tissues. Although MR2 did not directly transactivate gene expression, it increased corticosteroid-dependent transcriptional activity of MR1. Our results suggest that MR2 might function as a novel regulator of MR1 activity to fine-tune MR signaling in naked mole-rats. - Yoshinao Katsu, Islam M D Shariful, Xiaozhi Lin, Wataru Takagi, Hiroshi Urushitani, Satomi Kohno, Susumu Hyodo, Michael E BakerThe Journal of steroid biochemistry and molecular biology 210 105845 - 105845 2021/02/27 [Refereed][Not invited]
Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD + LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR. - Michael E Baker, Yoshinao KatsuBiochemical pharmacology 177 113976 - 113976 2020/07 [Refereed][Not invited]
The progesterone receptor (PR) mediates progesterone regulation of female reproductive physiology, as well as gene transcription in non-reproductive tissues, such as brain, bone, lung and vasculature, in both women and men. An unusual property of progesterone is its high affinity for the mineralocorticoid receptor (MR), which regulates electrolyte transport in the kidney in humans and other terrestrial vertebrates. In humans, rats, alligators and frogs, progesterone antagonizes activation of the MR by aldosterone, the physiological mineralocorticoid in terrestrial vertebrates. In contrast, in elephant shark, ray-finned fishes and chickens, progesterone activates the MR. Interestingly, cartilaginous fishes and ray-finned fishes do not synthesize aldosterone, raising the question of which steroid(s) activate the MR in cartilaginous fishes and ray-finned fishes. The simpler synthesis of progesterone, compared to cortisol and other corticosteroids, makes progesterone a candidate physiological activator of the MR in elephant sharks and ray-finned fishes. Elephant shark and ray-finned fish MRs are expressed in diverse tissues, including heart, brain and lung, as well as, ovary and testis, two reproductive tissues that are targets for progesterone, which together suggests a multi-faceted physiological role for progesterone activation of the MR in elephant shark and ray-finned fish. The functional consequences of progesterone as an antagonist of some terrestrial vertebrate MRs and as an agonist of fish and chicken MRs are not fully understood. The physiological activities of progesterone through binding to vertebrate MRs merits further investigation. - Nicole A McNabb, Melissa C Bernhard, Arnold Brunell, Russell H Lowers, Yoshinao Katsu, Demetri D Spyropoulos, Satomi KohnoJournal of applied toxicology : JAT 40 (2) 245 - 256 2020/02 [Refereed][Not invited]
During the Deepwater Horizon oil spill, vast quantities of a chemical dispersant Corexit 9500 were applied in remediation efforts. In addition to the acute toxicity, it is essential to evaluate Corexit further with a broader scope of long-term sublethal endocrine endpoints. The American alligator (Alligator mississippiensis) is an excellent organism for such an endeavor. It exhibits temperature-dependent sex determination, in which egg incubation temperatures during a thermosensitive period (TSP) in embryonic development determine the sex of embryos. Estrogen signals play a critical role in this process. For example, a single exposure to exogenous estrogen during the TSP overrides the effects of temperature and leads to skewed sex ratios. At a concentration of 100 ppm, Corexit significantly induced transcriptional activity of both alligator nuclear estrogen receptors 1 and 2 in vitro in reporter gene assays. To investigate the estrogenic effects of Corexit on gonadal development, alligator eggs were exposed to Corexit at environmentally relevant concentrations (0.25, 2.5 and 25 ppm) before the TSP in ovo. Exposure to Corexit at 0.25 and 25 ppm significantly delayed hatching and growth. Corexit exposure at any treatment level did not affect sex ratios or testicular mRNA abundance as measured at 1-week post-hatching, suggesting that the combination of Corexit components did not synergize enough to induce ovarian development in ovo. These results point to a need for further investigations on individual and combined components of Corexit to understand better their long-term effects on the development and reproductive health of alligators and other coastal aquatic wildlife. - Yoshinao Katsu, Satomi Kohno, Kaori Oka, Xiaozhi Lin, Sumika Otake, Nisha E Pillai, Wataru Takagi, Susumu Hyodo, Byrappa Venkatesh, Michael E BakerScience signaling 12 (584) 1945-0877 2019/06/04 [Refereed][Not invited]
The mineralocorticoid receptor (MR) is a nuclear receptor and part of a large and diverse family of transcription factors that also includes receptors for glucocorticoids, progesterone, androgens, and estrogens. The corticosteroid aldosterone is the physiological activator of the MR in humans and other terrestrial vertebrates; however, its activator is not known in cartilaginous fish, the oldest group of extant jawed vertebrates. Here, we analyzed the ability of corticosteroids and progesterone to activate the full-length MR from the elephant shark (Callorhinchus milii). On the basis of their measured activities, aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, 11-deoxcortisol, progesterone, and 19-norprogesterone are potential physiological mineralocorticoids. However, aldosterone, the physiological mineralocorticoid in humans and other terrestrial vertebrates, is not found in cartilaginous or ray-finned fish. Although progesterone activates MRs in ray-finned fish, progesterone does not activate MRs in humans, amphibians, or alligator, suggesting that during the transition to terrestrial vertebrates, progesterone lost the ability to activate the MR. Both elephant shark MR and human MR are expressed in the brain, heart, ovary, testis, and other nonepithelial tissues, suggesting that MR expression in diverse tissues evolved in the common ancestor of jawed vertebrates. Our data suggest that 19-norprogesterone- and progesterone-activated MR may have unappreciated functions in reproductive physiology. - Baker ME, Katsu YVitamins and hormones 109 17 - 36 0083-6729 2019 [Refereed][Not invited]
- Yukiko Ogino, Saki Tohyama, Satomi Kohno, Kenji Toyota, Gen Yamada, Ryohei Yatsu, Tohru Kobayashi, Norihisa Tatarazako, Tomomi Sato, Hajime Matsubara, Anke Lange, Charles R Tyler, Yoshinao Katsu, Taisen Iguchi, Shinichi MiyagawaThe Journal of steroid biochemistry and molecular biology 184 38 - 46 0960-0760 2018/11 [Refereed][Not invited]
Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARβ) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals. - Katsu Y, Oka K, Baker MEScience signaling 11 (537) 1945-0877 2018/07 [Refereed][Not invited]
Although multiple steroid ligands of the glucocorticoid, mineralocorticoid, and progestin families bind to and regulate the activity of mineralocorticoid receptors (MRs), the responses to these ligands differ across species. To understand how the different domains of MRs contribute to the ligand-induced activation or inhibition of MR activity, we studied the response to eight steroids (aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone, and spironolactone) of human, chicken, alligator, frog, and zebrafish full-length MRs and truncated MRs, which lacked the N-terminal domain (NTD) and DNA binding domain (DBD). Compared to full-length MRs, some truncated MRs were not activated by the steroids, and others required higher steroid concentrations for activation. Progesterone, 19-norprogesterone, and spironolactone did not activate full-length or truncated human, alligator, or frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs, whereas at 100 nM, these steroids had little activity for truncated chicken MRs, but they retained activity for truncated zebrafish MRs. This suggests that regulation of the activation of the chicken MR by progestin resides in the NTD-DBD and that of the zebrafish MR resides in the hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, which is required for the antagonist activity of progesterone for human MR, suggesting a previously uncharacterized mechanism of regulation of progestin activation of chicken and zebrafish MRs. These findings suggest that progesterone may be a physiological activator of chicken and zebrafish MRs. - Hiroshi Urushitani, Yoshinao Katsu, Hiroyuki Kagechika, Ana C.A. Sousa, Carlos M. Barroso, Yasuhiko Ohta, Hiroaki Shiraishi, Taisen Iguchi, Toshihiro HoriguchiAquatic Toxicology 199 103 - 115 1879-1514 2018/06/01 [Refereed][Not invited]
Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway. - Satomi Kohno, Yoshinao Katsu, Nicholas Cipoletti, Lina C. Wang, Zachary G. Jorgenson, Shinichi Miyagawa, Heiko L. SchoenfussJournal of Applied Toxicology 38 (5) 705 - 713 1099-1263 2018/05/01 [Refereed][Not invited]
Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17β, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erβ) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17β. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species. - Yoshinao Katsu, Michael E. BakerProceedings of the National Academy of Sciences of the United States of America 115 (13) E2908 - E2909 1091-6490 2018/03/27 [Refereed][Not invited]
- Williams CE, McNabb NA, Brunell A, Lowers RH, Katsu Y, Spyropoulos DD, Kohno SGeneral and comparative endocrinology 0016-6480 2017/12 [Refereed][Not invited]
- Michael E. Baker, Yoshinao KatsuJOURNAL OF ENDOCRINOLOGY 234 (1) T1 - T16 0022-0795 2017/07 [Refereed][Not invited]
The mineralocorticoid receptor (MR) is descended from a corticoid receptor (CR), which has descendants in lamprey and hagfish, cyclostomes (jawless fish), a taxon that evolved at the base of the vertebrate line. A distinct MR and GR first appear in cartilaginous fishes (Chondrichthyes), such as sharks, skates, rays and chimeras. Skate MR has a strong response to corticosteroids that are mineralocorticoids and glucocorticoids in humans. The half-maximal responses (EC50s) for skate MR for the mineralocorticoids aldosterone and 11-deoxycorticosterone are 0.07 nM and 0.03 nM, respectively. EC50s for the glucocorticoids cortisol and corticosterone are 1 nM and 0.09 nM, respectively. The physiological mineralocorticoid in ray-finned fish, which do not synthesize aldosterone, is not fully understood because several 3-ketosteroids, including cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone and progesterone are transcriptional activators of fish MR. Further divergence of the MR and GR in terrestrial vertebrates, which synthesize aldosterone, led to emergence of aldosterone as a selective ligand for the MR. Here, we combine sequence analysis of the CR and vertebrate MRs and GRs, analysis of crystal structures of human MR and GR and data on transcriptional activation by 3-ketosteroids of wild-type and mutant MRs and GRs to investigate the evolution of selectivity for 3-ketosteroids by the MR in terrestrial vertebrates and ray-finned fish, as well as the basis for binding of some glucocorticoids by human MR and other vertebrate MRs. - Osamu Nishimiya, Yoshinao Katsu, Hiroyuki Inagawa, Naoshi Hiramatsu, Takashi Todo, Akihiko HaraJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 165 (Pt B) 190 - 201 0960-0760 2017/01 [Refereed][Not invited]
One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ER beta Glade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes. (C) 2016 Elsevier Ltd. All rights reserved. - Ryohei Yatsu, Yoshinao Katsu, Satomi Kohno, Takeshi Mizutani, Yukiko Ogino, Yasuhiko Ohta, Jan Myburgh, Johannes H. van Wyk, Louis J. Guillette, Shinichi Miyagawa, Taisen IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 238 88 - 95 0016-6480 2016/11 [Refereed][Not invited]
Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates. (C) 2016 Elsevier Inc. All rights reserved. - Kaori Oka, Satomi Kohno, Yasuhiko Ohta, Louis J. Guillette, Taisen Iguchi, Yoshinao KatsuGENERAL AND COMPARATIVE ENDOCRINOLOGY 238 13 - 22 0016-6480 2016/11 [Refereed][Not invited]
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. (C) 2016 Elsevier Inc. All rights reserved. - Akira Sugimoto, Kaori Oka, Rui Sato, Shinji Adachi, Michael E. Baker, Yoshinao KatsuBIOCHEMICAL JOURNAL 473 (20) 3655 - 3665 0264-6021 2016/10 [Refereed][Not invited]
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 150 pM. For gar MR, EC50s were between 8 and 55 pM. 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 740 pM; for human MR, EC50s were between 65 pM and 2 nM. 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. - Yoshinao Katsu, Satomi Kohno, Kaori Oka, Michael E. BakerSTEROIDS 113 38 - 45 0039-128X 2016/09 [Refereed][Not invited]
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. (C) 2016 Elsevier Inc. All rights reserved. - Yoshinao Katsu, Paul A. Cziko, Charlie Chandsawangbhuwana, Joseph W. Thornton, Rui Sato, Koari Oka, Yoshio Takei, Michael E. Baker, Taisen IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 236 105 - 114 0016-6480 2016/09 [Refereed][Not invited]
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, Esrla and Esrlb 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 (ER alpha) and Esr2 (ER beta) within the jawed vertebrates. Reporter gene assays show that lamprey Esrla 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 Esrla. Unexpectedly, lamprey Esrlb does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esrlb 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 Esrlb. (C) 2016 Elsevier Inc. All rights reserved. - Ryohei Yatsu, Shinichi Miyagawa, Satomi Kohno, Shigeru Saito, Russell H. Lowers, Yukiko Ogino, Naomi Fukuta, Yoshinao Katsu, Yasuhiko Ohta, Makoto Tominaga, Louis J. Guillette, Taisen IguchiSCIENTIFIC REPORTS 5 18581 2045-2322 2015/12 [Refereed][Not invited]
Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual's genotypic background. In the American alligator (Alligator mississippiensis), eggs incubated during the TSP at 33 degrees C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 degrees C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive, and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators, and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel, and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism. - Kaori Oka, Andree Hoang, Daijiro Okada, Taisen Iguchi, Michael E. Baker, Yoshinao KatsuJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 154 112 - 119 0960-0760 2015/11 [Refereed][Not invited]
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. (C) 2015 Elsevier Ltd. All rights reserved. - Shinichi Miyagawa, Ryohei Yatsu, Satomi Kohno, Brenna M. Doheny, Yukiko Ogino, Hiroshi Ishibashi, Yoshinao Katsu, Yasuhiko Ohta, Louis J. Guillette, Taisen IguchiENDOCRINOLOGY 156 (8) 2795 - 2806 0013-7227 2015/08 [Refereed][Not invited]
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. - Saki Tohyama, Shinichi Miyagawa, Anke Lange, Yukiko Ogino, Takeshi Mizutani, Norihisa Tatarazako, Yoshinao Katsu, Masaru Ihara, Hiroaki Tanaka, Hiroshi Ishibashi, Tohru Kobayashi, Charles R. Tyler, Taisen IguchiENVIRONMENTAL SCIENCE & TECHNOLOGY 49 (12) 7439 - 7447 0013-936X 2015/06 [Refereed][Not invited]
Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research. - Maho Kodama, Mari Suda, Daiki Sakamoto, Takehiro Iwasaki, Yasuki Matsuo, Yoshinobu Uno, Yoichi Matsuda, Yoriko Nakamura, Shun Maekawa, Yoshinao Katsu, Masahisa NakamuraENDOCRINOLOGY 156 (5) 1914 - 1923 0013-7227 2015/05 [Refereed][Not invited]
The role of anti-Mullerian 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 Mullerian 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. - Satomi Kohno, Melissa C. Bernhard, Yoshinao Katsu, Jianguo Zhu, Teresa A. Bryan, Brenna M. Doheny, Taisen Iguchi, Louis J. GuilletteENDOCRINOLOGY 156 (5) 1887 - 1899 0013-7227 2015/05 [Refereed][Not invited]
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 (ER alpha) and ESR2 (ER beta). 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 were4,4',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-17 beta and PPT, but not WAY 200070, induced sex reversal at a MPT. PPT-exposed embryos exposed to the highest dose (5.0 mu g/g egg weight) exhibited enlargement and advanced differentiation of the Mullerian duct. These results indicate that ESR1 is likely the principal ESR involved in sex reversal as well as embryonic Mullerian duct survival and growth in American alligators. - Akane Hagiwara, Katsueki Ogiwara, Yoshinao Katsu, Takayuki TakahashiBIOLOGY OF REPRODUCTION 90 (6) 0006-3363 2014/06 [Refereed][Not invited]
We previously reported that the prostaglandin E-2 receptor subtype Ptger4b plays a role in ovulation in a teleost species, medaka and that ptger4b mRNA is drastically induced in preovulatory follicles prior to ovulation. The present study focuses on the hormonal regulation of ptger4b mRNA expression using this nonmammalian vertebrate model. Preovulatory follicles that had not been exposed to luteinizing hormone (Lh) in vivo were incubated in vitro with medaka recombinant Lh (rLh), which induced the ptger4b mRNA expression. The addition of trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase, strongly inhibited rLh-induced ptger4b expression, and trilostane-suppressed ptger4b expression was restored to the level observed in rLh-treated follicles when 17alpha, 20beta-dihydroxy-4-pregnen-3-one was included in the culture. We determined that the expression of the progestin-activated transcription factor nuclear progestin receptor (Pgr) was also induced by medaka rLh in the follicle and that its expression preceded ptger4b expression. Forskolin treatment induced both pgr and ptger4b mRNA expression in the follicle. Follicular ptger4b mRNA expression was drastically suppressed by RU486, which was demonstrated to compete with 17alpha, 20beta-dihydroxy- 4-pregnen-3-one for medaka Pgr in vitro, suggesting a role for Pgr in the expression of ptger4b mRNA. A chromatin immunoprecipitation assay with preovulatory follicles isolated from spawning medaka ovaries demonstrated direct binding of Pgr to the ptger4b promoter. These results indicate that ptger4b expression is regulated by a genomic mechanism involving Pgr. - Shinichi Miyagawa, Anke Lange, Ikumi Hirakawa, Saki Tohyama, Yukiko Ogino, Takeshi Mizutani, Yoshihiro Kagami, Teruhiko Kusano, Masaru Ihara, Hiroaki Tanaka, Norihisa Tatarazako, Yasuhiko Ohta, Yoshinao Katsu, Charles R. Tyler, Taisen IguchiENVIRONMENTAL SCIENCE & TECHNOLOGY 48 (9) 5254 - 5263 0013-936X 2014/05 [Refereed][Not invited]
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 ER alpha reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17 beta (E2) were similar to only a 3-fold difference in ER alpha sensitivity between species. Diethylstilbestrol was the most potent estrogen (similar to 10-fold that of E2) in transactivating the fish ER alpha s, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ER alpha s 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 ER alpha s. Triclosan had little or no transactivation of the fish ER alpha s. By constructing ER alpha 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 ER alpha 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. - Kazuki Takahashi, Tomoya Kotani, Yoshinao Katsu, Masakane YamashitaBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 448 (1) 22 - 27 0006-291X 2014/05 [Refereed][Not invited]
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 riaRNA 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 Pumiliol 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. (C) 2014 Elsevier Inc. All rights reserved. - A. Hagiwara, K. Ogiwar, Y. Katsu, T. TakahashiPROCEEDINGS OF THE 17TH INTERNATIONAL CONGRESS OF COMPARATIVE ENDOCRINOLOGY (ICCE 2013) 29 - 34 2014 [Refereed][Not invited]
Ovulation is the process by which one or more viable oocytes are shed from mature ovarian follicles of the ovary in response to the surge of leuteinizing hormone (LH). This process is accomplished by the coordinated actions of many proteins and enzymes, some of which are drastically induced by the gonadotropin. Prosataglandins (PGs) are a group of animal hormones and exhibit a variety of biological activities by binding to their respective receptors. PGs have been implicated in the ovulatory process of a wide variety of vertebrate aniimals. We previously reported that a prostaglandin E-2 receptor subtype EP4b (Ptger4b) plays a role in medaka ovulation and that ptger4b mRNA is drastically induced in the preovulatory follicles at ovulation. In the present study, we focused on the hormonal regulation of ptger4b mRNA expression using this non-mammalian vertebrate model. In vitro incubation of the preovulatory follicle, which had not yet undergone a surge of luteinizing hormone (Lh) in vivo, with medaka recombinant Lh induced expression of ptger4b mRNA. The expression of ptger4b mRNA was found to be inhibited by trilostane, a specific inhibitor of 3 beta-hydroxysteroid dehydrogenase. The inhibitory effect of the inhibitor on ptger4b mRNA expression was nullified by adding 17 alpha, 20 beta-dihydroxy-4-pregbeb-3-one (DHP), which is well known as maturation-inducing hormone (MIH) in medaka, suggesting a role of progestin in the expression of ptger4b mRNA. Two types of progestin receptors, membrane progestin receptors (mPRs) and nuclear PR (nPR), were found to be expressed in the medaka ovary. Among those, only nPR was induced by medaka recombinant Lh in the follicle and its expression preceded ptger4b mRNA expression. The expression of ptger4b mRNA in the follicle was significantly suppressed by RU486 (also known as mifepristone), which is known to exhibit anti-progestin activity. These results indicate that ptger4b expression is regulated by genomic mechanism involving nPR. - Hiroshi Urushitani, Yoshinao Katsu, Yasuhiko Ohta, Hiroaki Shiraishi, Taisen Iguchi, Toshihiro HoriguchiAquatic Toxicology 142-143 403 - 412 0166-445X 2013/10/15 [Refereed][Not invited]
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. © 2013 Elsevier B.V. - Tomohiro Oka, Naoko Mitsui-Watanabe, Norihisa Tatarazako, Yuta Onishi, Yoshinao Katsu, Shinichi Miyagawa, Yukiko Ogino, Ryohei Yatsu, Satomi Kohno, Minoru Takase, Yukio Kawashima, Yasuhiko Ohta, Yasunobu Aoki, Louis J. Guillette, Taisen IguchiJournal of Applied Toxicology 33 (9) 991 - 1000 1099-1263 2013/09/01 [Refereed][Not invited]
Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3: 10-11 M in TRα and 10-10 M in TRβ) and thyroxine (T4: 10-9 M in TRα and 10-8 M in TRβ). Analogs of thyroid hormone (3,5,3′,-triiodothyroacetic acid and 3,3′,5,5′-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4. These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3-stimulated transcriptional activity of the O. latipes TRα was inhibited by 10-5 M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10-5 M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation. © 2012 John Wiley & Sons, Ltd. - Yoshinao Katsu, Anke Lange, Shinichi Miyagawa, Hiroshi Urushitani, Norishisa Tatarazako, Yukio Kawashima, Charles R. Tyler, Taisen IguchiJOURNAL OF APPLIED TOXICOLOGY 33 (1) 41 - 49 0260-437X 2013/01 [Refereed][Not invited]
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; ERa, ER beta 1 and ER beta 2) 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, ERa, ER beta 1 and ER beta 2, 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% (a vs beta 1), 49% (a vs beta 2) and 53% (beta 1 vs beta 2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cER beta 2 showed a higher sensitivity to the natural steroid oestrogen, 17 beta-estradiol, than cERa. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptorenvironmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution. Copyright (C) 2011 John Wiley & Sons, Ltd. - Kaori Oka, Satomi Kohno, Hiroshi Urushitani, Louis J. Guillette, Yasuhiko Ohta, Taisen Iguchi, Yoshinao KatsuMOLECULAR AND CELLULAR ENDOCRINOLOGY 365 (2) 153 - 161 0303-7207 2013/01 [Refereed][Not invited]
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. (C) 2012 Elsevier Ireland Ltd. All rights reserved. - Takeshi Nakamura, Shinichi Miyagawa, Yoshinao Katsu, Takeshi Mizutani, Tomomi Sato, Takashi Takeuchi, Taisen Iguchi, Yasuhiko OhtaJOURNAL OF VETERINARY MEDICAL SCIENCE 74 (12) 1589 - 1595 0916-7250 2012/12 [Refereed][Not invited]
Female reproductive organs show organ-specific morphological changes during estrous cycles. Perinatal exposure to natural and synthetic estrogens including diethylstilbestrol (DES) or estrogenic chemicals induces estrogen-independent persistent proliferation of vaginal epithelium in mice. To understand the underlying mechanism of the estrogen-independent persistent vaginal changes induced by perinatal DES exposure, we examined global gene expressions in the vaginae of ovariectomized adult mice exposed neonatally to DES using a microarray. The cell cycle-related gene, p21, a cyclin-dependent kinase inhibitor, showed upregulation in the vagina, and p21 protein was localized in the basal layer of the vaginal epithelium in mice exposed neonatally to DES and an estrogen receptor a agonist, propyl pyrazole triol (PPT). The expressions of Notch receptors and Notch ligands were unchanged; however, the mRNAs of hairy-related basic helix-loop-helix (bHLH) transcription factor genes, Hes1, Hey1 and Heyl were persistently downregulated in the vagina, indicating estrogen-independent epithelial cell proliferation in mice exposed neonatally to DES and PPT. - Takeshi Nakamura, Shinichi Miyagawa, Yoshinao Katsu, Tomomi Sato, Taisen Iguchi, Yasuhiko OhtaIN VIVO 26 (6) 899 - 906 0258-851X 2012/11 [Refereed][Not invited]
Estrogen regulates morphological changes in reproductive organs, such as the vagina and uterus, during the estrous cycles in mice. Estrogen depletion by ovariectomy in adults results in atrophy accompanied by apoptosis in vaginal and uterine cells, while estrogen treatment following ovariectomy elicits cell proliferation in both organs. Sequential changes in mRNA expression of wingless-related MMTV integration site (Wnt) and Notch signaling genes were analyzed in the vagina and uterus of ovariectomized adult mice after a single injection of 17 beta-estradiol to provide understanding over the molecular basis of differences in response to estrogen in these organs. We found estrogen-dependent up-regulation of Wnt4, Wnt5a and p21 and down-regulation of Writ] 1, hairy/enhancer-of-split related with YRPW motif-1 (Hey1) and delta-like 4 (Dll4) in the vagina, and up-regulation of Wnt4, Wnt5a, Hey1, Heyl, Dll1, p21 and p53 and down-regulation of Wnt11, Hey2 and Dll4 in the uterus. The expression of Wnt4, Hey1, Hey2, Hey!, Dill and p.53 showed different patterns after the estrogen injection. Expression patterns for Wnt5a, Wnt11, Dll4 and p21 in the vagina and uterus were similar, suggesting that these genes are involved in the proliferation of cells in both those organs in mice. - Takeshi Nakamura, Shinichi Miyagawa, Yoshinao Katsu, Hajime Watanabe, Takeshi Mizutani, Tomomi Sato, Ken-Ichirou Morohashi, Takashi Takeuchi, Taisen Iguchi, Yasuhiko OhtaTOXICOLOGY 296 (1-3) 13 - 19 0300-483X 2012/06 [Refereed][Not invited]
Proliferation and differentiation of cells in female reproductive organs, the oviduct, uterus and vagina, are regulated by endogenous estrogen. In utero exposure to a synthetic estrogen, diethylstilbestrol (DES), induces vaginal clear-cell adenocarcinoma in humans. In mice, perinatal exposure to DES results in abnormalities such as polyovular follicles, uterine circular muscle disorganization and persistent vaginal epithelial cell proliferation. We reported the persistent gene expression change such as interleukin-1 (IL-1) related genes, insulin-like growth factor-I (IGF-I) and its downstream signaling in the mouse vagina exposed neonatally to DES. In this study, we found persistent up-regulation of Wnt4 and persistent down-regulation of Wnt11 in the vagina of mice exposed neonatally to DES and estrogen receptor a specific ligand. Also Wnt4 expression in vagina is correlated to the stratification of epithelial cells with the superficial keratinization of vagina, but not epithelial cell stratification only. (C) 2012 Elsevier Ireland Ltd. All rights reserved. - Ikumi Hirakawa, Shinichi Miyagawa, Yoshinao Katsu, Yoshihiro Kagami, Norihisa Tatarazako, Tohru Kobayashi, Teruhiko Kusano, Takeshi Mizutani, Yukiko Ogino, Takashi Takeuchi, Yasuhiko Ohta, Taisen IguchiCHEMOSPHERE 7 87 (7) 668 - 674 0045-6535 2012/05 [Refereed][Not invited]
The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L-1 of 17 alpha-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L-1 EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L-1 EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L-1 EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka. (C) 2011 Elsevier Ltd. All rights reserved. - Anke Lange, Yoshinao Katsu, Shinichi Miyagawa, Yukiko Ogino, Hiroshi Urushitani, Tohru Kobayashi, Toshiaki Hirai, Janice A. Shears, Masaki Nagae, Jun Yamamoto, Yuta Ohnishi, Tomohiro Oka, Norihisa Tatarazako, Yasuhiko Ohta, Charles R. Tyler, Taisen IguchiAQUATIC TOXICOLOGY 109 250 - 258 0166-445X 2012/03 [Refereed][Not invited]
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 ER alpha 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 (17 alpha-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 ER alpha was found to be the most responsive and carp and stickleback ER alpha the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ER alpha-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 ER alpha activation assays)> zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ER alpha compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ER alpha-mediated responses indicative of estrogenic responses (VTG induction) in vivo. (C) 2011 Elsevier B.V. All rights reserved. - Brandon C. Moore, Matthew R. Milnes, Satomi Kohno, Yoshinao Katsu, Taisen Iguchi, Teresa K. Woodruff, Louis J. GuilletteJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 1-2 127 (1-2) 58 - 63 0960-0760 2011/10 [Refereed][Not invited]
Environmental contaminant exposure can influence gonadal steroid signaling milieus; however, little research has investigated the vulnerability of non-steroidal signaling pathways in the gonads. Here we use American alligators (Alligator mississippiensis) hatched from field-collected eggs to analyze gonadal mRNA transcript levels of the activin-inhibin-follistatin gene expression network and growth differentiation factor 9. The eggs were collected from lake Woodruff National Wildlife Refuge, a site with minimal anthropogenic influence, and Lake Apopka, a highly contaminated lake adjacent to a former EPA Super-fund site. The hatchling alligators were raised for 13 months under controlled conditions, thus limiting differences to embryonic origins. Our data reveal sexually dimorphic mRNA expression in 13-month-old alligator gonads similar to patterns established in vertebrates with genetic sex determination. In addition, we observed a relationship between lake of origin and mRNA expression of activin/inhibin subunits a and beta B, follistatin, and growth differentiation factor 9. Our study suggests that embryonic exposure to environmental contaminants can affect future non-steroidal signaling patterns in the gonads of a long-lived species. (C) 2011 Elsevier Ltd. All rights reserved. - Hiroshi Urushitani, Yoshinao Katsu, Yasuhiko Ohta, Hiroaki Shiraishi, Taisen Iguchi, Toshihiro HoriguchiAQUATIC TOXICOLOGY 103 (1-2) 101 - 111 0166-445X 2011/05 [Refereed][Not invited]
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. (C) 2011 Elsevier B.V. All rights reserved. - Andrew D. Southam, Anke Lange, Adam Hines, Elizabeth M. Hill, Yoshinao Katsu, Taisen Iguchi, Charles R. Tyler, Mark R. ViantENVIRONMENTAL SCIENCE & TECHNOLOGY 45 (8) 3759 - 3767 0013-936X 2011/04 [Refereed][Not invited]
The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a "red list" pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach (Rutilus rutilus ) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish. - Tapas Chakraborty, Yoshinao Katsu, Lin Yan Zhou, Shinichi Miyagawa, Yoshitaka Nagahama, Taisen IguchiJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 123 (3-5) 115 - 121 0960-0760 2011/02 [Refereed][Not invited]
In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ER alpha, ER beta 1 and ER beta 2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17 beta-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ER beta 2 had higher activation potential than ER alpha and ER beta 1. To understand the molecular mechanism of estrogen action via ER, we also conducted E2 treatment where we observed a trigger in ER beta 2 expression upon E(2) exposure. The present data suggest that ER beta 2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka. (C) 2010 Elsevier Ltd. All rights reserved. - Tapas Chakraborty, Yasushi Shibata, Lin-Yan Zhou, Yoshinao Katsu, Taisen Iguchi, Yoshitaka NagahamaMOLECULAR AND CELLULAR ENDOCRINOLOGY 333 (1) 47 - 54 0303-7207 2011/02 [Refereed][Not invited]
In fish, estradiol-17 beta(E2) regulates various reproductive processes by acting through estrogen receptors (ERs). Here, we cloned three ER subtypes from medaka and examined their developmental expression in the gonads and liver of genetically females and males from embryos to adults. During embryogenesis, marked increases in the expression of ERN, but not either ER alpha or ER beta 1, were found in genetically female embryos during sex differentiation. E2 treatment induced marked up-regulation of ER beta 2 expression in genetically male embryos. In adult ovaries, ERa levels were high in follicles (granulosa cells) during oocyte growth. In the testis, ER beta 1 expression exhibited a distinct peak at 10 days post hatching (dph). In the liver, very high levels of ER beta 2 were found in both females and males throughout the sampling period with significantly higher levels in females during 30-50 dph. These findings suggest each action of E2 to be mediated by different types of ERs. (C) 2010 Elsevier Ireland Ltd. All rights reserved. - Hiroshi Urushitani, Yoshinao Katsu, Shinichi Miyagawa, Satomi Kohno, Yasuhiko Ohta, Louis J. Guillette, Taisen IguchiMOLECULAR AND CELLULAR ENDOCRINOLOGY 2 333 (2) 190 - 199 0303-7207 2011/02 [Refereed][Not invited]
Anti-Mullerian 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 Mullerian 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. (C) 2010 Elsevier Ireland Ltd. All rights reserved. - Yoshinao Katsu, Kazumi Matsubara, Satomi Kohno, Yoichi Matsuda, Michihisa Toriba, Kaori Oka, Louis J. Guillette, Yasuhiko Ohta, Taisen IguchiENDOCRINOLOGY 151 (12) 5710 - 5720 0013-7227 2010/12 [Refereed][Not invited]
In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution. (Endocrinology 151: 5710-5720, 2010) - L. K. Davis, Y. Katsu, T. Iguchi, D. T. Lerner, T. Hirano, E. G. GrauJOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 122 (4) 272 - 278 0960-0760 2010/10 [Refereed][Not invited]
Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ER alpha, ER beta 1, and ER beta 2 A primary function of 17 beta-estradiol (E(2)) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg) To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ER alpha and ER beta, and an antagonist for mammalian ER alpha, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-pipendino- pyrazole (MPP), respectively. ER alpha was more sensitive and responsive to PPT than ER beta 1 or ER beta 2 in transactivation assays All ER isoforms indicated equivalent responsiveness to DPN compared with E(2), although sensitivity to DPN was lower MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E2 effect on Vg and ERa 48 h post-injection DPN increased ERa and Vg expression and plasma Vg post-Injection, whereas PPT was without effect. DPN seems to stimulate Vg production through activation of ERa. The ligand binding domain of all tilapia ER forms shares only 60-65% amino acid identity with human ER alpha and ER beta. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs. (C) 2010 Elsevier Ltd All rights reserved. - Yoshinao Katsu, Ena Taniguchi, Hiroshi Urushitani, Shinichi Miyagawa, Minoru Takase, Kaoru Kubokawa, Osamu Tooi, Tomohiro Oka, Noriaki Santo, Jan Myburgh, Akira Matsuno, Taisen IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 168 (2) 220 - 230 0016-6480 2010/09 [Refereed][Not invited]
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 (ER alpha and ER beta) 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. (C) 2010 Elsevier Inc. All rights reserved. - Yoshinao Katsu, Satomi Kohno, Haruka Narita, Hiroshi Urushitani, Koudai Yamane, Akihiko Hara, Tonya M. Clauss, Michael T. Walsh, Shinichi Miyagawa, Louis J. Guillette, Taisen IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 168 (3) 496 - 504 0016-6480 2010/09 [Refereed][Not invited]
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 17 beta-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. (C) 2010 Elsevier Inc. All rights reserved. - Shinichi Miyagawa, Yoshinao Katsu, Yasuhiko Ohta, Tamotsu Sudo, Dennis B. Lubahn, Taisen IguchiBIOLOGY OF REPRODUCTION 82 (3) 497 - 503 0006-3363 2010/03 [Refereed][Not invited]
Development of the reproductive organs can be strongly affected by the hormonal environment. In the mouse, exposure to estrogens and androgens during the critical developmental period induces estrogen-independent cell proliferation and differentiation in the adult vaginal epithelium, which often results in cancerous lesions later in life. In the present study, we assessed the contributions of estrogen receptor 1 (alpha) (ESR1) to the developmental effects of the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) on female mouse vagina and external genitalia. The vagina of Esr1(-/-) mice treated neonatally with DHT showed atrophic epithelium, whereas the vaginal epithelium of Esr1(+/+) mice was stratified and keratinized even after ovariectomy. In addition, neonatal treatment with DHT led to persistent phosphorylation of ESR1 in the vaginae of 60-dayold ovariectomized mice. We infer from these data that ESR1 is obligatory for the induction and maintenance of persistent vaginal epithelial changes induced by neonatal administration of DHT. Neonatal DHT treatment also induced hypospadias in both Esr1(-/-) and Esr1(+/+) mice. In contrast, DHT-induced formation of an os penis-like large bone in the clitoris was found in Esr1(-/-) mice but not in Esr1(+/-) or Esr1(+/+) mice. These results shed light on mechanisms of the induction of developmental effects elicited by sex steroid hormones on the developing animals. - S. Kohno, Y. Katsu, H. Urushitani, Y. Ohta, T. Iguchi, L. J. GuilletteSEXUAL DEVELOPMENT 4 (1-2) 73 - 87 1661-5425 2010/03 [Refereed][Not invited]
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, HSP90 alpha, HSP90 beta, 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. Copyright (C) 2009 S. Karger AG, Basel - Yoshinao Katsu, Kaoru Kubokawa, Hiroshi Urushitani, Taisen IguchiENDOCRINOLOGY 151 (2) 639 - 648 0013-7227 2010/02 [Refereed][Not invited]
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 151: 639-648, 2010) - Brandon C. Moore, Matthew R. Milnes, Satomi Kohno, Yoshinao Katsu, Taisen Iguchi, Louis J. GuilletteBIOLOGY OF REPRODUCTION 82 (1) 194 - 201 0006-3363 2010/01 [Refereed][Not invited]
Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression ( female. male) observed in animals from the reference site ( Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka ( FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression ( ESR1. AR. ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5 degrees C expressed greater AR levels than females incubated at 30 degrees C; dimorphic expression was not observed in animals incubated at 32 degrees C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants. - Charles R. Tyler, Amy L. Filby, Lisa K. Bickley, Rob I. Cumming, Richard Gibson, Pierre Labadie, Yoshinao Katsu, Katherine E. Liney, Janice A. Shears, Vanessa Silva-Castro, Hiroshi Urushitani, Anke Lange, Matthew J. Winter, Taisen Iguchi, Elizabeth M. HillENVIRONMENTAL SCIENCE & TECHNOLOGY 43 (10) 3897 - 3904 0013-936X 2009/05 [Refereed][Not invited]
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 UK 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 17 beta-dihydroequilenin (17 beta-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 ER alpha 2.4-3490% of that for 17 beta-estradiol. Exposure of fish for 21 days to Eqn and 17 beta-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 ER alpha and ER beta 1 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. - Anke Lange, Gregory C. Paull, Tobias S. Coe, Yoshinao Katsu, Hiroshi Urushitani, Taisen Iguchi, Charles R. TylerENVIRONMENTAL SCIENCE & TECHNOLOGY 43 (4) 1219 - 1225 0013-936X 2009/02 [Refereed][Not invited]
Globally, feminization responses in wild male freshwater fish are caused by exposure to estrogenic chemicals, including natural and synthetic estrogens, contained in effluents from wastewater 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 17 alpha-ethinylestradiol (EE(2)) 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 EE(2) (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 EE(2) sensitized females to estrogen, as determined by the measurement of the responses of estrogen-sensitive genes in a further EE(2) challenge 398 days after the original exposure. In the wild, exposure to environmentally relevant concentrations of EE(2) during early life has significantly wider implications for the sexual physiology in fish than has thus far been determined. - Y. Katsu, E. L. Braun, L. J. Guillette, T. IguchiCYTOGENETIC AND GENOME RESEARCH 127 (2-4) 79 - 93 1424-8581 2009 [Refereed][Not invited]
Genome projects have revolutionized our understanding of both molecular biology and evolution, but there has been a limited collection of genomic data from reptiles. This is surprising given the pivotal position of reptiles in vertebrate phylogeny and the potential utility of information from reptiles for understanding a number of biological phenomena, such as sex determination. Although there are many potential uses for genomic data, one important and useful approach is phylogenomics. Here we report cDNA sequences for the c-Jun (JUN) and DJ-1 (PARK7) proto-oncogenes from 3 reptiles (the American alligator, Nile crocodile, and Florida red-belly turtle), show that both genes are expressed in the alligator, and integrate them into analyses of their homologs from other organisms. With these taxa it was possible to conduct analyses that include all major vertebrate lineages. Analyses of c-Jun revealed an unexpected but well-supported frog-turtle clade while analyses of DJ-1 revealed a topology largely congruent with expectation based upon other data. The conflict between the c-Jun topology and expectation appears to reflect the overlap between c-Jun and a CpG island in most taxa, including crocodilians. This CpG island is absent in the frog and turtle, and convergence in base composition appears to be at least partially responsible for the signal uniting these taxa. Noise reduction approaches can eliminate the unexpected frog-turtle clade, demonstrating that multiple signals are present in the c-Jun alignment. We used phylogenetic methods to visualize these signals; we suggest that examining both historical and non-historical signals will prove important for phylogenomic analyses. Copyright (C) 2010 S. Karger AG, Basel - Yoshinao Katsu, Satomi Kohno, Susumu Hyodo, Shigeho Ijiri, Shinji Adachi, Akihiko Hara, Louis J. Guillette, Taisen IguchiENDOCRINOLOGY 149 (12) 6300 - 6310 0013-7227 2008/12 [Refereed][Not invited]
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 alpha) and ESR2 ( ER beta),as previously reported for other vertebrate species, but a second type of ESR2 (ER beta 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 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'-dichloro-diphenyl-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 evolution. ( Endocrinology 149: 6300-6310, 2008) - Taisen Iguchi, Yoshinao KatsuBIOSCIENCE 58 (11) 1061 - 1067 0006-3568 2008/12 [Refereed][Not invited]
Several nuclear receptors have recently been identified as mediators of endocrine disruption as well as steroid hormone receptors. The ubiquitous environmental contaminant tributyltin chloride (TBT) is a ligand for retinoid X receptor (RXR) in rock shell at the nanomolar level, and it acts as a ligand for both the RXR and the peroxisome proliferator-activated receptor gamma in the frog Xenopus laevis and in humans. TBT which induces imposex in marine snails and promotes adipogenesis in X. laevis and in mice, is an example of an environmental endocrine disrupter that promotes adverse effects, from the snail to mammals, through common signaling. In addition, juvenile hormone agonists used as pesticides showed endocrine-disruptive effects on parthenogenic Daphnia magna, lowering rates of reproduction, and inducing 100% male offspring. In this article, we focus on commonality in signaling through nuclear receptors and newly endocrine disruption in D.magna. - Anke Lange, Yoshinao Katsu, Rie Ichikawa, Gregory C. Paull, Laura L. Chidgey, Tobias S. Coe, Taisen Iguchi, Charles R. TylerTOXICOLOGICAL SCIENCES 106 (1) 113 - 123 1096-6080 2008/11 [Refereed][Not invited]
Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17 alpha-ethinylestradiol (EE2) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE2 during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development. - Vinny Naidoo, Yoshinao Katsu, Taisen IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 159 (2-3) 188 - 195 0016-6480 2008/11 [Refereed][Not invited]
Seven of the nine vulture species in South Africa are listed as endangered on the international Union for the Conservation of Nature (IUCN) red list. From these, the Cape Griffon Vulture (Gyps corprotheres) is the most endangered species in the region. Although inadequate nutritional support has been blamed on the constant decline in populations, the process of vulture restaurants has failed to improve the population status over the last twenty years. One possible reason for the decline may be an underlying reproductive disorder as described in endocrine disruptive syndromes. Both DDT and p,p'-DDE have been detected previously at very high concentrations in the mid 1980s, with lower concentrations still being detectable as late as 2001. To establish the effect of DDT and DDE, the vulture estrogen receptor alpha (ER alpha) was sequenced from two species using 5' and 3' rapid amplification cDNA ends (RACE). Using transient transfected mammalian cell assays, vulture ER alpha estrogen-dependent transcription activity was validated using various estrogens and DDT derivatives. The receptor assay was sensitive to p,p'-DDT, o,p'-DDT and p,p'-DDE with EC(50) of 2.41 x 10(-6), 3.47 x 10(-7) and 3.81 x 10(-5) M. When compared to results obtained from human, zebrafish, chicken, salamander and turtle, the vulture ER alpha showed high sensitivity to o,p'-DDT and intermediately responsive to p,p'-DDE. Vulture ER alpha is, however, not responsive to the DDT and DDE levels reported in the plasma of vultures from the last population survey, indicating that the Southern African vulture are not currently exposed to disruptive levels of these contaminants. (c) 2008 Elsevier Inc. All rights reserved. - Takeshi Nakamura, Yoshinao Katsu, Hajime Watanabe, Taisen IguchiTOXICOLOGY 253 (1-3) 117 - 124 0300-483X 2008/11 [Refereed][Not invited]
Perinatal exposure to estrogens such as diethylstilbestrol (DES), and to estrogenic chemicals, induces persistent anovulation caused by a I teration of hypothalamic-pituitary-gonadal (HPG) axis, polyovular follicles, uterine abnormalities and persistent vaginal changes in mice, Most activities of estrogenic chemicals are mediated through estrogen receptora alpha (ER alpha) and/or ER beta. However, little was known about the relative contribution of the individual ER subtypes in induction of abnormalities. We tested the effects of neonatal exposure to ER selective ligands and DES on female mice. Transactivation assays using mouse ER alpha and ER beta showed that 10(-10) M DES activated both ER subtypes and that the ER alpha agonist (propyl pyrazole triol, PPT) and the ER beta agonist(diarylpropionitrile, DPN) selectively activated their respective ERs at 10(-9) M. Neonatal female mice were injected subcutaneously with DES, PPT or DPN and the animals were examined at 13 and 15 weeks of age, respectively. Persistent estrous smears and anovulation were induced in all mice by 0.025-2.5 mu g DES and 2.5-25 mu g PPT, but not by DPN, suggesting that the observed anovulation was primarily mediated through ER alpha. Disorganization Of Uterine Musculature and ovary-independent vaginal epithelial cell proliferation accompanied by persistent expression of EGF-related genes and interleukin-1-related genes were also mediated through ER alpha. In contrast, polyovular follicles were induced by neonatal treatment with both ER alpha and ER beta ligands, suggesting that ovarian abnormalities are mediated through both ER subtypes. (C) 2008 Elsevier Ireland Ltd. All rights reserved. - Taisen Iguchi, Hajime Watanabe, Yoshinao KatsuToxicogenomics: A Powerful Tool for Toxicity Assessment 143 - 158 2008/10/14 [Refereed][Not invited]
- Satomi Kohno, Yoshinao Katsu, Taisen Iguchi, Louis J. GuilletteINTEGRATIVE AND COMPARATIVE BIOLOGY 48 (4) 527 - 534 1540-7063 2008/10 [Refereed][Not invited]
Steroid hormones are essential for the normal function of most organ systems in vertebrates. Reproductive activities in females and males, such as the differentiation, growth and maintenance of the reproductive system, require signaling by sex steroid hormones. Although extensively studied in mammals and a few fish and bird species, the evolution and molecular mechanisms associated with the nuclear steroid hormone receptors are still poorly understood in amphibians and reptiles. Given our interest in environmental signaling of sex determination as well as a major interest in environmental contaminants that can mimic steroid hormone signaling, we have established an approach to study the molecular function (ligand binding and trans-activation) of steroid hormone receptors cloned from reptiles. This approach involves molecular cloning and sequencing of steroid hormone receptors, phylogenic analysis and in vitro trans-activation assays using endogenous or exogenous ligands. Comparing the in vitro trans-activation induced by different ligands with receptors cloned from different species would develop additional functional relationships (classification) among steroid hormone receptors. This approach can provide insight into understanding why each species could have different responses to exogenous ligands. Further, we have developed a novel and less invasive approach to obtaining mRNA for molecular cloning and sequencing of steroid hormone receptors in reptiles and other non-mammalian species, using blood cells as a source of genetic material. For example, white blood cells (WBCs) and red blood cells (RBCs) of the American alligator both express steroid hormone receptors and have adequate amounts of mRNA for molecular cloning. This approach would allow us to analyze components of endocrine function of steroid hormones without sacrificing animals. Especially in endangered species, this approach could provide an understanding of endocrine functions, elucidate the phylogenic relationships of various receptors in vitro, such as the steroid hormone receptors, and determine possible effects of environmental contaminants in a minimally invasive manner. - Matthew R. Milnes, Adriana Garcia, Emily Grossman, Felix Grun, Jason Shiotsugu, Michelle M. Tabb, Yukio Kawashima, Yoshinao Katsu, Hajime Watanabe, Taisen Iguchi, Bruce BlumbergENVIRONMENTAL HEALTH PERSPECTIVES 116 (7) 880 - 885 0091-6765 2008/07 [Refereed][Not invited]
BACKGROUND: Nuclear receptor subfamily 1, group I, member 2 (NR1I2), commonly known as steroid and xenobiotic receptor (SXR) in humans, is a key ligand-dependent transcription factor responsible for the regulation of xenobiotic, steroid, and bile acid metabolism. The ligand-binding domain is principally responsible for species-specific activation of NR1I2 in response to xenobiotic exposure. OBJECTIVES: Our objective in this study was to create a common framework for screening NR1I2 orthologs from a variety of model species against environmentally relevant xenobiotics and to evaluate the results in light of using these species as predictors of xenobiotic disposition and for assessment of environmental health risk. METHODS: Sixteen chimeric fusion plasmid vectors expressing the Gal4 DNA-binding domain and species-specific NR1I2 ligand-binding domain were screened for activation against a spectrum of 27 xenobiotic compounds using a standardized cotransfection receptor activation assay. RESULTS: NR1I2 orthologs were activated by various ligands in a dose-dependent manner. Closely related species show broadly similar patterns of activation; however, considerable variation to individual compounds exists, even among species varying in only a few amino acid residues. CONCLUSIONS: Interspecies variation in NR1I2 activation by various ligands can be screened through the use of in vitro NR1I2 activation assays and should be taken into account when choosing appropriate animal models for assessing environmental health risk. - Satomi Kohno, Dieldrich S. Bermudez, Yoshinao Katsu, Taisen Iguchi, Louis J. GuilletteAQUATIC TOXICOLOGY 88 (2) 95 - 101 0166-445X 2008/06 [Refereed][Not invited]
Reproductive and developmental abnormalities have been reported in the American alligator (Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17 beta (E-2) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor (3) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations ofT, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1(dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E-2 concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator. (C) 2008 Elsevier B.V. All rights reserved. - Matthew R. Milnes, Teresa A. Bryan, Yoshinao Katsu, Satomi Kohno, Brandon C. Moore, Taisen Iguchi, Louis J. GuilletteBIOLOGY OF REPRODUCTION 78 (5) 932 - 938 0006-3363 2008/05 [Refereed][Not invited]
A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka. hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5al) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature. - Yoshinao Katsu, Rie Ichikawa, Toshitaka Ikeuchi, Satomi Kohno, Louis J. Guillette, Taisen IguchiENDOCRINOLOGY 149 (1) 161 - 173 0013-7227 2008/01 [Refereed][Not invited]
Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ER alpha), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t) ER alpha, tAR, and tPR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tER alpha, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tER alpha protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tER alpha, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones. - Tomohiro Kobayashi, Yuko Takita, Atsuko Suzuki, Yoshinao Katsu, Taisen Iguchi, Yasuhiko OhtaJOURNAL OF VETERINARY MEDICAL SCIENCE 70 (1) 115 - 118 0916-7250 2008/01 [Refereed][Not invited]
ORP150 is a hypoxic stress-induced protein located in the endoplasmic reticulum. Transgenic mice overexpressing ORP150 (ORP-Tg) exhibit vacuolar degeneration in the heart. To determine whether vacuolization is present in skeletal muscle, we pathologically examined ORP-Tg mice. After 60 days of age, severe vacuolization was found in the soleus muscles of the hind legs of the ORP-Tg mice. Immunohistochemical staining of ORP150 revealed co-localization of ORP150 and vacuolization in the affected cells. Electron microscopy revealed a marked increase in the number of rough-surfaced endoplasmic reticula (rER) and distention of the cisterna. These findings suggest that overexpression of ORP150 causes accumulation of ORP150 in the rER, resulting in vacuolar degeneration in the skeletal muscle of ORP-Tg mice. - Yoshinao Katsu, Megumi Hinago, Kiyoaki Sone, Hiroshi Urushitani, Louis J. Guillette, Taisen IguchiMOLECULAR AND CELLULAR ENDOCRINOLOGY 276 (1-2) 10 - 17 0303-7207 2007/09 [Refereed][Not invited]
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, Gainbusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ER alpha; ESR1), ERbeta1 (ER beta 1) and ER beta 2 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 beta 1), 43% (alpha versus beta 2), and 52% (beta 1 versus beta 2). 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. (C) 2007 Elsevier Ireland Ltd. All rights reserved. - Jena L. Chojnowski, James Franklin, Yoshinao Katsu, Taisen Iguchi, Louis J. Guillette, Rebecca T. Kimball, Edward L. BraunJOURNAL OF MOLECULAR EVOLUTION 65 (3) 259 - 266 0022-2844 2007/09 [Refereed][Not invited]
Vertebrate genomes are mosaics of isochores, defined as long (> 100 kb) regions with relatively homogeneous within-region base composition. Birds and mammals have more GC-rich isochores than amphibians and fish, and the GC-rich isochores of birds and mammals have been suggested to be an adaptation to homeothermy. If this hypothesis is correct, all poikilothermic (cold-blooded) vertebrates, including the nonavian reptiles, are expected to lack a GC-rich isochore structure. Previous studies using various methods to examine isochore structure in crocodilians, turtles, and squamates have led to different conclusions. We collected more than 6000 expressed sequence tags (ESTs) from the American alligator to overcome sample size limitations suggested to be the fundamental problem in the previous reptilian studies. The alligator ESTs were assembled and aligned with their human, mouse, chicken, and western clawed frog orthologs, resulting in 366 alignments. Analyses of third-codon-position GC content provided conclusive evidence that the poikilothermic alligator has GC-rich isochores, like homeothermic birds and mammals. We placed these results in a theoretical framework able to unify available models of isochore evolution. The data collected for this study allowed us to reject the models that explain the evolution of GC content using changes in body temperature associated with the transition from poikilothermy to homeothermy. Falsification of these models places fundamental constraints upon the plausible pathways for the evolution of isochores. - Taisen Iguchi, Hajime Watanabe, Yoshinao KatsuGENERAL AND COMPARATIVE ENDOCRINOLOGY 153 (1-3) 25 - 29 0016-6480 2007/08 [Refereed][Not invited]
Chemicals released into the environment have the potential to disrupt the endocrine system in wild animals, mouse, and humans. To understand molecular mechanisms of chemical toxicity in various species, toxicogenomics/ecotoxicogenomics, describing the integration of genomics (trascriptomics. proteomics and metabolomics) into toxicology/ecotoxicology, needs to be established as a powerful tool for research. Ecotoxicogenomics is defined as the study of gene and protein expression in non-target organisms that is important in responses to environmental toxicant exposures. Estrogen-responsive genes and estrogen response element(s) in genes have been identified in the mouse reproductive tract by application of cDNA microarray technology. Additionally, functional mechanisms of tributyltin action via nuclear receptors such as retinoid X receptor alpha and peroxisome proliferator activated receptor gamma also have been identified using cDNA microarray. A microarray system has been established for Daphnia magna. Toxicogenomics/ecotoxicogenomics provide powerful tools to help us understand not only molecular mechanisms of chemical toxicity but also the basic biology of various animal species. (c) 2007 Elsevier Inc. All rights reserved. - Yoshinao Katsu, Anke Lange, Hiroshi Urushitani, Rie Ichikawa, Gregory C. Paull, Laura L. Cahill, Susan Jobling, Charles R. Tyler, Taisen IguchiENVIRONMENTAL SCIENCE & TECHNOLOGY 41 (9) 3368 - 3374 0013-936X 2007/05 [Refereed][Not invited]
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 17 alpha-ethinylestradiol, EE2 (up to 4 ng/L), during early life (from fertilization to 84 days post-hatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ER alpha and ER beta, 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 (E-1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E-2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ER beta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ER alpha. 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. - Urushitani H, Katsu Y, Kato Y, Tooi O, Santo N, Kawashima Y, Ohta Y, Kisaka Y, Lange A, Tyler CR, Johnson RD, Iguchi TEnvironmental sciences : an international journal of environmental physiology and toxicology 5 14 211 - 233 0915-955X 2007 [Refereed][Not invited]
- Tyler CR, Lange A, Paull GC, Katsu Y, Iguchi TEnvironmental sciences : an international journal of environmental physiology and toxicology 5 14 235 - 253 0915-955X 2007 [Refereed][Not invited]
- Edward F. Orlando, Yoshinao Katsu, Shinichi Miyagawa, Taisen IguchiJOURNAL OF MOLECULAR ENDOCRINOLOGY 37 (2) 353 - 365 0952-5041 2006/10 [Refereed][Not invited]
The mechanisms underlying sex determination and differentiation in fishes are labile in response to environmental parameters. Sex-specific phenotypes are largely regulated by sex steroids, and the inhibition or the stimulation of aromatase can reverse sex as well as alter secondary sexual characteristics in fishes. Among vertebrates, the mangrove rivulus is the only known self-fertilizing hermaphrodite. Throughout most of its range, rivulus appear to exist as clonally reproducing hermaphrodites. However, outcrossing has been documented in Belize, where up to 25% of rivulus collected are males. The direct development of (primary) males occurs when embryos are incubated at 18 degrees C and hermaphrodites develop into secondary males when held at 28 degrees C. Given the importance of sex steroids, their receptors, and aromatase in sex determination and differentiation of fishes, we cloned, sequenced, and quantified the expression of estrogen receptors (ER alpha, ER beta) and ovarian (AroA) and brain (AroB) aromatase genes. Hermaphrodites had increased ER alpha, ER beta, AroA, and AroB gene expression in the liver, gonad, gonad, and brain respectively, compared to males. These data are consistent with the gene expression data reported for other species and are reflective of the presence of ovarian tissue in the hermaphrodites. Interestingly, we show the elevated expression of brain aromatase in the hermaphrodite brain. The role of the dimorphic expression of brain aromatase in the regulation of sex-specific characteristics is intriguing and requires further research. Because of the uniqueness of its reproductive biology, rivulus is an excellent model for elucidating the mechanisms regulating vertebrate sex determination and sexual differentiation. - Yoshinao Katsu, Satomi Kohno, Tomohiro Oka, Naoko Mitsui, Osamu Tooi, Noriaki Santo, Hiroshi Urushitani, Yukio Fukumoto, Kazushi Kuwabara, Kazuhide Ashikaga, Shinji Minami, Shigeaki Kato, Yasuhiko Ohta, Louis J. Guillette, Taisen IguchiMOLECULAR AND CELLULAR ENDOCRINOLOGY 257-8 84 - 94 0303-7207 2006/09 [Refereed][Not invited]
Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ER alpha (ESRI) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERa mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan. (c) 2006 Elsevier Ireland Ltd. All rights reserved. - Hideo Kato, Tadakazu Furuhashi, Masami Tanaka, Yoshinao Katsu, Hajime Watanabe, Yasuhiko Ohta, Taisen IguchiREPRODUCTIVE TOXICOLOGY 22 (1) 20 - 29 0890-6238 2006/07 [Refereed][Not invited]
Male Sprague-Dawley rats (Crj:CD (IGS) were treated neonatally with bisphenol A (BPA) to evaluate effects on reproductive parameters. Animals were given BPA subcutaneously in corn oil to dosages of 0.002-97 mg/kg body weight, or 0.9 mg/kg 17 beta-estradiol (E2) once a day from postnatal day (PND) 0 to PND 9. Preputial separation, copulatory rate, fertility rate, sperm analysis, serum testosterone levels, and gene expression in the testis were assessed. Males in the E2 group showed a decrease in testis weight and alterations of estrogen-mediated gene expression in the testis on PND 10, and by PND 150 incomplete preputial separation, decreases in the copulatory rate, testicular and accessory organ weights and number of sperm. In contrast, males in all BPA groups showed normal reproductive parameters. These results indicate that in male rats, BPA given during the neonatal period neither affected reproductive function nor evoked estrogen-mediated gene responses in the testis. (c) 2005 Elsevier Inc. All rights reserved. - A Yamaguchi, Y Katsu, M Matsuyama, M Yoshikuni, Y NagahamaEUROPEAN JOURNAL OF CELL BIOLOGY 85 (6) 501 - 517 0171-9335 2006/06 [Refereed][Not invited]
The nuclear membranes surrounding fish and frog oocyte germinal vesicles (GVs) are supported by the lamina, an internal, mesh-like structure that consists of the protein lamin B3. The mechanisms by which lamin B3 is transported into GVs and is assembled to form the nuclear lamina are not well understood. In this study, we developed a heterogeneous microinjection system in which wild-type or mutated goldfish GV lamin B3 (GFLB3) was expressed in Escherichia coli, biotinylated, and microinjected into Xenopus oocytes. The localization of the biotinylated GFLB3 was visualized by fluorescence confocal microscopy. The results of these experiments indicated that the N-terminal domain plays important roles in both nuclear transport and assembly of lamin B3 to form the nuclear lamina. The N-terminal domain includes a major consensus phosphoacceptor site for the p34(cdc2) kinase at amino acid residue Ser-28. To investigate nuclear lamin phosphorylation, we generated a monoclonal antibody (C7B8D) against Ser-28-phosphorylated GFLB3. Two-dimensional (2-D) electrophoresis of GV protein revealed two major spots of lamin B3 with different isoelectric points (5.9 and 6.1). The C7B8D antibody recognized the pI-5.9 spot but not the pI-6.1 spot. The former spot disappeared when the native lamina was incubated with lambda phage protein phosphatase (lambda-PP), indicating that a portion of the lamin protein was already phosphorylated in the goldfish GV-stage oocytes. GFLB3 that had been microinjected into Xenopus oocytes was also phosphorylated in Xenopus GV lamina, as judged by Western blotting with C7B8D. Thus, lamin phosphorylation appears to occur prior to oocyte maturation in vivo in both these species. Taken together, our results suggest that the balance between phosphorylation by interphase lamin kinases and dephosphorylation by phosphatases regulates the conformational changes in the lamin B3 N-terminal head domain that in turn regulates the continual in vivo rearrangement and remodeling of the oocyte lamina. (c) 2006 Elsevier GmbH. All rights reserved. - Y Katsu, T IguchiEUROPEAN JOURNAL OF CELL BIOLOGY 85 (5) 345 - 354 0171-9335 2006/05 [Refereed][Not invited]
Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. We examined the temporal and spatial expression of DDV10, a novel C-type lectin during stratification and cornification of the vaginal epithelium. DDV10 was expressed in vagina but not uterus in ovariectomized mice treated with 17 beta-estradiol (E2). In mouse stomach, the expression of DDV10 was detected in pars proventricularis but not in pars glandularis. Furthermore, the DDV10 gene was found to possess two transcripts, a long form (DDV10) and a short form (OCILrP1, osteoclast inhibitory lectin-related protein 1). DDV10 mRNA but not OCILrP1 mRNA was expressed in the stratified and cornified epithelial tissues. DDV10 mRNA was first detected between 12 and 12 h after E2 treatment in the vaginal epithelium, and was detected in the vagina of the neonatally diethylstilbestrol (DES)-treated mouse. Recently, a unified name was registered in GenBank (C-type lectin domain family 2, member g; Clec2 g). Taken together, these data suggest that DDV10 is the long form of Clec2 g (Clec2g-L), and DDV10/Clec2g-L may play a role in the stratification and/or cornification of epithelial cells during differentiation. (c) 2006 Elsevier GmbH. All rights reserved. - Taisen Iguchi, Hajime Watanabe, Yoshinao KatsuENVIRONMENTAL HEALTH PERSPECTIVES 114 101 - 105 0091-6765 2006/04 [Refereed][Not invited]
Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans. Developing organisms are particularly sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in both reproductive and nonreproductive organs, including persistent molecular alterations. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, need to be identified for investigators to understand the molecular basis of estrogenic activity during embryonic development. For investigators to understand molecular mechanisms of toxicity in various species, toxicogenomics/ecotoxicogenomics, defined as the integration of genomics (transcriptomics, proteomics, metabolomics) into toxicology and ecotoxicology, need to be established as powerful tools for research. As the initial step toward using genomics to examine endocrine-disrupting chemicals, estrogen receptors and other steroid hormone receptors have been cloned in various species, including reptiles, amphibians, and fish, and alterations in the expression of these genes in response to chemicals were investigated. We are identifying estrogen-responsive genes in mouse reproductive tracts using cDNA microarrays and trying to establish microarray systems in the American alligator, roach, medaka, and water fleas (Daphnia magna). It is too early to define common estrogen-responsive genes in various animal species; however, toxicogenomics and ectotoxicogenomics provide powerful tools to help us understand the molecular mechanism of chemical toxicities in various animal species. - Y Katsu, J Myburgh, S Kohno, GE Swan, LJ Guillette, T IguchiCOMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 143 (3) 340 - 346 1095-6433 2006/03 [Refereed][Not invited]
Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor alpha (ER alpha) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ER alpha cDNA was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ER alpha showed high identity to the American alligator ER alpha (98%), caiman ER (98%), lizard ER (82%) and chicken ER alpha (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ER alpha was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ER alpha in crocodiles and expands our knowledge of estrogen receptor evolution. (c) 2005 Elsevier Inc. All rights reserved. - Hideo Kato, Kazuyoshi Naito, Yoshinao Katsu, Hajime Watanabe, Yasuhiko Ohta, Taisen IguchiOPHTHALMIC RESEARCH 38 (6) 361 - 365 0030-3747 2006 [Refereed][Not invited]
Purpose: To investigate ontogenic expression of estrogen receptor (ER)-alpha in female rat corneas, as part of basic studies to elucidate the mechanism of estrogenic effects on the corneas. Methods: The expression and localization of ER alpha were determined using quantitative reverse transcribed-polymerase chain reaction methodology and immunohistochemistry in the corneas of female rats on day 14 of gestation and postnatal days (PNDs) 0, 21, and 60. Results: Quantitative analysis of ER alpha mRNA revealed that ER alpha gene expression increased approximately 4 times on PND 21 and about 10 times on PND 60, as compared with expression levels detected on PND 0. Immunohistochemical analysis revealed that expression of ER alpha protein was evident only in nuclei of the corneal epithelial cells from PND 21 onward. Conclusion: Ontogenic expression of ER alpha occurred in female rat corneas. Copyright (c) 2006 S. Karger AG, Basel. - K Sone, M Hinago, M Itamoto, Y Katsu, H Watanabe, H Urushitani, O Tooi, LJ Guillette, T IguchiGENERAL AND COMPARATIVE ENDOCRINOLOGY 143 (2) 151 - 160 0016-6480 2005/09 [Refereed][Not invited]
Endocrine disrupting chemicals can affect normal hormone dependent processes through numerous mechanisms, including ligand mimicky. 17 beta-Trenbolone (TB), a pharmaceutical, androgenic, anabolic steroid, is a potent agonist of androgen receptors, and has been extensively used as a growth promoter for beef cattle in the US. The effects of TB on adult and newborn mosquitofish (Gambusia affinis affinis) were examined. Two forms of mosquitofish androgen receptor (AR), AR alpha and AR beta, were cloned. The mRNA expression levels of AR alpha and AR beta were transiently increased in the anal fin of adult females at day 3 following exposure to TB (1-10 mu g/L) or methyltestosterone (MT) (0.1-10 mu g/L), a pharmaceutical androgen used as a positive control. Gonopodium differentiation from the adult female anal fin was induced after 28 days of exposure to TB (1-10 mu g/L) or MT (0.1-10 mu g/L). Gonopodium differentiation also was induced in all mosquitofish fry exposed for 28 days to 0.3, 1 or 10 mu g/L TB. Furthermore, spermatozoa were observed histologically in the testes of male fry exposed for 28 days to I or 10 mu g/L TB; spermatozoa are normally observed only in the testes of mature males. Surprisingly, all female fry exposed for 28 days to I or 10 mu g/L TB displayed the formation of an ovotestis, as spermatozoa were found in the ovary. Thus, TB, like MT, induced masculinization of the anal fin accompanied by a transient up-regulation of AR alpha and AR beta in adult females. TB also induced differentiation of the anal fin into a gonopodium in fry of both sexes, stimulated precocious spermatogenesis in the testes of males and the formation of ovotestes in females. (c) 2005 Elsevier Inc. All rights reserved. - Akihiko Yamaguchi, Yoshinao Katsu, Michiyasu Yoshikuni, Michiya Matsuyama, Yoshitaka NagahamaCELL STRUCTURE AND FUNCTION 29 65 - 65 0386-7196 2004/05 [Refereed][Not invited]
- S. Miyagawa, A. Suzuki, Y. Katsu, M. Kobayashi, M. Goto, H. Handa, H. Watanabe, T. IguchiJ Mol Endocrinol 32 663 - 677 2004/04 [Refereed][Not invited]
- H Kato, T Iwata, Y Katsu, H Watanabe, Y Ohta, T IguchiJOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 52 (5) 1410 - 1414 0021-8561 2004/03 [Refereed][Not invited]
We used a modified yeast-based human estrogen receptor alpha (ERalpha) bioassay to determine the estrogenic activity in 22 kinds of diets for experimental animals. The estrogenic activity of each diet was reevaluated by comparison with a calibration curve of 17beta-estradiol. Almost all of the diets had estrogenic activity. The diets for rabbits and guinea pigs had the highest estrogenic activity compared to any other diets, including those for rats and mice. Estrogenic activity was found in dried skim milk, fishmeal, soybean meal, and alfalfa meal. In the NIH-07 diet opened for the ingredients, estrogenic activity was nearly all derived from the alfalfa meal. Multiple assays were performed to evaluate potential seasonal variations in the estrogenic potency in the raw materials of the rat and mouse diets. We found that the estrogenic activity in these raw materials changed throughout the year. - S Miyagawa, Y Katsu, H Watanabe, T IguchiONCOGENE 23 (2) 340 - 349 0950-9232 2004/01 [Refereed][Not invited]
Growth factors and estrogen receptor (ER) signaling cooperate to play essential roles in cell proliferation, differentiation and tumor progression in mouse reproductive organs. Treatment of neonatal mice with diethylstilbestrol (DES) induces an estrogen-independent persistent proliferation and cornification of the vaginal epithelium, which results in cancerous lesions later in life. However, the mechanisms of the estrogen-dependent and -independent pathways essentially remain unknown. We characterized the expression of epidermal growth factor (EGF)-like growth factors (EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (APR), epiregulin (EPR) and neuregulin (NRG) 1) and erbB receptors (EGF receptor (EGFR), erbB/neu, erbB3 and erbB4) in the vaginae of mice treated either neonatally (0-4 day) or as adults (55-59 day) with estrogens. EGFR and erbB2 were activated in the vaginal epithelium of mice by estrogen treatment. This activation was also encountered in vaginae from neonatally DES-exposed mice, along with the expression of EGF, TGF-alpha, HB-EGF, BTC, APR, EPR and NRG1. Immunohistochemical analysis indicated that erbB2 was primarily expressed in vaginal epithelium. Finally, we found that serine 118 and 167 located in the AF-1 domain of ERalpha were phosphorylated in these vaginae. AG825, AG1478 or ICI 182,780 administration blocked proliferation of vaginal epithelium induced by neonatal DES exposure. Thus, signal transduction via EGFR and erbB2 could be related to the estrogen-induced vaginal changes and persistent erbBs phosphorylation and sustained expression of EGF-like growth factors, leading to ERalpha activation that may result in cancerous lesions in vaginae from neonatally DES-exposed mice later in life. - C Seiwa, J Nakahara, T Komiyama, Y Katsu, T Iguchi, H AsouNEUROENDOCRINOLOGY 80 (1) 21 - 30 0028-3835 2004 [Refereed][Not invited]
We report studies on the mechanism of action of bisphenol A (BPA) on the differentiation of oligodendrocyte precursor cells (OPCs). Our results show that: ( 1) BPA inhibits the differentiation of OPCs induced by exposure to thyroid hormone (T-3). ( 2) The effect is mediated through various mechanisms via the thyroid hormone receptor (TRbeta1) which is considered to be responsible for OPC differentiation. ( 3) The action of BPA on OPC differentiation does not involve the FcRgamma-Fyn-myelin basic protein (MBP) cascade as an inducer of OPC differentiation nor does it suppress CREB phosphorylation, which is considered to be induced by the T-3-TR complex. (4) The presence of MBP isoforms (21.5, 18.5, 17.0 and 14.0 kDa) was detected in OPCs, and the expression of exon 2-containing isoforms (i.e. 17.0 and 21.5 kDa) was upregulated upon treatment with T-3. In contrast, expression of MBP was inhibited by BPA. Copyright (C) 2004 S. Karger AG, Basel. - S Nakahata, T Kotani, K Mita, T Kawasaki, Y Katsu, Y Nagahama, M YamashitaMECHANISMS OF DEVELOPMENT 120 (8) 865 - 880 0925-4773 2003/08 [Refereed][Not invited]
Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation. (C) 2003 Elsevier Ireland Ltd. All rights reserved. - Y Katsu, DB Lubahn, T IguchiENDOCRINOLOGY 144 (6) 2597 - 2605 0013-7227 2003/06 [Refereed][Not invited]
Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. However, the molecular mechanisms underlying estrogen-induced changes have not been elucidated. The goal of this study was to identify estrogen-responsive genes related to the proliferation and differentiation of mouse vaginal epithelial cells. We used differential display to reveal specific genes regulated by estrogens and identified a transcript that was designated DDV10. DDV10 encodes a membrane protein with a C-type lectin domain in the carboxyl-terminal region; thus, we inferred that it belongs to the C-type lectin family. We analyzed the temporal and spatial expression of DDV10 using RT-PCR, quantitative real-time RT-PCR, and in situ hybridization. Ovariectomy decreased DDV10 mRNA levels, whereas 17beta-estradiol treatment increased expression of DDV10 mRNA in vaginas of ovariectomized mice. DDV10 mRNA was first detected between 20 and 30 d after birth and was found in eye, tongue, stomach, and stratified and cornified vaginal epithelial cells, but not in stromal cells or uterus. DDV10 transcripts were not detected in vaginas of estrogen receptor alpha knockout mice. Taken together, these data suggest that DDV10 encodes a novel, 17beta-estradiol-regulated, C-type lectin in the mouse vagina. DDV10 may play a role in the stratification and/or cornification of epithelial cells during differentiation. - H Urushitani, M Nakai, H Inanaga, Y Shimohigashi, A Shimizu, Y Katsu, T IguchiMOLECULAR AND CELLULAR ENDOCRINOLOGY 203 (1-2) 41 - 50 0303-7207 2003/05 [Refereed][Not invited]
Developmental exposure to 17beta-estradiol (E-2) induced the death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones in the cyprinodont fish, the mummichog (Fundulus heteroclitus). To clarify the mechanism by which exogenous estrogens caused these developmental effects, we determined the sequence of an estrogen receptor (ER) coding region, encoded by 620 amino acid residues. This region shared 80% identity to that of ERalpha of medaka (Oryzias latipes). Northern blot analysis showed that two ERalpha mRNAs with 5.5 and 4 kb were expressed in the liver. These mRNAs were strongly induced by E2 stimulation. The 4 kb mRNA was expressed 8 h after treatment, whereas the 5.5 kb mRNA was not induced until 12 h after E2 stimulation. Vitellogenin (VTG) was expressed 8 It after E2 stimulation in the male liver. Receptor binding assays using the protein of F heteroclitus ERalpha (fhERalpha) ligand binding domain showed that alkylphenols bind to fhERalpha with a higher affinity (50 times or more) as compared with the human ERalpha. The present results demonstrate that the fhERalpha has a sequence very similar to that of medaka, and the mRNA for this receptor was induced by E-2-stimulation, followed subsequently by VTG expression. Furthermore, alkylphenols bind to fhERalpha more efficiently than to human ERalpha. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved. - H Urushitani, A Shimizu, Y Katsu, T IguchiJOURNAL OF EXPERIMENTAL ZOOLOGY 293 (7) 693 - 702 0022-104X 2002/12 [Refereed][Not invited]
Many chemicals released into the environment exhibit estrogenic activity, having the potential to disrupt development and the functioning of the endocrine system. In order to establish a model system to study the effects of such environmental chemicals on aquatic animals, we examined the effects of a natural estrogen, 17beta-estradiol (E-2), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8), and 10(-6) M E-2 throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner, and fry treated with 10(-6) M E-2 and 10(-8) M E-2 were dead by two weeks and 12 weeks after hatching, respectively. More than 85% of fry treated with 10(-8) M E-2 showed malformations: i.e., eye extrusion, crooked vertebral column, faded lateral-stripe pattern eight weeks after hatching. Body weight and head and body lengths were significantly reduced in E-2-treated fry when compared to controls. Ossification was not completed in vertebrae, cranial bones, and other bones in fry treated with 10(-8) M E-2 even 12 weeks after hatching. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E-2 were 100% female eight weeks after hatching. The present results demonstrate that exogenous estrogen induced death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones, which would result in shorter body and head lengths and in malformed vertebrae leading to a hunchback condition. (C) 2002 Wiley-Liss, Inc. - Y Katsu, E Takasu, T IguchiMOLECULAR AND CELLULAR ENDOCRINOLOGY 195 (1-2) 99 - 107 0303-7207 2002/09 [Refereed][Not invited]
Perinatal treatment of female mice with natural or synthetic estrogens including diethylstilbestrol (DES) results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. However, the molecular mechanisms of the estrogen-independent changes have not been elucidated. To analyze the mechanism of estrogen-independent cell proliferation and cornification of the vaginal epithelium, we used differential display and determined specific genes expressed in neonatally DES-treated vagina. A candidate clone that designated DDV5 was identical to the serine protease, neuropsin that is reportedly expressed in the mouse central nervous system. We then analyzed the expression pattern of DDV5/neuropsin using Northern blot analysis. We found: (1) DDV5/neuropsin mRNA is expressed in vaginae from neonatally DES-treated ovariectomized mice but not in vaginae from ovariectomized control mice, (2) its expression is not detected in uteri from neonatally DES-treated mice, (3) DDV5/neuropsin is expressed in vaginae from normal intact mice during estrus. Furthermore, we found that DDV5/neuropsin mRNA rapidly decreased in vaginae after ovariectomy. DDV5/neuropsin was detected in vaginae from ovariectomized mice 48 It after estrogen treatment. These results suggest that DDV5/neuropsin is expressed in estrogen-stimulated mouse vagina, and its gene expression is regulated by estrogen. Neonatal DES exposure affects transcriptional control of DDV5/neuropsin in the mouse vagina, which results in persistent expression of DDV5/neuropsin even after ovariectomy, thus, DDV5/neuropsin may play a role in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. Using in situ hybridization method, we found DDV5/neuropsin mRNA localized in epithelial cells but not stromal cells in vaginae. This is the first report on the gene expression of a serine-protease neuropsin in the mouse vagina, and as a marker of the estrogen-independent persistent proliferation and cornification of the vaginal epithelium. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved. - K. Uchida, A. Suzuki, Y. Kobayashi, D. L. Buchanan, T. Sato, H. Watanabe, Y. Katsu, J. Suzuki, K. Asaoka, C. Mori, K. Arizono, T. IguchiJournal of Health Science 公益社団法人日本薬学会 48 (6) 579 - 582 1344-9702 2002/04 [Refereed][Not invited]
Placental transfer of bisphenol-A (BPA) was studied in mice and Japanese monkeys (Macaca fuscata). BPA was found in maternal and fetal sera, liver, brain, uteri, testes and placenta as early as 30 min after a single subcutaneous (s.c.) injection to 17 days of pregnancy in mice. BPA was also found in fetal liver, kidney, and brain of Japanese monkeys 1 hr after a single s.c. injection to 150 days of pregnancy. These results clearly indicate that the maternal placental barrier can not protect the fetus from the consequences of BPA exposure in these species. - A Suzuki, A Sugihara, K Uchida, T Sato, Y Ohta, Y Katsu, H Watanabe, T IguchiREPRODUCTIVE TOXICOLOGY 16 (2) 107 - 116 0890-6238 2002/03 [Refereed][Not invited]
Reproductive tract development is influenced by estrogen. The aim of this study was to determine the effects of an environmental estrogenic chemical bisphenol-A (BPA) on prenatal and postnatal development of female mouse reproductive organs. In the prenatal treatment group, BPA or the synthetic estrogen diethylstilbestrol (DES) were given by subcutaneous (s.c.) injections to pregnant mice during gestational days 10-18. Some offspring treated prenatally with 10 and 100 mg/kg bw BPA or 0.67 and 67 mug/kg bw DES were ovariectomized at 30 days and sacrificed at 40 days of age. Vaginal smears were examined in the remaining offspring, then these offspring were mated with normal males. Prenatal exposure to 10 mg/kg BPA reduced the number of mice with corpora lutea compared to sesame oil controls at 30 days, but more than 80% of mice from either prenatally exposed BPA group were fertile at 90 days. Mice exposed prenatally to maternal doses of 67 mug/kg DES were sterile and showed ovary-independent vaginal and uterine epithelial stratification; however, mice exposed prenatally to BPA did not show ovary-independent vaginal and uterine changes. The number of offspring and litter sex ratio from mice exposed prenatally to BPA (10 or 100 mg/kg) or 0.67 mug/kg DES were not different compared to controls. In postnatal treatment group, female mice were given s.c. injections of BPA (15 or 150 mug/pup) or DES (0.3 or 3 mug/pup) for 5 days from the day of birth, then some mice were ovariectomized at 30 days and examined at 40 and 90 days. In the remaining mice, vaginal smears were examined from 61 to 90 days and ovarian histology was evaluated at 90 days. Mice exposed postnatally to 150 mug BPA exhibited ovary-independent vaginal epithelial stratification. Postnatal DES (0.3 and 3 mug) treatment also induced ovary-independent vaginal stratification. Polyovular follicles having more than one oocyte in a follicle were induced by postnatal injections of BPA (150 mug) or DES (0.3 or 3 mug) at 30 days. These findings indicate for the first time that a large dose of BPA can induce ovary-independent vaginal epithelial changes when given postnatally but not prenatally. (C) 2002 Elsevier Science Inc. All rights reserved. - S Honma, A Suzuki, DL Buchanan, Y Katsu, H Watanabe, T IguchiREPRODUCTIVE TOXICOLOGY 16 (2) 117 - 122 0890-6238 2002/03 [Refereed][Not invited]
In utero exposure to bisphenol-A (BPA) at doses relevant to human consumption has been reported to accelerate weight gain and puberty in female mice, but the effect of low dose BPA on female reproduction has not been described. In this study, we investigated low dose effects of BPA on sexual maturation and reproduction in female ICR/Jcl mice. Pregnant ICR mice (F0) were injected (s.c.) with BPA (2 and 20 mug/kg), diethylstilbestrol (DES; 0.02, 0.2, and 2 mug/ka) or oil vehicle once per (lay from gestational days 11-17. For both female and male offspring (F1), body weights were measured on postnatal day (PND) 0 (the day of birth), 11, 22, and 60, and anogenital distance (AGD) was measured on PNDs 22 and 60. Pups were weaned at PND 22 and males were caged separately from females. Vaginal smears were taken daily beginning the day of vaginal opening for 30 days. The age at vaginal opening was significantly earlier in all exposed females except for 2 mug/kg BPA females compared to oil controls. Body weight at vaginal opening was lower than controls in all exposed females. The first vaginal estrus was earlier in all exposed females except for the 2 mug/k, BPA group females compared to controls. From PND 90 to 120, gestationally exposed F1 female mice were mated with unexposed males. Total numbers of pups and sex ratio in F1 mice exposed to BPA or DES, and those of their offspring (F2) were not different from controls in any treatment group. The present results indicate that prenatal exposure to low doses of BPA and DES induces early vaginal opening, but does not affect reproductive functioning at the first breeding. (C) 2002 Elsevier Science Inc. All rights reserved. - Taisen Iguchi, Hajime Watanabe, Yoshinao Katsu, Takeshi Mizutani, Shinichi Miyagawa, Atsuko Suzuki, Satomi Kohno, Kiyoaki Sone, Hideo KatoCongenital anomalies 42 (2) 94 - 105 0914-3505 2002 [Refereed][Not invited]
Antenatal sex-hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long-term changes in reproductive as well as non-reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long-term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disruptors. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT-PCR, and differential display methods, and genes related to the estrogen-independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided. - Nakahata S, Mita K, Katsu Y, Nagahama Y, Yamashita MZoological Science 社団法人 日本動物学会 18 (3) 337 - 343 0289-0003 2001 [Refereed][Not invited]
Cytoplasmic polyadenylation regulates translational activation of dormant cyclin B1 mRNA stored in immature oocytes, a process required for the initiation of oocyte maturation in goldfish and Xenopus. As a first step towards understanding the molecular mechanisms of translational activation of cyclin B1 during oocyte maturation, we have isolated cDNA clones encoding proteins involved in cytoplasmic polyadenylation and produced specific antibodies against recombinant proteins. These include poly (A) polymerase (PAP), poly(A)-binding protein (PABP) and cytoplasmic polyadenylation element-binding protein (CPEB). Monoclonal antibodies raised against goldfish PAP recognized several forms of PAP in goldfish and Xenopus oocytes. Besides ordinary PAPs with high molecular weight (ca. 100 kDa), the antibodies also detected those with low molecular weight (ca. 40 kDa) that are present specifically in the cytoplasm, raising new players that might be responsible for cytoplasmic polyadenylation. An antibody against goldfish PABP showed for the first time in Xenopus oocytes the protein expression of PABPII, another PABP distinct from the well-characterized PABPI. Monoclonal antibodies raised against Xenopus CPEB recognized both unphosphorylated 62-kDa and phosphorylated 64-kDa forms but did not cross-react with goldfish CPEB, which was specifically detected by anti-goldfish CPEB monoclonal antibodies produced previously. The cDNAs, recombinant proteins and antibodies produced in this study are expected to provide useful tools for investigating the regulatory mechanisms of cyclin B1 translation during oocyte maturation in goldfish and Xenopus. - Nakahata S, Katsu Y, Mita K, Inoue K, Nagahama Y, Yamashita MJournal of Biological Chemistry 276 (24) 20945 - 20953 2001 [Refereed][Not invited]
- Y Katsu, N Minshall, Y Nagahama, N StandartDEVELOPMENTAL BIOLOGY 209 (1) 186 - 199 0012-1606 1999/05 [Refereed][Not invited]
During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs which are translationally dormant or masked until meiotic maturation. Fertilisation of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. Here we examine the protein kinase(s) that phosphorylates clam p82/CPEB using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. We show that p82/CPEB phosphorylation requires Ca2+ (<100 mu M) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. We propose a model of clam p82/CPEB phosphorylation in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed. (C) 1999 Academic Press. - Katsu Y, Yamashita M, Nagahama YMolecular and Cellular Endocrinology 158 (1-2) 79 - 85 1999 [Refereed][Not invited]
- Katsu Y, Yamashita M, Nagahama YEuropean Journal of Biochemistry 249 (3) 854 - 861 1997 [Refereed][Not invited]
- Tokumoto T, Yamashita M, Tokumoto M, Katsu Y, Horiguchi R, Kajiura H, Nagahama YJournal of Cell Biology 138 (6) 1313 - 1322 1997 [Refereed][Not invited]
- NAGAHAMA Y, YOSHIKUNI M, YAMASHITA M, TOKUMOTO T, KATSU YCurrent Topics in Develop. Biol. 30 103 - 145 1995 [Refereed][Not invited]
- KATSU Y, YAMASHITA M, HIRAI T, TOKUMOTO T, KAJIURA H, NAGAHAMA YDevelopmental Biology 170 (2) 616 - 625 1995 [Refereed][Not invited]
- KAJIURA H, YAMASHITA M, KATSU Y, NAGAHAMA YDevelopment Growth & Differentiation 35 (6) 647 - 654 1993 [Refereed][Not invited]
- KATSU Y, YAMASHITA M, KAJIURA H, NAGAHAMA YDevelopmental Biology 160 (1) 99 - 107 1993 [Refereed][Not invited]
- ONOE S, YAMASHITA M, KAJIURA H, KATSU Y, JIANG JQ, NAGAHAMA YBiomedical Research-Tokyo 14 (6) 441 - 444 1993 [Refereed][Not invited]
- M FUJISHIMA, Y KATSU, E OGAWA, M SAKIMURA, M YAMASHITA, Y NAGAHAMAJOURNAL OF PROTOZOOLOGY 39 (6) 683 - 690 0022-3921 1992/11 [Refereed][Not invited]
Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein. - M FUJISHIMA, KODAMA, I, M HORI, M HORI, Y KATSU, R IMAIEXPERIMENTAL CELL RESEARCH 193 (1) 155 - 160 0014-4827 1991/03 [Refereed][Not invited]
MISC
- 宮川信一, 勝義直, 井口泰泉 比較内分泌学 36- (139) 283 -285 2010 [Not refereed][Not invited]
- レポータージーンアッセイを利用した魚類エストロゲン受容体の種特異性.勝義直, 井口泰泉 環境ホルモン学会ニュースレター 10- (4) 6 2008 [Not refereed][Not invited]
- Iguchi, T, Katsu, Y BioScience 58- 1061 -1067 2008 [Not refereed][Not invited]
- Developmental reproductive effects of exposure to pharmaceutical steroids in the aquatic environment: Studies on mosquitofish (Gambusia affinis affinis), roach (Rutilus rutilus) and medaka (Oryzias latipes).Iguchi, T, Katsu, Y, Urushitani, H, Lange, A, Tyler, C.R J. Marine Sci. Technol. 15- 29 -36 2007 [Not refereed][Not invited]
- Katsu, Y, Hinago, M, Sone, K, Guillette, L.J.Jr, Iguchi, T Mol. Cell. Endocrinol. 276- 10 -17 2007 [Not refereed][Not invited]
- Endocrine disrupting organotin compounds are potent inducers of imposex in gastropods and adipogenesis in vertebrates.Iguchi, T, Katsu, Y, Horiguchi, T, Watanabe, H, Blumberg, B, Ohta, Y Mol. Cell. Toxicol. 3- 1 -10 2007 [Not refereed][Not invited]
- Ena Taniguchi, Yoshinao Katsu, Noriaki Santou, Osamu Tooi, Taisen Iguchi, Akira Matsuno ZOOLOGICAL SCIENCE 23- (12) 1213 -1213 2006/12 [Not refereed][Not invited]
- Yasuhiko Kato, Kaoru Kobayashi, Yoshinao Katsu, Hajime Watanabe, Taisen Iguchi ZOOLOGICAL SCIENCE 23- (12) 1182 -1182 2006/12 [Not refereed][Not invited]
- Yoshinao Katsu, Rie Ichikawa, Toshitaka Ikeuchi, Louis Guillette, Taisen Iguchi ZOOLOGICAL SCIENCE 23- (12) 1216 -1216 2006/12 [Not refereed][Not invited]
- Takeshi Nakamura, Yoshinao Katsu, Hajime Watanabe, Taisen Iguchi ZOOLOGICAL SCIENCE 23- (12) 1216 -1216 2006/12 [Not refereed][Not invited]
- Y Katsu, DS Bermudez, EL Braun, C Helbing, S Miyagawa, MP Gunderson, S Kohno, TA Bryan, LJ Guillette, T Iguchi GENERAL AND COMPARATIVE ENDOCRINOLOGY 136- (1) 122 -133 2004/03 [Not refereed][Not invited]
Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance, and responses to stress. The estrogens are essential for normal reproductive activity in female and male vertebrates and appear to have direct actions during sex determination in some vertebrates. To begin to understand the molecular mechanisms of estrogen action in alligators, we have isolated cDNAs encoding the estrogen receptors (ER) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify alligator ovary RNA. Two different DNA fragments (ERalpha and ERbeta) were obtained and the full-length alligator ERa cDNA was obtained using 5' and 3' RACE. The inferred amino acid sequence of alligator ERalpha (aERalpha) was very similar to the chicken ERalpha (91% identity), although phylogenetic analyses suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. We also isolated partial DNA fragments encoding ERbeta and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERbeta and PR. We examined the expression levels of these three steroid receptors (ERalpha, ERbeta, and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERalpha transcript levels in juvenile alligators decreased after E-2 treatment whereas ERbeta and PR transcripts were not changed. These results provide tools that will allow future studies examining the regulation and ontogenic expression of steroid receptors in alligators and expand our knowledge of vertebrate steroid receptor evolution. (C) 2003 Elsevier Inc. All rights reserved. - Estrogen Receptor Binding Assay for Evaluation of Endocrine Disrupting Chemicals on Marine FishNakai M, Yokota H, Urushitani H, Asai D, Katsu Y, Eto C, Yakabe Y, Iguchi T, Shimohigashi Y Marine Biotechnology 6- S137-141 2004 [Not refereed][Not invited]
- Kato Hideo, Ohta Yasuhiko, Katsu Yoshinao, Watanabe Hajime, Iguchi Taisen Journal of toxicological sciences 28- (4) 319 -319 2003/10/31
- URUSHITANI H., NAKAI M., INANAGA H., SHIMOHIGASHI Y., SHIMIZU A., KATSU Y., IGUCHI T. Proceedings of the Japan Society for Comparative Endocrinology (18) 63 -63 2003/08/08
- KATSU Yoshinao, GUNDERSON Mark, GUILLETTE JR. Louis J., IGUCHI Taisen Proceedings of the Japan Society for Comparative Endocrinology (18) 69 -69 2003/08/08
- A NOVEL C-TYPE LECTIN EXPRESSION IN STRATIFIED AND CORNIFIED EPITHELIAL CELLS(Endocrinology,Abstracts of papers presented at the 74^
Annual Meeting of the Zoological Society of Japan) :Katsu Yoshinao, Iguchi Taisen Zoological science 20- (12) 1611 -1611 2003 ESTROGEN-INDEPENDENT ACTIVATION OF ErbBs SIGNALING AND ESTROGEN RECEPTOR α IN THE MOUSE VAGINA EXPOSED NEONATALLY TO DIETHYLSTILBESTROL(Endocrinology,Abstracts of papers presented at the 74^Annual Meeting of the Zoological Society of Japan) :Miyagawa Shinichi, Katsu Yoshinao, Watanabe Hajime, Iguchi Taisen Zoological science 20- (12) 1606 -1606 2003 Katsu Yoshinao, Iguchi Taisen Zoological science 19- (12) 1491 -1491 2002Miyagawa Shinichi, Katsu Yoshinao, Watanabe Hajime, Iguchi Taisen Zoological science 19- (12) 1492 -1492 2002Iguchi T., Katsu Y., Watanabe H. Congenital anomalies 41- (3) 233 -234 2001/09Katsu Y., Takasu E., Watanabe H., Iguchi T. Zoological science 18- 13 -13 2001Nakahata Shingo, Mita Koichi, Katsu Yoshinao, Nagahama Yoshitaka, Yamashita Masakane Zoological science 18- 62 -62 2001Miyagawa S., Satoh M., Watanabe H., Katsu Y., Dobashi K., Oh-Ishi M., Maeda T., Iguchi T. Zoological science 18- 20 -20 2001Iguchi, T, Watanabe, H, Katsu, Y Horm. Behav. 40- (2) 248 -251 2001 [Not refereed][Not invited]Nakahata S., Katsu Y., Nagahama Y., Yamashita M. Zoological science 17- 61 -61 2000Yamaguchi Akihiko, Katsu Yoshinao, Nagahama Yoshitaka Zoological science 17- 57 -57 2000Katsu Y., Yamashita M., Nagahama Y. Zoological science 17- 61 -61 2000中畑新吾, 勝義直, 長浜嘉孝, 山下正兼 日本動物学会大会予稿集 70th- 1999Nakahata S., Katsu Y., Nagahama Y., Yamashita M. Zoological science 16- 62 -62 1999Katsu Y., Yamashita M., Ogawa K., Nagahama Y. Zoological science 16- 62 -62 1999Katsu Y., Cornall N., Nagahama Y., Standart N. Zoological science 15- 82 -82 1998勝義直, 山下正兼, 長浜嘉孝 日本動物学会大会予稿集 67th- 1996勝義直, 山下正兼, 長浜嘉孝 日本動物学会大会予稿集 65th- 1994勝義直, 山下正兼, 平井俊朗, 梶浦弘子, 長浜嘉孝 日本動物学会大会予稿集 64th- 1993梶浦弘子, 山下正兼, 勝義直, 長浜嘉孝 日本動物学会大会予稿集 64th- 1993尾上伸二, 山下正兼, 梶浦弘子, 勝義直, 長浜嘉孝 日本分子生物学会年会プログラム・講演要旨集 16th- 1993KATSU Y. Dev Biol 159- 1 -9 1993Katsu Y., Yamashita M., Kajiura H., Nagahama Y. Zoological science 9- (6) 1162 -1162 1992katsu Y., Fujishima M. Zoological science 7- (6) 1163 -1163 1990Association Memberships
Research Projects
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)Date (from‐to) : 2019/04 -2022/03Author : Katsu YoshinaoIn this study, we succeeded in isolating glucocorticoid, mineralocorticoid, and progesterone receptor genes from the cartilaginous fish elephant shark. This result suggests that the adrenal steroid receptor and progesterone receptor possessed in higher vertebrates such as humans are already expressed from the evolutionary stage of cartilage fish. Next, these isolated receptors were used to investigate hormone responsiveness in detail. As a result, we found some differences between cartilaginous fish receptors and human receptors, and then we identified the causative site. By analyzing the function of steroid receptors in cartilaginous fish in this way, we were able to obtain some findings that lead to the functional evolution of the receptors.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2014 -2016Author : Katsu YoshinaoThe adrenal steroid receptor binds to adrenal steroids such as glucocorticoids and mineralocorticoids secreted from the adrenal cortex and is involved in the mechanism of maintaining the homeostasis of animals and endocrine secretions. However, it is still unknown at which stage of biological evolution the mechanism of maintaining endocrine homeostasis by adrenal steroid receptors found in higher vertebrates such as humans. The purpose of this research project was to clarify the process of adrenal steroid receptor gene appearance and function acquisition in evolution process. As a result, ancient fishes, gar and sturgeon have the adrenal steroid receptor genes like humans, and the adrenal steroid receptors from ancient fishes have functionally equivalent to human receptors. These results can be expected to serve as important clues to elucidate the molecular evolution of the adrenal steroid receptor.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2011 -2013Author : KATSU YoshinaoThe estrogen receptor interacts with estrogen which is female sex hormone, and participates in mechanism of the homeostatic maintenance of the vertebrates. However, it is yet unknown that the molecular evolution of estrogen receptor gene and protein. This research theme was carried out for the appearance of the estrogen receptor gene with the organic evolution and the elucidation of the process of the gain of function. As a result, it became clear that the elasmobranchii has the estrogen receptor genes that encode a functional receptor like higher vertebrates. Our functional analysis suggest that the function of estrogen receptor as a hormone-dependent transcription factor might be gain through the evolutionary process. The above-mentioned result will become the important clue in elucidating the molecular evolution of the estrogen receptor.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2009 -2011Author : OHTA Yasuhiko, KATSU Yoshinao, TAKEUCHI Takashi, IGUCHI TaisenIn American alligator showing temperature-dependent sex determination(TSD), development of reproductive organs, ontogeny of expression of estrogen and progesteronereceptor and candidates involved in the TSD were identified during the thermo-sensitiveperiod. These results may contribute to the understanding of endocrine disrupters onreptiles.
- 性ホルモン受容体の分子進化科学研究費補助金Date (from‐to) : 2008 -2010性ホルモン受容体は、進化上いつから存在するのか、DNAとの結合能力やホルモンとの結合能力は、どの生物段階から備わったのかを調べる。
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 2005 -2006Author : 勝 義直, 井口 泰泉爬虫類である全てのワニ類と大部分のカメ類の性決定は、環境要因(外気の温度)によって決定する事が分かっている。しかし、この性染色体によらない温度依存的性決定の分子機構については、ほとんどわかっていない。本研究は、この爬虫類の性決定・性分化の分子機構を解析することを目的として行なわれた。本年度は、温度と密接に関連する遺伝子の候補として、熱ショックタンパク類の関連遺伝子のクローニングを行なった。これまでの研究からも予想されたが、鳥類のHSPとの相同性が高いと言う事が判明した。さらに、ワニは初期発生の一定の時期(ステージ19から24まで)の温度が重要で、この時期に性が決定する事が分かっている。オスになる温度、メスになる温度でそれぞれ処理した初期胚から生殖腺を摘出し、今回得られたHSP群の発現パターンを調べたところ、いずれのHSPもすでにステージ19から発現している事がわかった。さらに、その発現量は発生にしたがって増減することはなく、ほぼ一定のままであった。また、オスとメスでの差は観察できなかった。この結果は、温度依存性の性決定機構をもつワニでは、その性決定にはHSPは関与しないのではないかということを示唆している。しかし、メスへの分化に必要と考えられているエストロゲン受容体のか活性化機構にはHSPが関連することも知られているので、今後、さらに局在などを調べ性決定とHSPの関連を詳細に調べる必要があると考えている。さらに、オスへの分化に必要だと考えられる、抗ミュラー管ホルモン(AMH)遺伝子の全長のクローニングに成功した。また転写調節領域の単離も完了しており、今後どのように温度からの刺激がどのようにAMHの発現調節に繋がっていくのかを解析していく予定である。
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 2003 -2003Author : 勝 義直, 井口 泰泉, 渡邉 肇動物プランクトンであるミジンコは生態系の中でももっとも下位に位置し、生態系において非常に重要な位置を占めている。またミジンコは、その生活環の全てを水中で過ごすことから、環境の指標としても重要な生物であり、長年、環境や毒性の評価などにおいて、世界中の研究室で利用されてきている。特に近年、地球環境の重要性が認識されるにつれ、環境指標の生物としての重要性を増してきている。 ところが、ミジンコの重要性が認識されてきているにもかかわらず、その遺伝子情報は驚くほど乏しい。例えばNCBIのデータベースでは、Daphniaで登録されている遺伝子はわずか271遺伝子でその大半がリボソーマルRNAとヘモグロビン遺伝子であり、実際にアノーテーションされている遺伝子は数種類に過ぎない。様々な生物種のゲノムやEST情報が読まれているなかで、環境の指標として今後重要性を増して行くであろうミジンコにおけるこの遺伝子情報は皆無に近いといえる。 そこで本研究では、ミジンコについての基本的な遺伝情報を取得することを目的として、まずEST解析を行い、データの収集と解析を行った。この際の非常に大きな特色は、通常単為生殖をしているために作出が困難なオスのミジンコについての解析も行う点であった。我々は今まで報告のなかった100%のオスを作出する条件を見出しており、この条件により処理したミジンコからライブラリーを構築し、その遺伝子情報を解析することにより、通常発現している遺伝子の概要を把握すると同時に、生殖や性差に関連した遺伝子情報も取得した。
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 2002 -2003Author : 井口 泰泉, 大迫 誠一郎, 勝 義直, 渡邉 肇工業化時代の始まりと共に環境に放出された化学物質は野生生物の健康に大きな脅威となっており、それは現在も続いている。今までの環境問題は、致死作用、ガン化あるいは奇形といった健康影響のみに焦点が当てられてきた.しかし、最近見出された多くの事例から、内分泌撹乱と言った今までにはなかった新たな観点からの研究が必要となってきている。 特に環境中に放出されたダイオキシン,農薬,ポリカーボネートの原材料,界面活性剤をはじめとした化学物質がホルモンの受容体,特にエストロゲン受容体に結合することによって体内のエストロゲンと同じ作用を示し,野生動物種の性器異常,発生異常,個体数の減少,ヒトの乳癌、子宮内膜症、精巣癌の増加,精子数の減少を誘起している可能性が示唆された。欧米でこの問題に強く関心がもたれるようになったのは1990年代前半からで、日本では1997年になってからこの問題が広く取り上げられるようになった。以後、厚生省や環境庁を中心に、胎児を含めたヒトに対する内分泌撹乱物質の曝露状況調査が進められており、その結果、日本人の体内にも多くの内分泌撹乱物質が蓄積していることが判明してきた。そして環境中に放出された化学物質の多くが、ホルモン様あるいは抗ホルモン様に作用するという実験的な証拠、および野外での観察結果が蓄積されつつある。動物の生殖系の発達は内分泌撹乱物質の曝露によって大きな影響を受ける.野生動物の研究から、内分泌撹乱物質の曝露により、雌雄ともに生殖腺および生殖腺付属器官の発生、機能分化およびホルモンの代謝に影響が出ている。多くの野生動物種の生殖系で同様の異常が見られることから、ヒトに対する影響も懸念されている。 本研究では、モデル動物としてマウスへの影響に関する研究だけでなく、内分泌撹乱物質が水系に入ることから、魚類・両生類の発生・生殖に対しても着目し解析を行った。これにより、内分泌撹乱物質を簡便に検出する系を確立し、発生・生殖に対する作用機構の解明を試みた。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1999 -2000Author : IGUCHI Taisen, KATSU YoshinaoBisphenol-A (BPA), an estrogenic chemical, was given to pregnant mice and Japanese monkey and studied transplacental transfer. BPA was foun in fetal brain, liver and other organs in 30 min in mice and 1 h in monkey suggesting that BPA can be easily cross the placenta. BPA and a synthetic estrogen, diethylstilbestrol (DES), were given to pregnant mice and studied fertility and various abnormalities in female offspring. DES induced sterility, polyovular follicles in ovary and persistent stratification of vaginal epithelium, but not BPA.Hoxa-9 and a-10 genes in Mullerian duct were downregulated by DES in the fetal period suggesting the close correlation to uterine changes in DES-exposed mice. In order to study low dose issue of BPA, 2 and 20μg/kg body weight of BPA and 0.02-2μg/kg of DES were given to day 10 to 17 of pregnancy. In 20μg BPA-and all DES-exposed female offspring showed significantly early vaginal opening and lower body weight as compared to controls. This results suggest that there is a low dose effect of BPA, however, the mechanism underlie is still unclear. Neonatal DES treatment induced hypospadias in female mice, fusion of urethra and vaginal lumen at lower position of vagina, where apoptosis was observed. The critical period in induction of hypospadias was 7 days after birth. Neonatally BPA (150μg) treated female mice showed persistent estrus, and ovary-independent vaginal changes. In order to study estrogenic agents on development of fish, Fundulus heteroclitus embryos were exposed to estradiol. Various malformations, including sex ratio change were induced by estradiol. Estrogen receptor α and β were cloned and ER expression during development were analysed. FTZ-F1 gene was also cloned from Fundulus. In zebrafish, gonad develops to ovary by ca 20 days after hatching then a half of fish develop testis from the ovary. During testis formation, apoptosis of oocytes occurred in the ovary.