National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY

Professor

IGUCHI, Taisen

Assistant Professor:
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Staff:
Graduate Student:

Visiting Scientist:
Technical Assistant:
Secretary:

MIYAGAWA, Shinichi
OGINO, Yukiko
MIYAKAWA, Hitoshi
CHAKRABORTY, Tapas*
CHAKRABORTY, Tapas
HIRUTA, Chizue
MIYAKAWA, Hitoshi*
MIZUTANI, Takeshi
SATO, Masaru
TOYOTA, Kenji
SUMIYA, Eri
YATSU, Ryohei
ODA, Shigeto
HIRAKAWA, Ikumi
TOHYAMA, Saki
HAYASHI, Tomoko
IMAIZUMI, TaekoSynthetic chemicals found in the environment have thecapacity to disrupt the development and function of theendocrine system in both wildlife and humans. This has
drawn public concern since many of these chemicals maybind to estrogen receptors (ERs) and evoke estrogenic
effects. Early evidence that exposure to estrogenic chemicals
during development could pose a threat to human healthcame from studies of a synthetic hormone, diethylstilbestrol(DES), which was used to prevent premature birth andspontaneous abortion. Laboratory experiments showed that
exposure of animals to sex hormones during critical windowsof perinatal life caused irreversible alterations to theendocrine and reproductive systems of both sexes. The
immune and nervous systems, bone, muscle, and the liver
were also affected. Although many of these chemicals canbind to ERs in wildlife and humans, the molecular basis forthe action of environmental estrogens remains poorlyunderstood. Thus, understanding the molecular mechanisms
through which environmental estrogens and sex hormones
act during critical developmental windows is essential.

I. Developmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts
The emerging paradigm of the “embryonic/fetal origins ofadult disease” provides a powerful new framework forconsidering the effects of endocrine disrupters on human andanimal health. In 1971, prenatal DES exposure was found toresult in various abnormalities of the reproductive tract inwomen. This syndrome was named the DES syndrome.
Similar abnormalities have been demonstrated inexperimental animals exposed perinatally to estrogens.
Developmental estrogen exposure in mice, for example,
induces persistent proliferation of vaginal epithelial cells. We
found that the persistent changes in the vagina in mice
exposed neonatally to estrogens result from the persistentactivation of erbBs and ERα, and sustained expression ofEGF-like growth factors. Currently, we are analyzing themethylation status in the mouse vagina using MeDIP(methylated DNA immunoprecipitation) coupled with amicroarray (MeDIP-chip). We found several differentiallymethylated or demethylated DNA profiles in neonatally
DES-exposed mouse vaginae and controls. We thus consider
that neonatal DES exposure affects DNA methylation
profiles, resulting in persistent abnormalities in mouse
reproductive organs.

Figure 2. A hypothetical model for the estrogen-independent ER
activation pathway in mouse vaginae.

II. Estrogen receptors of birds, reptiles,
amphibians and fishes

Figure 1. Scheme of estrogen-dependent and -independent vaginal
epithelial cell proliferation in mice induced by perinatal estrogen
exposure.

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Steroid and xenobiotic receptors (SXR) have been cloned
from various animal species (fish, amphibians, reptiles, birds
and mammals) by our group and we have demonstratedspecies-specific differences in their responses to various
environmental and endogenous chemicals (receptor genezoo). Thus, simple predictions of chemical effects based ondata from a few established model species are not sufficient
to develop real world risk assessments. ER and ER-likegenes have been cloned from various animal species
including rockshell, Amphioxus, lamprey, lungfish, sturgeon,
gar, roach, stickleback, mosquitofish, mangrove Rivulus,

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

catshark, whale shark, Japanese giant salamander, Tokyo
salamander, newt, axolotl, toad, Silurana tropicalis,
American alligator, Nile crocodile, freshwater turtle,
Japanese rat snake, Okinawa habu, and vultures. Functional
studies showed that the Amphioxus ER sequence does notbind estrogen but Amphioxus steroid receptor and lamprey
ER exhibited ligand-dependent transactivation, proving that
invertebrate and primitive vertebrates, such as the Agnatha,
have a functional ER. We found that medaka ER subtypes
have their specific functions, and medaka, zebrafish andstickleback ERs are more sensitive to estrogen/estrogen-likechemical exposures than other fishes by reporter gene assay.
Thus, these approaches are efficient to evaluate therelationship between species and their sensitivities tochemicals.

III. Evolutionary history and functional
characterization of androgen receptor
genes in jawed vertebrates
Vertebrates show diverse sexual characteristics which areregulated by androgens. To elucidate the evolutionary history
and functional diversification of androgen receptor (AR)genes in vertebrates, we cloned the AR cDNAs from a shark,basal ray-finned fishes (Actinopterygii), namely bichir andsturgeon (Acipenseriformes), and teleosts including a basal
teleost, arowana (Osteoglossiformes). Molecularphylogenetic analysis revealed that a gene duplication event
gave rise to two different teleost ARs (α and β) and likely
occurred in the actinopterygian lineage leading to teleosts
after the divergence of Acipenseriformes but before the splitof Isteoglossiformes. Functional analysis revealed that theshark AR activates the target gene via androgen response
element by classical androgens. The teleost ARα showedunique intracellular localization with a significantly higher
transactivation capacity than that of teleost ARβ. These
results indicate that the most ancient type of AR, as activatedby the classic androgens as ligands, emerged before theChondrichthyes-Osteichthyes split and the AR gene was
duplicated during a teleost-specific gene duplication event.

IV. Gene zoo and receptor zoo
We are establishing cDNA library banks and receptor genebanks of animal species including lancelet, lamprey,sturgeon, lungfish, gar, mangrove Rivulus, whale shark
catshark, Japanese giant salamander, newt, Rana rugosa,
Silurana tropicalis, Japanese rat snake, Okinawa habu,Florida red berry turtle, American alligator, Nile crocodile,vulture and polar bear in collaboration with the University ofPretoria, South Africa, University of Florida, Medical
University of South Carolina, San Diego Zoo, USA, and theAsa Zoo in Hiroshima.

Figure 3. Evolutionary relationships of androgen receptor sequences.

database and developed an oligonucleotide-based DNA
microarray with high reproducibility and demonstrated theusefulness of the array for the classification of toxic
chemicals as well as for the molecular understanding ofchemical toxicity in a common freshwater organism. D.
magna reproduce asexually (parthenogenesis) when they are
in an optimal environment for food, photoperiod andpopulation density. Once environmental conditions become
sub-optimal, they alter their reproductive strategy from
asexual to sexual reproduction. Chemicals are able to affectthe sex determination of D. magna and we found thatjuvenile hormone (JH) agonists (insect growth regulators),
for example, induce the production of male offspring. The
molecular basis of environmental sex determination islargely unknown in D. magna. To understand the molecularmechanisms of this phenomenon we isolated sex
determination-related genes. Also, we have developed amethod to inject genes into D. magna embryos which willallow us to study gain- and loss-of function analyses in moredetail in this species. Using these techniques, wedemonstrated that DSX1 (double sex 1), one of theDM-domain genes, is essential for male differentiation in D.
magna. To further explore the signaling cascade of sexual
differentiation in D. magna, a gene expression profile ofV. Sex differentiation mechanism in Daphnids
Daphnia magna has been used extensively to evaluate theorganism- and population-based responses of toxicity orreproductive toxicity tests. These tests, however, provide no
information about the mode of action of the tested
compounds. Therefore, we applied an ecotoxicogenomic
assessment of D. magna. We established a Daphnia EST

Figure 4. Life cycle of Daphnia.

61National Institute for Basic Biology

Environmental Biology

JH-responsive genes is essential. Thus, DNA microarray
analysis has been performed in the gonads of D. magna
exposed to fenoxycarb (synthesized JH agonist widely used
as an insect growth regulator) and methyl farnesoate (JHidentified in decapods) at the critical timing of JH-induced
sex determination in D. magna. We are identifying
JH-responsive genes in the ovary. We are also identifying JH
receptor in daphnids.

John, J.A., Braun, E.L., Isberg, S.R., Miles, L.G., Chong, A.Y.,• St.Gongora,J., Dalzell, P., Moran, C., Bed’hom, B., Abzhanov, A.,

Publication List〔Original papers〕
E.K., Ogino, Y., Iguchi, T., Barber, D.S., and Denslow,• Brockmeier,
N.D. (2012). Effects of 17β-trenbolone on Eastern and Western•
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••••
•••
•

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mosquitofish (Gambusia holbrooki and G. affinis) and anal fin growthand gene expression patterns. Aquat. Toxicol. 128-129C, 163-170.
Goto, Y., Kajiwara, M., Yanagisawa, Y., Hirose, H., Yoshimi, T.,Uemura, M., Nakano, H., Takahashi, S., Shida, Y., Iguchi, T., Takahashi,Y., and Miura, T. (2012). Detection of vertebrate-type steroid hormones
and their converting activities in the neogastropod Thais clavigera(Kster, 1858). J. Molluscan Studies 78, 197-204.
Haraguchi, R., Matsumaru, D., Nakagata, N., Miyagawa, S., Suzuki, K.,
Kitazawa, S., and Yamada, G. (2012). The hedgehog signal induced
modulation of bone morphogenetic protein signaling: an essential
signaling relay for urinary tract morphogenesis. PLoS ONE 7, e42245.
Hirakawa, I., Miyagawa, S., Katsu, Y., Kagami, Y., Tatarazako, N.,
Kobayashi, T., Kusano, T., Mizutani, T., Ogino, Y., Takeuchi, T., Ohta,Y., and Iguchi, T. (2012). Gene expression profiles in the testis
associated with testis-ova in adult Japanese medaka (Oryzias latipes)
exposed to 17α-ethinylestradiol. Chemosphere 87, 668-674.
Kakuta, H., Tanaka, M., Chambon, P., Watanabe, H., Iguchi, T., andSato, T. (2012). Involvement of gonadotropins in the induction of
hypertrophy-hyperplasia in the interstitial tissues of ovaries inneonatally diethylstilbestrol-treated mice. Reprod. Toxicol. 33, 35-44.
Lange, A., Katsu, Y., Miyagawa, S., Ogino, Y., Urushitani, H.,
Kobayashi, T., Hirai, T., Shears, J.A., Nagae, M., Yamamoto, J.,
Ohnishi, Y., Oka, T., Tatarazako, N., Ohta, Y., Tyler, C.R., and Iguchi, T.
(2012). Comparative responsiveness to natural and synthetic estrogensof fish species commonly used in the laboratory and field monitoring.
Aquat. Toxicol. 109, 250-258.
Maekawa, T., Sakuma, A., Taniuchi, S., Ogo, Y., Iguchi, T., Takeuchi,
S., and Takahashi, S. (2012). Transforming growth factor-α mRNA
expression and its possible roles in mouse endometrial stromal cells.
Zool. Sci. 29, 377-383.
Myburgh, J.G., Huchzermeyer, F.W., Soley, J.T., Booyse, D.G.,
Groenewald, H.B., Bekker, L.C., Iguchi, T., and Guillette, L.J.Jr.
(2012). Technique for the collection of clean urine from the Nilecrocodile (Crocodylus niloticus). J. South African Vet. Assoc. 83, E1-6.
Nakajima, T., Iguchi, T., and Sato, T. (2012). Hedgehog signaling plays
roles in epithelial cell proliferation in the neonatal mouse uterus andvagina. Cell Tiss. Res. 348, 239-247.
Nakamura, T., Miyagawa, S., Katsu, Y., Mizutani, T., Sato, T., Takeuchi,T., Iguchi, T., and Ohta, Y. (2012). P21 and Notch signalings in thepersistently altered vagina induced by neonatal diethylstilbestrol
exposure in mice. J. Vet. Med. Sci. 74, 1589–1595.
Nakamura, T., Miyagawa, S., Katsu, Y., Watanabe, H., Mizutani, T.,
Sato, T., Morohashi, K.-I., Takeuchi, T., Iguchi, T., and Ohta, Y. (2012).
WNT family genes and their modulation in the ovary-independent andpersistent vaginal epithelial cell proliferation and keratinization inducedby neonatal diethylstilbestrol exposure in mice. Toxicology 296, 13-19.
Nakamura, T., Miyagawa, S., Katsu, Y., Sato, T., Iguchi, T., and Ohta, Y.
(2012). Sequential changes in expression of Wnt- and Notch-related
genes in the vagina and uterus of ovariectomized mice after estrogen
exposure. In Vivo 26, 899-906.
Oka, K., Kohno, S., Uruchitani, H., Guillette, L.J.Jr., Ohta, Y., Iguchi,T., and Katsu, Y. (2012). Molecular cloning and characterization of thecorticoid receptors from the American alligator. Mol. Cell. Endocrinol.
365, 153-161.

••

Burgess, S.C., Cooksey, A.M., Castoe, T.A., Crawford, N.G., Densmore,L.D., Drew, J.C., Edwards, S.V., Faircloth, B.C., Fujita, M.K.,Greenwold, M.J., Hoffmann, F.G., Howard, J.M., Iguchi, T., Janes,D.E., Khan, S.Y., Kohno, S., de Koning, A.J., Lance, S.L., McCarthy,F.M., McCormack, J.E., Merchant, M.E., Peterson, D.G., Pollock, D.D.,
Pourmand, N., Raney, B.J., Roessler, K.A., Sanford, J.R., Sawyer, R.H.,
Schmidt, C.J., Triplett, E.W., Tuberville, T.D., Venegas-Anaya, M.,
Howard, J.T., Jarvis, E.D., Guillette, L.J.Jr., Glenn, T.C., Green, R.E.,
and Ray, D.A. (2012). Sequencing three crocodilian genomes toilluminate the evolution of archosaurs and amniotes. Genome Biol. 13,415.
Takase, M., Shinto, H., Takao, Y., and Iguchi, T. (2012). Accumulationand pharmacokinetics of estrogenic chemicals in the pre- and posthatch embryos of the frog Rana rugosa. In Vivo 26, 913-920.
Taylor, J.A., Richter, C.A., Suzuki, A., Watanabe, H., Iguchi, T., Coser,K.R., Shioda, T., and vom Saal, F.S. (2012). Dose-related estrogeneffects on gene expression in fetal mouse prostate mesenchymal cells.
PLoS ONE 7, e48311.

〔Original paper (E-publication ahead of print)〕
I., Hirakawa, I., Miyagawa, S., Mitsui, N., Miyahara, M.,• Hirakawa,Onishi, Y., Kagami, Y., Kusano, T., Takeuchi, T., Ohta, Y., and Iguchi, T.
Developmental disorders and altered gene expression in the tropicalclawed frog (Silurana tropicalis) exposed to 17α-ethinylestradiol. J.
Appl. Toxicol. 2012 Nov 6.

〔Review articles〕
E.G., Nishioka, R.S., Bern, A., Hirano, T., Borski, R., Clarke, C.,• Grau,Foskett, K., Guillette, L.J., Iguchi, T., Jones, L.A., Loretz, C.,•••

McCormick, S., MacLachlan, A., Mason, C.A., Mills, K.T., Nagahama,Y., Nicoll, C.S., Richman, N.H., Sheridan, M., Specker, J.L., Sullivan,J.J., and Young, G. (2012). In memory of Professor Howard A. Bern.
Gen. Comp. Endocrinol. 176, 121-123.
Guillette, L.J.Jr., and Iguchi, T. (2012). Life in a contaminated world.
Science 337, 1614-1615.
Miyagawa, S., Yatsu, R., Sudo, T., Igarashi, K., Kanno, J., and Iguchi,T. (2012). Irreversible effect of diethylstilbestrol on reproductive organs
and current approach for epigenetic effects of endocrine disrupting
chemicals. In: Toxicology and Epigenetics. Ed. Sahu, S.C., John Wiley& Sons, Ltd., pp. 357-364.
Tatarazako, N., and Iguchi, T. (2012). Evaluation of toxicities ofherbicides using short-term chronic tests of alga, daphnid and fish. In:Herbiciddes – Environmental Impact Studies and Management
Approaches. Ed. Alvarez-Fernandez, R. ISBN: 978-953-307-892-2,
InTech.