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Aqua-BioScience Monographs Vol. 3, No. 2, pp. 39–72 (2010)
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Reproductive Biology of Salmoniform and
Pleuronectiform Fishes with
Special Reference to
Gonadotropin-Releasing Hormone (GnRH)
Masafumi Amano
School of Marine Biosciences, Kitasato University
Ofunato, Iwate 022-0101, Japan
e-mail: [email protected]
Abstract
A salmonid fish, masu salmon Oncorhynchus masou, has salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II), while a pleuronectiform fish, barfin flounder Verasper moseri, has sGnRH, cGnRH-II and seabream GnRH (sbGnRH). In masu salmon,
sGnRH-immunoreactive (ir) cell bodies are scattered from the olfactory nerve through the ventral telencephalon (VT) and the preoptic area (POA). cGnRH-II-ir cell bodies are located in
the midbrain tegmentum (MT). sGnRH but not cGnRH-II is detected in the pituitary. sGnRH
peptide levels and sGnRH mRNA levels in the VT and the POA increased during gonadal maturation. sGnRH neurons are derived from the olfactory epithelium and migrate into the brain.
In barfin flounder, sGnRH-ir, cGnRH-II-ir, and sbGnRH-ir cell bodies are located in the olfactory bulbs and the terminal nerve ganglion (TN), the MT, and the POA, respectively, and these
neurons do not migrate in the brain. sbGnRH is detected in the pituitary. sbGnRH mRNA
levels in the brain increased during gonadal maturation. Although three GnRH systems exist in
the barfin flounder, anatomical distinction between the TN- and the POA-GnRH systems is not
clear in masu salmon. Thus, it is suggested that sGnRH neurons in masu salmon play different
roles according to the location in the brain.
1. General introduction
Gonadotropin-releasing hormone (GnRH) is a
decapeptide originally isolated from pig and sheep
hypothalami as a physiologic regulator of luteinizing
hormone (LH) release from the pituitary (Matsuo
et al. 1971; Burgus et al. 1972). At present, it is
generally accepted that GnRH regulates synthesis
and release of pituitary gonadotropin (GTH) (see
King and Millar 1992; Sherwood et al. 1993). It
has been shown that two or three molecular forms of
GnRH exist, even within the same species (Oka 1997;
Okuzawa and Kobayashi 1999; Okubo and Nagahama
2008). In addition, GnRH can act as a neuromodulator
and has also been implicated in reproductive behavior in many species including teleost fish such as dwarf
gourami Colisa lalia (Yamamoto et al. 1997) and goldfish
Carassius auratus (Volkoff and Peter 1999).
© 2010 TERRAPUB, Tokyo. All rights reserved.
doi:10.5047/absm.2010.00302.0039
Received on
March 31, 2010
Accepted on
June 24, 2010
Online published on
August 31, 2010
Keywords
• GnRH
• brain
• pituitary
• gonad
• radioimmunoassay
• immunohistochemistry
• in situ hybridization
• quantitative PCR
• masu salmon
• sockeye salmon
• barfin flounder
To date, 15 forms of GnRHs have been identified based
on their primary structure or complementary DNAs
(cDNAs) in vertebrates, as shown in Fig. 1 (Okubo
and Nagahama 2008; Kavanaugh et al. 2008). GnRH
forms are traditionally named after the species from
which they were first identified. In addition to vertebrate
species, GnRH was isolated in invertebrates: e.g., in
the protochordate Ciona intestinalis (Powell et al. 1996;
Adams et al. 2003), octopus Octopus vulgaris (Iwakoshi
et al. 2002) and the sea hare Aplysia californica (Zhang
et al. 2008).
In mammalian, avian, reptilian, and amphibian
animals, GnRH is conveyed to the pituitary via the
hypothalamo-hypophyseal portal vessels (Fig. 2A).
In mammals, pulsatile release of mammalian GnRH
(mGnRH) by hypothalamic neurons stimulates GTH
secretion from the pituitary. However, teleost fishes
lack the median eminence. Instead, GnRH neurons are
found to directly innervate the pituitary (Fig. 2B). It is
therefore interesting to examine GnRH systems in teleost
fish in view of comparative endocrinology.
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 1. The primary structure of the 15 known molecular forms of GnRH in vertebrates. All GnRH forms are composed of 10 amino
acids and contain an N-terminal pyroglutamate and C-terminal glycinamide. GnRH forms are traditionally named after the species
from which they were first identified.
Fig. 2. Schematic drawing of the brain–pituitary–gonadal system
in (A) mammals and (B) teleost fishes.
Understanding of reproductive mechanism of teleost
fish is necessary for the establishment and development
of fish aquaculture. GnRH is considered to play an
important role in fish reproduction as in other vertebrate
species. This monograph will focus on the reproductive
biology of salmoniform and pleuronectiform fishes
with special reference to GnRH, with emphasis on the
author’s own work. Masu salmon, Oncorhynchus masou
(Figs. 3A–C), and sockeye salmon, Oncorhynchus
nerka, are used as a model of salmoniform. The masu
salmon used were offspring of wild fish which had migrated to the Shiribetsu River (Hokkaido). Wild masu
salmon migrate to the sea in the spring (1.5 years old),
and return to the river in May after a one-year stay
in the sea; they spawn in autumn and then die. The
masu salmon used in this experiment also smoltified
at 1.5 years old and matured at 3 years old in fresh
water, although the growth rate was not very rapid.
There is a landlocked form of masu salmon called
“yamame.” This variety for the most part does not
smoltify. Barfin flounder, Verasper moseri, is used as a
model of pleuronectiform (Fig. 3D). This species is a
large, multiple-spawning pleuronectiform fish inhabiting
cold sea basins around east Hokkaido, Japan, and is
promising for aquaculture and resource enhancement in
northern Japan due to its high commercial value. As for
the general information of GnRH in teleost fish, please
consult recent reviews (Lethimonier et al. 2004; Guilgur
et al. 2006; Okubo and Nagahama 2008; Oka 2009;
Zohar et al. 2010).
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
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Fig. 3. (A) Immature masu salmon, (B) precocious male masu salmon, (C) masu salmon smolt, and (D) barfin flounder. Bars indicate
5 cm.
2. Identification of GnRH forms in the brain
2-1. Identification of GnRH forms by HPLC and RIA
in masu salmon
Since two or three molecular forms of GnRH exist,
even within the same species (see Oka 1997; Okuzawa
and Kobayashi 1999; Okubo and Nagahama 2008), it is
quite important to identify GnRH forms in the brain to understand the reproductive biology of fish. The presence of
two forms of GnRH, salmon GnRH (sGnRH) and chicken
GnRH-II (cGnRH-II), in the teleost brain was first
reported in goldfish by employing reversed phase
high performance liquid chromatography (rpHPLC) in
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
conjunction with radioimmunoassay (RIA) (Yu et al.
1988). Thus, GnRH molecules present in the brain of
masu salmon were identified by rpHPLC in conjunction
with specific RIAs according to Okuzawa et al. (1990).
Major peaks were obtained in the fraction corresponding to the retention time of sGnRH and cGnRH-II in
sGnRH RIA and cGnRH-II RIA, respectively. A minor
peak was also obtained in the fraction corresponding to
the retention time of cGnRH-II in sGnRH RIA (Fig. 4).
These results indicate that masu salmon brain contained
a peptide chromatographically and immunologically
identical to sGnRH and cGnRH-II (Amano et al. 1992).
Later, sGnRH cDNA was cloned from masu salmon brain
(Suzuki et al. 1992).
Fig. 4. Reverse-phase HPLC of masu salmon brain extract followed by (A) sGnRH RIA and (B) cGnRH-II RIA. Arrows indicate the elution time of synthetic sGnRH and cGnRH-II. The
mobile phase was CH3CN (acetonitrile) containing 0.1% TFA.
Reprinted with permission from Zoological Science, 9, Amano
et al., Changes in salmon GnRH and chicken GnRH-II contents
in the brain and pituitary, and GTH contents in the pituitary in
female masu salmon, Oncorhynchus masou, from hatching
through ovulation, 375–386, Fig. 2, © 1992, Zoological Society of Japan.
2-2. Identification of GnRH forms by cDNA
cloning in barfin flounder
The cDNA coding for mGnRH was initially isolated
from human placenta (Seeburg and Adelman 1984) and,
subsequently, mGnRH genes have been cloned from several species (Adelman et al. 1986; Mason et al. 1986).
Chicken GnRH-I (cGnRH-I) gene has been cloned from
chicken (Dunn et al. 1993). In teleost fish, the cDNA
coding for sGnRH was first isolated from African cichlids Haplochromis burtoni (Bond et al. 1991). Then,
the cDNA for cGnRH-II has been isolated from several
teleost fish, e.g., African cichlids (White et al. 1994),
African catfish Clarias gariepinus (Bogerd et al. 1994)
and goldfish (Lin and Peter 1996), the cDNA for sGnRH
from several teleost fish, e.g., masu salmon (Suzuki et
al. 1992), Atlantic salmon Salmo salar (Klungland et al.
1992), red seabream Pagrus major (Okuzawa et al. 1994),
plainfin midshipman Porichthys notatus (Grober et al.
1995), sockeye salmon (Ashihara et al. 1995), goldfish
(Lin and Peter 1996), cDNA for catfish GnRH (cfGnRH)
from African catfish (Bogerd et al. 1994), and cDNA for
seabream GnRH (sbGnRH) from several teleost fish, e.g.,
African cichlid (White et al. 1995) and from red seabream
(Okuzawa et al. 1997). In general, a GnRH precursor is
composed of a signal peptide (SP), GnRH and a GnRHassociated peptide (GAP), which is connected to GnRH
by a Gly–Lys–Arg sequence. Here, GnRHs in barfin
flounder were identified by isolation of their cDNAs.
Single-strand cDNA was reverse transcribed from
barfin flounder brain poly (A) + RNA. A degenerate
forward primer was synthesized based on highly conserved amino acid sequences of GnRH. The 3′ end of
the GnRH cDNA was cloned by rapid amplification
of cDNA ends (RACE) using the degenerated primer.
The 5′ end of the GnRH cDNA was then cloned by
5′-RACE. PCR-amplified cDNA was inserted into pT7
Blue T (Novagen, Madison, WI) and sequenced using the
Dye Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems, Foster City, CA). The nucleotide
sequence was determined using a 373 DNA sequencer
(Applied Biosystems).
Barfin flounder had three molecular forms of GnRH;
sGnRH, cGnRH-II, and sbGnRH. Each GnRH cDNA encoded a SP, GnRH and a GAP. The sGnRH cDNA encoded a SP composed of 23 amino acids and a GAP composed of 54 amino acids (Fig. 5). The cGnRH-II cDNA
encoded a SP of 23 amino acids and a GAP of 49 amino
acids (Fig. 6). The sbGnRH cDNA encoded a SP of
26 amino acids and a GAP of 57 amino acids (Fig. 7)
(Amano et al. 2002a).
The existence of multiple forms of GnRH in the brain
of masu salmon and barfin flounder suggests that each
GnRH has a different distribution and function. Therefore, the physiological role of each GnRH form should be
clarified.
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
43
Fig. 5. Nucleotide sequences of the cDNA encoding the sGnRH precursor of barfin flounder brain and the deduced amino acid sequence
of the sGnRH precursor. Nucleotides are numbered from 5′ to 3′, beginning with the initiator codon (ATG) in the coding region for the
opening reading frame. Amino acid residues are numbered with the first residue (Met) in the open reading frame. The asterisk
indicates the stop codon. The nucleotides corresponding to the polyadenylation signal in the 3′-untranslated region (AATAAA) are
underlined. Reprinted from General and Comparative Endocrinology, 126, Amano et al., Molecular cloning of three cDNAs encoding
different GnRHs in the brain of barfin flounder, 325–333, © 2002, Elsevier Science (USA), with permission from Elsevier.
3. Distribution of GnRH in the brain and pituitary
3-1. Differential distribution of multiple forms of
GnRH in discrete brain areas
Differential distribution of multiple forms of GnRH in
discrete brain areas has been examined by RIA in several
fishes in order to clarify their functions in the brain.
Okuzawa et al. (1990) first measured the sGnRH and
cGnRH-II contents in the discrete brain regions of the
rainbow trout, Oncorhynchus mykiss, using specific
RIAs. These authors found that the contents of both
forms of GnRHs varied in different brain regions. The
levels of sGnRH were higher than those of cGnRH-II
in the olfactory bulbs (OB), the telencephalon including
preoptic area (POA), the hypothalamus, the optic tectumthalamus and the pituitary, whereas the cerebellum and
the medulla oblongata contained much more cGnRH-II
than sGnRH. Especially of note, cGnRH-II was undetectable in the pituitary. These results suggest that of the
two GnRHs only sGnRH is involved in GTH secretion.
In the goldfish, sGnRH was distributed in a larger amount
in the OB, the telencephalon, the hypothalamus, and the
pituitary than in the other regions, whereas cGnRH-II
was distributed widely throughout the brain with highest
concentrations in the medulla oblongata. The major
difference between salmonid fishes and goldfish is
that goldfish pituitary contains cGnRH-II (Kobayashi
et al. 1992, 1994). In the European eel, Anguilla
anguilla, mGnRH levels were higher than cGnRH-II
levels in the pituitary, the olfactory lobes together with
the telencephalon, and the diencephalon together with
the mesencephalon, while the opposite results were
obtained for the posterior part of the brain. Of interest,
cGnRH-II levels in the pituitary were slightly above
the detectable limit (Dufour et al. 1993). These studies
indicated that sGnRH (or mGnRH) and cGnRH-II are
differently distributed in the brain and also necessitated
the investigation of the localization of GnRH neurons and
the changes in GnRH levels during gonadal maturation.
Existence of multiple forms of GnRH was demonstrated
in the brain of masu salmon and barfin flounder, as shown
in the previous section. Thus, differential distribution of
multiple forms of GnRH in discrete brain areas of masu
salmon and barfin flounder was examined by RIA.
Extraction of GnRH from the discrete brain tissue
(Figs. 8A, 9A) was done according to Okuzawa et
al. (1990). sGnRH, cGnRH-II, and sbGnRH contents
were measured by respective RIAs established by
Okuzawa et al. (1990) and Senthilkumaran et al. (1999).
Concentrations of sGnRH and cGnRH-II (pg/mg tissue) in each region of the brain of ovulated female masu
salmon are shown in Fig. 8B. The concentrations of
sGnRH and cGnRH-II varied in different brain regions.
The levels of sGnRH were higher than those of cGnRH-II
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
44
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 6. Nucleotide sequences of the cDNA encoding the cGnRH-II precursor of barfin flounder brain and the deduced amino acid
sequence of the cGnRH-II precursor. For details, see the legend to Fig. 5. Reprinted from General and Comparative Endocrinology,
126, Amano et al., Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder, 325–333, © 2002,
Elsevier Science (USA), with permission from Elsevier.
Fig. 7. Nucleotide sequences of the cDNA encoding sbGnRH precursor of barfin flounder brain and the deduced amino acid sequence
of the sbGnRH precursor. For details, see the legend to Fig. 5. Reprinted from General and Comparative Endocrinology, 126, Amano
et al., Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder, 325–333, © 2002, Elsevier Science
(USA), with permission from Elsevier.
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 8. (A) Schematic diagram of a sagittal section of masu
salmon brain. The letters a–g represent the following brain areas: a, olfactory bulb; b, telencephalon including preoptic area;
c, hypothalamus; d, optic tectum-thalamus including midbrain;
e, cerebellum; f, medulla oblongata; g, pituitary. Reprinted with
permission from Zoological Science, 14, Amano et al., Distribution and function of gonadotropin-releasing hormone (GnRH)
in the teleost brain, 1–11, Fig. 2, © 1997, Zoological Society of
Japan. (B) The concentration (pg/mg tissue) of sGnRH and
cGnRH-II in discrete areas of the brain and pituitary of ovulated
masu salmon (mean ± SEM). The letters a–g represent the same
areas as indicated in (A).
in the OB, the telencephalon including POA, the hypothalamus, the optic tectum-thalamus and the pituitary,
whereas the medulla oblongata contained much more
cGnRH-II than sGnRH. Especially of note, cGnRH-II
was undetectable in the pituitary.
In barfin flounder, the dominant form of GnRH in
the pituitary was sbGnRH; sbGnRH levels were much
higher than the sGnRH and cGnRH-II levels (Fig. 9B).
sGnRH levels were high in the anterior part of the brain,
especially in the OB. cGnRH-II levels were high in the
posterior part of the brain, especially in the medulla
oblongata. Levels of sbGnRH were extremely low in all
regions of the brain compared to those of sGnRH and
cGnRH-II; sbGnRH was below the detectable limit in
the cerebellum and the medulla oblongata (Amano et
al. 2002b).
These results suggest that sGnRH and sbGnRH are involved in GTH secretion in masu salmon and barfin flounder, respectively. Judging from the wide distribution of
sGnRH and cGnRH-II in the brain of masu salmon and
barfin flounder, it is also suggested that sGnRH and
cGnRH-II function as a neuromodulator in the brain.
45
Fig. 9. (A) Schematic diagram of a sagittal section of barfin
flounder brain. The letters a–g represent the following brain areas: a, olfactory bulb; b, telencephalon including preoptic area;
c, hypothalamus; d, optic tectum-thalamus including midbrain;
e, cerebellum; f, medulla oblongata; g, pituitary. (B) The concentration (pg/mg tissue) of sGnRH, cGnRH-II and sbGnRH in
discrete areas of the brain and pituitary of barfin flounder
(mean ± SEM). The letters a–g represent the same areas as
indicated in (A). With kind permission from Springer
Science+Business Media: Cell and Tissue Research, Three GnRH
systems in the brain and pituitary of a pleuronectiform fish, barfin
flounder Verasper moseri, 309, 2002, 323–329, Amano et al.,
Figs. 1 and 2, © 2002, Springer-Verlag.
3-2. Localization of GnRH neurons in the brain
An approach requisite for clarification of the function
of each GnRH-immunoreactive (ir) neuronal group is to
examine its projection area in the brain. Immunohistochemistry (IHC) has been used for this purpose. Unfortunately, most of these studies used antiserum against
mGnRH. Furthermore, it was clarified that more than one
form of GnRH molecule exists, even within the same
species. Thus, the distribution of sGnRH- and cGnRH-IIir cell bodies and fibers in the brain of masu salmon and
that of sGnRH-, cGnRH-II-, and sbGnRH-ir cell bodies
and fibers in the brain of barfin flounder were examined
by IHC using specific antibodies for sGnRH, cGnRH-II,
and sbGnRH.
The distribution of sGnRH-ir cell bodies and fibers, and
that of cGnRH-II-ir cell bodies and fibers in the masu
salmon are summarized in Fig. 10. sGnRH-ir cell bodies were scattered in the olfactory nerve (ON) (Fig. 11A),
the OB (Fig. 11B), between the OB and the telencephalon
which corresponds to the terminal nerveganglion (TN)
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 10. (A) Schematic drawing of the distribution of sGnRH-ir cell bodies (closed circles) and fibers (lines) in a sagittal section of
masu salmon. (B) Schematic drawing of the distribution of cGnRH-II-ir cell bodies (closed circles) and fibers (lines) in a sagittal
section of masu salmon. C, cerebellum; M, medulla oblongata; MT, midbrain tegmentum; OB, olfactory bulb; ON, olfactory nerve;
OpN, optic nerve; OT, optic tectum; PIT, pituitary; SV, saccus vasculocus; T, telencephalon. Reprinted with permission of John Wiley
& Sons, Inc. from Journal of Comparative Neurology, 314, Amano et al., Immunocytochemical demonstration of salmon GnRH and
chicken GnRH-II in the brain of masu salmon, Oncorhynchus masou, 587–597, © 1991, Wiley-Liss, Inc., a Wiley Company.
(Figs. 11C, D), the ventral telencephalon (VT)
(Figs. 11E, F), and the POA (Figs. 11G, H). sGnRH-ir
fibers were distributed in various brain regions from
the OB to the spinal cord. sGnRH-ir fibers directly
innervated the pituitary (Fig. 11I). cGnRH-II-ir cell
bodies were found in the midbrain tegmentum (MT)
located rostral to the motoneurons of the oculomotor
nerve (Figs. 11J, K). The distribution of cGnRH-II-ir
fibers was basically similar to that of sGnRH-ir fibers
except for the absence of cGnRH-II-ir fibers in the
pituitary (Fig. 11L). The number of cGnRH-II-ir fibers
in the brain was much fewer than those of sGnRH. The
distribution of sGnRH-ir cell bodies in chum salmon,
Oncorhynchus keta, was later reported and was consistent
with that in masu salmon (Kudo et al. 1996). These
results suggest that, in salmonid, sGnRH not only regulates GTH secretion in the pituitary but also functions
as a neuromodulator in the brain, whereas cGnRH-II
functions only as a neuromodulator (Amano et al. 1991).
The distribution of sGnRH-, cGnRH-II-, and
sbGnRH-ir cell bodies and fibers in the barfin flounder
is summarized in Fig. 12. sGnRH-ir cell bodies were
located in the ventromedial part of the rostral OB and
in the TN, and distinct bundles of axons connecting
these two regions were observed (Fig. 13A). sGnRH-ir
fibers were observed throughout the brain excluding the
pituitary. cGnRH-II-ir cell bodies were located in the MT
(Fig. 13B) almost the same rostrocaudal levels as nucleus
of the medial longitudinal fasciculus (nMLF) neurons,
but they seem to constitute a separate cell group as in
dwarf gourami (Yamamoto et al. 1998). cGnRH-II-ir
fibers were observed throughout the brain excluding
the pituitary (Fig. 13B). sbGnRH-ir cell bodies were
located in the POA (Fig. 13C). sbGnRH-ir fibers were
localized mainly in the POA–hypothalamus–pituitary
which formed a distinctive bundle of axons projecting
to the pituitary (Fig. 13D), and were not distributed in
the other areas. In the pituitary, sbGnRH-ir fibers were
observed in the proximal pars distalis (PPD) (Fig. 13E).
Thus, it is strongly suggested that sbGnRH is by far the
best candidate GnRH that is involved in gonadal maturation by stimulating GTH secretion in barfin flounder.
Indeed, some sbGnRH-ir fibers were in close apposition
with GTH cells mainly in the PPD of the pituitary in
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
47
Fig. 11. (A) Sagittal section through the transitional area between the ON and the OB of masu salmon. sGnRH-ir cell bodies (arrowheads) are observed. (B) Sagittal section through the OB. sGnRH-ir cell body (arrowhead) is observed in the ventral part of the OB of
masu salmon. A bundle of sGnRH-ir fibers (double arrowhead) arises from the sGnRH-ir cell bodies in the OB. (C) Frontal section
through the ventral OB of masu salmon. sGnRH-ir cell bodies (arrowheads) and bundles of sGnRH-ir fibers (double arrowheads) are
observed. (D) Sagittal section through the transitional area between the OB and the telencephalon of masu salmon. sGnRH-ir cell
body (arrowhead) is observed in the most ventral part. (E) Sagittal section through the VT of masu salmon. sGnRH-ir cell bodies
(arrowheads) are observed. (F) Frontal section through the VT of masu salmon. sGnRH-ir cell bodies (arrowheads) are observed.
(G) Sagittal section through the POA of masu salmon. sGnRH-ir cell bodies (arrowheads) are observed in the nucleus preopticus
parvicellularis anterioris (PPa). (H) Frontal section through the POA of masu salmon. sGnRH-ir cell bodies (arrowheads) are observed
in the nucleus preopticus magnocellularis, pars magnocellularis (PMm). (I) Sagittal section through the pituitary of masu salmon.
sGnRH-ir fibers are observed in the vicinity of GTH cells of the proximal pars distalis of the pituitary. (J) Sagittal section through the
MT of masu salmon. cGnRH-II-ir cell bodies (arrowheads) are observed. (K) Frontal section through the MT of masu salmon. cGnRHII-ir cell body (arrowhead) is observed in the nMLF. (L) Sagittal section through the pituitary of masu salmon. No cGnRH-II-ir fibers
are observed. Bars indicate 100 µm. Reprinted with permission of John Wiley & Sons, Inc. from Journal of Comparative Neurology,
314, Amano et al., Immunocytochemical demonstration of salmon GnRH and chicken GnRH-II in the brain of masu salmon,
Oncorhynchus masou, 587–597, © 1991, Wiley-Liss, Inc., a Wiley Company.
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 12. (A) Schematic drawing of the distribution of sGnRH-ir
cell bodies (closed circles) and fibers (lines) in a sagittal section
of barfin flounder. (B) Schematic drawing of the distribution
of cGnRH-II-ir cell bodies (closed circle) and fibers (lines) in a
sagittal section of barfin flounder. (C) Schematic drawing of the
distribution of sbGnRH-ir cell bodies (closed circle) and fibers
(lines) in a sagittal section of barfin flounder. C, cerebellum;
M, medulla oblongata; MT, midbrain tegmentum; OB, olfactory
bulb; ON, olfactory nerve; OpN, optic nerve; OT, optic tectum;
PIT, pituitary; POA, preoptic area; T, telencephalon. With kind
permission from Springer Science+Business Media: Cell and
Tissue Research, Three GnRH systems in the brain and pituitary
of a pleuronectiform fish, barfin flounder Verasper moseri, 309,
2002, 323–329, Amano et al., Fig. 3, © 2002, Springer-Verlag.
a pleuronectiform fish Japanese flounder Paralichthys
olivaceus (Pham et al. 2007). It is also suggested that
sGnRH and cGnRH-II function as neuromodulators in
the brain, because both sGnRH-ir fibers and cGnRH-II-ir
fibers were distributed widely in the brain but not in the
pituitary (Amano et al. 2002b).
4. Effects of GnRH on GTH secretion
4-1. Effec ts of GnRH on GTH release in sockeye
salmon
In masu salmon, it is suggested that sGnRH not only
regulates GTH synthesis and release from the pituitary,
but also functions as a neuromodulator in the brain,
whereas cGnRH-II functions only as a neuromodulator,
because cGnRH-II was not detected in the pituitary
(Amano et al. 1991). sGnRH and cGnRH-II have been
detected also in sockeye salmon brain by rpHPLC and
RIA, and their distributions in the brain and pituitary
were similar to those of masu salmon (data not shown).
The absence of cGnRH-II in the pituitary in salmonid
species suggests that cGnRH-II would not be involved
in the stimulation of the pituitary GTH in physiological
conditions.
Two forms of teleostean GTHs, GTH-I and GTHII, was first isolated from chum salmon (Suzuki et
al. 1988a, b, c; Kawauchi et al. 1989). Then, they
have been isolated from several fish species including
salmonids such as coho salmon Oncorhynchus kisutch
(Swanson et al. 1989) and masu salmon (Gen et al. 1993;
Kato et al. 1993). GTH-I and GTH-II are now commonly
referred to as the follicle stimulating hormone (FSH) and
luteinizing hormone (LH), respectively, as in mammals
(Kawauchi and Sower 2006).
In goldfish, both sGnRH and cGnRH-II have the
potency to stimulate GTH (possibly LH) release in a
dose-dependent manner (Chang et al. 1990, 1991; Habibi
1991). In contrast to salmonids, goldfish possess both
forms of GnRH in the pituitary (Yu et al. 1987, 1988;
Kim et al. 1995). Therefore, it is interesting to examine
whether both GnRHs have the potency to stimulate
FSH and LH secretion in salmonid species. Thus, the
effects of sGnRH and cGnRH-II on GTH subunits,
FSH β and LHβ, release from the superfused pituitary of
mature female sockeye salmon were examined by newly
developed sensitive and specific RIAs for FSH β and
LH β.
Pituitaries were dissected into two parts for culture with
either sGnRH or cGnRH-II. Half of the pituitaries were
pooled and dissected into fragments, and were packed in
the superfusion medium. Fractions were collected every
10 minutes in an automatic fraction collector set at 13°C.
Fragments were exposed sequentially to 10 minute pulses
of six concentrations (0.1, 1, 10, 100, 1000, 10000 nM)
of GnRH administered at 80 minute intervals.
Exposure of 10 minute pulses of various concentrations of sGnRH and cGnRH-II resulted in a rapid increase of FSHβ and LHβ from perfused fragments of
the pituitary (Fig. 14). Thus, it is ascertained that both
GnRHs have the potency to stimulate the release of both
GTHs from sockeye salmon pituitary. However, cGnRHII was not detected in the pituitary of sockeye salmon by
RIA and IHC (data not shown). Therefore, it is possible
that cGnRH-II has no involvement in GTH release in this
species. It may be a pharmacological effect according to
the similarity of amino acid sequences.
It is reported in masu salmon that sGnRH stimulates release of LH but not FSH in spawning fish,
whereas sGnRH stimulates release of FSH and LH in
pre-spawning fish in vitro (Ando et al. 2004). Moreover,
sGnRH increases the amount of FSH β mRNAs but
not LHβ mRNAs in pre-spawning fish in vitro (Ando
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 13. (A) Sagittal section through the OB of barfin
flounder. sGnRH-ir cell bodies are observed in the
ventromedial OB (arrowhead on the left) and the TN
(arrowhead on the right). Double arrowhead indicates
sGnRH-ir fibers. (B) Sagittal section through the MT of
barfin flounder. cGnRH-II-ir cell bodies (arrowheads)
and fibers (double arrowhead) are observed. (C) Sagittal
section through the POA of barfin flounder. sbGnRH-ir
cell bodies (arrowheads) and fibers (double arrowhead)
are observed. (D) Sagittal section through the POA of
barfin flounder. Bundles (one on each side of the brain)
of sbGnRH-ir fibers (double arrowheads) arise from the
POA area and run toward the pituitary. (E) Sagittal section through the pituitary of barfin flounder. sbGnRH-ir
fibers are observed in the proximal pars distalis of the
pituitary. The bars indicate 50 µm. With kind permission from Springer Science+Business Media: Cell
and Tissue Research, Three GnRH systems in the brain
and pituitary of a pleuronectiform fish, barfin flounder
Verasper moseri, 309, 2002, 323–329, Amano et al.,
Fig. 5, © 2002, Springer-Verlag.
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49
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
4-2. Effects of GnRH antagonist on GTH levels in
masu salmon and sockeye salmon
Fig. 14. Changes in released (A) FSHβ (ng/ml) and (B) LHβ
(ng/ml) stimulated by various concentrations of sGnRH, and
(C) FSHβ (ng/ml) and (D) LHβ (ng/ml) stimulated by various
concentrations of cGnRH-II in sockeye salmon.
et al. 2004). Thus, it is suggested that the effects of
GnRH on GTH secretion depends on reproductive stage
of fish.
Unlike tetrapods, teleosts lack a hypothalamohypophysial portal vascular system (Fig. 2). The GnRH
neurons directly innervate the pituitary, and GnRH is
considered to be released either from nerve terminals
that are located near or in direct synaptic contact with
pituitary cells or from terminals separated from the pituitary cells by a basement membrane (Peter et al. 1990).
This renders it impossible to measure GnRH levels in the
hypothalamo-hypophysial portal blood, or to examine
GnRH release in vivo (Okuzawa and Kobayashi 1999).
Based on the changes of GTH in the pituitary and the
plasma in salmonid fishes, it is speculated that FSH stimulates early gonadal development and LH modulates the
later stages (Suzuki et al. 1988c; Naito et al. 1991; Tyler
et al. 1991; Prat et al. 1996). However, there is no direct
evidence for endogenous sGnRH-stimulated GTH secretion in salmonid fishes.
The two classical approaches used to study the role
of neuroendocrine factors in the regulation of pituitary
function are immunoneutralization (a method used to examine the role of a hormone by diminishing its potency
with the injection of its antibody) and antagonistic inhibition. However, it is not practical to use immunoneutralization in teleosts due to the direct innervation of gonadotrophs by neurosecretory fibers (Peter et al. 1990;
Kah et al. 1993).
Thus, GnRH antagonist treatment is considered to
be effective for examining the neuroendocrinological
function of endogenous GnRH. GnRH antagonist, [Ac∆3-Pro1, 4FD-Phe2, D-Trp3,6]-mGnRH was used. In late
September, underyearling precocious male masu salmon
that exhibited spermatogenesis were intraperitoneally
injected with saline, sGnRH (0.1 µ g/g body weight
(BW)), sGnRH (0.1 µ g/g BW) + GnRH antagonist
(0.5 µ g/g BW), and GnRH antagonist (0.5 µ g/g BW),
respectively. To measure plasma LH levels by RIA
(Kobayashi et al. 1987), blood samples were collected at
3 h postinjection.
GnRH antagonist treatment for 3 h significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration in underyearling precocious male masu salmon, indicating the
stimulatory effects of sGnRH on LH secretion (Fig. 15).
Considering the fact that sGnRH and not cGnRH-II has
been detected in the pituitary of the salmonid fish (Amano
et al. 1991), the effects of the GnRH antagonist on the LH
levels are considered to be the result of the inhibition of
sGnRH function.
Subsequently, the effect of the GnRH antagonist on LH
synthesis was examined in underyearling immature sockeye salmon that were pretreated with exogenous testosterone to increase the pituitary LH contents as a result of
positive feedback (Okuzawa 2002). The GnRH antagonist (50 µ l/20 g BW, i.e., 1.0 µ g/g BW) was intraperitoneally injected once a week for 6 weeks from early
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
51
Fig. 15. Changes in plasma LH concentrations (ng/ml) in underyearling precocious male masu salmon (mean ± SEM). In each
group (n = 8), means with different letters indicate significant
difference (P < 0.05). Reprinted with permission of John Wiley
& Sons, Inc. from Journal of Experimental Zoology Part A,
307A, Amano et al., Effects of a gonadotropin-releasing hormone antagonist on gonadotropin levels in masu salmon and
sockeye salmon, 535–541, © 2007, Wiley-Liss, Inc., a Wiley
Company.
August. The fish were fed testosterone 25 µ g/g-diet
(1.5% of BW) throughout the experiment. The LH
contents in the pituitary and plasma were measured by
RIA (Kobayashi et al. 1987).
GnRH antagonist treatment slightly but significantly
inhibited an increase in the testosterone-stimulated pituitary LH content levels (Fig. 16A). However, it had no
effect on the plasma LH levels (Fig. 16B). Assuming
that the pituitary LH content reflects the mRNA levels,
as observed in striped bass Morone saxatilis (Hassin et
al. 1999), these results indicate that the decrease in LH
contents following GnRH antagonist treatment was due
to a decrease in LH synthesis and not due to an increase in
LH release. Thus, it is suggested that endogenous sGnRH
stimulates LH synthesis in sockeye salmon. However,
considering that the GnRH antagonist decreased the pituitary LH contents only slightly, the GnRH antagonist partially inhibited an increase in testosterone-stimulated LH
synthesis (Amano et al. 2007). Moreover, GTH secretion
is controlled not only by GnRH but also by other factors
such as dopamine and sex steroids (Zohar et al. 2010).
Thus, it is necessary to take these factors into consideration for more precise analysis.
5. Changes in GnRH levels in the brain during
gonadal maturation
5-1. Changes in GnRH peptide levels during
gonadal maturation in female masu salmon
If GnRH is involved in gonadal maturation, GnRH
contents in the brain would be expected to change during
Fig. 16. Changes in (A) pituitary LH contents (ng) and
(B) plasma LH concentrations (ng/ml) in underyearling immature sockeye salmon (mean ± SEM). **(P < 0.01) indicates
statistical significance between the groups. In each group,
means with different letters indicate significant difference
(P < 0.05). Reprinted with permission of John Wiley & Sons,
Inc. from Journal of Experimental Zoology Part A, 307A,
Amano et al., Effects of a gonadotropin-releasing hormone
antagonist on gonadotropin levels in masu salmon and sockeye
salmon, 535–541, © 2007, Wiley-Liss, Inc., a Wiley Company.
gonadal maturation. Several studies were conducted
to measure brain GnRH contents by RIA in relation to
gonadal maturation of teleost fish, e.g., in European eel
(Dufour et al. 1982), platyfish Xiphophorus maculatus
(Schreibman et al. 1983), brown trout Salmo trutta
(Breton et al. 1986), caribe colorado Pygocentrus notatus
(Gentile et al. 1986), goldfish (Yu et al. 1987), and
chinook salmon Oncorhynchus tschawytscha (Lewis et
al. 1992). However, the results were discordant. A clear
correlation between brain GnRH contents and gonadal
maturity was observed only in caribe colorado (Gentile
et al. 1986). Such discrepancies may be, in part, due
to the use of RIAs employing nonspecific antibodies.
Thus, changes in sGnRH and cGnRH-II contents in
the brain and pituitary of female masu salmon from
hatching through gonadal maturation for three years
were examined by using specific RIAs for sGnRH and
cGnRH-II. For purposes of this study, masu salmon eggs
were artificially fertilized in October. The eggs hatched
in December, and the fish were reared under natural
photoperiod in spring water of constant temperature
(9–10°C) throughout the experiment. Brain and pituitary
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52
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
were sampled for the measurement of GnRHs and GTHs.
Ovarian weights were measured to calculate gonadosomatic index (GSI) as follows: gonad weight/BW
× 100. Extraction of GnRH from the brain tissue and
measurements of sGnRH and cGnRH-II by respective
RIAs were done according to Okuzawa et al. (1990).
Changes in BW and GSI are shown in Figs. 17A and
17B. GSI rapidly increased from July through October
of 2 year olds, in accordance with the advancement of
vitellogenesis and ovulation. Ovulation was observed in
October. sGnRH concentrations (pg/mg tissue) in the OB
and the telencephalon including POA increased significantly during vitellogenesis and ovulation (Figs. 18A, B).
sGnRH concentrations in the hypothalamus also showed
a similar tendency (Fig. 18C). To the contrary, no significant changes were seen in the optic tectum-thalamus
and cerebellum-medulla oblongata during vitellogenesis
and ovulation (Figs. 18D, E). Pituitary sGnRH contents
(pg) showed a stepwise increase every summer for three
years, and significantly increased prior to ovulation
(Fig. 19A). During gonadal maturation, LH levels in the
pituitary and those in the plasma correlatively increased
with the elevation in sGnRH levels in the brain and
pituitary (Figs. 19B, C). cGnRH-II was undetectable in
the pituitary throughout the experiment (data not shown).
Further, no significant changes in the concentration of
cGnRH-II were found in discrete brain areas during vitellogenesis and ovulation (data not shown). These results,
together with those of immunohistochemical studies, suggest that sGnRH in the telencephalon including POA and
the hypothalamus is involved in gonadal maturation in
masu salmon (Amano et al. 1992).
It has been demonstrated that GnRH fibers originating
from the TN is not involved in GTH secretion and further
in gonadal maturation in goldfish. The sGnRH contents
in the brain except the OB markedly decreased by olfactory tract sectioning (OTX), whereas the cGnRH-II contents in the brain showed no clear changes. Despite large
decreases in the brain sGnRH contents, gonadal maturation was not inhibited (Kobayashi et al. 1992, 1994;
Kim et al. 2001). In dwarf gourami, sGnRH neurons in
the TN may be the most extensively projecting GnRH
neurons in the brain except in the pituitary (Oka and
Matsushima 1993). These results indicate that most of
the sGnRH fibers in the brain originates from the TN and
that sGnRH neurons in the TN do not project to the pituitary. Thus, it is possible that changes in the sGnRH
contents measured by RIA reflect the activities of GnRH
neurons in the TN. It should be noted again that the levels
of GnRH measured by RIA must be considered as a summation of synthesis, release, and degradation of GnRH at
any point in development. Therefore, it is necessary to
examine the expression of GnRH gene in order to clarify
function of GnRH in reproduction.
5-2. Changes in sGnRH mRNA expression during
gonadal maturation in female masu salmon
Fig. 17. Changes in (A) BW (g) and (B) GSI (%) during
gonadal maturation of female masu salmon (mean ± SEM).
***(P < 0.001) and *(P < 0.05) indicate statistical significance.
Reprinted with permission from Zoological Science, 9, Amano
et al., Changes in salmon GnRH and chicken GnRH-II contents
in the brain and pituitary, and GTH contents in the pituitary
in female masu salmon, 375–386, Fig. 4, © 1992, Zoological
Society of Japan.
In female masu salmon, sGnRH peptide levels in the
telencephalon, including POA, and the hypothalamus increased during gonadal maturation as mentioned above
(Amano et al. 1992). The widespread distribution of
sGnRH-ir cell bodies in the ventral part of the brain suggests that sGnRH has several functions in the brain in
addition to the stimulation of GTH secretion (Amano et
al. 1991); however, it still remains obscure which sGnRH
neurons are involved in gonadal maturation via GTH secretion. In situ hybridization (ISH) is useful to detect
GnRH mRNA levels when certain GnRH neurons, such
as sGnRH neurons in masu salmon, are scattered in the
brain. Thus, the changes of sGnRH mRNA expression
during ovulation in 2-year-old female masu salmon were
examined by ISH.
Sampling was carried out at 2-monthly intervals from
April through October (month of ovulation). Brain tissue was fixed for ISH. Serial sagittal sections were cut at
8 µm. Procedure of ISH is illustrated in Fig. 20. The
number of neurons expressing sGnRH mRNA in the OB,
the TN, the VT, and the POA were measured (Amano et
al. 1994).
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
53
Fig. 19. Changes in (A) pituitary sGnRH contents (pg),
(B) pituitary LH contents (ng), and (C) plasma LH concentration (ng/ml) during gonadal maturation of female masu
salmon (mean ± SEM). ***(P < 0.001) and *(P < 0.05) indicate statistical significance. (A) and (B) are reprinted with
permission from Zoological Science, 9, Amano et al., Changes
in salmon GnRH and chicken GnRH-II contents in the brain
and pituitary, and GTH contents in the pituitary in female
masu salmon, 375–386, Figs. 8 and 5A, respectively, © 1992,
Zoological Society of Japan.
Fig. 18. Changes in sGnRH concentrations (pg/mg tissue) in
the (A) OB, (B) telencephalon including POA, (C) hypothalamus,
(D) optic tectum-thalamus, and (E) cerebellum and medulla
oblongata during gonadal maturation of female masu salmon
(mean ± SEM). ***(P < 0.001), **(P < 0.01) and *(P < 0.05)
indicate statistical significance. Reprinted with permission from
Zoological Science, 9, Amano et al., Changes in salmon GnRH
and chicken GnRH-II contents in the brain and pituitary, and
GTH contents in the pituitary in female masu salmon, 375–386,
Fig. 7, © 1992, Zoological Society of Japan.
Changes in BW and GSI are shown in Figs. 21A and
21B, respectively. GSI rapidly increased from April
through October, in accordance with vitellogenesis and
ovulation. The number of neurons expressing sGnRH
mRNA in the VT and the POA increased with gonadal
maturation (Fig. 21C). Plasma LH levels remained
low until August, but rapidly increased in October
(Fig. 22A). These results indicate that sGnRH neurons
in the VT and the POA are involved in the regulation
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54
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 20. Procedure of in situ hybridization using 35S labeled oligonucleotide probe.
of gonadal maturation possibly through GTH secretion.
The plasma levels of estradiol-17β (E2) (Fig. 22B) and
testosterone (Fig. 22C) increased in August, whereas
17α20β-dihydroxy-4-pregnen-3-one (DHP) (Fig. 22D)
and LH levels suddenly increased in October. This
suggests that increased E2 and/or testosterone stimulated sGnRH synthesis in the POA and VT as a result
of positive feedback (Amano et al. 1995a). This is
supported by the fact that sGnRH gene expression
in the POA was activated by aromatizable androgen,
17 α-methyltestosterone (17α -MT) in future precocious
male (Amano et al. 1994) and 2-year-old female masu
salmon which are just before the initiation of gonadal
maturation (Amano et al. 1997a).
No significant changes of the sGnRH synthetic activity
were observed in the OB and the TN (Fig. 21C), suggesting that sGnRH neurons in the OB and the TN are not
involved in the regulation of gonadal maturation. In the
dwarf gourami, GnRH originating from the TN does not
affect GTH secretion but rather has a function as a neuromodulator in the brain (Oka and Ichikawa 1990; Oka
1992; Yamamoto et al. 1995). sGnRH originating from
the TN is also not considered to be essential for gonadal
development in goldfish (Kobayashi et al. 1992, 1994;
Kim et al. 2001). It is also possible that sGnRH neurons
in the TN function to modulate neuronal activity in masu
salmon. sGnRH neurons in the ON and the OB are suggested to have some important roles in seaward migration
of chum salmon (Kudo et al. 1994; Parhar et al. 1994).
Wild masu salmon migrate to the sea in the spring after
smoltification. The fish used were offspring of wild fish
and they smoltified in the spring at 1.5 years old. Thus, it
may be that sGnRH neurons in the OB are also involved
in seaward migration in masu salmon.
5-3. Changes in brain GnRH mRNA and pituitary
GnRH peptide levels during gonadal maturation in barfin flounder
Barfin flounder first mature in early spring at 2
(male) and 3 years (female), respectively. The barfin
flounder has sGnRH, cGnRH-II, and sbGnRH (Amano
et al. 2002a). To clarify the possible functions of GnRH
in the brain of barfin flounder, we have previously
examined the distribution of the three forms of GnRH in
various areas of the brain by RIA, and the localization of
GnRH-ir cell bodies and fibers in the brain and pituitary
by IHC. As a result, it appears that sbGnRH is involved in
gonadal maturation through stimulating GTH secretion;
sbGnRH-ir cell bodies located in the POA send fibers
into the pituitary, and levels of sbGnRH in the pituitary
were much higher than those of sGnRH and cGnRH-II
(Amano et al. 2002b). To confirm that sbGnRH is
involved in gonadal maturation in barfin flounder, it is
necessary to show that sbGnRH levels in the brain and
pituitary change during gonadal maturation.
ISH is useful to detect GnRH mRNA levels when certain GnRH neurons, such as sGnRH neurons in masu
salmon, are scattered in the brain. In the barfin flounder,
GnRH neurons were localized in certain brain regions;
sbGnRH, sGnRH, and cGnRH-II neurons were located in
the POA, in the ventromedial part of the rostral OB and
the TN, and in the MT, respectively (Amano et al. 2002a,
b). In this case, it is useful to measure GnRH mRNA levels by real-time quantitative PCR rather than by ISH.
Therefore, to clarify the physiological roles of the
respective GnRH forms during gonadal maturation of
the barfin flounder, changes in brain GnRH mRNA
levels were examined by real-time quantitative PCR. We
also measured GnRH peptide levels in the pituitary by
time-resolved fluoroimmunoassay (TR-FIA) for sGnRH,
cGnRH-II, and sbGnRH. We also examined the changes
in plasma levels of testosterone, E2, and DHP.
In male barfin flounder, GSI remained low until autumn of 1-year-olds and then rapidly increased in January
and fish spermiated in March (Fig. 23A). The amount
of sbGnRH mRNA per brain significantly increased in
January and remained at high levels in March (Fig. 23B).
The amounts of sGnRH and cGnRH-II mRNA per brain
did not show significant changes during the experimental periods (Figs. 23C, D). Pituitary sbGnRH peptide
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 21. Changes in (A) BW (g), (B) GSI (%), and (C) the number
of neurons expressing sGnRH mRNA in the OB, TN, VT and
POA during gonadal maturation of 2-year-old female masu
salmon (mean ± SEM). In each group, means with different letters indicate significant difference (P < 0.05). (B) and (C) are
reprinted from General and Comparative Endocrinology, 99,
Amano et al., Salmon GnRH synthesis in the preoptic area and
the ventral telencephalon is activated during gonadal maturation
in female masu salmon, 13–21, © 1995, Academic Press, Inc.,
with permission from Elsevier.
content significantly increased in March (Fig. 23E).
Pituitary sGnRH peptide and cGnRH-II peptide contents
were extremely low compared to sbGnRH peptide levels
and showed no significant changes during the experiment (data not shown). In female barfin flounder, GSI
remained low until August of 2-year-olds and increased
thereafter until April when the fish began to ovulate
(Fig. 24A). The sbGnRH mRNA levels per brain
increased significantly from August to April (Fig. 24B).
Pituitary sbGnRH peptide levels also increased significantly during this period (Fig. 24C). These results in
55
Fig. 22. Changes in plasma concentrations (ng/ml) of (A) LH,
(B) E2, (C) testosterone, and (D) DHP during gonadal maturation of 2-year-old female masu salmon (mean ± SEM). In each
group, means with different letters indicate significant difference (P < 0.05). Reprinted from General and Comparative
Endocrinology, 99, Amano et al., Salmon GnRH synthesis in
the preoptic area and the ventral telencephalon is activated during gonadal maturation in female masu salmon, 13–21, © 1995,
Academic Press, Inc., with permission from Elsevier.
both sexes indicate that sbGnRH is involved in gonadal
maturation in barfin flounder via the synthesis of steroid
hormones (Amano et al. 2004a, 2008).
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56
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 24. Changes in (A) GSI (%), (B) sbGnRH mRNA levels
(× 106 copies/brain), and (C) pituitary sbGnRH contents
(ng/pituitary) during gonadal maturation of female barfin
flounder (mean ± SEM). Means with differing letters differ
significantly (P < 0.05). Reprinted from General and Comparative Endocrinology, 158, Amano et al., Changes in brain
seabream GnRH mRNA and pituitary seabream GnRH peptide
levels during ovarian maturation in female barfin flounder,
168–172, © 2008, Elsevier Inc., with permission from Elsevier.
Fig. 23. Changes in (A) GSI (%), (B) sbGnRH mRNA
levels (× 107 copies/brain), (C) sGnRH mRNA levels (× 107
copies/brain), (D) cGnRH-II mRNA levels (× 107 copies/brain),
and (E) pituitary sbGnRH contents (ng) during gonadal maturation of male barfin flounder (mean ± SEM). Means with
differing letters differ significantly (P < 0.05). Reprinted
from Comparative Biochemistry and Physiology, Part B, 138,
Amano et al., Changes in brain GnRH mRNA and pituitary
GnRH peptide during testicular maturation in barfin flounder,
435–443, © 2004, Elsevier Inc., with permission from Elsevier.
6. Effects of photoperiod on the brain–pituitary–
gonadal axis in masu salmon and sockeye salmon
6-1. Stimulation of precocious maturation in male
masu salmon by short photoperiod treatment
It is well established that photoperiod is of primary importance in the induction of the initiation and modulation
of reproductive development in salmonid fish. For example, in Atlantic salmon parr, precocious male reared
under short photoperiod mature earlier than those reared
under long photoperiod (Lundqvist 1980). In landlocked
masu salmon “yamame”, rapid vitellogenesis is induced
by a short photoperiod, although maturation is initiated
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
under a long photoperiod (Takashima and Yamada 1984).
However, the endocrinological mechanisms of such phenomena were not fully understood.
In masu salmon reared under constant water temperature, sGnRH concentrations in the discrete brain areas
showed seasonal changes: high during autumn–winter
and low in summer. In addition, pituitary sGnRH contents showed a stepwise increase every summer for three
years (Amano et al. 1992). These results suggest that
synthesis of sGnRH in the brain increases under a short
photoperiod and sGnRH produced is transported from
cell bodies to the pituitary to induce gonadal maturation.
However, direct evidence is lacking. Therefore, the effects of the photoperiod on sGnRH mRNA levels in the
brain, GTH levels in the pituitary, and testicular maturation of 1-year-old male masu salmon were examined in
order to ascertain that short photoperiod induces increases
in sGnRH and GTH.
Underyearling male masu salmon were reared under a
short photoperiod (8L16D; lights on 0900–1700 h) and a
long photoperiod (16L8D; lights on 0400–2000 h) from
June through September. Fish were sampled in July, August, and October.
Changes in GSI are shown in Fig. 25A. Spermiation was observed in August in the 8L16D group and
in September in the 16L8D group, respectively. sGnRH
mRNA levels in the VT and the POA increased when
the fish spermiated; the activity increased in August in
the 8L16D group, and in September in the 16L8D group,
respectively (Figs. 25B, C). Moreover, the increase of
sGnRH mRNA levels was in accordance with the increase of pituitary FSHβ (Fig. 25D) and LHβ contents
(Fig. 25E). No significant changes in sGnRH mRNA levels in relation to gonadal maturation were observed in the
OB and the TN (data not shown). These results indicate
that sGnRH neurons in the VT and the POA are influenced by photoperiod, and are involved in the regulation
of gonadal maturation through GTH secretion (Amano et
al. 1995b).
It has been shown that sGnRH synthetic activity is also
considered to be influenced by gonadal steroids; sGnRH
mRNA levels in the POA were activated by oral 17α -MT
treatment in 1-year-old future precocious males (Amano
et al. 1994) and in 2-year-old female masu salmon, which
initiated ovarian development (Amano et al. 1997a).
These results suggest that sGnRH neurons in the VT and
the POA also respond to changes in steroid hormone
levels. From these results, it is suggested that sGnRH
synthetic activity is influenced by both photoperiod
and steroid hormones. To further assess the effects of
photoperiod on sGnRH synthetic activity, underyearling
precocious male masu salmon were castrated to remove
the effects of steroid hormones, and the changes in
sGnRH mRNA levels in the brain were examined by
manipulating the photoperiod.
In early August, underyearling male masu salmon
(mean BW 9.4 g; mean GSI 3.35%) were castrated
57
according to methods reported (Aida et al. 1984). Upon
recovery, the fish were divided into short photoperiod
(8L16D; lights on 0900–1700 h) and long photoperiod
(16L8D; lights on 0400–2000 h) groups. The fish were
sampled 30 days after the castration. Blood was collected
to measure plasma testosterone levels by a TR-FIA
(Yamada et al. 1997). For ISH, brains were fixed with
4% PFA and 1% picric acid in 50 mM PB (pH 7.3).
Plasma testosterone concentrations decreased compared to initial levels (1.3 ng/ml) and no significant
differences were seen between the groups (Fig. 26A),
indicating the validity of castration. The number of neurons expressing sGnRH mRNA in the POA was greater
in the 8L16D group than the 16L8D group (Fig. 26B).
No significant differences were observed in the VT
(Fig. 26C). These results indicate that sGnRH neurons
in the POA receive short photoperiodic signals and that
either short photoperiod or steroid hormone secretion
is required for the activation of sGnRH synthesis in
1-year-old precocious male masu salmon (Amano et
al. 1999).
6-2. Incomplete development of the brain–pituitary–gonadal axis in male sockeye salmon
It is well known that precocious maturation occurs in
male salmonid fishes including Atlantic salmon, amago
salmon Onvorhynchus rhodurus, masu salmon, and sockeye salmon. However, the age at which fish mature precociously differs according to species. For example, most
male masu salmon precociously mature in the first autumn, while precocious males first appear in the second
summer in sockeye salmon landlocked in Japanese lakes.
Although the cause of this species-specific difference in
the initiation of precocious maturation is of much interest,
fundamental mechanisms have not yet been elucidated.
Since gonadal maturation of teleost fish is regulated
by brain–pituitary–gonadal axis, there is a possibility
that the periodic difference in the appearance of precocious maturation between masu salmon and sockeye
salmon is due to differences in the development of the
brain–pituitary–gonadal axis. Indeed, precocious maturation of underyearling male masu salmon can be induced
by short photoperiodic treatment (Amano et al. 1995b).
Thus, effects of photoperiod on brain–pituitary–gonadal
axis of underyearling sockeye salmon were examined.
In June, underyearling sockeye salmon (mean BW
8.0 g) were divided into the short photoperiod (8L16D;
lights on 0900–1700 h) group and the long photoperiod
(16L8D; lights on 0400–2000 h) group. Fish were reared
until October.
No significant differences in GSI were observed between both groups throughout the experiment (Fig. 27A).
Fish were still immature in October. sGnRH contents in
the telencephalon including POA tended to be higher in
the 8L16D group than in the 16L8D group from late August (Fig. 27B). sGnRH contents in the hypothalamus
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
58
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 25. Changes in (A) GSI (%), (B) the
number of neurons expressing sGnRH mRNA
in the POA, (C) the number of neurons expressing sGnRH mRNA in the VT, (D) pituitary
FSH β contents (ng/pituitary), and (E) pituitary
LHβ contents (ng/pituitary) during gonadal
maturation of underyearling male masu salmon
reared under short and long photoperiods
(mean ± SEM). In each group, means with
differing letters differ significantly (P < 0.05).
***(P < 0.001), **(P < 0.01) and *(P < 0.05)
indicate statistical significance between the
groups. (A)–(C) are reprinted from General
and Comparative Endocrinology, 99, Amano et
al., Short photoperiod accelerates preoptic and
ventral telencephalic salmon GnRH synthesis
and precocious maturation in underyearling
male masu salmon, 22–27, © 1995, Academic
Press, Inc., with permission from Elsevier.
were significantly higher in the 8L16D group in September (Fig. 27C). sGnRH contents in the pituitary were
significantly higher in the 8L16D group in September and
October (Fig. 27D). Pituitary FSHβ contents were significantly higher in the 8L16D group in early and late August and October (Fig. 27E). In the 8L16D group, a positive correlation was observed between GSI and pituitary
FSH β content (correlation coefficient r = 0.8250, P <
0.001, n = 34). LHβ was undetectable in the pituitary in
all individuals. These results suggest that synthesis and
storage of sGnRH increased under the short photoperiod
and then sGnRH stimulates FSHβ synthesis. It is reported
that FSH is involved in the early phases of spermatogenesis in salmonid fishes (Kawauchi et al. 1989; Swanson et
al. 1989). Indeed, FSHβ was positively correlated with
GSI in the short photoperiod group. However, levels of
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 26. (A) Plasma testosterone (ng/ml), (B) the number of neurons expressing sGnRH mRNA in the POA, and (C) the number
of neurons expressing sGnRH mRNA in the VT of castrated precocious male masu salmon reared under short and long
photoperiods (mean ± SEM). **(P < 0.01) indicates statistical significance. Reprinted from General and Comparative
Endocrinology, 115, Amano et al., Effects of photoperiod on salmon
GnRH mRNA levels in brain of castrated underyearling precocious male masu salmon, 70–75, © 1999, Academic Press, with
permission from Elsevier.
pituitary LHβ were not increased. These results suggest
that synthesis and release of LH are required for gonadal
maturation. Furthermore, it is suggested that the development of the brain–pituitary–gonadal axis of underyearling
sockeye salmon is inactive. This species-specific difference in the development of the brain–pituitary–gonadal
axis may underlie differences in the initiation of precocious maturation (Amano et al. 1997b).
7. Steroid feedback on GnRH levels in the brain of
masu salmon and sockeye salmon
7-1. Effects of steroid hormone administration on
GnRH synthesis in masu salmon
Accumulation of GTH in the pituitary can be stimulated by aromatizable androgen or estrogen in juvenile
fish; this is a known positive feedback system (see Goos
1987; Okuzawa 2002). The involvement of GnRH in this
59
mechanism is speculated because sex steroid administration increased the amount of GnRH in the hypothalamus
of the European silver eel (Dufour et al. 1985) and rainbow trout (Goos et al. 1986). On the other hand, the
stimulatory effects of testosterone on the expression of
pituitary LHβ gene were demonstrated in vitro in juvenile rainbow trout (Xiong et al. 1993, 1994a, b). Thus,
whether steroids have direct actions on the pituitary, or
act indirectly via GnRH was unclear.
Therefore, the effects of 17α-MT on sGnRH mRNA
expression in 1-year-old masu salmon (mean BW 44 g)
were examined. Fish were fed pellets containing 17αMT 25 µg/g-diet (1.5% of the BW) for 41 days. The fish
used in this study consisted of future precocious males
which will mature in autumn of that year and of immature
females. Blood was sampled for determination of plasma
LHβ levels. Pituitary contents of FSHβ and LHβ, and
plasma LHβ level were measured by respective RIAs as
described in Section 4-1. Pituitary sGnRH was extracted
and measured by RIA (Okuzawa et al. 1990). Brains were
fixed for ISH.
Oral 17α-MT application markedly increased pituitary LHβ, but not FSHβ concentrations in both sexes
(Figs. 28A, B). In future precocious males, 17α -MT
treatment further increased the number of neurons
expressing sGnRH mRNA in the POA (Fig. 28C) but not
in the OB and the VT (Figs. 28D, E). However, sGnRH
mRNA levels were not changed by 17α -MT in immature
females (Figs. 28C, D, E). These results suggest that
sGnRH is involved in the positive feedback system at
least in future precocious males, and that the difference
in the responsiveness of preoptic sGnRH neurons to
17 α-MT is based on the maturational stage of the fish
(Amano et al. 1994).
Thus, whether sGnRH mRNA levels were influenced
by 17α-MT in 2-year-old females which are just before
the initiation of gonadal maturation was further examined. 1-Year-old and 2-year-old masu salmon were randomly selected from a stock in May. At the start of the
experiment, mean BW was 29 and 94 g, and mean GSI
was 0.33 and 0.53%, for 1-year-old and 2-year-old fish,
respectively. Fish were divided into the control group and
17 α-MT 25 µg/g-diet treated group. The 17 α-MTtreated group was fed pellets containing 17α-MT (1.5%
of the BW) for 31 days. Brains were fixed for ISH.
The number of neurons expressing sGnRH mRNA
in the POA increased by 17α -MT in 2-year-old female
masu salmon, not in 1-year-old female masu salmon
(Fig. 29A). No significant difference was seen in the
number of neurons expressing sGnRH mRNA in the VT
by 17 α-MT treatment in both ages (Fig. 29B). These
results support the previous hypothesis that the difference in the responsiveness of preoptic sGnRH neurons
to 17α -MT is based on the maturational stage of the
fish (Amano et al. 1997a). Of note, studies on pubertal
development have been conducted in a number of teleost
fish species such as the rainbow trout (Gielen et al. 1982;
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60
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 27. Changes in (A) GSI (%), (B) sGnRH
contents (pg) in the telencephalon including POA,
(C) sGnRH contents (pg) in the hypothalamus,
(D) sGnRH contents (pg) in the pituitary, and
(E) pituitary FSHβ contents (ng) during gonadal
maturation of male sockeye salmon reared under
short and long photoperiods (mean ± SEM). In
each group, means with differing letters differ significantly (P < 0.05). **(P < 0.01) and *(P < 0.05)
indicate statistical significance between the groups.
Reprinted with permission from Fisheries Science,
63, Amano et al., Incomplete development of the
brain–pituitary–gonadal axis may underlie the
delay in the initiation of precocious maturation in
male sockeye salmon, 873–876, Figs. 2–4, © 1997,
the Japanesel Society of Fisheries Science.
Goos et al. 1986), the eel (Dufour et al. 1985; Counis
et al. 1987), the platyfish (Schreibman et al. 1986), and
African catfish (Schulz et al. 1994a, b). These studies
suggest that sex steroids initiate and/or accelerate the
development of the brain–pituitary–gonadal axis. Taken
together, the response of preoptic sGnRH neurons to
steroids may be a necessary condition for gonadal maturation or the development of the brain–pituitary–gonadal
axis (Amano et al. 1997a).
7-2. Low GnRH levels in the brain and pituitary in
triploid female sockeye salmon
Triploid female fish are known to be sterile. One
possible explanation is that oocytes cannot undergo
normal meiotic division to produce euploid gamates.
Another is that triploid fish have a defective reproductive
endocrine system, which regulates gonadal development.
It is well established that positive feedback system of sex
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
61
Fig. 29. Changes in the number of neurons expressing sGnRH
mRNA in (A) the POA and (B) VT of 1-year-old and
2-year-old female masu salmon of control and 17α-MT-treated
groups (mean ± SEM). *(P < 0.05) indicates statistical significance. With kind permission from Springer Science+Business
Media: Fish Physiology and Biochemistry, The maturation of
the salmon GnRH system and its regulation by gonadal steroids
in masu salmon, 17, 1997, 63–70, Amano et al., Fig. 2, © 1997,
Kluwer Academic Publishers.
Fig. 28. (A) Pituitary FSHβ contents (ng), (B) pituitary LHβ
contents (ng), the number of neurons expressing sGnRH mRNA
in (C) the POA, (D) VT and (E) OB of masu salmon of control
and 17α-MT-treated groups (mean ± SEM). ***(P < 0.001) and
**(P < 0.01) indicate statistical significance. Reprinted from
General and Comparative Endocrinology, 95, Amano et al.,
Activation of salmon gonadotropin-releasing hormone synthesis by 17α-methyltestosterone administration in yearling masu
salmon, Oncorhynchus masou, 374–380, © 1994, Academic
Press, with permission from Elsevier.
steroids exists in the brain–pituitary–gonadal system in
juvenile fish (Okuzawa 2002). There are some studies on
plasma levels of steroid hormones in triploid salmonids
and it has been clarified that triploid fish have low levels
of steroid hormones. Triploid females are known to have
much lower E2 levels than those of maturing diploid of
the same age in rainbow trout (Lincoln and Scott 1984;
Nakamura et al. 1987; Benfey et al. 1989a), pink salmon
Oncorhynchus gorbuscha (Benfey et al. 1989a), coho
salmon (Benfey et al. 1989b), and ayu Plecoglossus
altivelis (Iguchi et al. 1991). Thus, the triploid female
fish is considered to be a good model for examining the
feedback system in the context of low steroid hormone
levels. No information is available on brain and pituitary
GnRH contents of triploid salmonid fish, whereas it has
been reported that triploid salmonids have low plasma
LH concentration and low pituitary LH content (Benfey
et al. 1989b). Therefore, levels of sGnRH in the brain
and pituitary of 1-year-old diploid and triploid female
sockeye salmon were compared.
Eggs of sockeye salmon were artificially fertilized in
September, and triploidy was induced by heat shock treatment (30°C, 10 min). Diploid fish were also obtained
from artificially fertilized eggs of the same stock used for
triploids. Fish were reared under natural photoperiod using natural spring water at constant temperature (9–10°C).
Sampling was conducted at 22 months later.
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62
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 30. Levels of (A) GSI (%), (B) plasma testosterone (ng/ml), (C) plasma LH (ng/ml), and (D) pituitary LH content (ng) of diploid
and triploid sockeye salmon (mean ± SEM). **(P < 0.01) and *(P < 0.05) indicate statistical significance. Data was derived with
permission from Fisheries Science, 64, Amano et al., Low GnRH levels in the brain and the pituitary in triploid female sockeye
salmon, 340–341, Table 1, © 1998, the Japanesel Society of Fisheries Science.
Triploid fish (mean BW 76 g) had significantly lower
GSI than did diploid fish (mean BW 63 g) (Fig. 30A).
Diploids had oocytes in the early and late perinucleolus
stage, and in some fish, oocytes were in the oil droplet
stage, whereas triploids had only oogonia and lacked follicle cells (data not shown). Plasma levels of E2 were
below detectable limit (0.03 ng/ml) in both diploid and
triploid fish. Plasma levels of testosterone were significantly lower in triploids (Fig. 30B). Plasma LH concentration of diploid fish was 2.24 ng/ml, whereas this
was below the detectable limit (0.88 ng/ml) in triploid
fish (Fig. 30C). Although no significant differences in
pituitary LH contents were observed, they tended to be
lower in triploid fish (Fig. 30D). Triploid had significantly lower sGnRH concentrations in the telencephalon
including POA, hypothalamus, and pituitary, whereas no
significant differences of sGnRH concentration were seen
in the other parts of the brain (Fig. 31). These results suggest that the low sGnRH concentrations cause low plasma
LH and steroid hormone levels in triploid fish (Amano
et al. 1998a). The reason for the low brain sGnRH concentrations is not known; it is not likely to be due to
active release of sGnRH in the pituitary but to be due
to low sGnRH synthesis in the brain, since plasma LH
levels were lower in triploid fish. Low testosterone levels caused by low sGnRH and LH production or by incomplete development of ovarian follicular cells may not
stimulate further production of sGnRH in the triploid fish
brain.
8. Ontogenic development of GnRH neurons
8-1. Ontogenic development of GnRH neurons
in masu salmon
At present, three GnRH neuronal systems are proposed in teleost fish judging from the location of GnRH
neuronal somata and their projections (Oka 1997);
the TN-GnRH system, the MT-GnRH system, and
Fig. 31. Levels of sGnRH concentrations (pg/mg tissue) in
diploid and triploid sockeye salmon (mean ± SEM). The letters
a–g represent the following brain areas: a, olfactory bulb;
b, telencephalon including preoptic area; c, hypothalamus;
d, optic tectum-thalamus including midbrain; e, cerebellum;
f, medulla oblongata; g, pituitary. ***(P < 0.001) and
*(P < 0.05) indicate statistical significance. Reprinted with
permission from Fisheries Science, 64, Amano et al., Low
GnRH levels in the brain and the pituitary in triploid female
sockeye salmon, 340–341, Fig. 1A, © 1998, the Japanese
Society of Fisheries Science.
the POA-GnRH system (in some species POA-GnRH
neurons do not form a well-defined cell cluster and, in
this monograph, the author defines the ventral forebrainGnRH system excluding the TN-GnRH system as the
POA-GnRH system). The MT-GnRH system is the
most conserved among the vertebrate species; it produces
cGnRH-II and is present in all the teleost fish examined to
date. Among the three GnRH systems, the POA-GnRH
system is considered to regulate GTH secretion because
it is the main system that projects directly to the pituitary
(Oka 1997). Perciform fish have three forms of GnRH,
sGnRH, cGnRH-II and sbGnRH. With very few exceptions such as European sea bass Dicentrarchus labrax
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
(González-Martínez et al. 2001), the TN-GnRH system
and the POA-GnRH system are clearly distinguishable
and the GnRH forms produced in these two systems are
different; sGnRH is produced in the TN and sbGnRH
in the POA. The medaka Oryzias latipes has sGnRH,
cGnRH-II and medaka GnRH (mdGnRH), expressed in
the TN, the MT, and the POA, respectively (Okubo et
al. 2000). In contrast, in the other teleost species that
have two forms of GnRH in the brain (e.g., salmonid
fishes, eel, catfish), clear anatomical identification of the
TN-GnRH system and POA-GnRH system is difficult,
because the GnRH neurons located in the ventral forebrain are consecutive and the GnRH form(s) produced in
these neurons are the same; sGnRH in salmonid fishes,
mGnRH in the eel and cfGnRH in the catfish (Okuzawa
and Kobayashi 1999).
Studies of ontogeny of different GnRH systems are
considered to be helpful in understanding the functional role of each GnRH system. The ontogeny of the
GnRH system was first studied by IHC in mammalian
(Schwanzel-Fukuda and Pfaff 1989; Wray et al. 1989).
It was later reported in amphibian (Muske and Moore
1990; Murakami et al. 1992) and avian brains (Murakami
et al. 1991). Results indicate that GnRH neurons are
derived from the olfactory placode (OP) and migrate into
the forebrain during prenatal development. In teleost
fish, it has been reported by IHC in chum salmon (Chiba
et al. 1994) and sockeye salmon (Parhar et al. 1995) that
GnRH (probably sGnRH) neurons originate from the OP
and then migrate into the brain along the ON. However,
little is known about the differential ontogeny of sGnRH
and cGnRH-II systems in the brain of salmonid fish.
Thus, the ontogenic development of sGnRH and cGnRHII systems in the brain of masu salmon was examined by
IHC and ISH.
Masu salmon eggs were artificially fertilized and were
reared under natural photoperiod in spring water of constant temperature (9–10°C). Fish were sampled at embryonic stage, alevin stage (still have yolk sacks and stay
at the bottom of the tank), and fry stage (initiate feeding
and swimming) (Kubo 1980). The procedure for IHC and
ISH were basically similar to that described in Amano et
al. (1991, 1994).
Distribution of sGnRH neurons in the brain is summarized in Fig. 32. sGnRH-ir fibers were initially detected by IHC in the vicinity of the OP of the embryo
36 days after fertilization (Day 36) (Figs. 33A, B), and
were distributed widely in the brain as well as the pituitary of alevin just after hatching (Day 80) (Fig. 33C).
sGnRH-ir cell bodies were first detected about six months
after fertilization in the rostroventral brain area ranging
from the ON to the POA. sGnRH neuronal somata were
detected earlier by ISH than IHC. Neuronal somata expressing sGnRH mRNA were first detected in the vicinity of the olfactory epithelium (OE) in embryo on Day
40 (Fig. 33D), and then were seen to be migrating from
63
Fig. 32. Microprojection drawing of a series of sagittal sections
through whole heads of masu salmon on days (A) 40, (B) 60,
(C) 80, and (D) 152 showing the distribution of neuronal somata expressing sGnRH (solid circle). F forebrain, OB olfactory bulb, OE olfactory epithelium, ON olfactory nerve, POA
preoptic area, T telencephalon, VT ventral telencephalon. Bar
indicates 0.5 mm. With kind permission from Springer Science+Business Media: Cell and Tissue Research, Ontogenic development of salmon GnRH and chicken GnRH-II systems in
the brain of masu salmon, 293, 1998, 427–434, Amano et al.,
Fig. 4, © 1998, Springer-Verlag.
the OE, along the ON of alevin (Day 60) (Fig. 33E), and
in the transitional areas between the ON and the OB of
alevin (Day 80) (Fig. 33F). In the brain, these neurons
were first detected in the ventral OB on Day 80, and thereafter they were detected also in the caudal brain regions
(data not shown). cGnRH-II system was detected later
than sGnRH system; cGnRH-II-ir fibers were first detected of alevin (Day 67) (data not shown). cGnRH-II-ir
neuronal somata were not detected during the present observation period. These results suggest that sGnRH neurons derive from the OP and then migrate into the brain,
and that sGnRH is synthesized first and cGnRH-II later
(Amano et al. 1998b).
Among sGnRH neurons distributed from the ON
through the POA, those in the VT and the POA are
indicated to regulate GTH secretion. It is of interest
whether all the sGnRH neurons originate from the OE
or not. Thus, whether sGnRH neurons are present in the
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64
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 33. (A) Sagittal section through the OP of masu
salmon embryo on Day 36. (B) Higher magnification
of “A”. sGnRH-ir fibers (arrowheads) are observed.
(C) Sagittal section through the pituitary of masu
salmon just after hatching (Day 80). Arrowhead
indicates sGnRH-ir fiber. (D) Sagittal section of masu
salmon embryo on Day 40 showing neuronal somata
expressing sGnRH mRNA in the vicinity of the OE.
This figure corresponds to Fig. 32A. (E) Sagittal section through a fish at Day 60 showing neuronal somata
expressing sGnRH mRNA as a few small clusters,
emerging from the OE, along the ON. This figure
corresponds to Fig. 32B. (F) Sagittal section through
the ON of masu salmon on Day 80 showing neuronal
somata expressing sGnRH mRNA at transitional
area between ON and OB. This figure corresponds to
Fig. 32C. Bar indicates 20 µm. With kind permission
from Springer Science+Business Media: Cell and
Tissue Research, Ontogenic development of salmon
GnRH and chicken GnRH-II systems in the brain of
masu salmon, 293, 1998, 427–434, Amano et al.,
Figs. 2, 3, © 1998, Springer-Verlag.
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M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
VT-POA of fish whose OE including sGnRH clusters
were cauterized just after hatching, was examined by
ISH.
Just hatched larvae (Day 44) were collected and were
anesthetized. For the olfactory epithelium lesioned
(OEL) group, both nares including sGnRH clusters were
cauterized using an extra fine hot needle attached to a
handy type soldering iron for about 2 seconds under a
binocular microscope. Then, larvae were revived under
aerated running water. For the control group, fish were
also anesthetized and were put on the desk for about
30 seconds and then revived in aerated running water.
Fish were sampled 7 months after the operation.
Neurons expressing sGnRH mRNA were detected in
the ON, ventral OB and TN in all fish of the control group
(9 fish). In the OEL group (12 fish), however, a few neurons expressing sGnRH mRNA were detected in these regions in 3 fish. The rate of appearance of neurons expressing sGnRH mRNA in the ON-TN was significantly
lower in the OEL group than the control group. Neurons expressing sGnRH mRNA were detected in the VT
and the POA in 5 fish of the control group. However,
neurons expressing sGnRH mRNA were not detected in
these regions in any of the OEL group fish. The rate of
appearance of neurons expressing sGnRH mRNA in the
VT-POA was significantly lower in the OEL group than
the control group. Pituitary sGnRH content in the OEL
group was just above the detectable limit (1.85 pg) and
was significantly lower than the corresponding control
value both in males and in females (Fig. 34). These results indicate that sGnRH neurons in the VT-POA are derived from the OE in masu salmon, although the possibility cannot be ruled out that sGnRH neurons in the
VT-POA arise from the VT-POA but were delayed in expressing sGnRH because of the trauma of cauterization
(Amano et al. 2002c).
8-2. Ontogenic development of GnRH neurons in
barfin flounder
The barfin flounder experiences a metamorphosis
early in development. Barfin flounder expresses sGnRH,
cGnRH-II, and sbGnRH (Amano et al. 2002a). In the
barfin flounder, the TN-GnRH system, the MT-GnRH
system and the POA-GnRH system are clearly distinguishable, as in perciform fish and medaka. sbGnRH
is physiologically the most important hypophysiotropic
factor for the reproduction of the barfin flounder (Amano
et al. 2002a, b, 2004a, 2008).
To elucidate the ontogenic origin of the neurons that
produce these GnRH molecules, the development of three
GnRH systems was examined by IHC and ISH. Barfin
flounder larvae used are shown in Fig. 35. Distribution
of GnRH neurons in the brain is summarized in Fig. 36.
cGnRH-II mRNA-expressing neuronal somata were
first identified in the MT near the ventricle on Day 7
65
Fig. 34. Pituitary sGnRH content (pg) in male and female masu
salmon of the control and OEL groups. ***(P < 0.001) and
**(P < 0.01) indicate the level of statistical difference between
the two groups. Reprinted from General and Comparative Endocrinology, 127, Amano et al., Ontogenic origin of salmon
GnRH neurons in the ventral telencephalon and the preoptic area
in masu salmon, 256–262, © 2002, Elsevier Science (USA),
with permission from Elsevier.
(Fig. 37A). cGnRH-II-ir fibers were first found in the
brain on Day 7 (data not shown). Neuronal somata that
express sGnRH mRNA were detected first in the vicinity
of the OE on Day 21 (Fig. 37B), and then in the transitional area between the ON and OB (Fig. 37C) and the
TN on Day 28 (Fig. 37D). sbGnRH mRNA-expressing
neuronal somata were first detected in the POA on Day
42 (Fig. 37E). sbGnRH-ir fibers were localized in the
POA-hypothalamus and formed a distinctive bundle
of axons projecting to the pituitary on Day 70 (data
not shown). These results indicate that three forms of
GnRH neurons have separate embryonic origins in the
barfin flounder as in other perciform fish such as tilapia
Oreochromis niloticus and red seabream (Amano et
al. 2004b). In contrast, it has been reported that sbGnRH
neurons derive from the olfactory placode and migrate
into the brain in African cichlid (White and Fernald
1998) and European sea bass (González-Martínez et
al. 2002). Although the possibility cannot be ruled
out that sbGnRH neurons migrate without expressing
sbGnRH in the barfin flounder, the origin of sbGnRH
neurons (POA-GnRH system) may differ according to
the fish species.
9. Conclusion and future direc tions
Currently, 15 forms of GnRH molecules have been
characterized throughout the vertebrates and the existence of multiple forms of GnRH in the brain was
also observed in teleost fish. A salmonid fish, masu
salmon, has sGnRH and cGnRH-II, and a pleuronectiform fish, barfin flounder, has sGnRH, cGnRH-II
and sbGnRH. In masu salmon, sGnRH neurons are
scattered from the ON and the TN through the VT
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
66
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 35. Barfin flounder larvae of (A) 7 days after hatching (yolk-sack larva), (B) 21 days after hatching (flexion larva), (C) 28 days
after hatching (postflexion larva, onset of metamorphosis), and (D) 42 days after hatching (postflexion larva, late metamorphosis).
Bars indicate 5 mm.
and the POA. sGnRH neurons in the VT and the POA
are considered to be involved in gonadal maturation
via stimulation of GTH secretion. The other sGnRH
neurons and cGnRH-II neurons are suggested to have a
neuromodulatory function in the brain. In barfin flounder, sbGnRH neurons, which are located in the POA,
are considered to be involved in gonadal maturation,
and sGnRH and cGnRH-II are suggested to function
as a neuromodulator in the brain. It has recently been
reported that cGnRH-II modifies food intake in the musk
shrew Suncus murinus (Kauffman and Rissman 2004);
cGnRH-II administration in the brain reduced 24-h
ad libitum food intake. Supposing that melaninconcentrating hormone (MCH) also stimulates food
intake in the musk shrew, it is reasonable to speculate
that MCH and cGnRH-II interact in the brain and
regulate food intake. Indeed, in the barfin flounder, it
is indicated that cGnRH-II stimulates food intake by
interacting with MCH; food intake regulation might be
a novel function of cGnRH-II in teleost fish (Amiya et
al. 2008). Furthermore, interaction between GnRH and
other neuropeptide (e.g., neuropeptide Y, orexin) should
be examined in detail.
It has been clarified that three GnRH systems exist
in the barfin flounder, judging from the location of cell
bodies and their projections; the TN-, the MT-, and the
POA-GnRH systems. However, in masu salmon, clear
anatomical identification of the TN- and the POA-GnRH
system is difficult because the GnRH neurons located in
the ventral forebrain are consecutive and the GnRH form
produced in these neurons is the same (sGnRH). Thus,
it is suggested in masu salmon that sGnRH neurons are
derived from the OE, migrate into the brain, and play different roles according to the location in the brain.
Very recently, the KiSS1/GPR54 system was discovered in mammals (Ohtaki et al. 2001; de Roux et
al. 2003; Seminara et al. 2003). It is now confirmed
that KiSS1/GPR54 signaling is central to the regulation
of GnRH and, consequently, FSH and LH secretion
(Zohar et al. 2010). In teleost fish, the identification and
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
Fig. 36. Microprojection drawing of a series of sagittal sections
through whole head of barfin flounder larvae on (A) Days 7,
(B) 21, (C) 28, and (D) 42, showing the distribution of neuronal
somata expressing sGnRH (blue circle), cGnRH-II (red circle)
and sbGnRH (yellow circle); MT midbrain tegmentum, OB olfactory bulb, OE olfactory epithelium, ON olfactory nerve, OT
optic tectum, POA preoptic area, T telencephalon. Bars indicate 0.5 mm. Reprinted with permission from Zoological Science, 21, Amano et al., Ontogenic development of three GnRH
systems in the brain of a pleuronectiform fish, barfin flounder,
311–317, Figs. 1A, C, D, E, © 2004, Zoological Society of
Japan.
characterization of a KiSS1 gene has been reported in
zebrafish Danio rerio (van Aerle et al. 2008) and medaka
(Kanda et al. 2008). In future, the relationship between
GnRH system and the KiSS1/GPR54 system in teleost
fish should be clarified to understand the reproductive
biology thoroughly.
Acknowledgments
I thank Professor Katsumi Aida for giving me the opportunity
to write this monograph. I also extend my thanks to the following persons for their contributions to the original research:
Prof. Makito Kobayashi, Dr. Koichi Okuzawa, Dr. Naoto
Okumoto, Dr. Shoji Kitamura, Dr. Kazumasa Ikuta,
Prof. Yoshihisa Hasegawa, Prof. Yoshitaka Oka, Prof. Hiroshi
Kawauchi, Prof. Kunio Yamamori, Prof. Akiyoshi Takahashi,
Prof. Akihisa Urano, Dr. Susumu Hyodo, Dr. Masayuki Iigo,
67
Fig. 37. (A) Sagittal section through the brain of barfin flounder on Day 7. Neuronal somata expressing cGnRH-II mRNA
are observed in the MT near the ventricle (single arrowheads).
(B) Sagittal section through the OE of barfin flounder on Day
21. Neuronal somata expressing sGnRH mRNA are observed
in the vicinity of the OE (single arrowhead). (C) Sagittal section through the ON and OB of barfin flounder on Day 28. Neuronal somata expressing sGnRH mRNA are observed in the TN
(single arrowheads). (D) Sagittal section through the terminal
nerve ganglion of barfin flounder on Day 28. Neuronal somata
expressing sGnRH mRNA are observed in the TN (single arrowheads). (E) Sagittal section through the POA of barfin flounder
on Day 42. Neurons expressing sbGnRH mRNA are observed
(single arrowheads). Bars indicates 20 µm. Reprinted with permission from Zoological Science, 21, Amano et al., Ontogenic
development of three GnRH systems in the brain of a pleuronectiform fish, barfin flounder, 311–317, Figs. 3B, 2B, 2D, 2F, 4B,
© 2004, Zoological Society of Japan.
Dr. Arimune Munakata, Dr. Kataaki Okubo, Mr. Takeshi
Yamanome, and Dr. Noriko Amiya.
doi:10.5047/absm.2010.00302.0039 © 2010 TERRAPUB, Tokyo. All rights reserved.
68
M. Amano / Aqua-BioSci. Monogr. 3: 39–72, 2010
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