ISSN 0913-9036

PROCEEDINGS OF THE JAPAN SOCIETY FORCOMPARATIVE ENDOCRINOLOGY
No.22, 2007

Abstracts of the 32nd Annual Meeting and the 32nd Symposium on“Progress in Comparative Endocrinology for 10 Yearsand Perspectives in Future”,
andThe 8th Joint Symposium of Three Japanese Societies of Comparative
Biology on “Comparative Biology of Regeneration”
Nikko
October 12–13, 2007

Edited by

S. Tanaka and M. Suzuki

PROCEEDINGS OF THE JAPAN SOCIETY FORCOMPARATIVE ENDOCRINOLOGY
No. 22, 2007

Abstracts of the 32nd Annual Meeting and the 32nd Symposium on“Progress in Comparative Endocrinology for 10 Yearsand Perspectives in Future”,
andThe 8th Joint Symposium of Three Japanese Societies of Comparative
Biology on “Comparative Biology of Regeneration”
Nikko
October 12–13, 2007

Edited by

Shigeyasu Tanaka
Integrated Bioscience Section
Graduate School of Science and Technology
Shizuoka University
Ohya, Shizuoka
andMasakazu Suzuki
Department of Biology
Faculty of Science
Shizuoka University
Ohya, Shizuoka

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2007
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Proceedings of the Japan Society
for Comparative Endocrinology

Japan Society for Comparative Endocrinology
since 1975

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Tsutsui, Kazuyoshi
Abe, Shin-ichi
Ando, Hironori
Kagawa, Hirohiko
Kikuyama, Sakae
Kubokawa, Kaoru
Kobayashi, Tetsuya
Sakuma, Yasuo
Shioda, Seiji
Takei, Yoshio
Tanaka, Shigeyasu
Nagasawa, Hiromichi
Nakajima, Terumi
Hyodo, Susumu
Maeda, Kei-ichiroMori, Yuji
Yashiro, Takashi
Yasuhara, Tadashi
Uchiyama, Minoru
Takahashi, Akiyoshi
Park, Min Kyun
Kikuchi, Motoshi
Murakami, Shizuko（Waseda Univ.）（Kumamoto Univ.）（Kyushu Univ.）（Miyazaki Univ.）（Waseda Univ.）（Tokyo Univ.）（Saitama Univ.）（Nippon Medical School）（Showa Univ.）（Tokyo Univ.）（Shizuoka Univ.）（Tokyo Univ.）（Hoshi Univ.）（Tokyo Univ.）（Nagoya Univ.）（Tokyo Univ.）（Jichi Medical School）（Tokyo Univ. of Agri.）（Toyama Univ.）（Kitasato Univ.）（Tokyo Univ.）（Jichi Medical School）（Juntendo Medical School）

2007 PROGRAM COMMITTEE

Takashi Yashiro
Motoshi Kikichi
Shu Takigami
Ken Fujiwara
Tom Kouki(Jichi Med. Univ. School of Medicine)(Jichi Med. Univ. School of Medicine)(Jichi Med. Univ. School of Medicine)(Jichi Med. Univ. School of Medicine)(Jichi Med. Univ. School of Medicine)CONTENTS

ABSTRACTS OF THE 32ND SYMPOSIUM<SYMPOSIUM> “Progress in Comparative Endocrinology for 10
Years and Perspectives in Future”
Organizer: Shin-ichi Abe (Kumamoto Univ.)
Motofumi Kikuchi (Jichi Med Univ.)
Hormone regulation of oocyte maturation in teleosts and starfish- comparative to general Y.Nagahama1

Neuroendocrinology of instinctive behavior told by anuran amphibians and salmonids
A.Urano2

Identification of hormonal signal essential for mammalian hibernation
N. Kondo

3

Special Lecture
Proposing of a novel regulation system of the anterior pituitary gland in the rat
T. Soji

4

Abstracts of the 32ND annual meeting
Analysis of the relationship between the expressions of Ad4BP/SF-1 andsteroidogenic enzymes in the developing gonads of a squamata species withtemperature-dependent sex determination.
D. Endo, Y-I. Kanaho, and M.K. Park

5

Expression analysis of BDNF in the ventromedial hypothalamic nucleus of the chickembryoS. Maehiro, D. Endo, and M.K. Park6

The expression analysis of dmrt1 mrna in the differentiating testis of the leopard
gecko, squamata species with temperature- dependent sex determination
E. Kurakata, D. Endo, and M.K. Park7

Migration of GnRH neurons in three-dimensional culture of embryonic chickolfactory nerve bundle
Y. Kanaho, M.K. Park, and S. Murakami

8

Expression patterns of axon guidance molecules and their receptors during GnRH
neuronal migration from nose to the basal forebrain
S. Murakami and K. Ono

9

Short- and long-term synaptic plasticity in area CA1 of the hippocampus in adultrdw/rdw rats with congenital hypothyroidism
Oh-Nishi, N.Suzuki, S. Azuma, Y. Ishihara, R. Katada,and S. Furudate

10pacap induces differentiation of mouse embryonic neural stem cells into astrocytes
in vitro and in vivo
J. Watanabe, T. Nakamachi, M. Nakamura, S. Nakajo,
and S. Shioda

11Effect of pituitary adenylate-cyclase activating polypeptides (PACAP) on adultneural stem cells
T. Nakamachi, R. Matsuno, D. Hayashi, S. Yofu, H. Ohtaki, J. Watanabe,and S. Shioda

12Expression of retinaldehyde dehydrogenase (RALDH) mRNA in the chicken
embryonic pituitary gland
K. Nagashima, H. Takagi, M. Inoue, J. Zheng, and T. Sakai

13

Changes in expression of gastric ghrelin, aromatase and leptin in the postnatal rat
stomachT. Yanaka, Z. Zheng, C. Tsutsui, K. Koike, and T. Sakai

ii

14

mRNA expression patterns of IGFBPs in developing limb
K. Yamada, K. Tahara, I. Yamamoto, A. Tsukada, M. Tanaka,
S. Mizushima, T.Ono, N. Saito, and K. Shimada

15

Effect of estrogen and prolactin on the perception of sodefrin in female newts,
Cynops pyrrhogaster
T. Nakada, T. Iwata, F. Toyoda, K. Yamamoto, K. Tsutsui,and S. Kikuyama

16

Isolation of molecules which interact with HSP90 in germ cell apoptosis in newt testis
K. Fukaura, K. Eto, and S. Abe

17

Absence of genitalin (gonad-stimulating hormone)-induced 1-methyladenine
production in ovarian follicle and testicular interstitial cells of starfish gonads
during gametogenesis
M. Mita, M. Shinme, S. Yamamoto, and K. Yamamoto18

cDNA cloning, gene structure, and expression of Kiss1 and GPR54 in medaka
K. Kasahara, Y. Saito, M. Oyama, M. Endo, S. Fujita, T. Abe,
T.Yanagisawa, and M. Iigo

19

GnIH neurons project to GnRH neurons in the avian brain
V.S. Chowdhury, T. Ubuka, G. E. Bentley, T. Osugi, and K. Tsutsui

20

Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system
Y. Morita, G. E. Bentley, T. Ubuka, V. S. Chowdhury, I. Hasunuma,
T. Yano, and K. Tsutsui

21

In vivo action of octopus GnRH: pharmacological effects and immunocytochemistryin hatchling brains
H. Minakata and S. Shigeno

22

DES induces fish oocyte maturation through membrane progestin receptor
T. Tokumoto, M. Tokumoto, and P. Thomas

iii

23

Isolation and characterization of cDNA encoding neuromedin u (NMU) receptor
from the brain of goldfish
K. Maruyama, K. Ishiguro, T. Wakasugi, M. Uchiyama, S. Shioda, andK. Matsuda

24

Purification and properties of ghrelin from the intestine of goldfish, Carassius auratus
T. Miura, K. Maruyama, S. Shimakura, M. Uchiyama, H. Kaiya,
K. Kangawa, S. Shioda, and K. Matsuda

25

Effect of goldfish LPXRF-amide peptides on food intake in goldfish, Carassius
auratusK. Nakamura, K. Maruyama, S. Shimakura, M. Uchiyama, K. Tsutsui,
and K. Matsuda

26

Inhibitory effect of melanin-concentrating hormone on food intake in goldfish
S. Shimakura, K. Wada, K. Maruyama, T. Miura, T. Nakamachi,
M. Uchiyama, H. Kageyana, S. Shioda, A. Takahashi, and K. Matsuda

27

Diazepam-binding inhibitor-derived peptides-induced anorexigenic action is mediatedthrough the metabotropic endozepine receptor in goldfish
K. Wada, T. Miura, K. Maruyama, S. Shimakura, M. Uchiyama,
J. Leprince, M-C. Tonon, H. Vaudry, and K. Matsuda

28

Distribution of melanocortin receptors and effects of feeding on its expression withinbarfin flounder, pleuronectiformes
Y. Kobayashi, S. Okada, K. Tsuchiya, T. Yamanome, and A. Takahashi

29

Effects of advance of age in the postnatal period and fasting on ghrelin expression in
the rat duodenum
K. Koike, Z. Zheng, C. Tsutsui, T. Yanaka, and T. Sakai

30

The orexigenic effect of gonadotropin-inhibiting hormone is mediated by opioidsytem in the brain of chicks
T. Tachibana, N. Masuda, K. Ukena, K. Tsutsui, and H. Ueda

iv

31

Morphological observation of neuropeptide W neuron network in the rat  
hypothalamus
S. Shioda, F. Takenoya, and H. Kageyama

32

cDNA cloning and gene structure of hypocretin and hypocretin receptor in medaka
Oryzias latipes
K. Egashira, N. Amiya, M. Amano, K. Yamamori, T. Yanagisawa,
and M. Iigo

33

Identification of the reptilian uncoupling proteins in the leopard gecko, Eublepharismacularius
K. Kato and M.K. Park

34

Hemolymph major anionic peptide (HemaP) derived from the silkworm, Bombyx
mori, CAN influence on the larval feeding behavior
S. Nagata and H. Nagasawa

35

Identification and characterization of the vertebrate calcitonin orthologous gene in the
ascidian, Ciona intestinalis
T. Sekiguchi, T. Kawada, T. Sakai, M. Aoyama, M. Fujie, M. Ogasawara,
N. Satoh, and H. Satake36

Role of neurohypophyseal peptides in controlling the drinking behavior of the eel
acclimated to sea water
M. Ando, T. Mukuda, Y. Watanabe, and T. Sakihara

37

Salinity tolerance and expressions of neurohypophysial hormone genes in four speciesof puffers
E. Motohashi, S. Hasegawa, K. Mishiro, Y. Sugiyama, H. Doi,and H. Ando

38

Identification and physiological role of vasotocin precursor and its receptors in
African lungfish, Protopterus annectens
N. Konno, S. Hyodo, K. Matsuda, and M. Uchiyama39Electrophysiological analysis of two types of channels activated by cGMP inepithelial cells from the urinary bladder of Japanese tree frog, Hyla japonica
S. Kitani, T. Yamada, K. Matsuda, and M. Uchiyama

40

Effects of angiotensin II and arginine vasotosin on water absorption response inJapanese tree frog, Hyla japonica
S. Maejima, N. Konno, K. Matsuda, and M. Uchiyama

41

Diversity of arginine vasotocin-dependent aquaporin expressed in the ventral pelvic
skins of the anuran amphibians
Y. Ogushi, G. Akabane, M. Suzuki, and S. Tanaka

42

Characterization of an aquaporin, AQP-h3BL, expressed in melanotrophs in thebullfrog pars intermedia
Y. Komiyama, M. Suzuki, S. Kikuyama, and S. Tanaka

43

Possible hormonal regulation on dissolution of calcium carbonate crystals in thebullfrog endolymphatic sac
M. Sakimura, M. Ueda, M. Suzuki, H. Tomura, Y. Sasayama,
and S. Tanaka

44

Changes in prolactin gene expression in an amphidromous migratory teleost, ayu
Plecoglossus altivelis
T. Yada, S. Yamamoto, H. Sakano, K. Uchida, T. Takasawa, N. Abe,K. Katsura, and S. Hyodo

45

Elevation of the plasma levels of insulin-like growth factor-I (IGF-I) triggers homingof chum salmon of Japanese stocks
H. Katsumata, T.A. Onuma, K. Makino, S. Sato, M. Fukuwaka,
T. Azumaya, P. Swanson, and A. Urano

46

Gene expression for gonadotropin-releasing hormones in discrete brain loci of
pre-migratory and homing chum salmon
T.A. Onuma, A. Jodo, H. Ando, M. Ban, M. Fukuwaka, T. Azumaya,
and A. Urano

vi47

Adult chum salmon possess fresh water adaptability in the summer Bering Sea
K. Makino, M. Ban, K. Morita, S. Sato, and A. Urano

48

Measurement of melatonin and melatonin-related indoleamines in cyanobacteria
M.Takane, K. Inoue, and A. Hattori

49

Analysis of osteoblast specific messenger RNA expression in the regenerating scaleof goldfish, Carassius auratus
R. Furuya, M. Kobayashi, M. Nakamura, N. Suzuki, and A. Hattori

50

Establishment of experimental model for bone disease using the scale ofgoldfish, Carassius auratus
T. Thamamongood, J. Tabata, M. Nakamura, N. Suzuki, and
A. Hattori

51

Effect of tributyltin on the calcium metabolism in teleosts and attempt tobiodegradate by marine bacteria
N. Suzuki, F. Kobayashi, M. Matada, S. Ito, Y. Oshima, and A. Hattori

52

Direct role for growth hormone in regulating insulin-like growth factor binding
protein 4 (IGFBP4) expression in chicken
K. Tahara, T. Ohkubo, M. Tanaka, M. Akiba, K. Sato, N. Saito,
K. Shimada, and A. Tsukada53

A novel pituitary transcription factor, PRX2 was activated by GnRH and plays
synergistically with egr-1 on luteinizing hormone beta subunit gene
A. Sano, T. Sato, T. Kato, and Y. Kato

54

DNA-binding characteristics of transcription factors expressing in the anterior
pituitary grand
M. Nakayama, F. Kimoto, T. Kato, and Y. Kato

55

Neuronatin localizes in secretory granule of gonadotrope and is regulated by steroid
hormones
S. Murakami, L. Cai, T. Susa, S. Izumi, T. Kato, and Y. Kato
vii

56

Molecular evolution of prepro-thyrotropin-releasing hormone
Y. Aoki, Y. Saito, M.K. Park, T. Yoshimura, G. Nishi, M. Iigo, andT. Yanagisawa

57

Agnathan pituitary gland and its evolutionary implication
M. Nozaki, T. Shimotani, K. Uchida, S. Moriyama, A. Takahashi,
H. Kawauchi, and S.A. Sower

58

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) andmelanin-concentrating hormone (MCH) on somatolactin (SL) release from thegoldfish pituitary in vitro
M. Tanaka, M. Azuma, Y. Nejigaki, M. Uchiyama, A. Takahashi, andK. Matsuda

59

Molecular cloning and characterization of cDNA encoding pituitary adenylate
cyclase-activating polypeptide from the brain of far eastern brook lamprey
K. Ishiguro, K. Maruyama, Y. Yamazaki, M. Uchiyama, andK. Matsuda

60

Changes of corticotropin-releasing factor expression in the brain of the bullfrog,
Rana catesbeiana during metamorphosis
K. Hashimoto, R. Okada, H. Mochida, M. Uchiyama, S. Kikuyama, andK. Matsuda

61

Opposite myoactivity of novel neuropeptides (FRamides) encoded in the same
preprohormone gene in Hydra
T. Takahashi, E. Hayakawa, C. Nishimiya-Fujisawa, and T. Fujisawa

62

Evolutionary relationship of LPXRFa peptides and PQRFa peptides - insights fromnovel cartilaginous fish RF-amide peptides
T. Osugi, S. Hyodo, and K. Tsutsui

63Physiological effects of prolactin-releasing peptide (PrRP) on blood pressure andimmune reaction
A. Mochiduki, T. Yamada, T. Takeda, and K. Inoue

viii

64

Structures and distribution of proenkephalin mRNAs in banded houndshark Triakis
scyllium
A. Takahashi, R. Goto, Y. Kobayashi, S. Moriyama, and S. Hyodo

65

The circadian regulation of in vitro melatonin release from the fish pineal organ: acomparative study
T. Abe, T. Masuda, K. Mizusawa, S. Fujita, S. Kitamura, T. Azuma,
Y. Takagi, T. Yanagisawa, and M. Iigo

66

Convenient method for preparation of biologically active recombinant CHH of the
kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system
C. Nagai, H. Asazuma, S. Nagata, and H. Nagasawa

67

Insulin secretion ability of barfin flounder, Verasper moseri
T. Andoh

68

Histological analysis of the distribution of clusterin in the rainbow trout
R. Fukui, T. Nakakura, S. Tanaka, and M. Suzuki

69

Parafibromin tumor suppressor has oncogenic property in the cells expressing SV40
large T antigen
T. Iwata, N. Mizusawa, Y. Taketani, M. Itakura, and K. Yoshimoto

70

Visualizing exocytosis in rat anterior pituitary cell cluster using two-photon
extracellular polar tracer (TEP) method
J. Yasufuku-Takano, K. Takano, N. Takahashi, H. Kasai, and T. Fujita

71

Observation of the gap junction and lhrh projection on folliculo-stellate cells in thepars tuberalis of a rat pituitary gland by confocal laser microscopy
K. Horiguchi, K. Fujiwara, M. Kikuchi, T. Yashiro, and T. Soji

72

Effect of exogenous E-cadherin over-expression on the primary cultured cells of
the rat anterior pituitary gland
K. Kusumoto, M. Kikuchi, and T. Yashiro

ix

73

Molecular structure and evolution of gonadotropin receptors from mummichog
Fundulus heteroclitus
M. Ohkubo and A. Shimizu

74

Molecular evolution of thyrotropin-releasing hormone receptors
Y. Saito, N. Kobayashi, M. Kimura, M. Mekuchi, Y. Aoki, T. Azuma,
M. Iigo, and T. Yanagisawa

75

The functional analysis of the transcripts derived from human type ii GnRH receptor
gene
T. Kato, Y. Kanaho, M. Enomoto, and M.K. Park

76

Functions of GnRH receptor heterodimers of the ascidian, Ciona intestinalis
T. Sakai, M. Aoyama, T. Kusakabe, M. Tsuda, and H. Satake

77

Identification of cDNAs encoding thyrotropin-releasing hormone receptor from thebullfrog
A. Minagawa, I. Hasunuma, M. Nakano, R. Okada, K. Yamamoto,
S. Kikuyama, T. Kobayashi, and T. Machida

78

Involvement of D2 dopamine receptor subtype in inhibition of prolactin releasefrom the bullfrog pituitary gland
M. Nakano, I. Hasunuma, R. Okada, K. Yamamoto, S. Kikuyama,
T. Kobayashi, and T, Machida

79

Localization of arginine vasotocin V3/V1b type receptor in the newt pituitary andbrain
I. Hasunuma, K. Yamamoto, F. Toyoda, H. Namiki, and S. Kikuyama

80

A melanin-concentrating hormone receptor is expressed in the brain but not in theskin of zebrafish
K. Mizusawa and A. Takahashi

81

Identification of ghrelin receptor-like receptor and its gene structure in rainbow trout
H. Kaiya, T. Mori, M. Miyazato, and K. Kangawa

82

Molecular characterization of chicken motilin receptor (MTL-R)
I.Yamamoto, H. Kaiya, C. Tsutsui, T. Sakai, A. Tsukada, M. Miyazato,
and M. Tanaka

83

Characterization of signaling pathways through proton-sensing G-protein-coupled
receptors
H. Tomura, M. Tobo, C. Mogi, and F. Okajima

84

Cloning and characterization of the ecdysone receptor and ultraspiracle protein fromthe water flea, Daphnia magna
Y. Kato, K. Kobayashi, S. Oda, N. Tatarazako, H. Watanabe,
and T. Iguchi

85< Joint Symposium of Three Japanese Societies OF Comparative Biology >“Comparative Biology of Regeneration”
Organizers: Seiji Shioda (Showa Univ.)
Takeshi Yoshida (Spiritual Care Insti.)
Masayuki Iigo (Utsunomiya Unv.)
The Japanese Association for Developmental & Comparative Immunology
Prelude to regeneration - mechanisms of cell deathE. Furuta

86

The origin of stem cells in the regenerating marine and freshwater planarians
S. Ishida

87

The Japanese Society for Comparative Physiology and Biochemistry
Cellular and molecular mechanisms of nerve regereration of Hydra
O. Koizumi

88

Adult newt retinal regeneration: is FGF an induction factor of regeneration?
C. Chiba

89

xi

The Japan Society for Comarative Endocrinology
Neurogenesis and regeneration in the mammalian brain
T. Seki

90

Regeneration and resorption of the goldfish scale and its regeneration by melatonin
A. Hattori

91

xii

S-1
HORMONAL REGULATION OF OOCYTE MATURATION IN TELEOSTS AND
STARFISH - COMPARATIVE TO GENERAL Yoshitaka Nagahama
Laboratory of Reproductive Biology, Department of Developmental Biology, National
Institute for Basic Biology, Okazaki 444-8585, Japan.
Oocyte maturation is the process by which the oocyte completes the first meiotic
division and undergoes several maturational changes, and progresses to metaphase II.
Studies from our laboratory using teleosts and starfish have indicated that oocyte
maturation is regulated by three major mediators, namely gonadotropin (LH in teleosts andgonad-stimulating substance, GSS in starfish), maturation-inducing hormone (MIH), andmaturation-promoting factor (MPF).
In fish, LH acts on ovarian follicle cells to produce fish MIH (17α, 20β-dihydroxy-4pregnen-3-one, 17α, 20β-DP). 17α, 20β-DP is synthesized by a two-step process involving
the thecal and granulosa cell layers. A novel type of P450c17 lacking the lyase activity is
responsible for the shift from estradiol-17β to 17α, 20β-DP in ovarian follicles occurring
immediately prior to oocyte maturation. We recently purified GSS from the radial nervesof starfish (Asterina pectinifera). The purified GSS is a heterodimeric peptide composed of
A- and B-chains, with disulfide cross-linkages, and was named “genitalin” as a novel
insulin-like neuropeptide hormone (Mita et al., unpublished). Genitalin acts on ovarianfollicle cells to produce starfish MIH (1-methyladenine, 1-MeAde).
The site of 17α, 20β-DP and 1-MeAde is the surface of the oocytes. A distinct familyof membrane-bound progestin receptors having characteristics of G-protein-coupled
receptors seems to mediate non-genomic actions of 17α, 20β-DP. The MIH signal received
on the oocyte surface is transduced into the oocyte cytoplasm for the formation andactivation of MPF, the final mediator of oocyte maturation. Although MPF exhibits auniversal molecular structure as a complex of cdc2 and cyclin B in any species, themechanisms of MPF formation and activation have been modified to various extents from
species to species. In¸fish, the 17α, 20β-DP signal leads to the de novo synthesis of cyclin
B, the regulatory component of MPF, which activates a preexisting 35-kDa cdc2 kinase via
phosphorylation of its threonine 161 by a threonine kinase, thus producing the 34 kDa
active cdc2.
In summary, our findings appear to show that the basic mechanisms involved in oocyte
maturation are the same in vertebrates (teleosts) and invertebrates (starfish), despite thediffering chemical nature of the hormonal agents involved.

––

S-2
NEUROENDOCRINOLOGY OF INSTINCTIVE BEHAVIOR TOLD BY ANURAN
AMPHIBIANS AND SALMONIDS
Akihisa Urano
Section of Biological Science, Faculty of Science, Hokkaido University, Sapporo 060-0810,
Japan.
Instinctive behavior is defined as that for survival of individuals and maintenance ofspecies, and is considered to be genetically programmed in the genome. In vertebrates, theneuroendocrine systems located in the hypothalamus are involved in the controls of almost
all types of instinctive behaviors. Then, how do hypothalamic neurons, in particular
neurosecretory neurons (NSCs) which regulate endocrine functions, control instinctive
behavior? Because of complex interactions between the higher brain center and the
hypothalamus in higher vertebrates, a use of lower vertebrates, such as amphibians andteleosts, would be better to clarify general principles of neuroendocrine control
mechanisms of instinctive behavior. Another advantage for the use of amphibian andteleost brains is availability of in vitro imaging analysis of a hemisected brain preparation.
The studies of calling behaviors in leopard frog (Rana pipiens) and Japanese toad (Bufojaponicus) indicated an interaction of NSCs with the activating reticular systems. When aleopard frog was acoustically stimulated with conspecific mating calls, neurons in theanterior preoptic nucleus (APON), which is the triggering center of male mating behavior,
were excited under the influence of injection of pituitary homogenate. Golgi-electron
microscopic and retrograde HRP analyses, and immunocytochemistry showed that APON
neurons receive various types of afferents including projections from neurons containing
gonadotropin-releasing hormone (GnRH) and vasotocin. Iontophoretically applied GnRH
and vasotocin immediately excited APON units, whereas intracerebroventricular (icv)
GnRH enhanced amplitudes and frequencies of electroencephalogram almost 24 hrs after
the treatment. Similar icv treatments with GnRH elevated the plasma testosterone levels
also after considerable lag times. These results suggest dual controls by NSCs of thenervous system and the endocrine system to motivate instinctive behavior.
On the basis of abovementioned idea, we attempted to clarify neuroendocirnemechanisms of salmon spawning migration, or homing migration to the natal river. In thesalmonid brain, GnRH and vasotocin neurons also send their immunoreactive fibers tovarious brain loci and the neurohypophysis, indicating that NSCs are involved in the dual
control of nervous and endocrine systems in terms of motivation of instinctive behavior.
Actually, application of GnRH analogue facilitated homing behavior, and enhanced
expression of genes for gonadotropin subunits, in sockeye salmon. Thus, detailed
quantitative analyses of molecular events in the neuroendocrine systems were carried out
in aquacultured salmonids as model fishes and oceanic and homing chum salmon as wild
fish. The molecular events seen in pre-spawning fishes, both model and wild, weresimilarly under the influence of the hypothalamus-pituitary-gonadal axis, however, theevents in the high sea were additionally under the control of IGF-I, suggesting animportance of interaction between the neuroendocrine and the visceral systems.

––

S-3
IDENTIFICATION OF HORMONAL SIGNAL ESSENTIAL FOR MAMMALIANHIBERNATION
Noriaki Kondo
Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511, Japan.
Mammals are characterized by maintaining a constant body temperature (Tb) near
37 C, by which high metabolic activities are kept independently of environmental changes.
When Tb is decreased, principle organs, such as the central nervous system and hearts,
dysfunction, resulting in death. However, hibernators have a unique ability to survive
potentially lethal low Tb during the hibernation season. During hibernation, Tb isdecreased below 10oC with a marked reduction of metabolic rate (about 100 times lessthan that in euthermic states), indicating that the ability to tolerate nearly arrested
metabolic states is developed in association with hibernation. Furthermore, hibernation has
been suggested to protect organisms from various lethal diseases and harmful factors. A
factor that controls hibernation has long been explored, but not found. Here, a recent
identification of hormonal factor essential for hibernation is summarized.
In chipmunks (Tamias sibiricus), a rodent hibernator, hearts normally function even ato
5-6 C of body temperature only during the hibernation season. This was due to modulationof Ca2+ source for cardiac excitation-contraction coupling; Ca2+ source was changed fromexternal Ca2+ influx to internal Ca2+ release of cytoplasmic store sites (1). This modulation
was suggested to prevent lethal Ca2+ overload of myocardium induced at low Tb. This
study led us to assume endogenous factors that modulate organisms for hibernation, thenhibernation-specific protein (HP) complex was discovered in the blood (2). HP complex
was produced in the liver and downregulated during the hibernation season by an
endogenously generated circannual rhythm independently of Tb and environmental
changes. In correlation with this downregulation, HP complex was transported into thebrain through the blood-cerebrospinal fluid barrier and played an essential role inhibernation (3). Thus, HP complex carries an essential signal for hibernation from the liverto the brain under control of a circannual rhythm signaling.
oReferences(1) Kondo, N. and Shibata, S. (1984) Science, 225, 641-643.
(2) Kondo, N. and Kondo, J. (1992) J. Biol. Chem., 267, 473-478.
(3) Kondo, N. et al. (2006) Cell, 125, 161-172.

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S
PROPOSING OF A NOVEL REGULATION SYSTEM OF THE ANTERIORPITUITARY GLAND IN THE RAT

Tsuyoshi Soji
Department of Anatomy, School of Med, Nagoya City University, Nagoya, Japan.
It was demonstrated a few fluorescent LH-RH fibers in the pars tuberalis as well as on
the primary plexus and in the ependymal layer. The distribution of the S-100 protein cell(folliculo-stellate cell) is very important to our understanding of the regulation of theanterior pituitary. The distribution of the cells observed over 50% of the total cell
population in the so called “transitional zone”. They then decreased in number with
distance from this region to mid-way towards the sagittal axis before increasing again in
the tail of the gland. The population of cells also decreased with increasing distance fromthe "transitional zone" to the wing and with distance from the basal zone. Portal vessels
entered the anterior lobe through the "transitional zone" as thick capillaries, ran through thebasal surface of the anterior lobe. The distribution pattern of the folliculo-stellate cells
and the capillaries showed good agreement and the spatial relationship between these two
is detailed so as to better understand hypophyseal histophysiology.
Clusters of S-100 protein cells and numerous connexin 43 positive sites on S-100
protein cells were clear in the “transitional zone” as well as in the pars distalis. Random
penetration of electrode showed that larger populations of cell (~80 %) had membranepotentials with - 55.6 ± 5.1 mV, and less than 20 % of cells had the resting membranepotential with - 36.0 ± 4.4 mV. There were two types of cell couplings; one major groupfor the recordings from cells with similar deep resting membrane potentials and the otherfor the recordings from cells with different resting membrane potentials. The former
indicated that two cells were electrically coupled while the latter no electrical couples wereobserved. Carbenoxolone depolarized the membrane by 12.3 ± 5.5 mV and reduced theamplitude of electrotonic potentials, and the response recovered by removal ofcarbenoxolone by the superfusate.
The results clearly indicated that the folliculo-stellate cell system deeply participatedin the regulation of the anterior pituitary parallel with the portal vessel system, which was
the main regulatory system for pituitary hormone secretion.

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P1
ANALYSIS OF THE RELATIONSHIP BETWEEN THE EXPRESSIONS OF
AD4BP/SF-1 AND STEROIDOGENIC ENZYMES IN THE DEVELOPINGGONADS OF A SQUAMATA SPECIES WITH TEMPERATURE-DEPENDENTSEX DETERMINATION
Daisuke Endo, Yoh-Ichiro Kanaho, and Min Kyun Park
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Tokyo 113-0033, Japan.
Sex steroid hormones play an important role in sex determination and sex
differentiation in squamate species with temperature-dependent sex determination, as wellas in squamate species with genotypic sex determination. In the leopard gecko,
Eublepahris macularius, it is known that estrogen treatment can make the sex of embryo
female at any incubation temperature and we had reported female specific expression of
estrogen synthetase, P450arom in the gonad of hatchlings. However, it remains unknown
what mediates between the effect of incubation temperature and the sex steroid hormone
signaling system in this species. Ad4BP/SF-1 is one candidate for the mediator. Thistranscription factor is reported to regulate the expression of steroidogenic enzymes in thedeveloping gonad of the mouse and chicken in sex specific manner. Thus, we tried to
identify the cDNA of Ad4BP/SF-1 and conducted the expression analysis in the gonads ofthe leopard gecko embryos and hatchlings.
The length of the leopard gecko Ad4BP/SF-1 cDNA identified in this study was 1490
bp with open reading frame encoding a 468 amino acid protein. The deduced amino acidsequence showed moderate identity with that of other vertebrates: 84% with turtle, 84%zebra finch, 70% mice, and 77% newt.
The mRNA expression levels of Ad4BP/SF-1 were significantly higher in the gonads
of male hatchlings than in those of female, while the expression of P450arom was higher
in those of female than male. No sex difference was observed in the expression levels ofP450scc. In the embryonic gonads, no significant effect of incubation temperature on theexpression of these three genes was detected in all stages examined.
In summary, we identified the cDNA of the leopard gecko Ad4BP/SF-1 and conducted
the expression analysis in the developing gonads. The male specific expression of thisgene was detected in the gonads of hatchling, when morphological sex differentiation wasstarted to be observed. This male specific expression was inconsistent with the expression
pattern of two steroidogenic enzymes examined in this species. Thus, it is suggested thatleopard gecko Ad4BP/SF-1 plays different role from the regulation of the expression of
P450arom or P450scc in male sexual differentiation of the gonad.

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P2
EXPRESSIONANALYSIS
OF
BDNF
IN
THE
HYPOTHALAMIC NUCLEUS OF THE CHICK EMBRYOVENTROMEDIAL

Sayaka Maehiro, Daisuke Endo, and Min Kyun Park
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
113-0033 Japan.
Though brain sexual differentiation has been thought to be regulated by the gonadal
steroid hormones after sex determination of gonads, the direct effect of sex determining
factor on the brain was suggested in some species. The female leopard gecko, a lizard withtemperature-dependent sex determination, from different incubation temperature showed
different sexual behavior such as aggressiveness and attractiveness to male. In this species,
it has been reported that the mRNA expression of steroidogenic enzymes (P450scc andP450arom) were affected by incubation temperature. Because estrogen synthetase,
P450arom expression was not observed in the gonads before hatching, this result strongly
suggests that the brain can differentiate independently from the gonadal steroid. In thisstudy, we will demonstrate that the sex difference of a gene expression level in the brain of
the chicken, amniotic species with genotypic sex determination.
In the chick brain, the expression of cholesterol side chain cleavage enzyme (P450scc),
P450arom and sex steroid hormone receptors was observed by RT-PCR from days 4.5 ofincubation without sexual difference. Ad4BP/SF-1, a transcription factor of steroidogenic
enzymes was also expressed in the brain from days 4.5 of incubation, however its
expression at days 5.5 of incubation was significantly higher in the male brains than in thefemale brains. This expression was exclusively localized in the ventromedial hypothalamic
nucleus (VMH). In the chick gonad, sex specific expression of estrogen synthetase(P450arom) was detected from days 6.5 of incubation. Therefore the expression of
Ad4BP/SF-1 in the chick brain at days 5.5 of incubation strongly suggests the involvementof a sex steroid hormone signaling system in brain-autonomous sex differentiation in birds.
VMH is a region important for adult sexual behavior, and we analyzed brain-derived
neurotropic factor (BDNF) in this brain area. BDNF has been expected as an importantfactor in the formation of VMH in rodents. Analysis in the chick embryonic brain by RTPCR, its expression was detected from days 4.5, and this expression was observed in the
VMH by in situ hybridization. This result suggests that BDNF is able to be a usefulmolecular marker of VMH in the chicken.
In this study, we clearly demonstrated the existence of a sex steroid hormone signaling
system in the developing brain, and involvement of this system in brain-autonomous sex
differentiation of both temperature-dependent and genetic-sex determination amniotes.

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P3
THE EXPRESSION ANALYSIS OF DMRT1 mRNA IN THE DIFFERENTIATINGTESTIS OF THE LEOPARD GECKO, SQUAMATA SPECIES WITHTEMPERATURE- DEPENDENT SEX DETERMINATION
Erina Kurakata, Daisuke Endo, and Min Kyun Park
Department of Biological Science, Graduate School of Science, The University of Tokyo,
Tokyo, 113-0033, Japan.
Dmrt1 (double sex and mab-3 related transcription factor 1) is expressed exclusively inthe developing and adult testis of many vertebrates. For example, in adult mouse, like othervertebrates, the expression of Dmrt1 was found only in the testis (2) and this expression
was restricted to the Sertoli and germ cells (1). During the sexual differentiation of themouse, Dmrt1 was upregulated in the testis and its expression is also restricted in the
Sertoli and germ cells (2). However the expression of Dmrt1 in the testis of sqamate
species with temperature-dependent sex determination has not been reported. In squamate
species, there are two types of sex determination, sex chromosome- and temperaturedependent sex determination even in the same family. Therefore it is important to compare
the mechanism of sex determination of these species.
For this reason, we identified Dmrt1 mRNA in the leopard gecko (Eublepharismacularius) squamate species with temperature dependent sex determination as anexperimental animal. We also examined the expression of Dmrt1 mRNA in various
tissues of adults and differentiating testis of the leopard gecko.
As a result, Dmrt1 mRNA is expressed only in the testis of the adult leopard gecko,
and in situ hybridization analysis of adult testis showed that the germ cells only
expressed Dmrt1 mRNA. This is contrary to the results of other vertebrates where theSertoli cells also expressed Dmrt1 mRNA. After thermo-sensitive period of sex
determination, the expression became high in the gonads of some embryos incubated at
male-biased temperature. Furthermore, just after hatching, when the testis cords begin to
be visible, Dmrt1 mRNA level was high in all male gonads. These results suggest thatDmrt1 may play a role in the differentiation of male gonads as other vertebrates.
This study suggests that in the leopard gecko, Dmrt1 also play important role in thetestis. In the future, whether Dmrt1 mRNA is expressed in the Sertoli cells during testis
differentiation should be analyzed.
References(1) De Grandi, A. et al. (2000) Mech. Dev., 90, 323-326.
(2) Raymond, C. S. et al. (1999) Dev. Biol., 215, 208-220.

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P4
MIGRATION OF GnRH NEURONS IN THREE-DIMENSIONAL CULTURE OFEMBRYONIC CHICK OLFACTORY NERVE BUNDLE
Yoh-Ichiro Kanaho1, Min Kyun Park1, and Shizuko Murakami2
1

Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Tokyo, 113-0033, Japan.
2
Department of Anatomy, Juntendo University School of Medicine, Tokyo, 113-8421,
Japan.
Gonadotropin-releasing hormone (GnRH), which plays a pivotal role in regulating the
reproductive system, is synthesized in neurons located in the hypothalamus. In a variety ofvertebrate species, GnRH neurons originate in the olfactory placode and migrate along theterminal, olfactory, and/or vomeronasal nerves into the forebrain during embryonicdevelopment (1). Vigorous researches have been performed to understand the mechanism
underlying this phenomenon, uncovering factors involved in GnRH neuronal migration (2).
However, many points still remain to be clarified, as the mechanism regulating the entranceof GnRH neurons to the brain or the role of GnRH secreted from those neurons. As one
step to examine these issues, we established an in vitro model system which would beappropriate for morphological and pharmacological analysis of GnRH neuron migration.
A piece of olfactory nerve bundle (ON) along which GnRH neurons migrate wasobtained from embryonic days 7.5-8 of chick, and embedded in a mixture of collagen gel
and Matrigel. The ON explants were maintained for up to 5 days. After 2-3 days in vitro,
GnRH immunoreactive (-ir) cells migrated out of the ON explants into the gel matrix.
These GnRH-ir cells often contacted with each other forming cord-like structure, whilesome migrated independently along either GnRH-ir or non-GnRH-ir fibers. In addition,
GnRH-ir neurites were observed to extend from the explants. Morphological characteristics
of migrating GnRH-ir cells in this system resembled that of in vivo, thus this explant
culture system will allow direct examination and manipulation of GnRH cell movement.

References(1) Wray, S. (2001) J. Neuroendocrinol., 13, 3-11.
(2) Tobet, S. A. et al. (2006) Endocrinology, 147, 1159-1165.

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P5
EXPRESSION PATTERNS OF AXON GUIDANCE MOLECULES AND THEIRRECEPTORS DURING GnRH NEURONAL MIGRATION FROM NOSE TO THE
BASAL FOREBRIAN
Shizuko Murakami1 and Katsuhiko Ono2
1

Department of Anatomy, Juntendo University School of Medicine, Tokyo 113-8421,
Japan.
2
Division of Neurobiology and Bioinformatics, National Institute for Physiology Science,
Okazaki 444-8787, Japan.
GnRH (gonadotropin-releasing hormone or luteinizing hormone-releasing hormone;
LHRH) neurons originate in the olfactory placode and migrate into the forebrain during
development. Examination of the normal development of GnRH neurons indicates that two
major pathways of GnRH neuronal migration exist within the brain of chick embryos.
The main pathway of GnRH neuronal migration largely corresponds to the course of asubset of the olfactory fibers. At the most caudal level of this pathway, many of themdetach from these fibers and migrate ventrally, suggesting that the migration mode taken bythese GnRH neurons is a kind of axon-independent manner (1). Attractive or repulsive
chemical cues are implicated in regulating the ventral-directed migration of GnRH neurons.
Different molecules with chemotrophic functions and their receptors have been
identified to play a role in axon guidance and cell migration during development of thenervous system. In this study, we have performed in situ hybridization analysis of theexpression patterns of netrin-1, semaphorin (sema) 3A, slit-1 and their receptors in an
attempt to identify guidance substrates that GnRH neurons guide to their final destinations.
Netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal
region and the most rostral forebrain. A few GnRH neurons expressed netrin-1 receptor
neogenin. Slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in
GnRH neurons. Sema3A expression was detected in the olfactory-forebrain region, but wasabsent in the medial forebrain surface where GnRH neurons migrated in association with asubset of olfactory fibers. At the caudal end of this pathway, sema3A was observed in the
more dorsal region at which GnRH neurons changed their course ventrally. Many GnRH
neurons expressed sema3A receptor neuropilin-1, suggesting that sema3A may act as arepellent during the migration of GnRH neurons.
Reference(1) Murakami, S. et al. (2002) Prog. Brain Res., 141, 31-44.

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P7
SHORT– AND LONG-TERM SYNAPTIC PLASTICITY IN AREA CA1 OF THE
HIPPOCAMPUS
IN
ADULT rdw/rdw
RATS
WITH
CONGENITAL
HYPOTHYROIDISM
Arata Oh-Nishi1, Nobuyuki Suzuki2, Sadahiro Azuma1, Yuko Ishihara1, Risa Katada1, and
Sen-ichi Furudate1
1

Division of Reproductive Biotechnology, Graduate School of Medical Sciences, Kitasato
University, Japan.
2
Division of Brain Science, Graduate School of Medical Sciences, Kitasato University,
Japan.
Although various behavioral deficits have been described in hypothyroid animals,rodent hypothyroidism models have shown impaired spatial learning and memory (1-2), in
addition to deficiency in attention and hyperactivity. However, molecular mechanisms ofthis cognitive dysfunction have not been well studied. On the other hands, it is well known
that hippocampal circuits are crucially involved in cognitive functions, such as learningand memory, in humans and other animals and express a very high degree of functionaland structural plasticity. Thus, we investigated short and long synaptic plasticity including
long-term potentiation (LTP) and long-term depression (LTD), paired pulse facilitation(PPF) in area CA1 of the hippocampus slice in adult rdw/rdw rats. The rdw rat shows
hypothyroidism due to a missense mutation in thyroglobulin and secondary dwarfism (3).
At 60 min after high- frequency stimulation, the LTP of field excitatory postsynaptic
potentials slope (S-fEPSPs) was significantly reduced to 107.3 ± 4.5 % in the hippocampus
from adult hypothyroid rdw rats, as compared to 151.6 ± 38.3 % in that from adult normal
rats, although the S-fEPSPs at 0 min were not significantly different between normal andrdw rats. The LTD of the S-fEPSPs 40 min after low-frequency stimulation was 92.3 ±
3.4% and 86.2 ± 3.6% in rdw hypothyroid and normal rats, respectively. There was no
significant difference between rdw and normal rats. The paired-pulse ratios were recorded
at various interstimulus intervals ranging from 30 to 950 ms, significantly lowered mean
PPF at interstimulus intervals between 120 and 200 ms in rdw rats as compared to thatfrom normal rats. This result suggests that presynaptic glutamate releases were increased
by rdw rats.
Reduction of LTP and PPF ratio observed in rdw hypothyroid rats in this work
implicates that cognitive dysfunction including learning » memory, attention andhyperactivity, may occur in rdw hypothyroid rats.
References(1) Akaike, M. et al. (1991) Neurotoxicol. Teratol., 13, 317-322.
(2) Anthony, A. et al. (1993) Horm. Behav., 27, 418-433.
(3) Hishinuma, A. et al. (2000) Endocrinology, 141, 4050-4055.

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P8
PACAP INDUCES DIFFERENTIATION OF MOUSE EMBRIONIC NEURALSTEM CELLS INTO ASTROCYTES IN VITRO AND IN VIVO
Jun Watanabe1, 2, 4, Tomoya. Nakamach1, Masahisa Nakamura2, Shigeo Nakajo1,3, and Seiji
Shioda1
1

Department of Anatomy, School of Medicine, Showa University, Tokyo, 142-8555, Japan.
Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda
University, Tokyo, 162-0051, Japan.
3
Laboratory of Biochemistry, Yokohama College of Pharmacy, Kanagawa, 245-0066,
Japan.
4
Research Fellow of the Japan Society for the Promotion of Science
2

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide thatexerts a wide range of effects on different cell types in brain as early as fetal developmental
stage. Distribution and localization of PACAP and its specific receptor (PAC1-R) mRNAduring development suggests that PACAP is associated with proliferation anddifferentiation of neural stem cells (NSCs) (1). However, few is known on the functionalrole of PACAP in NSCs.
We have already shown that PACAP induces differentiation of cultured mouseembryonic NSCs into astrocytes. When NSCs were exposed to 2 nM PACAP instead ofbFGF for 8 days, 55% of these cells were immunostained with anti-glial fibrally acidic
protein (GFAP) antibody. This phenomenon was completely diminished with PACAP6-38,
PAC1-R antagonist treatment (2). The signal transduction pathway of PACAP signaling
was further investigated in detail. This differentiation was mimicked by protein kinase C(PKC) activator (PMA) but suppressed by phospholipase C inhibitor (U73122),
intracellular calcium chelator (BAPTA-AM), and selective inhibitor of PKC (chelerythrine).
Furthermore, overexpression of PKCβII with adenovirus vector synergistically enhanced
differentiation in the presence of 1 nM PACAP, whereas expression of the dominantnegative mutant of PKCβΙΙ proved inhibitory, suggesting that the β isoform of PKC plays acrucial role in the PACAP-induced NSCs differentiation (3).
Functional significance of PACAP on astroglial differentiation in vivo was studied withimmunohistochemical analysis at E14, 16, 18, P1 and P3 in mouse brain. PAC1-R was colocalized with nestin in the neural progenitor cells of E14 mouse ventricular zone. GFAP
immunopositive cells appeared in the region where PAC1-R was strongly expressed. These
results strongly suggest that PACAP play very important roles in astroglial differentiationfrom NSCs during brain development.
References(1) Watanabe, J. et al. (2007) Peptides, 28, 1713-19.
(2) Ohno, F. et al. (2005) Regul Pept., 126, 115-22.
(3) Watanabe, J. et al. (2006) J. Neurosci. Res., 84, 1645-55.

–11–

P9
EFFECTOFPITUITARYADENYLATE-CYCLASE
POLYPEPTIDES (PACAP) ON ADULT NEURAL STEM CELLSACTIVATING

Tomoya Nakamachi, Ryosuke Matsuno, Daisuke Hayashi, Sachiko Yofu, Hirokazu Ohtaki,
Jun Watanabe, and Seiji Shioda
Department of Anatomy, Showa University School of Medicine, Shinagawa-Ku, Tokyo
142-8555, Japan.
Mammalian adult brain was thought to be completely incapable of neurogenesis, thebirth of new neuron. In the last decade, it was defined that neurogenesis normally occurredin specific region, existed neural stem cells (NSCs), of the adult mammalian brain. Recent
findings show that it is possible to induce the neurogenesis, and the generated neuron may
replace lost or damaged neuron. However, the regulatory systems of neurogenesis are notwell understood. Pituitary adenylate cyclase-activating polypeptide (PACAP) is apleiotropic neuropeptide, which was mainly distributed in the hypothalamus, and thespecific receptor (PAC1R) was widely expressed in the brain (1, 2). It has been reported
that PACAP stimulates a proliferation or differentiation of NSCs derived from fetal brain
in vitro. However, effects of PACAP on neurogenesis in adult mouse brain are poorly
known. Therefore, we first observed the distribution of PAC1R immunoreactivity in thetwo neurogenic region, subventricular zone (SVZ) and subgranular layer (SGLâof dentate
gyrus of hippocampus. PAC1R immunoreactivity was co-localized with NSC marker,
nestin in SVZ, but not with neuroblast marker, doublecortin, suggesting PACAP affects
NSCs (3). Subsequently, to make clear the effect of PACAP on adult NSCs, thymidine
analog, BrdU was intraperitoneally injected to label a proliferating nucleus following
intraventricularly infusion of PACAP for 1 week. The number of BrdU immunopositive
cells in the SGL of dentate gyrus of hippocampus was significantly increased in PACAP
injected mouse brain rather than vehicle injected group. Moreover, neurogenesis inducedby ischemic model, common carotid artery occlusion (CCAO) was compared with wild
type and PACAP heterogeneous knockout (PACAP+/-KO) mice by BrdU labeling. BrdU
immunopositive cells in SGL increased at 3 to 7days after CCAO; however the number ofthe cells did not change in PACAP+/-KO mice after ischemia. These results suggest that
PACAP is a key factor for proliferation of NSCs in mouse brain.

References(1) Nakamachi, T. et al. (2007) Regul. Pept. In press(2) Ohtaki, H., Nakamachi T. et al. (2006) Proc. Natl. Acad. Sci. USA, 103:7488-93.
(3) Matsuno, R. et al. (2007) Regul. Pept . In press

–12–

P10
EXPRESSION OF RETINALDEHYDE DEHYDROGENASE (RALDH) mRNA IN
THE CHICKEN EMBRYONIC PITUITARY GLAND
Keiko Nagashima, Hiroyasu Takagi, Makiko Inoue, Zheng Jun, and Takafumi Sakai
Area of Regulatory Biology, Division of Life Science, Graduate school of Science andEngineering, Saitama University, 255 Shimo-Ohkubo, Saitama, Saitama 338-8570, Japan.
Retinoic acid (RA), the active derivative of vitamin A, is known to be an important
regulator of essential aspects of organogenesis. Previous studies on the temporal andspatial expression patterns of retinaldehyde dehydrogenase (RALDH) mRNA in thepituitary gland (1,2) and effects on hormone expression in a cultured cell line havesuggested that RA plays an important role in pituitary development and differentiation of
hormone-producing cells. However, a direct role of RA in development of the pituitary
gland remains obscure. This issue must be investigated by using chicken embryos because
chicken embryos can develop independently from maternal vitamin A and can be easily
cultured. In this study, we investigated the spatial and temporal expression patterns of
RALDH2 or 3 mRNA in embryonic chicken anterior pituitary glands using RT-PCR and insitu hybridization. RALDH3 mRNA-expressing cells were observed in the primordium of
the anterior pituitary gland from embryonic day 3 (E3), and the expression of RALDH3mRNA was consistently strong in the dorsal part of the gland close to the base of the
diencephalon and was weak in the ventro-caudal part of the pituitary gland. Interstingly,
the intensity of RALDH3 mRNA expression in the anterior pituitary gland gradually
decreased after E12 and then strong RALDH3 mRNA expression appeared in the pars
tuberalis. To identify the hormone-producing cells that express RALDH3 mRNA, we
performed double staining for RALDH3 mRNA and anterior pituitary hormones, and we
found that POMC-, αGSU-, LHβ-, FSHβ-and TSHβ-producing cells were not overlappedwith RALDH3 mRNA-expressing cells. Moreover, RALDH2 mRNA expressing-cells were
found in the primordium of the pars tuberalis at E8. These results suggest that RA is
generated by RALDH2 and RALDH3 in the chicken embryonic anterior pituitary glandand that RA may be closely associated with the pars tuberalis.
References(1) Niederreither, K. et al. (1997) Mech. Dev., 62, 67-78.
(2) Fujiwara, K. et al. (2007) Cell Tissue Res., 328, 129-35.

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P11
CHANGES IN EXPRESSION OF GASTRIC GHRELIN, AROMATASE AND
LEPTIN IN THE POSTNATAL RAT STOMACH
Takatsugu Yanaka, Zhao Zheng, Chihiro Tsutsui, Kanako Koike, and Takafumi Sakai
Area of Regulatory Biology, Division of Life Science, Graduate School of Science andEngineering, Saitama University, Saitama 338-8570, Japan.
Ghrelin, a 28-amino-acid peptide isolated from the rat stomach as an endogenous
ligand for growth hormone secretagogue receptor, has been shown to stimulate GH
release, food intake and gastric acid secretion. In an adult rat, it has been reported that
gastric ghrelin expression and release are regulated by estrogen, somatostatin, leptin andinsulin of the stomach. However, the relationships between gastric ghrelin and theseregulatory factors in postnatal development remain obscure. In this study, weinvestigated the changes in gastric ghrelin and these factors at each postnatal stage by
real-time quantitative PCR and double staining for ghrelin-producing cells andaromatase- or leptin- expressing cells.
Postnatal ghrelin mRNA expression level significantly increased between 2 weeks (2wk) and 4 wk and between 4 wk and 8 wk in male and female rats respectively. The
expression levels of gastric estrogen synthases (aromatase, P450 17α, 17β HSD typé)
also significantly increased between 2 wk and 4 wk in male and female rats. On the other
hand, expression level of gastric leptin mRNA level decreased between 2 wk and 4 wk.
Double staining for expression of aromatase or leptin mRNA by in situ hybridization andghrelin-producing cells by immunohistochemistry revealed that these cells were locatednear each other, and ghrelin-producing cells were usually found adjacent to leptin- andaromatase-expressing cells in the gastric mucosa through the period of postnatal
development.
The results suggest that the gradual increase in gastric ghrelin expression level in the
postnatal rat is caused by the inverse changes in production of gastric leptin and estrogenand that release of these factors regulate ghrelin expression in a paracrine manner.

–14–

P12
mRNA EXPRESSION PATTERNS OF IGFBPS IN DEVELOPING LIMB
Kikumi Yamada1, Ken-ichi Tahara1, Ichiro Yamamoto2,3, Akira Tsukada1, Minoru Tanaka2,3,
Shusei Mizushima1, Tamao Ono4, Noboru Saito1, and Kiyoshi Shimada1
1

Department of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya, 464-8602, Japan.
2High-tech Research Center, 3Department of Animal Science, Faculty of Applied Life
Science, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602,
Japan.
4
Department of Food Production Science, Shinshu University, Nagano, 399-4598, Japan.
Insulin-like growth factor (IGF) is mainly secreted from liver and partly by anautocrine or paracrine fashion. IGF is important for both fetal and postnatal development.
In addition, it also controls tissue homeostasis via regulation of cell proliferation andapoptosis. There are several types of IGF-specific binding protein, insulin-like growth
factor binding proteins (IGFBPs). They have great affinity to IGF and render the IGF
system much more complex. However, the mechanism of IGF-IGFBPs axis has not been
fully elucidated. The formation of the digits in amniota embryos is accomplished by the
proliferation of the digital segment and the apoptotic cell death of the interdigital zone
simulutaneously. We focused on the formation of the limb and investigated the IGFBPs
expression in that of Japanese quail. In this study, by whole mount in situ hybridization,
IGFBP2 and IGFBP4 were detected in the outgrowth region and in apoptotic zone
respectively. These results indicate that IGFBPs are important for characterizing the IGF
function in avian¸morphogenesis. Then, we treated the embryos with BrdU which inhibit
apoptosis in interdigital zone. The treated quail hatched with webbed-limb. Though the
inhibition of the apoptosis was observed in interdigital zone, there were no differences in
expressions of IGFBPs. Therefore, the inhibiting apoptosis by the BrdU might not be
concerned with IGFBP. Hence, we are planning to compare the IGFBPs expressions of thequail with that of waterfowl which has webbed-limb.

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P13
EFFECT OF ESTROGEN AND PROLACTIN ON THE PERCEPTION OF
SODEFRIN IN FEMALE NEWTS, Cynops pyrrhogasterTomoaki Nakada1, Takeo Iwata2, Fumiyo Toyoda3, Kazutoshi Yamamoto4, Kazuyoshi
Tsutsui1, and Sakae Kikuyama4, 5
1

Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, School of Science and Engineering, Waseda University,
Tokyo 169-8050, Japan.
2
Department of Pharmacology, Institute of Health Biosciences, The University ofTokushima Graduate School, Tokushima 770-8504, Japan.
3
Department of Physiology, Nara Medical University, Kashihara, Nara 634-8521, Japan.
4
Department of Biology, School of Education, Waseda University, Tokyo 169-8050, Japan.
5
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
Sodefrin, a female-attracting pheromone of the newt Cynops pyrrhogaster, has beenpresumed to be perceived by the sensory neurons of the lateral nasal sinus (LNS) of female
newts (1). LNS is a diverticulum situated lateral part of the main chamber of the nasal
cavity, which has been considered to correspond to the vomeronasal organ (VNO) in otherquadrupeds (2), where pheromones are mainly detected.. In VNO of rodents, intracellular
Ca2+ ([Ca2+]i) levels are known to be elevated by the stimulations with urinary extractcontaining potential pheromones (3). As a step to study the mechanisms of sodefrin
perception, measurement of [Ca2+]i in the dispersed LNS cells following the exposure to
sodefrin by microfluorometry with fura-2 AM was carried out. Increase of [Ca2+]i was
detected in a small number of LNS cells from sexually developed female newts after theapplication of sodefrin (>10-10 M), whereas sodefrin-responsive cells were very rarely
detected in the males or sexually undeveloped females. This coincides well with ourprevious observation that responsiveness of LNS cells to sodefrin as detected by electroolfactogram is sex-dependent and hormone (estrogen and prolactin)-dependent. In fact, it
was confirmed that LNS cells were equipped with both estrogen and prolactin receptors. In
ovariectomized and hypophysectomized sexually developed females, sodefrin-responsive
cells, as detected by Ca2+ imaging method, decreased in number. This decrease wasrestored by the supplementation with estradiol and prolactin. It has also been known thattreatment of female newts with estradiol and prolactin elicits proliferation of LNS cells.
Thus, it was concluded that in female newts, this combination of hormones enhances the
responsiveness to sodefrin and increases the population of LNS cells.

References(1)Toyoda, F. et al. (1999). In "Advances in chemical signals in vertebrates", pp. 607-615.
(2)Eisthen, HL. et al. (1991). Chemical senses. 16:517-518.
(3) Spehr, M. et al. (2002). Journal of neuroscience. 22:8429-8437

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P14
ISOLATION OF MOLECULES WHICH INTERACT WITH HSP90b IN GERM
CELL APOPTOSIS IN NEWT TESTIS
Kazuyuki Fukaura, Ko Eto, and Shin-ichi Abe
Department of Biological Sciences, Graduate School of Science and Technology,
Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.

We have shown in vivo and in vitro that prolactin induces apoptosis in the 7th
generation of spermatogonia during newt spermatogenesis, but the underlying molecularmechanism remained unknown. Western blotting has detected Tyrosine phosphorylation ofheat shock protein (HSP) 90β, a molecular chaperone for client proteins in signaltransduction and transcriptional regulation, by prolactin in newt testis, and coimmunoprecipitation has demonstrated that HSP90β associated constitutively withprolactin receptor on the plasma membrane of germ cells.
To determine the role of HSP90β in prolactin-induced apoptosis, we performed coimmunoprecipitation with an antibody against C terminal region of nHSP90β that we
produced. HSP90β was demonstrated to associate with some proteins and undergo
constitutive phosphorylation in newt testis. Furthermore, injection of newts with prolactincaused dissociation of HSP90β from prolactin receptor, while injection with folliclestimulating hormone (FSH) caused dephosphorylation of a protein (approx. 38 kDa of themolecular weight) associated with HSP90β. Taken together, these results suggest that
HSP90β is involved in signaling prolactin-induced apoptosis through the receptor and in
signaling FSH-induced initiation of meiosis. In future, we will identify the proteinsassociated with HSP90β by proteomics, and then examine their expressions in newt testis.

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P15
ABSENCE OF GENITALIN (GONAD-STIMULATING HORMONE)-INDUCED 1METHYLADENINEPRODUCTION
IN
OVARIANFOLLICLE ANDTESTICULAR INTERSTITIAL CELLS OF STARFISH GONADS DURINGGAMETOGENESIS
Masatoshi Mita1, Mari Shinme1, Sayaka Yamamoto1, and Kazutoshi Yamamoto2 ¸
1

Department of Biology, Faculty of Education, Tokyo Gakugei University, Tokyo 1848501, Japan.
2
Department of Biology, School of Education, Waseda University, Tokyo 169-8050, Japan.
Recently, we and collaborators purified and identified the gonad-stimulating hormone
in starfish, Asterina pectinifera (1). The starfish gonadotropic hormone was a dimeric
peptide like insulin. Here, we propose the hormone to be named “genitalin” as a novel
gonad-stimulating insulin-like neuropeptide hormone, instead of “gonad-stimulating
substance (GSS)”. To obtain further information about hormonal action of genitalin, thisstudy examined effect of the synthetic genitalin on 1-methyladenine (1-MeAde) production
in ovarian follicle and testicular interstitial cells obtained from growing and ripe gonads ofstarfish A. pectinifera. The synthetic genitalin stimulated ripe follicle cells as well as ripeinterstitial cells to produce 1-MeAde through arise in cyclic AMP (cAMP). However,neither 1-MeAde nor cAMP production occurred in follicle and interstitial cells at thegrowing stage under the presence of genitalin. According to competitive experiments (2)using radioiodinated and radioinert genitalin in ovarian follicle cells, Kd value in ripe stage
was lower than that in growing stage, though almost the same Bmax value was obtained inboth stages. The result suggests that the affinity of receptors for genitalin in ovarian follicle
cells becomes high enough to activate adenylyl cyclase in the breeding season. In contrast,
Kd and Bmax values in interstitial cells at the growing stage were almost the same as thoseat the ripe stage. It is unclear why genitalin is failed to produce 1-MeAde and cAMP ininterstitial cells at the growing stage. It may be possible that G-protein or adenylyl cyclasein these cells is not coupled with the genitalin receptors.
References(1) Mita, M. et al. (2007) in preparation.
(2) Mita, M. et al. (2007) Gen. Comp. Endocrinol., 153, 299-301.

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P16
cDNA CLONING, GENE STRUCTURE, AND EXPRESSION OF KISS1 AND
GPR54 IN MEDAKA
Kazuhito Kasahara1, Yuichi Saito1, Makoto Oyama1, Makoto Endo1, Shiho Fujita1,
Tomotaka Abe1,2, Tadashi Yanagisawa1,2, and Masayuki Iigo1,2
1

Department of Applied Biochemistry, Utsunomiya University, Tochigi 321-8505, Japan.
United Graduate School of Agricultural Science, Tokyo University of Agriculture andTechnology, Tokyo 183-8509, Japan.
2

KISS1 was initially identified as a metastasis suppressor gene. Recent studies havedemonstrated that the KISS1 products metastin/kisspeptin are endogenous agonists for a G
protein-coupled receptor, GPR54. In mammals, the KISS1-GPR54 system is a key
regulator of the reproductive system that regulates the hypothalamic gonadotrophinreleasing hormone. However, the roles of the KISS1-GPR54 system in nonmammalianvertebrates remain to be elucidated.
To obtain molecular basis on the roles of the KISS1-GPR54 system in teleost, in thepresent study, we cloned cDNA encoding KISS1 and GPR54 in medaka Oryzias latipes
and their gene structures were clarified. In addition, effects of photoperiod on theexpression of KISS1 and GPR54 genes were examined.
The medaka KISS1 cDNA (759 bp) is encoding 100 amino acid residues. The medaka
KISS1 gene is composed of three exons and two introns. The coding region of the medaka
GPR54 cDNA (1131 bp) is encoding 376 amino acid residues. The GPR54 gene iscomposed of five exons and four introns.
RT-PCR analyses demonstrated the expression of KISS1 and GPR54 in all tissues
examined (brain, retina, gonads, and so on). Quantitative real-time PCR analyses
demonstrated that there is no difference in the amount of KISS1 and GPR54 transcripts
between the whole brain samples collected under long day and short day both in females
and males.
Further studies should be required to elucidate the roles of the KISS1-GPR54 system inthis species.

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P17
GnIH NEURONS PROJECT TO GnRH NEURONS IN THE AVIAN BRAIN
Vishwajit S. Chowdhury1, Takayoshi Ubuka2, George E. Bentley2, Tomohiro Osugi1, andKazuyoshi Tsutsui1
1

Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan.
2
Department of Integrative Biology, University of California, Berkeley, California 947203140, USA.
The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor
responsible for the hypothalamic control of gonadotropin secretion. Until recently, no
neuropeptide acting at the level of the pituitary to regulate gonadotropin secretion
negatively had been identified in vertebrates, although gonadal sex steroids and inhibin
inhibit gonadotropin secretion via feedback from the gonads. In 2000, we discovered anovel hypothalamic dodecapeptide which acts on the pituitary to inhibit gonadotropin
release in the Japanese quail and termed it gonadotropin-inhibitory hormone (GnIH; 1).
The action of GnIH is mediated by a novel G protein-coupled receptor (2). This
gonadotropin-inhibitory system may be widespread in vertebrates, at least birds andmammals (3-6). In these higher vertebrates, histological evidences suggest contact of GnIH
immunoreactive axon terminals with gonadotropin-releasing hormone (GnRH) neurons (6,7), thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study weinvestigated the interaction of GnIH and GnRH-I and -II neurons in European starling
brain. Cloned starling GnIH precursor cDNA encoded three peptides which possesscharacteristic LPXRF-amide (X = L or Q) motifs at the C-termini. Starling GnIH wasfurther identified as SIKPFANLPLRF-NH2 by mass spectrometry combined withimmunoaffinity purification. GnIH neurons, identified by in situ hybridization (ISH) andimmunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus.
GnIH immunoreactive fiber terminals were present in the external layer of the medianeminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II
neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons
were shown by GnIH and GnRH double-label ICC. Further, the expression of starling
GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by ISH
combined with GnRH ICC. Thus GnIH may regulate reproduction of vertebrates bydirectly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing thepituitary gland.
References(1) Tsutsui, K. et al. (2000) Biochem. Biophys. Res. Commun., 275, 661-667.
(2) Yin, H. et al. (2005) J. Endocrinol., 184, 257-266.
(3) Ubuka, T. et al. (2005) Proc. Natl. Acad. Sci. USA, 102, 3052-3057.
(4) Tsutsui, K., and Ukena, K. (2006) Peptides, 27, 1121-1129 (review).
(5) Ubuka, T. et al. (2006) Endocrinology, 147, 1187-1194.
(6) Kriegsfeld, L.J. et al. (2006) Proc. Natl. Acad. Sci. USA, 103, 2410-2415.
(7) Bentley, G.E. et al. (2003) J. Neuroendocrinol., 15, 794-802.

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P18
GONADOTROPIN-INHIBITORY HORMONE AND ITS RECEPTOR IN THE
AVIAN REPRODUCTIVE SYSTEM
Yoshihiro Morita1,2, George E. Bentley3, Takayoshi Ubuka3, Vishwajit S. Chowdhury1,
Itaru Hasunuma1, Tetsu Yano2, and Kazuyoshi Tsutsui1
1

Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan.
2
Graduate School of Medicine, University of Tokyo 113-0033, Japan.
3
Department of Integrative Biology, University of California, Berkeley, California 947203140, USA.
We recently identified a novel hypothalamic dodecapeptide which acts on the pituitary
to inhibit gonadotropin release in the quail and termed it gonadotropin-inhibitory hormone(GnIH; 1). GnIH inhibits gonadal development and maintenance by decreasing
gonadotropin synthesis and release in birds (1-7). Many hormones that are classified asneuropeptides are synthesized in vertebrate gonads in addition to the brain. Receptors forthese hormones are also expressed in gonadal tissue; thus there is potential for a highly
localized autocrine or paracrine effect of these hormones on a variety of gonadal functions.
In the present study, we therefore investigated whether GnIH and its receptor are expressedin the avian reproductive system including gonads and accessory reproductive organs bystudies on two orders of birds: Passeriformes and Galliformes. Binding sites for GnIH were
initially identified via in vivo and in vitro receptor fluorography, and were localized in
ovarian granulosa cells along with the interstitial layer and seminiferous tubules of thetestis. Furthermore, species-specific primers produced clear PCR products of GnIH and
GnIH receptor (GnIH-R) in songbird and quail gonadal and other reproductive tissues,
such as oviduct, epididymis and vas deferens. Sequencing of the PCR products confirmed
their identities. Immunocytochemistry detected GnIH peptide in ovarian thecal andgranulosa cells, testicular interstitial cells and germ cells and pseudostratified columnar
epithelial cells in the epididymis. In situ hybridization of GnIH-R mRNA in testes
produced a strong reaction product which was localized to the germ cells and interstitium.
In the epididymis, the product was also localized in the pseudostratified columnar
epithelial cells. In sum, these results indicate that the avian reproductive system has thecapability to synthesize and bind GnIH in several tissues. The distribution of GnIH and its
receptor suggest a potential for autocrine/paracrine regulation of gonadal steroid
production and germ cell differentiation and maturation.
References(1) Tsutsui, K. et al. (2000) Biochem. Biophys. Res. Commun., 275, 661-667.
(2) Satake, H. et al. (2001) Biochem. J., 354, 379-385.
(3) Bentley, G.E. et al. (2003) J. Neuroendocrinol., 15, 794-802.
(4) Ciccone, N.A. et al. (2004) J. Neuroendocrinol., 16, 999-1006.
(5) Osugi, T. et al. (2004) J. Endocrinol., 182, 33-42.
(6) Ubuka, T. et al. (2005) Proc. Natl. Acad. Sci. USA, 102, 3052-3057. Nature Reviews
Highlight(7) Ubuka, T. et al. (2006) Endocrinology, 147, 1187-1194.

–21–

P20
IN VIVO ACTION OF OCTOPUS GNRH: PHARMACOLOGICAL EFFECTS ANDIMMUNOCYTOCHEMISTRY IN HATCHLING BRAINS
Hiroyuki Minakata1and Shuichi Shigeno2
1

Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan.
Department of Neurobiology, Pharmacology and Physiology, The University of Chicago,
Chicago 60637, USA.
2

Previous studies suggest that oct-GnRH acts not only as a hormone regulating gonadal
maturation of octopus, but also as a multifunctional modulatory factor in the central andautonomic nervous systems (1). To understand in vivo effects, we injected the peptide into
the optic blood sinus of hatchlings (Octopus bimaculoides), and their behaviors wereobserved.

Action of oct-GnRH caused several abnormalities such as unstable crawl,

postures, abnormal movements of arms, hyperactivities of mantle, hearts, andchromatophores, and ejection of yolk. These effects continued at least 1 hour, then these
specimens recovered the normal behavior 1 day after.
Oct-GnRH-like immunoreactive neural cell bodies were distributed in the anterior
subesophageal mass (movement of arms), posterior subesophageal mass (chromatophore,
visceral, and funnel movements), optic tract lobe and basal lobe (posture control) of thebrain.

Immunoreactive fibers were distributed in neuropils of these mass and lobes.

Effects of oct-GnRH on behaviors can be interpreted as pharmacological activities on these
immunoreactive neurons and fibers. Yolk is considered to be a part of content in digestiveorgans of hatchlings. So that, ejection of yolk may be attributed to intestinal contractions
caused by oct-GnRH. Peripherally, oct-GnRH exhibited contractile activities on the small
intestine and rectum. Furthermore, the immunoreactivites were also observed in theolfactory lobe and superior buccal lobe (higher center of taste and touch), inferior frontal
lobe (higher center of touch), superior frontal lobe (higher center of vision), and verticallobe (higher center of vision and touch), suggesting that oct-GnRH may have importantroles for higher brain functions as a neurotransmitter and/or neuromodulator.
Reference(1) Iwakoshi-Ukena, E. et al. (2004) J. Comp. Neurol., 477, 310-323.

–22–

P21
DES INDUCES FISH OOCYTE MATURATION THROUGH MEMBRANE
PROGESTIN RECEPTOR
Toshinobu Tokumoto1, Mika Tokumoto2, and Peter Thomas2
1

Department of Biology, Faculty of Science, National University Corporation Shizuoka
University, Shizuoka 422-8529, Japan.
2
Marine Science Institute, University of Texas at Austin, 750 Channel View Drive, Port
Aransas, TX 78373, USA.
.
The potency of endocrine-disrupting chemicals (EDCs) to induce or prevent theresumption of meiotic cell cycle by maturation-inducing steroid (MIS) was examined invitro oocyte culture. We found that exposing fish oocytes to diethylstilbestrol (DES) at adose within a range similar to that used in experimental exposure to a natural MIS: 17α,20β-dihydroxy-4-pregnen-3-one (17, 20β-DHP) (1). The result was the first reportshowing that EDC can potentially induce oocyte maturation like an endogenous MIS.
Maturation inducing activity was also evaluated by the detection of synthesis of cyclin B
protein. A synergistic action of DES on 17, 20β-DHP-induced oocyte maturation wasobserved. These results suggested that DES acted on MIS receptor.
Recently a membrane bound progestin receptor, named as membrane progestin receptor(mPR), was identified as a strong candidate for MIS receptor. To investigate possible
interaction between mPR and DES, cDNAs for mPR proteins were isolated from goldfish
ovarian cDNA library (2). The specific antibody for goldfish mPRα was raised against
recombinant mPRα expressed in E.coli. Both 17, 20β-DHP- and DES-induced oocyte
maturation was inhibited by an antibody against mPRα. The ability of DES and its
analogues to interact with the progestin receptor mediating oocyte maturation wasinvestigated in receptor binding assays using plasma membranes from goldfish ovaries andbreast cancer cells transfected with goldfish mPRα. Membranes prepared from bothovaries and mPRα−transfected cells showed high affinity, saturable, displaceable, single
binding sites specific for the 17, 20β-DHP. DES and DES analogues (DP-DES and HEX),
which induce oocyte maturation in goldfish, bound to the receptor and caused
concentration-dependent displacement of [3H]-17, 20β-DHP (3).
The relative binding affinities of various steroids and EDCs to these two receptor
preparations were similar, supporting the idea that mPRα is a MIS receptor in vivo. The
close correspondence between binding of DES and its analogues to the mPRα protein andtheir oocyte maturation-inducing activities suggests a mechanism of endocrine disruption
mediated by binding to mPRα, thereby mimicking the nongenomic action of the 17, 20βDHP.

References(1) Tokumoto, T. et al. (2004) Proc. Natl. Aca.d Sci. USA. 101, 3686-3690.
(2) Tokumoto, M. et al. (2006) Gen. Comp. Endocrinol. 145, 101-108.
(3) Tokumoto, T. et al. (2007) Endocrinology 148, 3459-3467.

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P22
ISOLATION AND CHARACTERIZATION OF cDNA ENCODING NEUROMEDINU (NMU) RECEPTOR FROM THE BRAIN OF GOLDFISH
Keisuke Maruyama1, Koutaro Ishiguro1, Tatsuya Wakasugi1, Uchiyama Minoru1, Seiji
Shioda2, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering
University of Toyama, Toyama 930-8555, Japan.
2
Department of Anatomy, Showa University School of Medicine, Tokyo 142-8555, Japan.

In rodents, neuromedin U (NMU) is a multifunctional neuropeptide implicated in theregulation of the circulatory and digestive systems and energy homeostasis, especiallyappetite (1). However, there is no available information on the nature and physiologicalroles of NMU and its receptor in fish. In the present study, we identified four cDNAs
encoding three NMU orthologs from the goldfish brain and gut. Putative peptides
consisting of 21, 25 and 38 amino acid residues (NMU-21, -25 and -38) were deduced
from their nucleotide sequences. In the brain, mRNA for NMU-21 was strongly expressed.
Intracerebroventricular (ICV)-injected synthetic NMU-21 suppressed food intake in adose-dependent manner. We also isolated two cDNAs encoding NMU receptor orthologs(NMU-R1 and NMU-R2) from the brain. The amino acid sequences of goldfish NMU-R1
and -R2 have 49% and 43% homology between that of human NMU-R1 and -R2,
respectively. These mRNAs were expressed in the brain, pituitary, gill, kidney, testis andovary while in the gut, NMU-R2 mRNA expressed strongly. These results suggest thatNMU orthologs and their receptors exist in fish, and that the NMU-21 thus deduced canpotently inhibit food intake in goldfish (2).

References(1) Brighton et al. (2004) Phamacol. Rev., 56, 231-248.
(2) Maruyama et al. (2007) J. Neuroendcrinol., in press.

–24–

P23
PURIFICATION AND PROPERTIES OF GHRELIN FROM THE INTESTINE OF
GOLDFISH, Carassius auratus
Tohru Miura1, Keisuke Maruyama1, Sei-Ichi Shimakura1, Minoru Uchiyama1, Hiroyuki
Kaiya2, Kenji Kangawa2, Seiji Shioda3, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,

University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan.
2

Department of Biochemistry, National Cardiovascular Center Research Institute, Suita

565-8565, Japan.
3

Department of anatomy, Showa University, School of Medicine, Shinagawa-ku, Tokyo

142-8555, Japan.
Ghrelin is a 28-amino acid peptide identified in rat and human stomach as an
endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [1]. Ghrelin is
now recognized to be a multifunctional peptide that is involved in the regulation of somatic
growth, feeding behavior and energy homeostasis in mammals. In goldfish, cDNA
encoding the ghrelin precursor has been identified [2]. Ghrelin mRNA is mainly expressedin the intestine (given that goldfish lack a stomach), and few in the brain. We haveindicated that, in the goldfish intracerebroventricular (ICV) and intraperitoneal
administrations of synthetic goldfish ghrelin (12 amino acid-residues form with an octanoic
acid modification at the third N-terminal serine residue (Ser3), 12C8) stimulate food intake
and locomotor activity [3]. However, the native mature ghrelin generated from the ghrelin
precursor has not yet been identified in goldfish. In the present study, we purified andcharacterized ghrelin from the intestine of goldfish. Ghrelin thus obtained from theintestine has 11 molecular forms consisting of 14, 17, 18 and 19 amino acid-residues. Ser3
was acylated by n-octanoic, n-nonanoic or n-decanoic acid. Since the 17 amino acidresidues form was much yielded, we synthesized 17 amino acid-residues with or without
the octanoic or decanoic acid modification at Ser3 (17C0, 17C8 and 17C10), and examined
their biological activity. 17C8 and 17C10, but not 17C0, increased intracellular calcium
concentrations in a cell line stably expressing rat GHS-R with similar potency to 12C8.
ICV administration of 17C8, but not 17C0 and 17C10, increases food intake as well as
12C8 in the goldfish.
References(1) Kojima et al. (1999) Nature, 402, 656-660.
(2) Unniappan et al. (2002) Endocrinology, 143, 4143-4146.
(3) Matsuda et al. (2005) Peptides, 27, 1335-1340.

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P-24
EFFECT OF GOLDFISH LPXRF-AMIDE PEPTIDES ON FOOD INTAKE IN
GOLDFISH, Carassius auratus
Kodai Nakamura1, Keisuke Maruyama1, Sei-Ichi Shimakura1, Minoru Uchiyama1,
Kazuyoshi Tsutsui2, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan.
2
Department of Biology, School of Education, Waseda University, Shinjuku-ku 169-0649,
Japan.

Novel neuropeptides, which are characterized by a similar structure including the Cterminal LPXRF-NH2 motif (X = L or Q residue), have been isolated from tetrapods (1). In
the goldfish brain, a cDNA encoding the precursor of three LPXRF-NH2 peptides(gfLPXRFa-1, -2 and -3) has also been identified (2). Their mRNA is specifically
expressed in the diencephalon, and neuronal cell bodies expressing the precursor are
located in the hypothalamus, the nucleus posterioris periventricularis (NPPv). The NPPv isone of centers implicated in the regulation of feeding behavior in goldfish, however it isunknown whether gfLPXRFa-1, -2 and -3 affect food intake. In the present study, we
examined the effect of intracerebroventricular (ICV) administration of gfLPXRFa-1, -2 and-3 on food consumption in goldfish. ICV injection of graded doses of gfLPXRFa-2 (0.1-10
pmol/g BW) induced a dose-dependent inhibition of food intake, a significant decrease incumulative food intake during the 60-min period after feeding being observed at doses of 1
and 10 pmol/g BW. On the other hand, ICV-injected gfLPXRFa-1 and -3 at same doses
have no evident changes in food intake. These results suggest that gfLPXRFa-2 has apotency to act as an anorexigenic factor involved in the feeding regulation of goldfish.

References(1) Tsutsui et al. (2006) Peptides, 27, 1121-1129.
(2) Sawada et al. (2002) Eur. J. Ciochem., 269, 6000-6008.

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P25
INHIBITORY EFFECT OF MELANIN-CONCENTRATING HORMONE ON
FOOD INTAKE IN GOLDFISH
Sei-Ichi Shimakura1, Kohei Wada1, Keisuke Maruyama1, Tohru Miura1, Tomoya
Nakamachi2, Minoru Uchiyama1, Haruaki Kageyana2, Seiji Shioda2, Akiyoshi Takahashi3,
and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
2
Department of Anatomy, Showa University School of Medicine, Tokyo 142-8555, Japan
3
Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University,
Iwate 022-0101, Japan.
Melanin-concentrating hormone (MCH) was originally isolated from the chum salmon
pituitary as a hormone involved in body color regulation (1). Recently, it has been
indicated that MCH is implicated in regulation of feeding behavior and energy homeostasis
in mammals (2). However, the role of MCH in appetite has not yet been well studied in fish.
We have indicated that MCH influences feeding behavior in the goldfish, and exerts ananorexigenic action, unlike its orexigenic effect in mammals (3, 4). This raises the questionof why MCH acts as an anorexigenic factor in goldfish. Therefore, the aim of the present
study was to investigate the involvement of anorexigenic or orexigenic neuropeptides in
the anorexigenic action of MCH in goldfish. Because α-melanocyte-stimulating hormone(α-MSH) and corticotropin-releasing hormone (CRH) are both powerful and key
anorexigenic neuropeptides in goldfish, we examined the effects of intracerebroventricular(ICV) injection of an α-MSH receptor (melanocortin 4 (MC4) receptor) antagonist, HS024,
and a CRH 1/2 receptor antagonist, α-helical CRH(9-41), on the anorexigenic action of ICVadministered MCH. ICV injection of HS024, but not α-helical CRH(9-41), suppressed
MCH-induced anorexigenic action for a 60 min observation period. We also examined,
using a real-time PCR method, whether ICV-injected MCH affects the expression oforexigenic neuropeptide mRNAs, such as neuropeptide Y (NPY), orexin, ghrelin andagouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH
at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for
NPY and ghrelin, but not for orexin and AgRP. In addition, we investigated the neuronal
relationship between MCH-containing nerve fibers and α-MSH- or NPY-containing
neuronal cell bodies in the hypothalamus using a double-immunofluorescence method.
MCH-containing nerve fibers were located in close proximity to α-MSH- or NPYcontaining neuronal cell bodies in the hypothalamus. These results indicate that theanorexigenic action of MCH in the goldfish brain is mediated by α-MSH signaling
pathway, perhaps through the MC4 receptor system, with inhibition of hypothalamic NPY
and ghrelin synthesis.

References(1) Kawauchi et al. (1983) Nature, 305, 321-323.
(2) Qu et al. (1996) Nature, 380, 243-247.
(3) Matsuda et al. (2006) Neurosci. Lett., 399, 259-263.
(4) Matsuda et al. (2007) Cell Tissue Res., 328, 375-382.

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P26
DIAZEPAM-BINDING
INHIBITOR-DERIVED
PEPTIDES-INDUCEDANOREXIGENIC ACTION IS MEDIATED THROUGH THE METABOTROPICENDOZEPINE RECEPTOR IN GOLDFISH
Kohei Wada1, Tohru Miura1, Keisuke Maruyama1, Sei-Ichi Shimakura1, Minoru Uchiyama1,
Jérôme Leprince2, Marie-Christine Tonon2, Hubert Vaudry2, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
2
Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS,
European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 MontSaint-Aignan Cedex, France.
An endogenous ligand of central-type benzodiazepine receptors (CBR), the endozepine
octadecaneuropeptide (ODN), is a very potent inhibitor of food intake in rodents (1-3).
Although endozepines have been localized and characterized in the trout hypothalamus, sofar, the action of these neuropeptides on feeding behavior has never been investigated infish. In the present study, we have examined the effect of ICV administration of synthetic
rat ODN, its C-terminal octapeptide (OP) and the head-to-tail cyclic analog cyclo1-8OP(cOP) on feeding behavior in the goldfish model. ICV injection of graded doses of ODN(2.5-10 pmol/g BW) induced a dose-dependent inhibition of food intake, a significant
decrease in cumulative food intake during the 60-min period after feeding being observedat doses of 5 and 10 pmol/g BW. The inhibitory effect of a 10 pmol/g BW dose of ODN on
food consumption (-39%) was mimicked by an equimolar dose of OP (-42%) and cOP (53%). The food intake-suppressing activity of ODN (10 pmol/g BW) was not affected by
pre-injection of the CBR antagonist flumazenil (200 pmol/g BW). In contrast, theanorexigenic effect of ODN (10 pmol/g BW) was totally suppressed by a selective
antagonist of metabotropic endozepine receptors, cyclo1-8[DLeu5]OP. These data indicate
that, in goldfish as in rodents, ODN is a potent inhibitor of food consumption, and that theanorexigenic effect of ODN is not mediated through CBR but through the metabotropic
endozepine receptor (4).

References(1) Ferrero et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 827_831.
(2) Patte et al. (1995) FEBS Lett., 362, 106_110.
(3) Leprince et al. (2001) Eur. J. Biochem., 268, 6045_6057.
(4) Matsuda et al. (2007) Neurosci., in press.

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P27
DISTRIBUTION OF MELANOCORTIN RECEPTORS AND EFFECTS OF
FEEDINGON ITS
EXPRESSION WITHIN BARFIN FLOUNDER,
PLEURONECTIFORMES
Yuki Kobayashi1, Saori Okada1, Keisuke Tsuchiya1, Takeshi Yamanome2, and Akiyoshi
Takahashi1
12School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.
Iwate Fisheries Technology Center, Kamaishi, Iwate 026-0001, Japan.

Melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) arecollectively termed melanocortin (MC). These peptides are derived from a precursor
protein, proopiomelanocortin (POMC). MSH and ACTH participate in body color changes
and stress responses, respectively. The functions of MC are mediated through the MC
receptor (MCR) family, which are G protein-coupled receptors with seven transmembrane
domains, showing a specific tissue distribution and pharmacological properties. Five MCR
subtypes have been identified in mammals and six and four in pufferfish and zebrafish,
respectively. The goal of our studies on MCR is the comprehensive understanding of
melanocortin systems consisting of MC and MCR in fish. So far, we have identified threesubtypes of POMC from barfin flounder Verasper moseri, Pleuronectiformes (1, 2).
Herein, we report the molecular cloning, tissue distribution, and expression of MCR
subtypes in barfin flounder.
We determined the entire nucleotide sequences of four MCR subtype cDNAs. The
deduced amino acid sequence length is 323 for MC1R, 300 for MC2R, 325 for MC4R, and342 for MC5R. The reverse transcription polymerase chain reaction showed a higher
expression of these four subtypes in the brain and testis than in other tissues examined.
Furthermore, prominent expression was also observed in the eyeball and skin for MC1R,
head kidney for MC2R, eyeball, liver, and ovary for MC4R, and skin for MC5R. The
diverse tissue distribution of MCRs indicates the multifunctional nature of MC systems in
addition to classical functions such as melanin synthesis in skin and cortisol release in thehead kidney. The lipolytic activity of MSH has been shown in the liver of rainbow trout(3). In barfin flounder liver, we found four-fold higher expression levels of MC4R in fasted
than fed fish. MC seems to be associated with energy metabolism through MC4R in theliver.
References(1) Takahashi, A. et al. (2005) Gen. Comp. Endocrinol., 141, 291-303.
(2) Takahashi, A. et al. (2006) Gen. Comp. Endocrinol., 145, 280-286.
(3) Yada, T. et al. (2000) Zool. Sci., 17, 1123-1127.

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P28
EFFECTTS OF ADVANCE OF AGE IN THE POSTNATAL PERIOD AND
FASTING ON GHRELIN EXPRESSION IN THE RAT DUODENUM
Kanako Koike, Zheng Zhao, Chihiro Tsutsui, Takatsugu Yanaka, and Takafumi Sakai
Area of Regulatory Biology, Division of Life Science, Graduate School of Science andEngineering, Saitama University, Saitama 338-8570, Japan.
Ghrelin, an endogeneous ligand for the growth hormone secretagogue receptor, is a 28amino-acid peptide and is produced predominantly in the stomach mucosa (1). However,after gastrectomy, nealy 30% of ghrelin remains in plasma (2), and the major source of theremaining ghrelin is thought to be the small and large intestines. Recently, several studies
have shown that the gastric ghrelin mRNA expression and secretion is up-regulated by
gastric estrogen (3) and down-regulated by somatostatin and leptin (4, 5). However, theregulatory mechanism of ghrelin mRNA expression level in the intestine, the most ghrelin
producing region in the intestines, is still unclear. In this study, we focused on the
duodenum, the most large part of ghrelin producing region in the small intestine, andstudied the effect of fasting on the duodenum ghrelin expression and ghrelin mRNA
expression levels at each neonatal and postnatal stage (1 week old and 2 weeks old;
lactation period, 4 weeks old; puberty, 8 weeks old; maturation period) by quantitative
PCR, in situ hybridization (ISH) and immunohistochemistry (IHC).
The quantitative RT-PCR results showed that ghrelin mRNA was expessed in the
duodenum from embryonic day 18 and that the expression level increased remarkbly justafter birth. Although gastric ghrelin expression level gradually increased in the
postneonatal rat and then the duodenum ghrelin expression was maintained at a high level
until the lactation period, the ghrelin expression level significant decreased at puberty andthe maturation period. The number of ghrelin-expressing and producing-cells detected by
ISH and IHC showed the same changes as those found by quantitative RT-PCR. After 48
hrs of fasting, unlike in the stomach, the duodenal ghrelin mRNA expression level was notchanged. Moreover, expression levels of aromatase, leptin and somatostatin in the
duodenum were not changed throughout the period of postnatal development.
The results suggested that rat duodenal ghrelin expression is controlled by mechanisms
different to those that control gastric ghrelin expression.
References(1) Kojima et.al. (1999) Nature, 402, 656-660.
(2) Ariyasu et. al. (2001) J.Clin Enodcrinol Metab, 86, 4753-58.
(3) Sakata et. al. (2006) J. Endocrinol, 190, 749-57.
(4) Shimada et. al. (2003) Biochem. Biophys. Res. Commun., 302, 520-525.
(5) Kamegai et al. (2004) Regulatory Peptides, 119, 77-81.

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P29
THE OREXIGENIC EFFECT OF GONADOTROPIN-INHIBITING HORMONE IS
MEDIATED BY OPIOID SYTEM IN THE BRAIN OF CHICKS
Tetsuya Tachibana1, Naoto Masuda1, Kazuyoshi Ukena2, Kazuyoshi Tsutsui3, and Hiroshi
Ueda1
1

Laboratory of Animal Production, Faculty of Agriculture, Ehime University, Matsuyama
790-8566, Japan.
2
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima
University, Higashi-Hiroshima 739-8521, Japan.
3
Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and IntegratedArts and Sciences,Waseda University, Tokyo 169-8050, Japan.
Gonadotropin-inhibiting hormone (GnIH) is a neuropeptide which suppresses
gonadotropin release from the anterior pituitary (1). Recently, we found that
intracerebroventricular (ICV) injection of GnIH stimulates feeding behavior in chicks (2).
However, the mechanism underlying the orexigenic effect has not been clarified. In thepresent study, we investigated whether the orexigenic effect of GnIH is mediated by opioid,
nitric oxide or noradrenergic systems, which are well known as feeding stimulatory factors
in the brain of chicks. The GnIH-induced feeding behavior was significantly attenuated by
ICV co-injection of opioid mu-receptor antagonist but not delta- and kappa-antagonists,indicating that the orexigenic effect of GnIH is mediated by opioid mu-receptor. On theother hand, ICV co-injection of nitric oxide synthase inhibitor did not affect the orexigenic
effect of GnIH. Adrenergic alpha2 receptor antagonist showed the tendency to attenuate the
GnIH-induced feeding behavior although the significant effect was not observed. In
conclusion, the present study revealed that the orexigenic effect of GnIH is related to
opioid system in the brain of chicks.

References(1) Tsutsui, K, et al. (2000) Biochem. Biophys. Res. Commun., 275, 661-667.
(2) Tachibana, T. et al. (2005) Brain Res., 1050, 94-100.

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P30
MORPHOLOGICALOBSERVATIONOFNEUROPEPTIDEW

NEURONNETWORK IN THE RAT HYPOTHALAMUS
Seiji Shioda1, Fumiko Takenoya1, 2, and Haruaki Kageyama1
1

Department of Anatomy, Showa University School of Medicine, Tokyo 142-8555, Japan
Department of Physical Education, Hoshi University School of Pharmacy andPharmaceutical Science, Tokyo 142-8501, Japan.
2

Neuropeptide W (NPW) is a novel neuropeptide which was recently isolated from theporcine hypothalamus. NPW is shown to be an endogenous ligand for GPR7 and GPR8 of
the orphan G protein-coupled receptors (1, 2). GPR7 and GPR8 are expressed in brain
regions, especially in the hypothalamus, which are related to feeding regulation.
Intracerebroventricular (icv) infusion of NPW into the rat cerebral ventricle increases foodintake for 2 h in the light phase and stimulates prolactin and corticosterone release (1). On
the other hand, in the dark phase, icv infusion of NPW reduces food intake for 48 h with
up-regulation of energy expenditure and decrease of body weight (3). However
immunohistochemical studies on the distribution and localization of NPW have revealed
differing results concerning whether or not NPW-containing cell bodies and their processes
are present in the hypothalamus (4, 5). To determine the distribution of NPW in rat brain,
we used specific and high-affinity antiserum against NPW to demonstrate
immunohistochemically the distribution and localization of NPW-containing neurons in the
rat hypothalamus. We found that NPW-like immunoreactive cell bodies were intensely
found in the hypothalamus especially abundant in the arcuate and paraventricular nuclei
and lateral hypothalamic area. Moreover, NPW-containing nerve fibers were found inapposition to orexin- and MCH-containing neurons in the lateral hypothalamus (6). Theseand our findings strongly suggest that NPW-containing neurons interact with orexinand/or MCH-containing neurons in the hypothalamus and NPW participates in theregulation of feeding behavior in harmony with other feeding-regulating peptides.
References(1) Shimomura, Y. et al. (2002) J. Biol. Chem., 277, 35826–32.
(2) Tanaka, H. et al. (2003) Proc. Natl. Acad. Sci., 100, 6251–56.
(3) Mondal, M.S. et al. (2003). Endocrinology, 144, 4729–33.
(4) Kitamura, Y. et al. (2006) Brain Res., 1093, 123–34.
(5) Dun, SL. et al. (2003) Neurosci. Lett., 349, 71–74.
(6) Takenoya, F, Shioda S. et al. (2007) Regul Pept. (Aug 25 [Epub ahead of print])

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P31
cDNA CLONING AND GENE STRUCTURE OF
HYPOCRETIN RECEPTOR IN MEDAKA Oryzias latipes

HYPOCRETIN

AND

Kouji Egashira1, Noriko Amiya2, Masafumi Amano2, Kunio Yamamori2,
Tadashi Yanagisawa1, and Masayuki Iigo1
1
2

Department of Applied Biochemistry, Utsunomiya University, Tochigi 321-8505, Japan.
School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Hypocretin/orexin (HCRT) is a neuropeptide that is involved in the regulation of

feeding and sleep in mammals (1,2). Recently, we immunohistochemically localized
HCRT-like immunoreactivity in the brain and pituitary gland of medaka (3). However, little
is known on the HCRT system in nonmammalian vertebrates including fish.
To obtain molecular basis on the roles of HCRT system in teleost, in the present study,
we cloned cDNA encoding HCRT and HCRT receptor (HCRTR) in medaka and their genestructures were clarified.
The medaka HCRT cDNA (640 bp) is encoding 139 amino acid residues including
HCRT-1 (orexin A) and HCRT-2 (orexin B). The medaka HCRT gene is composed of two
exons and one intron.
HCRTR is a member of G protein-coupled receptor family with seven transmembrane
domains. Two subtypes (HCRTR1 and HCRTR2) of HCRTR were classified to date.
However, we found only a single HCRTR gene (HCRTR2) in the medaka genome. The
medaka HCRTR2 cDNA (1348 bp) is encoding 420 amino acid residues. The HCRTR2
gene is composed of seven exons and six introns.
The site of HCRT and HCRTR2 expression and the effect of starvation on theexpression of HCRT and HCRTR2 genes in medaka are now under investigation.

References(1) de Lecea L et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 322–327.
(2) Sakurai et al. (1998) Cell, 92, 573-585.
(3) Amiya et al. (2007) Neurosci. Lett.. In press

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P32
IDENTIFICATION OF THE REPTILIAN UNCOUPLING PROTEINS IN THE
LEOPARD GECKO, Eublepharis macularius
Keisuke Kato and Min Kyun Park
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
The ground living vertebrates had been adapted to the annual changes of thetemperature in many ways. The most distinctive adaptation among them is nonshivering
thermogenesis observed in endotherms. In mammals, this thermogenesis arises from thebrown adipose tissues in response to cold exposure, and in birds, it occurs in the skeletal
muscles. The main factors of these phenomenon are the uncoupling proteins (UCP), which
are proton transporters localized in the mitochondrial inner membrane (1). To elucidate themolecular evolution of the endothermic mechanism, we focused on the UCP of the reptiles,
which are ectothermic amniotes.
In this study, we identified partial sequences of leopard gecko (lg) UCP2 and UCP3.
The amino acid sequences deduced from lgUCP2 and UCP3 cDNA show high similarities
with their corresponding peptides of mammals and birds. The purine nucleotide-bindingsites, which are involved in the control of the uncoupling activity of UCP, andmitochondrial carrier signature motives are well conserved in these deduced sequences. In
the phylogenetic tree drawn with these sequences and their corresponding sequences ofmammals, birds, reptiles and fishes, the leopard gecko clustered with reptiles and birds. By
the RT-PCR, lgUCP2 was detected in the whole brain, liver, kidney, abdominal adipose
tissue, and axillary adipose tissue. Whereas lgUCP3 was expressed in the whole brain,
abdominal muscle and tail muscle. These expressional patterns are similar with mammals.
In addition, synteny analysis of the UCP genes was performed using the squamate genome(green anole, Anolis carolinensis), and revealed the possibility of the deletion of reptilian
UCP1. In conjunction with the recent view that thermogenic proton transport of
mammalian UCP1 seems to be a late evolutionary characteristic and that ancestral UCPs
may carry other substrates (2), these results suggested that the reptilian UCPs are goodcandidates for pleiotropic mitochondrial activities.
References(1) Ledesma. A. et al. (2002) Genome Biology, 3(12) reviews3015.1-3015.9(2) Emre. Y. et al. (2007) Journal of Molecular Evolution, Online First

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P33
HEMOLYMPH MAJOR ANIONIC PEPTIDE (HemaP) DERIVED FROM THE
SILKWORM, Bombyx mori, CAN INFLUENCE ON THE LARVAL FEEDING
BEHAVIOR
Shinji Nagata and Hiromichi Nagasawa
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Regularly occurring feeding behavior in insects such as phytophagous caterpillars
provided us some important questions “why and how insects start meal?”, even though
they live on their diet.

In the silkworm, Bombyx mori, the patterned feeding behavior was

also observed in approximately two-hour rhythmicity (1).

When some fraction of thepartially purified Bombyx larval hemolymph by reverse-phase HPLC was injected,
disturbance in the behavioral pattern was observed.

Purification and structural

determination revealed a peptidyl factor consisting of 62 amino acid residues, whichshowed no homologous sequences in the database.

Then, we designated this peptide,

Hemolymph major anionic peptide, HemaP.
Injection of HemaP into Bombyx larvae increased the levels of feeding, approximate
digestibility (AD; a generally used digestive index), and duration in exploring around an
artificial diet block.

These results indicate that the hemal concentration of HemaP isrelated to feeding behavior in Bombyx larvae.
In addition, quantification of HemaP in the larval hemolymph by LC-ESI-TOF/MS
revealed that HemaP level was increased by starvation.

Interestingly, refeeding diet after

starvation returned to the similar level of HemaP to that in the larvae fed ad libitum.
Those results strongly indicate that the concentration of HemaP in the hemolymph can
influence on the motivation of feeding behavior in Bombyx larvae by.

Reference(1) Nagata, S. and Nagasawa, H. (2006) Journal of Insect Physiology, 52. 807-815.

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P34
IDENTIFICATION AND CHARACTERIZATION OF THE VERTEBRATECALCITONIN ORTHOLOGOUS GENE IN THE ASCIDIAN, Ciona intestinalis
Toshio Sekiguchi1,2, Tsuyoshi Kawada2, Tsubasa Sakai2, Masato Aoyama2, Manabu Fujie3,
Michio Ogasawara4, Nori Satoh5, and Honoo Satake2
1

JSPS research fellow, 2Suntory Institute for Bioorganic Research, 3CREST JST, 4Chiba

University, Graduate School of Advanced Integration Science, 5Department of Zoology,
Graduate School of Science, Kyoto University, Japan.
Calcitonin (CT) is a hormone peptide essential for bone metabolism in mammals. The
CT family peptides, including CT, CGRP, Adrenomedulin, Amylin, and CRSP, areconserved in vertebrates. However, no CT family peptides have ever been identified ininvertebrates. The ascidian, Ciona intestinalis, which belongs to protochordates as a basal
chordate, is closely related to vertebrates. The CT family signaling system is regulated bycomplicated combination of five CT family peptide ligands, two receptors, and three
receptor-activity-modifying proteins. In this study, we identified Ciona-CT (Ci-CT) and, its
putative receptor, Ci-CTR as the original components of CT family signaling pathway on
the Ciona genomic database. Ciona has no paralogous genes, indicating that these genes areprototype genes of vertebrate counterparts.
RT-PCR analysis revealed that Ci-CT was expressed in the neural complex, endostyle,branchial sac, heart, and ovary, and that Ci-CTR was distributed in the neural complex,
endostyle, intestine, and branchial sac. In situ hybridization analysis showed that Ci-CT
was expressed in the whole of neural gland, whereas Ci-CTR was detected in theepithelium region of neural gland. Maircoarray analysis of Ci-CT-regulated genes in theneural complex revealed that several hormones, neuropeptides, their receptors, andneurotransmitter receptors were induced by Ci-CT. In contrast, Ci-CT was also shown to
down-regulate the osmoregulatory gene and exocrine-associated gene, which are
anticipated to be expressed in the neural gland.

Functional analysis of these Ci-CT-

regulated genes, will lead to elucidation of the functional evolution of the CT family.
Keywords: calcitonin, ascidian, Ciona, evolution

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P35
ROLE OF NEUROHYPOPHYSEAL PEPTIDES IN CONTROLLING THE
DRINKING BEHAVIOR OF THE EEL ACCLIMATED TO SEA WATER
Masaaki Ando, Takao Mukuda, Yohei Watanabe, and Takashi Sakihara
Laboratory of Integrative Physiology, Graduate School of Integrated Arts and Sciences,
Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
Role of teleost neurohypophysial peptides, vasotocin (VT) and isotocin (IT), in
controlling the drinking behavior was examined. When VT was administrated into theposterior cardinal vein of the seawater eel, the drinking rate was suppressed significant- ly,
while IT enhanced the drinking rate. Since the drinking rate of the eel is limited bycontraction or relaxation of the upper esophageal sphincter (UES) muscle, effects of VT
and IT were examined in the isolated UES muscle preparations. VT constricted and IT
relaxed the UES muscle in a concentration-dependent manner, and the former effect was
blocked by V1-receptor antagonist (H-5350) and the latter by oxytocin receptor antagonist(H-9405), suggesting presence of VT and IT receptors in this UES preparation. Similar
relaxation were observed after treatments with IBMX, forskolin and 8BrcAMP and effectof 8BrcGMP was less than 1/4 of that of 8BrcAMP, suggesting that IT acts via cAMP
production and that the relaxation via NO is unlikely. Even in scraped UES preparations,
IT relaxed the UES. To clarify whether muscle cells or nerve terminals are target for IT,
nerve activity was completely blocked by TTX. Even after blocking nerve activity with
TTX, IT relaxed the UES muscle concentration-dependently, indicating direct action of IT
on the muscle cells. On the other hand, IT enhanced the UES contraction evoked by nerve
stimulation. This effect was also blocked by H-9405, suggesting that such enhancement isalso mediated by IT receptor. Similar enhancements were observed after application of
IBMX, forskolin and 8BrcAMP. However, carbachol- induced contraction was notenhanced by IT. In addition, when nerve activity and nerve-muscular transmission were
blocked by TTX and curare respectively, the UES can be constricted by a stronger
electrical stimulation (DC current) but the DC-current induced contraction was notenhanced by IT. These results suggest that IT acts nerve terminals and enhances release ofacetylcholine. All together, it is plausible that IT may open the UES when the vagal nerve,
which comes from the glossopharyngeal-vagal motor complex (GVC) in the medulla
oblongata, is inhibited, while IT may close the UES when the GVC neurons are activated.

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P36
SALINITY TOLERANCE AND EXPRESSIONS OF NEUROHYPOPHYSIALHORMONE GENES IN FOUR SPECIES OF PUFFERS
Eiji Motohashi1, Sanae Hasegawa2, Kenzo Mishiro3, Yukiko Sugiyama4, Hiroyuki Doi4,and Hironori Ando1
1

Laboratory of Advanced Animal and Marine Bioresources, Graduate School of
Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581,
Japan.
2
Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639, Japan.
3
Hayashikane Sangyo Co., Ltd, Shimonoseki 750-8517, Japan.
4
Shimonoseki Marine Science Museum "Kaikyokan", Shimonoseki 750-0036, Japan.
Two neurohypophysial hormones, vasotocin (VT) and isotocin (IT), are involved inosmoregulation and reproduction in teleosts. Our understanding of osmoregulatory
mechanisms of VT and IT in fish is still fragmentary and needs a good animal model for
elucidation of the mechanism. We chose puffer fish as an experimental model, because
genomic resources of Takifugu rubripes and Tetraodon nigroviridis can be utilized toelucidate molecular mechanisms of osmoregulation.
To examine the adaptability of four species of pufferfish (Takifugu rubripes, Takifugu
niphobles, Tetraodon nigroviridis, Tetraodon fangi) to different salinity environments,
fishes were transferred to fresh water (FW), 10, 33 and 100% seawater (SW) and were kept
for 3 days. Changes in blood osmolality, concentrations of Na+ and Cl-, Na+/K+-ATPase
activity in the gill and kidney and expressions of VT and IT genes in the brain were
examined during adaptation to different salinities.
In Takifugu rubripes transferred to the hypoosmotic conditions, blood osmolality,
concentrations of Na+ and Cl- were decreased on 1 day after transfer (Day 1), but weremaintained in the physiological levels during Day 2-3. The Na+/K+-ATPase activity in thekidney tended to increase in FW and 10% SW. The levels in expression of VT gene in FW
tended to be higher than 100% SW on Day 1, while no significant changes were observedin the levels of IT mRNA. In Takifugu niphobles, the levels of VT and IT mRNAs in
33% SW were significantly lower than 100% SW at 12 hours after transfer. In Tetraodon
nigroviridis, the levels of IT but not VT mRNAs were higher in 100% SW than FW and
33% SW. In Tetraodon fangi, all fish transferred to 100% SW died at 3 hours after transfer.
The present results show that the expressions of VT and IT genes as well as salinity
tolerance are different among the four puffer species, indication that puffer fish provides auseful experimental model for studying the osmoregulatory mechanisms of VT and IT.

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P37
IDENTIFICATION AND PHYSIOLOGICAL ROLE OF VASOTOCINPRECURSOR AND ITS RECEPTORS IN AFRICAN LUNGFISH, Protopterus
annectens
Norifumi Konno1, Susumu Hyodo2, Kouhei Matsuda1, and Minoru Uchiyama1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama, 930-8555, Japan.
2
Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Tokyo, 1648639, Japan.
African lungfish, Protopterus annectens, living in freshwater, estivates to avoid
dehydration of their body fluids in a subterranean mud cocoon during severe dry season. To
clarify the mechanism of water and electrolyte balance of the lungfish maintained under
the freshwater and estivation conditions, we focused on mechanisms of arginine vasotocin(AVT) known as an antidiuretic hormone in terrestrial tetrapods. We identified AVT
precursor and its receptors (VT1R and VT2R) from the brain and kidney, respectively, andexamined the mRNA expression of AVT precursor and the receptors in the hypothalamus
and kidney of the lungfish maintained in the freshwater and estivation conditions.
Lungfish AVT precursor identified from whole brain was structurally similar to those of
tetrapods and was higher identity with amphibian AVT precursor (68%) than that of teleost(57%). Phylogenetic analysis also showed that VT1R and VT2R of the lungfish wereclosely related to those of amphibian. The VT1R mRNA was abundantly expressed in theextrarenal tissues such as gill, lung and heart, moderately or low expressed in the brain,
intestine and kidney. On the other hand, the VT2R mRNA was abundantly expressed in thekidney and heart, weakly expressed in the brain and ventral skin. Expression level of VT2RmRNA was a 100-fold than that of VT1R mRNA in the kidney. When lungfish were kept
under dry condition for above 3 months, estivated lungfish significantly lost body weight,
suggesting that their body fluid were decreased. Plasma osmolality, Na+ and urea
concentrations were significantly elevated relative to those of the lungfish maintained in
freshwater. Furthermore, expression level of AVT mRNA was markedly increased in the
hypothalamus and the changes in the expression level were correlated with significant
increases of plasma osmolality in the lungfish kept in dry condition. Expression levels of
VT1R and VT2R mRNA tended to decrease, but not significant, relative to those of thelungfish maintained in freshwater.
In the present study, we cloned for the first time the lungfish V2-type AVT receptor
although the presence of the receptor has been questioned in fish. These results revealed
that the AVT precursor and its receptors of the lungfish are structurally similar to those of
tetrapods than teleost. Furthermore, the present study showed that the hypothalamic AVT
mRNA expression might be stimulated in response to increase of plasma osmolality and/or
cellular dehydration during estivation. Thus, osmoregulatory function of AVT in lungfish isprobably related to the terrestrial adaptation of the ancestral tetrapods.

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P38
ELECTROPHYSIOLOGICAL ANALYSIS OF TWO TYPES OF CHANNELSACTIVATED BY cGMP IN EPITHELIAL CELLS FROM THE URINARYBLADDER OF JAPANESE TREE FROG, Hyla japonica
Shohei Kitani, Toshiki Yamada, Kouhei Matsuda, and Minoru Uchiyama
Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
Atrial natriuretic peptide (ANP) is probably involved in osmoregulation of amphibian.
In our previous study, it is suggested that frog ANP (fANP) and cGMP stimulateamiloride-blockable Na+ absorption through PKA-dependent pathway in the urinary
bladder of the Japanese tree frog, Hyla japonica, using Ussing-type voltage clamp andwhole-cell patch clamp experiments (1).
In the present study, transepithelial ion transport activated by both fANP and cGMP
was investigated electrophysiologically using a patch-clamp technique in freshly obtained
epithelial cells from the urinary bladder of H. japonica. Na+ transport in the cell was
confirmed to activate by both fANP and cGMP. Na+ channel exhibited a low conductancefor inward currents of 4.8 ± 0.2 pS, long open and closed times (c.a. 200 msec), and apositive reversal potential. The channel activity was decreased under presence of 10-6 M
amiloride in the pipette solution. These characteristics were similar to those of amiloridesensitive epithelial Na+ channel (ENaC). Addition of 10-9 M fANP significantly increased
the ENaC channel activity (NPo) from 0.72 ± 0.22 to 1.88 ± 0.46. On the other hand, mean
amplitude and conductance of single channel did not change significantly after the additionof fANP. Addition of 10-5 M 8-Br-cGMP also significantly increased NPo from 0.56 ±0.10 to 2.00 ± 0.33. The addition of fANP or 8-Br-cGMP failed to activate the ENaC underpresence of 10-6 M amiloride. 8-Br-cGMP also failed to activate the ENaC under presenceof a specific PKA inhibitor, KT-5720. On the other hand, 8-Br-cGMP activated K+ channel
that exhibits a high conductance (more than 20 pS). Addition of 10-5 M 8-Br-cGMP
significantly increased NPo from 0.0007 ± 0.0004 to 0.0228 ± 0.0088. The channel isprobably the cyclic nucleotide-gated (CNG) channel, which is non-selective cation channel.
These results suggested that fANP and cGMP might activate Na+ and K+ transport via
ENaC and CNG channel, respectively, in the epithelial cells of frog urinary bladder.

Reference(1) Yamada et al., (2006) J. Comp. Physiol. B, 176, 203-212.

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P39
EFFECTS OF ANGIOTENSIN II AND ARGININE VASOTOSIN ON WATERABSORPTION RESPONSE IN JAPANESE TREE FROG, Hyla japonica.
Sho Maejima, Norifumi Konno, Kouhei Matsuda, and Minoru Uchiyama
Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
Anuran amphibians do not drink orally but absorb water osmotically across the highpermeable ventral skin. During water absorption frogs display a characterized behavior
termed the water absorption response (WAR). Previous studies demonstrated thatangiotensin II (Ang II) facilitates water absorption in anuran species (1, 2). In dehydrated
toads, plasma Ang II and arginine vasotocin (AVT) concentrations significantly increased
along with rise of the plasma osmolality (3). However, it is less well clarified whatmechanisms are involved in the WAR in anurans. The aim of the present study was toinvestigate effects of Ang II and AVT on the WAR in H. japonica.
Intracerebroventricular (ICV) injection of Ang II (1, 3, 10 pmol/g bw) significantly
increased the duration of WAR in dose-dependent manner. Ang II type-1 receptor (AT1R)
antagonists, Candesartan and Losartan, inhibited the increasing of WAR caused by Ang II.
These results suggest that Ang II promotes WAR through AT1R mediated action. ICV
injection of AVT (5, 10, 20 pmol/g bw) also increased the duration of WAR, and this effect
was prevented by preinjection of mammalian vasopressin type-1 receptor (V1R) antagonist
OPC21268. Thus, AVT on the WAR probably functions via V1-type AVT receptor (VT1R).
In the next experiment, we examined hormonal interrelationships between AVT and Ang II
on the WAR in H. japonica. Preceding injection of Candesartan to AVT inhibited the WAR
caused by AVT. In adverse condition, OPC21268 did not inhibit the effect of Ang II. In thepresent study, both Ang II and AVT are involved in regulation of the WAR in H. japonica.
It is suggested that the WAR induced by AVT may go through angiotensinergic pathways.
References(1) Propper, C.R. and Johnson, W.E. (1994) Horm. Behav., 28, 41-52.
(2) Viborg, A.L. and Rosenkilde. P. (2001) Horm. Behav., 39, 225–231.
(3) Konno et al. (2005) Gen. Comp. Endocrinol., 140, 86-93.

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P40
DIVERSITY OF ARGININE VASOTOCIN-DEPENDENT AQUAPORIN
EXPRESSED IN THE VENTRAL PELVIC SKINS OF THE ANURAN
AMPHIBIANS
Yuji Ogushi1, Gen Akabane2, Masakazu Suzuki2, and Shigeyasu Tanaka1,2
1

Integrated Bioscience Section, Graduate School of Science and Technology, andDepartment of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
2

We have cloned three kinds of arginine vasotocin (AVT)-dependent aquaporins (AQPs)
from the osmoregulatory organs from the tree frog, Hyla japonica. AQP-h2 protein was
expressed in the ventral pelvic skin and urinary bladder, but not in the kidney (1) , whereas
AQP-h3 displayed a specific distribution that was restricted to the ventral pelvic skin (2).
We also revealed that AQP-h2K was predominantly expressed in the apical membrane orcytoplasm of the collecting duct principal cells (3). In response to AVT-stimulation, AQPh2, AQP-h3, and AQP-h2K proteins are translocated to the apical plasma membrane forabsorbing water through the membrane.
The response to antidiuretic hormone tends to be greater in the anuran species normallyoccupying drier habitats than those from wet habitats. Anurans are divided into four groups
according to their habitats, i.e., aquatic, semiterrestrial, terrestrial and arboreal species.
AQP-h2 or AQP-h2-like protein was detected in the urinary bladder of all the species
examined. The AQP-h2 homologue was detected in the pelvic skin of the terrestrial toad,
Bufo japonica, as in the tree-adapted frog, H. japonica, but not in the other species. On theother hand, expression of the AQP-h3-like cDNA was identified in the ventral skin of all
the frogs examined, from aquatic species to terrestrial dwellers, by molecular cloning.
Therefore, AQP-h2 seems to be a urinary bladder-type AQP, while AQP-h3 seems to be aventral pelvic skin-type AQP. It is possible that as anurans evolved into drier terrestrial
environments, the urinary bladder-type AQP might have occurred in the pelvic skin, andresulted in absorbing water together with the ventral pelvic skin-type AQP.
Recently, our phylogenetic analyses of AQP proteins from anurans and mammals foundthat AQP-h2K belongs to the same clusters as mammalian AQP2, while AQP-h2 and
AQP-h3 belong to an anuran-specific cluster (3).
In the present study, we also found that mRNA for the pelvic skin-type AQP (AQP-x3)
in Xenopus is expressed in the pelvic skins, but the protein level is not detectable. The
amino acid sequence of AQP-x3 protein is longer than the other pelvic type AQPs: theprotein has the additional 10 amino acids in the C-terminal. When cRNA of AQP-x3C272stop mutant or AQP-x3-C272S mutant was injected into Xenopus oocytes, AQP-x3
protein was translated and the volume size of Xenopus oocytes increased because theoocytes absorbed water. This finding suggests a possibility that normal AQP-x3 protein isdegradated owing to the extension of the C-terminal 10 amino acid residues.

References(1) Hasegawa, T. et al. (2003) Endocrinology, 144, 4087-4096.
(2) Tanii, H. et al. (2002) J. Membrane Biol., 188, 43-53.
(3) Ogushi, Y. et al. (2007) Endocrinology, 148, 5891-5901.

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P41
CHARACTERIZATION OF AN AQUAPORIN, AQP-H3BL, EXPRESSED IN
MELANOTROPHS IN THE BULLFROG PARS INTERMEDIA
Yuta Komiyama1, Masakazu Suzuki1, Sakae Kikuyama1, and Shigeyasu Tanaka1,2
1

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,

Japan.
2Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka

University, Shizuoka 422-8529, Japan.
Previously we have cloned one cDNA encoding a new aquaporin (AQP) homologous
to mammalian AQP3 from the ventral pelvic skin of the tree frog, termed AQP-h3BL, andgenerated anti-peptide antibody (ST-184) against C-terminal region of the predicted protein(1). Immunolabels using this antibody was observed in the basolateral plasma membrane ofseveral epithelial cells in osmoregulatory organs. We also found that positive labels for
AQP-h3BL were present in melanotrophs of the pars intermedia in the bullfrogs.
We hypothesized that expression of AQP-h3BL protein is regulated by dopamine, andperformed the following experiments. In the present study, we investigated the effects of
the agonist (CB154) and antagonist (pimozide or haloperidol) of dopamine on expressionof AQP-h3BL protein in the melanotrophs. Further, we examined the effect of HgCl2, an
inhibitor of AQP function, to α-MSH synthesis. The pituitary glands of adult bullfrogs
were fixed with periodate-lysine-paraformaldehyde fixative, dehydrated with ethanol series,and embedded in Paraplast. Thin (4 µm) sections were cut and mounted on gelatin-coatedslides. Immunoflurorescence staining was performed essentially as described previously(2) using rabbit anti-Hyla AQP-h3BL serum.
Immunopositive labels for AQP-h3BL protein were obtained in the pars intermedia ofbullfrogs kept in a white background, but not in those of the bullfrogs kept in a black
background. The black-adapted bullfrogs after the agonist-injection displayed white bodycolor with Melanophore Index (MI) 1, and melanotrophs in the pars intermedia werelabeled for AQP-3BL. In additon, after injected with antagonists, white-adapted bullfrogs
showed black color (MI 5), but labels for AQP-3BL for AQP-3BL were still observed in
the melantrophs. Consequently, the expression of AQP-h3BL proteins may not be regulatedby dopamine. Next, when the pars intermedia taken from the white-adapted bullfrogs (MI1) were incubated with a cell medium containing HgCl2 (0.3 mg/ml), the expression ofproopiomelanocortin mRNA was not significantly different from that in the control group,suggesting that the expression of AQP-h3BL gene is not involved in synthesis of α-MSH.
Other factors regulating the synthesis of α-MSH such as serotonin, noradrenalin, and
GABA may be involved in the expression of AQP in the melanotrophs.
References(1) Akabane, G. et al. (2007) Am. J Physiol. Regul. Integr. Comp. Physiol., 292,
R2340-R2351.
(2) Tanaka, S. et al. (1997) J. Histochem. Cytochem., 45, 425-436.

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P42
POSSIBLE HORMONAL REGULATION ON DISSOLUTION OF CALCIUMCARBONATE CRYSTALS IN THE BULLFROG ENDOLYMPHATIC SAC
Munenori Sakimura1, Makoto Ueda1, Masakazu Suzuki1, Hideaki Tomura2, Yuichi
Sasayama3, and Shigeyasu Tanaka1,4
1

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
2
Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512,
Japan.
3
Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa 9270553, Japan.
4Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka
University, Shizuoka 422-8529, Japan.
The amphibian endolymphatic sac (ELS) not only enlarges to form processes aroundthe brain but also extends caudally along the vertebral canal and protrudes between thevertebratae, where it is referred to as the paravertebral lime sac. The lumen of these sacs
contains many tiny crystals consisting of calcium carbonate, which are formed byotoconin-22 (OC22) protein. Previously, we cloned and sequenced a cDNA encoding
bullfrog OC22 and found that calcitonin (CT) regulates the expression of OC22 mRNA in
the ELS, thereby stimulating the formation of calcium crystals in the lumen of the ELS (1).
In the present study, we investigated whether expression of vacuolar H+-ATPase (VATPase) is regulated by any hormones, because the dissolution of the crystals is considered
to be derived from a decrease in pH, adjusted by V-ATPase localized in mitochondria-richcells in the follicular epithelium of the ELS.
To examine this issue, we used bullfrogs kept for one week under a container withwater containing 0.8% CaCl2 for forming the many crystals in the sac. After the treatment,
ultimobranchial gland (UB) or parathyroid gland (PTG) was removed from the bullfrogs,
and injected with CT or parathyroid hormone (PTH) plus vitamin D (Vit. D) for 3
successive days. Twelve hours after the final injection, we collected the ELS andperformed RT-PCR and Western blot analyses to measure OC22 (1) and V-ATPase Esubunit (2) levels.
OC22 mRNA levels decreased after removal of UB, while the levels recovered after the
supplement of CT. We failed to measure the OC22 protein levels, because the protein in the
crystals was eliminated. However, the size of ELS recovered to the same levels as the
sham-operated controls, suggesting OC22 protein was also expressed higher than that in
the UB-removed controls. On the other hand, the removal of PTG also induced a decreasein the mRNA and protein expression of OC22, but a slight increase after supplement with
PTH plus Vit. D, suggesting that expression of OC22 may increase by synergetic
interactions of CT, PTH, and Vit. D. On the other hand, V-ATPase E-subunit mRNA levels
were changed as similar to OC22mRNA, but the protein levels did not show a significant
change. This finding may be related with the turnover of V-ATPase E-subunit protein.
There was a significant difference in V-ATPase mRNA levels between PTH plus Vit. Dinjected and control bullfrogs, suggesting a possibility that subunits other than E-subunit
play a role as a regulator for expression of the V-ATPase function.
References(1) Yaoi, Y. et al. (2003) Endocrinology, 144, 3287-3296.
(2) Yajima, S. et al. (2007) Zol. Sci., 24, 147-157.

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P43
CHANGES IN PROLACTIN GENE EXPRESSION IN AN AMPHIDROMOUSMIGRATORY TELEOST, AYU Plecoglossus altivelis
Takashi Yada1, Shoichiro Yamamoto1, Hiroyuki Sakano1, Kazuo Uchida1, Toshihide
Takasawa2, Nobuhiko Abe3, Kazuhiko Katsura4, and Susumu Hyodo5
1Freshwater Fisheries Research Division, National Research Institute of Fisheries Science,Tochigi 321-1661, Japan.
2
Yamagata Prefecture Inland Fisheries Experiment Station, Yamagata 992-0063, Japan.
3
Yamagata Prefecture Fisheries Experiment Station, Yamagata 997-1204, Japan.
4
Yamagata Prefectural Office, Yamagata 990-8570, Japan.
5
Ocean Research Institute, University of Tokyo, Tokyo 164-8639, Japan.
Prolactin (PRL) is well known as a hyper-osmoregulatory hormone which promotes
adaptation to fresh water but inhibits that to seawater, in a wide range of fish species. Ayu
Plecoglossus altivelis is an annual and amphidromous fish, spending early life in coastal area
over winter and ascending river in spring as juvenile. This fish has been used in anatomical,
genetic, immunological, and physiological studies, repeatedly. Osmoregulatory action of PRL
has been certified using this species as a hypernatremia after transfer from fresh water toseawater (1). To know the role of PRL in this migratory fish, we examined changes in mRNAlevels of PRL during their down- and up-stream migrations in comparison with those of growth
hormone (GH), which is known to be derived from a common ancestral molecule with PRL andpossesses an opposite role in osmoregulation, seawater adaptation in euryhaline fishes.
In October, fries of ayu caught in the shore of Nezugaseki River (Tsuruoka, Yamagata)showed lower whole body contents of PRL mRNA, which were measured by real-time PCR
and standardized by total RNA contents as pico mol/g RNA, than those in the stream. In
contrast, GH mRNA contents increased significantly after the entrance from the river to thecoastal area. In the shore, PRL mRNA maintained at low levels from March to mid April.
Before the start of up-stream migration, the juvenile fish still remained in the coastal area
showed ten-times elevations of PRL mRNA levels in late April and May. After the migrationback to fresh water, mRNA levels of PRL were higher than those in seawater. The fish caught inthe shore in late May and June showed moderate levels of PRL mRNA. During the up-stream
migration, there was no significant change in GH mRNA contents. Results in this study suggest
the osmoregulatory roles of PRL in ayu, inhibiting adaptation to seawater but promoting that to
fresh water. Furthermore, a transient elevation of PRL mRNA levels just before the entrance
into the river implied that PRL gene expression was already stimulated in the coastal area toprepare for the adaptation from seawater to fresh water.

Reference(1) Hasegawa, S. et al. (1986) Gen. Comp. Endocrinol., 63, 309-317.

1

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P44
ELEVATION OF THE PALSMA LEVELS OF INSULIN-LIKE GROWTH
FACTOR-I (IGF-I) TRIGGERS HOMING OF CHUM SALMON OF JAPANESESTOCKS
Hiroshi Katsumata 1, Takashi A. Onuma 1,2, Keita Makino 1, Shunpei Sato 3, Masa-aki
Fukuwaka 4, Tomonori Azumaya 4, Penny Swanson 5, and Akihisa Urano 1
1Section of Biological Science, Faculty of Science, Hokkaido University, Sapporo 0600810, Japan.
2
Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
Fukuoka 812-8581, Japan.
3
National Salmon Resources Center, Sapporo 062-0922, Japan.
4
Hokkaido National Fisheries Research Institute, Kushiro 085-0802, Japan.
5
Northwest Fisheries Science Center, NOAA Fisheries, Seattle, WA 99164, USA.
The previous study in masu salmon (Oncorhynchus masou), which has been used by us
as a model fish in the neuroendocrine research of the molecular mechanisms of spawningmigration, showed that IGF-I is important for the initiation of development of gonads,
including gametogenesis and steroidogenesis, in early spring or rather late winter. Since theinformation from masu salmon provided many useful ideas on the neuroendocrine
mechanisms of growth and sexual maturation, and spawning migration is instinctive
behavior related to reproduction, we expected that IGF-I is also important for the initiationof gonadal development in oceanic chum salmon (O. keta). Further, such information
would help understand when and how transition from growth to sexual maturation, or
puberty, occurs in chum salmon in late winter and early spring, the pre-migratory period.
Chum salmon were sampled in the Gulf of Alaska in February, 2006, and in the Bering
Sea during summer and autumn, 2003. On the basis of histology of gonads, they weredivided into immature and maturing adult fish. The testes of maturing males showedspermatogenesis, whereas ovaries of females deposition of vittelogenin. Plasma wascollected to determine the plasma levels of gonadotropins, IGF-I and steroid hormones,
pituitaries for determination of gonadotropin contents and gene expression, and livers fordetermination of gene expression for IGF-I. Proteinaceous hormones were determined byheterologous coho salmon RIA systems, steroid hormones by EIAs, and gene expression
was assessed by the amounts of mRNAs determined by quantitative real-time PCR assays.
The most important finding in the present study was that, in the Gulf of Alaska in
February, chum salmon was dividable into immature fish and maturing adults even within
the same age groups. In addition, body sizes of maturing adults were larger than those ofimmature fish, indicating those fish whose growth rates are high mature at least one year
earlier than slowly growing fish. Coincidently, the plasma levels of IGF-I in the maturing
fish in the winter Gulf of Alaska and the early summer Bering Sea were significantly
higher than those in the immature fish in the winter Gulf of Alaska and the autumn Bering
Sea. However, changes in the expression of IGF-I gene in the liver did not coincide withthose in the plasma levels of IGF-I, probably because the liver is not a sole source ofplasma IGF-I. Elevation of plasma levels of sex steroid hormones seemed to partiallyreflect initiation of activation of the pituitary-gonadal axis. IGF-I can be involved in thisinitiation of activation, and further decision whether homing or not homing in winter fish.

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P45
GENE EXPRESSION FOR GONADOTROPIN-RELEASING HORMONES INDISCRETE BRAIN LOCI OF PRE-MIGRATORY AND HOMING CHUMSALMON
Takeshi A. Onuma1,2, Aya Jodo2, Hironori Ando1, Masatoshi Ban3, Masa-aki Fukuwaka4,
Tomonori Azumaya4, and Akihisa Urano2
1

Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
Fukuoka 812-8581, 2Section of Biological Sciences, Graduate School of Life Science,
Hokkaido University, Sapporo 060-0810, 3National Salmon Resources Center, Fisheries
Research Agency, Sapporo 062-0922, 4Hokkaido National Fisheries Research Institute,Fisheries Research Agency, Kushiro 085-080.
Neurons producing gonadotropin-releasing hormone (GnRH) are typicalneuroendocrine cells that integrate information and regulate the pituitary-gonadal axis (PGaxis) and reproductive behavior (1). In chum salmon, activity of the PG-axis in terms of theamounts of gonadotropin mRNAs elevated by summer for motivation of spawningmigration from the Bering Sea (2). In the present study, we examined expression of GnRH
genes in discrete brain loci in pre-migratory chum salmon in the Bering Sea.
Chum salmon were caught along the 180o-longitude line in the Bering Sea in summer
and autumn from 2001 through 2003. They were divided into immature fish and maturing
adult I and II on the basis of histological aspects of gonads (2). Brains were frozen in liquid
nitrogen, and cut into 25µm thick serial transverse sections from the olfactory bulb (OB)through the saccus vasculosus with a cryostat. Every tenth sections were Nissl-stained toconfirm cutting region, and other sections were separated into nine loci. Total RNAs wereextracted from the brain sections and reverse transcribed. The amounts of mRNA encoding
a precursor of salmon GnRH (sGnRH-II), which was dominantly synthesized in theforebrain of chum salmon (1), were determined by quantitative real time PCR. Furthermore,
the amounts of mRNA encoding chicken GnRH-II (cGnRH-II) were determined.
The fish in the Bering Sea were mixtures of immature fish and maturing adults from
June to July, while almost all of the fish were immature fish in September (2), indicatingthat the maturing adults left for the natal river by the end of summer. The amounts ofsGnRH-II mRNA in all forebrain loci of the maturing adults were nonetheless not
significantly different from those of immature fish in all years examined. The amounts ofcGnRH-II mRNA in the midbrain tegmentum were also similar between the immature andmaturing adults.
The present results indicated that expression of GnRH gene was not different between
the immature fish and maturing adults in the Bering Sea during summer. We consider that
activation of the PG-axis and the onset of spawning migration occur through interactions ofvarious hormones including GnRH, sex steroid hormones (3) and somatotropic signals
such as insulin-like growth factor-I (4). Future investigation of winter chum salmon in the
Gulf of Alaska should be necessary, because the amounts of sGnRH mRNA elevated fromwinter through early spring in aquacultured-masu salmon (3).
References(1) Onuma, T. et al. (2005) J. Neurobiol., 63, 126-145.
(2) Onuma, T. et al. (2006) Proc. Jpn Comp. Endocrinol., 21, 74.
(3) Ando, H. et al. (2005) Zool. Sci., 22, 379-389.
(4) Ando, H. et al. (2006) Gen. Comp. Endocrinol.,149, 21-29.

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P46
ADULT CHUM SALMON POSSESS FRESH WATER ADAPTABILITY IN THE
SUMMER BERING SEA
Keita Makino1, Masatoshi Ban2, Kentaro Morita3, Shunpei Sato2, and Akihisa Urano1
1

Biosystems Science Course, Graduate School of Life Science, Hokkaido University,
Sapporo 060-0810, Japan.
2
National Salmon Resources Center, Fisheries Research Agency, Sapporo 062-0922, Japan.
3
Hokkaido National Fisheries Research Institute, Fisheries Research Agency, Kushiro 0850802, Japan.
The timing of runs of maturing Pacific salmon into the natal river varies among species
and migration distances in the river. Masu salmon home to their natal river from March toMay, when prolactin gene expression is stimulated prior to upstream migration (1). Since
salmonids are not phylogenetically so distant, high sea salmons are expected to commonly
possess hyperosmoregulatory ability prior to initiation of spawning migration. We therefore
carried out FW-challenge test using adult chum salmon (Oncorhynchus keta) during the2007 summer cruise of R/V Hokko-maru in the Bering Sea (25 June to 9 August).
Fish collected by angling were placed in an outdoor tank (1,000 L) on board, suppliedwith a continuous flow of seawater (SW, 34‰) under natural photoperiod. Running SW
was replaced with FW (6‰) in FW-challenged group after sampling at 0 h, and thereafter acontinuous flow of water was stopped and 200 L of water was renewed every 6 hours.
Blood and gill samples were taken at -6 (initial control), 0, 12 and 24 h after replacementof water to analyze changes in plasma levels of electrolytes, testosterone (T), estradiol-17β(E2) and cortisol, and gill Na+, K+-ATPase activity.
Five out of 7 fish in FW-challenged group survived until 24 h after water replacement.
The plasma levels of electrolytes in the FW-challenged group decreased within 12 h after
the replacement, compared to the initial control and the control SW groups. The plasma
cortisol levels increased in the FW-challenged group and decreased in the control groupduring 12 to 24 h. The plasma T levels transiently increased during 0 to 12 h, followed bydecrease to the initial level by 24 h after the treatment. There were, on the other hand, no
significant changes or differences in the plasma levels of E2 and the gill Na+, K+-ATPase
activity in the control and the FW-challenged groups.
In conclusion, our present results indicate that summer oceanic chum salmon cansurvive for at least 24 h, suggesting that adult chum salmon possess FW adaptability evenin the summer Bering Sea, although it is not clear whether the changes in the circulatinglevels of T and cortisol are involved in FW adaptation of summer oceanic chum salmon.

Reference(1) Bhandari, R. K. et al. (2003) Gen. Comp. Endocrinol., 130, 55-63.

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P47
MEASUREMENT OF MELATONIN AND
INDOLEAMINES IN CYANOBACTERIAMELATONIN-RELATED

Masayuki Takane1, Kazuhito Inoue2, and Atsuhiko Hattori1
Ō

Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental
University, Chiba 272-0827, Japan.
ōDepartment of Biological Sciences, Kanagawa University, Kanagawa 221-8686, Japan.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), an indoleamine that was originallyfound in the vertebrate pineal gland, is synthesized enzymatically from serotonin (5hydroxytryptamine, 5HT) by the sequential action of arylalkylamine N-acetyltransferase(AANAT) and hydroxyindole-o-methyltransferase (HIOMT). It is now widely accepted that,in vertebrates, MEL acts as an endogenous mediator of photoperiodic information and as amolecular component of the circadian time-keeping system.
Many previous studies have shown that MEL is not restricted to vertebrates, and is alsopresent in various arthropods and recently higher plants. However, all of these organisms areeukaryotes. Then, to examine the presence of MEL and the synthetic pathway of MEL in
cyanobacteria of prokaryotes, we used two cyanobacteria: Nostoc punctiforme PCC73102(N.73102) and Synechococcus elongatus PCC7942 (S.7942). In these cyanobacteria, MEL
and MEL-related indoleamines were measured by the reverse-phase HPLC withfluorescence detection. MEL was detected in the culture medium under normal conditions in
N. 73102, on the other hand, 5HT as well as MEL was detected in S.7942. The amount of
MEL in the medium of N. 73102 was much higher than that of S.7942.
To study enzymatic activities of AANAT and HIOMT in N.73102 and S.7942, therespective precursor compounds (5HT or 5-methoxytryptamine (5MTP) for AANAT; Nacetylserotonin for HIOMT) were added into the culture medium. In N.73102 AANAT
activity was detected, while HIOMT activity was undetectable. On the other hand, both
AANAT and HIOMT activities were detected in S.7942. In one hour incubation after
addition of 5MTP, we found that MEL production (NAT activity) increased at 850 foldcompared with the control medium in N. 73102. In S.7942, MEL production (NAT activity)in the medium containing 5MTP was at 10 fold higher than that in the control medium.
These results indicate that cyanobacteria (N.73102 and S.7942) have an ability of MEL
production. Further study needs to elucidate the detail synthetic pathway of MEL in
cyanobacteria.

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P48
ANALYSIS OF OSTEOBLAST SPECIFIC MESSENGER RNA EXPRESSION IN THE
REGENERATING SCALE OF GOLDFISH, Carassius auratus
Ryo Furuya1, Masaki Kobayashi1, Masahisa Nakamura1, Nobuo Suzuki2, and Atsuhiko Hattori3
1

Department of Biology, School of Education, Waseda University, Tokyo 169-0051 Japan.
Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa
University, Ishikawa 927-0553, Japan.
3
Department of Biology, Collage of Liberal Arts and Sciences, Tokyo Medical and Dental
University, Chiba 272-0827, Japan.
2

In most teleosts, it is known that the scale is a better potential internal calcium reservoir thanthe bone during periods of increased calcium demand, such as sexual maturation and starvation.
Furthermore, teleost scale contains osteoblasts and osteoclasts similar to those found in avian
and mammalian bone. We previously reported that osteogenesis in the regenerating scale is very
similar to that of mammalian membrane bone, judging from morphological observations of thescale in goldfish (Carassius auratus). However, basic data concerning mRNAs expression of
the osteoblast-specific genes in the regenerating scale are few.
To investigate changes of osteoblast specific mRNA expression during the scale
regeneration of goldfish, we cloned several major osteoblastic markers and then examined theexpression patterns of these osteoblastic markers using a quantitative real-time PCR. As a result,
we succeeded in cloning of the osteoblastic markers: runt-related transcription factor 2 (Runx 2),
osterix (OSX), type 1 collagen, alkaline phosphatase (ALP) and osteocalcin cDNAs from thenormal scale of goldfish. This is the first report of the full coding sequence of OSX, ALP andosteocalcin molecules in goldfish. Then, the expression of these mRNAs was analyzed in theregenerating goldfish scale. We found that these markers were expressed in the regenerating
scale and that the expression of OSX, type 1 collagen, ALP and osteocalcin mRNAs increased
remarkably during the scale regeneration although Runx 2 mRNA did not change. Taking theseresults into consideration, the obtained osteoblastic markers in the present study should be
useful for the study of osteoblasts in the teleost scale, especially during the scale regeneration.

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P49
ESTABLISHMENT OF EXPERIMENTAL MODEL FOR BONE DISEASE USINGTHE SCALE OF GOLDFISH, Carassius auratus
Thiparpa Thamamongood1, Jun Tabata2, Masahisa Nakamura3, Nobuo Suzuki4, Atsuhiko
Hattori1
1

Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental
University, Chiba 272-0827, Japan.
2
Department of Biostructural Science, Faculty of Dentistry, Tokyo Medical and Dental
University, Tokyo 113-0034, Japan.
3
Department of Biology, School of Education, Waseda University, Tokyo169-0051, Japan.
4
Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa
University, Ishikawa 927-0553, Japan.
Teleost scales are essentially composed of type I collagen and calcium phosphateorganized into hydroxyapatite. Similar to that found in human bone, both osteoblasts andosteoclasts existed on the scale regulate calcium homeostasis in teleosts.
This research focuses on two prospective experimental models of bone disease, i.e.,
autotransplantation of protein-denatured scales fixed by methanol and autotransplantation ofscales with reversal of their polarity (upside down) in goldfish (Carassius auratus).
Autotransplantion of the normal scales was used as a control group. On the fifth, tenth andfifteenth day after autotransplantation, tartrate-resistant acid phosphatase (TRAP) staining
was performed on these scales to examine the differentiation of osteoclasts. Data collectedon the fifth day reveal that osteoclasts appeared on the autotransplanted scales of bothexperimental groups. On the tenth day, in both groups, new regenerated scales were foundadjacent to dermal side of each scale pocket and became larger in their sizes on the fifteenthday. However, protein-denatured autotransplanted scales were continuously resorbed byosteoclasts until the fifteenth day, while the upside-down autotransplanted scales were not.
In the control group, few osteoclasts were found and none of regenerated scales were formed.
Furthermore, using a quantitative real-time PCR the expression of the osteoclast-specificgenes, i.e., nuclear factor of activated T cell c1, TRAP and cathepsin K, was examined in theprotein-denatured scales on the third day after autotransplantation. The expression amountsof three osteoclast-specific genes were significantlyáp<0.001âhigher than those of the
pre-autotransplanted scales.
These results suggest that autotrasplantation of protein-denatured scales is a possiblemodel for osteolytic diseases.

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P50
EFFECT OF TRIBUTYLTIN ON THE CALCIUM METABOLISM IN TELEOSTS
AND ATTEMPT TO BIODEGRADATE BY MARINE BACTERIA
Nobuo Suzuki1, Fumihisa Kobayashi2, Masahiro Matada1, Sei Ito3, Yuji Oshima3, andAtsuhiko Hattori4
1

Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa
University, Ogi, Noto-cho, Ishikawa 927-0553, Japan.
2
Division of Material Engineering, Graduate School of Natural Science and Technology,
Kanazawa University, Kakuma-machi, Kanazawa-city, Ishikawa 920-1192, Japan.
3
Department of Bioscience and Biotechnology, Graduate School of Bioresource andBioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 8128581, Japan.
4
Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental
University, Kohnodai, Ichikawa-city, Chiba 272-0827, Japan.
Tributyltin (TBT) has been extensively used in antifouling paints on ships and fishnets.
This causes widespread contamination of the marine environment. In marine Molluscan, forexample, masculinization (imposex) has been observed on various seashores and adapted as abiomonitoring tool of marine pollution by TBT. In teleosts, as well as in Molluscan, it has
recently been reported that TBT induced sex reversal of genetically female flounder tophenotypic males. As the sex hormone affects bone metabolism and participates inosteoblastic differentiation, we examined the effect of TBT on calcium metabolism in teleosts.
Furthermore, biodegradation of TBT by marine bacteria was studied.
To examine the direct effects of tributyltin acetate (TBTA) on osteoclasts and osteoblasts,
teleost scale, which has both osteoclasts and osteoblasts and is similar to mammalian
membrane bone, was used in the present study. The activities of tartrate-resistant acidphosphatase and alkaline-phosphatase, as respective indicators of activity in osteoclasts andosteoblasts, were used. In freshwater teleosts (goldfish) and marine teleosts (nibbler andwrasse), the osteoclastic activity in the scales did not change as a result of TBTA treatment(10-9 to 10-5 M). On the other hand, the osteoblastic activity decreased in goldfish, nibbler,
and wrasse after 6 hrs of incubation. In goldfish, even 10-10 M of TBTA significantly
inhibited osteoblastic activity. The inhibitory activity in goldfish was stronger than that innibbler and wrasse. Therefore, the mechanism was examined in detail using goldfish. The
mRNA expressions of the estrogen receptor and insulin-like growth factor-I, which
participate in osteoblastic growth and differentiation, decreased in the TBTA-treated scales.
The mRNA expression of metallothionein, a metal-binding protein that protects the organism
from heavy metal, increased much less than those of cadmium and methyl-mercury.
Furthermore, we showed that the plasma calcium and hypocalcemic hormone (calcitonin)level increased in goldfish kept in water containing TBTA (10-10 and 10-8 M). The current
data demonstrate that, in teleosts, TBT inhibits osteoblastic activity without affecting
osteoclastic activity and disrupts the calcium metabolism, including the calcemic hormone, ingoldfish.
In addition, we indicate the possibility that the strains of SK-2 (Pseudoalteromonas sp.)and SK-3 (Pseudoalteromonas sp.) degraded TBT when these bacteria were cultured in aseawater medium containing TBT (10 mg/L), peptone (0.1 %), and yeast extract (0.05 %).
Plans are underway to examine in detail the biodegradation process of TBT by these marine
bacteria and determine their culture conditions.

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P51
DIRECT ROLE FOR GROWTH HORMONE IN REGULATING INSULIN-LIKE
GROWTH FACTOR BINDING PROTEIN 4 (IGFBP4) EXPRESSION IN CHICKEN
Ken-ichi Tahara1, Takeshi Ohkubo2, Minoru Tanaka3,4, Masao Akiba5, Kan Sato5, Noboru Saito1,
Kiyoshi Shimada1, and Akira Tsukada1
1

Laboratory of Animal Physiology Graduate school of Bioagricultural Sciences Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
2
Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan
3
High-tech Research Center, 4Department of Animal Science, Faculty of Applied Life Science,
Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan.
5
Animal Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555,
Japan.

We report the cDNA clone for the growth hormone (GH) dependent chicken insulin-like growth
factor binding protein 4 (IGFBP4). Expression levels of the IGFBP4 mRNA in the chicken tissues
was analyzed by Northern blot analysis. In the chicken IGFBP4, relatively high level expressions
were observed in the liver, heart, lung, and testis. The expression level of IGFBP4 mRNA was
negligible in the liver of sex-linked dwarf chicken, in which the GHR gene is defective because of apoint mutation resulting in the generation of aberrantly spliced GHR mRNA. This result indicatesthat IGFBP4 mRNA is induced by GH in chicken. In addition to this in vivo studies, we employedcultured hepatocyte from chicken to demonstrate a possible direct regulation of IGFBP4 by GH.
Significant effects on IGFBP4 mRNA was detected in GH-cultured hepatocyte. These results indicatethat the expression of IGFBP4 mRNA in the chicken liver is depend on a GH signaling through
GHR.

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P52
A NOVEL PITUITARY TRANSCRIPTION FACTOR, PRX2 WAS ACTIVATED BY
GNRH AND PLAYS SYNERGISTICALLY WITH EGR-1 ON LUTEINIZINGHORMONE BETA SUBUNIT GENE
Akiko Sano1, Takanobu Sato1, 2, Takako Kato3, and Yukio Kato1, 2, 3
1

Division of Life Science, Graduate School of Agriculture, Meiji University, Kanagawa
214-8571, Japan.
2
Department of Life Science, School of Agriculture, Meiji University, Kanagawa 214-8571,
Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571,
Japan.
We are currently attempting the cloning of transcription factors for porcine folliclestimulating hormone β subunit (pFSHβ) gene by the Yeast One-Hybrid System with aporcine anterior pituitary cDNA library using the Fd2 region (-852/-746 b of pFSHβ
promoter) (1) as a bait sequence. And finally, we had cloned paired-related homeobox
factor 2, Prx2. Our recent studies revealed that Prx2 modulates the glycoprotein hormone α
subunit and luteinizing hormone β subunit (LHβ) genes in addition to the FSHβ gene,indicating that Prx2 regulates the synthesis of FSH and LH at the transcriptional level as acommon transcription factor. The present study, by focusing on a synergistic role of Prx2
with Egr-1 (early growth response factor-1) in the LHβ gene expression, and examined
whether Prx2 responses to GnRH signaling.
In the pLHβ promoter, Prx2 binds to three regions composing an AT-rich sequence.
One of the Prx2 binding sites, which is present at -90/-79 b from the transcription start site,
locates between two Egr-1 binding elements (-101/-92 and -49/-41 b; essential elements for
extracellular GnRH-action and are highly conserved in mammalian LHβ promoters) (2).
Co-transfection assay using expression vector of Prx2 and Egr-1 showed synergistic
activation of reporter genes fused with the region containing -1284/+7 by 40-fold, in CHO
cells. Moreover, this activation required binding site of Prx2 or Egr-1.
We further tested whether GnRH could stimulate the endogeneous gonadotropin
subunit, Egr-1 and Prx2 gene expression in LβT2 cells. The expression of all genes tested
significantly increased by GnRH.
In summary, Prx2 plays synergistic activation with Egr-1 and its expression was
regulated by GnRH. It is expected that Prx2 participates in a novel mediator of GnRH
signaling.
References(1) Kato et al. (1999) Mol. Cell. Endocrinol., 158, 69-78.
(2) Tremblay et al. (1999) Mol. Cell. Bio., 19, 2567-2576.

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P53
DNA-BINDING CHARACTERISTICS OF TRANSCRIPTIONEXPRESSING IN THE ANTERIOR PITUITARY GRAND

FACTORS

Michie Nakayama1, Fuyuko Kimoto1, Takako Kato3, and Yukio Kato1,2,3
1Division of Life Science, Graduate School of Agriculture, Meiji University, Kanagawa
214-8571, Japan.
2
Department of Life Science, School of Agriculture, Meiji University, Kanagawa
214-8571, Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571,
Japan.
We have reported that Prop-1, Lhx2, Prx2 are candidates for transcription factors of the
FSHβ gene. These three factors are predicted to play essential roles in the expansion of thepituitary primordium and the differentiation and development of the hormone-producing
cells.
They are also homeobox factors which have homeodmain (HD) for DNA binding. In
vitro DNA binding studies have demonstrated that most HD proteins bind to similar shortconsensus sequences containing a TAAT motif.
In this study, we have employed Systematic Evolution of Ligands by EXponential
enrichment (SELEX) using random N15 oligomers and Electophoretic Mobility Shift
Assay (EMSA) to expand and characterize the DNA binding sequences of these threefactors and another pituitary transcription factor, Hesx1.
We found that Prop-1, Prx2 and Hesx1 which are paired class HD factors bind to
TAATT/AATTA but only strongly Prop-1 binds to DNA by forming cooperative dimer.
Lhx2 is a LIM class HD factor and requires at least 5-6 bp of AT repeated sequence for
DNA binding.
These results indicate that these factors well recognize similar AT-rich sequences, buthave different DNA-binding characteristics from each other.
Further in vivo studies are required to determine these transcription factors’ bindingsites on target genes.

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P54
NEURONATIN
LOCALIZESIN
SECRETORYGRANULEGONADOTROPE AND IS REGULATED BY STEROID HORMONES

OF

Sanae Murakami1, Li-yi Cai1,2, Takao Susa1,2, Shun-ichiro Izumi4 , Takako Kato3 , andYukio Kato1,2,3
1Division of Life Science, Graduate School of Agriculture, Meiji University,
Kanagawa 214-8571, Japan.
2
Department of Life Science, School of Agriculture, Meiji University, Kanagawa 2148571, Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571,
Japan.
4
Department of Obstetrics and Gynecology, Tokai University, School of Medicine,
Boseidai, Isehara, Kanagawa 259-1193, Japan.
Neuronatin is a member of proteolipid protein family and two splicing variants (αand β-form) are expressed in various tissues including the pituitary gland. The functionis yet unclear, but a recent study reported that neuronatin is involved in glucosemediated insulin secretion (1). This study performed the immunohistochemistry andimmunoelectron microscopy to clarify the cellular and subcellular localization of
Neuronatin in the pituitary. In addition, the effect of steroid hormones on theexpression of neuronatin gene was examined by real-time PCR.
Immunohistochemistry showed that neuronatin localizes in cytoplasm of
gonadotropecells.

Immunoelectron

microscopy

demonstrated

that

positive

immunoreactants were only observed in the secretory granule of gonadotrope. Realtime PCR using total RNAs from rat pituitaries showed that the expression of
Neuronatin gene was decreased by castration and was further decreased by theadditional treatment of estrogen. On the other hand, the testosterone treatment after
castration recovered its expression to the level of intact rat.
As described above, this study demonstrated that neuronatin localizes in thesecretory granule of gonadotrope cells. Two steroid hormones regulate this gene
positively (testosterone) and negatively (estrogen), indicating the presence ofregulatory axis between pituitary and gonad.

Reference(1) Chu and Tsai (2005) Diabetes., 54, 1064-1073.

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P55
MOLECULARHORMONEEVOLUTIONOFPREPRO-THYROTROPIN-RELEASING

Yasuhiro Aoki1, Yuichi Saito2 , Min Kyun Park3 , Takashi Yoshimura4 , Genjirou
Nishi5,6 Masayuki Iigo1,2, and Tadashi Yanagisawa1,2
1

Department of Biotechnology, United Graduate School of Agricultural Science, Tokyo
University of Agriculture and Technology, Tokyo 183-8509, Japan.
2
Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya University,
Tochigi 321-8505, Japan.
3
Department of Biological Sciences, Graduate School of Science, The University ofTokyo, Tokyo 113-0033, Japan.
4
Division of Biomodeling, Graduate School of Bioagricultural Sciences, and Institutefor Advanced Research, Nagoya University, Nagoya 464-8601, Japan.
5
School of Marine Science and Technology, and Institute of Oceanic Research andDevelopment, Tokai University, Shizuoka 424-8610, Japan.
6
Marine Science Museum, Tokai University, Shizuoka 424-8620, Japan.
Thyrotropin-releasing hormone (TRH) is derived from a precursor protein, preproTRH (ppTRH) that contains multiple copies of the TRH progenitor sequence (-Lys/
Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg-) and proteolytic cleavage at dibasic residues
yields multiple copies of TRH. It is interesting to note that numbers of TRH progenitor
sequences vary among vertebrates studied to date: chicken, 4; rat and mouse, 5; humanand common carp, 6; Xenopus laevis and zebrafish, 7; sockeye salmon and goldfish, 8.
In the present study, we isolated ppTRH cDNA and characterized their deduced
amino acid sequences from three classes of vertebrate, Chondrichthyes (brownbanded
bambooshark Chiloscyllium punctatum),

Reptilia (leopard gecko Eublepharismacularius) and Aves (zebra finch Taeniopygia guttata). Each ppTRH cDNA encodes adifferent number of TRH progenitor sequences (brownbanded bambooshark, 5; leopard
gecko, 7; zebrafinch, 4). Comparison of deduced amino acid sequences among known
vertebrate ppTRHs suggests that the ancestral ppTRH might have had eight TRH
progenitor sequences. Several of these might be lost during the evolution due tomutation and/or deletion in the TRH-progenitor coding regions of ppTRH.

–57–

P56
AGNATHAN PITUITARY GLAND AND ITS EVOLUTIONARY IMPLICATION
Masumi Nozaki1, Toyokazu Shimotani1, Katsuhisa Uchida1, Shunsuke Moriyama2,
Akiyoshi Takahashi2, Hiroshi Kawauchi2, and Stacia A. Sower 3
1

Sado Marine Biological Station, Faculty of Science, Niigata University, Tassha, Sado,
Niigata, 952-2135, Japan.
2
Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University,
Sanriku, Iwate, 022-0101, Japan.
3
Department of Biochemistry and Molecular Biology, University of New Hampshire,
Durham, New Hampshire 03824, USA.
Lampreys and hagfish are particularly important animals to investigate the origin and theevolution of pituitary hormones, because they are only two extant representatives of theoldest class of vertebrates, Agnatha, arose about 530 million years ago. In this poster
presentation, current knowledge on the agnathan adenohypophysial hormones is outlined.
The pituitary gland of lampreys exhibits more advanced features than that of hagfish, andresembles to that of gnathostome vertebrates. To date, four adenohypophysial hormones
have been identified or cloned from the pituitary gland of sea lampreys. They are
adrenocorticotropin (ACTH), melanotropins (MSHs), growth hormone (GH) andgonadotropin (GTH) (1-2). The topographic distribution of these four hormones within thelamprey pituitary gland is also comparable to that of gnathostome vertebrates (3). On theother hand, the pituitary gland of the hagfish exhibits very primitive features and is notwell developed. It remains an enigma whether the hagfish pituitary gland contains tropic
hormones of any kind. However, our recent study has revealed that at least GTH and
ACTH are present in the hagfish pituitary gland (4-6). Namely, we have cloned cDNAs ofboth α- and β-subunits of GTH from the pituitary gland of brown hagfish, Paramyxine
atami (6). Morphological exploration demonstrated that both GTH α- and β-subunits werecompletely synthesized in the same cells of the hagfish adenohypophysis (6). ACTH-likecells are also demonstrable in the pituitary gland of the brown hagfish. They were stainedwith anti-lamprey ACTH as well as several lectins (5). In the hagfish, GTH-like and
ACTH-like cells are packed together in the same cell cluster, and thus each cell cluster
appears to serve as a separate functional unit. These conditions of the hagfish pituitary
gland seem to represent a primitive state before the differentiation of the pars intermedia(5). Finally, GTH and ACTH appear to be the original adenohypophysial hormones thatappeared first during the evolution of the earliest vertebrates.
Rerefences(1) Kawauchi and Sower (2006) Gen. Comp. Endocrinol., 148, 3-14.
(2) Sower et al. (2006) Gen. Comp. Endocrinol., 148, 22-32.
(3) Nozaki et al. (2007) Gen. Comp. Endocrinol., In press(4) Miki et al. (2006) Gen. Comp. Endocrinol., 148, 15-21.
(5) Nozaki et al. (2007) Cell Tissue Res., 328, 563-572.
(6) Nozaki et al. (unpublished, see Honda et al. Proc. Jap. Soc. Comp. Endocrinol., 21, pp.
52, 2006).

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P57
EFFECTSOFPITUITARYADENYLATECYCLASE-ACTIVATINGPOLYPEPTIDE (PACAP) AND MELANIN-CONCENTRATING HORMONE(MCH) ON SOMATOLACTIN (SL) RELEASE FROM THE GOLDFISHPITUITARY IN VITRO
Mio Tanaka1, Morio Azuma1, Yumika Nejigaki1, Minoru Uchiyama1, Akiyoshi
Takahashi2, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
2
Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato
University, Iwate 022-0101, Japan.
In the teleost pituitary, pituitary adenylate cyclase-activating polypeptide (PACAP)
plays a role in mediating prolactin, growth hormone and gonadotropin release (1-3), andmelanin-concentrating hormone (MCH) influences secretion of α-melanocyte stimulating
hormone (4). In the present study, we examined the immunohistochemical relationshipbetween PACAP and MCH nerve fibers and somatolactin (SL)-producing cells in the
goldfish pituitary. Nerve fibers with PACAP- or MCH-like immunoreactivity wereidentified throughout the neurohypophysis, with their nerve endings also identified in theregions of the adenohypophysis, especially the pars intermedia that SL-producing cells
exist. We also examined the effects of PACAP and MCH on SL release in goldfish
pituitary using a cell immunoblot assay. This method allowed for the semiquantitative
measurement of SL release from the cells. Incubation of the cultured pituitary cells for 3 hwith graded concentrations of PACAP at doses of 10-9-10-7 M provoked a dose-dependent
increase in the immunoblot area for SL-like immunoreactivity (SL-LI). Treatment of
pituitary cells with 10-8 and 10-7 M PACAP induced a significant increase in the
immunoblot area for SL-LI. On the other hand, incubation of the cultured pituitary cells for
3 h with graded concentrations of MCH at doses of 10-11-10-7 M provoked a dosedependent decrease in the immunoblot area for SL-LI. These results suggest that PACAP
and MCH can potentially function as a hypophysiotropic factor mediating SL release in the
goldfish pituitary.
References(1)Matsuda et al. (2007)Regul. Pept., in press.
(2)Wong et al. (1998) Endcrinology, 139, 3465-3479.
(3)Chang et al. (2001) J. Neuroendcrinol., 13, 540-550.
(4)Gröneveld et al. (1995) Peptides, 16, 945-949.

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P58
MOLECULAR CLONING AND CHARACTERIZATION OF cDNA ENCODINGPITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE FROM THE
BRAIN OF FAR EASTERN BROOK LAMPREY
Koutaro Ishiguro, Keisuke Maruyama, Yuji Yamazaki, Minoru Uchiyama, and Kouhei
MatsudaLaboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
Pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormonereleasing hormone (GHRH) are encoded by separate genes in mammals: PACAP is
encoded with PACAP-related peptide (PRP), whereas GHRH and cryptic peptide arepresent in the same transcript. In non-mammalian vertebrates and protochordates, PACAP
and GHRH have been believed to be encoded by the same gene and hence processed fromthe same transcript (1). Recently, genes encoding authentic GHRH were isolated andcharacterized from the zebrafish, goldfish and African clawed frog (2), suggesting that
PRP-PACAP gene structure is conserved from teleosts to mammals. However, PACAP
gene structure in the agnathans has not yet been examined. In this study, we isolated the
PACAP cDNA from the brain of Far Eastern brook lamprey (Lampetra reissneri) to
examine its structural characterization using RT-PCR method. PACAP and another peptide
were deduced from the isolated PACAP cDNA. The primary structure of PACAP shareshigh sequence identity among known primary structures of PACAP in vertebrates andprotochordate. Another peptide shares much lower level of sequence identity with known
PRPs and GHRHs. These results suggest that in Far Eastern brook lamprey, PACAP cDNA
also encodes another peptide.

References(1) Sherwood et al. (2000) Endocr. Rev., 21, 619-670.
(2) Lee et al. (2007) Proc. Natl. Acad. Sci. USA, 104, 2133-2138.

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P59
CHANGES OF CORTICOTROPIN-RELEASING FACTOR EXPRESSION IN THE
BRAIN OF THE BULLFROG, Rana catesbeiana DURING METAMORPHOSIS
Kazumasa Hashimoto1, Reiko Okada2,3, Hiroshi Mochida4, Mimoru Uchiyama1, Sakae
Kikuyama2,4, and Kouhei Matsuda1
1

Laboratory of Regulatory Biology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan.
2
Department of Biology, School of Education, Waseda University, Tokyo 169-8050,
Japan.
3
Depariment of Regulation Biology, Faculty of Science, Saitama University, Saitama
338-8570, Japan.
4
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
In submammalian vertebrates, corticotropin-releasing factor (CRF) acts as a potent
stimulator of thyrotropin release. During metamorphosis in amphibians, plasma thyroidal
and adrenocortical hormone levels are known to elevate almost simultaneously. Since
adrenal corticoids potentiate the action of thyroid hormone, the increase of both hormone
levels has been considered to facilitate metamorphic changes. To examine whether CRF is
involved in the synchronized elevation of these two hormones through the enhancement of
the release of thyrotropin and corticotropin, changes of CRF mRNA expression level and
CRF-like immunoreactivity in the brain of the bullfrog larvae during metamorphosis werestudied. While CRF mRNA levels in the whole brain of bullfrog were already high at
premetamorphic stage, the expression of CRF mRNA in the diencephalon was same levels
during the premetamorphic and metamorphic stages and it decreased at the end ofmetamorphosis. CRF-like immunoreactive neuronal cell bodies were observed in themecencephalic (thalamic) regions invariably throughout the larval development. CRF-like
immunoreactive cells were also found in the hypothalamus. The number of CRF-like
immunoreactive cells in hypothalamus, but not in mecencepphalic region, showed asignificant increase as metamorphosis progresses, and it reached a peak at metamorphic
climax. The immunoreactive nerve fibers were observed mainly in the median eminence
region and it became abundant as metamorphosis proceeds. The number of CRF-like
immunoreactive cells in the hypothalamus as well as the density of immunoreactive fibers
in the median eminence decreased simultaneously after metamorphosis was completed.
These results suggest the involvement of CRF which was synthesized in the hypothalamus
in the progression of metamorphosis.

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P60
OPPOSITE MYOACTIVITY OF NOVEL NEUROPEPTIDESENCODED IN THE SAME PREPROHORMONE GENE IN Hydra(FRamides)

1

Toshio Takahashi, 2Eisuke Hayakawa, 2Chiemi Nishimiya-Fujisawa, and 2Toshitaka
Fujisawa
1

Suntory Institute for Bioorganic Research, Shimamoto-cho, Osaka 618-8503, Japan.
Department of Developmental Genetics, National Institute of Genetics, Mishima,
Shizuoka 411-8540, Japan.
2

In the course of systematic identification of peptide signaling molecules combined with
EST database from Hydra, we have identified a novel neuropeptide family that consists of
two members with FRamide at the C-terminus; FRamide-1 (IPTGTLIFRamide) andFRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained asingle copy each of FRamide-1 and FRamide-2 precursor sequences. Expression analysis
by whole-mount in situ hybridization showed that the gene was expressed in a
subpopulation of neurons that were distributed throughout the body from tentacles to basal
disk. Double in situ hybridization analysis showed that the expressing cell population was
further subdivided into one population consisting of neurons expressing both the FRamide
and Hym-176 (neuropeptide) genes and the other consisting of neurons expressing only the
FRamide gene. FRamide-1 evoked elongation of the body column of epithelial Hydra thatis composed of epithelial cells and gland cells but lacking all the cells in the interstitialstem cell lineage, including neurons. In contrast, FRamide-2 evoked body column
contraction. The structure-activity relationship analysis showed that mature structures of
FRamide-1 (9mer) and FRamide-2 (8mer) are important for their myoactivities. These
results suggest that both of the neuropeptides directly act on epithelial cells as
neurotransmitters and regulate body movement in an axial direction.

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P61
EVOLUTIONARY RELATIONSHIP OF LPXRFa PEPTIDES AND PQRFa
PEPTIDES - INSIGHTS FROM NOVEL CARTILAGINOUS FISH RF-AMIDEPEPTIDES
Tomohiro Osugi1,2, Susumu Hyodo3, and Kazuyoshi Tsutsui1
1

Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan.
2
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima
University, Higashi-Hiroshima 739-8521, Japan.
3
Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Tokyo 1648639, Japan.
In the RFamide peptide family, LPXRFa (X = L or Q) peptides act as hypophysiotropic
hormones (1, 2). On the other hand, PQRFa peptides are known to act as neuromodulatorsof the opioid system (3). Interestingly, LPXRFa peptides and PQRFa peptides share similar
C-terminal structures: LPXRFa peptides have C-terminal Leu-Pro-Leu/Gln-Arg-Phe-NH2
motif and PQRFa peptides have C-terminal Pro-Gln-Arg-Phe-NH2 motif. Furthermore,their receptors showed high identity at the seven transmembrane domain (about 70%) (4,5). These observations suggest that LPXRFa peptides and PQRFa peptides may havediverged from a common ancestral gene. LPXRFa peptides and PQRFa peptides arepresent in the brain of teleosts and tetrapods. Our recent study demonstrated that at least
PQRFa peptides are present in the brains of agnathans, the most ancient vertebrates (6). On
the other hand, there is no evidence about the presence of neither LPXRFa peptides nor
PQRFa peptides in cartilaginous fish. Because they serve as a critical outgroup incomparisons of tetrapods and teleosts, this study aimed to identify genes encoding
LPXRFa peptides and PQRFa peptides in the brain of cartilaginous fish. We also
demonstrated genome database analyses and provided some insights into the evolutionary
relationship of LPXRFa peptide group and PQRFa peptide group.

References(1) Tsutsui, K. et al. (2000) Biochem. Biophys. Res. Commun., 275, 661-667.
(2) Tsutsui, K. and Ukena, K. (2006) Peptides, 27, 1121-1129 (review).
(3) Yang, H-Y. et al. (1985) Proc. Natl. Acad. Sci. USA, 82, 7757-7761.
(4) Yin, H. et al. (2005) J. Endocrinol., 184, 257-266.
(5) Ikemoto, T. and Park, M.K. (2005) J. Reprod. Dev., 51, 359-377.
(6) Osugi, T. et al. (2006) FEBS J., 273, 1731-1743.

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P62
PHYSIOLOGICAL EFFECTS OF PROLACTIN-RELEASING PEPTIDE (PrRP)
ON BLOOD PRESSURE AND IMMUNE REACTION
Akikazu Mochiduki, Tomo Yamada, Takuya Takeda, and Kinji Inoue
Depatment of Regulation Biology, Graduate School of Science and Engineering, Saitama
University, 255 Shimo-ohkubo, Sakuraku, Saitama 338-8570, Japan.
Prolactin-releasing peptide (PrRP) is an endogenous ligand of an orphan G-protein
coupled receptor, hGR3/GPR10 and its immunoreactive cells are found to exist in
dorsomedial hypothalamus (DMH), ventrolateral reticular nucleus (VLRN) and nucleus
tractus solitarius (NTS) in the brain, and in the peripheral tissues of adrenal gland. PrRP isalready known to have some functions like stress response, food intake regulation andprolactin secretion. Here, we would like to focus on its implication in the cardiovascularsystem as well as cell-mediated immunity as followed.
1), PrRP has already been reported to increase blood pressure. However, the mechanism
remains to be defined. Therefore, we set out to study this aspect of PrRP. PrRP icv injection
increases c-Fos expression in paraventricular nucleus (PVN) and locus coeruleus (LC), andwe found c-Fos expressing cells in LC were noradrenalin cells, a good candidate forincreasing blood pressure. This result shows PrRP acts on blood pressure throughnoradrenalin cells. Furthermore, AVP (arginine vasopressin) antagonist blocked AVPinduced increase in blood pressure and heart rate, but had no effect on PrRP-induced one.
This means PrRP is not related to AVP in increasing blood pressure and heart rate.
2), PrRP is known to activate HPA axis and glucocorticoid level increase thereby. This
glucocorticoid is known to have anti-inflammatory effect in the body. Therefore, we
examined the effect of PrRP on cellular immunity. We induced allergic contact dermatitis(ACD) by oxazorone administration. In consequence, PrRP knock out mice (PrRP-KO)
showed more ear swelling than wild type (WT) mice. This indicates PrRP suppressed
inflammation through controlling cellular immunity.

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P63
STRUCTURES AND DISTRIBUTION OF PROENKEPHALIN mRNAS IN
BANDED HOUNDSHARK Triakis scyllium
Akiyoshi Takahashi1, Ryo Goto1, Yuki Kobayashi1, Shunsuke Moriyama1, and Susumu
Hyodo2
1
2

School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.
Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639, Japan.

Met-enkephalin (Met-ENK), Leu-ENK, and β-endorphin (β-END) are opioid peptides
exhibiting analgesic effects. Met-ENK, Leu-ENK, and β-END are derived from theprecursor proteins proenkephalin (proENK), prodynorphin (proDYN), andproopiomelanocortin (POMC), respectively. These opioid peptides are suggested to have acommon ancestry. We cloned POMC cDNA from the banded houndshark Triakis scyllium
as a first step to elucidate the biological significance of opioid peptides in fish (1). Here,
we undertook the molecular cloning of proENK and proDYN cDNA and examined the
distribution of opioid peptide mRNAs in hypothalamus-pituitary systems.
Tissues were taken from Triakis captured off Misaki and kept for several weeks at the
Ocean Research Institute, University of Tokyo. ProENK cDNA was amplified from brain
cDNA with the polymerase chain reaction (PCR). ProDYN cDNA was cloned from a
pituitary cDNA library prepared using the pGCAP10 vector or amplified from brain cDNA
via PCR. PCR products were subcloned into the pT7 Blue-T vector. Nucleotide
sequences were determined using a 3100-Avant Genetic Analyzer.
Triakis proENK cDNA was composed of 1150 bp excluding the poly A tail.
PreproENK was deduced to comprise 265 aa, in which seven Met-ENKs were contained.
The location of these Met-ENKs in Triakis proENK was similar to those in the galeoid
shark, lungfish, and Amia. Triakis proDYN cDNA was composed of 1146 bp excluding thepoly A tail. PreproENK was duduced to be 257 aa long, in which two Leu-ENK, two MetENK, and one Leu-ENK-like sequence were contained. The location of these hormonal
segments in proDYN was similar to those in lungfish, bichir, eel, zebrafish, and tilapia.
Reverse transcription PCR showed the expression of proENK in the hypothalamus,
whereas the expression of proDYN was observed in the hypothalamus and pituitary. In the
Triakis brain, both proENK and proDYN may serve as precursors for opioid peptides in thecentral nervous system as in other vertebrates. In addition, proDYN seems to provide
ENKs as pituitary hormones for peripheral roles. Thus, the Triakis pituitary possesses adual opioid system consisting of proDYN and POMC.
Reference(1) Takahashi, A. et al. (2006). Proc. Japan Soc. Comp. Endocrinol. No. 21, p. 39.

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P64
THE CIRCADIAN REGULATION OF IN VITRO MELATONIN RELEASE FROM
THE FISH PINEAL ORGAN: A COMPARATIVE STUDY
Tomotaka Abe1,2, Tomohiro Masuda3, Kanta Mizusawa4, Shiho Fujita2, Shoji Kitamura5,
Teruo Azuma6, Yasuaki Takagi7, Tadashi Yanagisawa1,2, and Masayuki Iigo1,2
1

United Graduate School of Agricultural Science, Tokyo University of Agriculture andTechnology, Tokyo 183-8509, Japan.
2
Graduate School of Agriculture, Utsunomiya University, Tochigi 321-8505, Japan.
3
Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
4
School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
5Japan International Research Center for Agricultural Sciences, Ibaraki 305-8686, Japan.
6
National Research Institute of Fisheries Science, Tochigi 321-1661, Japan.
7
Graduate School of Fisheries Sciences, Hokkaido University, Hokkaido 0441-8611, Japan.
In many fishes, the photoreceptive pineal organ harbors the circadian clock that
regulates melatonin release in the pineal organ itself. However, the pineal organ of somesalmonids did not exhibit circadian rhythms in melatonin release when maintained underconstant darkness (DD) in vitro, suggesting that the pineal organs of all salmonids lack thecircadian regulation of melatonin production. To test this hypothesis, the pineal organ ofnine salmonids (Coregonus lavaretus, Thymallus thymallus, Hucho perryi, Salvelius
leucomaenis, Salvelius fontinalis, Oncorhynchus mykiss, O. keta, O. nerka, and Salmo
trutta), closely related osmerids (Plecoglossus altivelis and Hypomesus nipponensis) andtwo model teleosts (medaka Oryzias latipes and torafugu Takifugu rubripes) wereindividually maintained in flow-through culture under several light conditions.
Under light-dark cycles, the pineal organ of all species showed a rhythmic melatoninrelease with high rates during the dark phase. Under DD, pineal organs of osmerids,
medaka and torafugu exhibited circadian rhythms in melatonin release with high rates
during the subjective-night but the salmonid pineal organs constantly released melatonin athigh rates. Under constant light, melatonin release was suppressed in all species. Thus,
melatonin release from the pineal organ of osmerids, medaka and torafugu is regulated byboth light and circadian clock but the circadian regulation is lacking in salmonids.
These results indicate that ancestral salmonids lost the circadian regulation of melatonin
production during the evolution after the divergence from the other fish species.

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P65
CONVENIENT METHOD FOR PREPARATION OF BIOLOGICALLY ACTIVE
RECOMBINANT CHH OF THE KURUMA PRAWN, Marsupenaeus japonicus,
USING THE BACTERIAL EXPRESSION SYSTEM
Chiaki Nagai, Hideaki Asazuma, Shinji Nagata, and Hiromichi Nagasawa
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Crustacean hyperglycemic hormone (CHH) is a neuropeptide synthesized by andsecreted from the X-organ/sinus gland complex in medulla terminalis of the eyestalk incrustaceans. CHH exerts several biological functions, involving glucose metabolism,
reproduction, molting, lipid metabolism, and stress response (1). CHHs and structurally
related peptides comprise a peptide family, CHH-family. CHH-family peptides have beenfound only in arthropods so far. Among six CHHs characterized in Marsupenaeus
japonicus (Penaeus japonicus-sinus gland peptide [Pej-SGP] -I-III, V-VII) (2), an
expression system for recombinant Pej-SGP-VII (rPej-SGP-VII) has not yet been
established. Therefore, we groped for a convenient preparation method of biologically
active rPej-SGP-VII.
Here, we constructed an expression plasmid containing Pej-SGP-VII cDNA, by whichrPej-SGP-VII was expressed in Escherichia coli as a fusion protein with N-terminal Histag and Nus-tag. Through affinity-purification by a Ni-column, removal of N-terminal
tags, and amidation at C-terminus, rPej-SGP-VII was obtained. Analysis of arrangementof intramolecular disulfide bonds revealed that the structure of rPej-SGP-VII was identicalwith native Pej-SGP-VII. Comparison of the CD spectrum of rPej-SGP-VII with that ofnative Pej-SGP-VII showed that they were almost identical. In addition, an in vivo assay
confirmed that rPej-SGP-VII had authentic hyperglycemic activity. These data indicatethat rPej-SGP-VII was successfully synthesized. Furthermore, this expression system forrPej-SGP-VII may be applicable to preparation of the other recombinant CHH-family
peptides.
References(1) Chan, S.M. et al. (2003) Gen. Comp. Endocrinol., 134, 214-219.
(2) Nagasawa, H. et al. (1999) Peptide Science-Present and Future (Shimonishi, Y. ed.),453-454.

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P66
INSULIN SECRETION ABILITY OF BARFIN FLOUNDER, Verasper moseri
Tadashi Andoh
Hokkaido National Fisheries Research Institute, Fisheries Research Agency, 116
Katsurakoi, Kushiro 085-0802, Japan.
Like in mammals, feeding induces insulin secretion in fish, but unlike in mammals,amino acids in fish are stronger insulinotropins than glucose (1). This suggests that theinsulin secretion system in β-cells is different between fish and mammals. Plasma insulinlevel after feeding is related to neither the mRNA level of the insulin gene nor to theconcentration of total insulin in principal islets (PIs) (2). The present study assessed thequantitative ability of insulin secretion to confirm the functional importance of thesecretion stage in the regulation of insulin secretion compared to the transcriptional andtranslational stages of insulin production in fish.
PIs from flounders kept under various conditions were weighed and the concentrationsof insulin in the PIs were measured using a time-resolved fluoroimmunoassay (TR-FIA)for barfin flounder insulin. Plasma insulin levels at various times after feeding in flounders
fed fish or artificial feed were also measured using the TR-FIA. The half-life of barfin
flounder insulin in plasma was measured using alloxan-treated barfin flounders injected
intramuscularly with barfin flounder insulin.
The weight of PI was 76.3±23.8 mg/kgBW. Insulin concentration in PIs varied from
0.94±0.13 to 1.04±0.13 µg/mg. These results indicated that the amount of insulin in PIs
of barfin flounder was about 80 µg/kgBW. The maximum plasma insulin level in flounders
was 110 ng/ml. Plasma volume was thought to be less than 20 ml/kgBW in flounder;
suggesting that the maximum insulin amount in plasma was less than 2.2 µg/kgBW.
Therefore, barfin flounder stores insulin in PIs at over thirty-times higher than the
maximum plasma level. The half-life of insulin in plasma was 4.1±0.4 hrs, which istwenty-times longer than that of mammals. The results in this study suggested theoverwhelming dominancy of the secretion stage in the regulation of plasma insulin levels
compared with transcriptional and translational stages in the insulin production system offlounder.
References(1) Andoh, T. (2007) Gen. Comp. Endocrinol., 151, 308-317.
(2) Andoh, T. (2003) Proc. Japan. Soc. Comp. Endocrinol., 18, 46.

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P67
HISTOLOGICAL ANALYSIS OF THE DISTRIBUTION OF CLUSTERIN IN THE
RAINBOW TROUT
Ryoji Fukui1, Takashi Nakakura2, Shigeyasu Tanaka2, and Masakazu Suzuki1
1

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
2Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka
University, Shizuoka 422-8529, Japan.
In the rainbow trout (Oncorhynchus mykiss), the ultimobranchial gland secretes
calcitonin (CT), which is involved in calcium metabolism. We have been studying thisendocrine organ as a model system to understand molecular mechanisms for activating aparticular gene and producing a peptide hormone abundantly. In the present study, we havecharacterized mRNAs expressed in the trout ultimobranchial gland by suppressionsubtractive hybridization. Sequence analysis of 110 cDNA fragments indicated theexistence of three types of keratins, Is (S8), IIe (E3), and IIs (S2), and ictacalcin, acalcium-binding protein reported to be expressed in the epidermis in mammals, suggestingthat the CT cell may have characteristics of the epithelial cell. Cytoskeletal protein 4.1G
was also identified. Furthermore, we found a secretory glycoprotein, clusterin, which is
implicated in various physiological processes in other animals. RT-PCR analysis showed
an amplified band for clusterin cDNA in each of the tissues examined in the rainbow trout,such as the ultimobranchial gland, thyroid, brain, pituitary, and intestine. In situ
hybridization histochemistry localized strong expression of clusterin gene in theultimobranchial gland, with the highest level in the sub-basal cytoplasm of theparenchymal cells located at the outermost region. Immunofluorescence staining with ananti-clusterin antibody revealed positive labels not only in the ultimobranchial gland, butalso in the thyroid gland, gills, and intestine. At the cellular level, intense immunoreativity
was detected at parenchymal cells in the ultimobranchial gland. Although immunopositive
labels were observed at the follicular epithelial cells of the thyroid, at goblet cells in theintestine, and at mucous cells in the gills, clusterin mRNA has not yet been detected inthese cells by in situ hybridization. The results suggest that the ultimobranchial gland is amajor site of clusterin production. More study is needed to clarify the production sites ofclusterin and to elucidate the physiological role of clusterin in the ultimobranchial gland.

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P68
PARAFIBROMIN TUMOR SUPPRESSOR HAS ONCOGENIC PROPERTY IN
THE CELLS EXPRESSING SV40 LARGE T ANTIGEN
Takeo Iwata1, Noriko Mizusawa1, Yutaka Taketani2, Mitsuo Itakura3, and Katsuhiko
Yoshimoto1
1

Department of Medical Pharmacology, Institute of Health Biosciences, The University ofTokushima Graduate School, Tokushima, Japan.
2
Department of Clinical Nutrition, Institute of Health Biosciences, The University ofTokushima Graduate School, Tokushima, Japan.
3
Division of Genetic Information, Institute for Genome Research, The University ofTokushima, Tokushima, Japan.
Primary hyperparathyroidism usually results from a single parathyroid adenoma, but ina minority of cases is part of hereditary syndromes, namely multiple endocrine neoplasia
types 1 and 2A, familial isolated hyperparathyroidism, and hyperparathyroidism-jaw tumor(HPT-JT) syndrome. HPT-JT syndrome is characterized by parathyroid tumors, fibroosseous lesions of the mandible and maxilla, and renal cysts and tumors. The gene whose
inactivation is directly associated with the pathogenesis of HPT-JT syndrome has been
identified as the tumor suppressor gene HRPT2 (1). Parafibromin is a 531-amino acidprotein encoded by HRPT2, a putative tumor suppressor gene recently implicated in theautosomal dominant hyperparathyroidism-jaw tumor familial cancer syndrome andsporadic parathyroid carcinoma. To investigate effects of parafibromin's overexpression oncell proliferation, we performed assays in four different cell lines. The transient
overexpression of parafibromin inhibited cell growth in HEK293 and NIH3T3 cells, butenhanced cell growth in the SV40 large T antigen expressing-cell lines such as 293FT and
COS7 cells. In 293FT cells, parafibromin was found to interact with SV40 large T antigen
and its overexpression promoted entry into the S phase, implying that the interaction
enhanced progression through the cell cycle. The tumor suppressor protein parafibromin
acts as a positive regulator of cell growth like an oncoprotein in the presence of SV40 large
T antigen.
Reference(1) Carpten, J.D. et al. (2002) Nat. Genet. 32, 676-680.

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P69
VISUALIZING EXOCYTOSIS IN RAT ANTERIOR PITUITARY CELL CLUSTERUSING TWO-PHOTON EXTRACELLULAR POLAR TRACER (TEP) METHOD
Junko Yasufuku-Takano1, Koji Takano1, Noriko Takahashi2, Haruo Kasai2, and Toshiro
Fujita1
1

Department of Nephrology and Endocrinology, University of Tokyo Faculty of Medicine,
Bunkyo-ku, Tokyo 113-8655, Japan.
2

Division of Biophysics, Center for Disease Biology and Integrative Medicine, Graduate
School of Medicine, the University of Tokyo, Tokyo 113-0033, Japan.
Exocytosis is the final step in peptide hormone secretion. Although this step is crucial
in understanding the regulation of hormone secretion, it has been difficult to observe
exocytosis in intact cells. TEP method (1,2) that combines post-fusion labeling for staining
secretory granules and two-photon fluorescence microscopy for detection enables
visualizing individual exocytotic events in intact cells by obviating cell manipulation
including transfection. Single exocytotic event appears as a bright spot near the plasma
membrane by this method. This spot represents the entry of extracellular fluorescence
into granule through fusion pore of granule membrane and plasma membrane.
Pituitaries from male Wistar rats (8-10 week-old) were dispersed into cell clusters
by collagenase treatment and mild pipetting. Normal human pituitary tissue was obtainedwith patient’s consent during surgery for Rathke’s cleft cyst. This normal tissue had to be
removed to access the lesion. Human prolactin-secreting pituitary adenomas were obtainedwith patient’ consent who underwent surgical treatment. These cell clusters were immersed
in 0.5mM sulphorohdamine B for TEP. Rat pituitary cells had no spontaneous exocytosis at
6 hours after dissociation. GHRH (10-7M) induced exocytosis in some cells. The secretory
granules full-fused rapidly (few seconds). Most exocytosis occurred abvascularly rather
than advascularly. These findings were similar to those in human normal pituitary clusters.
LHRH (10-7M) induced exocytosis in some cells in rat cell clusters. Full-fusion and kissand-stay mode were observed. Some human prolactin-secreting adenomas showedspontaneous exocytosis that was mostly kiss-and-stay type. It appears that each anterior
pituitary cell type has peculiar pattern in terms of the mode of exocytosis.

References(1) Nemoto, T. et al. (2001) Nat. Cell Biol. 3, 253–258.
(2) Takahashi, N. et al. (2007) Endocr. J. 54, 337-346.
Key words: exocytosis, pituitary cells, rat, human

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P70
OBSERVATION OF THE GAP JUNCTION AND LHRH PROJECTION ON
FOLLICULO-STELLATE CELLS IN THE PARS TUBERALIS OF A RATPITUITARY GLAND BY CONFOCAL LASER MICROSCOPY
Kotaro Horiguchi1, Ken Fujiwara1, Motoshi Kikuchi1, Takashi Yashiro1, and Tsuyoshi Soji2
1
2

Department of Anatomy, Jichi Medical University School of Medicine, Tochigi, Japan.
Functional Morphology, Nagoya City University Medical Science, Aichi, Japan.
Luteinizing hormone-releasing hormone (LHRH) stimulates gonadotrophs via thehypophyseal portal vein system. Recently, Soji et al. (2004) have been proposing anotherpathway in which a network system of folliculo-stellate cells (FS cells) that are connectedby the gap junction (GJ) modulates and/or supports the portal vein system (1). However, nomorphological evidence that the FS cells are connected by the GJ from the pars tuberalis
through the pars distalis has been shown.
Recently, Itakura et al. (2007) succeeded in generating transgenic rats (S100b-GFPrats) expressing green fluorescent protein under the promoter of the S100b protein genethat is a marker of FS cells (2). Utilizing these transgenic rats, the present study was
designed to show the distribution of the GJ on FS cells by immunohistochemistry forconnexin 43 (Cx43) and confocal laser microscopy. Furthermore, to determine whether the
GJ is reconstructed between FS cells in vitro, we immunocytochemically observed Cx 43
in a primary culture of S100b-GFP rat’s anterior pituitary cells.
Our observation indicated that FS cells interconnected the pars tuberalis and the pars
distalis and that the GJ existed throughout these FS cells. We observed that the LHRH
nerve projected not only onto the portal vein but also onto FS cells in the pars tuberalis.
Furthermore, we identified that the GJ was constructed between FS cells in vitro. Our
results supported Soji et al.’s hypothesis that FS cells have a role in transmitting LHRH
signal from the hypothalamus to the gonadotrophs in the pars distalis.
References(1) Mabuchi et al. (2004) Cell Tissue Res., 317, 79-90.
(2) Itakura et al. (2007) Endocrinology, 148, 1518-1523.

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P71
EFFECT OF EXOGENOUS E-CADHERIN OVEREXPRESSION ON PRIMARYCULTURE CELLS OF THE RAT ANTERIOR PITUITARY GLAND
Kenji Kusumoto, Motoshi Kikuchi, and Takashi Yashiro
Department of Anatomy, Jichi Medical University School of Medicine, Tochigi, Japan.
Cadherins are a family of cell surface glycoproteins that mediate calcium-dependentcell-to-cell adhesion, including epithelial (E-) cadherin, neuronal (N-) cadherin, andplacental cadherin.We reported that E- and N-cadherin were coexpressed in the
Rathke’s pouch through the early development of the rat (1). However, in the adult rat,
hormone-producing cells express only N-cadherin, while folliculo-stellate (FS) cells
express only E-cadherin (2). We hypothesized that the change of cadherin types relates
to the differentiation of hormone-producing cells and/or effects on the topography of
hormone-producing cells and FS cells. In the present study, we observed the effect ofexogenous overexpression of E-cadherin on hormone-producing cells and the
topography of anterior pituitary cells in primary culture.
We used transgenic rats that express green fluorescence protein under the S-100b
protein gene promoter, a marker of FS cells, donated by Professor Inoue of Saitama
University. We cloned the full-length gene of rat E-cadherin and constructed E-cadherin
expression-plasmids (p-rECDH). Primary culture anterior pituitary cells of thetransgenic rats were transfected with p-rECDH by Lipofectamine 2000 (Invitrogen),dispersed by EDTA, and re-cultured. Expression of E-cadherin was determined
immunocytochemically.
Folliculo-stellate cells and hormone-producing cells aggregated in clusters. Hormoneproducing cells expressing E-cadherin were affected in their shape, but not in cellattachment. We may conclude that the expression of E-cadherin does not affect
topographic cell affinities of hormone-producing cells.
References(1) Kikuchi, M. et al. (2007) Anat. Rec., 290, 486-90.
(2) Kikuchi, M. et al. (2006) Anat. Re.c A Discov. Mol. Cell Evol. Biol., 288, 1183-9.

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P72
MOLECULAR STRUCTURE AND EVOLUTION OF
RECEPTORS FROM MUMMICHOG Fundulus heteroclitus

GONADOTROPIN

Makoto Ohkubo and Akio Shimizu
National Institute of Fisheries Science, Fisheries Research Agency, Yokohama 236-8648,
Japan.
Gonadotropin receptors (GTHRs) belong to the glycoprotein hormone receptor, afamily of G-protein coupled receptors. GTHRs have important roles in regulation of
gonadal function by mediating gonadotropin signals to gonadal cells. In the present study,
we determined the cDNA sequences and the deduced primary structures of the follicle
stimulating hormone receptor (FSHR) and the luteinizing hormone receptor (LHR) fromthe mummichog (Fundulus heteroclitus), a useful model fish for reproductive biology, andcompared them with those of various fish GTHRs. Both the mummichog FSHR (fhFSHR)
and the LHR (fhLHR) had typical structural characteristics of the glycoprotein hormone
receptor, such as the large extracellular domain (ECD), the transmembrane domain (TMD),
and the intercellular domain (ICD). However, unlike most other vertebrate GTHRs, thefhFSHR had 10 leucine-rich repeats (LRRs). This unique structural characteristic wasshared with perciform fish FSHRs. The phylogenetic tree based on the amino acidsequences of fish GTHRs showed that fish GTHRs are divided into 2 clusters, the FSHR
cluster and the LHR cluster. The topology of the tree based on a whole amino acidsequence of the fish GTHRs was consistent with the well accepted taxonomicalclassification in the case of the FSHR cluster. However, it was not consistent in the case of
the LHR cluster. Although the tree of the C-terminal half sequences (TMD and ICD)
showed similar topology to that of whole sequences, the tree of the N-terminal half of fish
GTHR sequences (ECD) showed different topology: the fhFSHR and perciform FSHRs
were diverged from other fish FSHRs as soon as the fish FSHR was diverged from the fish
LHR. These results suggest that the acanthopterygian ECD, which is responsible for thebinding of the hormone, might have more rapidly evolved than other regions along withdiversification of physiological properties of reproduction.

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P73
MOLECULAR EVOLUTION OF THYROTROPIN-RELEASING HORMONE
RECEPTORS
Yuichi Saito1, Noriaki Kobayashi1, Makoto Kimura1, Miyuki Mekuchi1, Yasuhiro Aoki2,
Teruo Azuma3, Masayuki Iigo1,2, and Tadashi Yanagisawa1,2
1

Department of Applied Biochemistory, Utsunomiya University, Tochigi 321-8505, Japan.
Department of Biotechnology, United Graduate School of Agricultual Science, Tokyo
University of Agriculture and Technology, Tokyo 183-8509, Japan.
3
National Research Institute of Fisheries Science, Tochigi 321-1661, Japan.
2

Thyrotropin-releasing hormone receptor (TRHR) is a member of the G protein-coupled
receptor family with seven transmembrane domains. Although the structure of the ligand,thyrotropin-releasing hormone, is conserved among vertebrates, the presence of threesubtypes of TRHR (TRHR1, TRHR2 and TRHR3) is known to date. Thus, it is interesting
to understand the molecular evolution of TRHR in vertebrates.
In this study, we isolated and characterized cDNA encoding TRHR in sockeye salmon(Oncorhynchus nerka) and medaka (Oryzias latipes). In addition, we predicted thestructure of TRHR in the sockeye salmon by homology modeling.
Four different TRHR (TRHR1, TRHR2a, TRHR2b and TRHR3) were cloned from thesockeye salmon. The TRHR2b with only five transmembrane domains might be a splicevariant of the TRHR2a. TRHR1a, TRHR1b, TRHR2 and TRHR3 were cloned frommedaka. Analyses of deduced amino acid sequences and homology modeling predicted thethree-dimensional structure of TRHR1, TRHR2a and TRHR3 of sockeye salmon.
Molecular phylogenetic analyses revealed that TRHR2 were first evolved form theancient TRHR, and then TRHR1 and TRHR3 were differentiated. Furthermore, only
mammals lacked the TRHR3 subtype, suggesting that mammals lost the TRHR3 during theevolution.
Further studies should be required to elucidate the difference in the ligand-receptor
interaction among the three TRHR subtypes in vertebrates.

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P74
THE FUNCTIONAL ANALYSIS OF THE TRANSCRIPTS DERIVED FROMHUMAN TYPE II GNRH RECEPTOR GENE
Tomoko Kato1, Yoh-Ichiro Kanaho1, Masahiro Enomoto1, 2, and Min Kyun Park1
1

Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Tokyo, 113-0033, Japan.
2
Present address; Laboratory for Developmental Neurobiology, RIKEN Brain Science
Institute, Hirosawa, Wako-City, Saitama, 351-0198, Japan.
Gonadotropin releasing hormone (GnRH) was originally identified as a hypothalamic
decapeptide and stimulates pituitary gonadotropes to synthesize and release gonadotropins.
Then it has the central role for controlling the reproductive function. Human possesses two
GnRH ligands (GnRH-1, GnRH-2) and GnRH receptor genes (GnRH-R1, GnRH-R2). In
the previous study, we used RNAi method to clarify the role of each receptor subtypes. It isnoteworthy that at least two transcripts derived from GnRH-R2 exists (GnRH-R2a, GnRHR2b) and the established knocked-down cells (GnRH-R2a (-), GnRH-R2b (-)) showedaltered effects of GnRH on cell proliferation. These results suggested that transcripts
derived from GnRH-R2 are functional although GnRH-R2 was thought to be a pseudogene
in human (1).
In this study, we examined the effect of two major transcripts derived from GnRH-R2
to clarify GnRH receptor function, using the cell line which is enable to be knocked down
the GnRH-R2a mRNA and GnRH-R2b mRNA simultaneously using RNAi method(GnRH-R2a/b (-)). As a model, we selected the TSU-Pr1, derived from human prostatecancer cell. And colony-forming efficiency assay was employed, which was established in
our laboratory to easily evaluate the activity of GnRH on cell proliferation. In GnRHR2a/b (-) cell, the effect of GnRH-2 was detected and Cetrorelix, which is known to be a
GnRH-R1 antagonist, worked as an agonist. These results suggest that there are transcripts
derived from GnRH-R2 other than GnRH-R2a and GnRH-R2b. In future, it is required to
examine the ligand selectivity on GnRH-R2a/b (-) cell and reconsider possible functionaltranscripts from GnRH-R2.
Reference(1) Enomoto et al. (2004) Zool. Sci., 21,763-770.

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P75
FUNCTIONS OF GnRH RECEPTOR HETERODIMERS OF THE ASCIDIAN,
Ciona intestinalis
Tsubasa Sakai1, Masato Aoyama1, Takeshiro Kusakabe2, Motoyuki Tsuda2, and Honoo
Satake11Suntory Institute for Bioorganic Research, Osaka, Japan; 2Graduate School of Life
Science, University of Hyogo, Hyogo, Japan.
GnRH is a ten-amino acid peptide hormone that plays pivotal roles in reproduction invertebrates and octopus, and GnRH receptors are member of Class A (rhodopsin-like) G
protein-coupled receptors (GPCRs). Recently, six GnRH forms (t-GnRH-3-8) and four
GnRH receptor subtypes (Ci-GnRHR-1-4) were identified in the protochordate, Ciona
intestinalis. t-GnRHs exhibit multiple cell-signaling activities including elevation of
intracellular calcium ion and production of cAMP in HEK293 cells expressed individual
Ci-GnRHR subtypes in ligand-receptor specific manners. Over the past decade, there is a
growing body of evidence that GPCRs in native tissue in vivo form heterodimers acquiring
ligand-binding affinity or selectivity, signal transduction, and internalization kinetics which
are distinct from those of the corresponding monomers or homodimers. In the presentation,
we show the functional modulation of Ci-GnRHR-1 via heterodimerization with the orphan
receptor subtype, Ci-GnRHR-4. The dimerization between Ci-GnRHR-1 and R-4 in themembranes of the transfected HEK293 cells and Ciona tissues was detected by coimmunoprecipitation and immunoblot analysis. Binding assays using the membranes of thetransfected HEK293 cells confirmed the binding of t-GnRHs to Ci-GnRHR-1 but not to R4, and verified no alternation in ligand-binding affinity between Ci-GnRHR-1 homodimer
and Ci-GnRHR1&4 heterodimer. Moreover, the heterodimer was found to elicit theelevation of intracellular calcium, extension of ERK phosphorylation, and up-regulation ofcell proliferation in ligand specific manners, compared with the Ci-GnRHR-1 homodimer.
In combination, these results indicated that Ci-GnRHR-4 is not an inactive receptor, but amodulatory component for Ci-GnRHR-1 in C. intestinalis.

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P76
IDENTIFICATION

OF

cDNAS

ENCODING

THYROTROPIN-RELEASING

HORMONE RECEPTOR FROM THE BULLFROG
Atsuko Minagawa1, Itaru Hasunuma2, Mmaki Nakano1, Reiko Okada1, Kazutoshi
Yamamoto2, Sakae Kikuyama2, Tetsuya Kobayashi1, and Takeo Machida11Department of Regulation Biology, Faculty of Science, Saitama University, Saitama 338-

8570, Japan and 2 Department of Biology, School of Education, Waseda University, Tokyo
169-8050, Japan.
Thyrotropin-releasing hormone (TRH) has been proposed as a prolactin (PRL)releasing factor in amphibians as well as in mammals. Indeed, TRH has been shown tostimulate the release of PRL from the bullfrog pituitary gland both in vitro and in vivo.
Until now, however, the intracellular mechanisms that regulate PRL release and thereceptor structures of TRH in amphibians are not known. The aims of the present study
were to clarify the contribution of extracellular Ca2+ on TRH-induced PRL release and tocharacterize the cDNAs encoding the bullfrog TRH receptors (TRHRs).
The effect of TRH on the release of PRL was studied using a modified perifusion
technique. Anterior lobes of the male bullfrog (Rana catesbeiana) pituitary glands were
continuously perifused with a medium containing TRH as a secretagogue. The amount of
PRL released was measured using a newly developed homologous enzyme immunoassay.
TRH was found to stimulate the release of PRL from the perifused pituitary glands in adose-dependent manner. In the presence of EGTA (2 mM) or Ni2+ (non-selective Ca2+channel blocker, 3 mM), the basal PRL release was decreased and the stimulatory effect of
TRH (3 x 10-9 M) on PRL release was suppressed. In the presence of nifedipine (L-type
Ca2+-channel blocker, 10-5M) also, the basal PRL release was decreased. These results
suggest that both the basal PRL release and TRH-evoked PRL release from the bullfrog
pituitary depend on the extracellular Ca2+, and that L-type Ca2+-channels may be involvedin at least basal PRL release.
On the other hand, we isolated three distinct cDNA species encoding TRHR from thebrain of the bullfrog. These three species were designated as fTRHR1, fTRHR2 andfTRHR3. Amino acid sequence and phylogenetic analyses revealed that the structure offTRHR3 is closer to that of fTRHR1 than to that of fTRHR2. RT-PCR analysis revealed
that the three bullfrog TRHR mRNAs have distinct patterns of expression. Theseobservations suggest that each of the three TRHRs have specific functions that remain tobe elucidated.

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P77
INVOLVEMENT OF D2 DOPAMINE RECEPTOR SUBTYPE IN INHIBITION OF
PROLACTIN RELEASE FROM THE BULLFROG PITUITARY GLAND
Maki Nakano1, Iataru Hasunuma2, Reiko Okada1, Kazutoshi Yamamoto2, Sakae
Kikuyama2, Tetsuya Kobayashi1, and Takeo Machida11Department of Regulation Biology, Faculty of Science, Saitama University, Saitama 3388570, Japan and 2 Department of Biology, School of Education, Waseda University, Tokyo
169-8050, Japan.

Thyrotropin-releasing hormone (TRH) has been proposed as a prolactin (PRL) releasing factor in amphibians as well as in mammals. Indeed, TRH has been shown tostimulate the release of PRL from the bullfrog pituitary gland both in vitro and in vivo. In
mammals dopamine (DA) plays an inhibitory role in the control of PRL release from thepituitary gland. In vitro studies have revealed that DA likewise suppresses PRL releasefrom the bullfrog pituitary gland. As receptor for DA, two types (D1 receptor and D2
receptor) have been reported. Until now, the intracellular mechanisms that regulate PRL
release and the structure of DA receptors have not been elucidated in amphibians. The aimsof the present study were therefore to clarify the subtypes of DA receptors that are involvedin the DA-induced inhibition of PRL release and to characterize the gene structures of thebullfrog DA receptors.
The effect of the test substances on the release of PRL was studied by using a modified
perifusion technique. The anterior lobes of the male bullfrog (Rana catesbeiana) pituitary
glands were continuously perifused with a medium containing the test substances. The
amount of PRL released was measured using a homologous enzyme-immunoassay. DA
suppressed the TRH-inducible elevation of PRL release in a dose-dependent manner.
Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on therelease of PRL. The inhibitory effect of DA on the TRH-inducible PRL release from thepituitary was nullified by the addition of L-741,626 (a D2 receptor antagonist) to themedium. In contrast, addition of SKF38393 (a D1 receptor agonist) was not associatedwith any suppressive effect on the TRH-induced PRL release, and that of SCH23390 (a D1receptor antagonist) had no influence on the inhibitory effect of DA on the TRH-inducible
stimulation of PRL release. These results suggest that the D2 receptor subtype mediates theinhibitory effect of DA on the TRH-induced PRL release from the pituitary glands.
On the other hand, two isoforms of the D2 receptor have already been identified in
mammals. In this study, we isolated the mRNAs of three distinct D2 receptor isoforms
from the brain of the bullfrog. RT-PCR analysis revealed that mRNAs of these three
isoforms were apparently expressed in the bullfrog anterior pituitary. Further studies are
required to elucidate the function of each isoform of D2 receptors in relation to theinhibitory effect of DA on PRL release.

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P78
LOCALIZATION OF ARGININE VASOTOCIN V3/V1b TYPE RECEPTOR IN
THE NEWT PITUITARY AND BRAIN
Itaru Hasunuma1, Kazutoshi Yamamoto1, Fumiyo Toyoda2, Hideo Namiki1, and Sakae
Kikuyama1,3
1

Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda
University, Tokyo 169-8050, Japan.
2
Department of Physiology, Nara Medical University, Nara 634-8521, Japan.
3
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
In mammals, vasopressin V3/V1b receptor is predominantly found in the anterior
pituitary and involved in adrenocorticotropic hormone (ACTH) release from corticotrophs(1). This receptor is also known to be expressed in the brain area, being involved in thecontrol of social behavior.
Recently, we cloned newt (Cynops pyrrhogaster) arginine vasotocin (AVT) V3/V1btype receptor cDNA for the first time in an amphibian. RT-PCR revealed that V3/V1b-type
receptor mRNA was expressed mainly in the pituitary and brain, and this expression
pattern was similar to that of mammalian vasopressin V3/V1b receptor (2).
As a step to elucidate the action of AVT via this type of receptor in the pituitary andbrain, in situ hybridization using V3/V1b-type receptor specific probe was conducted. In
the anterior lobe of the pituitary, V3/V1b-type receptor mRNA was specifically expressedin the ACTH cells. In the intermediate lobe, expression of this type of receptor gene was
not observed. In the brain, V3/V1b type receptor mRNA was localized in several area ofthe diencephalon (ventral thalamus, dorsal hypothalamic nucleus, nucleus of theperiventricular organ) and raphe nucleus of the medulla oblongata. Notably, in the raphe
nucleus, mRNAs of V3/V1b type receptor and serotonin transporter that is known to be amarker of serotonin neuron were revealed to be colocalized in the raphe nucleus.
The results obtained in the newt indicate that V3/V1b-type receptor in the pituitary
mediates the release of ACTH as in the case of mammals and raise the possibility that in
the medulla oblongata it regulates serotonin neuron functions.
References(1) de Keyzer et al. (1996) J. Clin. Invest., 97, 1311-1318.
(2) Hasunuma et al. (2007) Gen. Comp. Endocrinol., 151, 252-258.

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P79
IDENTIFICATION OF GHRELIN RECEPTOR-LIKE RECEPTOR AND ITS
GENE STRUCTURE IN RAINBOW TROUT
Hiroyuki Kaiya, Tsukasa Mori1, Mikiya Miyazato, and Kenji Kangawa
Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1
Fujishirodai, Suita, Osaka 565-8565, Japan.
1

Laboratory of Marine Molecular Biochemistry, College of Bioresource Sciences, Nihon

University, Kameino 1866, Fujisawa 252-8510, Japan.
Ghrelin is a GH-releasing and orexigenic peptide released from the stomach. The
receptor is a G-protein-coupled, growth hormone secretagogue-receptor (GHS-R), and two
types of receptor, a functional GHS-R1a and function-unknown splice variant GHS-R1b,
have been known. In non-mammalian vertebrates, these receptors have been identified inchicken, pufferfish, seabream and zebrafish. In this study, we identified ghrelin receptorlike receptor (GHSR-LR) in rainbow trout. We isolated two mRNA: one encodes a 387amino acid protein, which is similar to GHS-R1a (GHSR1a-LR), and the other encodes a
300-amino acid GHS-R1b-like protein (GHSR1b-LR). Trout GHS-R1a-LR showed 71, 67,
60, 58% identity with seabream, pufferfish, chicken and rat GHS-R, respectively. GHSR1a-LR mRNA predominantly expressed in the pituitary, and followed by the brain,
intestine, head kidney and heart. GHSR1b-LR mRNA also expressed predominantly in thepituitary, and followed by the brain, stomach, spleen, intestine and head kidney. The
GHSR-LR gene was composed of two exons separated by an intron, as seen in otheranimal species, and GHS-R1b-LR was considered to be generated an alternative splicing of
the gene. Functional analyses of the GHSR1a-LR were conducted using human embryonickidney (HEK) 293 cells, but expected intracellular calcium increase could not observe. We
found that full-length of receptor proteins might be not generated in the cells because
unexpected mRNA splicing was occurred. In conclusion, further studies is necessary toconclude our identified GHS-R1a-LR as functional ghrelin receptor in rainbow trout,
although the gene structure and characterisation of protein sequence are highly conserved
among other GHS-R.

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P80
IDENTIFICATION OF GHRELIN RECEPTOR-LIKE RECEPTOR AND ITS
GENE STRUCTURE IN RAINBOW TROUT
Hiroyuki Kaiya, Tsukasa Mori1, Mikiya Miyazato, and Kenji Kangawa
Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1
Fujishirodai, Suita, Osaka 565-8565, Japan.
1

Laboratory of Marine Molecular Biochemistry, College of Bioresource Sciences, Nihon

University, Kameino 1866, Fujisawa 252-8510, Japan.
Ghrelin is a GH-releasing and orexigenic peptide released from the stomach. The
receptor is a G-protein-coupled, growth hormone secretagogue-receptor (GHS-R), and two
types of receptor, a functional GHS-R1a and function-unknown splice variant GHS-R1b,
have been known. In non-mammalian vertebrates, these receptors have been identified inchicken, pufferfish, seabream and zebrafish. In this study, we identified ghrelin receptorlike receptor (GHSR-LR) in rainbow trout. We isolated two mRNA: one encodes a 387amino acid protein, which is similar to GHS-R1a (GHSR1a-LR), and the other encodes a
300-amino acid GHS-R1b-like protein (GHSR1b-LR). Trout GHS-R1a-LR showed 71, 67,
60, 58% identity with seabream, pufferfish, chicken and rat GHS-R, respectively. GHSR1a-LR mRNA predominantly expressed in the pituitary, and followed by the brain,
intestine, head kidney and heart. GHSR1b-LR mRNA also expressed predominantly in thepituitary, and followed by the brain, stomach, spleen, intestine and head kidney. The
GHSR-LR gene was composed of two exons separated by an intron, as seen in otheranimal species, and GHS-R1b-LR was considered to be generated an alternative splicing of
the gene. Functional analyses of the GHSR1a-LR were conducted using human embryonickidney (HEK) 293 cells, but expected intracellular calcium increase could not observe. We
found that full-length of receptor proteins might be not generated in the cells because
unexpected mRNA splicing was occurred. In conclusion, further studies is necessary toconclude our identified GHS-R1a-LR as functional ghrelin receptor in rainbow trout,
although the gene structure and characterisation of protein sequence are highly conserved
among other GHS-R.

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P81
MOLECULAR CHARACTERIZATION OF CHICKEN MOTILIN RECEPTOR(MTL-R)
Ichiro Yamamoto1, Hiroyuki Kaiya3, Chihiro Tsutsui4, Takafumi Sakai4, Akira Tsukada5,
Mikiya Miyazato3, and Minoru Tanaka1,2
1High-tech Research Center, 2Department of Animal Science, Faculty of Applied Life
Science, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602,
Japan.
3
Department of Biochemistry, National Cardiovascular Center Research Institute, Suita,
Osaka, 565-8565, Japan.
4
Laboratory of Cell Biology, Department of Regulation Biology, Faculty of Science,
Saitama 338-8570, Japan.
5
Department of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya, 464-8602, Japan.
G-protein coupled receptor 38 (GPR38 / MTL-R) belongs to the ghrelin receptor
superfamily (1), and has been characterized as specific receptor of gastrointestinal peptide,motilin (2). However, molecular characterization of MTL-R has been performed only inmammals and fish. In this study, we performed molecular characterization for chicken
MTL-R (cMTL-R) and profiled its mRNA expression in chicken tissues. Total RNA wasextracted from chicken tissues and a fragment of cMTL-R cDNA was cloned after theamplification by RT-PCR using primers designed from the putative cMTL-R genesequence appeared in the chicken genome database. The cDNAs for 3’- and 5’- regions
were cloned by RACE PCR. The cloned full-length cMTL-R cDNA was about 1.7 kb inlength and encoded 349 amino acids. The amino acid sequence showed a significant
homology to mammalian MTL-Rs (>59%). Realtime PCR analysis revealed that cMTL-R
mRNA was expressed in all tissues examined with high levels in digestive organs andoviduct. The expression levels of cMTL-R in the proventriculus and duodenum wererapidly decreased after hatching. Functional analysis revealed that chicken motilinincreased intracellular Ca2+ concentration in HEK293 expressed cMTL-R, but chicken
ghrelin did not. These results indicate that identified cMTL-R is specifically activated bymotilin and might be involved in peristaltic motion in digestive and reproductive ducts inchicken.
References(1) McKee et al. (1997) Genomics, 46, 426-434.
(2) Feighner, S.D. et al. (1999). Science, 284, 2184-2188.

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P82
CHARACTERIZATION OF SIGNALING PATHWAYS THROUGH PROTONSENSING G-PROTEIN-COUPLED RECEPTORS
Hideaki Tomura, Masayuki Tobo, Chihiro Mogi, and Fumikazu Okajima
Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma
University, Maebashi 371-8512, Japan.
Extracellular acidification has been shown to induce a variety of responses at the wholeanimal, tissue, and cellular levels. In vascular systems, for example, acidosis causes
vasodilation of systemic circulation at whole animal levels, relaxation in isolate vessels andalteration of a variety of cellular activities at the cell levels, including cAMP accumulationand changes in intracellular Ca2+ concentration ([Ca2+]i). However, the mechanism bywhich extracellular acidification modulates the cellular activities has not fully beencharacterized. OGR1, GPR4, G2A, and TDAG8 share about 50 % homology with eachother and form a family of G-protein-coupled receptors (GPCRs). Recent studies haverevealed that the GPCRs sense extracellular protons and modulate some cellular activities.
In this study, we examined the signaling pathways of GPR4, which is a member of protonsensing GPCRs.
Cellular activities, including inositol phosphate production, cAMP accumulation, theepidermal growth factor (EGF)-induced extracellular signal-regulated kinase (ERK)
acitiviy, the serum response element (SRE) promoter activity and nuclear factor of
activated T cell (NFAT) promoter activity, in response to extracellular acidification were
measured in HEK293 cells or RH7777 cells, which were transfected with human GPR4
plasmid. Specific inhibitors or dominant negative constructs for intracellular signaling
pathways were employed to characterize the acidification-induced cellular activities.
Extracellular acidic pH reduced EGF-induced ERK activity in association withincreased cAMP accumulation. Acidification stimulated the SRE activity in a mannerdependent on G13 protein/Rho signaling pathway. Acidification also stimulated the NFAT
activity in association with inositol phosphate production. Our experimental results suggestthat GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in
response to extracellular acidification.
Reference(1) Tobo, M. et al. (2007) Cell. Signal., 19, 1745-1753.

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P83
CLONING AND CHARACTERIZATION OF THE ECDYSONERECEPTOR AND ULTRASPIRACLE PROTEIN FROM THE WATER
FLEA, Daphnia magna
Yasuhiko Kato1, Kaoru Kobayashi1, Shigeto Oda2, Norihisa Tatarazako2, Hajime
Watanabe1, and Taisen Iguchi1
1

Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology,

National Institutes of Natural Sciences, Japan.
2

National Institute for Environmental Studies, Japan.

By screening the Daphnia magna cDNA library (1) and combination of 5’-RACE,
cDNAs encoding the ecdysone receptor (EcR) and ultraspiracle (USP) protein wereobtained. We isolated three isoforms of EcR that differ in the A/B domain (EcR-A, B1, B2).
Phylogenetic analysis indicated that Daphnia EcR is most similar to EcRs from othercrustaceans, and that Daphnia USP is most similar to mammalian RXRs rather than to
USPs from insects such as the Diptera or Lepidoptera. Quantitative PCR analysis
demonstrated that the expression of EcR-A correlated well with the timing of molt.
We constructed a two-hybrid system as a reporter for ligand-dependent activation of
EcR from Daphnia and Drosophila. The gene fusions encoded the EcR lignad-binding
domain (LBD) fused to the Gal4 DNA binding domain, and the USP-LBD fused to the
Vp16 activation domain. These chimeric genes were transfected into Chinese hamster
ovary (CHO) cells with a luciferase reporter gene. Dose-dependent activation of thereporter gene could be observed for both Daphnia and Drosophila EcR reporters whentransfectants were exposed to ecdysone analogs such as ponasterone A, muristerone A andtebufenozide. This two-hybrid system may represent a useful reporter system for further
examination of hormonal and chemical effects on Daphnia at the molecular level. This
study was supported by Grants-in-Aid for Scientific Research from Ministry of Education,
Culture, Sports, Science and Technology, and grants from the Ministry of Environment,
Japan, and a grant of LRI by JCIA.
Reference(1) Watanabe, H. et al. (2005) Genome, 48, 606-609.

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3S-1
PRELUDE TO REGENERATION----MECHANISMS OF CELL DEATH----

Emiko Furuta
The Research Institute for Comparative Immunology, Japan.
Regeneration can be classified based on morphological criteria into two categories :
pathological(accidental or traumatic) and physiological(repeat or repair) regeneration. In
either category, the first reaction is cell-death and cell-elimination in the wound area .
There are two types of cell death: necrosis and programmed cell death. Necrosis ischaracterized by cell swelling and membrane bursting. Programmed cell death plays animportant role for the formation and maintenance of tissue architecture in multicellularorganisms. It regulates the sculpting of structures, deletion of vestigial organs, andmaintenance of proper cell number. Programmed cell death may be classified on the basisof morphological changes into two types. The most extensively studied one, called
apoptosis, is characterized by membrane smoothing, cell shrinkage, DNA fragmentationand preservation of organelles. The other one, autophagic cell death or type II programmed
cell death, is characterized by the appearance of vacuoles engulfing bulk cytoplasm andorganelles such as mitochondria and endoplasmic reticulum. Allografts in human or mouse,
for instance, are chronically rejected by apoptosis with DNA fragmentation, while rejectionof allografts in terrestrial molluscs, degeneration of insect muscle, degeneration of eye or
copulatory apparatus in planarians are induced by autophagic cell death. In the final
process of autophagic cell death, pycnotic nucleus left in hemocoel is cleared away byheterophagy. Cell –death¸genes exist from nematode to human with high homology. This
fact indicates that the genes may be conserved across phylogenic lineages. Thus,
programmed cell death in regeneration system is considered to occur in two different
pathways: apoptosis and autophagy.

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3S-2
THE ORIGIN OF STEM CELLS IN THE REGENERATING MARINE ANDFRESHWATER PLANARIANS
Sachiko Ishida
Department of Biofunctional Science, Faculty of Agriculture and Life Science, Hirosaki
University, Hirosaki 036-8561, Japan.
Planarians have high regenerative ability. It has been reported by many researchers that
this ability resides in the neoblasts (pluripotent/totipotent stem cells). Neoblasts are
considered to be the only proliferating cells in asexual organisms, and they can renew bythemselves and undergo differentiation to any cell types. Electron microscopic studies haveshown that the neoblast has many chromatoid bodies in the cytoplasm.
Recently our group found that the intestinal cells of Pseudostylochus intermedius(polyclad) include mitochondrial large ribosomal RNA (mtlrRNA) , mitochondrial small
ribosomal RNA (mtsrRNA), chromatoid bodies and abundant free ribosomes, and haveproliferative ability. Furthermore, we obtained the figures that the neoblast-like cells withsignals for mtlrRNA and mtsrRNA might have derived from the regenerating intestine anddispersed in the new tissue. Using EM in situ hybridization technique, we previously
revealed that the mtlrRNA and mtsrRNA were localized on the surface of the chromatoid
bodies in the embryos of P. intermedius (1). MtlrRNA and mtsrRNA have been identified
as the factor required for the formation of the germline progenitors in Drosophila (2).
Regarding to the origin of neoblast, there exist two theories, that is, the embryonicstock cell theory and the dedifferentiated cell theory. I support both theories and consider
as follows. 1. In the species reserving neoblasts abundantly in the parenchyma, thededifferentiated cells may not play the role of cell supply in epimorphosis. However, in thespecies not reserving so much, the dedifferentiated cells may also take part in theirregeneration. 2. There is a possibility that the intestinal cells have a pluripotency and havethe ability to regenerate themselves, and can supply the neoblasts in marine planarianpolyclads.
References(1) Sato, K. et al. (2001) Develop. Growth Differ., 43, 107-114.
(2) Kobayashi and Okada (1989) Development, 107, 733-742.

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3S-3
CELLULAR AND MOLECULAR MECHANISMS OF NERVE REGENERATION OF
HYDRA
Osamu Koizumi
Neuroscience Laboratory, Department of Environmental Science, Fukuoka Women's
University, Fukuoka 813-8529, Japan.

The developmental dynamics of the nerve cells are unique.

Neurons are produced

continuously by differentiation from interstitial multipotent stem cells. These neurons arecontinuously displaced outwards along with epithelial cells and are sloughed off at theextremities. However, the spatial distribution of each neural subset is maintained.
Mechanisms related to these phenomena, of the position-dependent changes of neuralphenotypes were proposed.
Hydra possesses several advantages for the study of nerve net formation. We can examine
nerve net formation in various unique experimental systems: the regenerating, repopulating,budding, and normal system. Nerve net formation progresses under different conditions
depending on the system. Therefore, we can clarify important factors in the cellular level bycomparing nerve net formations.
In the course of large scale screening of peptide signal molecules, peptide molecules
related to nerve differentiation have been identified. The LPW family, composed of four
members sharing common N-terminal, L(or I)PW, inhibits nerve differentiation in hydra. In
contrast, Hym355 (FPQSFLPRG-NH3 ) activates nerve differentiation in hydra. LPWs areepitheliopeptides, whereas Hym355 is a neuropeptide. Our preliminary results about study toclarify the exact site of the action suggest that it is commitment, but neither migration, nordifferentiation.

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3S-4
ADULT NEWT RETINAL REGENERATION: IS FGF AN INDUCTION FACTOR
OF REGENERATION?

Chikafumi Chiba

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai
1-1-1, Tsukuba, Ibaraki 305-8572, Japan.

The retina is a part of the central nervous system in the eye, and disease or traumatic
injury of the retina has a serious impact on normal life. Recent biomedical studies haverevealed that pigment epithelium (PE) cells of the adult eye, which transdifferentiate into
retinal cells in vitro, are one of the most hopeful cell sources for future cell therapy ofdegenerated retinas. However, unfortunately, we do not have clinical strategies toregenerate the whole retina from PE cells in the eye of a patient. In contrast, some urodele
amphibians such as newts can regenerate the entire retina through transdifferentiation of
the retinal PE (RPE) cells even when the neural retina is completely lost. Therefore,
urodelian retinal regeneration can be an in vivo transdifferentiation model of PE cells togain critical information aimed at establishing new clinical strategies for retinal
regeneration. Thus far, we have studied the cellular and molecular mechanisms of adultnewt retinal regeneration, and currently focus on the induction mechanism of retinal
regeneration. In embryonic and larval vertebrates, an FGF2/FGFR-2/MEK/Pax6 pathway
has been suggested to be involved in the induction of RPE transdifferentiation into a neuralretina. Here we show that 1) the adult newt RPE cells transdifferentiate into neural cells in
vitro through activation of an MEK pathway, 2) FGF2 promotes de-differentiation andproliferation of RPE cells but is unlikely a primary induction factor, 3) FGFR-2 and Pax6
genes do not express in the adult newt RPE cells, 4) both genes are expressed upon
removal of the neural retina, 5) neural transdifferentiation of RPE cells is inhibited in theinitial process of retinal regeneration. These results suggest that the retinal regeneration of
adult newt is induced via a mechanism different from embryonic/larval one and the RPEtransdifferentiation is controlled elaborately in vivo.

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3S-5
NEUROGENESIS AND REGENERATION IN THE MAMMALIAN BRAIN

Tatsunori Seki
Department of Anatomy, Juntendo University School of Medicine, Tokyo 113-8421,
Japan.
For more than 100 years, it was believed that neurons are never generated in the adultbrain. This paradigm changed in the late 90’s and most neuroscientists now believe thatneurons continue to be generated in two restricted adult brain regions: the hippocampus
and the subventricular zone of the forebrain. The phenomenon of adult neurogenesis itself
had been discovered in the early 1960’s by Altman. However, his studies were notappreciated and unregarded. In the early 90’s we found that newly generated anddeveloping neurons in the adult hippocampus specifically express polysialic acid (PSA), acarbohydrate

portionofneuralcell

adhesionmolecules(NCAM).

The

immunohistochemistry for PSA can visualize the detailed morphology of the dendrites andaxons of developing neurons. In the late 90’s the adult neurogenesis was demonstrated in
the human hippocampus, and the concept of the adult neurogenesis is widely accepted.
Recently, progenitor cells of the adult neurogenic regions have been reported to possess
astrocytic features and express GFAP. However, the questions remain how astrocyticprogenitors divide and differentiate into neurons, and how the neuroblasts migrate to the
GCL. To observe these developmental courses, we developed a time-lapse imaging systemwith mouse GFAP promoter-controlled enhanced green fluorescent protein (GFAPp-EGFP
Tg mice, kindly provided by Dr. Shioda, Showa Univ.) or with an EGFP-retrovirus
labeling. These results show that GFAP-positive neural progenitor cells differentiate into
neurons via intermediate cells expressing both astrocytic and neuronal markers.
Furthermore, neuronal proliferating progenitors make clusters, and most postmitotic
neuroblasts migrate horizontally from the clusters, extending tangentially oriented
processes, and finally extend apical dendrites. These results suggest that the neuralprogenitor cells have a systematic cellular arrangement and intercellular interaction during
their development.

–90–

3S-6
REGENERATION AND RESORPTION OF THE GOLDFISH SCALE AND ITSREGENERATION BY MELATONIN
Atsuhiko Hattori
Department of Biology, Collage of Liberal Arts and Sciences, Tokyo Medical and Dental
University, Chiba 272-0827, Japan.
Different from mammals, most teleosts have the scales which contain as much as 20% of
the total body calcium and are a better potential internal calcium reservoir than the vertebral
bone during periods of increased calcium demand, such as sexual maturation and starvation.
Furthermore, the teleost scale contains osteoblasts and osteoclasts similar to those found in
mammalian bone. In addition, we recently demonstrated that the osteogenesis of theregenerating scale is very similar to that of mammalian membrane bone. Judging from thesemorphological studies, we strongly suggest that the teleost scale should be a good model forthe study of osteogenesis and calcium metabolism.
To clarify the function of osteoclasts and osteoblasts in the scale of goldfish (Carassius
auratus) during the scale resorption and regeneration, we cloned several osteoclastic markers:
nuclear factor of activated T cell c1 , tartrate-resistant acid phosphatase (TRAP), cathepsin K
and matrix metalloprotease 9, and several osteoblastic markers: runt-related transcription
factor 2 (Runx 2), osterix (OSX), type 1 collagen (COL-1), alkaline phosphatase (ALP) andosteocalcin. The expression patterns of these osteoclastic and osteoblastic markers were
examined during the scale resorption and regeneration using a quantitative real-time PCR. As
a result, we found that these osteoclastic markers were expressed in the multinucleatedosteoclasts and increased remarkably during the scale resorption after intramuscular
autotransplantation of the scale. On the other hand, these osteoblastic markers were expressedin the regenerating scale, and the expression of OSX, COL-1, ALP and osteocalcin mRNAs
increased remarkably during the scale regeneration although Runx 2 mRNA did not change.
Melatonin is a secretory product of the vertebrate pineal gland which is synthesized during
darkness and exhibits a conspicuous circadian rhythm. Using the cultured normal scale ofgoldfish, we recently demonstrated that melatonin suppressed osteoclastic activity. Then, we
determined whether melatonin suppresses 1) a differentiation of single-nucleate preosteoclasts
from hematopoietic stem cells and 2) an induction of multinucleated osteoclasts from thesingle-nucleate preosteoclasts. We cloned receptor activator of nuclear factor κB (RANK)
expressed on osteoclast precursors and RANK ligand (RANKL) expressed on osteoblasts inthe scale of goldfish. RANK-RANKL system plays a key role in the osteoclast differentiation.
In autotransplanted scales after 1day by the treatment with melatonin, RANK and RANKLmRNA expressions were significantly lowered compared¸with those of autotransplanted
control. In autotransplanted scales after 7days, TRAP activity significantly increased and lotsof multinucleated osteoclasts appeared; however, by the treatment with melatonin from day 3
to day 7, the TRAP activity significantly decreased and lots of single-nucleate preosteoclasts
were still observed.
These findings indicate for the first time that physiological concentrations of melatonin
suppress the differentiation of osteoclasts and the induction of multinucleated osteoclasts invivo in the teleost scale.

–91–

AUTHOR INDEX
Abe, Nobuhiko	45
Abe, Shin-ichi	17Abe, Tomotaka	19, 66
Akabane, Gen	42
Akiba, Masao	53
Amano, Masafumi	33
Amiya, Noriko	33
Ando, Hironori	38, 47
Ando, Masaaki	37
Andoh, Tadashi	68
Aoki, Yasuhiro	57, 75
Aoyama, Masato	36, 77
Asazuma, Hideaki	67
Azuma, Morio	59
Azuma, Sadahiro	10
Azuma, Teruo	66, 75
Azumaya, Tomonori	46, 47
Ban, Masatoshi	47. 48
Bentley, George E.,	20, 21

Hasunuma, Itaru	21, 78, 79, 80
Hattori, Atsuhiko	49, 50, 51, 52, 91
Hayakawa, Eisuke	62
Hayashi, Daisuke	12
Horiguchi, Kotaro	71
Hyodo, Susumu	39, 45, 63, 65
Iguchi, Taisen	85
Iigo, Masayuki	19, 33, 57, 66, 75
Inoue, Kazuhito	49
Inoue, Kinji	64
Inoue, Makiko	13
Ishida, Sachiko	87
Ishiguro, Koutaro	24, 60
Ishihara, Yuko	10
Itakura, Mitsuo	70
Ito, Sei	52
Iwata, Takeo	16, 70
Izumi, Shun-ichiro	56
Jodo, Aya	47

Cai, Li-yi	56
Chiba, Chikafumi	89
Chowdhury, Vishwajit S.	20, 21
Doi, Hiroyuki	37
Egashira, Kouji	33
Endo, Daisuke	5, 6, 7
Endo, Makoto	19
Enomoto, Masahiro	76
Eto, Ko	17
Fujie, Manabu	36
Fujisawa, Toshitaka	62
Fujita, Shiho	19, 66
Fujita, Toshiro	71
Fujiwara, Ken	72
Fukaura, Kazuyuki	17
Fukui, Ryoji	69
Fukuwaka, Masa-aki	46, 47
Furudate, Sen-ichi	10
Furuta, Emiko	86
Furuya, Ryo	50
Goto, Ryo	65
Hasegawa, Sanae	38
Hashimoto, Kazumasa	61

Kageyama, Haruaki	27
Kaiya, Hiroyuki	25, 82, 83
Kanaho, Yoh-Ichiro	5, 8, 76
Kangawa, Kenji	25, 82
Kasahara, Kazuhito	19
Kasai, Haruo	71
Katada, Risa	10
Kato, Keisuke	34
Kato, Takako	54, 55, 56
Kato, Tomoko	76
Kato, Yasuhiko	85
Kato, Yukio	54, 55, 56
Katsumata, Hiroshi	46
Katsura, Kazuhiko	45
Kawada, Tsuyoshi	36
Kawauchi, Hiroshi	58
Kikuchi, Motoshi	72, 73
Kikuyama, Sakae	16, 43, 61, 78, 79, 80
Kimoto, Fuyuko	55
Kimura, Makoto	75
Kitamura, Shoji	66
Kitani, Shohei	40
Kobayashi, Fumihisa	52
Kobayashi, Kaoru	85
Kobayashi, Masaki	50
Kobayashi, Noriaki	75
Kobayashi, Tetsuya	78, 79–93–

Kobayashi, Yuki	28, 65
Koike, Kanako	14, 30
Koizumi, Osamu	88
Komiyama, Yuta	43
Kondo, Noriaki	3
Konno, Norifumi	39, 41
Kurakata, Erina	7
Kusakabe, Takeshiro	77
Kusumoto, Kenji	73
Leprince, Jérôme	28
Machida, Takeo	78, 79
Maehiro, Sayaka	6
Maejima, Sho	41
Makino, Keita	46, 48
Maruyama, Keisuke	24, 25, 26, 27, 28, 60
Masuda, Naoto	31
Masuda, Tomohiro	66
Matada, Masahiro	52
Matsuda, Kouhei	24, 25, 26,
	27, 28, 39, 40, 41, 59, 60, 61
Matsuno, Ryosuke	12
Mekuchi, Miyuki	75
Minagawa, Atsuko	78
Minakata, Hiroyuki	22
Mishiro, Kenzo	38
Mita, Masatoshi	18
Miura, Tohru,	25, 27, 28
Miyazato, Mikiya	82, 83
Mizusawa, Kanta	65, 81
Mizusawa, Noriko	70
Mizushima, Shusei	15
Mochida, Hiroshi	61
Mochiduki, Akikazu	64
Mogi, Chihiro	84
Mori, Tsukasa	82
Morita, Kentaro	48
Morita, Yoshihiro	21
Moriyama, Shunsuke	58, 65
Motohashi, Eiji	38
Mukuda, Takao	37
Murakami, Sanae	56
Murakami, Shizuko	8, 9
Nagahama, Yoshitaka	1
Nagai, Chiaki	67
Nagasawa, Hiromichi	35, 67
Nagashima, Keiko	13

Nagata, Shinji	34, 67
Nakada, Tomoaki	16
Nakajo, Shigeo	11
Nakakura, Takashi	69
Nakamachi, Tomoya	11, 12, 27
Nakamura, Masahisa	11, 50, 51
Nakano, Maki	69, 78, 79
Nakayama, Michie	55
Namiki, Hideo	80
Nejigaki, Yumika	59
Nishi, Genjirou	57
Nishimiya-Fujisawa, Chiemi	62
Nozaki, Masumi	58
Oda, Shigeto	85
Ogasawara, Michio	36
Ogushi, Yuji	42
Ohkubo, Makoto	74
Ohkubo, Takeshi	53
Oh-Nishi, Arata	10
Ohtaki, Hirokazu	12
Okada, Reiko	61, 78, 79
Okada, Saori	29
Okajima, Fumikazu	84
Ono, Katsuhiko	9
Ono, Tamao	15
Onuma, Takeshi A.	46, 47
Oshima, Yuji	52
Osugi, Tomohiro	20, 63
Oyama, Makoto	19
Park, Min Kyun	5, 6, 7, 8, 34, 57, 76
Saito, Noboru	15, 53
Saito, Yuichi	19, 57, 75
Sakai, Takafumi	13, 14, 30, 83
Sakai, Tsubasa	36, 77
Sakano, Hiroyuki	45
Sakihara, Takashi	37
Sakimura, Munenori	44
Sano, Akiko	54
Sasayama, Yuichi	44
Satake, Honoo	36, 77
Sato, Kan	53
Sato, Shunpei	46, 48
Sato, Takanobu	54
Satoh, Nori	36
Seki, Tatsunori	90
Sekiguchi, Toshio	36–94–

Shigeno, Shuichi	22
Shimada, Kiyoshi	15, 53
Shimakura, Sei-Ichi	25, 26, 27, 28
Shimizu, Akio	74
Shimotani, Toyokazu	58
Shinme, Mari	18
Shioda, Seiji	11, 12, 23, 24, 25, 26, 31
Soji, Tsuyoshi	4, 72
Sower, Stacia A.	58
Sugiyama, Yukiko	38
Susa, Takao	55
Suzuki, Masakazu	42, 43, 44, 69
Suzuki, Nobuo	50, 51, 52
Suzuki, Nobuyuki	10
Swanson, Penny	46
Tabata, Jun	51
Tachibana, Tetsuya	31
Tahara, Ken-ichi	15, 53
Takagi, Hiroyasu	13
Takagi, Yasuaki	66
Takahashi, Akiyoshi	27, 29, 58, 59, 65, 81
Takahashi, Noriko	71
Takahashi, Toshio	62
Takane, Masayuki	49
Takano, Koji	71
Takasawa, Toshihide	45
Takeda, Takuya	64
Takenoya, Fumiko	32
Tanaka, Minoru	15, 53, 83
Tanaka, Mio	59
Tanaka, Shigeyasu	42, 43, 44, 69
Tatarazako, Norihisa	85
Thamamongood, Thiparpa	51
Tobo, Masayuki	84
Tomura, Hideaki	44, 84
Tonon, Marie-Christine	28
Toyoda, Fumiyo	16, 80
Tsuchiya, Keisuke	29
Tsuda, Motoyuki	77
Tsukada, Akira	15, 53, 83
Tsutsui, Chihiro	14, 30, 83
Tsutsui, Kazuyoshi	16, 20, 21, 31, 63

Vaudry, Hubert	28
Wada, Kohei	27, 28
Wakasugi, Tatsuya	24
Watanabe, Hajime	85
Watanabe, Jun	11, 12
Watanabe, Yohei	37
Yada, Takashi	45
Yamada, Kikumi	15
Yamada, Tomo	64
Yamada, Toshiki	40
Yamamori, Kunio	33
Yamamoto, Ichiro	15, 83
Yamamoto, Kazutoshi	16, 18, 78, 79, 80
Yamamoto, Sayaka	18
Yamamoto, Shoichiro	45
Yamanome, Takeshi	29
Yamazaki, Yuji	60
Yanagisawa, Tadashi	19, 33, 57, 66, 75
Yanaka, Takatsugu	14, 30
Yano, Tetsu	21
Yashiro, Takashi	72, 73
Yasufuku-Takano, Junko	71
Yofu, Sachiko	12
Yoshimoto, Katsuhiko	70
Yoshimura, Takashi	57
Zheng, Jun	13
Zheng, Zhao	14, 30

Uchida, Katsuhisa	58
Uchiyama, Minoru	24, 25, 26,
	27, 28, 39, 40, 41, 59, 60, 61
Ueda, Makoto	44–95–

SUBJECT INDEX
1-methyladenine	18
Ad4BP/SF-1	5, 6
Adrenocorticotropin	58
Agnatha	60
α-MSH	27
Amiloride-sensitive Na+ channel	40
Angiotensin II	41
Anorexigenic action	24, 26, 27, 28
Anuran	42
Apoptosis	17, 86
Aquaporin	42, 43
Arginine vasotocin (AVT)	39, 41, 42
Arginine vasotocin (AVT) receptor	39, 80
Arylalkylamine N-acetyltransferase	49
Ascidian	36, 77
Astrocyte	11, 90
AT1R	41
Atrial natriuretic peptide (ANP)	40
Autophagy	85
Autotransplantation	51
Ayu	45Bacterial expression system	67
Banded houndshark	65
Barfin flounder29
BDNF	6
Bering Sea	47
Blood pressure	64
Blood-cerebrospinal fluid barrier
3
Bombyx mori	35
Brain	12, 63, 80, 81
Bullfrog	43, 44, 61, 78, 79
Calcitonin	36, 44
Calcium	3
Calcium Metabolism	52
Cartilaginous fish
63
cDNA cloning	83
Cell culture	73
Cell immunity	64
cGPR38	83
Chick	31
Chick embryo	6, 8, 9
Chicken	13, 34, 83
Chum salmon	46 47, 48
Ciona	36
Ciona intestinalis	77
Circadian clock	66

Circannual rhythm	3
Clusterin	69
cMTL-R	83
Cnidaria	62
Cortisol	48
CRF	61
Crustacean hyperglycemic hormone	67
Culture	8
Cyanobacteria	49
Cyclic nucleotide-gated channel (CNG Channel)	40
Daphnia	85
DES	23Development	5, 11, 15
Developmental neurobiology	88
Distribution	29
Dmrt1	7DNA-binding	55
Dopamine	43, 79
Dopamine Receptor	79
Drinking behavior	37
Duodenum	30
E-cadherin	73
Ecdysone	85
Egr-1	54
Electrical couple	4
Endolymphatic sac	44
Endozepines	28
Estivation	39
Estrogen	14, 16
European starling	20
Evolution	36, 66
Exocytosis	71
Expression	19
Fasting	30
Feeding behavior	31, 35
Feeding regulation	32
FGF2	89
Fish	66
Fish reproduction	74
Flounder	68
Folliculo-stellate cell	72
FRamide	62
Fresh water adaptation	48
Frog urinary bladder	40
Gap junction	4, 72–96–

Gene expression	38
Gene structure	19, 33
Germ cell	21
gfLPXRFa	26
GH	53
Ghrelin	14, 25, 30, 82
GHRH	60
GHS-R	25
GnIH	20, 21
GnIH receptor	21
GnRH	76, 77
GnRH induction	55
GnRH neuron	8, 9
GnRH receptor	76
GnRH-I and-II	20
Goldfish24, 25, 26, 27, 28, 59
Gonad	5, 21
Gonadotroph	56
Gonadotropin	1, 54, 58
Gonadotropin receptor	74
Gonadotropin-inhibiting hormone	31
Gonadotropin-releasing hormone	47
Gonad-stimulating hormone	18
Gonad-stimulating substance	1
GPCR	77
GPR4	84
GPR54	19
G-protein	84
G-protein-coupled receptor	84
Growth	46
Growth hormone secretagogue-receptor	82
Guidance molecules	9
Gulf of Alaska	46
Hagfish58
Half-life	68
Hemolymph	35
Heterodimer	77
Hibernation-specific protein complex
3
Hippocampus	10, 90
Homeobox	54
Homing	2
Hormonal signal	3
Hormone	85
HSP90β	17
Human	70
Hydra	62, 88
Hydroxyindole-O-methyltransferase	49
Hypocretin/orexin	33

Hypocretin receptor	33
Hypothalamus	32
Hypothyroidism	10
IGF	15IGF-I	46
IGFBP	15
IGFBP4	53
Immunocytochemistry	22
Immunoreactivity	61
In situ hybridization	9, 13
Instinctive behavior	2
Insulin	68
Intestinal cell	87
Intracerebroventricular injection	26, 27
Isotocin	37,38
KISS1	19
Lamprey	58, 60
Lateral nasal sinus	16
Learning and memory	10
Leopard gecko	7, 34
Leptin	14
LHRH	72
Limb	15
Lower vertebrates	2
LPXRFa peptide	63
LPXRF-NH2 motif	26
Lungfish39
Marine bacterium	52
Marsupenaeus japonicus	67
Maturation-inducing hormone	1
Maturation-promoting factor	1
MCH	27, 59
Medaka	19, 33, 75
Melanin-concentrating hormone	81
Melanocortin receptor	29
Melantroph	43
Melatonin	49, 66, 91
Membrane potential	4
Membrane progestin receptor	23
Metamorphosis	61
Migration	8, 9, 45, 46
Molecular cloning	57
Molecular evolution	57, 74, 75
Motilin	83
Mouse	12–97–

mPR	23mRNA	47, 61
mtrRNAs	87
Mummichog	74
Myoactivity	62
Neoblast	87
Nerve net formation	88
Neural progenitor	90
Neural stem cells	11, 12
Neuroendocrine systems	2
Neurogenesis	90
Neurohypophyseal peptides	37
Neuromedin U	24
Neuronatin	56
Neuropeptide	62
Neuropeptide W	32
Newt	17, 80, 88
NMU receptor	24
NNAT	56
Nongenomic action	23
Nucleotide sequence	65
Octadecaneuropeptide	28
Octapeptide	28
oct-GnRH	22
Octopus hatchlings	22
Oocyte maturation	1, 23
Opioid	31
Orexigenic action	25
Osmoregulation	38, 45
Osteoblasts	50, 51, 52, 91
Osteoclasts	91
Osteolytic disease	51
Ovarian follicle cells	18
PACAP	12, 59, 60
Parafibromin
70
Parathyroid	70
Parathyroid hormone	44
Pars intermedia	43
Pars tuberalis	72
Peptide	35, 88
Pheromone perception	16
Pineal organ	66
Pituitary	5, 56, 59, 73, 80
polypeptide (PACAP)	11
Pituitary development	13
Pituitary gland	4

Pituitary-gonadal axis	46
Planarian	87
Postnatal	14
PQRFa peptide	63
Principal islet	68
Prodynorphin	65
Proenkephalin	65
Programmed cell death	86
Prolactin (PRL)	16, 45, 78, 79
Prolactin receptor	17
Prolactin-releasing peptide (PrRP)	64
Proliferation	12
Protein kinase C (PKC)	11
Proton	84
Protopterus annectens	39
PRP	60Prx2	54
Puffer	38
Purification
25
Quail	15
Rainbow trout	69, 82
Rat	4, 14, 30, 32, 56, 71
rdw/rdw	10
Real-time PCR	50
Receptor	81, 85
Regeneration	50, 88, 89, 91
Regeneration system	86
Reproduction	20
Reptile	34
Resorption	91
Retina	89
Retinaldehyde dehydrogenase	13
Retinoic acid	13
RNAi method	76
RPE	89
Scale	50, 51, 52
Secretory granule	56
Sex differentiation	6, 7
Sex-linked dwarf chicken	53
Signal transduction	84
Single channel recording	40
Skin	81
Sockeye salmon	75
Sodefrin	16
Somatolactin	59
Spawning migration	47–98–

Spermatogenesis	17
Squamata	5
Starfish18
Stem cell	87
Steroid	21
Suppression-subtractive hybridization	69
SV40 large T antigen	70
Synaptic plasticity	10
Temperature-dependent sex determination	5
Testicular interstitial cells	18
Testis	7
Testosterone	48
Thermogenesis	34
Thyroid	10
Thyrotropin-releasing hormone	57
Transcripts	76
Transfection	73
TR-FIA	68
TRH	78, 79
TRHR	75, 78
Tributyltin	52
Tumor suppressor	70
UCP	34Ultimobranchial gland	69
Upper esophageal sphincter muscle	37
Vasotocin	37, 38
Ventral pelvic skin	42
V-H+-ATPase	44
VMH	6
VT1R	41
Water absorption response	41
Xenopus laevis	42
Zebrafish

81–99–