National nstitute or asic iology

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Professor

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aisen

Assistant Professor:
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Staff:
SOKENDAI Graduate Student:
Visiting Graduate Student:
Visiting Scientist:
Technical Assistant:
Secretary:

MIYAGAWA, Shinichi
OGINO, Yukiko
MIYAKAWA, Hitoshi
HIRUTA, Chizue
MIYAKAWA (OKAMOTO), Misato
MIZUTANI, Takeshi
TOYOTA, Kenji
SUMIYA, Eri
YATSU, Ryohei
TOHYAMA, Saki
EVENSETH, Linn
DOHENY, Brenna
OMORI, Akiko
HAYASHI, Tomoko
INABA, KayoIMAIZUMI, TaekoSynthetic  chemicals  found  in  the  environment  have  the capacity  to  disrupt  the  development  and  function  of  the endocrine  system  in  both  wildlife  and  humans.  This  has drawn  public  concern  since  many  of  these  chemicals  may bind  to  estrogen  receptors  (ERs)  and  evoke  estrogenic effects. Early evidence that exposure to estrogenic chemicals during  development  could  pose  a  threat  to  human  health came from studies of a synthetic hormone, diethylstilbestrol (DES),  which  was  used  to  prevent  premature  birth  and spontaneous  abortion.    Laboratory  experiments  showed  that exposure of animals to sex hormones during critical windows of  perinatal  life  caused  the  immune  and  nervous  systems, bone, muscle, and the liver to be affected. Although many of these chemicals can bind to ERs in wildlife and humans, the molecular  basis  for  the  action  of  environmental  estrogens remains  poorly  understood.  Thus,  understanding  the molecular  mechanisms  through  which  environmental estrogens  and  sex  hormones  act  during  critical developmental windows is essential. Figure  1.  Scheme  of  estrogen-  dependent  and  -independent  vaginal epithelial  cell  proliferation  in  mice  induced  by  perinatal  estrogen exposure.

I. Developmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts

The  emerging  paradigm  of  the  embryonic fetal  origins  of adult  disease   provides  a  powerful  new  framework  for 2Figure  2.  A  hypothetical  model  for  the  estrogen-independent  ER activation pathway in mouse vaginae.

considering the effects of endocrine disrupters on human and animal health. In 1 1, prenatal DES exposure was found to result  in  various  abnormalities  of  the  reproductive  tract  in women.  This  syndrome  was  named  the  DES  syndrome. Similar  abnormalities  have  been  demonstrated  in experimental  animals  exposed  perinatally  to  estrogens. Developmental  estrogen  exposure  in  mice,  for  example, induces  persistent  proliferation  of  vaginal  epithelial  cells (Figure 1). We found that the persistent changes in the vagina in  mice  exposed  neonatally  to  estrogens  result  from  the persistent  activation  of  erbBs  and  ERa,  and  sustained expression of EGF-like growth factors (Figure 2). Currently, we are analyzing the methylation status in the mouse vagina using  a  microarray  (MeDIP-chip).  We  found  several differentially  methylated  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.
 II. Estrogen receptors of birds, reptiles,
amphibians, and fishes

Steroid  and  xenobiotic  receptors  (S R)  have  been  cloned from various animal species (fish, amphibians, reptiles, birds, and  mammals)  by  our  group  and  we  have  demonstrated species-specific  differences  in  their  responses  to  various environmental  and  endogenous  chemicals  (receptor  gene zoo). Thus,  simple  predictions  of  chemical  effects  based  on data from a few established model species are not sufficient to  develop  real  world  risk  assessments.  ER  and  ER-like genes  have  been  cloned  from  various  animal  species including rockshell, Amphioxus, lamprey, lungfish, sturgeon, gar,  roach,  stickleback,  mosquitofish,  mangrove,  Rivulus, 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  not bind  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  and stickleback ERs are more sensitive to estrogen estrogen-like chemical exposures than other fishes by reporter gene assay. Thus,  these  approaches  are  efficient  to  evaluate  the relationship  between  species  and  their  sensitivities  to chemicals.

III. Evolutionary history and functional
characterization of androgen receptor
genes in jawed vertebrates

ertebrates  show  diverse  sexual  characteristics  which are regulated 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  and  sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana  (Osteoglossiformes).  Molecular  phylogenetic analysis  revealed  that  a  gene  duplication  event  gave  rise to two different teleost ARs (a and b) and likely occurred in the actinopterygian lineage leading to teleosts after the divergence  of Acipenseriformes  but  before  the  split  of Osteoglossiformes.  Functional  analysis  revealed  that  the shark AR activates the target gene via androgen response element by classical androgens. The teleost ARa showed unique  intracellular  localization  with  a  significantly higher  transactivation  capacity  than  that  of  teleost ARb. These results indicate that the most ancient type of AR, as activated  by  the  classic  androgens  as  ligands,  emerged before  the  Chondrichthyes-Osteichthyes  split  and  the  AR gene  was  duplicated  during  a  teleost-specific  gene duplication event (Figure  ). IV. Papillary process formation in medaka

Androgens  play  key  roles  in  the  morphological specification  of  male  type  sex  characteristics  and reproductive  organs,  whereas  little  is  known  about  the developmental  mechanisms.  Medaka  show  a  prominent masculine  sexual  character,  papillary  processes  in  the  anal fin,  which  has  been  induced  in  females  by  exogenous androgen  exposure.  We  have  identified  androgen-dependent expressions  of  Bmp7  and  Lef1  are  required  for  the  bone nodule  outgrowth  leading  to  the  formation  of  the  papillary process  in  the  postal  region  of  the  anal  fin.  We  have  also developed  a  testing  method  for  screening  of  chemicals having  androgenic  and  anti-androgenic  activity  using  the anal fin in juvenile medaka.

Figure  . Evolutionary relationships of androgen receptor sequences.

microarray  with  high  reproducibility  and  demonstrated  the usefulness  of  the  array  for  the  classification  of  toxic chemicals  as  well  as  for  the  molecular  understanding  of chemical  toxicity  in  a  common  freshwater  organism.  D.
magna  and  D. pulex  reproduce  asexually  (parthenogenesis) when  they  are  in  an  optimal  environment  for  food, photoperiod  and  population  density.  Once  environmental conditions become sub-optimal, they alter their reproductive strategy  from  asexual  to  sexual  reproduction  (Figure  ). Chemicals  are  able  to  affect  the  sex  determination  of daphnids and we found that juvenile hormone (J ) agonists (insect  growth  regulators),  for  example,  induce  the production  of  male  offspring.  The  molecular  basis  of environmental  sex  determination  is  largely  unknown  in daphnids.  To  understand  the  molecular  mechanisms  of  this phenomenon,  we  isolated  sex  determination-related  genes. Also,  we  have  developed  a  method  to  inject  genes  into  D.
magna  and  D. pulex  embryos  which  will  allow  us  to  study gain-  and  loss-of  function  analyses  in  more  detail  in  these species. Using these techniques, we demonstrated that DS 1 (double sex 1), one of the DM-domain genes, is essential for male  differentiation  in  D. magna.  We  have  developed  an RNAi  method  and  a  TALEN  method  using  D. pulex.  To further explore the signaling cascade of sexual differentiation in  D. magna,  a  gene  expression  profile  of  J -responsive genes is essential. We are identifying J -responsive genes in the  ovary  of  D. magna and  D. pulex  exposed  to  J   agonist and  methyl  farnesoate  (J   identified  in  decapods)  at  the critical timing of J -induced sex determination in D. magna and D. pulex. We have identified J  receptor (heterodimer of methoprene-tolerant  and  steroid  receptor  co-activator)  in daphnids  and  the  function  of  ecdysone  in  molting  and V. Environmental sex differentiation in
Daphnids and American alligators

Daphnia magna  has  been  used  extensively  to  evaluate  the organism-  and  population-based  responses  of  toxicity  or reproductive toxicity tests. These tests, however, provide no information  about  the  mode  of  action  of  the  tested compounds.  Therefore,  we  applied  an  ecotoxicogenomic assessment  to    D. magna.  We  established  a  Daphnia  EST database  and  developed  an  oligonucleotide-based  DNA Figure  . Life cycle of Daphnia.

National nstitute or asic iologyBovulation in D. magna.
Sex determination mechanisms can broadly be categorizedby either a genotypic or environmentally driven mechanism.
Temperature-dependent sex determination (TSD), an
environmental sex determination mechanism mostcommonly observed among vertebrates, has long been
observed especially among reptiles. However, thetemperature-dependent triggering mechanism of TSD and thesubsequent differentiation cascade has long remained
unknown. We are working on the thermosensitive cation
channel, TRP vanilloid subtype 4 (TRPV4) as a malecascade trigger for the American alligator, Alligator
mississippiensis, in response to high environmental
temperature. We have successfully isolated and cloned the
TRPV4 channel, and demonstrated its thermal activation attemperatures proximate to TSD-related temperature inalligators. Furthermore, using pharmacological exposure tomanipulate TRPV4 channel activity, we have demonstrated
that TRPV4 channel activity has a direct relationship withmale differentiation gene expression, suggesting that
AmTRPV4 is involved in the male differentiation cascade,and propose a novel mechanism for the sex determination
pathway.

Publication List

〔Original papers〕

hatia,  ., Kumar, A., Ogino, Y., Gregg, A., Chapman, J., McLaughlin,  BM.J., and  Iguchi,  T.  (201 ).  Di-n-butyl  phthalate  causes  estrogenic effects  in  adult  male  Murray  rainbowfish  (Melanotaenia fluviatilis). Aquat. Toxicol. 149C, 10 -115.
Bhatia,  .,  Kumar,  A.,  Ogino,  Y.,  Du,  J.,  Gregg,  A.,  Chapman,  J., McLaughlin,  M.,  and  Iguchi,  T.  (201 ).  Effects of the commercial
antiandrogen flutamide on the biomarkers of reproduction in male
Murray rainbowfish (Melanotaenia fluviatilis).  Environ.  Toxicol. Chem. 33, 10 -110 .
Hamlin, H.J., Lowers, R.H., Kohno, S., Mitsui-Watanabe, N., Amano,
H., Hara, A., Ohta, Y., Miyagawa, S., Iguchi, T., and Guillette, L.J.Jr.
(2014). The reproductive hormone cycle of adult female American
alligator from a Barrier island population. Reproduction 147, 855-863.
Hiruta, C., Ogino, Y., Sakuma, T., Toyota, K., Miyagawa, S., Yamamoto,T., and Iguchi, T. (2014). Targeted gene disruption by use of transcription
activator-like effector nuclease (TALEN) in the water flea Daphnia
pulex. BMC Biotechnol. 14, 95.
Ichikawa, M., Murai, E., Hashiguchi, Y., Iguchi, T., and Sato, T. (2014).
Effects of diethylstilbestrol on luteinizing hormone-producing cells in
the mouse anterior pituitary. Exp. Biol. Med. 239, 311-319.
Ihara, M., Ihara, M.O., Kumar, V., Narumiya, M., Hanamoto, S.,
Nakada, N., Yamashita, N., Miyagawa, S., Iguchi, T., and Tanaka, H.
(2014). Co-occurrence of estrogenic and anti-estrogenic activities inwastewater: quantitative evaluation of balance by in vitro ERα reporter
gene assay and chemical analysis. Environ. Sci. Technol. 48, 63666373.
Miyagawa, S., Harada, M., Matsumaru, D., Tanaka, K., Inoue, C.,
Nakahara, C., Haraguchi, R., Matsushita, S., Suzuki, K., Nakagata, N.,
Ng, R.C., Akita, K., Lui, V.C., and Yamada, G. (2014). Disruption of thetemporally regulated cloaca endodermal b-catenin signaling causes
anorectal malformation. Cell Death Differ. 21, 990-997.
Miyagawa,  S.,  Lange,  A.,  irakawa,  I.,  Tohyama,  S.,  Ogino,  Y., Mizutani, T., Kagami, Y., Kusano, T., Ihara, M., Tanaka,  ., Tatarazako,N.,  Ohta,  Y.,  Katsu,  Y.,  Tyler,  C.R.,  and  Iguchi,  T.  (201 ).  Differing
species responsiveness of estrogenic contaminants in fish is conferredby the ligand binding domain of the estrogen receptor. Environ. Sci.
Technol. 48, 5254-5236.
Nakamura, A., Takanobu, H., Tamura, I., Yamamuro, M., Iguchi, T., and

 
 
 
 
 
 
 
 

Tatarazako, N. (2014). Verification of responses of Japanese medaka(Oryzias latipes) to antiandrogens, vinclozolin and flutamide, in shortterm assays. J. Appl. Toxicol. 34, 545-553.
Ogino, Y., Hirakawa, I., Inohaya, K., Sumiya, E., Miyagawa, S.,
Tatarazako, N., Denslow, N., Yamada, G., and Iguchi, T. (2014). Bmp7
and Lef1 are the downstream effectors of androgen signaling in
androgen-induced sex characteristics development in medaka.
Endocrinology 155, 449-462.
Omori, A., Miyagawa, S., Ogino, Y., Harada, M., Ishii, K., Sugimura,Y., Ogino, H., Nakagata, N., and Yamada, G. (2014). Essential roles of
epithelial bonee morphogentic protein signaling during prostatic
development. Endocrinology 155, 2534-2544.
Sébillot, A., Damdimopoulou, P., Ogino, Y., Spirhanzlova, P.,
Miyagawa, S., Du Pasquier, D., Mouatassim, N., Iguchi, T., Lemkine,G., Demeneix, B.A., and Tindall, A.J. (2014). Rapid fluorescentdetection of (anti-)androgens with spiggin-gfp medaka. Environ. Sci.
Technol. 48, 10919-10928.
Sumiya, E., Ogino Y., Miyakawa, H., Hiruta, C., Toyota, K., Miyagawa,S., and Iguchi, T. (2014). Roles of ecdysteroids for progression of
reproductive cycle in the fresh water crustacean Daphnia magna. Front.
Zool. 11, 60.
Toyota, K., Kato, Y., Miyakawa,  ., Yatsu, R., Mizutani, T., Ogino, Y., Miyagawa, S., Watanabe,  ., Nishide,  ., Uchiyama, I., Tatarazako, N., and Iguchi, T. (201 ). Molecular impact of juvenile hormone agonists on neonatal Daphnia magna. J. Appl. Toxicol. 34, 5 -5 .

 
 
 
 
 

〔Review articles〕

iruta, C., Toyota, K., Miyakawa,  ., Sumiya, E., and Iguchi, T. (201 ).  Sexual reproduction is a key element in the life history strategy of water fleas, Daphnia magna and Daphnia pulex - Casting a spotlight on male induction and its morphology -. In  El-Doma, M (ed.) Daphnia  Biology and Mathematics Perspectives, Nova Science Publishers, pp. 261-278.
Kohno, S., Parrott, B.B., Yatsu, R., Miyagawa, S., Moore, B.C., Iguchi,T., and Guillette, L.J. Jr. (2014). Gonadal differentiation in reptiles
exhibiting environmental sex determination. Sex. Devel. 8, 208-226.
Miyakawa, H., Iguchi, T., and Miura, T. (2014). Developmental processof defensive morph in Daphnia pulex. In: El-Doma, M. (ed). Daphnia:
Biology and Mathematics Perspectives. Nova Science Publishers, pp.
245-260.
Miyakawa, H., Toyota, K., Sumiya, E., and Iguchi, T. (2014).
Comparison of JH signaling in insects and crustaceans. Curr. Opinion
Insect Sci. 1, 81-87.
Zoeller, R.T., Bergman, ., Becher, G., Bjerregaard, P., Bornman, R.,
Brandt, I., Iguchi, T., Jobling, S., Kidd, K.A., Kortenkamp, A.,
Skakkebaek, N.E., Toppari, J., and Vandenberg, L.N. (2014). A pathforward in the debate over health impacts of endocrine disrupting
chemicals. Environ. Health 13, 118.