ISSN 0913-9036

PROCEEDINGS OF THE JAPAN SOCIETY FORCOMPARATIVE ENDOCRINOLOGY
No.21, 2006

Abstracts of the 31st Annual Meeting and the 31st Symposium on“Nanobioscience of Chemical Mediation:
New Applications in Comparative Endocrinology”
and“New World of Chemical Mediation Revealed bySingle Molecule and Cell Analyses”
SapporoNovember 3-4, 2006

Edited by

S. Tanaka and M. Suzuki

PROCEEDINGS OF THE JAPAN SOCIETY FORCOMPARATIVE ENDOCRINOLOGY
No. 21, 2006

Abstracts of the 31st Annual Meeting and the 31st Symposium on“Nanobioscience of Chemical Mediation:
New Applications in Comparative Endocrinology”
and“New World of Chemical Mediation Revealed bySingle Molecule and Cell Analyses”
SapporoNovember 3-4, 2006

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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2005
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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
Yamamoto, Kazutoshi（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）（Waseda Univ.）

2006 PROGRAM COMMITTEE
Akihisa Urano
Takayuki Takahashi
Atsushi Kikura
Katsueki Ogiwara（Hokkaido Univ.）（Hokkaido Univ.）（Hokkaido Univ.）（Hokkaido Univ.）

CONTENTS

ABSTRACTS OF THE 31ST SYMPOSIUM<SYMPOSIUM I> “Nanobioscience of Chemical Mediation: New
Applications in Comparative Endocrinology”
Organizer: Susumu Hyodo (Tokyo Univ.)
Hitoshi Aonuma (Hokkaido Univ.)
Future of nano-biotechnology: lesson from history of neurobiology
H. Aonuma

1Studies of biomolecular function in living cell nucleus using fluorescence correlation spectroscopy
M. Kinjo

2Real-time imaging of corticosteroid receptors in living cells
M. Nishi and M. Kawata3

Glass microcapillary osmometer that can measure real time changes in the intracellular osmolarity: its development and application
Y. Naitoh

4Aggressive behavior regulation in a Japanese carpenter ant
M. Ozaki

5

Recent advances of ethological physiology for free-living animals by micro-devices
K.Q. Sakamoto and K. Sato

6<SYMPOSIUM II> “New World of Chemical Mediation Revealed bySingle Molecule and Cell Analyses”
Organizer: Hirotoku Ando (Kyushu Univ.)
Atsushi Kimura (Hokaido Univ.)
Gene expression in a single neuron
E. Ito7

Identification of novel G-protein coupled receptors in laser-captured single GnRH neurons
S. Parhar

8Chromatin regulation at the human growth hormone gene cluster
A. Kimura9Dynamics and modification of histones in living cells
H. Kimura

10Single-molecule analyses of neurotrophin signalling in the growth cones of sensory neurons during
the activation of axonal outgrowth
T. Tani11

Single-neuron molecular physiological analysis of the multifunctional GnRH peptidergic systems
Y. Oka12

ABSTRACTS OF THE 31ST ANNUAL MEETING
Molecular cloning and gene expression pattern of ecdysone 20-monooxygenase in the silkworm,
Bombyx mori
Y. Ito, S. Maeda, A. Nakashima, Y. Fujimoto and H. Sonobe

13

Cloning and expression of genes encoding four different subtypes of melatonin receptors in goldfish
T. Ikegami and H. Ando

14

Cloning of the cdnas encoding bone morphogenetic proteins from rainbow trout
R. Ohkado, S. Toki, S. Tanaka and M. Suzuki

15

Cloning of the receptor for frog growth hormone-releasing peptide in the brain of bullfrogs, Ranacatesbeiana
T. Osugi, K. Ukena and K. Tsutsui

16

Expression of novel isoform of bullfrog prolactin receptor
I. Hasunuma, K. Yamamoto and S. Kikuyama

17

Analysis of mrnas expressed in the parathyroid gland of the bullfrog
M. Ueda, S. Tanaka and M. Suzuki

18

Tissue distribution and development of chicken motilin cells
C. Tsutsui, I. Sakata and T. Sakai

19ii

cDNA cloning and expression profile of mrna for chicken gpr39
Yamamoto, M. Numao, Y. Sakaguchi, N. Tsushima and M. Tanaka

20

Gonadotropes express the receptor for gonadotropin-inhibitory hormone (GnIH) in quail
V.S. Chowdhury, H. Yin, T. Ubuka, K. Ukena and K. Tsutsui

21

Expression of retinaldehyde dehydrogenase (raldh) in the embryonic anterior pituitary glands of rats
K. Fujiwara, M. Kikuchi, S. Takigami, T. Kouki and T. Yashiro

22

Inverse relationship between ghrelin and leptin mrna expression levels in the fasted rat stomach
Z. Zhao, I. Sakata, Y. Okubo, K. Koike and T. Sakai

23Transcription factors Prop-1, lhx2 and Lhx3 activate fsh β gene expression
K. Kitahara, T. Sato, T. Kato and Y. Kato

24

In vitro selection of pituitary transcription factor Prop-1 responsive elements
M. Nakayama, T. Sato, K. Kitahara, T. Kato and Y. Kato

25

Distinction between Lim homeodomain transcription factors Lhx2 and Lhx3
T. Sato, K. Kitahara, A. Sano, T. Kato and Y. Kato

26

A novel pituitary transcription factor, paired-related homeobox factor 2, regulates gonadotropin
subunit genes
A. Sano, T. Sato, K. Kitahara, T. Susa, T Kato and Y. Kato

27

Expression and chromatin structure of granulosa-cell specific genes in the mouse ovary
V. Ribeiro and A. Kimura

28

Expressed sequence tags from the ovary of the red sea urchin (Pseudocentrotus depressus)
K. Yamano and T. Unuma

29

Survey of pituitary hormones on the Hatschek’s pit of amphioxus
Y. Tando, M. Inaba and K. Kubokawa

30

Molecular Identifications of three isoforms of PPARs (Peroxisome Proliferator-Activated Receptors) in the Leopard Gecko, Eublepharis macularius.
K. Kato and M.K. Park

31
iii

Mass analysis of secretory proteins expressed in the early formation of the pituitary of fetal rats
T. Nakakura, M. Suzuki and S. Tanaka

32

Selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses
H. Nakamura, Y. Ishii, Y. Sato, K. Kobayashi and K. Itoi

33

Comparative analysis of the gonadal and pituitary gnrh systems in chordates
T. Ikemoto and M.K. Park

34

Cloning and characterization of cdnas encoding pituitary adenylate cyclase-activating polypeptide
from the teleost and elasmobranch fish
K. Ishiguro, K. Maruyama, Y. Yamazaki, M. Uchiyama and K. Matsuda

35

Nucleotide sequences of proopiomelanocortin subtype genes in barfin flounder, Pleuronectiformes
Y. Kobayashi, Y. Yamamoto and A. Takahashi

36

Relationship between anal respiration and neurosubstances in the crayfish, Procambarus clarkii
A. Yasuda and Y. Yasuda-Kamatani

37

Purification, kinetic characterization, and molecular cloning of a novel enzyme ecdysteroid 22-kinase
H. Sonobe, R. Yamada, T. Ohira, K. Ieki, S. Maeda, Y. Ito, A.
Nakashima, M. Ajimura, K. Mita and M. N. Wilder

38

Primary structure and post-translational processing of proopiomelanocortin in dogfish, Triakis
scyllium
A. Takahashi, R. Goto, S. Moriyama and S. Hyodo

39

Operation of the tissue-type plasminogen activator /plasmin system in the medaka ovary
K. Minagawa, K. Ogiwara and T. Takahashi

40

Analysis of medaka kallikrein like protein the reproductive organs
T. Suzuki, S. Rajapakse, K. Ogiwara and T. Takahashi

41

Processing of gonadotropin-inhibitory hormone in the quail hypothalamus
K. Ukena and K. Tsutsui

42iv

Detailed analysis of localization of acylated and des-acylated ghrelin immunoreactivity in rat gastric ghrelin cells
Y. Okubo, I. Sakata, K. Kangawa and T. Sakai

43

Identification of steroidogenic enzymes in the ovary of amphioxus
T. Mizuta, M. Suzuki, K. Asahina, and K. Kubokawa

44

The rare occurrence of a spontaneously maturing eel in captivity
H. Matsubara, H. Tanaka, K. Nomura, K. Murashita, T. Kobayashi,
P. M. Lokman, T. Matsubara, N. Ohkubo, H. Kagawa and H. Ohta

45

Structure of medaka collagen and its expression in the ovary: comparison with mammalian ovary
M. Horiguchi, K. Ogiwara and T. Takahashi

46

Gene expression pattern in tilapia gonads during sex differentiation
S. Ijiri, H. Kaneko, D. Wang, S. Adachi and Y. Nagahama

47

HSP90β is involved in signaling prolactin-induced apoptosis in newt testis
K. Eto, B. Saribek, Y. Jin, M. Saigo and S-I Abe48

Ultrastructural abnormality of spermatozoa and age-dependent loss of spermatogenesis in transgenic rat
L.-Y. Cai, T. Susa, M. Nakayama, S. Murakami, T. Kato, S-I Izumi and Y. Kato

49

Analysis of testis-specific serine proteases, tessp-3 and tessp-4 in mouse testes: comparison withother tessps
S. Fukumoto and T. Takahashi

50

Delayed development of the testis in growth-retarded (grt) mice
K. Kobayashi, H. Kubota and J. Saegusa

51

Identification of gonadotropin producing cells in the adenohypophysis of brown hagfish,
Paramyxine atami
K. Honda, K. Uchida, T. Shimotani, S. Moriyama and M. Nozaki

52

Identification of growth hormone-releasing hormone, somatostatin and growth hormone in elasmobranch, dogfish, Triakis scyllium
S. Moriyama, H. Kumada, A. Nishino, C. Maeda, N. Amiya,
K. Amano, S. Hyodo and H. Kawauchi53

igf-i stimulates synthesis and release of gth in masu salmon pituitary cells at early stages of gametogenesis
S. Furukuma, T. Onuma, A. Urano and H. Ando

54

Immunohistochemical localization of prolactin-releasing peptide in the brain of the ovoviviparousfish species Poecilia reticulata (guppy)
M. Amano, N. Amiya, M. Matsuki, H. Itoh, Y. Oka and K. Yamamori

55

Retrograde and Golgi staining study of medaka fish brain: vasotocin and/or isotocin may act as neuromodulator
T. Ohya and S. Hayashi

56

Immunohistochemical localization of orexin peptide in fish brain
N. Amiya, M. Amano, M. Iigo, S. Kitamura, Y. Oka,
T. Sunuma, A. Takahashi and K. Yamamori

57

Mechanisms of trh-induced prl release from the bullfrog pituitary gland
Minagawa, I. Hasunuma, K. Yamamoto, S. Kikuyama,
T. Kobayashi and T. Machida

58

Expression of the glucocorticoid receptor in rat corticotrophs during postnatal development is involved in changes in proopiomelanocortin processsing patterns
K. Ito, M. Suzuki, H. Ozawa, M. Kawata and S. Tanaka

59

Higher fractal dimension (FD) of C6 glioma cells on the fractal AKD surface
P. Wang , W. Fang , T. Onuma, N. Birukawa, K. Tsujii, J. Li and A. Urano

60

Onset of metamorphosis of conger eel leptocephali in laboratory-reared conditions
H. Kurogi, K. Yamano and N. Mochioka

61

Central actions of angiotensin II on spontaneous baroreflex sensitivity in the trout Oncorhynchus
mykissF. Lancien and J.-C. Le Mevel

62

The dual mode of action of cortisol on cell turnover of osmoregulatory esophagi in euryhaline fishes
C. Takagi, H. Takahashi and T. Sakamotovi63

Physiological role and hormonal regulation of urea transporter expressed in the urinary system of
the marine toads, Bufo marinus
N. Konno, S. Hyodo, K. Matsuda and M. Uchiyama

64

Water passageway by AVT-dependent and -independent aquaporin (AQP) in several water-permeable epithelial cells in the osmoregulatory organs of the tree frog
G. Akabane, Y. Ogushi, T. Hasegawa, M. Suzuki and S. Takana

65Electrophysiological analysis of atrial natriuretic peptide action on Na channels in epithelial cells
from the urinary bladder of Japanese tree frog, Hyla japonica
T. Yamada, S. Kitani, K. Matsuda and M. Uchiyama

66

What is physiological significance of increased ghrelin level in plasma?
H. Kaiya, E-S Saito, T. Tachibana, M. Furuse and K. Kangawa67

The role of human type ii GnRH receptor gene in the regulation of cell proliferation
Y. Kanaho, M. Enomoto, M. Utsumi, T. Kato and M.K. Park

68

Comparison of serum sex steroid profiles between four spine sculpin Cottus kazika and Japanese
sculpin Cottus sp. se reared in captivity
R. Hatano, D. Tahara, H. Hashimoto, R. Fujii, Y. Koya,
Y. Hayakawa, H. Yambe and T. Seikai

69

Sex difference in the synthesis of 7a-hydroxypregnenolone in the brain of breeding newts
S. Haraguchi, M. Matsunaga, K. Ukena and K. Tsutsui

70

The effect of incubation temperature on the sex steroid hormone signaling systems in the brain andgonad of the squamata species with temperature-dependent sex determination
D. Endo and M.K. Park

71

Circadian change in the biosynthesis of 7a-hydroxypregnenolone stimulating locomotor activity
and its mechanism mediated by melatonin in the brain of male quail
S. Suzuki, H. Miyabara, K. Inoue, S. Haraguchi, K. Ukena and K. Tsutsui

72

Involvement of neuropeptide in thyroxine surge in smolting kokanee salmon
H. Chiba, Y. Satoh, T. Yahata, D. Ojima, S. Moriyama, H. Kudo and M. Iwata

vii

73

Elevation of the pituitary contents of gonadotropins and plasma levels of follicle-stimulating hormone in chum salmon prior to initiation of spawning migration from the Bering sea
T.A. Onuma, S. Sato, W. Hu, A. Jodo, N. Davis, M. Fukuwaka,
T. Azumaya, P. Swanson and A. Urano

74

Relationship of homing behavior from seawater to fresh water with plasma levels of sex steroid
hormones in pre-spawning chum salmon
K. Makino, T.A. Onuma, T. Kitahashi, H. Ando, M. Ban and A. Urano

75

Isolation and characterization of neuromedin U from the goldfish
K. Maruyama, T. Miura, M. Uchiyama, S. Shioda and K. Matsuda

76

Inhibitory effect of melanin-concentrating hormone on feeding behavior in the goldfish
S. Shimakura, K. Maruyama, T. Miura, M. Uchiyama,
H. Kawauchi, S Shioda, A. Takahashi and K. Matsuda

77

Regulation of food intake by ghrelin in the goldfish
T. Miura, K. Maruyama, S. Shimakura, H. Kaiya, M. Uchiyama,
K. Kangawa and K. Matsuda

78

Effect of octadecaneuropeptide on food intake and locomotor activity in the goldfish, Carassius auratus
K. Wada, Y. Inaoka, T. Miura, K. Maruyama, S. Shimakura, M. Uchiyama,
J. Leprince, M-C. Tonon, H. Vaudry and K. Matsuda

79

The role of androgen in protogynous sex change of grouper
M.A. Alam and M. Nakamura

80

Functional associations among positive emotion, central nervous, peripheral autonomic nervous,
endocrine, and immune systems
M. Matsunaga, T. Konagaya and H. Ohira81

Sex difference in the PEP-19 expression in song nuclei of zebra finches during early brain development
K Inoue, M Kato, K. Okanoya, H. Sakaguchi, K. Wada,
E.D. Jarvis and K. Tsutsui

viii

82

SI-1
FUTURE OF NANO-BIOTECHNOLOGY: LESSON FROM HISTORY OF
NEUROBIOLOGY
Hitoshi Aonuma
Laboratory of Neuro-Cybernetics, Research Institute for Electronic Science, Hokkaido
University, Sapporo 060-0812, Japan.
Galvani L. (1737-1789) was an anatomy biologist. His contribution to physiology,
neurobiology and electronic technology was remarkable. His finding of actions of nerve
and muscle in 1791 gave great idea for finding a battery of Volta A. in 1780. Thesefindings were the first contribution of biology to modern technology. Studying biology is
not only to understand the lives or evolution but also to understand the design principlethat exist in our world. Recently, every field of technology, mathematics, chemistry andphysics etc have been developed, which accelerates developing every field of biology.
However, still studying biology is not enough to understand where we were from andwhere we are going. In most cases, the strategy of biologists is focusing on the element of
the lives to understand each interest. This might be one of the reasons why biology isdeveloping slowly although now rather quicker than before. To overcome this situation I
would like to suggest adopting modeling technique and computational simulation toinvestigate biology systematically. Hodgkin A.L. and Huxley A.S. (1952) built a modelthat demonstrates events of a neuron (1). This model is excellent and accelerates to develop
neurobiology in particular electrophysiology. Still after half a century, this model
demonstrates many neuronal events even new findings are appeared. This strongly
suggests that modeling and simulation techniques are powerful to accelerate developing
neurobiology. Grillner P. has also adopted modeling and computational simulation in his
research (2). Adopting modeling and simulation to investigate neurobiology must provide
us better idea of design principle of nervous system, which in turn accelerates developing
many kinds of technology.
Recent development of nanotechnology is remarkable. Nanotechnology also
accelerates developing every field of biology. To understand nature of lives and common
design principle of lives, it is necessary to reconstruct every hierarchy of system fromelements found. Therefore, modeling and simulation techniques must be powerful tools forthe future of nanobiotechnology.
(1) Hodgkin AL. and Huxley AS. (1952) J. Physiol., 117, 500-544.
(2) Grillner S. and Wallén P. (2002) Brain Res. Rev., 40, 92-106.
1: modeling, 1: computer simulation, 1: electrophysiology, 1: behavior, 1: nervous system

––

SI-2
STUDIES OF BIOMOLECULAR FUNCTION IN LIVING CELL NUCLEUS
USING FLUORESCENCE CORRELATION SPECTROSCOPY
Masataka Kinjo
Laboratory of a Supramolecular Biophysics, Research Institute for Electronic Science,
Hokkaido University, Sapporo 060-0812, Japan.
E-mail: kinjo@imd.es.hokudai.ac.jp
Fluorescence correlation spectroscopy (FCS) is a unique method to determine
diffusional properties and number concentrations of fluorescent molecules with single
molecule sensitivity in aqueous solution by monitoring the fluctuations in fluorescenceintensity in a very tiny detection area. We are applying FCS to investigation of thephysiological microenvironments of subcellar compartments (organelles) in living cells.
The cell nucleus contains many macromolecules that form multimolecular complexes andstructures. These molecules are involved in dynamic cell activities such as transcription
and molecular transportation from and to the nucleus. The local environment in the
nucleus is expected to vary during different cell activities. To analyze the nuclear
microenvironment in the living cell, we have constructed tandem-oligomers of green
fluorescent proteins (EGFPn: EGFP1~EGFP5) as molecular rulers and measured the
diffusion of EGFPns in living cells using fluorescence correlation spectroscopy (1).
The glucocorticoid receptor (GR) is one of transcriptional regulators that control
broad physiological gene networks and pathological effects in a range of diseases, andoffer excellent targets for therapeutic intervention. GR is associated with several proteinsin the cytoplasm. Upon ligand binding, GR is driven into the nucleus, and regulates
transactivation by association with specific genomic glucocorticoid response elements(GRE). Using FCS, we analyzed diffusion properties of green fluorescence proteintagged GR (GR-GFP) before and after addition of ligands in the nucleus of the living cell.
The diffusion of GR-GFP did not change in the cytoplasm with addition of dexamethasone(Dex), which is a transactivation agonist. On the other hand, we detected a slow-moving
component in the nucleus with Dex addition as well as with RU486, which acts as anantagonist. To elucidate this slow component, we constructed a deletion mutant of theligand-binding domain (LBD) existing in the C-terminal of GR. These results indicatethat the slow-moving component is not originated by binding to GRE. Moreover, theysuggest that GR can bind not only to specific cofactors but also other nonspecific bindingsites in DNA because its diffusion constant is excessively large compared to that of GR
associated with all cofactors currently known.
(1) Pack, C. et al. (2006) Biophys J., 91, 3921-3936.
2: tandem EGFP; 2molecular ruler; 2 microenvironment; 2 diffusion constant;
2 Glucocorticoid receptor

––

SI-3
REAL-TIME IMAGING OF CORTICOSTEROID RECEPTORS IN LIVING
CELLS
Mayumi Nishi and Mitsuhiro Kawata
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine,
Kyoto 602-8566, Japan.
Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert
numerous effects in the central nervous system that regulate the stress response, mood,learning and memory, and various neuroendocrine functions. Corticosterone (CORT)
actions in the brain are mediated by two corticosteroid receptors, glucocorticoid receptor(GR) and mineralocorticoid receptor (MR), and they show a high degree of colocalizationin the hippocampal region. These receptors are predominantly resided in the cytoplasm
without ligand and translocated into the nucleus upon ligand binding to act astranscriptional factors. Thus their subcellualr localizations are important component of
their biological activity. Given the differential action of MR and GR in the central nervous
system, it is important to elucidate how the trafficking of these receptors between
cytoplasm and nucleus and their interaction are regulated by ligand or othermolecules to exert transcriptional activity. In this symposium, we focus on the
nucleo-cytoplasmic and subnuclear trafficking of GR and MR in neural cells and nonneural cells. Dynamics of MR and GR in living cultured hippocampal neurons and COS-1
cells were presented using real time imaging technique with green fluorescent protein(GFP) and its color variants including fluorescence recovery after photobleach (FRAP) (1).
Effects of various kinds of effectors on the nucleo-cytoplasmic trafficking of thesereceptors, such as ligands, cytoskeletal elements, HSP90, and carrier proteins of importin α
and β, were investigated. Furthermore, we will discuss possible heterodimerization of MR
and GR by using fluorescence resonance energy transfer (FRET) in living cells (2).
(1) Nishi et al. (2001) Mol. Endocrinol., 15, 1077-1192.
(2) Nishi et al. (2004) J. Neurosci., 24, 4918-4927.

––

SI-4
GLASS MICROCAPILLARY OSMOMETER THAT CAN MEASURE REALTIME CHANGES IN THE INTRACELLULAR OSMOLARITY: ITS
DEVELOPMENT AND APPLICATION
Yutaka Naitoh
Faculty of Pharmaceutical Sciences at Kagawa Campus Tokushima Bunri University,
1314-1Shido, Sanuki, Kagawa 769-2193, Japan.
A novel microcapillary osmometer that could measure the osmolarity of the cytosolwith a time constant of less than a second was developed. The osmometer consists of an
osmotic pressure sensor probe and an electronic pressure feedback system. The sensor
probe was made of a glass microcapillary with a tip diameter of ~1µm. The tip was
plugged by semipermeable cupric ferrocyanite. An osmotic pressure reference solution was
introduced into the capillary. Mineral oil was then introduced into the capillary to make ameniscus with the reference solution. When the tip of the probe was inserted into a cell,water moves across the plug according to an osmotic pressure difference between thereference solution and the cytosol, and the meniscus was thereby shifted. The meniscus
shift was detected by a photo-sensor that produced an electrical signal proportional to theshift. The electrical signal was amplified and fed into a pressure generator to generate ahydraulic pressure in the probe that counteracts the water movement. The hydraulicpressure was monitored by using a pressure sensor in the probe. The osmotic pressure of
the cytosol could be determined as a difference between the osmotic pressure of thereference solution and the counter pressure (1).
When the osmometer probe is inserted into a cell together with a microelectrode anda micro-ion-selective electrode, the time course of change in the cytosolic osmolarity of
the cell after changing the external osmolarity can be determined simultaneously withchanges in the membrane potential and the ionic strength of the cytosol. Thesephysiological parameters are indispensable for understanding the mechanisms ofosmoregulation.
If the semipermeable plug is removed from the osmometer probe, the osmometer can
now be used as a manometer that measures the hydraulic pressure of the cytosol. Bulk
modulus of the cell (2) and the amount of osmotically non-active portion of the cytosol canbe determined based on the hydraulic pressure of the cytosol.
(1) Naitoh (2002) Molecul. Biol. Cell., Suppl., 13, 223a.
(2) Iwamoto et al. (2005) J. Exp. Biol., 208, 523-537.
SI-4: microcapillary osmometer, osmotic pressure, cytosolic pressure, osmoregulation,bulk modulus.

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SI-5
AGGRESSIVE BEHAVIOR REGULATION IN A JAPANESE CARPENTER ANT
Mamiko Ozaki
Department of Biology, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan.
The struggle to maintain order in societies has led social animals including humanbeings to evolve and developed various means of communication. Ants have developed asophisticated chemical communication system that enables them to reject non-nestmate
conspecifics and to accept nestmates. Our goal is to understand the underlying mechanisms
involved in these critical decisions. Elucidation of this problem has been elusive andsubject to much speculation, especially at the neurophysiological level. Our study
addresses this problem and suggests a novel stepwise mechanism from the peripheral to thecentral nervous systems via a newly discovered sensory organ that acts to filter pheromonal
information(1).
The newly discovered sensory organ on the antennae of Camponotus japonicus
looked like an olfactory sensillum with multiporous surface, but is actually sensitive tocontact stimulation with cuticular hydrocarbon (CHC) blends (Here we certified that the
CHCs are pheromonal chemical cues for aggressive behavior of the ant.) of non-nestmates
but not nestmates. Thus, it functions as a nestmate-information-cut filter to higher
neuronal systems. Considering adaptation properties in other sensory systems, visualcells for example, we tried to discuss on presumable mechanisms for such a particularfiltering function of this sensory organ.
Considering behavioral data on aggression correlated to the activities of this organ,
we found regulative effect of octopamine on aggressiveness of the ants; octopamine
increases the threshold of aggressive behavior. We constructed ant-octopamine receptor
antiserum and attempted to found the histological localization of the octopamine receptor
around the mushroom bodies and antennal lobes.
(1) Ozaki et al. (2005) Science, 309, 311-314.
SI-5: agressive behavior, pheromone, nestmate recognition, octopamine, ant

––

SI-6
RECENT ADVANCES OF ETHOLOGICAL PHYSIOLOGY FOR FREE-LIVINGANIMALS BY MICRO-DEVICES
Kentaro Q Sakamoto1and Katsufumi Sato2
1

Department of Environmental Veterinary Sciences, Graduate School of Veterinary
Medicine, Hokkaido University, Sapporo 060-0818, Japan.
2International Coastal Research Center, Ocean Research Institute, The University of Tokyo,
Iwate 028-1102, Japan.
Recently there has been remarkable progress in the development of sensory system
coupled with loggers that have been attached to free-living animals, which have named
bio-logging devices. Increases in sensor resolution, sensor diversity and memory size havebeen coupled with decreases in unit size. We have investigated diving behavior andphysiology of penguins by bio-logging devices. It is well observed that a group of Adélie
penguins synchronize the timing of diving and of surfacing. We employed the loggers withtime depth recorder and still camera system to study underwater group behavior of Adélie
penguins and revealed that several birds descended and ascended synchronously andgathered at the bottom depth to feed preys (1). Dive for foraging is essentially importantfor penguins. However it is still little known how penguins make it possible to perform
deep and prolonged dives. To investigate air usage of penguins during dives, we evaluatedthe air volume of diving penguins using a newly developed logger to measure acceleration.
When comparing the passive ascents for shallow and deep dives, there was a positive
correlation between air volume and the depth of the dive. It was suggested that penguins
regulated their air volume to optimize the costs and benefits of buoyancy (2). In case ofdeep dive, the time budget of diving might be modulated by the blood concentration ofcarbon dioxide. Further, it was suggested that penguins decreased metabolic rate andconsumed oxygen in air sac during dive to perform prolonged dives by newly developed
bio-logging devices equipped with oxygen electrode (3). Bio-logging will play a more
important role to understand animals in extreme environment.
(1) Takahashi et al. (2004) J. Ethol., 22, 5-11.
(2) Sato et al. (2002) J. Exp. Biol., 205, 1189-1197.
(3) Stockard et al. (2005) J. Exp. Biol., 208, 2973-2980.
SI-6: Bio-logging, dive, penguin, data logger––SII-1
GENE EXPRESSION IN A SINGLE NEURON
Etsuro Ito
Laboratory of Functional Biology, Faculty of Pharmaceutical Sciences at Kagawa Campus,
Tokushima Bunri University, Sanuki 769-2193, Japan.
Gene expression is differently regulated in every cell even though the cells are includedin the same tissue. We therefore developed a procedure for isolating a single, identifiable
cell and determining the exact copy numbers of mRNAs within it (1). We first isolated the
cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in
the process of memory consolidation of a feeding behavior. We then determined the copy
numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs).
The protocol uses two techniques in concert with each other: a technique for isolating asingle neuron with newly developed micromanipulators coupled to an assay of mRNAs byquantitative real-time reverse transcription-polymerase chain reaction (real-time PCR). The
molecular assay determined the mRNA copy numbers, each of which was compared to a
standard curve prepared from cDNA solutions corresponding to the serially diluted
solutions of Lymnaea CREB mRNA. The standard curves were linear within a range
between 10 to 105 copies, and the intra-assay variation was within 15%. Each neuron
removed from the ganglia was punctured to extract the total RNA directly and was used forthe assay without further purification. Using this two-step procedure, we found that themRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant
cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay(< 25). These results suggest that the CREB cascade is regulated by an excess amount of
CREB2 in the cerebral giant cells. Next, the amounts of CREB1 and CREB2 mRNAs werechanged by an RNA interference method. We thus injected the small interfering RNA(siRNA) of CREB1 or CREB2 into the cerebral giant cells, and then examined the changes
in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons(2). The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereasthat was augmented 60 min after injection of CREB2 siRNA.
(1) Wagatsuma et al. (2005) J. Exp. Biol., 208, 2389-2398.
(2) Wagatsuma et al. (2006) J. Neurosci. Res., 84, 954–960.
SDII-1: CREB, mRNA, real-time PCR, RNAi––

SII-2IDENTIFICATION OF NOVEL G-PROTEIN COUPLED RECEPTORS IN
LASER-CAPTURED SINGLE GnRH NEURONS
Ishwar S. Parhar
School of Medicine and Health Sciences, Monash University, 46150 Petaling Jaya,
Malaysia.
We have developed a novel single cell real-time quantitative PCR technique, which
incorporates harvesting marker-identified single cells using laser-capture. Here, for the
first time in a vertebrate species, using this innovative single cell gene profiling technique,
we report the presence of G-protein coupled receptors in individual gonadotropin-releasing
hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis
niloticus.
The differential expression of multiple combinations of three GnRH receptor types(R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates
cellular and functional heterogeneity. The differential use of GnRH receptors incorticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive
behaviors suggests new roles for these hormones.
Further, we provide evidence of the structure of a novel nonmammalian G-protein
coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly
conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We
hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We
speculate, the use of this method will allow the identification and quantification of known
and unknown genes in single cells, which would greatly facilitate our understanding of thecomplex interactions that govern the physiology of individual cells in vertebrate species.
SII-2: GnRH, G-protein coupled receptor, single-neuron, puberty, neuroendocrinology––SII-3
CHROMATIN REGULATION AT THE HUMAN GROWTH HORMONE GENE
CLUSTER
Atsushi Kimura
Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo
060-0810, Japan.
The human growth hormone (hGH) gene cluster, located on chromosome 17q22-24,
is composed of five structurally conserved genes: hGH-N, chorionic somatomammotropinlike (hCS)-L, hCS-A, hGH-variant (hGH-V), and hCS-B. Although the primary structures
are highly conserved among those five genes, they show mutually exclusive tissue
specificities. hGH-N is expressed only in anterior pituitary whereas expression of theother four genes is restricted to the syncytiotrophoblast (STB) layer of placental villi.
Our previous data have demonstrated that the activation of the hGH cluster in the twotissues is dependent on a set of remote locus control region (LCR) including five DNase I
hypersensitive sites (HS) located between 15 and 32 kb 5’ to the cluster. The HSI at -15
kb plays key roles for establishing a large histone acetylation domain and the LCR domainof transcription required to activate the hGH-N gene in pituitary. In contrast, themolecular mechanisms for activation of the placental genes are not clear.
In order to gain insight into the placental gene activation, we investigated epigenetic
modifications at the hGH locus using several placental model systems. In human term
placental STB, which express the cluster genes at maximum levels, we identified highlevels of histone acetylation and methylation at the LCR and within the cluster region
encompassing the four placental genes. In the BeWo cell line, which barely expressed theplacental genes, only histone H3K4 methylation domains were observed. In freshly
prepared cytotrophoblast (CTB) cells, discrete sites of the LCR and the cluster region were
methylated at histone H3K4 and no histone acetylation was detected. During the
spontaneous differentiation of the CTB to STB, the histone methylation domains werecompletely established and the histone acetylation levels increased at the LCR and within
the cluster region. These data suggest that activation of the hGH cluster in the placenta isdependent on a series of epigenetic modifications and that histone acetylation andmethylation have different and dynamic roles in this pathway.
SII-3: histone modification, placenta, differentiation, growth hormone, locus control region––SII-4
DYNAMICS AND MODIFICATION OF HISTONES IN LIVING CELLSHiroshi Kimura
Nuclear Structure and Function Unit, HMRO, Graduate School of Medicine, Kyoto
University, Kyoto 6060-8501, Japan.
Eukaryotic nuclear DNA is wrapped around a protein octamer composed of the core
histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units ofchromatin. These core histones are highly conserved from yeast to mammals, and play
key roles in chromatin function, such as transcription, DNA replication, repair, andchromosome segregation, through the regulation by post-transcriptional modification andreorganization of nucleosome structure.
We have studied the kinetics of core hisotnes in living cells by photobleaching
analysis and cell fusion, and revealed that H2B in euchromatin is exchanged more rapidly
compared to H3.1 and H4 (1, 2). To understand the molecular mechanisms of histone
exchange found in living cells, we developed an in vitro system using permeabilized cells,
allowing the assembly and exchange of histones in situ. H2A and H2B, each tagged withthe green fluorescent protein (GFP), are incorporated into euchromatin by exchange
independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin.
By fractionating cellular factors assisting the incorporation of H2A–H2B into
permeabilized-cell chromatin, we identified protein phosphatase 2C gamma-subtype(PP2Cgamma) as a histone chaperone that binds to and dephosphorylates H2A–H2B, in
addition to nucleosome assembly protein 1 and 2. These data provide a novel link
between histone deposition and dephosphorylation through a protein phosphatase, further
emphasizing the importance of histone modification.
Towards understanding histone modification signals in living cells, we are generating
specific antibodies against modified histones.
(1) Kimura and Cook (2001) J. Cell Biol., 153, 1341-1353.
(2) Kimura (2005) DNA Repair, 4, 939-950.
(3) Kimura et al. (2006) J. Cell Biol., 175, 389-400.
SII-4: chromatin/gene expression/living cell/nucleosome/protein phosphatase–10–

SII-5SINGLE-MOLECULE ANALYSES OF NEUROTROPHIN
SIGNALLING IN THE GROWTH CONES OF SENSORYNEURONS DURING THE ACTIVATION OF AXONALOUTGROWTH
Tomomi Tani
Research Institute for Electronic Science, Hokkaido University, Kita 12, Nishi 6, Kitaku, Sapporo 060-0812, Japan. E-mail: tani@es.hokudai.ac.jp
Imaging of fluorescently labeledbiomolecules has been used to observe inreal time the interactions between ligands
and receptors on various kinds of living
cells. This technique allows us to analyze
the local
signaltransductionandamplification processes in confined subcellular domains of neurons such as axons,dendrites, synapses and cell bodies. We
employ this method to reveal themechanisms by which neural axons anddendrites detect and reach the target tissues DIC (left) and fluorescent (right) micrographs ofduring development. The growth cone is a the growth cone after the application of
motile structure located at the distal tip of fluorescent NGF. Bar; 10 µm
the nerve fiber. During embryogenesis, the growth cone serves to guide the nerve fiber
to an appropriate target through the aid of guidance cues and neurotrophins. The 2.5S
nerve growth factor (NGF), the founding member of neurotrophins, promotes the axonal
growth of sensory and sympathetic neurons. We observed the interaction of NGF withreceptors on the growth cone of chick sensory neurons using a novel fluorescentconjugate of NGF, Cy3-NGF. Upon application of 400 pM of fluorescent-NGF, thegrowth cones responded within one minute of adding the stimulus by expanding theirlamellipodia. Imaging of fluorescent NGF enabled us to analyze the in situ kinetics of
NGF and the receptor interaction on the growth cone. Based on these analyses, theestimated number of high affinity receptors on a single growth cone was approximately
200 and that of low affinity receptors was 700. Surprisingly, only 40 molecules ofbound NGF, which occupied less than 5% of the estimated total binding sites on asingle growth cone, could initiate the motile responses of a single growth cone. Afterbinding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion, which was
followed by a one-directional rearward movement toward the central region of thegrowth cone. The one-directional movement of Cy3-NGF displayed the same rate as
the rearward flow of actin. Molecules of Cy3-NGF were internalized in the vicinity of
the central region of the growth cone during this rearward trafficking. These results
suggested that actin-driven trafficking of the NGF-receptor complex is an essential stepto the accumulation and endocytosis of NGF at the growth cone. We are currently
combining this single molecule imaging of neurotrophins with fluorescent protein-based
imaging technology for simultaneous observations of activated receptors as well as
intracellular signaling molecules.
SII-5: Single molecule imaging, nerve growth factor, TrkA, growth cone, dorsal root
ganglion–11–

SII-6SINGLE-NEURON MOLECULAR PHYSIOLOGICAL ANALYSIS OF THE
MULTIFUNCTIONAL GNRH PEPTIDERGIC SYSTEMS
Yoshitaka Oka
Department of Biological Sciences, Graduate School of Science, the University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
The GnRH peptidergic neuron systems consist of hypothalamic neuroendocrine andextrahypothalamic neuromodulatory GnRH neurons. Here, I introduce our recent studies
on the physiological properties of neuroendocrine preoptic (POA) GnRH neurons and theneuromodulatory terminal nerve (TN-) and tegmental (TEG-) GnRH neurons, and theircontrol by metastin peptidergic neuron system1. We use a fish model system in which we
can easily identify all of them in intact or GnRH-GFP transgenic fish brain preparations invitro, which is advantageous over brains of most other vertebrates for the single-neuron
molecular physiological analysis of multifunctional GnRH peptidergic systems. In contrast
to the regular pacemaker activities of TN- and TEG-GnRH neurons, the POA-GnRH
neurons showed alternating active and silent phases of firing activities. Now that we havevarious single-neuron molecular physiological experimental approaches (electrochemicalmeasurement of GnRH release, Ca2+ imaging after single-cell electroporation, single-cell
RT-PCR, double patch clamp recording, etc.) and experimental systems (in vitro
preparations of the dwarf gourami and three lines of GnRH-GFP transgenic medaka) inhand, simultaneous multidisciplinary approaches can be applied to study the physiology ofmultifunctional GnRH neurons. We also describe our recent studies on a new metastin
peptidergic neuronal system. We found that the metastin-ir cell bodies are distributed in
two distinctive brain regions. Double immunocytochemistry showed that metastin-ir fibers
project not only near the POA-GnRH neurons but also near TEG- and TN-GnRH neurons.
Furthermore, the metastin neurons project to wide areas in the brain (mainly sensoryrelated brain areas), suggesting that it may modulate the sensory information processing,while it stimulates LH surge.
(1) Abe, H. and Oka, Y. (2007) Neuromodulatory functions of terminal nerve-GnRH
neurons. In : Fish Physiology Vol. 25, Sensory Systems Neuroscience, pp. 455-503.
SII-6: GnRH, physiology, imaging, single-neuron, neuroendocrinology

–12–

P-1
MOLECULAR CLONING AND GENE EXPRESSION PATTERN OF ECDYSONE
20-MONOOXYGENASE IN THE SILKWORM, Bombyx mori
Yoichi Ito1, Sayaka Maeda1, Asuka Nakashima1, Yoshinori Fujimoto2 and Haruyuki
Sonobe11Department of Biology, Konan University, Kobe, 658-8501, Japan.
Department of Chemistry and Material Science, Tokyo Institute of Technology, Tokyo
152-8550, Japan.
2

In silkworm, Bombyx mori, it is known that 20-hydroxyecdysone (20E) is
indispensable for embryonic development (1). 20E was demonstrated to be formed byboth dephosphorylation of physiologically inactive ecdysteroid-phosphates and de novo
synthesis. The hydroxylation of ecdysone (E) at the C-20 position, which is catalyzed byecdysone 20-monooxygenease (E20OHase), is a rate-limiting step in the de novo
synthesis (2). In this study, a cDNA sharing a high sequence similarity with known
E20OHases was isolated from eggs by using 3’and 5’-RACEs (Maeda et al., 2005, Genebank accession No.: 236417). Deduced amino acid sequence of obtained clone has 48%
and 81% identity with Drosophila melanogaster and Munduca sexta E20OHases,
respectively. We assume that this clone is B. mori E20OHase by using phylogenetic
analysis. To confirm the enzyme activity of the cloned B. mori E20OHase, therecombinant E20OHase was expressed using baculovirus expression system. Sf9 cells
were infected with recombinant baculovirus containing the whole open reading frame of
B. mori E20OHase. The infected cell extract was incubated with E, and the product was
analyzed using reverse-phase HPLC. HPLC profile demonstrated that the product is 20E.
Semi-quantitative reverse transcription-PCR analysis revealed that E20OHase mRNAlevels increased 2 days after oviposition and reached a peak 3-4 days after oviposition,and then decreased gradually to the basal level in nondiapause eggs. In diapause eggs,
E20OHase mRNA remained at a low level. This result coincided with the pattern ofchanges in E20OHase activity (3), suggesting that the gene transcription is required forthe induction of E20OHase activity.
(1) Makka et al. (2002) Arch. Insect Biochem. Physiol., 51, 111-120.
(2) Sonobe et al. (1999) Zool. Sci., 16, 935-943(3) Horike and Sonobe (1999) Arch. Insect Biochem. Physiol., 41, 9-17.
P1: keyword: silkworm, ecdysone, P450, diapause, monooxygenase

–13–

P-2
CLONING AND EXPRESSION OF GENES ENCODING FOUR DIFFERENTSUBTYPES OF MELATONIN RECEPTORS IN GOLDFISH
Taro Ikegami and Hironori Ando
Laboratory of Advanced Animal and Marine Bioresources, Graduate School of
Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581,
Japan.
Melatonin is synthesized primarily in pineal gland and retina, where its synthesis andrelease exhibit a diurnal rhythm with peak activity during the dark period. Melatonin is
involved in multiple physiological functions including circadian rhythms and seasonal
reproduction. Melatonin receptors (Mel) belong to the G-protein coupled receptor
superfamily. In vertebrates, there are three types of Mels, termed as Mel1a (MT1), Mel1b(MT2), and Mel1c. In teleosts, two different subtypes of Mel1a were identified, and termed
as Mel1a1.4, and Mel1a1.7. Mel1b and Mel1c were termed as Mel1b2.6 and Mel1c2.3,
respectively. The distribution of Mel1a1.4 and Mel1a1.7 transcripts has been reported
mainly in the brain and pituitary (1), while that of Mel1b2.6 transcript has been reported in
the brain and various peripheral organs, such as retina, liver, spleen, heart, and kidney (2).
However, the distribution of Mel1c2.3 transcript has never been reported yet. As a firststep to clarify molecular mechanisms of action of fish Mels, we determined partial
sequences of the four subtypes of Mel genes in goldfish, and tissue distribution of thesetranscripts were examined by reverse transcription (RT)-PCR.
Four partial DNA fragments were amplified from genomic DNA by PCR using
primers that correspond to the transmembrane domain 2 and 7 of zebrafish Mel genes.
They were cloned into a pCR-Script plasmid vector and their sequences were determined.
The similarities of the deduced amino acid sequences between goldfish and zebrafish were
96-100%. Total RNA was extracted from the brain, pituitary, eyeball, gill, atrium, bulbus
arteriosus, intestine, spleen, kidney, and scale of goldfish. RT-PCR was carried out using
four sets of specific primers. The transcripts of Mel1a1.4 and Mel1a1.7 were distributed in
the brain, gill, bulbus arteriosus, spleen, and kidney. Mel1a1.7 transcript was also detectedin the eyeball. Mel1b2.6 transcript was detected in all tissues examined. Mel1c2.3
transcripts were detected only in the kidney. These results indicate that four subtypes ofMel genes are differentially expressed in the examined tissues of goldfish.
(1) Qiong et al. (2004) Gen. Comp. Endocrinol., 136, 311-321.
(2) Park et al. (2006) J. Pineal Res., 41, 166-174.
P2: Melatonin, Melatonin receptor, Goldfish, Gene expression

–14–

P-3
CLONING OF THE cDNAS ENCODING BONE MORPHOGENETIC PROTEINSFROM RAINBOW TROUT
Rei Ohkado, Shingo Toki, Shigeyasu Tanaka and Masakazu Suzuki
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
The thyroid hormones play important roles in the regulation of development,
metabolism, homeostasis, and reproduction. These hormones are synthesized in the
thyroid follicles, which are scattered over the ventral aorta in the rainbow trout. To
understand the molecular mechanisms underlying the development of the thyroid, we
previously carried out histological analysis of thyroidogenesis in the rainbow trout, using
Nkx2.1 and Pax8 as molecular markers. As a result, in situ hybridization histochemistryof trout embryos showed the thyroid primordium to be surrounded by larger blood vessels,
suggesting that paracrine factors, secreted from the blood vessels, might be involved inthyroidogenesis in the trout. In mice, bone morphogenetic protein (BMP) 4 is known to
be expressed in the outflow-tract myocardium of the developing heart (1). BMPs can also
induce formation of adipose tissue and cartilage, both of which are observed around thethyroid (2). In the present study, we have identified partial sequences of three distinct
BMP cDNAs from a ZAP Express cDNA library prepared from the ventral aorta with thethyroid of the adult trout. PCR was performed using degenerate primers, and sequence
analysis of the amplified products revealed the partial sequences of three types of cDNAs
encoding rainbow trout BMPs, rtBMP2/4, rtBMP5/6, and rtBMP7. The rtBMP2/4 cDNA
is composed of 150 bp, and the deduced amino acid sequence has 78% and 75% similarity
to mouse BMP2 and BMP4, respectively. The rtBMP5/6 cDNA is composed of 64 bp,
and the predicted amino acid sequence shows 74% and 78% similarity to mouse BMP5 and
BMP6, respectively. The rtBMP7 cDNA is composed of 64 bp, and the predicted aminoacid sequence exhibits 86% similarity to mouse BMP7. Because these clones wereobtained from the ventral aorta with the thyroid, it is possible that BMPs might have someeffects on thyroid gland in the rainbow trout.
(1) Liu, W. et al. (2004) Proc. Natl. Acad. Sci., 101, 4489-4494.
(2) Rebbapragada, A. et al. (2003) Mol. Cell. Biol., 23, 7230-7242.
Keywords: Thyroid, BMP, Pax8, Nkx2.1, rainbow trout

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P-4
CLONING OF THE RECEPTOR FOR FROG GROWTH HORMONERELEASING PEPTIDE IN THE BRAIN OF BULLFROGS, Rana catesbeiana
Tomohiro Osugi1,2, Kazuyoshi Ukena2 and Kazuyoshi Tsutsui1,2
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.
We previously identified a novel hypothalamic neuropeptide in the brain of bullfrogs.
This peptide stimulated the release of growth hormone (GH) from the pituitary of bullfrogs
and therefore was named frog GH-releasing peptide (fGRP) (1). The precursor cDNA
encoded fGRP and three fGRP-related peptides (fGRP-RPs; fGRP-RP-1, -RP-2 and -RP-3)
that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln;LPXRFamide peptides) (2). These related peptides were also identified as endogenous
ligands in the bullfrog brain (3). Interestingly, fGRP-RPs have various functions such as
pain modification (fGRP-RP-1) (4), stimulation of GH release (fGRP-RP-2) (3) andstimulation of prolactin release (fGRP-RP-2) (3). Identification of the receptor for fGRP is
crucial to elucidate the mode of action of fGRP. We therefore cloned a putative receptor for
fGRP in the bullfrog brain. We first amplified a partial fragment of the putative fGRP
receptor using PCR primers designed from the sequence of the receptor of other
LPXRFamide peptides. Then we amplified the 5' end and 3' end of the fragment by a
combination of 3' and 5' rapid amplification of cDNA ends (RACE) using PCR primers
specific for the partial sequence of the putative fGRP receptor. The nucleotide and deduced
amino acid sequences of the putative fGRP receptor revealed a full length of 1503 bp andan open reading frame of 1299 bp encoding 433 amino acid residues with a calculated
molecular mass of 49.6 kDa. Hydrophobic analysis revealed that the putative fGRP
receptor possessed seven transmembrane domains, indicating a new member of the Gprotein-coupled receptor superfamily.
(1) Koda, A. et al. (2002) Endocrinology, 143, 411-419.
(2) Sawada, K. et al. (2002) J. Endocrinol., 269, 6000-6008.
(3) Ukena, K. et al. (2003) Endocrinology, 144, 3879-3884.
(4) Kanetoh, T. et al. (2003) Comp. Biochem. Physiol., 134, 259-266.
P4: LPXRFamide peptide, fGRP, GPCR, cloning

–16–

P-5
EXPRESSION OF NOVEL ISOFORM OF BULLFROG PROLACTIN RECEPTOR
Itaru Hasunuma1, Kazutoshi Yamamoto1 and Sakae Kikuyama1,2
1

Department of Biology, School of Education, Waseda University, Tokyo 169-8050, Japan.
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
2

It is known that prolactin receptor (PRLR) belongs to class I cytokine receptor
superfamily. In mammalian species, existence of several isoforms of PRLR such as long,intermediate, and short forms has been reported (1). Previously, we obtained bullfrog
PRLR cDNA encoding 617 amino acids that exhibited the feature of mammalian long form
PRLR (2). In the present study, a novel isoform cDNA of bullfrog PRLR was cloned by the
3'-RACE method using bullfrog kidney cDNA as a template. This isoform cDNA encoded
242 amino acids. The sequence of extracellular domain of the isoform (from translation
initiation Met to Gly321 ) was completely identical with bullfrog long form PRLR, but thetransmembrane domain and intracellular domain were lacking. In the C-terminus, it
contained an isoform-specific amino acid sequence comprising 11 amino acids(NDLIYITHHII). This novel PRLR isoform structure was similar to PRL- binding protein
identified in mammalian species (3). Expression of mRNA of this isoform was observed in
the brain, lung, liver, kidney, testis, and ventral and dorsal skin of adult frog. To confirm
that this isoform binds PRL, PRL-binding assay was performed. A cDNA fragment
containing open reading frame was obtained by RT-PCR, and ligated into pCS2+
mammalian expression vector. Transient expression assays were conducted by using COS7 monkey kidney cells. Cells were harvested at 48 h after the transfection, and then scraped,
lysed by four repeated freeze-thaw cycles. Specific binding of 125I-labeled bullfrog PRL towater-soluble protein fraction of cell lysate was observed. Furthermore, the specificbinding activity was also detected in the medium in which COS-7 cells were transiently
transfected with a construct containing the isoform cDNA were harvested. The result
suggests that novel PRLR isoform protein was secreted from COS-7 cells to the culture
medium.
(1) Bole-Feysot et al. (1998) Endocrine Rev., 19, 225-268.
(2) Hasunuma et al. (2004) Gen. Comp. Endocrinol., 138, 200-210.
(3) Trott et al. (2003) J. Mol. Endocrinol., 30, 31-47.
P5: prolactin, prolactin receptor, isoform, bullfrog

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P-6
ANALYSIS OF mRNAS EXPRESSED IN THE PARATHYROID GLAND OF THE
BULLFROG
Makoto Ueda, Shigeyasu Tanaka and Masakazu Suzuki
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
The parathormone (PTH), derived from the parathyroid gland, is of prime importancefor the control of calcium homeostasis in mammals, and well known to increase circulatinglevels of calcium. Phylogenetically, the parathyroid first occurs in amphibians, but PTH
gene already exists in fish genome. In zebrafish embryogenesis, PTH is found in lateralline neuromasts (1), whereas glial cells missing 2 (Gcm2), a marker gene for theparathyroid, is expressed in the pharyngeal epithelium and gill filament buds (2). For theunderstanding of the molecular mechanisms underlying the occurrence of the parathyroidin vertebrates, it is indispensable to identify more molecules specific to this endocrine
gland, and further to investigate the cellular localization and function of these molecules.
In the present study, we have examined mRNAs expressed in the parathyroid of thebullfrog, Rana catesbeiana, using molecular cloning and suppression subtractive
hybridization (SSH). By RT-PCR and subsequent sequence analysis of the amplified
products, we first tried identifying GCM2 mRNA to confirm that the collected organ was
the parathyroid. As a result, not only GCM2 cDNA but also GCM1 cDNA was cloned
partially. The partial amino acid sequences of bullfrog GCM1 and GCM2 showed 82%
and 89% similarity to Xenopus GCM1 and GCM2, respectively. Although only Gcm2 is
detected in mammalian parathyroid, both Gcm1 and Gcm2 seem to be expressed in thebullfrog parathyroid. The SSH analysis between the parathyroid and fat body further
revealed the expression of transcripts similar to transposase/inhibitor of apoptosis protein 1,
reverse transcriptase, transport permease, elongation factor 1, etc., in the parathyroid. 10
novel transcripts were also isolated. Among these transcripts, however, PTH mRNA
could not be identified presumably because the amino acid sequence of PTH is not highly
conserved between vertebrate classes. It is also possible that PTH might not be produced
abundantly in bullfrog parathyroid.
(1) Hogan, B.M. et al. (2005) Endocrinology, 146, 547-551.
(2) Hogan, B.M. et al. (2004) Dev. Biol., 276, 508-522.
Keywords: parathyroid hormone, GCM1, GCM2, suppression subtractive hybridization,
bullfrog

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P-7
TISSUE DISTRIBUTION AND DEVELOPMENT OF CHICKEN MOTILIN
CELLS
Chihiro Tsutsui, Ichiro Sakata and Takafumi Sakai
Area of Regulatory Biology, Division of Life Science, Graduate School of Science andEngineering, Saitama University, Saitama 338-8570, Japaan.
It is well established that motilin, a 22-amino-acid peptide, is mainly produced in themucosal layer of the mammalian duodenum. Although the sequence and structure ofchicken motilin have been determined, the tissue distribution and morphologicalcharacteristics of motilin-producing cells (motilin cells) in the chicken gastrointestine arestill unknown. In this study, we investigated motilin expression and distribution of motilin
cells in the central and peripheral tissues of thatching (3 days) and adult (125 days)
chickens by RT-PCR and in situ hybridization (ISH).
The results of RT-PCR showed that motilin mRNA was slightly expressed in the
hypothalamus, cerebellum and pituitary gland and was abundantly expressed in the
medulla oblongata, duodenum and large intestineof the hatching chicken. Although motilinmRNA expression was also found in the duodenum and ileum in the adult chicken, theexpression levels of motilin mRNA were lower than these in the hatching chicken. In the
medulla oblongata, motilin mRNA was transiently expressed at the hatching stage but was
not observed at any other stages.
ISH analysis revealed that motilin mRNA-expressing cells (motilin-ex cells) in thehatching chicken were distributed in the mucosal layer but not in the myenteric plexus in
the duodenum and existed only as closed-type cells. On the other hand, no motilin-ex cells
were found in the adult chicken duodenum. These results indicate that motilin mRNA
expression level in the chicken duodenum gradually decreases during development andsuggest that motilin in the medulla oblongata plays an important role in some functions of
the central nervous system in the hatching chicken.
P7: Motilin, Chicken, in situ hybridization

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P-8
cDNA CLONING AND EXPRESSION PROFILE OF mRNA FOR CHICKEN
GPR39
Ichiro Yamamoto1, Makoto Numao2, Yuka Sakaguchi2, Nobumichi Tsushima2 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.
GPR39 belongs to the subfamily of G protein-coupled receptors containing 7transmembrane domains, and shows high homology to ghrelin receptor, GHSR (1).
GPR39 has recently been characterized as a specific receptor for a novel anorexic peptide
hormone, obestatin, isolated from rat stomach (2). However, negative results for theanorexic action and GPR39 binding of obestatin have also been reported. In this study,
we performed cDNA cloning for chicken GPR39 and characterized expression profiles of
its mRNA in chicken tissues. Total RNA was extracted from chicken tissues and GPR39
cDNA was cloned by RT-PCR using primers designed from the putative GPR39 genesequence appeared in chicken genome database. The cDNAs for 3’- and 5’- regions werecloned by RACE. The cloned GPR39 cDNA was about 3.0 kb in length and encoded 462
amino acids. The amino acid sequence showed significant homology to mammals
GPR39 (>60%). Realtime PCR analysis for GPR39 mRNA expression was carried out
using primers derived from Exon 1 (sense) and Exon 2 (antisense) of GPR39 gene.
Realtime PCR analysis revealed that chicken GPR39 mRNA was expressed in all thetissues examined with high levels in digestive organs and oviduct. The expression levelof GPR39 in duodenum was rapidly increased during post-hatch period. These findings
suggest that GPR39 might be involved in peristaltic motion in digestive and reproductive
ducts in chicken.
(1) McKee et al. (1997) Genomics, 46, 426-434.
(2) Zhang et al. (2005) Science, 310, 996-999.
P8: Chicken, GPR39, Obestatin, Duodenum, Oviduct

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P-9
GONADOTROPES EXPRESS THE RECEPTOR FOR GONADOTROPININHIBITORY HORMONE (GNIH) IN QUAIL
Vishwajit S. Chowdhury1,2, Hong Yin1, Takayoshi Ubuka3, Kazuyoshi Ukena1 andKazuyoshi Tsutsui1,2
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, Graduate School of Integrated Arts and Sciences, Hiroshima
University, Higashi-Hiroshima 739-8521, Japan.
3
Department of Integrative Biology, University of California, Berkeley, California 94720,
USA.
Recently, we identified a novel hypothalamic dodecapeptide, Ser-Ile-Lys-Pro-Ser-AlaTyr-Leu-Pro-Leu-Arg-Phe-NH2, in the brain of Japanese quail (Coturnix japonica) (1).
This avian neuropeptide was shown to inhibit gonadotropin release from the cultured quail
anterior pituitary (1). We therefore termed it gonadotropin-inhibitory hormone (GnIH) (1).
This is the first hypothalamic peptide inhibiting gonadotropin release reported in any
vertebrate. GnIH was also effective in inhibiting LH in vivo (2). Interestingly, chronic
treatment with GnIH inhibited gonadal development and maintenance by inhibiting
gonadotropin release and synthesis in quail (2). Because GnIH was shown to be located inneurons of the paraventricular nucleus and their terminals in the median eminence (1, 3, 4),
GnIH may act directly on the pituitary to inhibit gonadotropin release and synthesis. To
elucidate the mode of action of GnIH, we then identified a novel G protein-coupled
receptor for GnIH in quail (5). Southern blotting analysis of reverse-transcriptase mediated
PCR products revealed the expression of GnIH receptor mRNA in the quail pituitary (5). In
this study, we characterized the localization of GnIH receptor in the quail pituitary by insitu hybridization of GnIH receptor combined with immunocytochemistry for LH. GnIH
receptor was located in gonadotropes in the quail pituitary. Thus, it appears that GnIH acts
directly on gonadotropes via GnIH receptor to inhibit gonadotropin release and synthesis.
(1) Tsutsui, K. et al. (2000) Biochem. Biophy. Res. Commun., 275, 661-667.
(2) T. Ubuka, T. et al. (2006) Endocrinology, 147, 1187-1194.
(3) K. Ukena, K. et al. (2003) Cell Tissue Res., 312, 73-79.
(4) Ubuka, T. et al. (2003) J. Endocrinol., 178, 311-318.
(5) Yin, H. et al. (2005) J. Endocrinol., 184, 257-266.
P9: GnIH, GnIH receptor, Gonadotropes, Quail pituitary.

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P-10
EXPRESSION OF RETINALDEHYDE DEHYDROGENASE (RALDH) IN THE
EMBRYONIC ANTERIOR PITUITARY GLANDS OF RATS
Ken Fujiwara, Motoshi Kikuchi, Shu Takigami, Tom Kouki and Takashi Yashiro
Department of Anatomy, Jichi Medical University, School of Medicine, Tochigi, Japan.
Retinoic acid (RA) is a signaling molecule essential for both normal embryonicdevelopment and tissue differentiation in vertebrates. RA is also thought to act as adifferentiation factor in pituitary cells. RA receptors are expressed in the mouse embryonic
pituitary gland (1). RA acts on non-hormone-producing cells among pituitary cells andinduces growth hormone-producing cell differentiation in vitro (2). RA was suggested toinduce pituitary development. However, whether RA is generated in the pituitary gland andplays a role as a paracrine and/or autocrine hormone is generally unknown. RA is
synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze
oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH)
family. In this study, we examined the expression of RALDH1, RALDH2, and RALDH3mRNA in the rat embryonic pituitary gland. Using in situ hybridization with digoxigeninlabeled cRNA probes, we detected mRNA expression of RALDH2 and RALDH3 but not
RALDH1 in Rathke’s pouch and later, in the developing anterior pituitary gland. The
RALDH2 and RALDH3 mRNA signals were faint in the anterior pituitary gland of
neonates. At E14.5, neither RALDH2 nor RALDH3 was expressed in cells positive forexpression of αGSU mRNA. Our findings suggest that RALDH2 and RALDH3 areexpressed in undifferentiated developing pituitary cells. We also used quantitative realtime PCR to analyze RALDH2 and RALDH3 mRNA expression levels during
development of the pituitary gland. As expected, RALDH2 and RALDH3 mRNA levels in
the pituitary gland gradually decreased through the embryonic stages to the postnatal
stages. These results suggest that RA is generated by RALDH2 and RALDH3 in thedeveloping anterior pituitary glands of rats. RA may play a role as a paracrine and/or
autocrine signaling molecule in the developing anterior pituitary gland.
(1) Dolle. et al. (1990) Development, 110, 1133-1151.
(2) Mogi. et al. (2005) J. Endocrinol., 184, 41-50.
P10: Retinoic acid, Rat, Pituitary, Development, In situ hybridization

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P-11
INVERSE RELATIONSHIP BETWEEN GHRELIN AND LEPTIN mRNA
EXPRESSION LEVELS IN THE FASTED RAT STOMACH
Zheng Zhao, Ichiro Sakata, Yusuke Okubo, 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, an endogenous ligand for the growth hormone secretagogue receptor, is a
peptide produced predominantly in the stomach (1). It has been reported that endogenous
ghrelin levels are increased by fasting and decreased after re-feeding (2). It has also beenreported that estrogen upregulates ghrelin expression and production (3) and thatsomatostatin inhibits ghrelin secretion (4), whereas leptin has a paradoxical effect (5,6).
Recently, several studies have shown that estrogen, somatostatin and leptin are produced in
the stomach. However, the direct effects of these gastric hormones on ghrelin expression ina fasting state remain obscure. In this study, we examined the mRNA expression levels ofghrelin, aromatase (estrogen synthetase), leptin and somatostatin in the fasted male rat
stomach by quantitative RT-PCR. Then incubation of stomach tissue was carried out toanalyze the direct effect of leptin on in vitro ghrelin mRNA expression. Finally, double
staining of the male rat stomach for leptin and ghrelin was also performed. After 48 hrs offasting, although gastric ghrelin mRNA expression level was significantly increased, leptinmRNA expression level was decreased. On the other hand, expression levels of aromataseand somatostatin mRNA were not changed after fasting. In vitro ghrelin mRNA expression
was significantly inhibited by leptin at a dose of 100 nM. Double staining showed that
leptin mRNA-expressing cells were adjacent to ghrelin-immunopositive cells. The results
suggest that an elevated gastric ghrelin expression level in a fasting state is due todecreased gastric leptin level.
(1) Kojima et al. (1999) Nature, 402, 656–660.
(2) Lee et al. (2002) Endocrinology, 143, 185–90.
(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.
(6) Toshinai et al. (2001) Biochem. Biophys. Res. Commun., 281, 1220–1225.
P11: Ghrelin; Leptin; Fasting; mRNA

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P-12
TRANSCRIPTION FACTORS PROP-1, LHX2 AND LHX3 ACTIVATE FSHβGENE EXPRESSION
Kousuke Kitahara1, Takanobu Sato1, 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, 2148571, Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa, 214-8571,
Japan.
Recently, we reported that a transcription factor Prop-1 stimulates the promoter
activity of the porcine FSHβ and αGSU genes (1,2). In addition, we cloned Lhx2 as
another transcription factor of the FSHβ gene and analyzed its function. In this meeting,
we report the comparison of binding sequences and transcriptional activity of Prop-1, Lhx2and Lhx3.
To determine binding sites and sequences of Prop-1, Lhx2 and Lhx3 in the FSHβpromoter, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis
using fluorescence-labeled FSHβ upstream fragments were carried out with recombinant
protein of Prop-1, Lhx2 and Lhx3. Transfection assay in LβT2 cells was carried out using
reporter vectors fused with several truncated FSHβ upstream regions (-2320/+10 bp) to
pSEAP2-Basic and expression vectors of Prop-1, Lhx2 and Lhx3 in pcDNA3.1.
EMSA and DNaseI footprinting analysis showed that Prop-1 and Lhx2 bind not only
Fd2 region (-852/-746 bp) but also several regions of porcine FSHβ promoter, whichcorresponded mostly to the regions as reported for Lhx3. Extensive comparison of theresults represented that Prop-1, Lhx2 and Lhx3 share the same binding regions differing in
the binding span and DNase I sensitivity. In addition, unique binding sites each for Prop-1,
Lhx2 and Lhx3 were found. Transfection assay in LβT2 cells showed that Prop-1 and Lhx2
enhance the porcine FSHβ promoter activity as well as Lhx3.
The present study demonstrated that Prop-1, Lhx2 and Lhx3 bind common andunique sequences in the FSHβ promoter and stimulate its transcription. In consequence, the
FSHβ gene is likely to be modulated with several homeodomain transcription factors by
sharing the common and unique regulatory regions.
(1) Aikawa et al. (2004) Biochem. Biophys. Res. Commun., 324, 946-952(2) Sato et al. (2006) J.Mol.Endocrinol., 37, 341-352
P12: Prop-1, Lhx2, Lhx3, FSHβ, Pituitary

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P-13
IN VITRO SELECTION OF PITUITARY TRANSCRIPTION FACTOR PROP-1
RESPONSIVE ELEMENTS
Michie Nakayama1, Takanobu Sato1, Kousuke Kitahara1, Takako Kato3 and Yukio Kato1,2,3
1Division of Life Science, Graduate School of Agriculture, Meiji University, Kanagawa
214-8571, Japan.
2
Division of Life Science, School of Agriculture, Meiji University, Kanagawa
214-8571, Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571,
Japan.
Transcription factor Prop-1 plays an essential role in the expansion of the pituitaryprimordium and the differentiation and development of the hormone-producing cells (1).
Recently, we reported that Prop-1 binds to the upstream region of the porcine FSHβ and
αGSU genes and stimulates their promoter activities (2, 3). Notably, the upstream regionsof porcine FSHβ nor αGSU genes do not contain a consensus sequence for Prop-1 binding,
PRDQ9 (5’-TAATtgaATTA-3’). The present study aimed to reinvestigate the DNA binding
specificity of Prop-1 by employing the SELEX (Systematic Evolution of Ligands by
EXponential enrichment) using random N15 oligomers. Binding ability to Prop-1 was
confirmed by electophoretic mobility shift assay (EMSA) and cis-acting activity wasevaluated by trasnsient transfection assay using GH3 cells.
We found that Prop-1 binds to not only PRDQ9 but also various AT-rich sequencescomposing of AAAT-, AAAC-, TTAT-, TAAA-, TAAC-, and CAAT-N3-ATTA. EMSA andtransfection assay revealed that Prop-1 bound to the identified sequences as a dimmer andactivated the transcriptional level. On the other hands, Prop-1 also binds to a sequence
containing a single ATTA motif as a monomer but cannot enhance promoter activity.
The present results indicate that Prop-1 participates in the regulation of genes bybinding to various AT-rich sequences containing ATTA motif, a consensus motif forhomeodomain transcription factor, supplying with a couple of binding site.
(1) Sornson. et al. (1996) Nature, 384.327-333(2) Aikawa et al. (2004) Biocem. Biophys. Res. Commun., 324,946-952(3) Sato et al. (2006) J. Mol. Endocrinol., 37, 341-352
P13: SELEX, Prop-1, Pituitary, PRDQ9, Homeodomain

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P-14
DISTINCTION BETWEEN LIM HOMEODOMAIN TRANSCRIPTION FACTORS
LHX2 AND LHX3
Takanobu Sato 1, Kousuke Kitahara 1, Akiko Sano 2, Takako Kato 3 and Yukio Kato 1, 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 2148571, Japan.
3
Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571,
Japan.
Recently, we cloned a LIM homeodomain transcription factor Lhx2 as a trans-acting
factor for the porcine follicle-stimulating hormone β subunit (FSHβ) gene. As Lhx2 hasbeen to regulate the pituitary glycoprotein hormone α subunit (αGSU) gene expression (1),
our finding represents that Lhx2 regulates the FSH production at a transcriptional level. On
the other hand, it is acknowledged that Lhx3 has similar effects on αGSU and FSHβ genes(2, 3). In this study, we attempt to determine the Lhx2 and Lhx3 binding sequence in the
αGSU and FSHβ promoters, and to evaluate the cis-acting activity by transient transfection
assay using LβT2 cells (gonadotrope cell line) and CHO cells (Chinese hamster ovary cellline).
DNase I footprinting analysis revealed that the binding sites of both factors are
somewhat different. For example, in the αGSU promoter, Lhx2 and Lhx3 bound to–340/–329 and –340/–326 bp, respectively. The sequence AGCTAATTAA (–340/–329 bp)
was common to them and contained TAAT and ATTA, a core motif of homeodomaintranscription factors. DNase I footprinting analysis of both factors for FSHβ promoter gave
similar but subtle different profiles. Transient transfection assay showed that Lhx2 andLhx3 activate αGSU and FSHβ promoters in CHO cells. Notably, in LβT2 cells, Lhx2
represses αGSU promoter, while Lhx2 activates FSHβ promoter and Lhx3 activated bothpromoters.
In summary, LIM homeodomain transcription factors Lhx2 and Lhx3 share the target
sites in the same regions with differences in the binding mode and the effectiveness of gene
expression.
(1) Roberson et al. (1994) Mo. Cell. Biol., 14, 2985-2993.
(2) Bach et al. (1997) Genes. Dev., 11, 1370-1380.
(3) West et al. (2004) Endocrinology, 145, 4866-4879.
P14: Lhx2, FSH, Pituitary, Pig, Homeodomain

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P-15
A NOVEL PITUITARY TRANSCRIPTION FACTOR, PAIRED-RELATEDHOMEOBOX FACTOR 2, REGULATES GONADOTROPIN SUBUNIT GENES
Akiko Sano 1, Takanobu Sato 2, Kousuke Kitahara 2, Takao Susa 1, 2, Takako Kato 3 andYukio Kato 1, 2, 3
1

Department of Life Science, School of Agriculture, Meiji University, Kanagawa 214 8571,
Japan.
2
Division of Life Science, Graduate 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 (1) as a bait sequence. We
previously cloned porcine (p) Prop-1 (2) and pLhx2, and recently succeeded in cloning of a
porcine paired-related homeobox factor 2, pPrx2, as a transcription factor for pFSHβ gene.
Our recent studies revealed that pProp-1 regulates porcine glycoprotein hormone α
subunit (pαGSU) gene, but not porcine luteinizing hormone β subunit (pLHβ) gene (3).
This study focused whether pPrx2 regulates the gene expression of pαGSU and/or pLHβ in
addition to that of pFSHβ.
Transient transfection assay using -1059/+12 bp of pαGSU gene, -2320/+10 bp of
pFSHβ gene and -1284/+7 bp of pLHβ gene fused to pSEAP2-Basic showed an activationof promoter activities by pPrx2. Electrophoretic mobility shift assay and DNase I
footprinting analysis using recombinant pPrx2 and FAM-labeled DNA fragments ofpαGSU, pFSHβ and pLHβ promoters revealed that pPrx2 binds to all gonadotropin
subunit promoters by recognizing the AT-rich sequences. Following transient transfection
assay using a series of deletion mutants of three promoter regions in CHO cells
demonstrated that some of the binding sites are responsive to pPrx2.
In summary, we succeeded in cloning of pPrx2 as a transcription factor which
regulates FSH and LH at a transcriptional level. It is expected that pPrx2 provides novel
clues to understand the mechanisms of gonadotropin subunit gene regulation.
(1) Kato et al. (1999) Mol. Cell. Endocrinol., 158, 69-78.
(2) Aikawa et al. (2004) Biochem. Biophys. Res. Commun., 324, 946-952.
(3) Sato et al. (2006) J. Mol. Endocrinol., 37, 341-352.
P15: Prx2, Gonadotropin, Pig, Pituitary, Homeobox

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P-16
EXPRESSION AND CHROMATIN STRUCTURE OF GRANULOSA-CELL
SPECIFIC GENES IN THE MOUSE OVARY
Vanessa Ribeiro and Atsushi Kimura
Division of Biological Sciences, Graduate School of Life Science, Hokkaido University,
Sapporo. Japan.

The aim of this research is to investigate the mechanisms of gene expression in the
reproductive organs. As a first step, we set to investigate the mechanisms of specific gene
expression in reproductive tissues. We based our studies on the hypothesis that genes
which are expressed in the same reproductive tissue present, at least to some extent, similar
activation mechanisms.
This time, we focused our studies on the granulosa cells in the ovary. We cloned theAmhr2, inhibinβ B, and Scd2 genes and their expression patterns showed that they arehighly expressed in the reproductive organs. Moreover, the genes Amhr2 and Scd2 showed
to be expressed specifically in the granulosa cells in the ovary. Since recent studies haveshown that the regulation of chromatin structure plays an important role in gene expression,
we investigated the chromatin structure of these three genes. Investigation on the granulosa
cells detected DNase I HS sites on the promoter region of Inhibinβ B gene and on the
intron 6 of Amhr2 gene.
The comparison of the mouse Amhr2 gene sequence to the human and the rat
sequences showed high homology between DNase I HS sequence on the mouse Amhr2
gene and the correspondent sequences on the human and rat genes. We found that the HS
sequences in the Amhr2 include binding sites to the transcription factors Sp1, NF-1, YY1,
ISGF-3, NF-ATc, NF-ATp, NF-ATx, HNF-3alpha, RXR-alpha, VDR, ER-alpha, ER-beta,
LF-1, and LXR-alpha that are also present in the Inhibin β B promoter. The current results
suggest that these transcription factors may play an important role in gene regulation and in
the activation of genes in granulosa cells.

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P-17
EXPRESSED SEQUENCE TAGS FROM THE OVARY OF THE RED SEA URCHIN(Pseudocentrotus depressus)
Keisuke Yamano1 and Tatsuya Unuma2
1

National Research Institute of Aquaculture, Fisheries Research Agency (FRA), Minami-ise,Mie 516-0193, Japan.
2
Japan Sea National Fisheries Research Institute, FRA, Suido-cho, Niigata 951-8121, Japan.
The molecular nature of the gonad-stimulating substance (GSS) of the starfish has
recently been clarified, and the goal of our study is to identify the GSS of the sea urchin, whichis expected to be involved in oocyte growth and maturation. The sensitive methods for
examining the effects of GSS candidates on oogenesis are essential to find the sea urchin GSS,
and the microarray method for monitoring the expression levels of genes in the ovary is usefulfor this purpose. Here, cDNAs for spotting on microarray slides were isolated from the ovaryof the red sea urchin (Pseudocentrotus depressus) and characterized.
Total RNA prepared from the ovary of the red sea urchin at the stage of mid oogenesis
was reversetranscribed to cDNA. The redundancy of cDNA formed by abundant mRNA
was reduced (normalization) by the Trimmer-Direct cDNA Normalization Kit (Evrogen JCS),
and then cDNAs were amplified by PCR based on the SMART technique (Clontech
laboratories. Inc.). A directional cDNA library was constructed, from which 960 clones wererandomly one-pass sequenced from the 5'-end of the cDNAs.
The average length of
nucleotide sequences (ESTs) obtained was 644 bases, and 941 ESTs contained more than 100
bases. The 941 ESTs, assembled to create contigs from overlapping ESTs with the use of thegenetyx-mac ATSQ program, formed 60 contigs from 132 ESTs; the remaining 809 ESTs were
singletons, and the contigs were made from 4 ESTs at most.
Thus, 869 independent
sequences were obtained, which strongly suggesting that the library was successfully
normalized. The public database of DNA sequences was searched by the blast programs tofind counterparts of ESTs. About 70 % of ESTs showed significant homologies (E-value,less than 1E-20) to deposited sequences. The highest matches were seen mostly againstsequences from the purple sea urchin (Strongylocentorotus purpuratus) whose whole genome
sequence has been completed. The variety of cDNAs obtained in this study could be of greatuse in the establishment of a microarray method for evaluating the gene expression profiles
during oogenesis in the red sea urchin.
This work was partly supported by the Program of Basic Research Activities forInnovative Biosciences (PROBRAIN).
P17: EST, sea urchin, ovary, oogenesis

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P-18
SURVEY OF PITUITARY HORMONES ON THE HATSCHEK’S PIT OFAMPHIOXUS
Yukiko Tando, Mayumi Inaba and Kaoru Kubokawa
Group of Marine System Analysis, Center for Advanced Marine Science, Ocean ResearchInstitute, The University of Tokyo, Nakano, Tokyo 163-8639, Japan.
Amphioxus, which is categorized into subphylum Cephalochordata, is known as thephylogenetically closest invertebrate to vertebrates. Hatschek’s pit, which exists in a rightside of notochord in the amphioxus head, is considered as the putative homologue of thepituitary of vertebrates because of the presences of secretary granules and immunoreactive
cells against a human luteinizing hormone. Furthermore, the role of Hatschek’s pit has
been suspected to be important in the reproductive functions, which might be occurred bythermal, chemical, or pheromonal cues, based on its structure opening to the water currents
in the mouth cavity(1). However, direct evidence on the pituitary function has never been
demonstrated. In order to study on the function of the Hatschek’s pit, we attempted tosurvey the tissue specific genes expressed in the pit of amphioxus Branchiostoma belcheri.
Hatchek’s pit is very small organ which is 300µm in length, and strongly connectedwith epithelial tissues. It is difficult to anatomically remove the only pit by using ordinary
dissection methods. We used a novel technology of Laser-microdissection (LMD), which isable to dissect selected tissues, to collect the only pit. One hundred forty pieces of crosssections of frozen amphioxus heads were prepared on a few slide glasses from fifteen
animals. Whole tissues of Hatchek’s pit were cut by using LMD and collected in a tube.
Total RNA was extracted from the collected sample and we constructed cDNA library by amethod of subtractive-hybridization, which using suppression PCR method. By sequencing
two hundreds of subtracted clones, five clones which belonged in different gene families
were obtained as candidates for genes specifically expressed in the pit. These clones
showed homologies to cDNAs of amphioxus Branchiostoma floridae which are placed in
an EST database. However, the specific expression of these clones did not demonstrate
only in Hatchek’s pit. We continue to survey the specific genes on the pit by sequencing
more number of remaining clones, and try a differential cloning method.
(1) Gorbman, A. et al. (1999) Gen. Comp. Endocrinol., 113, 251-254.
P18: amphioxus, Hatschek’s pit, Laser-microdissection

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P-19
MOLECULAR IDENTIFICATIONS OF THREE ISOFORMS OF PPARS(PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS) 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.
In gekkonid lizards, a tail is an important site for lipid storage and is indispensablefor their survival in desert environments (1). In order to reveal the molecular mechanism oflipid metabolism in tails of the lizards, we focused on peroxisome proliferator-activatedreceptors (PPARs). PPARs are the members of superfamily of the nuclear hormone
receptors. A great number of studies in rodent and human have shown that PPARs wereinvolved in the lipids metabolism.
In this study, we identified a sequence of leopard gecko (lg) PPARγ cDNA andpartial sequences of PPARα and PPARβ cDNA containing a DNA binding domain. In
addition, we identified a sequence of PPARγ2 variant. The amino acid sequence deduced
from lgPPARγ cDNA shows a high similarity with its corresponding peptides of crocodile,turtle, mammals, and birds. In the phylogenetic tree drawn with this sequence and its
corresponding sequences of mammals, birds, reptiles and fishes, the leopard gecko
clustered with reptiles and birds. The RT-PCR analysis revealed that all three isoforms of
PPARs was detected in all tissues examined, except that PPARγ2 was found only in theabdominal fat body and tail adipose tissue. The competitive PCR assay revealed thatlgPPARγ mRNA was highly expressed in the abdominal fat body, tail adipose tissue andtail muscle, whereas lgPPARγ2 shows less expression in the adipose tissue in comparison
with total expression of lgPPARγ. lgPPARα showed high expression in the liver andlgPPARβ showed high expression in the brain and liver. These results may reflect thedifference of their physiological functions. PPARγ is mainly associated with lipid storagein adipose tissues, while PPARα and PPARβ are associated with fatty acid oxidation.
(1) Bustard. H.R. (1967) Science, 158, 1197-1198.
P19: PPAR¹Lipid¹Metabolism¹Reptile¹Tail

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P-20
MASS ANALYSIS OF SECRETORY PROTEINS EXPRESSED IN THE EARLYFORMATION OF THE PITUITARY OF FETAL RATS
Takashi Nakakura1, Masakazu Suzuki2 and Shigeyasu Tanaka1,2
1

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka
University, Shizuoka 422-8529, Japan.
2
Deptartment of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
The hypothalamic-pituitary axis plays a pivotal role in vertebrate endocrine systems
by integrating signals from the environment and the brain. The pituitary gland is composedof an adenohypophysis (the pars distalis, pars intermedia, and pars tuberalis) and aneurohypophysis (the pars nervosa). The former is derived from an invagination of the oral
ectoderm of the stomodeum, known as Rathke’s pouch, whereas the latter is an
infundibulum of the diencephalon. Hormones in the pars distalis (PD) are regulated byhypothalamic neurohormones that are produced in the hypothalamic area and transported
to the PD via portal vessels.The PD is vascularized by hypophysial portal vessels that arise
from the capillary beds in the median eminence of the hypothalamus, and this hypophyseal
portal system provides an important link for carrying hormonal information from thecentral nervous system to the pituitary. The capillaries of the PD are characterized byrichly fenestrated endothelia.
The results of a previous study by our group suggest that vascular endothelial growth
factor (VEGF-A), which is known to have a mitogenic effect or guidance effect on theendothelial cells, is involved in the development of the primary capillaries and in thevascularization of the PD, but not in the development of the portal vessels between
hypothalamus and pituitary. This latter conclusion is based on the observation that theformation of portal vessels begins at E13.5, before the expression of VEGF-A in the rostral
region of the pars distalis (1). The assumption drawn, therefore, was that other signal
molecules may be involved in the formation of the pituitary portal vessels. In the present
study, we extensively analyzed secretory proteins by the Signal Sequence Trap method
using rat pituitary anlage at E13.5, the pint at which the formation of the portal vessels
starts. We cloned and characterized about 500 clones, eventually identifying several
unknown molecules. Spatiotemporal expression of unknown molecules was examined byin situ hybridization and RNA dot blot analysis. We expect that novel growth factors otherthan VEGF-A will be identified in near future.
(1) Nakakura et al. (2006) Cell Tissue Res., 324, 87-95.
Key words: pituitary gland, hypophyseal portal system, formation of portal vessels, VEGFA, Signal Sequence Trap method,

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P-21
SELECTIVE COLLECTION OF CATECHOLAMINERGIC (CA) NEURONS IN
THE BRAIN AND ITS APPLICATION TO GENE EXPRESSION ANALYSES
Hiroaki Nakamura1, Yoshiyuki Ishii1, Yasufumi Sato2, Kazuto Kobayashi3 and Keiichi Itoi1
1

Laboratory of Information Biology, Graduate School of Information Sciences, Tohoku
University, Sendai 980-8579, Japan.
2
Department of Vascular Biology, Institute of Development, Aging and Cancer Tohoku
University, Sendai 980-9575, Japan.
3
Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical
University, Fukushima 960-1295, Japan.
In the brain, catecholaminergic (CA) neurons are localized in discrete regions
including the olfactory bulb (OB), the ventral tegmental area (VTA), the substantia nigara(SN), the arcuate nucleus of the hypothalamus, the locus coeruleus (LC), and the nucleusof the solitary tract (NTS). Among these neurons, those in the brain stem and/or the
hypothalamus are implicated in physiological functions including feeding, sleep andarousal, emotional actions, and reproductive functions, as well as stress responses. We
have been studying the functional roles of the CA neurons in these regions using animalmodels. In the present study, the tyrosine hydroxylase (TH)-green fluorescence protein(GFP) transgenic mouse (TH-GFP mouse) was used in order to characterize the gene
expression profile of CA neurons in the brain stem. The GFP-labeled NA neurons in the
fetal LC were collected with high precision, i.e., 95% or more, both in specificity andselectivity, using flow cytometry (FACS). We made comprehensive analysis of transcripts
expressed in the NA neurons in the LC using GeneChipTM (Affymetrix), and identified
transcripts that are specifically expressed in the LC. The result of the present study
provides us with a new method for analyzing extensively the gene expression profiles inthis single species of NA neurons in a highly specific manner.
(1) Sawamoto et al. (2001) Pro.Natl.Acad.Sci.USA., 98, 6423-6428.
Key wards: Micro array, flow cytometry, catecholamines, green fluorescence protein

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P-22
COMPARATIVE ANALYSIS OF THE GONADAL AND PITUITARY GNRH
SYSTEMS IN CHORDATESTadahiro Ikemoto 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.
GnRH was originally identified as a hypothalamic decapeptide, gonadotropinreleasing hormone, which stimulates pituitary gonadotropes to synthesize and release
gonadotropins. GnRH and its receptor are expressed in various extrapituitary sites,suggesting that GnRH has diverse physiological functions. We identified genes encoding
GnRH ligands and receptors from various animal classes of chordates. The results revealed
that gene numbers of both ligands and receptors remarkably differ among and in somecases within animal classes. This indicated that comparative approaches are indispensable
to clarify the complex physiology of GnRH systems so as to understand generality andspecies specificity of physiological functions of GnRH. In these situations, we found that
despite differences in gene numbers, the expression of GnRH receptor in gonads is wellconserved in many chordate classes including Ascidiacea. It was hypothesized thatphysiological roles of GnRH in gonads would be an early evolved function originating
before the emergence of the pituitary gland and have been conserved significantly. In thisstudy, we characterized GnRH systems in the anterior pituitary gland and gonads inchordates by focusing on the leopard gecko Eublepharis macularius, a species with two
GnRH isoforms and three GnRH receptor subtypes. The quantitative RT-PCR analysis
revealed that only one receptor subtype was expressed in the anterior pituitary gland.
Meanwhile, all the three receptor subtypes were expressed in gonads with high levels.
Seasonal and developmental changes in the expression of GnRH receptors were observedin ovarian follicles. In situ hybridization showed spatial expression patterns of ovarian
receptors and suggested co-expression of multiple receptor subtypes in granulosa cells. Cotransfections of multiple receptor subtypes showed distinct pharmacologies in COS-7 cells
compared with those of single transfections, possibly due to interaction and signaling
crosstalk between multiple receptor subtypes. These findings combined with results inother chordate species suggest that distinct signaling mechanisms are involved in the
gonadal and pituitary GnRH systems. Molecular diversity of GnRH systems andcombination of GnRH ligands and receptors may largely contribute to the functional
diversity of GnRH.
P22: GnRH, leopard gecko, reptile, pituitary gland, evolution

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P-23
CLONING AND CHARACTERIZATION OF cDNAS ENCODING PITUITARYADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE FROM THE TELEOSTAND ELASMOBRANCH FISH
Kotaro 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) is widely distributed in
the central nervous system (CNS) and peripheral tissues, and is involved in many
physiological processes, such as cell proliferation, differentiation, cell death,neurotransmission and neuroprotection in mammals (1-3). On the other hand, PACAP
plays a role in mediating pituitary hormone release in the teleost fish (4). The aim of thepresent study was thus to use the teleost and elasmobranch fish, fish models employed to
study the neuronal actions of PACAP, to examine the structural characterization of PACAP
in fish. We examined the nature of PACAP cDNAs isolated from the goldfish, Japanesedace, stargazer and stingray. The primary structure of PACAP deduced from its cDNA ishighly conserved among these species, while that of growth hormone releasing hormone
which is also encoded in PACAP cDNA has a few variation. We also observed the
distribution of PACAP mRNA in fish. PACAP mRNA expression was mainly found in the
CNS and visceral organs such as the heart, stomach, intestine and gonads.
(1) Arimura (1998) Jpn. J. Physiol., 48, 301-331.
(2) Sherwood et al. (2000) Endocr. Rev., 21, 619-670.
(3) Vaudry et al. (2000) Pharmacol. Rev., 52, 269-324.
(4) Montero et al. (2000). J. Mol. Endocrinol., 25, 157-168.
Keyword: PACAP, GHRH, cDNA, teleost, elasmobranch

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P-24
NUCLEOTIDE SEQUENCES OF PROOPIOMELANOCORTIN SUBTYPE GENES
IN BARFIN FLOUNDER, PLEURONECTIFORMES
Yuki Kobayashi, Yushi Yamamoto and Akiyoshi Takahashi
School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Proopiomelanocortin (POMC) is a precursor of melanocortins (MCs) such as
melanophore-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH),
which contains an essential sequence, His-Phe-Arg-Trp, and a single β-endorphin (βEND).
POMC is mainly produced in the corticotropes of pars distalis and the melanotropesof pars intermedia of the pituitary, in which tissue-specific cleavage and modification takeplace; ACTH and β-END are preferentially generated in the pars distalis and MSHs and Nterminally acetylated β-END in the pars intermedia (1). One transcription factor, theTbox factor Tpit, is critical for the differentiation of two POMC lineages and expression of
the POMC gene (2). Recently, we found that the barfin flounder, Pleuronectiformes,
expresses three subtypes of POMC (A, B, and C) in the pituitary (3). The expression of
POMC-A was restricted in the pituitary, while POMC-B and -C were expressed in thebrain and many peripheral tissues. The present study was undertaken to determine thegene structure of barfin flounder POMCs as a first step to examine functional
differentiation of the POMCs.
The 5’-flanking regions of POMC subtype genes were amplified from genomic DNA
prepared from barfin flounder liver by cassette polymerase chain reaction (PCR).
POMC-A (-648 to -1) contained one TATA box, two cAMP response element-like elements(CRE-like), two Eboxes and one retinoic acid-inducible element. POMC-B (-958 to -1)
contained one TATA box, one CCAAT box, four E boxes, one CRE-like, and one bindingsite for Tpit. POMC-C (-584 to -1) contained one TATA box, two CCAAT boxes, two E
boxes, and one binding site for Tpit. There were no similarities in the distribution
patterns of these cis-elements among the three POMC genes. Nucleotide sequences of
introns were determined using DNA amplified from genomic DNA by PCR. POMC-A, B,
and C genes were composed of three exons and two introns. In these genes, an open
reading frame was encoded in the second and third exons, and all MSH and β-END
segments were encoded on the third exon. These structural characteristics are similar to
POMC genes of other vertebrates (1). In the barfin flounder, intron B of POMC-A and -B
genes contained microsatellites consisting of a CA repeat. The molecular architecture of
the POMC gene may have been established in early vertebrates, and present vertebrates
have inherited it. In the barfin flounder strain, at least two duplications may haveoccurred on the POMC gene; ancestral POMC-A/B and POMC-C occurred at the first
duplication, and POMC-A/B split into POMC-A and POMC-B at the second duplication.
The CA repeat may have been inserted into intron B of ancestral POMC-A/B. The
difference in tissue distribution of POMC genes may result from the diversification of ciselements in the 5’-flanking region.
(1) Takahashi and Kawauchi (2006) In: M. Reinecke (Eds.), Fish Endocrinology, pp. 325-392.
(2) Drouin. (2006) In: A. Arzt et al. (eds.), Pituitary Today, vol. 35, pp. 74-87.
(3) Takahashi et al. (2005) Gen. Comp. Endocrinol., 141, 291-303.

Key word:proopiomelanocortin, intron, exon, cis-elements, barfin flounder.

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P-25
RELATIONSHIP BETWEEN ANAL RESPIRATION AND NEUROSUBSTANCESIN THE CRAYFISH, Procambarus clarkii
Akikazu Yasuda and Yoshimi Yasuda-Kamatani
Suntory Institute for Bioorganic Research, Shimamoto, Osaka 618-8503, Japan.
“Anal respiration” in the crayfish has been described in the book "The Crayfish: An
Introduction to the Study of Zoology" published in 1880. The observation has been notedthat water is alternately taken into and expelled from the anus fifteen to seventeen times ina minute and it seemed to be regulated by the sixth abdominal ganglion.
To investigate neurosubstances included in the sixth abdominal ganglion, topologicalmass spectrometry analysis was available. Direct matrix-assisted laser
desorption/ionization with time-of-flight mass spectrometry with slice of the sixth
abdominal ganglion was resulted in the presence of orcokinin and its gene-related peptides,
crustacean-SIFamide, decapod tachykinin-related peptide, and crustacean cardioactive
peptide. These peptides, together with acetylcholine, γ-amino butyric acid and L-glutamic
acid, were examined in vivo on bioassays for rhythmic anal contraction and anal water flux.
The activity of the peptides on the isolated intestine was also examined in vitro. Rhythmic
anal contraction was caused by orcokinins, decapod tachykinin-related peptide, andacetylcholine, when each sample of 100 pmol was injected into the crayfish. Crustacean
cardioactive peptide had most potent activity of anal opening that was kept for fifteen
seconds or more. Water flux through the anus was observed by injection of crustacean
cardioactive peptide or decapod tachykinin-related peptide. Interestingly, the action of theneurosubstances on the isolated intestine was diversified. The contraction of hindgut was
activated by orkokinins and acetylcholine, but not by crustacean cardioactive peptide.
Decapod tachykinin-related peptide showed repetitive action alternating between
contraction and relaxation on the rectum. Crustacean-SIFamide caused complex
movements of the hindgut, and induced most potent activity of defecation.
The crayfish can take water from the anus, but each function of neurosubstances
identified in sixth abdominal ganglion remains unclear. We think that “anal respiration”
might not be a normal phenomenon, since the action which is alternately taken into andexpelled from the anus fifteen to seventeen times in a minute was able to be reproduced by
the overdose of a combination with crustacean-SIFamide and decapod tachykinin-related
peptide.
P25: crayfish, anal respiration, neuropeptide

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P-26
PURIFICATION, KINETIC CHARACTERIZATION, AND
CLONING OF A NOVEL ENZYME ECDYSTEROID 22-KINASE

MOLECULAR

Haruyuki Sonobe1, Ryoichi Yamada1, Tsuyoshi Ohira2, Katsunori Ieki1, Sayaka Maeda1,
Yoichi Ito1, Asuka Nakashima1, Masahiro Ajimura3, Kazuei Mita3 and Marcy N. Wilder2
1

Department of Biology, Konan University, Kobe 658-8501, Japan.
Japan Int’l Research Center for Agricultural Sciences, Tsukuba 305-8686, Japan.
3
Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba
305-8635, Japan.
2

It has been suggested that the ovaries of most insect species have the capacity toaccumulate ecdysteroid phosphates, which are physiologically inactive, and theecdystreoid phosphates function as a source of free active ecdysteroid before theprothoracic glands differentiate during embryonic development (1). Recently, in thesilkworm B.mori eggs, a novel enzyme ecdysteroid-phosphate phosphatase (EPPase),
which is specifically involved in dephosphorylation of ecdysteroid phosphates, wasisolated, characterized and revealed to be a vital enzyme that may control theßon/off
switchà of embryonic development (2). In this study, ecdysteroid 22-kinase (EcKinase)was purified from the cytosol of the B. mori ovaries, and the biochemically characterized.
Results obtained indicated that the reciprocal conversion of free ecdysteroids andecdysteroid 22-phosphates by two enzymes, EcKinase and EPPase, plays an important rolein ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial
amino acid sequence obtained from purified EcKinase, the nucleotide sequence of thecDNA encoding EcKinase was determined. The full-length cDNA of EcKinase wascomposed of 1,850 bp with an open reading frame encoding a protein of 386 amino acidresidues. A database search showed that EcKinase has an amino acid sequencecharacteristic of phosphotransferases (Brenner’s motif), but there are no functional proteinsthat share high identity with the amino acid sequence of EcKinase (3).
(1) Makka T. et al. (2002) Arch. Insect Biochem. Physiol., 51, 111-120.
(2) Yamada, R. and Sonobe, H. (2003) J. Biol. Chem., 278, 26365-26373.
(3) Sonobe H. et al. (2006) J. Biol. Chem., 281, 29513-29524.
P26: Molting hormone, Ecdysteroid, Ecdysteroid 22-kinase, Phosphorylation of steroid
hormone, Silkworm

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P-27
PRIMARY STRUCTURE AND POST-TRANSLATIONAL PROCESSING OF
PROOPIOMELANOCORTIN IN DOGFISH, TRIAKIS SCYLLIUM
Akiyoshi Takahashi1, Ryo Goto1, Shunsuke Moriyama1 and Susumu Hyodo2
1School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 1648639, Japan.
2

In tetrapods, proopiomelanocortin (POMC) is composed of adrenocorticotropin(ACTH), β-lipotropin (β-LPH) and N-POMC segments. Each segment contains one
MSH; α-MSH in ACTH, β-MSH in β-LPH, and γ-MSH in N-POMC. β-LPH also
contains β-endorphin (β-END). The pituitary is a major tissue producing POMC;
however, POMC-derived peptides of the pars distalis (PD) differ from those of the pars
distalis (PI). Final products in the PD are N-POMC, ACTH and β-END, and those in the
PI are MSHs and N-terminally acetylated and C-terminally truncated β-END. In
cartilaginous fish, the primary structure of POMC has been shown in Holocephalans(Chimaeriformes) (1) and Elasmobranchs (Heterodontiformes, Squaliformes, andRajiformes) (2-4). They characteristically have an additional MSH (termed δ-MSH) in
the β-LPH segment; however, no information is available for the post-translational
processing of POMC. In the present study, we first determined the primary structure of
POMC by cDNA cloning in dogfish, Triakis scyllium, of Lamniformes, one of the fiveorders of Elasmobranchii, and then screened POMC-derived peptide in PD and PI withMALDI-TOF mass spectrometry to evaluate post-translational processing of POMC in
cartilaginous fish.
Triakis pituitary cDNA library was prepared using the pGCAP10 vector. POMC
cDNA was identified with BLAST analysis of 1536 clones. Triakis prePOMC contained
four MSHs and a single β-END as in other cartilaginous fish POMCs and showed thehighest sequence identity (72%) with Squalus and Heterodontus prePOMC. All
hormonal segments flanked by basic amino acids also showed the highest sequence
identity with Squalus peptides excluding β-MSH, which was most similar to theHeterodontus peptide. MALDI-TOF mass spectrometry was performed using extracts ofrostral PD (RPD) and PI. As a result, ACTH, β-END, and joining peptide (JP) weredetected in the extract of the RPD, and α-MSH, β-MSH, β-MSH1-16, γ-MSH, δ-MSH, βEND, β-END1-30, JP, and some other POMC-derived peptides were identified in the extractof PI; however, neither acetylated α-MSH or acetylated β-END was detected in PI.
In summary, Triakis POMC has the same molecular architecture as othercartilaginous fish. Post-translational processing of Triakis RPD differs from that of PI as
shown by the identification of differential final products. These tissue-specific processing
mechanisms are similar to other vertebrate pituitaries. No detection of acetylated
peptides suggests the lack of an acetylation system in melanotrophs of the Triakis pituitary.
(1) Amemiya et al. (1999) Gen. Comp. Endocrinol., 114, 387-395.
(2) Amemiya et al. (2000) Gen. Comp. Endocrinol., 118, 105-112.
(3) Dores et al. (2003) Gen. Gomp. Endocrinol., 133, 71-79.
(4) Takahashi et al. (2004) Gen. Comp. Endocrinol., 138, 159-165.
P27:

proopiomelanocortin, post-translational processing, cartilaginous fish, Triakis.

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P-28
OPERATION OF THE TISSUE-TYPE PLASMINOGEN ACTIVATOR /PLASMINSYSTEM IN THE MEDAKA OVARY
Kazuto Minagawa, Katsueki Ogiwara and Takayuki Takahashi
Laboratory of Molecular and Cellular Interactions, Faculty of Advanced Life Science,
Hokkaido University, Sapporo 060-0810, Japan.
It is well known that tissue-type plasminogen activator /plasmin (tPA/Pln) system
participates in fibrinolysis. Plasminogen is mainly expressed in the liver and secreted into
the blood stream. Plasminogen is converted to plasmin by tPA or urokinase-type
plasminogen activator (uPA). Previous reports have demonstrated the existence of thetPA/Pln system in reproductive organs in mammals. However, their physiological functionsin the reproduction remain to be elucidated.
In this study, to understand the physiological roles of the tPA/Pln system in
reproduction, we used medaka ovary. We first cloned medaka plasminogen cDNA andgenerated its specific antibody using a recombinant protein. Western blot analysis showedthat plasminogen protein is constantly expressed in the medaka ovary during the daily
ovulatory cycle except for immediately before ovulation. In contrast, the activity ofplasmin, the mature enzyme form of plasminogen, was detected at the 5 and 9 hours after
ovulation by gelatin zymographic analysis. This suggests that the medaka plasmin is
involved in follicular degradation after ovulation. Because mammalian plasmin effectively
degrades extracellular matrix proteins like fibronectin and laminin, we examined whether
medaka plasmin degrades extracellar matrix in the follicle. The results showed that plasmin
could degrade type I collagen, fibronectin, laminin, and fibrinogen. These data suggest thatplasmin may participate in follicle degradation after ovulation.
P28: Plasminogen , tissue-type Plasminogen Activator, follicular degradation

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P-29
ANALYSISO̧F¸
MEDAKAĶALLIKREINĻIKEP̧ROTEINTHEŖEPRODUCTIVEORGANS
Takashi Suzuki, Sanath Rajapakse, Katsueki Ogiwara and Takayuki Takahashi
Laboratory of molecular and cellular interaction, Faculty of Advanced Life Science,
Hokkaido University, Sapporo, Japan.
Kallikreins are a subgroup of serine protease and these proteolytic enzymes havediverse physiological functions in many tissues. Growing evidence suggests that manykallikreins are potential biomarkers for many human diseases. Most of them are found to
be under hormonal regulation. Studies on kallikreins have been extensively done on
mammals such as human, mouse, and rat. Lower vertebrate species like medaka could
serve as good experimental models for the study of unresolved problems in biology
common to vertebrates.
Recently we cloned a novel serine protease from a medaka ovary cDNA library by
screening followed by 5’–RACE. The primary amino acid sequence showed a 59%
homology with a serine protease from brook trout and nearly 30% with mouse tissue
kallikreins. Northern blot analysis revealed that the novel protease is heavily expressed in
the ovary and testis of the adult medaka. RT-PCR analysis demonstrated that, in the ovary,
the gene is expressed in all stages of follicular development and in ovulated follicles.
In situ hybridization analysis detected the transcript in theca cells of the ovarianfollicles and in somatic cells of the testis.
We produced the recombinant protein of this enzyme in E.coli and raised a polyclonal
antibody against the protein in rats. Western blot analysis done using the whole ovary
extracts after ovulation could detect a study expression of the protein for a long period, andthe protein level significantly decreased prior to the next ovulation. Subsequent
immunohistochemical analysis detected the protein in the theca cells of the ovulated
follicles. These results suggest that this protease may have a function in the theca cells of
the follicle that has released the ovum.
P29: medaka, serine protease, kallikrein, ovulation.

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P-30
PROCESSING OF GONADOTROPIN-INHIBITORY HORMONE IN THE QUAILHYPOTHALAMUS
Kazuyoshi Ukena1 and Kazuyoshi Tsutsui1,2
1

Laboratory of Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima
University, Higashi-Hiroshima 739-8521, Japan.
2
Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, and Major in Integrative Bioscience and Biomedical
Engineering, Graduate School of Science and Engineering, Waseda University, Tokyo 1698050, Japan.
Recently, we identified a novel hypothalamic neuropeptide with a C-terminal
LPLRFamide sequence in the quail brain (1). This avian neuropeptide was shown to inhibit
gonadotropin release from the cultured anterior pituitary. This peptide is the first
hypothalamic peptide that inhibited gonadotropin release reported in vertebrates. We,
therefore, termed it gonadotropin-inhibitory hormone (GnIH). After this finding, we foundthat GnIH-related peptides were present in the brains of other vertebrates, such as
mammals, birds, amphibians, and fish (2-8). These GnIH-related peptides possessed a
LPXRFamide (X=L or Q) motif at their C-termini in all investigated animals.
We further characterized the GnIH cDNA from the quail brain and found that the
deduced GnIH precursor polypeptide encoded one GnIH and two gene-related peptides(GnIH-RP-1 and GnIH-RP-2). These peptides possessed a LPXRFamide (X=L or Q) motif
at their C-termini. Subsequently, GnIH-RP-1 and GnIH-RP-2 were identified as matureendogenous peptides using mass spectrometric analyses. In addition, N-terminallyextended forms of GnIH and GnIH-RP-2 were found to be present in the quail
hypothalamus.
In the present study we found that GnIH and GnIH-RP-2 were processed from theirN-terminally extended forms in the hypothalamus by biochemical andimmunohistochemical analyses. This result suggests that the unique N-terminal processing
mechanism may be present in the precursor polypeptide of GnIH in the quail
hypothalamus.
(1) Tsutsui et al. (2000) Biochem. Biophys. Res. Commun., 275, 661-667.
(2) Ukena et al. (2002) FEBS Lett., 512, 255-258.
(3) Sawada et al. (2002) Eur. J. Biochem., 269, 6000-6008.
(4) Koda et al. (2002) Endocrinology, 143, 411-419.
(5) Ukena et al. (2003) Endocrinology, 144, 3879-3884.
(6) Osugi et al. (2004) J. Endocrinol., 182, 33-42.
(7) Ubuka et al. (2005) Proc. Natl. Acad. Sci. USA., 102, 3052-3057.
(8) Ukena and Tsutsui (2005) Mass Spectrom. Rev., 24, 469-486.
P30: Brain, Hypothalamus, Neuropeptide, Avian, GnIH

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P-31
DETAILED ANALYSIS OF LOCALIZATION OF ACYLATED AND DESACYLATED GHRELIN IMMUNOREACTIVITY IN RAT GASTRIC GHRELIN
CELLSYusuke Okubo1, Ichiro Sakata1, Kenji Kangawa2 and Takafumi Sakai1
1Area of Regulatory Biology, Division of Life Science, Graduate School of Science andEngineering, Saitama University, Japan.
2
Department of Biochemistry, National Cardiovascular Center Research Institute, Suita,
Osaka, Japan.
Ghrelin was isolated as an endogenous ligand for growth hormone secretagogue
receptor (GHS-R) from the rat and human stomach (1) and it exists as two forms: acylated
ghrelin (AG) and des-acylated ghrelin (DG) (2). Exogenous administration of fatty acidincreased AG production in the stomach (3). However, the intracellular region of ghrelin
modification is still unclear. To clarify this point, we studied the detailed localization of
AG and DG in gastric ghrelin cells by immunofluorescence double staining.
AG and DG were distributed in a heterogeneous pattern in the cytoplasm of ghrelincells. Strong immunochemical staining with AG was found in the region under the plasma
membrane, whereas DG was localized throughout the cytoplasm with strong signals in thejuxtanucleus region. Interestingly, AG and DG were colocalized in the intermediate region
between the plasma membrane and nucleus. These results indicate that the two forms
ghrelin localize in different intracellular regions and that modification of DG by fatty acidmay occur during transport from the juxtanucleus region to the plasma membrane.
(1) Kojima, M. et al. (1999) Nature, 402, 656-660.
(2) Hosoda, M. et al. (2000) Biochem. Biophys. Res. Commun., 279, 909-913.
(3) Yamato, M. et al. (2005) Biochem. Biophys. Res. Commun., 333, 583-589.
P31: acylated ghrelin, des-acylated ghrelin, immunofluorescence

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P-32
IDENTIFICATION OF STEROIDOGENIC ENZYMES IN THE OVARY OF
AMPHIOXUS
Takanobu Mizuta1, Miwa Suzuki2, Kiyoshi Asahina2, and Kaoru Kubokawa1
1

Group of Marine System Analysis, Center for Advanced Marine Science, Ocean ResearchInstitute, The University of Tokyo, Nakano, Tokyo164-8639, Japan.
2
Department of Marine Science and Resources, College of Bioresearch Sciences, Nihon
University, Fujisawa, Kanagawa 252-8510, Japan.
Sex steroids play the essential roles on the reproductive phenomena of vertebrates.
The synthetic pathway of sex steroids is commonly distributed in vertebrates, from agnate
to mammals, but has not found in invertebrates. The origin of sex steroidogenesis invertebrates is considered to be evolved from the reproductive system in ancestral animal ofvertebrate such as protochordate. The urochordate is a candidate of the animal in which
the sex steroids regulate the reproduction. However, in the genomic sequences ofascidian Ciona intestinalis, have not found any genes related to the sex steroid
biosynthesis.
In the molecular phylogenetic analysis, cephalochordata is known to be the closest
invertebrate relative to vertebrate, in comparison with urochordate. We examined thepresence of sex steroidogenic pathway in amphioxus Branchiostoma belcheri, which
belongs to the subphylum cephalochordata of the phylum chordate. We isolated thecDNAs encoding steroid metabolic enzymes such as cytochrome P450 side chain cleavage
enzyme (CYP11A), cytochrome P450 17α-hydroxylase/17, 20-lyase (CYP17) andcytochrome P450 aromatase (CYP19) from ovary. By the results of multiple alignment andmolecular phylogenetic analysis, amphioxus CYP enzymes were considered to be theancestral forms of those in vertebrates.
To confirm the presence of sex steroids metabolizing pathway, in vitro conversion of
14
C-labeled precursor steroids was examined by the extracts of amphioxus ovaries. The
conversions from pregnenolone (P5) to progesterone (P4), P4 to androstenedione (AD),and AD to testosterone (T), estrone (E1) and estradiol-17β (E2) were clearly detected byusing a thin layer chromatography. These results demonstrated that CYP17 and CYP19
which we cloned from amphioxus ovary act on sex steroid synthesis. Furthermore, 3βhydroxysteroid dehydrogenase (3β-HSD) and 17β-HSD which we have not cloned yet alsopresent and act in amphioxus.
It is strongly suggested that serial sex steroid synthetic pathway from cholesterol toE2 via P5, P4, AD and T is present in the ovary of amphioxus as same as in vertebrates.
This is the first finding of the vertebrate-like synthetic pathway of sex steroids ininvertebrate species.
P32:

amphioxus, ovary, sex steroid, steroidogenic enzyme

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P-33
THE RARE OCCURRENCE OF A SPONTANEOUSLY MATURING EEL IN
CAPTIVITY
Hajime Matsubara1, Hideki Tanaka1, Kazuharu Nomura1, Koji Murashita1, Toru
Kobayashi2, P. Mark Lokman3, Takahiro Matsubara4, Nobuyuki Ohkubo4, Haruhiko
Kagawa5 and Hiromi Ohta6
1

National Research Institute of Aquaculture, Minaiise, Mie 516-0193, Japan.
National Research Institute of Aquaculture, Tamaki, Mie 516-0423, Japan.
3
Department of Zoology, University of Otago, POBOX 56, Dunedin, New Zealand
4
Hokkaido National Fisheries Research Institute, Kushiro, Hokkaido 085-0802, Japan. .
5
Faculty of Aguriculture, University of Miyazaki, Miyazaki, 889-2192, Japan
6
Department of Fisheries, Kinki University, Nara, 631-8505, Japan.
2

Unlike many fish, eels (Anguilla spp.) do not normally undergo gonadal development
under culture conditions and gonadal maturation has therefore been induced artificiallyusing injections of gonadotropin-like substances. We recently stumbled across on
exception to this dogma as a Japanese eel, (A. japonica) which had been reared long-termin sea water, matured without exogenous hormone treatment. Here, we report on thefertility and appearance of the gonads from this spontaneously maturing eel.
Eight 3-year-old eels were purchased from a commercial company and reared inambient sea water under natural light conditions for 2.5 years. One fish matured
spontaneously, and its gonads consisted of a number of testicular lamella and a total of 7
lumps of gonadal tissue. The presence of spermatozoa in the testicular compartment wasapparent. To investigate fertility, gonads were minced into small pieces with scissors andmilt was collected. The milt was maintained in artificial seminal plasma (1) at 4 degrees,
and used to determine motility and to fertilize ovulated eggs when obtained from
artificially matured female eels. The survival rate of larvae 10 days after hatching was
subsequently recorded.
Gonadosomatic index (GSI) of the spontaneously maturing eel was 18.4%, whereasthat of the other 7 immature fish, all male, was less than 0.18%. Histologically, the gonadsof the maturing eel had male germ cells in all stages of development, as well as a large
number of early vitellogenic oocytes. Sperm motility was 87.1%, and gametes had
fertilizing capability as reflected in the acquisition of hatched larvae. The larvae grewnormally and some of them underwent metamorphosis into glass eel. Albeit rare, we haveshown that a naturally maturing eel under cultivation conditions could produce milt that
had fertilizing capability.
(1) Ohta et al. (1997) J. Exp.Zool., 277, 171-180.
P33: natural maturation, eel,spermatogenesis,oocyte

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P-34
STRUCTURE OF MEDAKA COLLAGEN AND ITS EXPRESSION IN THE
OVARY: COMPARISON WITH MAMMALIAN OVARY
Maya Horiguchi, Katsueki Ogiwara and Takayuki Takahashi
Laboratory of Molecular and cellular Interactions, Faculty of Advanced Life Science,
Hokkaido University, Sapporo, 060-0810, Japan.
Ovulatory process is an essential prerequisite for fertilization and subsequent
embryonic development. The climax of ovulation is the rupture of follicle wall, leading to
the release of a fertilizable ovum. However, it has not been known over the past century
what is the mechanism of follicle wall rupture. Previous studies on mammalian ovaries
demonstrated that the follicle wall abundantly contains extracellular matrix (ECM) proteins.
Therefore, degradation of those ECM embedded in the wall is thought to be essential forfollicle rupture during ovulation. Recently, we have demonstrated that follicle rupture in
the medaka involves the cooperation of at least three matrix metalloproteinases (MMPs)
and the tissue inhibitor of metalloproteinase-2b protein. In this study, we have attempted todetermine the distribution of type I collagen in the ovary of the fish and a correlation
between type I collagen α1 degradation and follicle rupture during ovulation.
A cDNA clone for type I collagen α1 was isolated from a medaka ovary by RT-PCR
using degenerate primers synthesized based on the available partial nucleotide sequence of
medaka type I collagen α1 chain followed by 5’ and 3’-RACE. The clone for medaka type
I collagen α1 (5124bp) was found to code for a protein of 1446 amino acids. The primary
sequence showed 80% homology with human type I collagen α1 and nearly 88% with
Paralichthys. RT-PCR analysis conducted for various tissues demonstrated abundant
expression of type I collagen α1 in ovary of the mature medaka. In situ hybridization
analysis detected the transcript in the follicle layer. Type I collagen α1 was observed in thetheca cells by immunohistochemistry. The requirement of degradation of type I collagen
α1 during ovulatory process was examined by the analysis of the protein in follicle layers
collected before and after ovulation. Western blot analysis using a specific antibody for
type I collagen α1 indicated that the amount of type I collagen α1 protein was higher infollicle layers before ovulation compared to that of after ovulation. These observations are
the first direct evidence that the degradation of type I collagen in the follicle layer ofovulating follicles is essential for ovulation.
P34: follicle rupture; ovulation; type I collagen degradation; medaka fish

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P-35
GENE EXPRESSION
DIFFERENTIATIONPATTERNIN

TILAPIA GONADSDURING

SEX

Shigeho Ijiri1, Hiroyo Kaneko2, De-shou Wang2, Shinji Adachi1 andYoshitaka Nagahama2
1 Graduate school of Fisheries Science, Hokkaido University, Hakodate, Hokkaido
2 National Institute for Basic Biology, Okazaki, Aichi, Japan.
The molecular mechanism underlying gonadal sex differentiation has been little
known throughout vertebrates. This study was aimed to demonstrate gene expression
patterns which were involved in gonadal sex differentiation in XX and XY gonads during
early developmental stages of the Nile tilapia. In this study, all genetic XX group and XY
group were used, then gonadal ridges were isolated surgically throughout experimental
period. Total RNA was extracted from each gonads, then used for one-step real time RTPCR analyses. In the XX gonads, foxl2 and aromatase (cyp19a1) were upregulated after 5
to 7 days post hatching (dph). All other steroidogenic enzymes, such as cholesterol sidechain cleavage, 3β-hydroxysteroid dehydrogenase (3β-HSD), steroid 17α-hydroxylase /c17-20 lyase, 17β-HSD type1, were gradually upregulated in both XX and XY gonads
during this period. These results indicated that estrogen production began only in the XX
gonads after 5 dph, and which was regulated by cyp19a1 expression that was undercontrolled by foxl2 expression. The XY gonads also gradually increased an ability for
producing androgen during this period as same as XX gonads did. However, after 10 dph,
the expression levels of all steroidogenic enzymes in the XY gonads maintained relatively
low until 35 dph, in contrast to the fact that XX gonads further increased expressions of all
steroidogenic enzymes during this period. These results probably indicated that thesteroidogenic activity was much more active in the XX gonad during 10 to 35 dph. In
contrast to dimorphic expression pattern in steroidogenic enzymes between XX and XY
gonads, expression levels of steroid receptors, such as estrogen receptor α, β1 and β2, andandrogen receptor α and β did not show significant differences between the sexes. Taking
all together, tilapia ovarian differentiation is regulated by estrogen production during early
developmental stages from 5 to 35 dph, however XY gonads may not be necessary to
produce androgen during this period for differentiating toward testis.
P35: Nile tilapia, gonad, steroidogenesis, sex differentiation, steroid receptor

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P-36
HSP90β IS INVOLVED IN SIGNALING PROLACTIN-INDUCED APOPTOSIS IN
NEWT TESTISKo Eto, Buget Saribek, Yuji Jin, Mikiko Saigo and Shin-ichi Abe
Department of Biological Sciences, Graduate School of Science and Technology,
Kumamoto University, Kumamoto860-8555, Japan.
We have shown in vivo and in vitro that prolactin induces apoptosis in the 7th
generation of spermatogonia just before the initiation of meiosis during newt
spermatogenesis, but the underlying molecular mechanism remained unknown. The aim of
this study is to determine the role of heat shock protein (HSP) 90β in prolactin-induced
apoptosis. HSP90β is a molecular chaperone for client proteins in signal transduction andtranscription to regulate cell proliferation and survival. Several lines of evidence indicatethat HSP90β protects cells from apoptosis. First, in order to explore if HSP90β is
expressed in newt testis, we cloned HSP90 cDNA, which is highly homologous with thoseof other species, from newt testis by RT-PCR. To reveal the relationship of HSP90β andprolactin receptor in prolactin-induced apoptosis, we examined the expressions of theirproteins in the spermatogenic stages and cell types of newt testis by Western blotting andimmunofluorescence staining with antibodies to the amino terminal region of HSP90 andthe extracellular domain of prolactin receptor. HSP90β was detected highly atspermatogonial stage and in both the membrane and cytosol fractions of germ cells, while
prolactin receptor was expressed in the membrane fraction of only germ cells, suggestingco-localization and co-operation of HSP90β and prolactin receptor in the execution of
spermatogonial apoptosis induced by prolactin. To investigate how HSP90β participates in
the apoptosis, we examined the interaction of HSP90β with prolactin receptor by coimmunoprecipitation with an antibody to prolactin receptor from the organ cultures of newt
testis treated with or without prolactin. HSP90β was demonstrated to associateconstitutively with prolactin receptor on the plasma membrane of germ cells, suggestingthat prolactin receptor is also one of the client proteins for HSP90βýthat was newly
identified from newt testis. Because the association of HSP90β with prolactin receptor
implied their functional relevance, we examined the effect of an inhibitor of HSP90β
functionýgeldanamycin, on prolactin-induced apoptosis. Dissociated testicular cells werereaggregated and cultured for 2 days in the absence or presence of prolactin or/and
geldanamycin to evaluate apoptotic cells with a crescent-like nucleus stained by eosin andhematoxylin and with a TUNEL positive nucleus. Treatment of the cultures with prolactin
or geldanamycin induced apoptosis. In the cultures treated with prolactin andgeldanamycin, inhibition of HSP90β function by geldanamycin was shown to potentiate
prolactin-induced spermatogonial apoptosis synergistically, suggesting that the apoptosis ismediated by a defect in HSP90β unction. Taken together, these results suggest that
HSP90β is involved in signaling prolactin-induced apoptosis through the receptor.
Therefore, we speculate that, like geldanamycin, prolactin could inhibit or/and change
HSP90β function, causing the changes in varieties and statuses (i.e. phosphorylation) of
the client proteins for HSP90β interacted with prolactin receptor to induce apoptosis in the
7th generation of spermatogonia.
P36: HSP90β, prolactin receptor, prolactin, apoptosis, spermatogenesis

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P-37
ULTRASTRUCTURAL ABNORMALITY OF SPERMATOZOA AND
DEPENDENT LOSS OF SPERMATOGENESIS IN TRANSGENIC RAT

AGE-

Li-yi Cai1,2,4, Takao Susa1,2, Michie Nakayama1, Sanae Murakami2, Takako Kato3, Shunichiro Izumi4 and Yukio Kato1,2,3
1)Division of Life Science, Graduate School of Agriculture, 2)Department of Life Science,
School of Agriculture, 3)Institute of Reproduction and Endocrinology, Meiji University,
Kanagawa, 214-8571, Japan.
4)
Department of Obstetrics and Gynecology, Tokai University, School of Medicine,
Kanagawa, 259-1193, Japan.
We recently established a transgenic rat (1) using the construct of porcine FSHβpromoter, –852/+10 bp region, fused to the Herpes simplex virus thymidine kinase (HSVTK) gene. The transgenic rats showed the tissue-specific expression of the HSV-TK genein the pituitary cells producing FSH, and ectopic expression of theTK gene under thecontrol of cryptic promotor in the testes with abnormal spermatogenesis, resulting maleinfertility. In this study, histology, immunohistochemistry, and electron microscopy
techniques were employed to disclose the causes for the infertility in transgenic male.
Immunohistochemical analysis of HSV-TK and WT1 proteins showed that the TK
protein is located in the postmeiotic germ cells, but not in spermatogonia, spermatocyte
and Sertoli cells. Evaluation of spermatogenesis with electron microscopy demonstrated
that both normal germ cells development and ultrastructural abnormality of testicularspermatozoa were present in the transgenic rats. In addition, age-dependent loss ofspermatogenesis has also been found when compared different age of transgenic andnormal rats.
The accumulations of the HSV-TK protein in the postmeiotic germ cells not only
directly disrupt the spermiogenesis, but also indirectly damage the spermatogenesis. Since
the Sertoli cell plays an essential role in development of spermatogenesis, those functions
are affected by interaction with germ cells. Thus, this transgenic rat may be a useful animalmodel for studies on germ cell-Sertoli cell interaction and male infertility.
(1) Li-yi Cai et al. J. Reprod. Dev., In press.
P37: HSV-TK, Infertility, spermatogenesis, spermatozoa, Transgenic rat

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P-38
ANALYSIS OF TESTIS-SPECIFIC SERINE PROTEASES, TESSP-3 AND TESSP-4
IN MOUSE TESTES: COMPARISON WITH OTHER TESSPs
Shunsaku Fukumoto and Takayuki Takahashi
Laboratory of Molecular and Cellular Interactions, Faculty of Advanced Life Science,
Hokkaido University, Sapporo 060-0810, Japan.
The testis is a reproductive organ that serves two crucial functions: synthesis andsecretion of steroid hormones and production of spermatozoa. Spermatogenesis is acomplex process that involves formation of spermatogonia from germline stem cells;
spermatogonial renewal and differentiation into primary spermatocytes; meiosis, by which
diploid spermatocytes develop into haploid spermatids; and spermiogenesis, in whichround spermatids mature into spermatozoa (1). Various germ-specific serine proteases havebeen reported in previous studies and thought to play important roles during
spermatogenesis. We are particularly interested in genes encoding intracellular or
extracellular proteolytic enzymes.
We cloned cDNAs for novel serine proteases from a mouse testis cDNA library by
RT-PCR using degenerate primers directed at conserved sequence motif within the
catalytic region of serine proteases. As a result, four novel serine proteases were isolated
and designated as testis-specific serine protease-1 (TESSP-1), TESSP-2, TESSP-3 andTESSP-4. Transcripts of these clones were restricted in the testis as observed by Northernblot analysis. The clones for TESSP-1 (1,128bp), TESSP-2 (1,595bp), TESSP-3 (1,995bp)
and TESSP-4 (1,839bp) were found to code for proteins of 323, 336, 383 and 373 aminoacids, respectively. In situ hybridization analysis revealed that TESSP-1 mRNA wasexpressed in type B spermatogonia and spermatocytes (2), TESSP-3 mRNA was inspermatocytes and spermatids whereas mRNA of TESSP-2 and TESSP-4 were mainly
expressed in spermatocytes. Immunostaining analysis showed that TESSP-1 was localized
in Golgi apparatus, TESSP-2 was in ER and TESSP-3/TESSP-4 were in subcellular
membrane structures. These results suggest that TESSPs play different roles during
spermatogenesis in the mouse testis.
(1) Baarends WM and Grootegoed JA. (2003) Male gametogenesis, In: Fauser BCJM, (ed.),Reproductive medicine. Parthenon Publishing, London, pp. 31–399.
(2) Takano, N. et al. (2004) Mol. Reprod. Develop., 70, 1-10.
P38: protease; TESSP; spermatogenesis; testis; mouse

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P-39
DELAYED DEVELOPMENT OF THE TESTIS IN GROWTH-RETARDED (grt)
MICE
Kenichi Kobayashi, Hisayo Kubota and Junzo Saegusa
National Institute of Occupational Safety and Health, Kawasaki 214-8585, Kanagawa,Japan.
Growth-retarded (grt) mice are congenitally hypothyroid and exhibit an autosomal
recessive pattern of inheritance (1). In grt mice, the plasma level of thyroxine issignificantly lower but that of thyroid-stimulating hormone (TSH) is significantly higher
than those in control mice (1, 2). Our previous works suggested that a defect might exist in
TSH receptor–mediated signaling, including the expression and function of TSH receptor
and TSH receptor–guanine nucleotide-binding protein coupling in the thyroid gland (2, 3).
During holding grt mice in our animal facility, we noticed that male mice are infertileat the young adult stage (~13 weeks) but fertility is partially restored after 26 weeks-old.
To understand the delayed maturity of testis in grt mice, we assessed the developmental
pattern of their testes compared with normal mice. The testicular weight of grt mice wassignificantly slighter than age-matched normal mice until 8 weeks-old but was comparable
at 13 and 26 weeks-old. The epididymis of grt mice was smaller before 13 weeks-old but
similar to normal mice thereafter. While normal mice had mature sperms in both testes andepididymis at 5 weeks-old, any mature sperms were not seen in neither seminiferous
tubules nor epididymal duct, but a lot of degenerative germinal cells were noted in thelatter in 5 weeks-old grt mice. Mature sperms were detected after 8 weeks-old in grt mice.
Both diameter and area of seminiferous tubules in testes of grt mice were significantly
smaller than those of normal mice before 13 weeks-old, but they were comparable at 26
weeks-old. Additionally, the number of Leydig cells in grt mice was significantly smaller
than those of normal mice until 8 weeks-old, but they were comparable after 13 weeks-old.
The findings in this chronological study indicated that male grt mice might need more than
13 weeks to develop structurally mature testis. Further studies on sperm function are in
progress.
(1) Yoshida, T. et al. (1994) J. Endocrinol., 142, 435-446.
(2) Kobayashi, K. et al. (2001) Zool. Sci., 18, 955-961.
(3) Kobayashi, K. et al. (2005) Congenit. Anom., 45, 89-92.
P39: growth-retarded mouse, hypothyroid, testis, seminiferous tubule, sperm

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P-40
IDENTIFICATION OF GONADOTROPIN PRODUCING CELLS IN THE
ADENOHYPOPHYSIS OF BROWN HAGFISH, PARAMYXINE ATAMI
Kaori Honda1, Katsuhisa Uchida1, Toyokazu Shimotani1, Shunsuke Moriyama2 and Masumi
Nozaki1
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.
The hagfish is the extant representatives of the oldest class of vertebrates, Agnatha(Jawless vertebrates). Not only the external body features but also the pituitary glandremains in very primitive conditions. For instance, hypophysectomy in hagfish(Eptatretus stouti) did not show any changes in gonadal or thyroidal functions (1), andthus it remains enigma whether adenohypophysial hormones similar to those of moreadvanced vertebrates are present in the hagfish pituitary or not. However, our recent
study has revealed that immunoreactive gonadotropin (GTH)-like material wasdemonstrable in the pituitary gland of brown hagfish, Paramyxine atami, and further that
accumulation of GTH-like material was strongly correlated to the degrees of thedevelopmental conditions of gonads (2). It was suggested that GTH is present in thehagfish pituitary gland. The present study aimed to identify a GTH molecule and its
cellular activities in the adenohypophysis of brown hagfish.
First, we constructed cDNA library from pituitary of brown hagfish, and isolated
two kinds of pituitary-related genes using an expressed sequence tag analysis. Based on
their putative amino acid sequences, especially the positions of cysteine (Cys) residues,
and molecular phylogenetic analyses, the isolated two clones were considered to be
cDNAs encoding the α- and β-subunits of hagfish GTH. Hagfish GTHβ contained 12
Cys residues at the homologous position to gnathostome GTHβ and showed 30-43%
sequence identities. On the other hand, hagfish GTHα contained 8 out of 10 Cys residues
at the homologous positions to gnathostome GTHα and showed 39-45% sequence
identities. In phylogenetic trees, α- and β-subunits of hagfish GTH are far removed fromthose of gnathostomes, and take positions as out groups, respectively. Thus, it seems
most likely that there is a single GTH molecule in the hagfish representing an ancestralfrom that has given rise gnathostome GTHs.
Secondly, we examined the localization of GTH producing cells in the
adenohypophysis of brown hagfish using immunohistochemical technique and in situ
hybridization. Specific antisera were raised against the synthetic peptides of hagfish
GTH α-subunit, putative amino acid positions 58-74, and β-subunit, positions 86-99. As
a template for in situ hybridization, the hagfish GTH α-subunit cDNA (nt 69 to nt 358;
290bp) was amplified by PCR, generated the digoxigenin (DIG)-labeled RNA probes.
Morphological explorations demonstrated both GTH α- and β-subunits were completely
synthesized in the same cells of hagfish adenohypophysis. These cells were wellaccordance with our previously observed GTH-like cells which were identified with anantiserum raised against ovine LH β-subunit. Moreover, cellular activities of GTHproducing cells showed positive correlation with gonadal conditions of hagfish; low
expression in juveniles and high in adults possessing well-developed gonad. Therefore,
our present results clearly demonstrated that brown hagfish has a GTH molecule, which
is an ancestral molecule of gnathostome GTHs, and it has crucial functions in the
reproductive endocrine system of hagfish.
(1) Matty, A. J. et al. (1976) Gen. Comp. Endocrinol., 30, 500-516.
(2) Miki, M. et al. (2006) Gen. Comp. Endocrinol., 148, 15-21.
P40: Gonadotropin; Hagfish; Adenohypophysial hormones; Agnatha; Pituitary gland

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P-41
IDENTIFICATION OF GROWTH HORMONE-RELEASING HORMONE,
SOMATOSTATIN AND GROWTH HORMONE IN ELASMOBRANCH, DOGFISH,
Triakis scyllium

Shunsuke Moriyama1, Hikari Kumada1, Aiko Nishino1, Chisato Maeda1, Noriko Amiya1,
Katsufumi Amano1, Susumu Hyodo2 and Hiroshi Kawauchi1
12School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Ocean Research Institute, University of Tokyo, Tokyo 164 -8639, Japan.

The hypothalamus-pituitary-liver endocrine axis plays an important role in growth
regulation in fish as well as in mammals. In fish, growth hormone-releasing hormone(GHRH) and somatostatin (SS) in the hypothalamus, and growth hormone (GH) in thepituitary have been identified in a wide variety teleost fish. Biological functions of these
hormones have also been characterized. In contrast to teleosts, the mechanism of growth
regulation in elasmobranch is still unclear because SS and GH were identified only in
ratfish (1) and blue shark (2), but GHRH is not identified yet. To get a better understanding
about growth regulation in elasmobranch, we cloned GH, GHRH/PACAP, and SS cDNAs
from the pituitary cDNA library of dogfish, Triakis scyllium, and characterized the tissue
distributions of these hormones.
The pre-GH cDNA consists of a signal peptide of 27 amino acids (aa) and a mature
protein of 183 aa, which was confirmed by N-terminal sequencing of isolated GH from thepituitary. Dogfish GH contains 4 cysteine residues at positions homologous to those of
teleost GHs, and the residues involved in receptor binding (site 1 and 2) were highly
conserved as compared with human GH (3). The GHRH precursor cDNA encoded GHRH
and pituitary adenylate cyclase activating polypeptide (PACAP), the same as in teleosts.
GHRH and PACAP are estimated to consist of 47 and 38 aa, based on the sequencecomparison with teleosts counterparts. Dogfish GHRH showed lower sequence identity
than PACAP as compared with those of teleosts. Two molecular forms of SS, SS-14 andSS-26, are derived from the precursor cDNA. Both SS-14 and 26 were highly conserved at
C-terminus as compared with those of teleosts. The GH producing cells were located in the
rostral pars distalis of pituitary gland, similar to that of teleosts. The PACAP
immunoreactive cell bodies and fibers were located in the hypothalamus. No GHRH
immunoreactive cell bodies and fibers were observed using mammalian GHRH antibody.
The SS immunoreactive cell bodies and fibers were found in hypothalamus.
(1) Conlon (1990) Gen. Comp. Endocrinol., 80, 314-320.
(2) Yamaguchi et al. (1989) Gen. Comp. Endocrinol., 73, 252-259.
(3) Goffin et al. (1996) Endocrine Rev., 17, 385-410.
P41: cDNA cloning, Dogfish, GH, GHRH/PACAP, SS, Tissue Distribution

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P-42
IGF-I STIMULATES SYNTHESIS AND RELEASE OF GTH IN MASU SALMONPITUITARY CELLS AT EARLY STAGES OF GAMETOGENESIS
Shunji Furukuma1, Takeshi Onuma1,2, Akihisa Urano2 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
Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
Insulin-like growth factor I (IGF-I) is involved in the control of reproductive function at aparticular stage of sexual maturation, the onset of puberty. In salmonids, IGF-I increased
gonadotropin-releasing hormone (GnRH)-stimulated release of GTH at early gametogenesis(1). IGF-I had synergistic interaction with salmon GnRH (sGnRH) to regulate GTH synthesis,but the effects were different depending on the reproductive stages (2). The aim of this study
was to determine if IGF-I itself stimulates GTH synthesis and release at the early stage of
gametogenesis in salmonids. Direct effects of IGF-I on expression of GTH subunit genes andrelease of GTHs were examined using primary pituitary cells of masu salmon (Oncorhynchus
masou) at three reproductive stages.
Masu salmon of Mori hatchery strain were caught at early gametogenesis in February,
early sexual maturation in May, and spawning in September 2003. IGF-I (0.01, 0.1, 1, 10 and
100 nM) alone or IGF-I (100 nM) and sGnRH (100 nM) were added to the primary pituitarycell cultures. Amounts of mRNAs for glycoprotein α2, follicle-stimulating hormone (FSH) β
and luteinizing hormone (LH) β subunits were determined by real-time PCR, and levels of
FSH and LH in the plasma and the culture medium were determined by RIA. In addition, theplasma IGF-I levels were determined by RIA.
In males, IGF-I alone increased three GTH subunit mRNAs on day 3 of incubation in
February in dose-dependent manners. However, it did not affect the three GTH subunit
mRNAs in May and September. In females, stimulatory effects of IGF-I were not observed inall stages. On the other hand, IGF-I alone stimulated release of FSH and LH in February infemales, although it had no effects in males. Plasma IGF-I levels elevated from February
through May in both sexes. Our results suggested that IGF-I itself has stimulatory effects on
GTH synthesis and release at early gametogenesis and it functions as a metabolic signal thatinduces puberty in salmonids.
(1) Baker et al. (2000) Biol. Reprod., 63, 865-871.
(2) Ando et al. (2006) Gen. Comp. Endocrinol., 149, 21-29.
P42: anterior pituitary, GTH, insulin-like growth factor I, puberty, Salmon

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P-43
IMMUNOHISTOCHEMICALLOCALIZATION OF PROLACTIN-RELEASING
PEPTIDE IN THE BRAIN OF THE OVOVIVIPAROUS FISH SPECIES Poecilia reticulata(GUPPY)
Masafumi Amano1, Noriko Amiya1, Mio Matsuki1, Hiroshi Itoh1, Yoshitaka Oka2 and Kunio
Yamamori1
1

School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo,
Tokyo 113-0033, Japan.
2

Prolactin (PRL)-releasing peptide (PrRP) was first discovered in bovine hypothalamus [1].
In teleost fish, PrRP homologues have been isolated from brain extracts of the Japanese crucian carp,
tilapia, chum salmon, and Atlantic salmon. These teleost PrRPs potently facilitate PRL secretion
both in vitro and in vivo. It is also reported that intra-arterial injection of PrRP elevates PRL gene
expression and plasma PRL levels in the rainbow trout, and that PrRP is essential to maintain the PRLlevel and osmotic balance in the goldfish. However, knowledge of the function of PrRP in fish has
been restricted to certain fish species.
The guppy Poecilia reticulata is a euryhaline and viviparous teleost, and has been extensively
used for the analysis of reproductive phenomena. It is reported that oxytocin (OXT) and vasotocin
can induce premature parturition, and that gonadotropin is not involved in the induction of ovulationand parturition. Incidentally in rats, central administration of PrRP stimulates OXT release.
Therefore, it is possible that PrRP regulates parturition via OXT neurons in the guppy. Thus, we
examined the distribution of PrRP-immunoreactive (ir) cell bodies and fibers in the adult fish and theontogenic development of the PrRP and PRL system by immunohistochemistry. To examine theprojection of PrRP-ir fibers to OXT-ir cell bodies in the adult fish brain, double-staining
immunohistochemistry was conducted using the OXT antiserum that cross-reacts with isotocin.
In adult guppies, PrRP-ir cell bodies were detected in the posterior part of the hypothalamus.
In the pituitary, a small number of PrRP-ir fibers were observed adjacent to the PRL cells, whereas
numerous PrRP-ir fibers were detected not only in the hypothalamus but also widely throughout thebrain. PrRP-ir cell bodies and PRL cells were already detected on the birth day in the hypothalamus
and pituitary, respectively. The number of PrRP-ir fibers in the brain increased as the fish developed.
These results suggest that PrRP is involved in neuromodulation in the brain and that PrRP plays somephysiological roles in the early development of the guppy. Furthermore, some PrRP-ir fibers werein close contact with OXT-ir cell bodies in the nucleus preopticus, suggesting that PrRP is involved in
OXT release in the guppy.
(1) Hinuma et al. (1998) Nature, 393, 272-276.
P43: prolactin-releasing peptide, prolactin, immunohistochemistry, ontogeny, guppy

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P-44
RETROGRADE AND GOLGI STAINING STUDY OF MEDAKA FISH BRAIN:
VASOTOCIN AND/OR ISOTOCIN MAY ACT AS NEUROMODULATOR
Tamaki Ohya and Shinji Hayashi
Laboratory of Endocrinology, Graduate School of Integrated Science, Yokohama City
University, Yokohama 236-0027, Japan.
Previously we reported that Vasotocin/Isotocin-immunoreactive (VT/IT-ir) neurons are
only present in a cluster in the nucleus preopticus (NPO) of the preoptic area in the medaka
fish (Oryzias latipes) brain. VT/IT-ir neurons are sexually dimorphic and reduce in number
after spawning in the female. Further, VT/IT-ir fibers form a plexus in the medulla
oblongata (1). This plexus seems to be corresponding portion of the medulla oblongata inthe midshipman which is known for vocalization of courtship behavior. In the present
study, by injecting biocytin into the spinal cord of the medaka fish in vitro, we observed
fibers extending from the diencephalon, and fibers consisting the plexus in dorsal andventral portion of the medulla oblongata. Furthermore large neurons were labeled in the
nucleus of medial longitudinal fasciculus (nMLF). The present observation is in good
agreement with a previous report in the dwarf gourami (2), that the neurons of medial
longitudinal fasciculus (MLF) form several different clusters, since we detected at least
three kinds of cells in the MLF by golgi staining in the medaka fish brain.In the present
experiment we failed to obtain any direct evidence that shows direct projection of the NPO
neurons to the spinal cord. Since, the plexus in the medulla oblongata, which is labeled by
the retrograde tracer applied to the spinal cord, is consisted by the VT/IT-ir fibers, it ispossible that VT and/or IT detected in the plexus may play a roles as neuromodulators.
(1) Ohya and Hayashi (2006) Zool. Sci., 23, 23-29.
(2) Yamamoto et al. (1998) Neurosci. Lett., 240, 50-52.
P44: medaka , vasotocin, isotocin, biocytin, DiI.

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P-45
IMMUNOHISTOCHEMICAL LOCALIZATION OF OREXIN PEPTIDE IN FISH
BRAIN
Noriko Amiya1, Masafumi Amano1, Masayuki Iigo2, Shoji Kitamura3, Yoshitaka Oka4,
Toshikazu Sunuma1, Akiyoshi Takahashi1 and Kunio Yamamori1
1

School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.
Japan International Research Center for Agricultural Science, Tsukuba, Ibaraki 305-8686,
Japan.
3
Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya University,
Utsunomiya, Tochigi 321-8505, Japan.
4
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Bunkyo, Tokyo 113-0033, Japan.
2

Orexin/hypocretin (ORX) is a neuropeptide that is involved in the regulation of
feeding behavior and the sleep-wakefulness cycle in mammals (1). ORX interacts withmelanophore-stimulating hormone (MSH) and gonadotropin-releasing hormone (GnRH) in
mammals (2, 3). Recently it was reported that ORX is involved in food intake in goldfish
Carassius auratus (4). Thus, it is interesting to examine whether ORX regulates foodintake directly or indirectly via interacting with other neurons. However, the knowledgeof the function of ORX in fish has been restricted to certain fish species. Thus, in thepresent study, we first examined the localization of ORX-immunoreactive (ir) cell bodies
and fibers in the brain of medaka Oryzias latipes and masu salmon Oncorhynchus masou
by immunohistochemistry. We further examined the interaction of ORX and MCH/MSH
in medaka brain by double-staining immunohistochemistry to elucidate the neural networkfor food intake regulation.
ORX-ir cell bodies were detected in the nucleus posterioris periventricularis (NPPv)of the hypothalamus, and ORX-ir fibers were detected not only in the hypothalamus but
also widely throughout the brain in both fish species. In medaka, some ORX-ir fibers
were in close contact with MCH-ir and MSH-ir cell bodies in the hypothalamus revealed
by double-staining immunohistochemistry. These results suggest that ORX is involved inneuromodulation in the brain not only in mammals but also in fish and that the neuralconnections between ORX and MCH/MSH neurons exist in the brain.
(1) Huesa et al. (2005) J. Comp. Neurol., 488, 476-491.
(2) Elias et al. (1998) J. Comp. Neurol., 402, 442-459.
(3) Iqbal et al. (2001) J. Neuroendocrinol., 13, 1033-1041.
(4) Nakamachi et al. (2006) J. Neuroendocrinol., 18, 290-297.
P45: Orexin, MCH, MSH, Brain, Fish

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P-46
MECHANISMS OF TRH-INDUCED PRL RELEASE FROM THE
BULLFROG PITUITARY GLAND
Astuko Minagawa1, Itaru Hasunuma2, Kazutoshi Yamamoto2, Sakae Kikuyama2, Testuya
Kobayashi1 and Takeo Machida11Department of Regulation Biology, Faculty of Science, Saitama University, Saitama 338-8570,
Japan.
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 to stimulate the releaseof PRL from the bullfrog pituitary gland both in vitro and in vivo. Until now, intracellularmechanisms that regulate PRL release in amphibians and receptor structures of TRH have notbeen known. The aims of the present study were to clarify the contribution of extracellular Ca2+ on
TRH-induced PRL release and to characterize the gene structures of bullfrog TRH receptors( TRHRs ).
The effect of TRH on the release of PRL was studied by using a modified perifusion
technique. Anterior lobes of male bullfrog ( Rana catesbeiana ) pituitary glands were continuously
perifused with a medium containing TRH as a secretagogue. The amount of PRL released wasmeasured using a newly developed homologous enzyme-immunoassay. TRH stimulated therelease of PRL from perifused pituitary glands in a dose-dependent manner. In the presence of
EGTA ( 2 mM ) or Ni2+ ( non-selective Ca2+-channel blocker, 3 mM ), the basal PRL release wasdecreased and the stimulatory effect of TRH ( 3 x 10-9 M ) on PRL release was suppressed. In thepresence of nifedipine ( L - type Ca2+-channel blocker, 10-5M ), the basal PRL release wasdecreased. These results suggest that basal PRL release and TRH-evoked PRL release from thebullfrog pituitary depend on the extracellular Ca2+ and L - type Ca2+-channels are involved at leastin the basal PRL release.
On the other hand, of TRHRs, two subtypes in some species and three subtypes in Xenopus
have already been cloned. Here from the brain of the bullfrog, we isolated three distinct cDNAs
encoding TRHR. These three subtypes were designated as fTRHR1, fTRHR2, and fTRHR3.
Using RT-PCR, we investigated patterns of expressions of mRNAs of these subtypes. It was
revealed that fTRHR1 was found only in the brain, fTRHR2 was in the brain and the testis, andfTRHR3 in the brain, in the anterior and, intermediate lobes, and in the testis. These results showedthat three subtypes exist in the bullfrog TRHR. This is in good accordance with the result of
Xenopus TRHRs.
P46: PRL, TRH, TRH receptor, Ca2+, bullfrog

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P-47
EXPRESSION OF THE GLUCOCORTICOID RECEPTOR IN RAT
CORTICOTROPHS DURING POSTNATAL DEVELOPMENT IS INVOLVED IN
CHANGES IN PROOPIOMELANOCORTIN PROCESSSING PATTERNSKae Ito1, Masakazu Suzuki1, Hitoshi Ozawa2, Mitsuhiro Kawata3 and Shigeyasu Tanaka1
1

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,
Japan.
2
Department of Anatomy and Neurobiology, Nippon Medical School, Tokyo 113-8602,
Japan.
3
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine,
Kyoto 602-0841, Japan.
Two mammalian prohormone convertases, PC1 (also called PC3) and PC2, havebeen identified by cDNA cloning and shown to be involved in the cleavage ofproopiomelanocortin (POMC) at paired basic amino acid sites. Previous experimental
results revealed that the pattern of the proteolytic processing of POMC in the corticotrophschanges in a characteristic fashion during the postnatal period. In newborn rats, this pattern
is similar to the one occurring in the melanotrophs; however, as development proceeds,
this processing pattern changes to that of the adult rat, and the ability to cleave POMC
into α-MSH is lost. This conversion is evidenced by the decrease in PC2 mRNA levels as
postnatal development proceeds (1). Based on results with AtT-20 cells, a mouse pituitary
tumor cell line, it has been suggested that PC2 mRNA is regulated by corticosterone (2).
In the present study, we examined the expression patterns of PC2 mRNA andglucocorticoid receptor (GR) in anterior corticotrophs during the postnatal period by meansof immunocytochemistry with a specific antibody against the GR (3) and in situ RT-PCR
analysis, respectively. Wister male rats, aged 1, 3, and 8 weeks were sacrified and theirpituitaries fixed with 4% paraformaldehyde and cut into 4µm-Paraplast sections. In the 1week-old rats, numerous PC2 mRNA-positive cells were clearly visible in thecorticotrophs, whereas GR were scarcely expressed. In the 3-week- and 8-week-old rats,
the number of PC2 mRNA-positive cells decreased significantly, while the number of GRexpressing cells gradually increased. In addition, the number of GR-expressing and PC2
mRNA-positive corticotrophs increased significantly as development proceeded. The
expression of few GRs during the early postnatal period may be advantageous to thethymus-lymph system because corticosterone inhibits the establishment of the immune
system. The steadily decreasing level of PC2 mRNA in the corticotrophs with increasing
duration of the postnatal period, followed by the increase in the ratio of GR-expressing
corticotrophs, initiates the secretion of a large amount of ACTH instead of α-MSH andsubsequently, establishment of the
hypothalumus-pituitary corticotrophs-adrenal
corticosterone axis.
(1) Kato, H. et al. (2004) J. Histochem. Cytochem., 52, 943-957.
(2) Day, R. et al. (1992) Mol Endocrinol., 6, 485-497.
(3) Ozawa, H. et al. (1999) Cell Tissue Res., 295, 207-214.
P47: glucocorticoid receptor, PC2, POMC , Immunocytochemistry, in situ RT-PCR

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P-48
HIGHER FRACTAL DIMENSION (FD) OF C6 GLIOMA CELLS ON THE
FRACTAL AKD SURFACE
Ping Wang1, 2 , Wenjun Fang3, 4, Takeshi Onuma1, Naoko Birukawa1, Kaoru Tsujii3, Jicheng
Li2 and Akihisa Urano1
1

Division of Biological Science, Graduate School of Life Science, Hokkaido University,
Sapporo 060-0810, Japan.
2
Institute of Cell Biology, Zhejiang University, Hangzhou 310031, China.
3
Nanotechnology Research Center, Research Institute for Electronic Sciences, Hokkaido
University, Sapporo 001-0021, Japan.
4
Department of Chemistry, Zhejiang University, Hangzhou 310027, China.
Neurons and glial cells in the brain are surrounded by a fractal environment (1, 2).
Protoplasmic glial cells play a pivotal role in the CNS tissues in vivo, by their highly
complex morphology, whereas they often present flattened fibroblast-like aspects bearing
only a few simple processes on the traditional culture surface. Recent studies have
implicated that cell behavior and function were affected by the topography of culture
surface. However, little is known about the glial cell culture on an artifical fractal
surface in vitro.
The previous studies showed that a fractal alkylketene dimmer (AKD) surface
provided such an environment for glial cell culture (3). However, no information isavailable on effects of fractal surface on the complex cell morphology. In particular, themolecular mechanisms of action of the cytoskeletal gene expression remain to beelucidated.
In the present study, C6 glioma cells, a popular model in studies of glial cellmorphogenesis and differentiation were therefore grown on the fractal AKD, non-fractal
AKD and PLL-coated surfaces. Complexity of C6 cells was analyzed with a geometricalcalculational parameter (fractal dimension, FD), and arrangements of actin and tubulin
were visualized by double-fluorescent staining. The absolute amounts of actin andtubulin mRNAs were determined by quantitative real-time PCR (QRT-PCR) analyses, as
an index of cytoskeletal gene expression.
A fractal AKD surface induced C6 glioma cells with higher FD and the larger
numbers of copies of cytoskeletal mRNAs. The amounts of actin and tubulin mRNAs
increased gradually from 0-12 h after subculture, and then γ-actin mRNA peaked off,
despite continuous elevation of β-tubulin mRNA. The amounts of β-actin and α-tubulin
mRNAs were kept at the same values after reaching the maximum.
In conclusion, higher FD of cells provided by fractal AKD surface was closely
associated with cell differentiation. The present results from a use of fractal AKD surface
further suggest that the regulation of cytoskeletal gene expression precedes appearance ofmorphological changes associated with differentiation.
(1) Kiselve, V.G. et al. (2003) NeuroImage, 20, 1765.
(2) Zhang, L. et al. (2006) Journal of Neuroscience Methods, 50, 242.
(3) Yan, H. et al. (2006) Colloid Surface (A), 284, 490.
P48: fractal surface, C6 glioma, differentiation, cytoskeleton, gene expression

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P-49
ONSET OF METAMORPHOSIS OF CONGER EEL LEPTOCEPHALI IN
LABORATORY-REARED CONDITIONS
Hiroaki Kurogi1, Keisuke Yamano2 and Noritaka Mochioka3
1

Coastal Fisheries Laboratory, National Research Institute of Fisheries Science, Fisheries
Research Agency, Yokosuka, Kanagawa 238-0316, Japan.
2
National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Mie
516-0193, Japan.
3
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
Eel leptocephalus larvae show drastic morphological changes, such as the forward
movement of the anus and the reduction of a body length during metamorphosis.
Information about the onset of metamorphosis is important for successful artificial grasseel production; however, the mechanism of the metamorphosis is still unclear in both
ecological and physiological aspects. To clarify the factors influencing the onset ofmetamorphosis of eel leptocephali, the conger eel (Conger myriaster) at the final stage of
leptocephalus caught from wild were reared without feeding. The leptocephali divided into
two groups by 20 days after the start of rearing: about one-third of which started
metamorphosing (named “ON” individuals) and the rest remained at leptocephalus stage(“OFF” individuals), indicating that only ON individuals had been triggered for the onsetof metamorphosis when they were caught. No difference between two groups (ON/OFF)
was seen in the body morphology at the start of rearing but the otolith microstructure at thestart of rearing that is estimated by back-calculating for 20 days using daily growthincrements differed between two groups; a remarkable ring at an otoliths edge wasrecognized in ON individuals but not in the OFF individuals. Wild conger eel leptocephali
before metamorphosis could be discriminated between ON and OFF by this otolith feature.
Metamorphosis of conger eel leptocephalus coincides with a thyroid hormone surge(1). Whole body concentrations of thyroxine (T4) and triiodothyronine (T3) were comparedamong ON and OFF phase leptocephali to investigate the hormonal regulation of thetrigger for metamorphosis. The levels of T4 and T3 were quite low in both ON and OFF
leptocephali with no significant differences. Therefore, the specific ring formation for ON
individuals, which is presently the first indication of metamorphosis, may not be regulatedby thyroid hormones but by other unknown physiological mechanisms.
(1) Yamano et al. (1991) J. Comp. Physiol. B, 161, 371-375.
P49:

Eel, Leptocephalus, Metamorphosis, Otolith, Thyroid hormone

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P-50
CENTRAL ACTIONS OF ANGIOTENSIN II ON SPONTANEOUS BAROREFLEXSENSITIVITY IN THE TROUT Oncorhynchus mykiss
Frederic Lancien1 and Jean-Claude Le Mevel2
1

Laboratory of Physiology, Ocean Research Institute, The University of Tokyo, Japan.
Laboratory of Neurophysiology (LaTIM, INSERM U650), Faculty of Medicine, Brest,
France.
2

Cardiovascular baroreceptor control evolved early during evolution. In teleost fish,
both a reflex bradycardia and a reflex tachycardia can be observed following appropriate
pharmacologically induced changes in blood pressure. In addition, in teleosts as inmammals, a brain renin-angiotensin system could be involved in central cardiovascularregulation.
The goal of the present study was to investigate the central action of native
angiotensin II (ANG II) on the spontaneous baroreflex sensitivity (BRS) in unanaesthetized trout. The animals were equipped with two subcutaneous electrocardiographic (ECG)
electrodes, a dorsal aorta catheter and an intracerebroventricular (ICV) cannula which wasinserted within the third ventricle of the brain. The ECG and the systolic blood pressure(SBP) signals were recorded during a pre-injection period of five minutes and during fivepost-injection periods of five minutes. All injections were made at the fifth minute of thetest. The time-series were processed with a sequence technique in order to detect thesequences of three or more consecutive increases in the SBP pulse, or three or moredecreases in the SBP pulse correlated respectively with one delay beat increase of the RR
interval of the ECG signal or shortening of this interval. The slope of the average
regression line between the SBP and the RR intervals for each type of sequence was taken
as a measure of the spontaneous BRS.
Compared with pre-injection values, the ICV injection of vehicle (0.5 µl) had noeffect on heart rate (HR), SBP, the total number of positive or negative sequences or on the
spontaneous BRS during the post-injection periods. By contrast, ANG II at doses of 5 and50 pmol increased HR but only 50 pmol ANG II elevated SBP. For all doses, ANG II
depressed the spontaneous BRS, but the peptide had no effect upon the number of eachbaroreflex sequences.
This study determines for the first time the spontaneous BRS in a non mammalian
species and demonstrates an inhibitory action of ICV injection of ANG II upon thisvariable.
P50: Baroreflex, Angiotensin II, Fish Brain, Cardiovascular system, Signal processing

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P-51
THE DUAL MODE OF ACTION OF CORTISOL ON CELL TURNOVER OFOSMOREGULATORY ESOPHAGI IN EURYHALINE FISHES
Chiyo Takagi, Hideya Takahashi and Tatsuya Sakamoto
Ushimado Marine Laboratory, Faculty of Science, Okayama University, Setouchi,
Okayama 701-4303, Japan.
In the seawater (SW)-acclimated euryhaline fishes, the ion/water permeability of
the gastrointestinal tract is generally greater than that of freshwater (FW)-acclimated fish.
The esophageal epithelium of SW fishes is simple columnar in form, whereas that of FW
fishes is stratified. We have previously shown that esophageal epithelium of a euryhaline
goby and tilapia displays elevated cell proliferation in FW fish, but undergoes apoptosis
during SW acclimation (1). In-vivo cortisol treatment of the goby stimulated not only the
apoptosis but also cell proliferation in the esophageal epithelium, whereas 11deoxycorticosterone, the putative teleostean mineralocorticoid (2), did not have any impact
on cell proliferation or apoptosis (3). To understand the possible dual mode of action ofcortisol on the esophageal cell turnover, we have developed a method of culture tissue
explants of the esophagus from euryhaline medaka (Oryzias latipes). Oligonucleotide
detection assay and an oxidation-reduction indicator were used as indicators of apoptosis
and cell proliferations, respectively. Addition of cortisol (10 nM) to the culture medium
for 8 days stimulated apoptosis in the medaka esophagus, as a well-establishedglucocorticoid function. No effects were seen at higher doses (100 and 1000 nM). On
the other hand, addition of cortisol (1000 nM) for 8 days induced the cell proliferation.
The response of cell proliferation was dose-dependent within physiological range (10-1000nM). Thus, it is likely that cortisol induces directly not only apoptosis but also cellproliferation at the esophagus in euryhaline fishes, possibly via glucocorticoid receptor,
since glucocorticoid receptor shows lower sensitivity for cortisol than mineralocorticoid
receptor (2).
(1) Takahashi et al. (2006) J. Comp. Physiol. B, 176, 463-468.
(2) Prunet et al. (2006) Gen. Comp. Endocrinol., 147, 17-23.
(3) Takahashi et al. (2006) Life Sci., 79, 1873-1880.
P51: Cortisol; Apoptosis; Cell proliferation; Esophagus; Osmoregulation

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P-52
PHYSIOLOGICAL ROLE AND HORMONAL REGULATION OF
UREA TRANSPORTER EXPRESSED IN THE URINARY SYSTEM OF
THE MARINE TOADS, BUFO MARINUS
Norifumi Konno1, Susumu Hyodo2, Kouhei Matsuda1 and Minoru Uchiyama1
1

Department of Life and Environmental Science, Graduate School of Science andEngineering, University of Toyama, 3190 Gofuku, Toyama, 930-8555, Japan.
2
Laboratory of Physiology, Ocean Research Institute, University of Tokyo, 1-15-1
Minamidai, Nakano, Tokyo, 164-8639, Japan.
Anuran amphibians except for aquatic species usually utilize urea as a major end
product of nitrogen metabolism. However, they accumulate a large amount of urea in theirbody fluids and maintain hyper-osmolality to tolerate a severe dehydration under dry andhyper-saline environments. To clarify the mechanisms of urea retention and effects ofarginine vasotocin (AVT) on urea reabsorption in the urinary system, we characterized urea
transporter isolated from the toad kidney and examined the effect of AVT on urea uptake inthe cells isolated from the urinary bladder.
Bufo UT cDNA cloned from the kidney encodes a 390 amino acid residue protein,which belongs to homologue of mammalian UT-A2. The Bufo UT mRNA and protein wereabundantly expressed in the kidney and urinary bladder, but not in the skin. When
expressed in Xenopus oocytes by microinjection of the Bufo UT cRNA, the Bufo UT
induced more than 10-fold increase in [14C]urea uptake compared with water-injected
control oocytes. Phloretin, urea transport inhibitor, fully inhibited the increase of the urea
uptake. When toads were acclimated to dry and hyper-saline environments for 7 days, theplasma urea concentration and circulating AVT level were significantly elevated, and these
physiological changes were correlated with significant increases in the levels of Bufo UTmRNA in both the kidney and urinary bladder. In the epithelial cells isolated from the toadurinary bladder, addition of different AVT concentrations (10-12 to 10-8 M) to normal
medium increased the urea uptake in a concentration-dependent manner. To examine therelationship between the Bufo UT protein expression and an increase of urea
transportability, we analyzed time course of the Bufo UT expression levels and urea uptake
in the cells treated with 10-8 M AVT. Treatment of 10-8 M AVT increased the urea uptake inthe cells after 24 and 48 h incubation, but not after 12 h. According to the immunoblot
analysis, UT protein expression was coincident with the results of urea uptake in the AVTtreated cells.
These results suggest that the Bufo UT contributes to urea reabsorption in the urinary
system in response to hyperosmotic stresses and an increase of circulating AVT level. AVT
seems to play an important role for mechanism of urea retention in terrestrial amphibians.
P52: Urea transporter (UT), Urea, AVT, Kidney, Bufo marinus

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P-53
WATER PASSAGEWAY BY AVT-DEPENDENT AND -INDEPENDENTAQUAPORIN (AQP) IN SEVERAL WATER-PERMEABLE EPITHELIAL CELLSIN THE OSMOREGULATORY ORGANS OF THE TREE FROGGen Akabane1, Yuji Ogushi1, Takahiro Hasegawa2, Masakazu Suzuki1 and Shigeyasu
Takana1,3
1

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8522,
Japan.
2
Department of Anatomy and Cell Biology, Gunma University Graduate School of
Medicine, Maebashi 371-85111, Japan.
3
Integrated Bioscience, Graduate School of Science and Technology, Shizuoka University,
Shizuoka 422-8529. Japan.
E-mail: sbstana@ipc.shizuoka.ac.jp
An aquaporin (AQP) was cloned from a cDNA library constructed from the urinary
bladder of Hyla japonica. This AQP (Hyla AQP-h3BL) consisted of 292 amino acidresidues with high sequence homology to mammalian AQP3 (ca. 80%). The predicted
amino acid sequence contains the two conserved Asn-Pro-Ala motifs found in all major
intrinsic protein (MIP) family members and six putative transmembrane domains. The
sequence also contains a potential N-glycosylation site at Asn-141. In a swelling assay
using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a tenfold higher
permeability to water than oocytes injected with water. Immunofluorescence staining using
an antipeptide antibody (ST-184) against the AQP-h3BL protein revealed the presence of
immunopositive cells in the basolateral plasma membrane of granular cells in the ventral
pelvic and dorsal skins and in principal cells in the mucous glands. In the kidneys,immunopositive cells were found in the basolateral membrane of the principal cells in thecollected ducts and a portion of the late distal tubules. The principal cells in the urinary
bladder were also stained in the basolateral membrane. Both the homology of amino acidsequence and the immunostaining of the basolateral plasma membrane in water-permeable
epithelial cells imply that this AQP-h3BL is a homolog to mammalian AQP3. In theventral pelvic skins and urinary bladders, water enters into the cytoplasm through theapical plasma membrane at sites where AQP-h2, either in association with AQP-h3 oralone, is located in response to stimulation of arginine vasotocin (AVT) (1). A second AQP,
AQP-x5, has also been shown to be expressed in the apical membrane of principal cells in
the mucous glands (2). However, to data the water exit site for this AQP remains to beclarified. In the present study, we found specific AQP expression in the basolateral plasma
membrane of the water-permeable epithelial cells, as noted above. This finding suggeststhat AQP-h3BL is formed for the exit of water from the cytoplasm to the surrounding
capillaries in the granular cells in ventral skins and urinary bladders, whereas in the
mucous glands, water enters via this AQP-h3BL and exists through AQP-x5. However, wehave not yet identified any AQPs at the apical plasma membrane in the renal collected duct.
In addition, this AQP-h3L protein was observed to be expressed in the plasma and nuclear
membranes and some parts of cytoplasmic structures of gonadotropin (GTH) cells in thepituitary glands. This latter finding is very interesting in terms of the expression of AQPh3BL protein, and future studies will be necessary to clarify the physiological function of
the AQP in the pituitary glands.
1. Hasegawa, T. et al. (2003) Endocrinology, 144, 4087-4096.
2. Kubota, M. et al. (2006) J. Exp. Biol., 209, 3199-3208.
Key words: aquaporin, vasotocin, basolateral plasma membrane, exit of water, Hylajaponica

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P-54
ELECTROPHYSIOLOGICAL ANALYSIS OF ATRIAL NATRIURETIC PEPTIDEACTION ON NA CHANNELS IN EPITHELIAL CELLS FROM THE URINARYBLADDER OF JAPANESE TREE FROG, HYLA JAPONICA
Toshiki Yamada1, Shohei Kitani2, Kouhei Matsuda1 and Minoru Uchiyama1
1

Department of Life and Environmental Science, Graduate School of Science andEngineering, University of Toyama, Japan.
2
Department of Biology, Faculty of Science, University of Toyama, 3190 Gofuku, Toyama
930-8555, Japan.
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, Na+ transports activated by
ANP and cGMP were investigated electrophysiologically using a cell-attached patchclamp technique in primary culture epithelial cells from the urinary bladder. The channel
exhibited a low conductance for inward currents of 4.8 ± 0.2 pS, long open and closed
times (c.a. 200 msec), and positive reversal potential. The channel activity was decreased
under presence of 10-6 M amiloride in the pipette solution. These characteristics weresimilar to those of amiloride-sensitive Na+ channels (ENaC). Addition of 10-9 M fANP
activated and significantly increased the ENaC channel activity from 0.72 ± 0.22 to 1.88 ±0.46. On the other hand, mean amplitudes and conductance of single channel did notchange significantly after the addition of fANP. Addition of 10-5 M 8-Br-cGMP alsosignificantly increased the channel activity (NPo) from 0.56 ± 0.10 to 2.00 ± 0.33. The
addition of fANP failed to activate the ENaC under presence of 10-6 M amiloride. These
results suggested that ANP and cGMP activated Na+ transport via ENaC in the epithelial
cells of frog urinary bladder. On the other hand, 8-Br-cGMP failed to activate the ENaC
under presence of a specific PKA inhibitor, KT-5720.
In the next experiment, we measured both cGMP and cAMP production levels after
treatment of fANP on the frog urinary bladder cells using radioimmunoassay kits. Frog
ANP significantly increased cGMP production, but not the cAMP production. Taken
together, these results suggest that fANP activates ENaC through increases in cGMP
production and activation of PKA. It is also hypothesized that PKA might be activateddirectly by fANP via cGMP production in the epithelial cells of frog urinary bladder.
(1) Yamada et al. (2006) J. Comp. Physiol. B, 176, 203-212.
P54: Frog urinary bladder · Atrial natriuretic peptide · Amiloride-sensitive Na+ channel ·Single channel recording

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P-55
WHAT IS PHYSIOLOGICAL SIGNIFICANCE OF INCREASED GHRELINLEVEL IN PLASMA?
Hiroyuki Kaiya, Ei-Suke Saito1, Tetsuya Tachibana1, Mitsuhiro Furuse1 and Kenji
Kangawa
Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1
Fujishirodai, Suita, Osaka 565-8565, Japan.
1
Laboratory of Advanced Animal and Marine Bioresources, Graduate School of
Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581,
Japan.
Ghrelin stimulates feeding in rats when injected centrally or peripherally. On the other
hand, in chicks, an intracerebroventricular injection of ghrelin inhibits food intake (1,2).
We examined ghrelin levels in plasma and proventriculus, the glandular portion of theavian stomach, by using a specific radioimmunoassay for acylated ghrelin, as well as theexpression of ghrelin mRNA in the proventriculus after a 12-h fasting period followed by a
6-h feeding period with 6-day-old layer chicks. Furthermore, in order to examine the effectof increased circulating ghrelin on food intake, a bolus intravenous injection of chicken
ghrelin was given to 8-day-old chicks, and measured feeding.
Plasma ghrelin levels were increased by 12-h fasting, but once re-fed it returned to thecontrol value. Ghrelin mRNA and the peptide levels in the proventriculus were increasedby 12-h fasting, and once re-fed the ghrelin content returned to the control level, but
ghrelin mRNA levels remained high. The intravenous ghrelin injection (500 pmol) did notcause any significant changes in food intake during 120 min after injection.
These results indicate that the levels of ghrelin and its mRNA with layer chicks arealtered according to the feeding state. However, unlike in mammals, an increase incirculating ghrelin does not cause the promotion of food intake in chicks (3).
(1) Saito, E.-S. et al. (2002) Eur. J. Pharmacol., 453, 75-79.
(2) Saito, E.-S. et al. (2005) Regul. Pept., 125, 201-208.
(3) Kaiya et al. (2006) Domest. Anim. Endocrinol., In press
P55: ghrelin, chicken, fasting, refeeding

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P-56
THE ROLE OF HUMAN TYPE II GNRH RECEPTOR GENE IN THE
REGULATION OF CELL PROLIFERATION.
Yoh-Ichiro Kanaho1, Masahiro Enomoto1, 2, Mari Utsumi1, Tomoko Kato1 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.
GnRH is well known as a neuropeptide hormone, which is synthesized in
hypothalamus and stimulates the pituitary gland to secrete gonadotropin. Recently, theexpression of GnRH and GnRH receptor in several tissues other than pituitary have been
demonstrated, and GnRH has been suggested to possess several roles as controlling cell
growth, cell death, or gene expression. In our laboratory, in vitro assay was performed and
it indicated direct effects of GnRH on cell proliferation. In DU145, which is derived fromhuman prostate cancer, GnRH inhibited cell proliferation. On the other hand, in TSU-Pr1,
which is also derived from human prostate cancer, the effect was stimulatory (1). These
opposite effects are reported also in other cell lines, and the underlying molecularmechanisms are being examined in this study.
While the role of GnRH being clarifying bit-by-bit, intriguing issue has arisen fromour laboratory; using RNAi method, human type II GnRH receptor gene was suggested to
be functional, which was thought to be a pseudogene (2). In the experiment, colonyforming efficiency assay was employed, which was established in our laboratory to easily
detect the direct effect of GnRH on cell proliferation by performing it in extremely low celldensity. As there was a possibility that the effect on colony-forming efficiency does not
represent the effect on cell proliferation, in this study, we directly calculated the rate of cellproliferation in normal cell culture condition and clearly confirmed the relevance of type II
GnRH receptor to the regulation of cell proliferation by GnRH.
(1) Enomoto et al. (2004) Life Sci., 74, 3141-3152.
(2) Enomoto et al. (2004) Zool. Sci., 21, 763-770.
P56: GnRH, GnRH receptor, cell proliferation

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P-57
COMPARISON OF SERUM SEX STEROID PROFILES BETWEEN FOUR SPINESCULPIN Cottus kazika AND JAPANESE SCULPIN Cottus sp. SE REARED IN
CAPTIVITY
Ryo Hatano1, Daisuke Tahara1, Hiroshi Hashimoto2, Ryouji Fujii3, Yasunori Koya4, Youichi
Hayakawa5, Hidenobu Yambe6 and Tadahisa Seikai1
1Department of Mar. Sci., Fukui Prefectural University, Obama, 917-0003
Fukui Inland General Center, Fukui 441-3614, Japan.
3
Gifu Pref. Res. Inst. for Freshwater Fish and Aquatic Environ., Gero 509-2592, Japan.
4
Department of Biol., Gifu University, Gifu 501-1193, Japan.
5
Department of Biol., International Christian University, Mitaka, Tokyo181-8585, Japan.
6
Sesoko Station, Tropical Biosphere Res. Center, University of Ryukus, Okinawa 905-0227,
Japan.
2

The fourspine sculpin Cottus kazika is indigenous to Japan, but its abundance has
declined in almost all rivers. Therefore since 1988, techniques for seed production of thefour spine sculpin have been developed in Fukui Prefecture. However, procurement of
fertilized eggs of good quality has been highly variable because both male and female fish
do not mature normally in captivity. Recently the appearance of abnormally stripped eggs,
which did not fertilize in the artificial insemination, occurred in cultured broodstock
females. In contrast, cultured Japanese sculpin Cottus sp. SE mature and spawn withoutabnormal gonadal development. Studies on the reproductive physiology of these species
are limited and fragmentary. This study compares serum profiles of sex steroids between
both C. kazika and Cottus sp. SE reared in captivity.
The serum profiles of sex steroids in rearing sculpins were monitored for a full year.
In Japanese sculpins Cottus sp. SE, serum estradiol-17β (E2) and 11-ketotestosterone(11KT) levels were elevated significantly in the middle of vitellogenesis andspermatogenesis, respectively. Serum 17, 20β-dihydroxy-4-pregnen-3-one (DHP) in
female and male freshwater sculpins increased during the maturation period. However, infour spine sculpins, serum sex steroids levels remained low during the annual reproductive
cycle, throughout ovarian and testicular development, in captivity. These results suggestthat the serum sex steroids of four spine sculpins were disturbed by the captive rearing
conditions.
P57: freshwater sculpins, gonadal development, reproductive cycle, sex steroids

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P-58
SEX DIFFERENCE IN THE SYNTHESIS OF 7α-HYDROXYPREGNENOLONE IN
THE BRAIN OF BREEDING NEWTS
Shogo Haraguchi1,2, Masahiro Matsunaga3, Kazuyoshi Ukena2 and Kazuyoshi Tsutsui1,2
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
Department of Psychology, Nagoya University, Nagoya 464-8601, Japan.
It is becoming clear that steroids can be synthesized de novo by the brain and othernervous systems. Such steroids are called neurosteroids, and de novo neurosteroidogenesis
from cholesterol is a conserved property of vertebrate brains. Seasonal changes inneurosteroid levels in the brain have been demonstrated in seasonally breeding wild
animals, such as amphibians (1, 2) and birds (3). Recently, we identified in the newt brain anovel amphibian neurosteroid, 7α-hydroxypregnenolone that acts as a neuronal activator to
stimulate locomotor activity of breeding newts by means of the dopaminergic system (4). It
is well known that locomotor activity of male newts is higher than that of females during
the breeding season. To understand the sex difference in locomotor activity of newts during
the breeding season, we therefore investigated sex differences in the production andconcentration of 7α-hydroxypregnenolone in the newt brain. In this study, we show thatthe male brain actively produces 7α-hydroxypregnenolone during the breeding season,
unlike the female brain. The concentration of 7α-hydroxypregnenolone in the brain
markedly changed during the annual breeding cycle, with a maximal level in the spring
breeding season (April) in male newts. The 7α-hydroxypregnenolone concentration in themale brain was higher than that in the female brain in the breeding seasons (April andOctober). In the spring breeding season, the production of 7α-hydroxypregnenolone in thebrain of breeding males was higher than that of females. We then examined the expressionof cytochrome P4507α, which produces 7α-hydroxypregnenolone from pregnenolone, inthe male and female brains. Cytochrome P4507α mRNA was expressed highly in the malebrain than that of the female brain in the spring breeding season. These results indicate that
7α-hydroxypregnenolone is a key factor for the increase in locomotor activity of malenewts during the breeding season. This is the first demonstration of sex differences in the
production and concentration of neurosteroid in any vertebrate.
(1) Takase, M. et al. (1999) Endocrinology, 140, 1936-1944.
(2) Inai, Y. et al. (2003) Brain Res., 959, 214-225.
(3) Lea, R.W. et al. (2001) Microsc. Res. Tech., 55, 12-26.
(4) Matsunaga, M. et al. (2004) Proc. Natl. Acad. Sci. USA, 101, 17282-17287.
P58: neurosteroid, 7α-hydroxypregnenolone, sex difference, newt, brain

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P-59
THE EFFECT OF INCUBATION TEMPERATURE ON THE SEX STEROIDHORMONE SIGNALING SYSTEMS IN THE BRAIN AND GONAD OF THE
SQUAMATA SPECIES WITH TEMPERATURE-DEPENDENT SEX
DETERMINATION
Daisuke Endo and Min Kyun Park
Department of Biological Science, Graduate School of Sciences, The University of Tokyo.
Tokyo, 113-0033, Japan.
Some reptilian species have no sex chromosomes and their sex is determined byincubation temperature. Recently, it has been reported that incubation temperature affected
not only gonadal sex but also sexual behavior in a female leopard gecko, a lizard withtemperature-dependent sex determination (1). This suggests that the brain may differentiate
independently from the gonadal steroid hormones to some degree. To access thispossibility, the effect of incubation temperature on the expression of sex steroid hormonerelated genes was investigated in the brains and gonads of the developing leopard geckoes.
In the embryo of leopard gecko, the mRNAs of two sex steroidogenic enzymes,
cholesterol side chain cleavage enzyme (P450scc) and P450 aromtase (P450arom), wereexpressed only in the brains and gonad. The mRNAs of sex steroid hormone receptors
were also expressed in these two organs. Thus, it is suggested that in the developing
leopard gecko, the brain and gonad are the main steroidogenic organs and the sex steroid
hormones can affect their sexual differentiation as auto-para-crine factors. The mRNA
expression levels of P450arom were not affected by the incubation temperature during
development and the sex difference of the expression was observed only after hatch. In thebrain, however, the effect of incubation temperature on the mRNA expression levels of
seteroidogenic enzymes were observed before hatch. Those of P450scc were significantly
higher in the brains of the embryo incubated in male-biased temperature (32°C) than thosein female-biased temperature (26°C and 34°C) during temperature sensitive period (TSP).
On the other hand, the expression levels of P450arom in the developing brains, were highly
dependent on incubation temperature (34°C >32°C >26°C).
These results strongly suggested that incubation temperature affect on the brain
directly and the brain can differentiate independently from the gonad. Because of thedifference in the patterns of the effect of incubation temperature, the roles of P450scc andP450arom are different in the brain sexual differentiation.
(1) Rhen T. and Crews D. Endocrinology, 140, 4501-4508, 1999
P59: sex differentiation, sex steroid hormone, temperature-dependent sex determination,
reptiles, brain

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P-60
CIRCADIAN CHANGE IN THE BIOSYNTHESIS OF 7α-HYDROXYPREGNENOLONE STIMULATING LOCOMOTOR ACTIVITY AND ITS MECHANISM MEDIATED BY MELATONIN IN THE BRAIN OF MALE QUAIL
Saori Suzuki1, Hitomi Miyabara1, Kazuhiko Inoue1,2 , Shogo Haraguchi1,2,
Kazuyoshi Ukena1 and Kazuyoshi Tsutsui1,2
1

Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima
University, Higashi-Hiroshima 739-8521, Japan.
2
Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education
and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan.
We recently identified 7α-hydroxypregnenolone stimulating locomotor activity as anovel avian neurosteroid in the quail brain. The presence of this neurosteroid may be aconserved property of vertebrates (1). Because locomotor activity of male birds is known
to be higher than that of female birds, 7α-hydroxypregnenolone may be involved in theincrease of locomotor activity of male birds. To test this hypothesis, we therefore analyzed
sex differences in locomotor activity and 7α-hydroxypregnenolone synthesis using adultquail exposed to long-day photoperiods (16L 8D; lights on at 0700 h). Behavioral analysis
revealed that locomotor activity of males was much higher than that of females from thestarting of light on until noon. Locomotor activity of males decreased thereafter andreached to a lower level similar to females. Both the biosynthesis of 7αhydroxypregnenolone and expression of cytochrome P4507α, a steroidogenic enzyme of
7α-hydroxypregnenolone production, in the male diencephalon markedly changed during
the observed 24 h-period, with a maximal level at 1100 h when locomotor activity of males
was high. In contrast, locomotor activity of females and 7α-hydroxypregnenolone
synthesis and P4507α expression in the female diencephalon were constantly low during thesame observed 24 h-period. Thus, 7α-hydroxypregnenolone may be involved in the higher
locomotor activity of male birds. Subsequently, we investigated the mechanism that
regulates the circadian change in 7α-hydroxypregnenolone synthesis, in male quail.
Pinealectomy (Px) combined with orbital enucleation (Ex) (Px plus Ex) increased 7αhydroxypregnenolone synthesis in the diencephalon of males. Melatonin administration toPx plus Ex males caused a decrease in 7α-hydroxypregnenolone synthesis in the male
diencephalon. These results indicate that melatonin is a key factor for circadian change inthe biosynthesis of 7α-hydroxypregnenolone in male birds.
(1) Matsunaga, M. et al. (2004) Proc. Natl. Acad. Sci. USA., 101, 17282-17287.
P60: quail, 7α-hydroxypregnenolone, melatonin

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P-61
INVOLVEMENT OF NEUROPEPTIDE IN THYROXINE SURGE IN SMOLTING
KOKANEE SALMON
Hiroaki Chiba1, Yuya Satoh1, Tomoki Yahata1, Daisuke Ojima1, Shyunsuke Moriyama1,
Hyuma Kudo2 and Munehico Iwata1
1

School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Marine Products Division of Ofunato Regional Development Bureau, Iwate 022-8502,
Japan.
2

Involvement of thyrotropin-releasing hormone (TRH) in secretion of thyroxine (T4)through the release of thyroid-stimulating hormone (TSH) from the pituitary is wellestablished in mammals and birds. Prior to downstream migration, a sharp increase ofplasma thyroxine (T4 surge) has been observed in many species of salmonid fishes.
However, the hypothalamic regulation in thyrotropin secretion and subsequent thyroid
activity in teleost fish still remains obscure. Neuropeptides (NP) such as TRH(1),gonadotropin-releasing hormone (GnRH) (2), growth hormone-releasing hormone(GHRH)(2), and corticotrophin-releasing hormone (CRH) (3) have been proposed as
potential candidates for the hypothalamic regulation of TSH. The objective of the present
study was to investigate which NP is involved in the T4 surge during smoltification insalmonid fishes.
To assess the sensitive period of T4 secretion by environmental stimuli,
underyearing kokanee salmon (Oncorhynchus nerka) were exposed to clay-suspended
water under natural photoperiod from March to September (4). On the other hand, mTRH,
oCRH, sGnRH, or hGHRH were injected intraperitoneally (IP) and intracerebrally (IC) at
doses of 1µg/gBW and 1µg/µl/fish, respectively. The blood was collected from peduncle 3
hours after administration to determine the plasma T4 concentration by time-resolved
fluoroimmunoassay (TR-FIA).
Average plasma T4 levels of control (intact) fish increased from 3.8 ng/ml in March
to 9.7 ng/ml in mid June followed by a decrease in July, suggesting the mid June is a peakof smoltification. Exposure to the clay-suspended water caused distinctive T4 surges (11 –23 ng/ml) from April to July. While IP injections of all NP did not stimulate T4 secretion
throughout the experimental period, IC injections of GHRH or CRH caused significant
increases in plasma T4 (23 – 37 ng/ml) from April to June. These results suggest that
GHRH and/or CRH have a stimulatory effect on T4 surge via TSH in the pituitary ofsmolting kokanee salmon.
(1) Eales, J.G. and Himick, B.A. (1988) Gen. Comp. Endocrinol., 72, 333-339.
(2) Pahar, I. S. and Iwata, M. (1996) Neurosci. Res., 26, 299-308.
(3) Larsen, D. A. et al. (1998) Gen. Cmp. Endocrinol., 109, 276-285.
(4) Iwata, M. et al. (2003) Aquaculture, 222, 315-189.
P61: Neuropeptide, Thyroxine, Salmon, Smolt

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P-62
ELEVATION OF THE PITUITARY CONTENTS OF GONADOTROPINS AND
PLASMA LEVELS OF FOLLICLE-STIMULATING HORMONE IN CHUMSALMON PRIOR TO INITIATION OF SPAWNING MIGRATION FROM THE
BERING SEA
Takeshi A. Onuma1,2, Shunpei Sato3, WeiWei Hu2, Aya Jodo2, Nancy Davis4, Masa-aki
Fukuwaka5, Tomonori Azumaya5, Penny Swanson6 and Akihisa Urano2
1

Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
Fukuoka 812-8581, Japan.
2
Section of Biological Sciences, Graduate School of Life Sciences, Hokkaido University,
Sapporo 060-0810, Japan.
3
National Salmon Resources Center, Sapporo 062-0922, Japan.
4
School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, 981955020, USA.
5
Hokkaido National Fisheries Research Institute, Kushiro 085-0802, Japan.
6
Northwest Fisheries Science Center, NOAA Fisheries, Seattle, WA, 99164, USA.
The hypothalamus-pituitary-gonadal axis is a neuroendocrine system through whichhoming salmon concordantly regulate gonadal maturation and migratory behavior to thespawning ground. A line of previous evidence in aquacultured-salmon indicated that thepituitary-gonadal axis (PG-axis) was activated from late spring through summer, when
wild chum salmon initiate spawning migration (1). In the present study, we examined
whether similar activation occurs in the PG-axis of pre-migratory chum salmon in the
Bering Sea.
Chum salmon were caught along the 180o-longitude line in the Bering Sea insummer and autumn from 2001 through 2003.
We referred immature fish as stage I, andmaturing fish as stage II and III, on the basis of the gonadosomatic indices (GSI) andhistological aspects of gonads. The amounts of FSH and LH in hemisected pituitaries
were determined by radioimmunoassays (2), and GTH subunit mRNAs by quantitative
real-time polymerase chain reactions. Plasma levels of sex steroid hormones, such as
testosterone (T), 11-ketotestosterone (11KT) and estradiol-17β (E2), were determined byenzyme immunoassays. Mitochondrial DNA haplotypes of the fish were determined by
DNA microarray (3) to distinguish the genealogical clade A (Japanese population) andclade B (Japanese, Russian, Canadian and North American populations).
The fish in the Bering Sea were comprised of those in the stage I, II and III insummer, while almost all of the fish were in the stage I in September, indicating that thestage II and III fish left for the natal river by the end of summer. The amounts of FSH
and LH in stage II and III fish were 100- to 1000-fold those in stage I fish in all three years.
The amounts of α2 and FSHβ mRNAs in the stage II and III fish were 2- to 10-fold, while
those of LHβ mRNA were about 100-fold when compared to the amounts in the stage I
fish. Plasma levels of FSH in the stage II and III fish were 2- to 3-fold those of the stage I
fish. Plasma levels of T, 11KT and E2 were more than 10 nM in the stage II and III fish,
while about 1.0 nM in the stage I fish. Such increases were similarly confirmed in boththe clade A and clade B fish.
The present results indicated that the PG-axis was activated along with the onset of
spawning migration in the Bering Sea. The activation of the PG-axis at least partly by
GnRH neurons should be a programmed neuroendocrine event that is inseparable from theinitiation of gonadal maturation and homing behavior to the natal river.
(1) Ando, H. et al. (2005) Zool. Sci., 22, 379-389.
(2) Swanson, P. et al. (1989) Fish Physiol. Biochem., 7, 169-176.
(3) Moriya, S. et al. (2005) Mar. Biotechnol., 6, 430-434.
P62: chum salmon, Bering Sea, spawning migration, gonadotropin, follicle-stimulating

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P-63
RELATIONSHIP OF HOMING BEHAVIOR FROM SEAWATER TO FRESH
WATER WITH PLASMA LEVELS OF SEX STEROID HORMONES IN PRESPAWNING CHUM SALMON
Keita Makino 1 , Takeshi A. Onuma 1, 2 , Takashi Kitahashi 1 , Hironori Ando 2 , Masatoshi
Ban 3 and Akihisa Urano 1
1)Section of Biological Sciences, Graduate School of Life Science, Hokkaido University,
Sapporo 060-0810, Japan.
2)
Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University,
Fukuoka 812-8581, Japan.
3)
National Salmon Resources Center, Sapporo 062-0922, Japan.
Most salmonid species migrate from ocean through the natal river for spawning.
Our previous study showed that homing behavior of pre-spawning chum salmon(Oncorhynchus keta) in the Ishikari stock and the Sanriku stock was dependent on ocean
temperature, because salmon is typical cold-water fish. There were nonetheless few lines
of evidence to explain the relationship between migration from seawater (SW) to fresh
water (FW) and sexual maturation. We therefore investigated a temporal profile ofmigratory behavior of pre-spawning chum salmon, and examined changes in plasma levelsof sex steroid hormones during upstream migration from 2001 to 2005. We further
investigated spontaneous behavior of fish between SW and FW environments in aquaria.
Pre-spawning fish were collected at the estuary of the Ishikari River, released after
attachment of a micro data logger which can record salinity, temperature and water depth,
and re-captured at the hatchery in 2005. Pre-spawning fish in the Otsuchi Bay werereared in a large aquarium, which allows fish to migrate between separated SW and FW
streams. Plasma was collected from each fish every morning, and the relationship ofplasma sex steroid hormones with the ratio of time spent in FW was monitored.
Pre-spawning fish of Ishikari stock continued vertical movement between SW andFW environment at the mouth of the river for about 12 hours, followed by continuous
migration in FW. In aquarium experiment, plasma testosterone (T) levels in theindividuals which spent mainly in FW were higher, when compared to the levels in thosewhich stayed mainly in SW. Plasma estradiol-17_ (E2) levels in the males were lower inFW than in SW, while the levels in the females were higher in FW than SW. Plasma Tlevels increased during upstream migration and peaked on the midway of the river,followed by decreases at the hatchery in both sexes. The levels of E2 also reached theirpeaks at the estuary or on the midway of the river in all years examined.
In conclusion, migratory behavior from SW through FW is closely associated with
progress in gonadal maturation. These results suggested that sex steroid hormones
modulate preference of environmental salinity to FW and facilitate upstream migratory
behavior.
P63: Chum salmon; Upstream migration; Sex steroid hormone; Fresh water adaptation

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P-64
ISOLATION AND CHARACTERIZATION OF NEUROMEDIN U FROM THE
GOLDFISH
Keisuke Maruyama1, Tohru Miura1, Minoru Uchiyama1, 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.
Neuromedin U (NMU) was first isolated from the porcine spinal cord (1).
Subsequently, NMU has been identified in the central nervous system (CNS) andgastrointestinal tract of vertebrates including the human being, rat, chick and frog, andNMU can potentially act as an anorexigenic peptide in rats and Japanese quials (2-4).
However, in fish, NMU has never been found, and therefore, its role is unknown. In thepresent study, the aims were 1) to isolate NMU cDNA from the goldfish, and 2) to examine
its function. We identified four kinds of cDNAs encoding NMU precursors from the brain
and intestine, suggesting that deduced NMUs with 21, 25 and 38 residues are derived fromthese precursors. We observed the distribution of mRNAs for NMU precursors in goldfish.
The expression of these mRNAs was mainly found in CNS and intestine. Thus, the effectof feeding status upon expression of NMU precursor mRNA which is abundant in the brain
and encodes NMU with 21-residue (NMU-21) was examined. Fasting for 7 days decreased
significantly its mRNA expression in the brain. We synthesized NMU-21 with the Cterminal amide, and examined the effect of intracerebroventricular (ICV) administration of
NMU-21 on feeding behavior in the goldfish. ICV-injected NMU-21 suppressed foodconsumption and locomotor activity in a dose-dependent manner. These results suggestthat NMU expresses mainly in CNS and intestine, and that NMU-21 is involved in theregulation of food intake and locomotor activity in the goldfish.
(1) Minamino et al. (1985) Biochem. Biophys. Res. Commun., 130, 1078-1085.
(2) Kojima et al. (2000) Biochem. Biophys. Res. Commun., 276, 435-438.
(3) Nakazato et al. (2000) Biochem. Biophys. Res. Commun., 277, 191-197.
(4) Shousha et al. (2005) Gen. Comp. Endocrinol., 140, 156-163.
P64: neuromedin U, goldfish, mRNA, feeding behavior, anorexigenic action

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P-65
INHIBITORY EFFECT OF MELANIN-CONCENTRATING HORMONE ON
FEEDING BEHAVIOR IN THE GOLDFISH
Sei-Ichi Shimakura1, Keisuke Maruyama1, Tohru Miura1, Minoru Uchiyama1, Hiroshi
Kawauchi2, Seiji Shioda3, 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.
3
Department of Anatomy, Showa University School of Medicine, Tokyo 142-8555, Japan.
Melanin-concentrating hormone (MCH) is a hypothalamo-pituitary peptide, which
had been first identified in the salmon pituitary as a hormone affecting body color (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. Our recent research has indicated that intracerebroventricular(ICV) injection of MCH influences feeding behavior in the goldfish, and may exert ananorexigenic action, unlike its orexigenic action in mammals (3). The aim of the present
study was to investigate the involvement of endogenous MCH in feeding behavior in the
goldfish. We examined the distribution of MCH-like immunoreactivity (MCH-LI) in the
goldfish brain, and the effect of feeding status upon it. Neuronal cell bodies containing
MCH-LI were localized specifically to four areas of the hypothalamus. Nerve fibers with
MCH-LI were found mainly in the neurohypophysis, and a few in the mesencephalon anddiencephalon. The number of neuronal cell bodies containing MCH-LI in the dorsal areaadjoining the lateral recess of the third ventricle in the posterior and inferior lobes of the
hypothalamus showed a significant decrease in fasted fish as compared with that innormally fed fish. We also administered an antiserum against fish MCH (anti-MCH serum)by ICV injection and examined its immunoneutralizing effect on food intake. Cumulative
food intake was significantly increased by ICV injection of anti-MCH serum. These result
suggest that MCH influences the feeding behavior, and that MCH can potentially function
as an anorexigenic neuropeptide in the goldfish brain, unlike in mammals.
(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.
P65: Goldfish, MCH, Anorexigenic action, Immunohistochemistry, Immunoneutralization

–77–

P-66
REGULATION OF FOOD INTAKE BY GHRELIN IN THE GOLDFISH
Tohru Miura1, Keisuke Maruyama1, Sei-Ichi Shimakura1, Hiroyuki Kaiya2, Minoru
Uchiyama1, Kenji Kangawa2 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.
Ghrelin was originally isolated from rat and human stomachs as an endogenous ligand
for the growth hormone secretagogue receptor (1). Ghrelin is now recognized to be amultifunctional peptide that is involved in the regulation of somatic growth, feeding
behavior and energy homeostasis in mammals. It is known that peripheral ghrelinstimulates growth hormone release and appetite via the vagal afferent pathway, which issensitive to the neurotoxin capsaicin (2) and the orexigenic action of ghrelin is mediated by
the neuropeptide Y (NPY)/Agouti-related protein in mammals (3). However, in goldfish,
the possible involvement of afferent pathways in mediating the orexigenic action of
peripheral ghrelin, and the interactive effect of ghrelin and NPY on feeding, like that seen
in mammals, have been unclear. In this study, we examined the effect of capsaicin, which
destroys primary sensory (vagal and splanchnic) afferents, on the orexigenic activity
induced by intraperitoneal (IP)-injected ghrelin. Pretreatment with IP-injected capsaicin
cancelled the orexigenic action of IP-injected ghrelin, although IP-injected capsaicin alone
did not affect food intake. We also examined the effect of intracerebroventricular (ICV)
preinjection of a NPY Y1-receptor antagonist, BIBP-3226, on the orexigenic action of
ICV- or IP-administered ghrelin in goldfish. Food intake induced by ICV or IP injection ofghrelin was suppressed by ICV preinjection of BIBP-3226 for 1 h. Furthermore, we
studied whether either ICV or IP injection of ghrelin affects the expression of NPY mRNAin the brain using a real-time PCR method. ICV, but not IP, administration of ghrelin at adose sufficient to stimulate food intake increased the expression of brain NPY mRNA
obtained from 2 h after treatment. These results indicate that circulating ghrelin derived
from peripheral tissues acts via primary sensory afferent pathways on feeding centers in
the goldfish and that the orexigenic action of ghrelin is mediated by NPY pathway in the
goldfish brain.
(1) Kojima et al. (1999) Nature, 402, 656-660.
(2) Date et al. (2002) Gastroenterology, 123, 1120-1128.
(3) Nakazato et al. (2001) Nature, 409, 194-198.
P66: Goldfish, Ghrelin, NPY, BIBP3226, Capsaicin

–78–

P-67
EFFECT OF OCTADECANEUROPEPTIDE ON FOOD INTAKELOCOMOTOR ACTIVITY IN THE GOLDFISH, Carassius auratus

AND

Kohei Wada1, Yoko Inaoka1, Tohru Miura1, Keisuke Maruyama1, Sei-Ichi Shimakura1,
Minoru Uchiyama1, Jérôme Leprince2, Marie-Christine Tonon2, Hubert Vaudry2 andKouhei 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.
Octadecaneuropeptide (ODN) is a peptide derived from a precursor, the diazepambinding inhibitor protein (DBI) in rats. DBI and its processing peptides such as ODN are
also called endozepines (1). Endozepine has been identified in the central nervous systemof vertebrates such as fish, amphibians, birds and mammals, indicating that endozepine ismainly distributed in the hypothalamic region (2-4). Recently, behavioral studies revealed
that intracerebroventricular (ICV) injection of ODN affects feeding behavior andpsychomotor activity in rodents (5,6). However, the effect of ODN has never been clarified
in fish. The aim of the present study was to investigate the effect of ODN on feeding
behavior and spontaneous locomotor activity in the goldfish, and we therefore examinedthe effect of ICV administration of synthetic rat ODN on food intake and locomotor
activity using an automatic monitoring system.
ICV injection of ODN at 2.5-10 pmol/g body weight (BW) suppressed foodconsumption in a dose-dependent manner, and ICV-injected ODN at 5-10 pmol/g BW
induced a significant decrease in the cumulative food intakes during 60 min after feeding.
Locomotor activity was assessed by tracing individual swimming distances in the fish tank.
ICV injection of ODN at 10 pmol/g BW induced a significant increase in locomotor
activity for an observation period (60 min). These results suggest that ODN influences
feeding behavior and spontaneous locomotor activity in the goldfish.
(1) Tonon et al. (1994) Med. Sci., 10, 433-443.
(2) Alho et al. (1985) Science, 229, 179-182.
(3) Malagon et al. (1992) Brain Res., 576, 208-214.
(4) Malagon et al. (1992) Peptides, 13, 99-107.
(5) de Mateos-Verchere et al. (2001) Eur. J. Pharmacol., 414, 225-231.
(6) de Mateos-Verchere et al. (1998) Peptides, 19, 841-848.
P67: goldfish, octadecaneuropeptide, ICV injection, anorexigenic action, hypermotility

–79–

P-68
THE ROLE OF ANDROGEN IN PROTOGYNOUS SEX CHANGE OF GROUPER
Mohammad Ashraful Alam1 and Masaru Nakamura1,2
1

Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Sesoko
3422, Motobu, Okinawa 905-0227, Japan.
2Core Research for Evolutionary Science and Technology (CREST), Japan Science andTechnology Corporation, Saitama 332-0012, Japan.
Most groupers change sex from female to male during their life time. In order todetermine the role of androgen in the sex change, we first identified the sites of androgensynthesis in the gonads using immunohistological observations with specific antibody
against cytochrome P45011ß-hydroxylase (P45011ß), which is the key enzyme in 11ketotestosterone (11-KT) production, during the sex change in the honeycomb grouper,
Epinephelus merra. P45011ß-positive cells were observed near blood vessel (BV) in thetunica enclosing the outer periphery of the ovary, but not in theca layer of the oocyte infemale fish. In early and late transitional gonads, P45011ß-reactive cells appeared in thetheca cells enclosing degenerate oocytes, and near the BV in the tunica ovary. However, inthe testis after the sex change, immunopositive cells derived from theca cells weretransformed into Leydig cells. Cell clusters near the BV in the tunica ovary also showedstrong signals for P45011ß in the testis of the sex-changed male. The nuclei diameter of
immunopositive cells also increased with the plasma 11-ketotestosterone (11-KT) level as
the sex change progressed. These results suggest that cell clusters near BV in the tunica
ovary is the location of new androgen biosynthesis, which actively produces 11-KT andplays a critical role during the sex change (1). These findings also provide evidence thatthe theca cells enclosing degenerate oocytes transform into Leydig cells during the sex
change (2).
(1) Alam et al. (2005) Cell & Tissue Research, 320, 323-329(2) Alam et al. (2006) Molecular Reproduction and Development, 73, 206-14.
Key words: Tunica ovary, andorogen, sex change

–80–

P-69
FUNCTIONAL ASSOCIATIONS AMONG POSITIVE EMOTION, CENTRALNERVOUS, PERIPHERAL AUTONOMIC NERVOUS, ENDOCRINE, AND
IMMUNE SYSTEMS
Masahiro Matsunaga1,2, Toshihiro Konagaya2 and Hideki Ohira1
12Department of Psychology, Nagoya University, Chigusa 464-8601, Japan.
Division of Gastroenterology, Aichi Medical University, Nakakute 480-1195, Japan.

Recent studies in psychoneuroimmunology have revealed that the central nervous,
peripheral autonomic nervous, endocrine, and immune systems are interrelated throughcomplex biochemical pathways. Studies have also revealed that psychosocial stressors
such as public speaking, examinations, and even short-term mental arithmetic are sufficient
to bring about changes in immunological parameters. These stressors can stimulate theactivities of the sympathetic nervous system and hypothalamus-pituitary-adrenal (HPA)
axis, and increase circulating levels of catecholamines such as norepinephrine andepinephrine. Prolonged elevation of sympathetic nervous activity by such stressors induces
extremely high levels of circulating catecholamines, reduces the number of circulating
lymphocytes and activity of natural killer (NK) cells, which are a subgroup of lymphocytesthat play an essential role in the cellular immune defense against virus-infected cells,
bacteria or tumor cells by a β-adrenergic mechanism, and, as a result, decreases immune
defense.
Then, how is it in the case of positive emotion? For example, when we look at ourfavorite person, our heart will be filled with fortunate feelings, and occasionally we will
feel like ‘flying in the sky’. Everybody knows that such romantic feeling is cheerful, strong
and passionate. Accompanying romantic feeling, multiple responses in brain, peripheral
autonomic nervous, endocrine and immune systems may occur. To investigate such
associations among biological systems, we recorded simultaneously brain activity withpositron emission tomography (PET), heart rate, blood pressure, serum levels of several
hormones, and natural killer (NK) cell activity when twelve male healthy participants
watched the film of their favorite persons. Interestingly, accompanying self-reported
positive feelings, a component of innate immune system, NK cell activity, was activated.
Various brain regions, such as the medial prefrontal cortex (MPFC), orbitofrontal cortex(OFC) and hypothalamus, were also activated. Furthermore, mean blood pressure and theserum level of prolactin, which is related to positive emotions, also increased. The
activation in the OFC positively correlated with magnitudes of NK cell activity andprolactin response. This study is the first observation demonstrating that romantic feelingactivates innate immune functions and also the first observation showing functional
associations among romantic feeling, central nervous, peripheral autonomic nervous,
endocrine, and immune systems. It is suggested that the OFC might be one of pivotal
regions for top-down regulation over endocrine and immune activity accompanying
romantic feeling.
P69: positive emotion, orbitofrontal cortex, innate immune system

–81–

P-70
SEX DIFFERENCE IN THE PEP-19 EXPRESSION IN SONG NUCLEI OF ZEBRAFINCHES DURING EARLY BRAIN DEVELOPMENT
Kazuhiko Inoue1, 2, Masaki Kato3, Kazuo Okanoya3, Hironobu Sakaguchi4, Kazuhiro
Wada5, Erich D. Jarvis5 and Kazuyoshi Tsutsui1, 2
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 for Biolinguistics, RIKEN Brain Science Institute, Saitama 351-0198, Japan.
4
Department of Physiology and Biological Information, Dokkyo University, School of
Medicine, Mibu, Tochigi 321-0293, Japan.
5
Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA.
Courtship song in the zebra finch (Taeniopygia guttata) is produced by males but notby females. The brain regions controlling song learning and production are highly
developed in males, but greatly reduced or absent in females (1) (Fig.1). The factors that
initiate sexual differentiation of the brain are not known in the zebra finch. To investigate
the key factors initiating sexual differentiation in brain structure, a screen was conducted
for genes involved in sexually dimorphic development of the neural song system in zebra
finches. cDNA microarrays were initially used to compare gene expressions in thetelencephalon of hatchling males and females. We found gene expression differences
between the male and female in several song control nuclei several genes showing different
expressions in song control nuclei including the nucleus robustus archistriatum (RA).
Interestingly, a higher expression of PEP-19 was detected in the RA of males. PEP-19 is acalmodulin-regulatory protein known to suppress the apoptotic process in PC12 cells (2).
Accordingly, these results suggest that PEP-19 acts to decrease apoptosis in the RA of
males during early brain development. PEP-19 may be a factor initiating sexual
differentiation in the RA of zebra finches.
(1) Erhardt, J.A. et al. (2000) Neuroreport., 11, 3719-3723.
(2) Nottebohm, F. and Arnold, A.P. (1976) Science, 194, 211-213

P70: song nuclei, PEP-19, cDNA microarray, sex difference, zebra finch–82–

AUTHOR INDEX
Abe, Shin-ichi	
48
Adachi, Shinji	47
Ajimura, Masahiro
38
Akabane, Gen
65
Alam, Mohammad Ashraful
80
Amano, Katsufumi	
53
Amano, Masafumi	
55
Amiya, Noriko
53, 55, 57
Ando, Hironori	14, 54, 75
Aonuma, Hitoshi	
1Asahina, Kiyoshi	
44
Azumaya, Tomonori	74
		Ban, Masatoshi	
5Birukawa, Naoko
60
		Cai, Li-yi	
49
Chiba, Hiroaki	
73
Chowdhury, Vishwajit S.
21

Iigo, Masayuki	
Ijiri, Shigeho
Ikegami, Taro
Ikemoto, Tadahiro
Inaba, Mayumi	
Inaoka, Yoko
Inoue, KazuhikoIto, Etsuro
Ito, Kae
Ito, Yoichi	
Itoh, Hiroshi	Itoi, Keiichi	
Ishiguro, Kotaro
Ishii, Yoshiyuki	
Iwata, Munehico
Izumi, Shun-ichiro

Davis, Nancy
74
		
Endo, Daisuke
71
Enomoto, Masahiro
68
Eto, Ko
48
		Fang, Wenjun
60
Fujimoto, Yoshinori	
13
Fujiwara, Ken,
22
Fukumoto, Shunsaku
50
Fukuwaka, Masa-aki	
74
Furukuma, Shunji	
54
Furuse, Mitsuhiro
67		Goto, Ryo39		Haraguchi, Shogo70, 72
Hashimoto, Hiroshi	
69
Hasunuma, Itaru
17, 58
Hatano, Ryo
69
Hayakawa, Youichi	
69
Hayashi, Shinji	
56
Honda, Kaori	
52
Horiguchi, Maya
46
Hu, WeiWei	
74
Hyodo, Susumu
39, 53, 64		Ieki, Katsunori	
38

Kagawa, Haruhiko
Kaiya, Hiroyuki	
Kanaho, Yoh-Ichiro
Kaneko, Hiroyo
Kangawa, Kenji	Kato, Keisuke
Kato, Masaki	Kato, Takako
Kato, Tomoko
Kato, Yukio
Kawata, Mitsuhiro
Kawauchi, Hiroshi	
Kikuchi, Motoshi,
Kikuyama, Sakae
Kimura, Atsushi	
Kimura, Hiroshi	
Kinjo, Masataka
Kitahara, Kousuke
Kitahashi, Takashi	
Kitamura, Shoji	Kitani, Shohei	
Kobayashi, Kazuto
Kobayashi, Kenichi	
Kobayashi, Tetsuya
Kobayashi Toru
Kobayashi, Yuki	
Koike, Kanako
Konagaya, Toshihiro–83–

Jarvis, Erich D.
Jin, Yuji	

57
47
14
34
30
79
72, 82
7
59
13, 38
55
33
35
33
73
49
82
48
45
67, 7868
47
43, 67, 78
31
82
24, 25, 26, 27, 49
68
24, 25, 26, 27, 49
3, 59
53, 7722
17, 589, 28
102
24, 25, 26, 27
75
57
66
33
51
58
45
36
23
81

Konno, Norifumi	
64
Kouki, Tom
22
Kubokawa, Kaoru
30, 44
Kubota, Hisayo
50, 51
Kudo, Hyuma
73
Kumada, Hikari	
53
Kurogi, Hiroaki	
61
		
Lancien, Frederic
62
Le Mevel, Jean-Claude
62
Leprince, Jerome
79
Li, Jicheng
60
Lokman, P. Mark
45
		
Machida, Takeo
58
Maeda, Chisato
53
Maeda, Sayaka
13, 38Makino, Keita
75
Maruyama, Keisuke
35, 76, 77, 78, 79
Matsubara, Takahiro
45
Matsuda, Kouhei	
35, 64, 66, 76, 77, 78, 79
Matsubara, Hajime
45
Matsuki, Mio
55
Matsunaga, Masahiro
70, 81
Minagawa, A
58
Minagawa, Kazuto
40
Mita, Kazuei	
38
Miura, Tohru
76, 77, 78, 79
Miyabara, Hitomi	
72
Mizuta, Takanobu
44
Mochioka, Noritaka
61
Moriyama, Shunsuke
39, 52, 53, 73
Murakami, Sanae
49
Murashita, Koji	
45		Nagahama, Yoshitaka
47
Naitoh, Yutaka
4
Nakakura, Takashi	
32Nakamura, Hiroaki	
33
Nakamura, Masaru
80
Nakashima, Asuka
13, 38Nakayama, Michie
25, 49Nishi, Mayumi	
13
Nishino, Aiko
53
Nomura, Kazuharu
45
Nozaki, Masumi	
52
		
Ogiwara, Katsueki	
40, 41, 46–84–

Ogushi, Yuji	
Ohira, Hideki	Ohira, Tsuyoshi	
Ohkado, Rei	
Ohta, Hiromi	Ohya, Tamaki	Ojima, Daisuke
Oka, Yoshitaka
Okanoya, Kazuo
Okubo, Yusuke
Ohkubo, Nobuyuki	
Onuma, Takeshi A.
Osugi, Tomohiro
Ozaki, Mamiko
Ozawa, Hitoshi	

65
81
38
15
45
56
73
12, 55, 57
82
23, 4345
54, 60, 74, 75
16
5
59

Parhar, Ishwar S.
Park, Min Kyun

831, 34, 68, 71

Rajapakse, Sanath
Ribeiro, Vanessa

41
28

Saegusa, Junzo
Saigo, Mikiko
Saito, Ei-Suke
Sakaguchi, Hironobu
Sakaguchi, Yuka
Sakai, Takafumi	
Sakamoto, Kentaro Q
Sakamoto, Tatsuya
Sakata, Ichiro
Sano, Akiko
Saribek, Buget
Sato, Katsufumi	Sato, Shunpe
Sato, Takanobu
Sato, Yasufumi	Satoh, Yuya
Seikai, Tadahisa
Shimakura, Sei-Ichi	
Shimotani, Toyokazu
Shioda, Seiji	
Sonobe, Haruyuki	
Susa, Takao
Suzuki, Masakazu
Suzuki, Miwa
Suzuki, Saori	
Suzuki, Takashi	

50, 5148
67
82
20
19, 23, 43
6
63
19, 23, 43
26, 2748
674
24, 25, 26, 27
33
73
69
77, 78, 79
52
76, 7713, 3827, 4915, 18, 32, 59, 65
44
72
41

Swanson, Penny74
		
Tachibana, Tetsuya
67
Tahara, Daisuke
69
Takagi, Chiyo
63
Takahashi, Akiyoshi	
36, 39, 57, 77
Takahashi, Hideya
63
Takahashi, Takayuki	
40, 41, 46, 50
Takigami, Shu
22
Tanaka, Hideki	
45
Tanaka, Minoru
20
Tanaka, Shigeyasu
15, 18, 32, 59, 65
Tando, Yukiko
30
Tani, Tomomi	
11Toki, Shingo
15
Tonon, Marie-Christine
79
Tsujii, Kaoru
60
Tsushima, Nobumichi	
20
Tsutsui, Chihiro
19
Tsutsui, Kazuyoshi	
16, 21, 42, 70, 72, 82
Ubuka, Takayoshi	
Uchida, Katsuhisa
Uchiyama, Minoru
Ueda, Makoto
Ukena, Kazuyoshi	
Unuma, Tatsuya
Urano, Akihisa

21
52
35, 64, 66, 76, 77, 78, 79
18
16, 21, 42, 70, 72
29
54, 60, 74, 75

Wada, Kazuhiro
Wada, Kohei	Wang, De-shouWang, Ping
Wilder, Marcy N.

82
79
47
60
38

Yahata, Tomoki	
Yamada, Ryoichi	Yamada, Toshiki	Yamamori, Kunio
Yamamoto, Ichiro
Yamamoto, Kazutoshi	
Yamamoto, Yushi	
Yamano, Keisuke
Yamazaki, Yuji	
Yambe, Hidenobu
Yashiro, Takashi	

73
38
66
55, 5720
17, 5836
29, 61
35
69
22

–85–

Yasuda, Akikazu
Yasuda-Kamatani, Yoshimi	
Yin, Hong

37
37
21

Zhao, Zheng

23

SUBJECT INDEX
7a-hydroxypregnenolone
70, 72		Acylated ghrelin
43
Adenohypophysial hormones
52
Agnatha52
Agressive behavior
5
AMhr2
28
66
Amiloride-sentitive Na+ channel
Amphioxus30, 44
Anal respiration
37
Andorogen
80
Angiotensin II
62
Anorexigenic action
76, 77, 79
Ant
5Anterior pituitary
50, 54Apoptosis
48, 63Aquaporin
65
Arginine vasotocin (AVT)64
Atrial natriuretic peptide
66
Avian
42
		
Barfin flounder
36
Baroreflex
62
Basolateral plasma membrane
65
Behavior
1
Bering Sea
74
BIBP3226
78
Biocytin
56
Bio-logging
6
BMP
15
Brain
42, 57, 70, 71
Bufo marinus
64
Bulk modulus
4
Bullfrog
17, 18, 58

Chromatin structure
Chum salmon
Cis -elements
Cloning
Computer simulation
Cortisol
Crayfish
CREB
Cytoskeleton
Cytosolic pressure

28
74, 75
36
16
1
63
37
7
60
4

Data logger
Des-acylated ghrelin
Development
Diapause
Differentiation
Diffusion constant
DiI
Dive
DogfishDorsal root ganglion
Duodenum

6
43
22
13
9, 60
2
56
6
53
11
20

Ecdysone
Ecdysteroid
Ecdysteroid 22-kinase
Eel
ElasmobranchElectrophysiology
Esophagus
EST
Evolution
Exit of water
Exon

13
38
38
45, 61
35
1
63
29
34
65
36

C6 glioma
Ca2+
Capsaicin
Cardiovascular system
Cartilaginous fish
Catecholamines
cDNA
cDNA cloning
cDNA microarray
Cell proliferation
Central nervous system
Chicken
Chromatin

Fasting
Feeding behavior
fGRP
Fish
Fish brain
Flow cytometry
Follicle rupture
Follicle-stimulating hormone (FSH)
Follicle-stimulating hormone b (FSHb)
Follicular degradation
Formation of portal vessels
Fractal surface
Fresh water adaptation23, 67
76
16
57
62
33
46
26, 74
24
40
32
60
75

60
58
78
62
39
33
35
53
82
63
3
19, 20, 67
10

–86–

Freshwater sculpins
69
Frog urinary bladder
66
		
GCM1
18
GCM2
18
Gene expression
10, 14, 60
GH53
Ghrelin23, 67, 78
GHRH
35
GHRH/PACAP
53
Glucocorticoid receptor
2, 3, 59
GnIH
21, 42
GnIH receptor
21
GnRH
8, 12, 34, 68
GnRH receptor
68
Goldfish
14, 76, 77, 78, 79
Gonad
47
Gonadal development
69
Gonadal maturation
74
Gonadotropes
21
Gonadotropin (GTH)27, 50, 52, 54, 74
GPCR
16
G-protein coupled receptor
8
GPR39
20
Granulosa cells
28Green fluorescent protein
3, 33
Growth cone
11
Growth hormone
9Growth-retarded mouse
51
Guppy
55
		
Hagfish52
Hatschek’s pit
30
Histone modification
9
Homeobox
27
Homeodomain
25, 26
HSP90b48
HSV-TK
49
Hyla japonica
65
Hypermotility
79
Hypophyseal portal system
32
Hypothalamus
42
Hypothyroid51
		
ICV injection
79
Imaging12
Immunocytochemistry59, 77
Immunofluorescence
43

–87–

Immunohistochemistry
Immunoneutralization
Infertility
Inhibin b B
Innate immune system
In situ hybridization
In situ RT-PCR
Insulin-like growth factor I
IntronIsoform
Isotocin
Kallikrein
Kidney

55
77
49
28
81
19, 22
59
50, 54
36
17
56
41
64

Laser-microdissection
Leopard gecko
Leptin
Leptocephalus
Lhx2
Lhx3
Lipid
Living cells/Living cell
Locus control region
LPXRFamide peptide
Luteinizing hormone

30
34
23
61
24, 26
24
31
3, 10
9
16
74

MCH
MedakaMedaka fish
Melatonin
Melatonin receptor
Metabolism
Metamorphosis
Microarray
Microcapillary osmometer
Microenvironment
Mineralocorticoid receptor
Modeling
Molecular ruler
Monooxygenase
Molting hormone
MotilinmRNA
MSH

57, 7741, 56
46
14, 72
14
31
61
33
4
2
3
1
2
13
38
19
7, 23, 76
57

Natural maturation
Nerve growth factor

45
11

Nervous system
1
Nestmate recognition
5
Neuroendocrinology
8, 12
Neuromedin U
76
Neuropeptide
37, 42, 73
Neurosteroid
70
Newt
70
Nile tilapia
47
Nkx2.115
NPY
78
Nucleosome
10		Obestatin
20
Octadecaneuropeptide
79
Octopamine
5
Ontogeny
55
Oocyte45
Oogenesis
29
Orbitofrontal cortex
81
Orexin57
Osmoregulation
4, 63
Osmotic pressure
4
Otolith61
Ovary
28, 29, 44
Oviduct
20
Ovulation
41, 46
		
P450
13
PACAP
35
Parathyroid hormone
18
Pax8
15
PC2
59
Penguin6
PEP-1982
Pheromone5
Phosphorylation of steroid hormone
38
Physiology
12Pig26, 27
Pituitary
22, 24, 25, 26, 27, 74
Pituitary gland
32, 34, 52
Placenta
9
Plasminogen
40
Positive emotion
81
Post-translational processing
39
PPAR
31
PRDQ9
25
Prolactin (PRL)17, 48, 55, 58
		

–88–

Prolactin receptor
Prolactin-releasing peptide
Proopiomelanocortin (POMC)
Prop-1
Protein phosphatase
Prx2
PubertyQuail17, 4855
36, 39, 59
24, 2510
27
8, 50, 54
72

Rainbow trout
Rat
Real time imaging
Real-time PCR
Refeeding
Reproductive cycle
ReptileRetinoic acid
RNAi	

15
22
3
7
67
69
31, 34, 71
22
7SalmonScd2
SDII-1
Sea urchin
SELEX
Seminiferous tubule
Serine proteaseSex change
Sex differenceSex differentiation
Sex steroid (s)Sex steroid hormone
Silkworm
Signal processing
Signal sequence trap method
Single channel recording
Single molecule imaging
Single-neuron
Smolt
Song nuclei	
Spawning migration
Sperm
Spermatogenesis
Spermatozoa
SS
Steroidogenic enzyme
Steroidogenesis
Steroid receptor

50, 54, 73
28
7
29
25
51
41
80
70, 8247, 7144, 6971, 7513, 38
62
32
66
11
8, 12
73
82
74
51
45, 48, 49
49
53
44
47
47

Suppression subtractive hybridization

18

Tail
Tandem EGFP
TeleostTemperature-dependent sex determination
TestisThyroidThyroid hormone
Thyroxine
Tissue distribution
Tissue-type Plasminogen Activator
Transgenic rat
TRH
TRH receptor
Triakis
TrkA
Tunica ovary
Type I collagen degradation

31
2
35
71
51
15
61
73
53
40
49
58
58
39
11
80
46

Upstream migration
Urea
Urea transporter (UT)

75
64
64

Vasotocin
VEGF-A56, 6532

Zebra finch

82–89–