Blackwell Science, LtdOxford, UKFISFisheries Science0919 92682006 Blackwell Science Asia Pty LtdFebruary 20067218994Original ArticleGnRHs in the wild

Japanese flounder
KX Pham
et al.

FISHERIES SCIENCE 2006; 72: 89–94

Distribution of three GnRHs in the brain and pituitary of

the wild Japanese flounder Paralichthys olivaceus
Ky Xuan PHAM,1 Masafumi AMANO,1* Noriko AMIYA,1 Yutaka KURITA2 AND
Kunio YAMAMORI1

1
School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101 and 2Tohoku National Fisheries
Research Institute, Shiogama, Miyagi 985-0001, Japan

ABSTRACT: Wild adult maturing and immature female Japanese flounder Paralichthys olivaceus
were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-
releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II
(cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved
fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the
pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced
amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels
of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of
immature fish were very low in these regions. These results indicate that sbGnRH is mainly respon-
sible for gonadotropin secretion, and that sbGnRH in the anterior part of the brain is associated with
gonadal maturation in the Japanese flounder.

KEY WORDS: brain, GnRH, Japanese flounder, pituitary, time resolved fluoroimmunoassay
TR-FIA.

INTRODUCTION cichlid Haplochromis burtoni,7 pacu Piaractus

mesopotamicus,8 striped bass Morone saxatilis,9
The pivotal role of gonadotropin-releasing hor- red sea bream Pagrus major, black sea bream
mones (GnRHs) in the reproduction of vertebrates, Acanthopagrus schlegeli, Nile tilapia Oreochromis
especially in the regulation of gonadotropin (GTH) niloticus, striped knifejaw Oplegnathus fasciatus,10
secretion in teleosts, has been proved. Until now, sea bass Dicentrachus labrax,11 turbot Scoph-
14 forms of GnRH have been identified in verte- thamus maximus12 and barfin flounder Verasper
brates. Among them, eight GnRH forms, i.e. mam- moseri,13 hrGnRH in pacific herring Clupea har-
malian GnRH (mGnRH), salmon GnRH (sGnRH), engus pallasi,14 mdGnRH in medaka Oryzias
chicken GnRH-II (cGnRH-II), catfish GnRH latipes15 and wfGnRH in basal salmonid Coregonus
(cfGnRH), sea bream GnRH (sbGnRH), herring clupeaformis.16
GnRH (hrGnRH), medaka GnRH (mdGnRH) which Distribution of several GnRH forms in the brain
is also called pejerrey GnRH (pjGnRH), and white- and pituitary was investigated in some species by
fish GnRH (wfGnRH) have been found in the brain radioimmunoassay (RIA) or enzyme-linked immu-
of different teleosts.1 nosorbent assay (ELISA). For example, levels of
Two or three GnRH forms coexisting in the brain sGnRH, cGnRH-II and sbGnRH in perciforms10 and
of a single species were confirmed in several teleo- barfin flounder13 were detected by RIA. In addition,
sts. One is the ubiquitous cGnRH-II and another is levels of these hormones in gilthead sea bream,17
a species-specific form,2 while a third form was sea bass,18 striped bass19 and turbot12 were also
found in some representatives of advanced evolu- examined using ELISA. Among these GnRHs, levels
tionary teleosts. In the brain of salmonids and cyp- of sbGnRH were abundant in the pituitary and
rinids, only sGnRH and cGnRH-II are present.3–5 In were also detected in the telencephalon, hypothal-
some fish species, together with sGnRH and amus and optic tectum–thalamus region at low
cGnRH-II, a third form was identified: sbGnRH in levels, while sGnRH and cGnRH-II were detected
the gilthead seabream Sparus aurata,6 African in all areas of the brain.
GnRHs produced in the various neuronal sys-
*Corresponding author: Tel & Fax: 81-192-44-1904. tems are differentially distributed in the brain.
Email: amanoma@kitasato-u.ac.jp Each GnRH may express an independent role or
Received 2 May 2005. Accepted 4 August 2005. act in a synergic manner with other GnRHs in the
90 FISHERIES SCIENCE
reproductive regulation of fish. However, not all of
the physiological functions of GnRHs have been
clarified in teleosts. Among three GnRHs coexist-
ing within a single species, it is now accepted that
sGnRH and cGnRH-II are associated with neu-
rotransmission and/or neuromodulation, whereas
a third of GnRH produced in the preoptic area reg-
ulates reproduction in most fish.20
Japanese flounder Paralichthys olivaceus, order
Pleuronectiformes, is a commercially important
species for aquaculture in Japan. Wild mature fish
inhabit offshore areas (at ≤150 m depth), but
spawn in shallower waters (at 20–50 m depth).
Eggs and larvae develop from planktonic to
benthic life by metamorphosis and settle on near-
shore nursery grounds,21–23 then they gradually
migrate offshore. This species is a multi-spawner
which is easy to handle in artificial spawning, and
is considered as a good model for reproductive
studies. The presence of sGnRH, cGnRH-II and an
unknown form in the brain was confirmed by high
performance liquid chromatography and specific
RIA.24 After sbGnRH was found in gilthead sea
bream,6 the unknown form in the Japanese floun-
der was identified as sbGnRH. In addition, in
females, messenger ribonucleic acid (mRNA)
levels of gonadotrophin-Ιβ (GΤΗ-Iβ) and GTH-IIβ
in the pituitary were highly correlated with
gonadosomatic index (GSI), and estradiol-17β and
testosterone (T) levels. In males, only the mRNA
levels of GTH-Iβ increased with the increase in
GSI, 11-ketotestosterone (11-KT) and T.25 Levels
of plasma T, 11-KT and 17α,20β-dihydroxy-4-
pregnen-3-one (DHP) were also investigated with
testicular germ cells during the daily spawning
cycle in male fish at 4-h intervals; the presence of
a diurnal periodicity in spermatogenesis and
spermiation, and DHP production were demon-
strated.26 However, information on the GnRH
system has not yet been fully documented. This
study investigated the distribution pattern of three
GnRH forms in the brain and pituitary of maturing
and immature Japanese flounder to clarify pos-
sible roles of GnRHs in the reproduction of this
species. Female fish were examined because clari-
fication of the spawning property is important for
aquaculture and fisheries.
MATERIALS AND METHODS
Sampling
Wild Japanese flounder were obtained alive on 4
June 2004 and 28 January 2005 at Soma Fishing
Port, and transported to Tohoku National Fisheries
Research Institute, Miyagi Prefecture, with aera-
KX Pham et al.
Fig. 1 Schematic diagram of a sagittal section of the
Japanese flounder brain. (a) olfactory bulb, (b) telen-
cephalon including the preoptic area, (c) hypothalamus,
(d) optic tectum–thalamus including the midbrain, (e)
cerebellum, (f) medulla oblongata, and (g) pituitary.
tion. Fish were caught offshore from Soma city
(37°50′N, 140°59′E), Fukushima Prefecture by gill
net. Fish were anesthetized with ice. Total length
(TL) and body weight (BW) were measured. Pitu-
itaries and brains of decapitated fish were dis-
sected out. Brains were divided into six parts (the
olfactory bulb, telencephalon including the preop-
tic area, hypothalamus, optic tectum–thalamus,
cerebellum, and medulla oblongata) as shown in
Figure 1. They were quickly placed on dry ice and
stored at −85°C until extraction of GnRHs. Ovaries
were removed and weighed to calculate GSI =
(ovarian weight/[body weight −ovarian weight])
× 100. Small pieces of ovaries were fixed in Bouin’s
solution for histological observation.
Ovarian development
Ovaries fixed in Bouin’s solution were dehydrated
through serial ethanol concentrations and em-
bedded in paraplast. Sections at 12 µm thickness
were cut and stained with hematoxylin–eosin by a
routine procedure for microscopic examination.
The stages of ovarian development were identified
according to the method of Yamamoto.27
Measurement of GnRH
Extraction of GnRHs was conducted according to
Okuzawa et al.4 with a slight modification. Brain
tissues were homogenized with 1 mL 0.1 N HCl by
sonication and centrifuged at 4°C, 12 000 × g for
30 min. The supernatant was transferred into
another microtube, neutralized with 1 N NaOH,
lyophilized, and reconstituted in an assay buffer
(20 mM sodium phosphate buffer, 0.9% NaCl, 0.1%
bovine serum albumin, 20 M diethylenetriamine-
N,N,N′,N′′,N′′-pentaacetic acid, 0.01% Tween-40,
pH 7.2). After reconstitution, samples were re-
sonicated and centrifuged to yield a clear liquid,
and frozen at −85°C until time-resolved fluoro-
immunoassay (TR-FIA).
GnRHs in the wild Japanese flounder FISHERIES SCIENCE 91

Antiserums against sGnRH (lot 2), cGnRH-II cephalon and hypothalamus were significantly
(aCII6) and sbGnRH (AS-691) were diluted 50 000-, higher in maturing fish than in immature fish. On
25 000- and 5000-fold, respectively. The specificity the contrary, sGnRH contents (pg/tissue) in the
of these antibodies was previously confirmed by cerebellum, medulla oblongata and pituitary were
Senthilkumaran et al.10 and Amano et al.13 by RIA. significantly higher in immature fish than in
Measurement of sGnRH, cGnRH-II, and sbGnRH maturing fish (Fig. 3a). The medulla oblongata of
levels was carried out by TR-FIA.28,29 Parallelism of both groups contained an appreciable amount of
typical standard curves of sGnRH, cGnRH-II and cGnRH-II. The cGnRH-II content was extremely
sbGnRH and corresponding competition curves of low in the pituitary in both groups, and cGnRH-II
brain extracts of Japanese flounder were examined contents (pg/tissue) in the olfactory bulb and
with serially twofold-diluted standards and brain hypothalamus were significantly higher in matur-
extracts in an assay buffer. The GnRH levels were ing fish than in immature fish (Fig. 3b). The
expressed as content (pg/tissue) and concentra- sbGnRH contents (pg/tissue) in the telencephalon
tion (pg/mg tissue). and hypothalamus were significantly higher in
maturing fish than in immature fish. Pituitary
sbGnRH content (pg/tissue) was extremely high
Statistics compared to those of sGnRH and cGnRH-II in both
fish groups, and was higher in maturing fish
All data were expressed as mean ± standard error (Fig. 3c). Results of GnRH concentrations (pg/mg
(SE). Differences in TL, BW, GSI and GnRH levels tissue) were identical to those of GnRH contents
between two groups were tested by Student’s (pg/tissue). No significant difference was seen in
t-tests or unpaired t-test with Welch’s correction. pituitary sbGnRH concentrations between the two
Significance was accepted at P < 0.05. groups (Fig. 4).

RESULTS DISCUSSION

The TL, BW and GSI of the two groups are summa- The results not only showed the coexistence of
rized in Table 1. No statistical differences in either sGnRH, cGnRH-II and sbGnRH, but also con-
TL or BW between maturing and immature fish firmed that sbGnRH was abundant in comparison
were observed, but the GSI of maturing fish was
significantly higher than that of immature fish
(P < 0.05). Ovaries of maturing fish were at the sec- Table 1 Total length (TL), body weight (BW) and gona-
ondary or tertiary yolk stage, whereas ovaries of dosomatic index (GSI) of fish collected in June 2004 and
immature fish were at the perinucleolus stage (data January 2005
not shown). Collecting
Displacement curves of brain extracts of the times n TL (mm) BW (g) GSI (%)
Japanese flounder paralleled the corresponding
June 2004 6 604.3 ± 15.5 3251 ± 379 4.44 ± 0.88*
standard curves of sGnRH, cGnRH-II and sbGnRH
Jan. 2005 6 632.5 ± 12.5 2966 ± 214 1.47 ± 0.09
(Fig. 2). In both groups, all forms of GnRH were
found in the discrete brain at various levels. sGnRH Values are the mean ± standard error. *(P < 0.05) indicates the
contents (pg/tissue) in the olfactory bulb, telen- levels of significance for differences.

(a) (b) (c)

Serial dilution of extracts (x2) 100 Serial dilution of extracts (x2) Serial dilution of extracts (x2)
100 100
B/Bo (%)

75 75 75

50 50 50

25 25 25

0 0 0
0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.5 15.6 31.3 62.5 125 250 500 1000 2000 0.39 0.78 1.56 3.12 6.25 12.5 25 50
sGnRH (ng/ml) cGnRH-II (pg/ml) sbGnRH (ng/ml)

Fig. 2 Typical standard curves of (a) sGnRH (b) cGnRH-II, and (c) sbGnRH, and competition curves of serially twofold-
diluted brain extracts of the Japanese flounder. B/Bo(%), the binding rate.
92 FISHERIES SCIENCE KX Pham et al.

(a) (b) (c)

1000 500 8000
GnRH (pg/tissue)

*
* June 2004
* January 2005 *
400
750 6000
*
*
*
* 300 **
*
* *
500 * 4000 **
* *
* 200 *
250 ** 2000
100 **
0 0 0
a b c d e f g a b c d e f g a b c d e f g

Fig. 3 Content (pg/tissue) of (a) sGnRH (b) cGnRH-II, and (c) sbGnRH in the brain and pituitary of maturing fish in
June 2004 and immature fish in January 2005. Error bars, standard error of the mean; a–g as shown in Fig. 1; *(P < 0.05),
**(P < 0.01) and ***(P < 0.001) indicate the levels of significance for differences; n = 6.

(a) (b) (c)

GnRH (pg/mg tissue)

50 15 400
June 2004
January 2005
40
300
10 *
30
* * ** 200
*
* *
20
5 **
*
* 100
10 *
* *
*
0 0 0
a b c d e f g a b c d e f g a b c d e f g

Fig. 4 Concentration (pg/mg tissue) of (a) sGnRH (b) cGnRH-II, and (c) sbGnRH in the brain and pituitary of maturing
fish in June 2004 and immature fish in January 2005. Error bars, standard error of the mean; a–g as shown in Fig. 1;
*(P < 0.05), **(P < 0.01) and ***(P < 0.001) indicate the levels of significance for differences; n = 6.

with sGnRH and cGnRH-II in the pituitary of the
Japanese flounder. In maturing fish, the sbGnRH
level was high in the telencephalon including the
preoptic area and in the hypothalamus. In the pitu-
itary, the level of sbGnRH was much higher than
sGnRH and cGnRH-II levels, approximately 27.5-
and 172-fold, respectively. Further data for com-
parison was obtained by measuring levels of these
hormones in the brain and pituitary of immature
fish. The high level of sbGnRH compared with
sGnRH and cGnRH-II in the pituitary, around 9-
and 121-fold, respectively, but low level in the brain
of immature fish, confirmed that sbGnRH is the
major pituitary GnRH in the Japanese flounder.
Our observation is in agreement with previous
studies showing that sbGnRH was dominant in the
pituitary of perciform fish,10 barfin flounder,13 sea
bass,18 and striped bass.19 The significant amount
of pituitary GnRH in young and immature fish has
also been documented, including the sGnRH in
rainbow trout,4 and sbGnRH in striped bass19 and
barfin flounder.13 However, the function of pitu-
itary dominant GnRH in young and immature
teleosts is still unclear. It is suggested that the abso-
lute GnRH level was an indication of GnRH releas-
ing activity, and that the significant amount of
dominant pituitary GnRH was necessary for the
initiation of gonadal maturation, at least in striped
bass.19 In the Japanese flounder, pituitary sbGnRH
levels were high in both groups, and were higher in
maturing fish than that in immature fish. In the
pituitary of sexually mature gilthead sea bream, the
levels of sbGnRH were higher than those in recru-
descent fish.17 Therefore, it is possible that the high
level of sbGnRH in the pituitary of maturing
Japanese flounder is important for complete mat-
uration, while the appreciable amount of sbGnRH
in the pituitary of immature fish is necessary for
gonadal recrudescence.
The dominance of sbGnRH compared to other
pituitary GnRHs in the Japanese flounder could
compensate for low sbGnRH bioactivity for GTH
release, as observed in gilthead sea bream30 and sea
bass.31 Likewise, low potency to GTH release of
cfGnRH, the dominant form in the pituitary of
the African catfish Clarias gariepinus, was also
documented.32 Alternatively, in maturing Japanese
flounder in this study, sbGnRH levels were high in
the anterior part of the brain, including the telen-
cephalon and hypothalamus. The distributive ten-
dency of sbGnRH in the forebrain of maturing fish
was different from that of immature fish (present
study) and barfin flounder,13 but the same as in
maturing striped knifejaw with a high amount of
GnRHs in the wild Japanese flounder FISHERIES SCIENCE
sbGnRH in the anterior part of the brain.10 The high
accumulation of sbGnRH in the forebrain of
maturing Japanese flounder, but not in immature
fish, indicates that sbGnRH is not always dominant
in the brain, but only transient with the status of
the fish in early June. The notable amount of
sbGnRH in the forebrain of these fish only reflected
the temporary balance of production, transport
and release of this hormone. The different levels of
sbGnRH in the telencephalon and hypothalamus
between the two groups suggest that sbGnRH lev-
els in these regions change with the process of mat-
uration, similar to the variation in sbGnRH brain
levels during the spawning season in red sea
bream.10 The presence of sbGnRH and the precise
role of this hormone in the forebrain of the
Japanese flounder need to be investigated in
further studies.
The low level of cGnRH-II in the pituitary of
Japanese flounder is consistent with previous
reports showing that cGnRH-II was below the
detectable limit in the pituitary of rainbow trout,4
masu salmon,33 and perciform fish (red sea bream,
black sea bream, striped knifejaw, and Nile tila-
pia)10 and low in the barfin flounder.13 Like in gilt-
head sea bream,17 cGnRH-II in the pituitary of the
Japanese flounder was steady at low levels in both
maturing and immature fish, and the level of
cGnRH-II was much lower than that of sbGnRH in
this organ. Thus, although cGnRH-II is distributed
through the brain, its extremely low and
unchanged levels in the pituitary almost rule out
the responsibility of the cGnRH-II for the regula-
tion of GTH or other pituitary hormone secretion
in this species.
In contrast to cGnRH-II, a pronounced amount
of sGnRH was detected in the pituitary. Moreover,
sGnRH contents differed according to both the
brain region and the maturity of fish; sGnRH con-
tents in the pituitary, cerebellum and medulla
oblongata were higher in immature fish, whereas
those in the olfactory bulb, telencephalon and
hypothalamus were higher in maturing fish. In the
turbot, with sbGnRH as the dominant form of
GnRH in the pituitary, it is suggested that sGnRH
has a potential physiological role in reproduction;
over the spawning season, sGnRH levels in the
pituitary significantly decreased, in contrast to
sbGnRH levels.12 Thus, it is suggested that sGnRH
has a potential physiological role in the Japanese
flounder.
The distribution pattern of sGnRH and cGnRH-II
in the brain of Japanese flounder is almost in
agreement with that of rainbow trout.4 Like some
teleosts,34 the presence of sGnRH and cGnRH-II in
many areas of the brain of the Japanese flounder
could be considered as neurotransmitters and/or
93
neuromodulators. This possibility is supported by
the results of immunocytochemistry that revealed
the widespread distribution of sGnRH and cGnRH-
II-immunoreactive fibers in the brain (unpub-
lished data).
In conclusion, in the present study, among three
forms of GnRH detected in the brain and pituitary
of both maturing and immature Japanese flounder,
sbGnRH was the dominant pituitary GnRH. The
high level of sbGnRH in the pituitary indicates that
sbGnRH is associated with GTH secretion and is
mainly responsible for the regulation of reproduc-
tion in this fish. Alternatively, the appreciable level
of sbGnRH in the anterior part of brain in maturing
fish could be involved in maturation in the
Japanese flounder.
ACKNOWLEDGMENTS
Thanks to Ms. Hiroe Aoki and Ms. Mio Matsuki,
School of Fisheries Sciences, Kitasato University
for their help collecting samples.
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