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First published online December 16, 2008
Journal of Experimental Biology 212, 56-70 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.021352
Activity of the pituitary–gonadal axis is increased prior to the onset of spawning migration of chum salmon
1 Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu
University, Fukuoka 812-8581, Japan
2 Section of Biological Sciences, Graduate School of Life Sciences, Hokkaido
University, Sapporo 060-0810, Japan
3 National Salmon Resources Center, Fisheries Research Agency, Sapporo 062-0922,
Japan
4 School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA
98195, USA
5 Northwest Fisheries Science Center, NOAA Fisheries, Seattle, WA 99164,
USA
6 Hokkaido National Fisheries Research Institute, Fisheries Research Agency,
Kushiro 085-0802, Japan
* Author for correspondence (e-mail: takeshikiai{at}msn.com)
Accepted 14 October 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: spawning migration, salmon, follicle-stimulating hormone, luteinizing hormone, steroid hormone, pituitary–gonadal axis
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), and consists of several phases: initiation of the homing
behavior, approach to the coast, adaptation to fresh water (FW), and upstream
migration (Urano et al.,
1999). The hypothesis for the route of oceanic migration of chum
salmon indicates that winter chum salmon of the Japanese population leave the
Gulf of Alaska for the Bering Sea in spring
(Urawa, 2000). In summer
before the commencement of spawning migration, chum salmon originating in
Japan form mixed populations with stocks from other geographical areas of the
Pacific Rim in the Bering Sea, although Japanese populations are distributed
mainly in the central Bering Sea (Moriya
et al., 2007; Sato et al.,
2007). Homing adults, which leave the Bering Sea in early summer,
require two to three months to reach their natal rivers in Japan
(Tanaka et al., 2005). Then,
they adapt to FW and complete their final maturation during upstream migration
in autumn (Makino et al.,
2007).
Gonadal maturation of salmonids is regulated primarily by pituitary
gonadotropins (GTHs), i.e. follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), and sex steroid hormones such as testosterone (T),
11-ketotestosterone (11KT), estradiol (E2) and
17
-20β-dihydroxy-4-pregnen-3-one (DHP)
(Swanson et al., 2003). It is
also true in artificially reared masu salmon that served as a fish model to
understand seasonal changes in the pituitary–gonadal axis (PG axis) of
Japanese chum salmon (Kitahashi et al.,
2004; Jodo et al.,
2005). The absolute amounts of mRNAs encoding GTH subunits, i.e.
glycoprotein (GP) 2, FSHβ and LHβ, in the pituitary started
to be elevated during gametogenesis from spring through early summer
(Kitahashi et al., 2004). The
plasma levels of T, 11KT, and E2 were elevated during the same period
(Jodo et al., 2005).
Afterward, they peaked during the spawning period in autumn, followed by an
increase in the plasma levels of DHP and final maturation
(Jodo et al., 2005).
Therefore, we hypothesized that activity of the PG axis of oceanic chum salmon
changes similarly during their spawning migration.
In homing salmon, activity of the PG axis increased during the final phases
of the spawning migration. The amount of LHβ mRNA in the pituitary of
chum salmon has been shown to increase during upstream migration in the long
Ishikari River (Kitahashi et al.,
1998; Onuma et al.,
2005) and the short Otsuchi River
(Onuma et al., 2003b). The
plasma levels of GTHs also increase during upstream migration of pre-spawning
chum salmon (Ueda et al.,
1984), pink salmon (Dye et
al., 1986) and sockeye salmon
(Truscott et al., 1986),
although these studies detected mainly LH because of a lack of antisera that
specifically distinguishes salmon FSH from LH. The plasma levels of T, 11KT
and E2 in pre-spawning chum salmon peak during upstream migration from the
coast until the fish reach the midway to the upriver, then there is an
increase in the plasma levels of DHP and final maturation when the fish
arrived at the natal hatchery (Onuma et
al., 2003a). However, little information is available regarding
the activity of the PG axis during the period when chum salmon are migrating
in the Gulf of Alaska and also during the phase when fish initiate their
homing behavior from the Bering Sea. Understanding of the endocrine
mechanisms, which govern the spawning migration, requires comprehensive
information on changes in activity of the PG axis during the above-mentioned
behavioral phases.
In the present study, we tried to clarify changes in the activity of the PG
axis in chum salmon of Japanese populations at several locations along the
presumed pathway of their spawning migration. Pre-migratory fish were sampled
in the central Bering Sea in summer and autumn and the Gulf of Alaska in
winter (Fig. 1A). Japanese
populations were identified on the basis of mitochondrial DNA (mtDNA)
haplotypes that were determined with DNA microarray
(Moriya et al., 2004). In
autumn, homing adults were collected along their migratory pathway in the
Ishikari River–Ishikari Bay system in Hokkaido, Japan. Sampling sites
includes coastal areas, the estuary, midway and the upstream in the river, and
at or near the natal hatchery (Fig.
1B). The activity of the PG axis was assessed from the absolute
amounts of GP2, FSHβ and LHβ mRNAs, FSH and LH contents in
the pituitary, and plasma levels of GTHs, T, 11KT, E2 and DHP. We here report
increase in the activity of the PG axis prior to the initiation of spawning
migration from the Bering Sea.
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|
MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Sato et al., 2007). Summer fish in the central Bering Sea were fished by surface longline during cruises of the research vessel (RV) Wakatake-maru from late June to mid-July (Fig. 1A). The longline was set 30 min before sunset, and hauled 30 min after the sunset. After retrieval from the longline, salmon were transferred into a flow-through seawater (SW) tank, and actively swimming fish were sampled. A maximum of 10 individuals were sampled at each station. At the same stations, a series of gillnets was set in the evening and hauled in the morning. Chum salmon caught by the gill nets were used to obtain sufficient individuals for clarification of the proportion of immature and maturing fish from frequency distributions of the gonadosomatic index (GSI; gonad mass/body massx100). In September, fish were caught with a 50x50 m trawl net towed at the surface at 5 knots for 1 h usually starting at 9:00 h. Trawl operations were conducted in the Bering Sea by RV Kaiyo-maru of the Fisheries Agency of Japan in 2002 and 2003 (Fig. 1A). In 2001, fish on the presumed homing route to Japan were sampled using gill nets during a cruise of the RV Hokusei-maru of Hokkaido University from 39°N to 44° N in the northwest (NW) Pacific Ocean (Fig. 1A).
Winter chum salmon in the Gulf of Alaska were also caught by a 50x50 m trawl net towed at 5 knots for 1 h during the cruise of RV Kaiyo-maru that was operated under the auspices of the North Pacific Anadromous Fish Commission, in February, 2006. Live chum salmon were selected, and tissue and blood samples were collected.
We collected data from the Bering chum salmon for three years to confirm
that reproducible or typical data were obtained from ocean-migrating chum
salmon, because our previous endocrine analyses of homing chum salmon showed
considerable year-to-year variations
(Onuma et al., 2003a;
Onuma et al., 2003b;
Onuma et al., 2005). We
determined plasma levels of Cl– and cortisol in all fish used
in the present study and confirmed that the levels were not different between
fish captured by the trawl and longline gear (data not shown).
Homing chum salmon
Homing adults were captured along their migratory route in the Ishikari
River–Ishikari Bay system (Fig.
1B) to clarify changes in the activity of the PG axis during
upstream migration. In this river, about 30 million juvenile chum salmon are
released every year from the hatchery at the Chitose Field Station, hereafter
referred to as the hatchery, which is located on a tributary of the Ishikari
River (G in Fig. 1B). The
number of naturally spawning chum salmon is much lower than the
hatchery-released chum salmon, and more than 95% of hatchery-released fish
around the Ishikari Bay are from the Ishikari River. Thus, almost all fish
caught in the Ishikari River–Ishikari Bay water system can be regarded
as those released from the hatchery. The distance from the mouth of the
Ishikari River to the Chitose Field Station is about 70 km. Analyses of data
loggers attached to homing adults in the Ishikari Bay showed that fish spent a
few weeks to reach the hatchery from the mouth of the Ishikari River
(Kitahashi et al., 2000;
Makino et al., 2007).
In mid-September, fish were caught at Esashi on the NE coast of Hokkaido facing the Sea of Okhotsk (A in Fig. 1B) because tagging analyses of homing chum salmon showed a considerable portion of fish caught here migrated to the Ishikari Bay within a week. In addition, fish were sampled at six locations along the homing pathway from the Ishikari Bay to the hatchery from late September to mid-October, when homing chum salmon were abundant in the Ishikari River. Fish in SW areas were captured in the Ishikari Bay using set-nets commercially operated at Atsuta (B in Fig. 1B), 20 km from the river mouth, and Ishikari (C), 1.5 km from the river mouth. Fish in the Ishikari River estuary were captured by a gill net at Honmachi (D), the area between the river mouth and a wharf 2.5 km upstream from the river mouth. Fish midway in the FW were captured by a gill net at Ebetsu (E) at the junction of the mainstream and the Chitose River. Homing adults near the hatchery were captured by the fish wheel at Hanazono (F) 4 km downstream of the hatchery (G). Fish captured at Hanazono were transported to the hatchery and kept in a rearing pond at 12°C under natural photoperiod for about a week until fully mature. Fully matured adults were sampled in the hatchery.
Blood and tissue sampling
All sampling procedures were conducted under the animal care guidelines of
Hokkaido University. Following a blow to the head, fork length and body mass
of the fish were measured (Table
1), and blood samples were collected from the caudal vasculature.
Blood samples were temporarily kept on ice, centrifuged at 1870
g for 15 min to obtain the plasma, and stored at
–30°C until assays of hormones were conducted. A portion of the
blood clot was stored at –30°C for later extraction of DNA to
determine the mtDNA haplotypes. Pituitaries were collected and frozen in
liquid nitrogen and stored at –80°C until total RNA could be
extracted. In 2002 and 2003, the pituitaries were halved using a razor blade
along the mid-sagittal plane and stored in two different tubes to determine
the amount of GTH subunit mRNAs and GTHs from the same individuals.
Spermiation and ovulation of homing adults were checked by gentle massage of
the abdomen. Gonads were removed and weighed to calculate the GSI. A portion
of the testes and ovaries were fixed in Bouin's solution for 1 day, then
replaced with 70% ethanol and stored at 4°C for later histological
analyses. Fish age was determined from the number of annuli in their scales,
as previously reported (Davis et al.,
1990).
|
Classification of immature fish and maturing adults
Pre-migratory fish were classified as immature or maturing adults by
histological diagnosis of gonads. The testes were dehydrated in a graded
series of ethanol, embedded in Paraplast, sectioned at 10µm, and stained
with Delafield's Hematoxylin and Eosin. The ovaries of immature and early
maturing fish before initiation of vitellogenesis were sectioned as described
above, whereas ovaries in vitellogenesis were halved with a fine razor to
assess histological aspects, as previously described
(Onuma et al., 2003a). Stages
of gametogenesis were determined according to Campbell et al.
(Campbell et al., 2003;
Campbell et al., 2006). Male
chum salmon whose testes were mostly composed of primary A spermatogonia were
regarded as immature. Males with testes containing germ cells of late B
spermatogonia and spermatocytes were designated as maturing adult I fish.
Males with testes containing spermatozoon were designated as maturing adult II
fish. Female chum salmon whose ovaries contained oocytes with little yolk
globules were designated as immature fish. Females with ovaries containing
centrally located yolk globules were designated as maturing adult I fish.
Females with ovaries whose yolk globules were in the peripheral regions of
oocytes were designated as maturing adult II fish.
|
). An oligonucleotide microarray that detects these
SNPs was developed for rapid identification of these chum salmon haplotypes
(Moriya et al., 2004). In the
present study, the mtDNA haplotype of individual pre-migratory fish was
identified by this technique. Samples (2 µl) of the blood clots were
dissolved in 300 µl of Cell Lysis Solution (Gentra system, Minneapolis, MN,
USA), and DNA was recovered by ethanol precipitation. The extracted DNA was
used as a template for PCR amplification of about 500 bp in the 5' half
of the control region of mtDNA. Amplification was performed with a
biotinylated reverse primer for labeling of PCR products. The PCR products
were hybridized with oligonucleotide captures on a microarray, and
hybridization signals were visualized by the conventional ABC method with
tetramethylbenzidine as a chromogen. The mtDNA haplotypes of individuals were
then identified by patterns of hybridization signals. The fish in the present
study were then divided into the genealogical clade A (Japanese population),
clade B (Japanese, Russian and North American populations) and clade C
(Japanese and Russian populations) on the basis of the established category
(Sato et al., 2004). The
sequence of primer, condition of PCR and specificity of hybridization have
been described elsewhere (Moriya et al.,
2004).
|
). Internal
reference RNAs, such as those for β-actin and GADPH, were not used to
normalize the amounts of target RNAs, because expression of housekeeping genes
can vary depending on physiological status
(Bustin, 2000).
Total RNA was extracted from individual pituitary samples by the guanidium
thiocyanate hot phenol–chloroform method
(Chirgwin et al., 1979). Total
RNA was treated with 1 i.u. of deoxyribonuclease I (TaKaRa Biochemicals,
Shiga, Japan) to degrade genomic DNA, and then recovered by a
phenol–chloroform–isoamyl alcohol extraction, chloroform
extraction, and ethanol precipitation. Concentrations of total RNA were
measured with a NanoDrop ND-1000 Spectrometer (NanoDrop Technologies,
Wilmington, DE, USA). The integrity of total RNA was confirmed by agarose gel
electrophoresis. In samples obtained in 2002 and 2003, one side of the
hemisected pituitary was randomly selected and used for extraction of total
RNA. The amount of all GP subunit mRNAs was not significantly different
between the two sides of hemisected pituitary from a single fish in extra
pituitary samples of pre-spawning fish that were collected for the use as the
pooled standard sample through assays of mRNA. The recovery rate of these
extraction and precipitation protocols was confirmed to be about 80% using
known amounts of total RNAs from chum salmon livers and pituitaries
(Onuma et al., 2005). The
first strand cDNA was synthesized from 200 ng of total RNA as previously
described (Onuma et al.,
2005), diluted 1:10, and used as sample cDNAs.
Standard sense RNAs for GP2, FSHβ and LHβ sequences were
synthesized to prepare standard curves as previously described
(Ando et al., 2004). In
addition, sense RNA having the thyroid stimulating hormone β (TSHβ)
sequence was prepared to assess gene expression for other GP subunits. In
brief, plasmid DNAs containing a full cDNA insert for chum salmon GP2,
FSHβ, LHβ [a gift from Kyowa Hakko
(Sekine et al., 1989)] and
TSHβ [a gift from Kitasato University
(Ito et al., 1993)] were
digested with a restriction enzyme just after the 3' end of cDNA insert.
Sense RNAs were synthesized with a MAXIscriptTM kit (Ambion, Austin, TX,
USA), and absolute amounts were quantified with the NanoDrop ND-1000
Spectrometer. Afterwards, serially diluted standard RNA (from
2x102 to 2x109 copies) were reverse
transcribed to first strand cDNA, diluted 1:10, and used as the standards for
quantitative real-time PCR assays.
Quantitative real-time PCRs were carried out with an ABI Prism 7700
Sequence Detection System (PE Applied Biosystems, Foster, CA, USA). The
conditions of PCR and nucleotide sequences of primers and fluorogenic probes
for GTH subunit mRNAs were described previously
(Ando et al., 2004). For
TSHβ mRNA, the sequences of primers were
5'-GCTCAGCCACCACGGTCAC-3' (forward) and
5'-GGAAGTGGCCGTAGGCTGC-3' (reverse), and that of the probe was
5'-(Fam)-CCTTCGTCACGTCACCCATAAGCATG-(Tamra)-3' (TaqMan probe, PE
Applied Biosystems). A standard sample (pooled chum salmon pituitary cDNA) was
used, in triplicate, in each assay to estimate coefficients of variation (CV)
within and between assays. In the present study, the ranges of intra-assay CVs
were 0.8–14.2% for the GP2 mRNA assay, 3.2–14.4% for the
FSHβ mRNA assay, 3.2–15.8% for the LHβ mRNA assay and
2.8–9.3% for the TSHβ mRNA assay. The inter-assay CVs were 10.8%
for the GP2 mRNA assay, 13.1% for the FSHβ mRNA assay, 15.5% for
the LHβ mRNA assay and 6.9% for the TSHβ mRNA assay. We confirmed
that the single specific fragment was amplified in each assay system and the
curves obtained from serially diluted pituitary cDNA were parallel to the
standard curves, which were linear between 4x101 and
4x107 copies.
|
; Swanson et al.,
1989). Cross-reactivity of FSH in the LH assay was less than 0.01%
and that of LH in the FSH assay was 6.0%. Detectable cross-reactions with TSH,
the growth hormone prolactin (PRL) and somatolactin were not found. The
hemisected pituitaries, the other halves of which were used in mRNA assays,
were sonicated on ice following addition of 1 ml phosphate buffer (0.05 mol
l–1, pH 7.4) which contained 1 mmol l–1
phenylmethylsulfonyl fluoride. They were centrifuged at 7700 g
for 10 min at 4°C, supernatants were diluted with phosphate buffer
containing bovine serum albumin (BSA), and stored at 4°C until assayed
serial dilution of the pituitary extract and plasma showed displacement curves
parallel to those obtained from purified coho salmon GTHs in both the FSH and
LH assays (data not shown). Plasma levels of LH in the pre-migratory fish were
not assayed because preliminary tests showed that the levels were less than
the detection limit. Purified coho salmon GTHs, which served as standard
samples, were used to calculate assay CVs. The ranges of intra-assay CVs were
0.7–7.0% for the FSH assay and 0.6–14.4% for the LH assay. The
inter-assay CVs were 7.5% for the FSH assay and 13.4% for the LH assay.
Enzyme immunoassay of plasma sex steroid hormones
Plasma levels of T, 11KT, E2 and DHP were determined by enzyme
immunoassays, as previously described
(Onuma et al., 2003a). Steroid
hormones were extracted from plasma samples with diethyl ether, evaporated to
dryness with a gentle flow of nitrogen gas, and reconstituted with assay
buffer containing 0.2% BSA and 0.01% thimerosal in 0.05 mol
l–1 borate buffer, pH 7.8. Microtiter plates (MS-8496F;
Sumitomo Bakelite, Tokyo, Japan) were coated with 15
µgml–1 of anti-rabbit IgG (ICN Pharmaceuticals, Aurora,
CA, USA), 0.05% carbonate buffer, pH 9.7, and further coated with blocking
solution containing 0.1% BSA and 3% sucrose in 0.05 mol l–1
phosphate buffer, pH 7.4. The standards and samples were incubated with
anti-steroid antiserum and horseradish peroxidase-labeled steroid (T: FKA 102
and FKA 101; 11KT: FKA 118 and FKA 117; E2: FKA 236 and FKA 235; DHP: FKA 332
and FKA 331; Cosmo Bio Co. Ltd, Tokyo, Japan) at 4°C overnight. A
substrate solution (0.5 mg ml–1 o-phenylenediamine,
0.01%H2O2 in 0.2 mol l–1 citrate
buffer, pH 4.5) was added and incubated at room temperature for 30 min. After
washing with 0.9%NaCl, the absorbance at 492 nm was measured with a microplate
reader (MTP-300, Corona Electric Company, Hitachinaka, Japan). A standard
sample (pooled chum salmon plasma) was used to calculate assay CVs. In the
present assays, the sensitivities of assays were 30 pg ml–1
for T and 11KT, and 10 pg ml–1 for E2 and DHP
(Onuma et al., 2003a). The
ranges of intra-assay CVs were 1.8–13.0% for the T assay,
1.7–10.0% for the 11KT assay, 1.8–8.7% for the E2 assay, and
1.8–12.0% for the DHP assay. The inter-assay CVs were 12.5% for the T
assay, 7.7% for the 11KT assay, 15.1% for the E2 assay, and 6.2% for the DHP
assay. Recovery of steroid hormone extractions from plasma and specificities
of the assays were previously confirmed
(Onuma et al., 2003a).
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|
RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Identification of mtDNA haplotypes showed that about 30% of maturing adult I fish belonged to the genealogical clade A, i.e. the Japanese population (supplementary material Table S1). Other maturing adult I fish were identified as clade B, i.e. a mixture of Japanese, Russian and North American populations, and clade C., i.e. a mixture of the Japanese and Russian populations. About half of the maturing adult I fish are thus considered to belong to the Japanese population. The proportion of maturing adult II fish that belonged to clade A was much lower than that of maturing adult I fish.
Increases in the amount of GTH subunit mRNAs and GTH in the pituitaries of pre-migratory chum salmon in the Bering Sea
The absolute amounts of mRNAs encoding GTH subunits in the pituitaries of
the maturing adults were significantly higher than those of the immature fish
in June and July (Figs 3 and
4). The amounts of GP2
and FSHβ mRNAs in the maturing adults were two- to fivefold those
observed in the immature fish (Fig.
3A,B). Such differences were seen in the three years examined. In
2001, the amount of FSHβ mRNA in the maturing adults in the Bering Sea
was similar to that in the fish in the NW Pacific Ocean. There was also more
FSH in the pituitary of the maturing adults than in the immature fish
(Fig. 3C). In the immature
fish, the FSH content in the fish caught in June and July did not
significantly differ from that in the fish caught in September.
Pituitary LHβ mRNA was about 100-fold higher in the maturing adults that in the immature fish in all three years examined (Fig. 4A). LH in the pituitary of the maturing adults was also about 1000-fold higher that in the immature fish (Fig. 4B). Such differences were not observed in the amount of TSHβ mRNA (data not shown). In the immature fish, the amount of LHβ mRNA and LH in fish caught in June and July did not significantly differ from the amounts observed in fish caught in September.
Increases in the plasma levels of FSH, T, 11KT and E2 in pre-migratory chum salmon in the Bering Sea
The plasma levels of hormones related to the activity of the PG axis in the
maturing adults were higher than those in the immature fish, in accordance
with the above-mentioned higher synthesis of GTHs. In males, the plasma level
of FSH in the maturing adults was two- to threefold that found in the immature
fish (Fig. 5). T and 11KT in
the plasma of the immature fish were both about 1.0 nmol l–1,
whereas in the maturing adults 10 nmoll–1 were recorded in
the three years examined. In 2001, the levels of T and 11KT in the maturing
adults caught in the NW Pacific Ocean were more than 10 nmol
l–1, which was the same level as those observed in fish
caught in the central Bering Sea.
In females, the plasma level of FSH in the maturing adults were 1.5- to 2.5-fold the levels seen in the immature fish (Fig. 5). In 2001, the FSH level in the maturing females caught in the NW Pacific Ocean was higher than that observed in the immature fish. T and E2 levels in the plasma of the immature fish were both about 1.0 nmol l–1, whereas in the maturing adults they were more than 10 nmol l–1, in the three years examined.
The elevation of the PG-axis activity was observed regardless of the origins of chum salmon
There were positive correlations among the pituitary, plasma and gonadal
parameters for the activity of the PG axis in pre-migratory chum salmon of
both males (Table 2) and
females (Table 3). Maturing
adults that showed high pituitary GTHs were observed in both clade A and B
(Fig. 6). The plasma levels of
FSH, T and 11KT in males (Table
2) and FSH, T and E2 in females
(Table 3) correlated positively
(P<0.05) with the gonad masses and the GSI in both clades. The
plasma levels of T, 11KT or E2 correlated positively with the amount of GTH
subunit mRNAs and the contents of FSH and LH in the pituitaries. By contrast,
the amount of mRNA encoding another GP, i.e. TSHβ, did not correlate with
the GSI and other parameters related to sexual maturation.
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Elevation of the PG-axis activity was initiated in the winter chum salmon in the Gulf of Alaska
Maturing adults with higher activity of the PG axis were found even in the
Gulf of Alaska in February 2006 (Fig.
7). About 30% of males and 45% of females were maturing adult I
(Table 1). The levels of
pituitary FSH and LH in the maturing fish were significantly higher than in
the immature fish. The levels of plasma FSH and T in both sexes, 11KT in the
males and E2 in females were several to 10-fold those in the immature fish.
The levels of these PG-axis hormones correlated positively
(P<0.05) with the gonad masses and the GSI in both sexes (data not
shown).
|
The magnitude of increase in the activity of the PG-axis components in the maturing fish in the Gulf of Alaska were lower compared to those of the Bering Sea fish (Fig. 7). The absolute amounts of GTH subunit mRNAs and FSH and LH of the maturing fish in the Gulf of Alaska were less than one tenth those from the Bering Sea. The circulating levels of FSH, T and 11KT in the maturing adults in the Gulf of Alaska attained similar levels to those in the maturing fish in the Bering Sea.
|
|
; Onuma et al.,
2005).
In the homing adults, the amounts of GP2, FSHβ and LHβ
mRNAs peaked in fish caught at the fish wheel, followed by a decrease at the
hatchery (Fig. 8). In both
sexes, the pituitary FSH and LH contents in fully mature fish at or near the
hatchery were about twofold those observed in pre-spawning adults. The plasma
level of FSH in males peaked at the mid-point of the upstream river migration,
and that in females peaked when they reached or were near the hatchery. The
plasma LH level in fish at the hatchery showed three- to tenfold increases
over fish caught at the midway point. Such increases were observed in 2001,
2002 and 2003 (supplementary material Figs S1 and S2).
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|
DISCUSSION |
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|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Onuma et al., 2005). Our
present study showed the general pattern of changes in the PG axis of homing
chum salmon because the results were similar in the three years examined. The
magnitudes of significant differences in the amounts of mRNAs and the levels
of hormones were much greater than the assay CVs of 0.7–15.8%. The
elevation in the amounts of GTH subunit mRNAs in the maturing adults was not
due to changes in the sizes of pituitaries, because the magnitude of increases
in copy numbers of mRNAs was much larger than that in body mass (Tables
1 and
4). The present results are
thus reliable to discuss how the activity of the PG axis changes during
important phases of homing, such as migration from the Gulf of Alaska to the
Bering Sea, initiation of homing, entrance into FW, and upstream
migration. Chum salmon in the Bering Sea were a mixture of immature and maturing fish in June and July, whereas most fish were immature in September (Fig. 2). A concern is an influence of catching probability of immature and maturing fish as a result of different fishing methods, i.e. gill net and trawl net. Summer chum salmon in the Bering Sea were collected by the two different fishing methods in 2003, nonetheless the ratio of maturing adults were not different (data not shown). The patterns of distribution of the GSI were similar in the three years. Therefore, the present study showed the relationship of gonadal development to initiation of spawning migration. Chum salmon with gonads of advanced developmental stages left the Bering Sea for the natal river by the end of summer.
Activity of the PG axis during the spawning migration
In the Bering Sea, the maturing adults that were captured in June and July
showed increases in the parameters of activity of the PG axis (Figs
3,
4,
5). The maturing adults thus
left the Bering Sea after the increase in the activity of the PG axis. These
increases were commonly observed in both the clade A, i.e. the Japanese
population, and the clade B, i.e. a mixture of Japanese, Russian and North
American populations (Fig. 6).
Therefore, the increase in activity of the PG axis is considered as a general
endocrine phenomenon that is inseparable from initiation of the spawning
migration of chum salmon.
The increases in the endocrine parameters for activity of the PG axis had
already initiated in fish in the Gulf of Alaska in February
(Fig. 7). Such activation of
the PG axis in winter coincides with seasonal changes seen in hatchery-reared
male chinook salmon (Campbell et al.,
2003), coho salmon (Campbell
et al., 2006) and masu salmon
(Furukuma et al., 2008). The
previous studies showed that reproductive parameters such as GSI and the
plasma levels of FSH, 11-KT and E2 started to elevate from winter to spring.
The increase in the PG-axis activity should be triggered several months before
the initiation of spawning migration from the Bering Sea in summer. Future
investigations will clarify factors in the immature fish that interact with
the reproductive endocrine system and determine whether to mature or not to
mature in the coming spawning season.
The increase in the PG-axis activity was reflected by increases in the
amounts of mRNAs encoding GTH subunits and both FSH and LH content in the
pituitary. In salmonids, FSH is involved in the earlier phases of
gametogenesis, whereas LH is involved in the final maturation
(Swanson et al., 2003),
because LH was not detectable in plasma until the spawning period
(Prat et al., 1996;
Gomez et al., 1999). In
rainbow trout, the amount of FSH receptor mRNA in the testis was higher until
March, whereas LH receptor (LH-R) mRNA peaked in August
(Kusakabe et al., 2006). These
facts indicate that FSH is important for the increase in the GSI and levels of
sex steroid hormones in the plasma of chum salmon prior to initiation of
spawning migration.
The plasma levels of T, 11KT and E2 in maturing adults were about tenfold
those in immature fish. In maturing adults, the concentration exceeded 10 nmol
l–1, which is higher than the equilibrium dissociation
constants (Kd) of androgen receptors (ARs) and estrogen
receptors (ERs). The Kd for ARs and [3H]T was
estimated as 2–5 nmol l–1 in cytosolic and nuclear
extracts of goldfish forebrain (Pasmanik
and Callard, 1988) and the Kd for ERs and
[3H]E2 was estimated as 1.5–2.5 nmol l–1 in
extracts of rainbow trout forebrain
(Allison and Omeljaniuk, 2000).
Furthermore, implantation of T, 11KT or E2 induced migratory behavior in masu
salmon in artificial raceways (Munakata et
al., 2001). We consider that the levels of circulating sex steroid
hormones in maturing chum salmon migrating from the Gulf of Alaska to the
Bering Sea are sufficient to act on the forebrain to initiate migratory
behavior, although it is difficult to confirm this by behavioral experiments
using oceanic chum salmon.
The amount of LHβ mRNA and the LH in the pituitary of maturing adults were more than 100-fold those of immature fish (Figs 4 and 7). Synthesis of LH is thus stimulated in the pituitary of pre-migratory chum salmon in the Bering Sea before summer. Nonetheless, the increase in the GSI and levels of steroid hormones in the plasma cannot be explained by LH, because our preliminary test showed that the plasma level of LH was below the detection limit in the plasma of the maturing adults. Afterwards, the plasma LH level increased in the homing adults when they were near or at the natal hatchery during upstream migration in the spawning period (Fig. 8). Therefore, the synthesized and stored LH in the pituitary of the high sea chum salmon may be released for the final maturation during the last phase of spawning migration.
In homing adults, the amounts of FSHβ mRNA and FSH protein in the
pituitary, and the plasma levels of FSH were elevated during upstream
migration (Fig. 8). However,
increases in FSHβ mRNA were not apparent in the previous analyses of chum
salmon in the Ishikari River–Ishikari Bay system from 1993 to 1999
(Kitahashi et al., 1998;
Onuma et al., 2005). Although
we do not have any lines of evidence to explain this discrepancy, there are
several reports which showed increases in the pituitary FSH contents during
the spawning season (Nozaki et al.,
1990; Naito et al.,
1991; Amano et al.,
1992; Amano et al.,
1993). The plasma FSH level also increased before spermiation and
after ovulation in rainbow trout (Prat et
al., 1996; Gomez et al.,
1999). Cross reaction of FSH with LH-R was demonstrated by binding
of 125I-FSH and 125I-LH to sections of the testis of
coho salmon (Miwa et al.,
1994), and binding of them to membranes of ovarian
theca-interstitial layers and granulose cells of coho salmon
(Yan et al., 1992).
Stimulation of cAMP production by FSH was also seen in COS-7 cells that
expressed amago salmon LH-R (Oba et al.,
1999). These findings suggest an involvement of FSH in control of
the final maturation.
The plasma levels of T, 11KT and E2 were increased in homing adults during
migration from the coast to the natal river through the midway point or the
junction of the main Ishikari River and the tributary
(Fig. 9). Elevated levels of
sex steroid hormones have been found to be associated with the adaptation to
FW in pre-spawning chum salmon (Makino et
al., 2007). Makino et al. found that the plasma levels of T and/or
E2 were related to the amount of time spent in FW, when spontaneous behavior
of pre-spawning chum salmon was monitored in an aquarium that allowed fish to
migrate between separated SW and FW streams
(Makino et al., 2007). An
application of T, 11KT or E2 directly modulated prolactin (PRL) gene
expression, a FW adaptation hormone
(Hirano et al., 1987), in
primary pituitary cells of pre-spawning and mature masu salmon
(Onuma et al., 2007). In
homing salmon, the endocrine systems that govern salt and water homeostasis
are thus under the influence of activity of the PG axis.
Underlying mechanisms: season-dependent regulation of the PG axis
The amounts of GTH subunit mRNAs in the pituitary and the plasma levels of
T, 11KT and E2 in maturing adults were more than tenfold those in immature
fish in June and July. In masu salmon reared in aquaria, these parameters were
elevated between March and June, and peaked by the spawning season in late
September (Kitahashi et al.,
2004). Seasonal changes in the activity of the PG axis of
ocean-migrating chum salmon thus coincided with those in farmed masu salmon.
The regulatory mechanisms of the PG axis in ocean-migrating chum salmon
appears to be similar to those found in aquaculture condition
(Ando and Urano, 2005).
The activity of the salmonid PG axis is regulated primarily by
hypophysiotropic salmon gonadotropin-releasing hormone (sGnRH) neurons in the
ventral telencephalon and the preoptic area
(Amano et al., 1997). Salmon
GnRH directly elevated the amounts of GP2 and FSHβ mRNAs in
pituitaries of maturing coho salmon in vitro in May
(Dickey and Swanson, 2000). In
masu salmon, the amount of sGnRH mRNAs in the forebrain loci were already high
in the pre-pubertal stage from winter to spring, declined toward summer, and
then increased in the spawning period in autumn
(Ando et al., 2001;
Kitahashi et al., 2004). Such
changes corresponded to seasonal changes in the sGnRH content in the
forebrain, whereas the pituitary sGnRH was gradually elevated from spring to
autumn (Amano et al., 1992;
Amano et al., 1993). We
recently found that sGnRH mRNA increased in the ventral telencephalon and the
preoptic area of maturing chum salmon in the winter in the Gulf of Alaska (our
unpublished data). Since the amount of sGnRH mRNA was not different between
the immature fish and maturing adults in the summer in the Bering Sea
(Onuma et al., 2008), sGnRH
gene expression seemed to be increased prior to the arrival of the chum salmon
in the Bering Sea. The PG axis of pre-migratory chum salmon may be activated
by sGnRH neurons until early summer, prior to initiation of spawning
migration.
In terms of the neuroendocrine control of spawning migration, sGnRH neurons
have the appropriate neuroanatomical features to coordinate functions of the
PG axis and the central nervous system
(Urano et al., 1999;
Onuma et al., 2005). In
salmonids, sGnRH immunoreactive fibers are localized in the neurohypophysis
and the various brain loci including the optic tectum and the hypothalamic
neurosecretory neurons. In addition to its well-established function in
controlling the PG axis, sGnRH has been shown to directly increased electric
activities of magnocellular neurosecretory neurons in rainbow trout
(Saito et al., 2003), and
facilitated synaptic transmission of retinotectal neurons
(Kinoshita et al., 2007).
Behaviorally, administration of the GnRH analog (GnRHa) to pre-spawning chum
salmon stimulated a preference for FW, and shortened the duration of the
homing migration from the mouth of the Ishikari River to the hatchery
(Kitahashi et al., 2001).
These findings strongly support our hypothesis that the GnRH–GTH system
is essential for the control of migratory events, including coordination of
the initiation of gonadal development and the spawning migration.
The plasma levels of T, 11KT and E2 also increased in line with the
increases in the amounts of GTH subunit mRNA (Table
2 and
3). In salmonids, there is an
abundance of evidence to show that sGnRH and sex steroid hormones
synergistically stimulate syntheses of GTHs from spring to early summer
(Ando and Urano, 2005).
Co-incubation with both sGnRH and E2 increased the amounts of LHβ mRNA
and released FSH and LH from primary pituitary cells of masu salmon
(Ando et al., 2004). In March
and May, sGnRH, T or E2 elevated expression of the salmon fushi tarazu factor
1 homolog (sFF1-I) gene and estrogen receptor (ER) , which directly
activated the salmon LHβ promoter (Le
Drean et al., 1996), in primary pituitary cells of masu salmon
(Onuma et al., 2007).
Furthermore, T and E2 elevated the amounts of LHβ mRNA in primary
pituitary cells of immature rainbow trout
(Xiong et al., 1994), maturing
coho salmon in February and April (Dickey
and Swanson, 1998), and maturing masu salmon in May
(Ando et al., 2004). Such
stimulatory effects of sex steroids were attenuated after the late stages of
gametogenesis in late July (Xiong et al.,
1994; Ando et al.,
2004; Onuma et al.,
2007). Both transcription and translation of GTHs are thus
season-dependently upregulated from early spring to summer before the period
when chum salmon initiate spawning migration and leave the Bering Sea for
their natal river.
In conclusion, the activity of the PG axis was increased in chum salmon during migration from the Gulf of Alaska to the Bering Sea from winter to summer. The increase in activity of the PG axis is one of the seasonal endocrine events that are inseparable from the onset of spawning migration.
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Footnotes |
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Supplementary material available online at http://jeb.biologists.org/cgi/content/full/212/1/56/DC1
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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