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Different Modes of Gonadotropin-Releasing Hormone
(GnRH) Release from Multiple GnRH Systems as
Revealed by Radioimmunoassay Using Brain Slices of a
Teleost, the Dwarf Gourami (Colisa lalia)
MAMI ISHIZAKI, MASAYUKI IIGO, NAOYUKI YAMAMOTO,
Misaki Marine Biological Station (M.Is., Y.O.), Graduate School of Science, The University of Tokyo, Miura, Kanagawa 238-
0225, Japan; Department of Anatomy (M.Ii.), St. Marianna University School of Medicine, Miyamae-ku, Kawasaki,
Kanagawa 216-8511, Japan; and Department of Anatomy (N.Y.), Laboratory for Comparative Neuromorphology, Nippon
Medical School, Bunkyo-ku, Tokyo 113-8602, Japan
It has become a general notion that there are multiple GnRH
systems in the vertebrate brains. To measure GnRH release
activities from different GnRH systems, we conducted a static
incubation of brain-pituitary slices under various conditions,
and GnRH released into the incubation medium was mea-
sured by RIA. The slices were divided into two parts, one
containing GnRH neurons in the preoptic area and axon ter-
minals in the pituitary (POA-GnRH slices), and the other con-
taining the cell bodies and fibers of terminal nerve-GnRH
neurons and midbrain tegmentum-GnRH neurons (TN-TEG-
GnRH slices). We demonstrated that GnRH release was
evoked by high [Kⴙ]o depolarizing stimuli (in both POA-GnRH
and TN-TEG-GnRH slices) via Ca2ⴙ influx through voltage-
T HERE HAVE BEEN a number of studies on GnRH as a
peptide hormone that has critical functions in repro-
duction in vertebrates. The GnRH neurons in the preoptic
area (POA) (hypothalamo-hypophyseal GnRH neuron is a
more general term) release GnRH and facilitate gonadotro-
pin release from the pituitary. Recent studies have shown
that GnRH functions not only as a hypophysiotropic hor-
mone that controls reproductive functions but also as a neu-
romodulator that controls motivational or arousal states of
the animal (for review, see Refs. 1–3). However, our knowl-
edge about functions of GnRH other than the hypophysio-
tropic function in the brain is still limited at present.
Based mainly on the results obtained in the teleost brain,
it has been generally accepted that there are at least two or
three anatomically as well as functionally distinctive GnRH
systems in the brain of most vertebrates (1, 3– 6). GnRH
neurons exist in several brain areas such as POA, midbrain
tegmentum (TEG), and terminal nerve (TN). The POA-GnRH
system has been most intensively studied from neuroendo-
crinological viewpoints among the different GnRH systems.
Tegmental GnRH neurons (TEG-GnRH neurons) project
Abbreviations: cGnRH-II, Chicken II [His5Trp7Tyr8] GnRH; mGnRH,
mammalian GnRH; PBST, PBS containing 0.3% Triton X-100; POA, pre-
optic area; sbGnRH, seabream [Ser8] GnRH; sGnRH, salmon [Trp7Leu8]
GnRH; TEG, tegmentum; TN, terminal nerve.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
2092
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Endocrinology 145(4):2092–2103
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2003-0960
AND YOSHITAKA OKA
gated Ca2ⴙ channels. The most prominent result was the pres-
ence of conspicuous sexual difference in the amount of GnRH
release in the POA-GnRH slices. The GnRH release from TN-
TEG-GnRH slices also showed a small sexual difference,
which was by far more inconspicuous than that of POA-GnRH
slices. Immunohistochemical analysis using an antiserum
specific to the seabream GnRH (sbGnRH; suggested to be spe-
cific to POA-GnRH neurons) revealed the presence of a much
larger number of POA-GnRH neurons in males than in
females. This clear morphological sexual difference is sug-
gested to underlie that of GnRH release in the POA-GnRH
slices. (Endocrinology 145: 2092–2103, 2004)
their axons mainly to the posterior parts of the brain. TN-
GnRH neurons project their axons widely in the brain but not
to the pituitary. Recently it has been suggested that the TN-
GnRH system has neuromodulatory functions by releasing
GnRH in wide area of brain (for reviews, see Refs. 1–3).
Because the TEG-GnRH system also projects widely in the
brain, it is also suggested to have neuromodulatory func-
tions. The study of physiological control mechanisms of
GnRH release is critical for our understanding of each sys-
tem’s functions.
In mammals, it is extremely difficult to examine GnRH
release activities of GnRH neurons because GnRH neurons
are sparse and diffusely distributed in the brain. Among
vertebrate species the GnRH system is most developed and
best studied in teleost brains. We selected a tropical anaban-
tid teleost fish, the dwarf gourami (Colisa lalia) to study the
GnRH release in the present paper. This fish has many char-
acteristics advantageous for the study of GnRH neurons. The
brain of the dwarf gourami has a well-developed TN-GnRH
system (7, 8). TN-GnRH cell bodies are large and make tight
cell clusters beneath the ventral meningeal membrane. This
is especially of great advantage over brains of other verte-
brates, in which peptidergic neurons are small and diffusely
distributed and are extremely difficult to study individually.
We have already shown that TN- and TEG-GnRH neurons
in the dwarf gourami are immunoreactive to salmon
[Trp7Leu8] GnRH (sGnRH) and chicken II [His5Trp7Tyr8]
GnRH (cGnRH-II) (6), respectively. POA-GnRH cells also
 Ishizaki et al. • GnRH Release from Multiple GnRH Systems
make clusters in the dwarf gourami (9, 10), and their axons
project directly to the pituitary (general feature of hypophy-
siotropic neurons in teleosts lacking the portal system). Fur-
thermore, electrophysiological properties of TN-GnRH neu-
rons of the dwarf gourami have been most intensively
studied among vertebrate brains so far (8, 11–15). Thus, the
brain of the dwarf gourami may be one of the best experi-
mental systems to analyze physiological mechanisms of
GnRH release and its relationship with the electrical activity
of the GnRH neurons.
From electrophysiological studies, it has been shown that
GnRH, which is probably released by TN-GnRH system,
modulates the pacemaker activity of TN-GnRH neurons
themselves in the dwarf gourami (11, 15). These studies
suggest that TN-GnRH system may have some neuromodu-
latory functions (1–3). In accordance with this idea, there
have been some studies on the modulatory functions of TN-
GnRH neurons thus far. Walker and Stell (16) showed that
retinal ganglion cells of the goldfish (Carassius auratus) re-
spond to several compounds present in TN (including
GnRH), and Umino and Dowling (17) showed that retinal
ganglion cells of the goldfish change their responses to light
when they were perfused with GnRH. Yamamoto et al. (18)
reported that the lesion of the TN-GnRH cells changed the
threshold for initiation of one of the reproductive behavior
repertory, male nest-building behavior, in the dwarf
gourami. Eisthen et al. (19) found that GnRH affects ionic
channel properties of olfactory receptor neurons and sug-
gested neuromodulatory effects of the TN-GnRH neurons on
the olfactory receptor neurons in amphibians. Before con-
cluding that the TN-GnRH system exerts modulatory func-
tions in vertebrate brains, however, it is necessary to dem-
onstrate GnRH release activities from the TN-GnRH
neurons. GnRH release from the brain has been determined
by RIA in POA-GnRH system of the goldfish brain slices (20)
and the rat median eminence fragment (21). However, the
GnRH release from TN-GnRH neurons and its control mech-
anisms have not been studied thus far. It is also important to
study the difference in GnRH release activities between
POA- and other (TN- and TEG-) GnRH systems because they
are suggested to have quite different functions, e.g. neuroen-
docrine vs. neuromodulatory.
In the present study, we first measured GnRH release from
the brain-pituitary slices of the dwarf gourami separated into
functionally different two parts (POA- and TN-TEG-GnRH
slices), by a static incubation system and RIA using a single
antibody that almost equally recognizes various molecular
species of GnRH (see Materials and Methods). This way, we
wanted to compare the similarities and differences of GnRH
release from the two functionally different systems. We took
advantage of the distinctive anatomical features of the three
GnRH systems of the dwarf gourami (1, 6, 18) and separated
the slices into two, one containing the POA-pituitary region
(POA-GnRH slices) and the other containing TN-GnRH neu-
rons, TEG-GnRH neurons, and their projection areas in wide
areas of the brain (TN-TEG-GnRH slices). The GnRH release
from the former represents that related to the reproductive
functions, whereas that from the latter represents that related
to the neuromodulatory functions. We found a characteristic
sexual difference in GnRH release from POA-GnRH neurons.
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The sexual difference in GnRH release from TN-TEG-GnRH
slices was less prominent than that of POA-GnRH slices.
Because the preoptic GnRH molecular form is seabream
[Ser8] GnRH (sbGnRH) in the percomorph teleosts (22–25)
and the presence of sbGnRH has been suggested by an HPLC
analysis combined with RIA in the dwarf gourami (6), we
performed GnRH-immunohistochemistry with a sbGnRH
antiserum to examine sexual dimorphism in POA-GnRH
cells.
Materials and Methods
Animals
The dwarf gouramis were purchased from a local dealer and kept in
the aquaria at 25–28 C under a photoperiod of 12-h light/12-h dark cycle
until used. The animals were fed tropical fish food purchased commer-
cially. The animals were maintained in accordance with the guidelines
of The Physiological Society of Japan and the University of Tokyo for the
Use and Care of Experimental Animals.
Preparation of brain and pituitary slices
The male and female fish were killed by decapitation, and the brain
with pituitary was removed and immersed rapidly into a chilled Ringer
solution. The Ringer solution contained (in millimoles) NaCl 124, KCl 5,
CaCl2 2.4, KH2PO4 1.2, MgSO4 1.3, NaHCO3 26, glucose 10 (pH 7.4,
adjusted with NaOH). The brain with pituitary was sliced into sagittal
sections of 250 ␮m using a microslicer (DTK-1000, DOSAKA EM Co.
Ltd., Osaka, Japan). Four parasagittal slices per fish were obtained so
that each slice has a pituitary fragment connected to the brain. The slices
were divided into two parts under a dissection microscope using a razor
blade as shown in Fig. 1, the one containing the POA and the pituitary
(POA-GnRH slice), and the remaining part of the brain (TN-TEG-GnRH
slice), which contained the cell bodies of TN and TEG GnRH neurons,
and most of their fibers. Because the fibers of the both TN- and TEG-
GnRH systems are distributed in wide areas of the brain and are inter-
FIG. 1. Preparation of brain-pituitary slices for the measurement of
GnRH release with RIA. The brain-pituitary slice (250 ␮m) was di-
vided into two parts. The TN-TEG-GnRH slice contains the cell bodies
of TN-GnRH (denoted by star) and midbrain TEG-GnRH neurons
(denoted by triangle) and most of their axons distributed in wide areas
of the brain. The POA-GnRH slice contains the cell bodies of POA-
GnRH neurons (denoted by closed circle) and the pituitary where the
axon terminals of POA-GnRH neurons exist. Eight slices (from two
fish) were collected for each type of slice and incubated in Ringer
solution. The media were sampled using a static incubation system to
measure the GnRH release from slices.
 2094 Endocrinology, April 2004, 145(4):2092–2103
mingled extensively, it was technically impossible to separate the two
systems.
Each experimental replicate contained eight slices obtained from two
fish. That is, one replicate of POA-GnRH slices corresponds to the POA
regions and the entire pituitaries from two fish. Four to six replicates
were examined in each experiment.
Static incubation of brain-pituitary slices in vitro
The slices in each replicate were preincubated in an oxygenized
Ringer solution in a well of the 24-well plate over 50 min for recovery
from injury by brain slicing. After preincubation, slices were washed
with fresh Ringer solution. The medium was replaced with 200 ␮l of
experimental solution (see below). After incubation for 10 min (20 min
in thapsigargin experiment) at 27 C, the medium was transferred to
sample tubes, mixed with HCl (0.1 n in final concentration), frozen on
dry ice and stored at ⫺80 C until assayed for GnRH. After the collection
of experimental solutions, the slices were washed with the Ringer so-
lution three times, and each well was filled with fresh Ringer. The next
treatment was done after the slices were allowed to recover for 50 min.
One experimental series consisted of two to five different treatments.
Experimental design of static incubations
Experiment 1. Depolarization-induced GnRH release. We examined depo-
larization-induced GnRH release in response to high [K⫹]o Ringer so-
lutions [NaCl was partly replaced with KCl to make (K⫹)o ⫽ 20, 40, 60,
or 100 mm]. POA- and TN-TEG-GnRH slices from male and female fish
were incubated first in Ringer solutions for 10 min to measure sponta-
neous GnRH release. Next, the slices were incubated for 10 min in high
[K⫹]o Ringer solutions. Series of high [K⫹]o tests were done in the order
of increasing [K⫹]o.
Experiment 2. Contribution of extracellular Ca2⫹ to the GnRH release in
response to depolarization. Extracellular Ca2⫹ dependence of depolariza-
tion-induced GnRH release was tested. The slices were incubated with
Ringer solutions or high [K⫹]o solutions (100 mm) that contained nom-
inally no [Ca2⫹]o (Ca2⫹-free) or Ca2⫹-free Ringer solutions and high
[K⫹]o solutions (100 mm). Each treatment was done for 10 min.
To assess contribution of different types of Ca2⫹ channels to depo-
larization-induced GnRH release, the high [K⫹]o stimulation (100 mm)
was done in the presence of specific Ca2⫹ channel blockers. L-type (10
␮m nifedipine), N-type (1 ␮m ␻-conotoxin GVIA), and P/Q-type (250 nm
␻-agatoxin TK) Ca2⫹ channel blockers were used in this experiment. The
slices were incubated for 10 min with Ringer solutions, then high [K⫹]o
solutions (100 mm) and high [K⫹]o solutions (100 mm) with specific Ca2⫹
channel blockers. Nifedipine and ␻-conotoxin were tested in the same
series of experiment, and ␻-agatoxin was tested in another series.
Experiment 3. Effects of Ca2⫹ release from intracellular Ca2⫹ stores on spon-
taneous GnRH release. Effects on spontaneous (basal) GnRH release were
examined by using caffeine to induce Ca2⫹ release from intracellular
Ca2⫹ stores. First, the slices were incubated for 10 min with Ringer
solutions and next with Ringer solutions that contained 10 mm of
caffeine.
Experiment 4. Effects of store-operated Ca2⫹ influx on spontaneous GnRH
release. Store-operated Ca2⫹ influx (Ca2⫹ influx induced by depletion of
intracellular Ca2⫹ store, see Ref. 26) was induced by thapsigargin (an
inhibitor of endoplasmic reticulum Ca2⫹-ATPase so that it will even-
tually deplete the intracellular Ca2⫹ stores). The slices were first incu-
bated in a Ca2⫹-free Ringer solutions (control) for 20 min, and the
intracellular Ca2⫹ store was depleted by Ca2⫹-free Ringer with thapsi-
gargin (20 ␮m) for 20 min. Thereafter, the slices were returned to Ca2⫹-
containing [(Ca2⫹)o ⫽ 2.4 mm] normal Ringer for 20 min. If GnRH release
occurs when [Ca2⫹]o is restored, it is supposed to be due to a store-
operated Ca2⫹ influx. Then slices were incubated in Ringer that con-
tained 2 mm Zn2⫹ (inhibitor of store-operated Ca2⫹ influx, see Refs.
27–29) for 20 min.
Chemicals used in the static incubation
Stock solutions of ␻-conotoxin GVIA and ␻-agatoxin TK (Alomone
Labs, Jerusalem, Israel) were dissolved in double-distilled water. Stock
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Ishizaki et al. • GnRH Release from Multiple GnRH Systems
solutions of nifedipine (Alomone Labs) and thapsigargin (Sigma, St.
Louis, MO) were dissolved in dimethyl sulfoxide. The stock solutions of
␻-conotoxin, ␻-agatoxin, nifedipine, and thapsigargin were diluted in
Ringer solution before the experiments. The vehicle (dimethyl sulfoxide)
was added to control Ringer solutions in nifedipine and thapsigargin
experiments. Caffeine was obtained from Wako Pure Chemical Indus-
tries, Ltd. (Tokyo, Japan) and dissolved in Ringer solution.
RIA
The sGnRH, cGnRH-II, and mammalian GnRH (mGnRH) peptides
were obtained from Peninsula Laboratories, Inc. (Belmont, CA). The
sbGnRH peptide was kindly donated by Dr. K. Okuzawa (National
Research Institute of Aquaculture, Tamaki, Mie, Japan). [125I]-labeled
mGnRH (2200 Ci/mmol, NEN Life Science Products, Boston, MA) was
used as the tracer. The rabbit anti-GnRH serum (anti-GnRH, lot R-II, see
Ref. 30) and antirabbit ␥-globulin goat serum (HAC-RBA2– 05GTP91)
were obtained from Dr. Y. Hasegawa (Kitasato University, Towada,
Aomori, Japan) and Prof. K. Wakabayashi (Gunma University, Mae-
bashi, Gunma, Japan), respectively. BSA (fraction V, RIA grade) was
purchased from Sigma.
The amount of immunoreactive GnRH released into the medium
from brain slices was determined by RIA according to the procedure of
Okuzawa et al. (31), with slight modifications. A standard curve (4.9 –
2500 pg/ml) was constructed by using serial 2-fold dilutions of sGnRH
dissolved in PBS [50 mm phosphate buffer containing 140 mm NaCl and
0.1% sodium azide (pH 7.5)] containing 1% BSA (BSA-PBS). For the
initiation of RIA, anti-GnRH serum (1:20,000 dilution with PBS con-
taining 50 mm EDTA and 1% normal rabbit serum, 100 ␮l) were added
to test tubes [Eiken RIA tubes (no. 2), Eiken Kizai, Tokyo, Japan] con-
taining BSA-PBS (300 ␮l) and the standard or samples (100 ␮l) and
incubated for 5 d at 4 C. [125I]-mGnRH (approximately 10,000 cpm in
BSA-PBS, 100 ␮l) was added to each tube and further incubated for 24 h
at 4 C. Antirabbit ␥-globulin goat serum (1:50 dilution with PBS con-
taining 50 mm EDTA, 200 ␮l) was added to each tube. After incubation
for an additional 24 h at 4 C, these tubes were centrifuged (2000 ⫻ g, 30
min, 4 C) and the supernatant was aspirated. Radioactivity was then
determined with a ␥-counter (Aloka, Tokyo, Japan).
For the validation of the RIA, parallelism and quantitative recovery
studies were performed. The inhibition curve for serial 2-fold dilution
of the samples was parallel to the curve for the sGnRH standard. The
relationship between the quantity of sGnRH added (0 –312.5 pg/ml) to
the samples and the recovered amount was analyzed using linear re-
gression analysis. A significant correlation was obtained between the
two [Y ⫽ 1.094 ⫻ ⫹0.22 (r ⫽ 0.962, n ⫽ 7, P ⬍ 0.001); X: sGnRH added
(pg/ml); Y: sGnRH recovered (pg/ml)]. Intra- and interassay coeffi-
FIG. 2. The competition curves for GnRH RIA. Four GnRH subtypes
(mGnRH, sGnRH, sbGnRH, and cGnRH-II) were tested. This RIA
system using antiserum against cGnRH-II (lot R-II) recognized all
four GnRH subtypes.
 Ishizaki et al. • GnRH Release from Multiple GnRH Systems
cients of variation of the RIA were 5.0 –12.9% (n ⫽ 3–10) and 10.7% (n ⫽
5) at 98.7 pg/ml, respectively. The minimum detectable level defined as
2 sd from the buffer controls was 8.5 pg/ml (corresponding to 1.7
pg/sample). The antiserum used in this study is known to cross-react
with various molecular species of GnRH (6, 26). The displacement curves
for GnRHs were parallel to that of the sGnRH standard. The cross-
reactivities investigated in the present RIA system were as follows:
sGnRH, 100%; cGnRH-II, 117.9%; sbGnRH, 156.0%; mGnRH, 187.7%
(Fig. 2). Fifty percent inhibition doses for sGnRH were 40.8 ⫾ 0.8 pg/ml
(n ⫽ 16).
FIG. 3. GnRH release from POA- and TN-TEG-GnRH slices in re-
sponse to high K⫹ depolarizing stimuli. GnRH release (pg/sample;
note different scales in different graphs) was dependent on the ex-
tracellular [K⫹]o (P ⬍ 0.005 in POA-GnRH slices; P ⬍ 0.0001 in
TN-TEG-GnRH slices, two-way ANOVA). A, In POA-GnRH slices,
sexual difference in GnRH release was highly significant (P ⬍ 0.0001;
not marked by symbols) at every [K⫹]o, and the male (blank bars)
released larger amounts of GnRH than female (hatched bars). B, In
TN-TEG-GnRH slices, the sexual difference of GnRH release was
considerably smaller (P ⬍ 0.05) than in POA-GnRH slices. The bars
and vertical lines represent means ⫾ SEM (n ⫽ 4 in each group).
Asterisks indicate means significantly different from the control (*,
P ⬍ 0.05; **, P ⬍ 0.01; ANOVA followed by Dunnett’s test).
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Current clamp recording of electrical activity of terminal
nerve-GnRH cells
We examined the dose dependence in frequency of electrical activity
of TN-GnRH neurons on depolarizing stimuli. A whole-cell patch clamp
recording was carried out to record the pacemaker activity of TN-GnRH
neurons. The whole brain of male dwarf gourami was dissected and
pinned ventral side up to a trough, the ventral meningeal membrane was
removed, and the cluster of TN-GnRH cells were exposed. The prepa-
ration was perfused with Ringer solution or high [K⫹]o Ringer solutions
(20, 40, 60, and 100 mm). To quantify the increase of spike frequency, the
number of spikes during 10-sec periods before and after the perfusion
of high [K⫹]o solution was counted, and the ratios between them were
plotted. Whole-cell current-clamp recording was carried out with a
patch clamp amplifier (PC-501A, Warner Instruments, Inc., Hamden,
CT) and pCLAMP software (Axon Instrument, Inc., Union City, CA).
The direct visual identification and electrophysiological characterization
of TN-GnRH neurons have already been well established by our pre-
vious studies (2, 8, 11–15, 32), and there were no electrophysiological
differences between males and females.
Immunohistochemistry
Ten mature dwarf gourami (five males and five females) ranging 3– 4
cm in standard length were used for sbGnRH immunohistochemistry.
The fish were deeply anesthetized in tricaine methanesulfonate (300
mg/liter) and perfused through the conus arteriosus with 4% parafor-
maldehyde in 0.1 m phosphate buffer (pH 7.4). The brains were removed
from the skull and postfixed in a fresh solution of the same fixative at
FIG. 4. Electrical activities of TN-GnRH cells in response to high
[K⫹]o Ringer solution. A whole cell current clamp recording was car-
ried out from TN-GnRH cells using a whole brain preparation. A,
Recording of the pacemaker potential of TN-GnRH cells. High [K⫹]o
solution ([K⫹]o ⫽ 100 mM) was applied by perfusion during the period
indicated by the bar. TN-GnRH cells were depolarized in response to
the application of high [K⫹]o solution, and frequency of the pacemaker
activities was increased. B, The ratios of number of spikes between
before and after the perfusion of high [K⫹]o were plotted. The fre-
quency of the action potentials was increased in a dose-dependent
manner (P ⬍ 0.005, one-way ANOVA). The columns and the vertical
lines represent means ⫾ SEM of the ratios (n ⫽ 4). Asterisks indicate
that the number of spikes were significantly different from controls
by paired t test (*, P ⬍ 0.05; **, P ⬍ 0.01).
 2096 Endocrinology, April 2004, 145(4):2092–2103 Ishizaki et al. • GnRH Release from Multiple GnRH Systems

FIG. 5. Effects of extracellular Ca2⫹ on

GnRH release in response to high K⫹
depolarizing stimulus. GnRH release
(pg/sample; note different scales in dif-
ferent graphs) evoked by high K⫹ (100
mM) was abolished by eliminating ex-
tracellular Ca2⫹ (⫺Ca2⫹) in both slices
in both sexes. A, GnRH release from
POA-GnRH slices from male (A1) and
female (A2) fish. B, GnRH release from
TN-TEG-GnRH slices of male (B1) and
female (B2) fish. The bars and vertical
lines represent means ⫾ SEM (n ⫽ 4) of
control, high K⫹ solution (100K), Ca2⫹-
free solution (⫺Ca2⫹), Ca2⫹-free-high
K⫹ solution (100K-Ca2⫹), respectively.
Bars with different superscript letters
are significantly different (P ⬍ 0.05;
ANOVA followed by Student-Newman-
Keuls test).

4 C for 12 h. The brains were then immersed in 20% sucrose in 0.1 m
phosphate buffer (pH 7.4) for 6 h and embedded in 5% agar (Sigma, type
IX) containing 20% sucrose. Tissue blocks were frozen in n-hexane at
⫺60 C, and 30-␮m-thick serial sections were cut on a cryostat either
sagittally (one male and one female) or frontally (four males and four
females). Thaw-mounted sections on gelatin-coated slides were dried by
fans, washed with 0.05 m PBS containing 0.3% Triton X-100 (PBST), and
reacted with an affinity-purified anti-sbGnRH antiserum (generous gift
of Dr. I. S. Parhar, Nippon Medical School, Tokyo, Japan) for 20 h. The
primary antiserum was diluted 1:50,000 with PBST containing 1% nor-
mal goat serum. Sections were then washed three times in PBST and
reacted with biotin-labeled antirabbit IgG (1:200) for 2 h. After the
reaction with secondary antibody, the sections were incubated for 10
min in 0.3% H2O2 in methanol to block remaining endogenous perox-
idase activity. The sections were washed three times in PBST and reacted
with avidin-biotin-horseradish peroxidase complex solution [1:50;
Sigma (Vectastain), ABC elite kit] for 3 h. After three washes in 0.05 m
PBS, the sections were incubated with diaminobenzidine containing
0.04% nickel ammonium sulfate for 30 min. All the reactions were carried
out at room temperature. After dehydration through a series of graded
concentrations of alcohol, the sections were cleared and coverslipped,
and immunoreactive neurons were counted under the microscope. All
the sections were used for cell counting. A positive cellular structure
with a clearly identifiable nucleus (immunonegative, round structure in
the center of cytoplasm) was regarded as one cell.
To test the specificity of the primary antiserum and immunohisto-
chemical procedures of the present study, the following control exper-
iments were also performed: 1) use of primary antiserum preabsorbed
with 2.5 ␮g/ml sbGnRH peptide (generous gift of Dr. M. Kobayashi,
International Christian University, Tokyo, Japan) for 20 h at 4 C; 2)
omission of primary antiserum from the incubating solution; 3) omission
of secondary antiserum from the incubating solution; and 4) omission
of avidin-biotin-horseradish peroxidase complex from the incubating
solution. All of these tests resulted in no positive structures. Alternate
series of sections, which were processed normally, exhibited immu-
nopositive neurons as in specimens used for cell counting. These ob-
servations strongly support the sbGnRH-specific immunoreaction of the
present study.
Statistics
The data are expressed as the mean ⫾ sem. Effects of sex and drug
treatment were analyzed by two-way ANOVA in RIA experiments. In
Experiment 1, the dose-dependence of GnRH release on [K⫹]o was
analyzed by one-way ANOVA followed by Dunnett’s test. In Experi-
ments 2– 4, comparison among treatment groups was performed with

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 Ishizaki et al. • GnRH Release from Multiple GnRH Systems Endocrinology, April 2004, 145(4):2092–2103 2097

FIG. 6. Effects of Ca2⫹ channel blockers on

GnRH release in response to high K⫹ depolariz-
ing stimulus. A, In POA slices, GnRH release
(pg/sample; note different scales in different
graphs) evoked by high K⫹ solution (100 mM
[K⫹]o; 100K) was diminished by ␻-conotoxin (Ctx;
N-type Ca2⫹ channel blocker). Nifedipine (Nif;
L-type Ca2⫹ channel blocker) facilitated GnRH
release in both males (A1) and females (A2). B, In
TN-GnRH slices, GnRH release evoked by high
K⫹ solution was significantly reduced by ␻-cono-
toxin and nifedipine in both males (B1) and fe-
males (B2). The bars and vertical lines represent
means ⫾ SEM (n ⫽ 4). Bars with different super-
script letters are significantly different (P ⬍ 0.05;
ANOVA followed by Student-Newman-Keuls
test).

one-way ANOVA followed by Student-Newman-Keuls test. Effects of
high-[K⫹]o stimuli on electrical activities of TN-GnRH neurons were
analyzed by one-way ANOVA, and paired t test was used to analyze the
increase in the number of spikes. The number of positive cells in im-
munohistochemistry was analyzed by Student’s t test. Differences were
considered significant if P ⬍ 0.05.
Results
Depolarization-induced GnRH release
Figure 3 shows the GnRH release when the POA-GnRH
slices and TN-TEG-GnRH slices from male and female fish
were stimulated with high [K⫹]o Ringer solutions [(K⫹)o ⫽
20, 40, 60, and 100 mm]. The high [K⫹]o Ringer solutions
significantly increased the GnRH release from POA- and
TN-TEG-GnRH slices in a dose-dependent manner [Fig. 3,
two-way ANOVA, F(4,29) ⫽ 5.36; P ⬍ 0.005 in POA-GnRH
slices; F(4,30) ⫽ 33.22; P ⬍ 0.0001 in TN-TEG-GnRH slices].
In POA-GnRH slices, the GnRH release was by far larger in
male fish than in female, and the sexual difference was highly
significant [two-way ANOVA, sex: F(1,29) ⫽ 36.21; P ⬍
0.0001]. In TN-TEG-GnRH slices, the sexual difference of the
GnRH release was much smaller than in POA-GnRH slices,
but still the sexual difference was significant [two-way
ANOVA, sex: F(1,30) ⫽ 6.42; P ⬍ 0.05]. When we compared
TN-TEG-GnRH slices with POA-GnRH slices, POA-GnRH
slices released a larger amount of GnRH than TN-TEG-
GnRH slices [two-way ANOVA, type of slice: F(1,59) ⫽ 15.39;
P ⬍ 0.0005] despite the fact that POA-GnRH slices were much
smaller in size than TN-TEG-GnRH slices. It also should be
noted that there was a considerable amount of spontaneous
(basal) release of GnRH (GnRH release in controls) in both
POA- and TN-TEG-GnRH slices, and it was also sexually
different in POA- but not in TN-TEG-GnRH slices.
The electrical activity of TN-GnRH cells was also exam-
ined by whole-cell current clamp recording using a whole-
brain in vitro preparation of the dwarf gourami. The perfu-
sion of high [K⫹]o Ringer solution ([K⫹]o ⫽ 20, 40, 60, 100
mm) increased the frequency of pacemaker potentials of TN-
GnRH cells in a dose-dependent manner [Fig. 4, one-way
ANOVA; F(3,12) ⫽ 9.81, P ⬍ 0.005]. The results shown in
Figs. 3 and 4 suggest that the GnRH release in TN-TEG-

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 2098 Endocrinology, April 2004, 145(4):2092–2103 Ishizaki et al. • GnRH Release from Multiple GnRH Systems

FIG. 7. Effects of P/Q-type Ca2⫹ chan-

nel blockers on GnRH release in re-
sponse to high K⫹ depolarizing stimu-
lus. In POA-GnRH slices (A) and TN-
TEG-GnRH slices (B), GnRH release
(pg/sample; note different scales in dif-
ferent graphs) evoked by high K⫹ solu-
tion (100 mM [K⫹]o; 100K) was not af-
fected by ␻-agatoxin (Aga; P/Q-type
Ca2⫹ channel blocker); there was no sig-
nificant difference in the amount of
GnRH release between high K⫹-treated
slices and high K⫹ with ␻-agatoxin-
treated slices; A1, B1, males; A2, B2,
females. The bars and vertical lines rep-
resent means ⫾ SEM (n ⫽ 4). Bars with
different superscript letters are signifi-
cantly different (P ⬍ 0.05; ANOVA fol-
lowed by Student-Newman-Keuls test).

GnRH slices is dependent on the frequency of pacemaker
potentials of TN-GnRH cells.
Next, the dependence on extracellular Ca2⫹ of GnRH re-
lease was examined (Fig. 5). Figure 5 clearly shows that in
both POA- and TN-TEG-GnRH slices of males and females
the GnRH release evoked by the depolarizing stimulus is
dependent on [Ca2⫹]o. Spontaneous GnRH release was ob-
served in the controls in Fig. 5, A and B. The spontaneous
release from POA and TN-TEG-GnRH slices was indepen-
dent of the [Ca2⫹]o.
From these results, it was shown in both POA and TN-
TEG-GnRH slices that the depolarization-induced GnRH re-
lease was dependent on Ca2⫹ influx. Therefore, we assessed
contribution of different types of Ca2⫹ channels to the GnRH
release in response to depolarizing stimuli (Figs. 6 and 7). In
POA-GnRH slices, ␻-conotoxin almost completely abolished
the GnRH release in response to high K⫹ (100 mm), whereas
nifedipine increased the GnRH release significantly (Fig. 6A).
␻-Agatoxin caused no significant changes (Fig. 7A). In con-
trast, the GnRH release from TN-TEG-GnRH slices in re-
sponse to the high [K⫹]o stimuli was abolished by ␻-cono-
toxin in males, significantly decreased in females, and was
decreased by application of nifedipine in both sexes (Fig. 6B).
GnRH release from TN-TEG-GnRH slices in response to the
high [K⫹]o stimuli was not affected by agatoxin (Fig. 7B).
Thus, it is suggested that the GnRH release from POA-GnRH
slices induced by depolarization is mainly dependent on the
Ca2⫹ influx through N-type Ca2⫹ channels and that from
TN-TEG-GnRH slices induced by depolarization is mainly
dependent on both L- and N-type Ca2⫹ channels.
Effects of Ca2⫹ release from intracellular Ca2⫹ stores and
store-operated Ca2⫹ influx
The contribution to GnRH release of Ca2⫹ released from
intracellular Ca2⫹ stores was tested. The effects of caffeine (10

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 Ishizaki et al. • GnRH Release from Multiple GnRH Systems
FIG. 8. Effects of caffeine on GnRH release. GnRH release (pg/sam-
ple; note different scales in different graphs) was not affected signif-
icantly by caffeine treatment (10 mM) in either slice and in either sex.
A, POA-GnRH slices. B, TN-TEG-GnRH slices; A1, B1, males; A2, B2,
females. The bars and vertical lines represent means ⫾ SEM (n ⫽ 6)
of control and caffeine solution (Caf).
mm) and thapsigargin (20 ␮m) are shown in Figs. 8 and 9. No
significant increase of GnRH release was observed by the
application of caffeine for 10 min to either POA- or TN-TEG-
GnRH slices (Fig. 8). Thapsigargin application for 20 min
decreased the GnRH release in TN-TEG-GnRH slices (Fig. 9,
B1 and B2) but not in POA-GnRH slices (Fig. 9, A1 and A2).
Thus, it is suggested that Ca2⫹ release from Ca2⫹ store con-
tributes to the GnRH release only from TN-TEG-GnRH
slices. Then, we examined whether store-operated Ca2⫹ in-
flux affects GnRH release activities. GnRH release was in-
creased by this treatment in TN-TEG-GnRH slices of females
(Fig. 9, B2, SOC). This increase was abolished when 2 mm
Zn2⫹ (inhibitor of store-operated Ca2⫹ influx) was added to
the Ca2⫹-containing normal Ringer (Fig. 9, B2, SOC⫹Zn2⫹).
Immunohistochemistry
Anti-sbGnRH serum-labeled neurons in the POA and their
axon terminals in the pituitary. Minor populations of sb-
GnRH-immunoreactive cells were also observed in the area
ventralis telencephali pars ventralis and nucleus lateralis
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Endocrinology, April 2004, 145(4):2092–2103 2099
tuberis pars posterior of Peter et al. (33). Somata of TN-GnRH
cells were only faintly labeled, and faintly stained axons
could be traced for very short distances. TEG-GnRH cells and
their fibers were not labeled at all. This result supports sb-
GnRH production in this GnRH cell group as in other per-
comorph teleosts (22–25). Because we have already shown
that TN- and TEG-GnRH neurons are immunoreactive to
salmon and chicken II type GnRH (6), respectively, it is
concluded that there are at least three different types of
GnRH neuronal systems in the dwarf gourami brain.
Two major groups of sbGnRH-immunoreactive cells were
identified in the POA: rostral and caudal populations (Fig.
10A). The rostral population was darkly stained and present
in the most rostral part of the POA surrounding the preoptic
recess. No sexual differences were detected with regard to
this cell group (Fig. 10, B and C). The numbers of rostral
neurons were 26.9 ⫾ 4.8 neurons/side in males (mean ⫾ sem;
n ⫽ 5; see Fig. 10B) and 24.9 ⫾ 2.1 neurons/side in females
(n ⫽ 5; see Fig. 10C; P ⬎ 0.5 vs. male), and the staining
intensity and distribution of these cells were much the same
in both sexes. The caudal population appeared slightly cau-
dal to the rostral population and showed a marked sexual
difference (Fig. 10, D and E). In contrast to many moderately
stained neurons in males (170.6 ⫾ 19.6 neurons/side; n ⫽ 5;
see Fig. 10D), labeled cells were rarely encountered in the
corresponding area in females (4.4 ⫾ 0.9 neurons/side; n ⫽
5; see Fig. 10E; P ⬍ 0.005 vs. male). Also, positive cells in
females were only very faintly stained.
Discussion
In the present study, we found that control mechanisms of
GnRH release from POA- and TN-/TEG-GnRH systems
have some characteristics in common but that there are also
some characteristics different from each other. We found
prominent sex differences of GnRH release in POA-GnRH
slices but not in TN-TEG-GnRH slices. The Ca2⫹ mobilization
pathways in GnRH release mechanisms of POA- and TN-/
TEG-GnRH systems were also different.
Depolarizing stimuli caused GnRH release from both
POA- and TN-TEG-GnRH slices in a dose-dependent man-
ner. Interestingly, there was a characteristic sexual difference
in spontaneous and depolarization-induced GnRH release
from the POA-GnRH slices: the POA-GnRH slices from male
fish released, by far, a larger amount of GnRH than those of
female fish. This is consistent with our immunohistochemical
data: the number and staining intensity of sbGnRH-immu-
noreactive cells in the caudal population of POA showed a
marked sexual difference with an overwhelmingly larger
number in males than in females. This morphological sexual
difference most probably underlies the sexual differences in
GnRH releasing activities from the POA-GnRH slices. Less
pronounced but statistically significant sexual differences
were also observed in the GnRH release from TN-TEG-
GnRH slices. Because the male brains were slightly larger
than female brains (around 3%) and we did not make cor-
rections for this size difference, this sexual difference of TN-
TEG-GnRH slices may be simply because of such sexual
difference in the brain volume. In contrast, the sexual dif-
ferences in the GnRH release from POA-GnRH slices and in
 2100 Endocrinology, April 2004, 145(4):2092–2103 Ishizaki et al. • GnRH Release from Multiple GnRH Systems

FIG. 9. Effects of thapsigargin and

store-operated currents on GnRH re-
lease (pg/sample; note different scales
in different graphs). Thap, thapsigargin
treatment (20 ␮M); SOC, extracellular
Ca2⫹ restoration after depletion of in-
tracellular Ca2⫹ stores (to assess the
effect of store-operated currents);
SOC⫹Zn2⫹, restoration of extracellular
Ca2⫹ in the presence of Zn2⫹. Thapsi-
gargin treatment decreased GnRH re-
lease from TN-TEG-GnRH slices of fe-
males. The effects of Ca2⫹ restoration
and Ca2⫹ restoration in the presence of
Zn2⫹ were significantly different in TN-
TEG-GnRH slices of females. A, POA-
GnRH slices. B, TN-TEG-GnRH slices;
A1, B1, males; A2, B2, females. The
bars and vertical lines represent
means ⫾ SEM (n ⫽ 4 in males, n ⫽ 5 in
females). Bars with different super-
script letters are significantly different
(P ⬍ 0.05; ANOVA followed by Student-
Newman-Keuls test).

the number of GnRH cells in caudal POA are too large to be
explained by the brain volume.
Some previous studies have reported on sexual differences
in several aspects of POA-GnRH neurons. The size and num-
ber of GnRH cells were larger in males than in females in the
ballan wrasse (34). Sexual difference was observed in the
activation of GnRH synthesis by 17␣-methyltestosterone ad-
ministration in yearling masu salmon (35), and GnRH im-
munoreactivity was different between sexes in the musk
shrew brain (36). On the other hand, GnRH contents in brain
slices of the rainbow trout are apparently equivalent in both
sexes (31). It will be important in future experiments to de-
termine whether there are seasonal differences in GnRH
contents and/or GnRH release from the POA-GnRH system
of the dwarf gourami in both sexes. One possibility is that
males have larger amounts of GnRH in POA-GnRH neurons
and release higher amounts of GnRH in all seasons, but
females have GnRH fluctuations and release smaller
amounts of GnRH except for the spawning period.
Morphological analyses in the present study revealed
two populations of POA-GnRH cells: rostral and caudal
populations. The rostral population did not show sex-
dependent differences in immunostaining, whereas the
caudal population exhibited clear sexual dimorphism. On
the other hand, it has been shown that the gonadotropins
of teleosts consist of two types, FSH (GTHI) and LH
(GTHII) (37). It is thus an interesting possibility that each
preoptic GnRH cell group is involved in the control of
either FSH or LH specifically.
In POA-GnRH slices, GnRH release evoked by high [K⫹]o
depolarizing stimuli was dependent on extracellular Ca2⫹
and was dependent on Ca2⫹ influx mediated mainly by N-
type Ca2⫹ channels but not by L or P/Q type. In general,
L-type Ca2⫹ channels are found in cell types such as endo-
crine cells, muscles, and neurons, and the Ca2⫹ influx via
these channels can induce exocytosis in several cells such as
chromaffin cells (38) and GT1 cells (39). On the other hand,
N-, P-, and Q-type Ca2⫹ channels are generally found in

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 Ishizaki et al. • GnRH Release from Multiple GnRH Systems
FIG. 10. Immunohistochemistry of male and female dwarf gourami
brains using a specific antiserum to sbGnRH. A, Sagittal section of
male POA region. In the POA, two groups of sbGnRH immunoreactive
cells were stained. Rostral, Rostral cell group; caudal, caudal cell
group. B–E, Frontal sections. The rostral group of neurons (B, C) was
darkly stained in both sexes (B, male; C, female), and the number of
cells did not differ (P ⬎ 0.5; t test). On the other hand, the caudal group
of neurons (D, E) showed a marked sexual difference in number of cells
(P ⬍ 0.005; t test). There were many immunoreactive cells in the
caudal POA of males (D) but by far fewer immunoreactive cells in the
corresponding area of females (E). Scale bar, 100 ␮m.
neurons and are suggested to trigger transmitter release at
conventional synapses in the brain (40, 41).
The involvement of N-type Ca2⫹ channels for POA-GnRH
slices is different from previous studies that described that
Ca2⫹ channels involved in GnRH release from GnRH-secret-
ing cells are L-type Ca2⫹ channels, not N-type Ca2⫹ channels
(39, 42). This may be due to differences among different
vertebrate species or may be from differences in the degree
of maturity of GnRH cells. We used POA-GnRH slices from
adult mature dwarf gourami. It has been reported that the
types of Ca2⫹ channels coupled to secretion change with
degree of maturity (43– 45).
In the present study, there was an unexpected but signif-
icant increase of GnRH release from POA-GnRH slices by the
application of nifedipine, an L-type Ca2⫹ channel blocker. It
is possible that the L-type Ca2⫹ channel is not directly in-
volved in the depolarization-induced GnRH release from
POA-GnRH neurons per se and that high [K⫹]o depolariza-
tion stimulated release of factors that inhibit GnRH release
or some inhibitory synaptic transmitters that heavily de-
pends on L-type Ca2⫹ channels and that nifedipine removed
this inhibition.
It has not been reported which type of Ca2⫹ channels are
involved in GnRH release from TEG- or TN-GnRH neurons.
It has been suggested that TN-GnRH neurons release GnRH
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Endocrinology, April 2004, 145(4):2092–2103 2101
from various parts of the neurons including somatodendritic
areas and axonal varicosities (3, 46). The present results sug-
gest that GnRH release from these parts of TN-GnRH neurons
is dependent on Ca2⫹ influx via N- and L-type Ca2⫹ channels.
Ca2⫹ release from intracellular stores has been reported to
contribute to the exocytosis in several cell types (47– 49). In
the sympathetic neurons, intracellular Ca2⫹ and GnRH re-
lease are increased by agents that cause Ca2⫹-induced Ca2⫹
release such as caffeine (50, 51), whereas in GT1 cells, such
agents have no effect on Ca2⫹ signaling (52). In the present
study, caffeine application had little effect on the GnRH
release from POA- and TN-TEG-GnRH slices. Thapsigargin
treatment for 20 min decreased spontaneous GnRH release
from TN-TEG-GnRH slices of females. Because thapsigargin
is an inhibitor of endoplasmic reticulum Ca2⫹-ATPase, thap-
sigargin treatment should have reduced Ca2⫹ concentration
in the endoplasmic reticulum, which resulted in the decrease
of intracellular Ca2⫹ concentration in 20 min. In TN-TEG-
GnRH slices of females, when extracellular Ca2⫹ was re-
stored after depletion of intracellular Ca2⫹ stores with thap-
sigargin, GnRH release from TN-TEG-GnRH slices was
higher than when extracellular Ca2⫹ was restored in the
presence of Zn2⫹, a blocker of store-operated current. These
results suggest that store-operated Ca2⫹ entry may contrib-
ute to the GnRH release in TEG- and/or TN-GnRH neurons.
There are some studies that report on the contribution of
store-operated Ca2⫹ influx to exocytosis (53, 54). In the
present study, the amount of GnRH released possibly by
store-operated Ca2⫹ influx after depletion of intracellular
store did not exceed that of the control level in which intra-
cellular stores was not depleted. Therefore, it is possible that
Ca2⫹ in the intracellular Ca2⫹ store may be somehow related
to the spontaneous or basal GnRH release from TEG- and/or
TN-GnRH neurons.
It has been reported that peptidergic neurons and endo-
crine cells not only receive several inputs from other neurons
but they are also modulated by peptide hormones released
by themselves, which implies the possible role of the peptide
in modulating its own secretion (autocrine or paracrine con-
trol) (11, 55, 56; see also Ref. 2). In the present study, effects
of GnRH agonist on GnRH release were not investigated.
Simultaneous recording of the GnRH release and electrical
activities is necessary to test GnRH effects on GnRH release
and the relationship between pacemaker activity and GnRH
release activity. Recently we developed a method for real-
time electrochemical recording of GnRH release using car-
bon fiber electrodes (57). An amperometric current was
recorded in response to high [K⫹]o stimulation in a dose-
dependent manner. This method will provide much infor-
mation on the relationship between pacemaker activity and
GnRH release activity.
In summary, we found similarities as well as differences
in the GnRH release activities of TEG- and TN-GnRH neu-
rons and POA-GnRH neurons of the dwarf gourami. TEG-
and TN-GnRH neurons are considered to play neuromodu-
latory roles, and from the present study, we found multiple
Ca2⫹ mobilization pathways related to the GnRH release in
TN-TEG-GnRH slices. It is thus possible that these multiple
Ca2⫹ mobilization mechanisms are related to the neuro-
modulatory function of TEG- and TN-GnRH neurons. Ad-
 2102 Endocrinology, April 2004, 145(4):2092–2103
ditional future studies are needed to determine how GnRH
release from the POA-, TEG-, and TN-GnRH systems is dif-
ferently regulated and how this release may be involved in
reproductive functions of the POA-GnRH system and neu-
romodulatory functions of the TEG- and TN-GnRH systems.
Acknowledgments
We thank Professor K. Aida (University of Tokyo) for allowing us to
use the facilities for the RIA; Mr. T. Masuda for help in RIA; Dr. Y.
Hasegawa (Kitasato University School of Veterinary Medicine and An-
imal Sciences), Professor K. Wakabayashi (Gunma University), and Dr.
I. S. Parhar (Nippon Medical School) for their kind supply of the antisera
(anti-GnRH, lot R-II, HAC-RBA2-05GTP91 and anti-sbGnRH, respec-
tively); and Mr. K. Haneda for help in analysis of the data. We also would
like to thank Drs. K. Okuzawa (National Research Institute of Aqua-
culture), M. K. Park (University of Tokyo), M. Kobayashi (International
Christian University), and M. Amano (Kitasato University) for kind
technical advice and discussion, and K.-I. Maeda, H. Tsukamura, and S.
Tsukahara (Nagoya University) for their kind help in the preliminary
studies.
Received July 29, 2003. Accepted December 29, 2003.
Address all correspondence and requests for reprints to: Dr. Yoshi-
taka Oka, Department of Biological Sciences, Graduate School of Science,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan. E-mail: okay@biol.s.u-tokyo.ac.jp.
This work was supported by Grants-in-Aid from the MEXT of Japan
(to Y.O. and M.Ii.).
Present address for M.Is.: National Agricultural Research Center,
Kannondai, Tsukuba, Ibaraki 305-8666, Japan.
Present address of M.Ii.: Department of Applied Biological Chemis-
try, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi,
Utsunomiya, Tochigi 321-8505, Japan.
Present address for Y.O.: Department of Biological Sciences, Graduate
School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan.
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