Journal of Neuroendocrinology, 1999, Vol. 11, 181–186

Distribution and Seasonal Variations in Levels of Three Native GnRHsin the Brain and Pituitary of Perciform Fish

B. Senthilkumaran, K. Okuzawa, K. Gen, T. Ookura* and H. Kagawa†Inland Station, National Research Institute of Aquaculture, Tamaki, Japan.*Faculty of Bioresources, Mie University, Tsu, Japan.†National Research Institute of Aquaculture, Nansei, Japan.

Key words: GnRH, perciform fish, teleost, seasonal changes.

Abstract

Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types ofgonadotropin-releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH-II. Weemployed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study thepresence and regional distribution of these three GnRHs in the brains and pituitaries of fourperciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; stripedknifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonalchanges in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs werefound in brains of all fishes examined, with regional distributions in the brains of the three GnRHsbeing rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH-II wasconcentrated from the middle to posterior part of the brain and distributed throughout the brain.sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH-II.The dominant form of GnRH in the pituitary was sbGnRH, with levels 500- to 2400-fold higher thanthose of sGnRH, while cGnRH-II was undetectable in all four species. In the brain and pituitary offemale red seabream, levels of both brain and pituitary sbGnRH increased from October (immaturephase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomaticindex and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary ofcompletely regressed fish in June. Levels of both sGnRH and cGnRH-II in the brain were higher inthe regressed phase and remained lower during the spawning phase. From these and previousresults, it appears that sbGnRH is physiologically the most important form of GnRH in reproductionin red seabream and, probably, in other perciforms also.

Gonadotropin-releasing hormone (GnRH) plays an essential We have studied GnRH in the brains of several species ofperciform fish, focusing on red seabream, Pagrus major. Werole in vertebrate reproduction. Twelve different forms of

GnRH from vertebrates or protochordates have been identi- found sGnRH, cGnRH-II and an unidentified but immunore-active (ir) GnRH in three perciforms, namely, red seabream,fied and their amino acid sequences determined (1; for review,

see 2). In teleosts, reports of two or three different forms of black seabream (Acanthopagrus schlegeli ), and red spottedgrouper (Epinephelus akaara (8)); using a combination ofGnRH in a single species are becoming increasingly common

(3). For example, the presence of three forms of GnRH, high-performance liquid chromatography (HPLC) and radio-immunoassay (RIA). The presence of sGnRH in the brainnamely, salmon (s) GnRH, chicken (c) GnRH-II and seab-

ream (sb) GnRH, was first reported in two perciform species, of red seabream was confirmed and that of sbGnRH wasdemonstrated by cDNA cloning (9, 10). The unidentifiedgilthead seabream, Sparus aurata (4) and African cichlid,

Haplochromis burtoni (5), and subsequently in a scorpaeni- ir-GnRH in the three perciforms mentioned above is nowconsidered to be sbGnRH because the retention time ofform fish, rockfish, Sebastes rastrelliger (6) and a characiform

fish, pacu, Piaractus mesopotamicus (7). ir-GnRH is similar to that of synthetic sbGnRH (Okuzawa

Correspondence to: K. Okuzawa, Inland Station, National Research Institute of Aquaculture, 224-1, Hiruta, Tamaki, Watarai, Mie 519-0423,Japan (e-mail: kokuzawa@nria-tmk.afffrc.go.jp).

© 1999 Blackwell Science Ltd

182 Brain and pituitary levels of GnRHs in perciforms

et al., unpublished results). Our data and those of others in grams/100 g b.w.; mean±SEM) were 9.77±0.87, and11.9±1.27 (female) and 9.31 (male), respectively. The stripedindicate that brains of most perciform fish contain three

forms of GnRH, namely, sGnRH, cGnRH-II and sbGnRH. knifejaw and Nile tilapia were in the process of vitellogenesisor spermatogenesis and their GSIs were 1.67±0.13,Are some or all three forms of GnRH involved in secretion

of gonadotropin (GTH) from the pituitary? Some data 3.43±0.53 (female) and 0.48±0.19 (male), respectively.Brains were divided into five parts as shown in Fig. 1.suggest that sbGnRH is the main or sole form of GnRH in

terms of hypophysiotropic function in perciform fish. In red seabream (Fig. 1), sbGnRH was found at equallevels in the telencephalon (area b) and hypothalamus (areasbGnRH-immunoreactive neuronal cell bodies are localized

in the preoptic area (POA), with axons projecting mainly to c), with a very small amount in the optic tectum-thalamusregion (area d). cGnRH-II was distributed in all areas of thethe pituitary in red seabream (10). In African cichlid, only

sbGnRH was found in the pituitary (11). However, no brain extensively, with high levels in the posterior parts, optictectum-thalamus and cerebellum-medulla oblongata (area e).specific RIA for sbGnRH has been available and, thus, the

relationship between gonadal maturation and brain and Similarly, sGnRH was distributed throughout the brain buthigh levels were found in the olfactory bulb (area a).pituitary levels of sbGnRH remains to be characterized.

In the present study, we performed the following studies In black seabream (Fig. 1), sbGnRH was found at equallevels in the telencephalon and hypothalamus, whileto identify the pivotal hypophysiotropic form of GnRH in

perciform fish. We developed highly specific and sensitive cGnRH-II and sGnRH were found throughout the brain.RIAs for sbGnRH and cGnRH-II. We then analysed regionaldifferences in levels of three GnRHs in discrete areas of thebrains of four perciform fishes, namely, red seabream, blackseabream, striped knifejaw (Oplegnathus fasciatus) and Niletilapia (Oreochromis niloticus) using these two RIAs and apreviously reported RIA for sGnRH (12). Finally, we studiedseasonal variations in levels of the three forms of GnRH inthe brain and pituitary of red seabream.

Results

Pituitary levels of GnRHs

In red seabream and striped knifejaw, sbGnRH was foundto be the dominant form of GnRH in the pituitary, withlevels being much higher than those of sGnRH. Levels ofsbGnRH were 500- and 2400-fold higher than those ofsGnRH in striped knifejaw and red seabream, respectively.In black seabream and Nile tilapia, only sbGnRH wasdetected and levels of sbGnRH in the pituitary of Nile tilapiawere higher than in any other perciforms. cGnRH-II wasundetectable in the pituitaries of all perciforms examined(Table 1)

Regional differences in levels of GnRHs in the brain

We used five female specimens of red seabream and stripedknifejaw, four female and one male specimen of black seab-ream, and two female and three male specimens of Niletilapia. The red and black seabream were fully mature andtheir gonadosomatic indices (GSI; defined as gonadal weight

T 1. Concentrations (pg/mg protein) of Pituitarya b d e

(C) Nile tilapia

Bra

in G

nR

H (

pg

/mg

pro

tein

)

300

200

100

0c a b d e

(D) Knifejaw100

60

20

0c

(B) Black seabream100

60

20

0

(A) Red seabream300

200

100

0

80

40

80

40

b

a d

c

e

Pit

F. 1. Schematic representation of a sagittal section of the brain of aGnRHs in Four Perciform Fishes.perciform fish showing the dissections used for determination of levelsof GnRH (upper panel ) and the concentrations of salmon GnRH,Red Black Striped Nilechicken GnRH-II and seabream GnRH in discrete areas of the brainsseabream seabream knifejaw tilapia( lower panels) of four perciforms: () red seabream; () black seabream;(), Nile tilapia; ( ) striped knifejaw. The vertical bar on each column

sGnRH 2.75±0.75 ND 8.38±2.24 ND indicates the standard error of the mean (SEM). Letters represent thecGnRH-II ND ND ND ND following brain areas: a, olfactory bulb; b, telencephalon, includingsbGnRH 6824±746 726±76 4322±554 9938±2838 preoptic area; c, inferior lobe of hypothalamus; d, optic tectum and

thalamus, including anterior part of cerebellum; e, cerebellum andmedulla oblongata; Pit, pituitary gland. %, sGnRH; b, cGnRH-II;Values are the means±SEM. ND, hormone was below the limit of

detection. ba, sbGnRH.

© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 181–186

Brain and pituitary levels of GnRHs in perciforms 183

There were high levels of cGnRH-II in the caudal part of thebrain, in addition to the telencephalic region, and relativelyhigh levels of sGnRH were found in the olfactory bulb.

In Nile tilapia (Fig. 1), the distribution of sbGnRH wassimilar to that in red seabream, and sGnRH and cGnRH-IIwere found at high levels in the olfactory bulb and in thecerebellum-medulla oblongata region, respectively. Both theseforms of GnRH were found throughout the brain.

In striped knifejaw (Fig. 1 ), the distribution of sbGnRHwas different from that in other perciforms. Large amountsof sbGnRH were found in almost all areas apart from thecerebellum-medulla oblongata region and levels were higherin the olfactory bulb. As in the other perciforms mentionedabove, cGnRH-II and sGnRH were distributed all over thebrain of the striped knifejaw, with particularly high levels inthe posterior part of the brain and in the olfactory bulb,respectively.

Seasonal variations in brain and pituitary levels of GnRHs inred seabream

In the brain (Fig. 2) and pituitary (Fig. 2 ) of red seabream,levels of sbGnRH increased from October (immature phase)and reached a maximum in April (spawning phase) reflectingan increase in GSI (Fig. 2) and vitellogenesis. Brain levelsof sbGnRH decreased in June but the levels were still ratherhigh and similar to levels in March. The levels of sbGnRHdid not change and remained high in the pituitary of com-pletely regressed fish in June. One-way anova followed byDuncan’s multiple range test revealed that the levels ofsbGnRH in the brain in April, and in both April and Junein the pituitary were significantly different (P<0.05) from Calendar month

O J A

(E) Brain sGnRH

sGn

RH

(p

g/m

g p

rote

in) 40

30

20

10

0N F MD M J

spawningseason

bab

a aa

b

O J A

(F) Pituitary sGnRH

sGn

RH

(p

g/m

g p

rote

in)

6

4

2

0N F MD M J

spawningseason

aa

a a

(C) Brain sbGnRH

sbG

nR

H (

pg

/mg

pro

tein

)

10

6

4

2

0

bcab

d

e

a

cd

(D) Pituitary sbGnRH

sbG

nR

H (

ng

/mg

pro

tein

)

6

4

2

0

bab

cc

a

b

(A) Brain cGnRH-II

cGn

RH

-II (

pg

/mg

pro

tein

)

100

80

40

0

babab

a aab

(B) GSI

go

nad

oso

mat

ic in

dex

(G

SI)

8

4

2

0

ababc

a

c

a

bc

8 8

10

6

10

60

20

a

a

those in any other months studied. Conversely, levels of F. 2. Seasonal changes in the gonadosomatic index (GSI; ); in brainsGnRH in the brain (Fig. 2) were higher during immature levels of chicken GnRH-II (), seabream GnRH () and salmon GnRH

(); and pituitary levels of seabream GnRH ( ) and salmon GnRH ( )(October) and regressed (June) phases than during vitellog-in the female red seabream, Pagrus major from October 1996 to Juneenic (February and March) and spawning (April ) phases.1997. The number of samples in each group was five (for GnRH) or 10Statistical analysis revealed that levels were not significantly (for GSI). The levels of GnRH were subjected to an analysis of variance

different in June, October and December, and from December followed by Duncan’s multiple-range test, and GSIs were analysed bythe Kruskal–Wallis test. Means that are not significantly different areto April, respectively. Pituitary levels of sGnRH in eachindicated by common letters of the alphabet. Means with different lettersmonth exhibited considerable variations due to the very lowof the alphabet, for example a and bc, differ significantly (P<0.05) inlevel, which was near the limit of detection, and there wereeach series.

no significant differences among months throughout theexperiment (Fig. 2 ). Levels of brain cGnRH-II (Fig. 2)were higher in regressed fish (June) than in vitellogenic fish

sbGnRH is the dominant form of GnRH in the pituitaries(February and March). In all the pituitary samples, levels of of perciform species.cGnRH-II were below the limit of detection.

Estimations of levels of the three different GnRHs indiscrete regions of brains revealed that all three GnRHscoexist in the brains of individual species of perciforms. MostDiscussionof the sbGnRH was present in the telencephalon and hypo-thalamus, while the other two forms were found throughoutThe present study describes for the first time the use of

specific RIAs for three different GnRHs to measure levels of the brains of the four perciforms. Differential distribution ofmultiple forms of GnRH in discrete brain areas has beenGnRH in the brains and pituitaries of perciforms. The

pituitaries of all four perciforms contained very high levels examined by RIA in several teleosts (for review, see 3),however, the present study is the first report of the quantifica-of sbGnRH as compared to sGnRH, 500- to 2400-fold higher,

while cGnRH-II was undetectable in the pituitary. High tion of sbGnRH in different regions of the brain and pituitaryby homologous RIA. In salmonid and cyprinid, only sGnRHlevels of sbGnRH have been detected in the pituitary of

sexually mature gilthead seabream by a combination of and cGnRH-II were found in the brains. The distributionpatterns of these two forms of GnRH in discrete brain areasHPLC and heterologous RIA (4). Our observations, are in

agreement with previous results and indicate, moreover, that of salmonid (12) and cyprinid (13) were similar to those of

© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 181–186

184 Brain and pituitary levels of GnRHs in perciforms

perciforms. This indicates that sGnRH and cGnRH-II are considered to regulate GTH secretion. Both the fibres fromthe TN-sGnRH system and the cell bodies and fibres of thecommon and rather ancient GnRH forms among teleosts

and the distribution patterns of these two forms have been POA-sGnRH system are contained in discrete areas of brain,such as the telencephalon and hypothalamus. The levels ofunchanged during the evolution of teleost, while sbGnRH

is newly evolved and a specific GnRH for evolutionary the TN-sGnRH system is much higher than the POA-sGnRHsystem (13, 20), thus changes in POA-sGnRH which isadvanced teleosts, such as perciforms.

The quantitative distribution in the brain of three native expected to have a positive correlation with gonadal activityis probably occluded by changes in the TN-sGnRH systemGnRHs in red seabream is in good accord with our earlier

histological localization of populations of GnRH neurons in which is independent to gonadal maturation, as we revealedin the present study (see below).the brain of red seabream by in-situ hybridization (ISH) and

immunocytochemistry: sbGnRH-producing cell bodies were In contrast to the previous studies in salmonid and cyprinid,we found a distinct correlation between brain levels oflocalized only in the POA and projected mainly to the

pituitary, while sGnRH-and cGnRH-II-producing cell bodies sbGnRH and gonadal maturity in red seabream. This correla-tion suggests a dominant role for sbGnRH in the regulationwere observed only in the ganglions of the terminal nerve

(TN) and midbrain, respectively, and their fibres were widely of GTH(s) and in turn, of reproductive activities. Levels ofsGnRH and cGnRH-II were high during regressed or imma-distributed in the brain (10; Okuzawa et al. unpublished

observations). The similarities in the patterns of quantitative ture phases of the reproductive cycle. The absence of anycorrelation between levels of sGnRH and cGnRH-II, on thedistribution of GnRHs in the brain and pituitary among

perciform species, suggest that sbGnRH cells are localized in one hand, and GSI and reproductive stage on the othersuggests that the involvement of these forms of GnRH inthe POA and their axons probably project into the pituitary

in perciform species. In fact, ISH studies of other perciform regulation of GTH(s) or in the cycle of reproduction eventsis limited.species, namely African cichlid (5) and gilthead seabream

(14) revealed that mRNA for a precursor to sbGnRH was In the present study, brain levels of sbGnRH decreasedbut pituitary levels of sbGnRH remained high in regresseddetectable only in the POA. Furthermore, in African cichlid

(11) and gilthead seabream (4), sbGnRH was shown to be fish in June. No report in teleosts is available to date tocompare our data, and we do not know the exact mechanismthe sole or dominant form of GnRH stored in the pituitary.

These studies together suggested that sbGnRH might be the underlying this phenomenon. However, it can be speculatedthat during gonadal regression the release of sbGnRH frompivotal hypophysiotropic form in perciforms. This hypothesis

is supported by the present study, which showed that the nerve terminals contained in the pituitary is first decreased inaccordance with inactivation of sbGnRH neurons, which alsolevels of sbGnRH in the brain and the pituitary increased

with the sexual maturation of red seabream (see below). cause an attenuation of the synthesis of sbGnRH and inturn, levels of sbGnRH in the brain are decreased. AfterAlthough cGnRH-II and sGnRH were distributed through-

out the brain at high concentrations, their absence or very gonadal regression, increased clearance of sbGnRH from thepituitary (a decrease in the levels of sbGnRH levels in thelow levels in the pituitary almost rule out their involvement

in the regulation of GTH or any other pituitary hormone. pituitary) may occur due to a reduction of transportation ofsbGnRH from cell bodies. Quantitative studies of mRNAThe wide distribution of sGnRH and cGnRH-II in the brains

of perciforms indicates their involvement in neurotransmis- for sbGnRH will be required to support this hypothesis. Asimilar phenomenon was observed in an avian species, thesion and/or neuromodulation, as suggested in other teleosts

(for review, see 15). European starling Sturnus vulgaris (21): the number and sizeof GnRH cells decreased during gonadal regression, evenSeveral studies were conducted to measure brain GnRH

contents by RIA in relation to gonadal maturation in teleost though staining for GnRH on the median eminence (medianeminence is included in the pituitary in teleosts) remained(for review, see 3). Although only one report is available to

demonstrate positive correlation between brain GnRH con- intense.In summary, from the distribution of sbGnRH in the POAtents and gonadal maturity in caribe colorado, Pygocentrus

notatus (16), other studies failed to show clear correlation in the brain and the high levels in the pituitary, as well asthe distinct rise in the level of sbGnRH in both the brainbetween brain GnRH contents and gonadal maturity. For

example, when levels of sGnRH and cGnRH-II in the dis- and pituitary during the spawning phase of the reproductivecycle of red seabream, it is likely that sbGnRH is the pivotalcrete areas of brain and the pituitary of masu salmon,

Oncorhynchus masou were examined from hatching until hypophysiotropic form and plays a dominant role in theregulation of the hypothalamo-hypophyseal-gonadal axis ingonadal maturation, sGnRH in the pituitary increased in

parallel with gonadal maturation, while the relationship red seabream. This hypothesis can be extended to otherperciforms in view of resemblance of the quantitative patternsbetween levels of either sGnRH or cGnRH-II in brain and

gonadal maturation was not clear (17, 18). The reason for of distribution of sbGnRH and other GnRHs in brains andpituitaries to those in red seabream.the discrepancy between levels of GnRH in brain and gonadal

maturity observed in several teleosts such as masu salmon(17, 18) and goldfish (19) may be the heterogeneity ofsGnRH neuronal system. sGnRH neuronal system is consid- Materials and methodsered to have two distinct subsystems: one is a TN-sGnRH All the marine perciforms, red seabream, black seabream and striped knifejaw,system which is thought to be unrelated with GTH secretion were obtained from stocks maintained at the National Research Institute of

Aquaculture (NRIA), Nansei, Mie Prefecture, Japan. Specimens of the(20), and the other is a POA-sGnRH system which is

© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 181–186

Brain and pituitary levels of GnRHs in perciforms 185

freshwater perciform fish, Nile tilapia, were obtained from the stock main- to determine the recovery of sbGnRH. The recovery of standard sbGnRH inthe RIA was 84.4±5.3% (n=5).tained at the Inland Station of NRIA, Tamaki, Mie Prefecture, Japan. All

the fish were sampled between March and April, 1997. Prior to that time,Radioisotopic iodination of GnRHsfemale red seabream, reared under natural conditions in net pens in the

seawater in Gokasho Bay, Nansei, were collected in October and December All three GnRHs namely, sbGnRH, sGnRH and cGnRH-II, were radioiodin-ated as described elsewhere (12) with minor modifications. In brief, synthetic1996, and also in February, March, April and June 1997. These fish corre-

sponded to the regressed (June–July), immature (August–November), vitellog- GnRH (5 mg) in 0.25% acetic acid (10 ml ), 0.5 M phosphate buffer (pH 7.5;20 ml ), 18.5 MBq of Na 125I (5 ml; Amersham, Buckinghamshire, UK), andenic (December–March) and spawning (April–May) phases. Fish were deeply

anaesthetized with 2-phenoxyethanol (Nacalai tesque, Kyoto, Japan) before chloramine T (10 mg in 10 ml of 50 mM phosphate buffer) were mixed for10–15 s and the reaction was stopped by the addition of sodiumdeath by decapitation. Brains were quickly dissected out and placed on ice,

rinsed briefly with physiological saline (0.9% NaCl ) and divided into five metabisulfite(40 mg in 40 ml of 50 mM phosphate buffer). Purification andthe separation of iodinated peptides from non-iodinated peptides and freeparts, as shown in Fig. 1. The pituitaries were also collected. For seasonal

studies, we used the entire brains of red seabream without dividing them. All iodine were achieved by use of an isocratic reversed-phase column (TSK-ODS 80 TM column, 5 mm, 30 cm×0.46 cm i.d.; with a flow rate oftissue samples were frozen in liquid nitrogen and stored at −80 °C until

extraction for RIAs. The b.w. and length of each fish were recorded prior to 0.8 ml/min) with an HPLC (Shimadzu, Kyoto, Japan) LC-3 A system and25%, 27% and 35% acetonitrile as eluent for sbGnRH, cGnRH-II anddissection. Gonads were excised and weights were determined for calculation

of the GSI and parts of the gonads were fixed in Bouin’s solution for sGnRH, respectively.histological identification of reproductive stages.

RIAs of GnRHsPreparation of brain and pituitary extracts for RIA Preparation of antibodies against sGnRH (12), cGnRH-II (22) and sbGnRH

(10) has been described previously. The sbGnRH standard was dissolved inBrain and pituitary extracts of different fish were prepared as described byOkuzawa et al. (12). In brief, different regions of the brain and the pituitary BSA-PBS at 10 different concentrations from 9.76 to 5,000 pg/ml; sGnRH

and cGnRH-II were diluted from 500 to 1.9 pg/ml and from 500 to 3.9 pg/ml,were homogenized in 0.1 N HCl (1 ml/50 mg for brain tissues and 0.5 ml forpituitary samples) with a Ten Broeck homogenizer ( Wheaton, Millville, NJ, respectively. Antisera against sbGnRH (AS-691), sGnRH (Lot no. 2) and

cGnRH-II (aCII6) were individually diluted 40,000-, 300,000- andUSA) for brain tissue and with an Eppendorf tube homogenizer (Eppendorf,Hamburg, Germany) for each pituitary. Aliquots of 50 or 100 ml were removed 100,000-fold, respectively, in NRS-EDTA-PBS [50 mM phosphate buffer,

pH 7.5, containing 1% (v/v) normal rabbit serum, 140 mM NaCl, 50 mMfrom the homogenate of each sample for quantification of protein with a DCprotein assay kit (Bio-Rad, Richmond, CA, USA) with bovine serum albumin EDTA and 0.1% (w/v) sodium azide]. Samples and standards were always

analysed in parallel and assayed in duplicate. For RIA of GnRH, a double-(Sigma, St. Louis, MI, USA) as the standard. Each homogenate wascentrifuged at 10 000×g for 30 min at 4 °C. The supernatant was frozen on antibody method was used as described previously (12).dry ice, lyophilized and reconstituted in RIA buffer, namely, BSA-PBS

Validation of assays[50 mM phosphate buffer, pH 7.5, containing 1% (w/v) bovine serum albumin,140 mM NaCl, and 0.1% (w/v) sodium azide]. In the case of brain tissues, Extracts from the brains and pituitaries of four perciforms, namely, red

seabream, black seabream, striped knifejaw and Nile tilapia, gave competitionafter reconstitution, the samples were recentrifuged to yield a clear supernatantfor RIA. After the analysis of regional differences in levels of GnRHs in curves parallel to the standard curve for sbGnRH. Extracts of brains from

all four perciforms used in this study gave competition curves parallel to thevarious regions of the brain, extracts were pooled to give a whole-brainextract for analysis of parallelism to standard curves in RIAs. Seasonal standard curve for cGnRH-II, while pituitary extracts from these perciforms

caused no displacement of antibody-bound labelled cGnRH-II. Brain extractschanges in levels of GnRHs in the brain and pituitary of red seabream wereestimated similarly. Recovery of sGnRH and cGnRH-II in the respective from all four perciforms gave competition curves parallel to the standard

curve for sGnRH. Pituitary extracts from red seabream and striped knifejawRIAs had been estimated previously (12) and the same method was adopted

T 2. Specificity of the Antisera and Radioimmunoassay (RIA) Systems.

RIA system(Standard and labelledhormone) Antiserum Cross-reactivity with GnRH (%)

Salmon GnRH antisalmon GnRH (Lot No.2) S 100CII 1.58SB <0.01LI 0.08M <0.01CI <0.01CF <0.01

Chicken GnRH-II antichicken GnRH-II S 1.46(aCII6) CII 100

SB <0.01LI <0.01M <0.01CI <0.01CF <0.01

Seabream GnRH antiseabream GnRH (AS-691) S <0.01CII <0.01SB 100LI <0.01M <0.01CI <0.01CF <0.01

Cross-reactivity was measured at B/Bo=0.5. S, salmon GnRH; CII, chicken GnRH-II; SB, seabream GnRH; LI, lamprey GnRH-I; M, mammalianGnRH; CI, chicken GnRH-I; CF, catfish GnRH.

© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 181–186

186 Brain and pituitary levels of GnRHs in perciforms

gave competition curves parallel to the standard curve for sGnRH, but those NM. Primary structure of three forms of gonadotropin-releasing hormone(GnRH) from the pacu brain. Reg Pept 1997; 68: 189–195.from black seabream and Nile tilapia caused no displacement of labelled

8 Okuzawa K, Amano M, Aida K, Hasegawa Y, Tanaka H, Kagawa H.sGnRH (data not shown).Chromatographic and immunological identification of gonadotropin-Cross-reactivity measured at B/Bo=50% with several forms of GnRH inreleasing hormone in five marine teleosts. Fish Physiol Biochem 1993;the three RIA systems used in the present study were summarized on Table 2.12: 337–345.The three systems did not cross-react with thyrotropin-releasing hormone,

9 Okuzawa K, Araki K, Tanaka H, Kagawa H, Hirose K. Molecularhuman corticotropin-releasing hormone, human growth hormone-releasingcloning of a cDNA encoding the prepro-salmon gonadotropin-releasinghormone, somatotropin release-inhibiting factor (SS-14), arginine vasotocin,hormone of the red seabream. Gen Comp Endocr 1994; 96: 234–242.and isotocin even at 10 mg/ml.

10 Okuzawa K, Granneman J, Bogerd J, Goos HJT, Zohar Y, Kagawa H.The sensitivity of the assay defined as twice the standard deviation at zeroDistinct expression of GnRH genes in the red seabream brain. Fishdose (n=16) was found to be 0.37 pg/tube for sbGnRH RIA and 0.47 pg/tubePhysiol Biochem 1997; 17: 71–79.for cGnRH-II RIA. Intra-and interassay coefficients of variations for the

11 Powell JFF, Fischer WH, Park M, Craig AG, Rivier JE, White SA,RIA for sbGnRH were 4.28% (n=7) and 3.79% (n=5), respectively. Intra-Francis RC, Fernald RD, Licht P, Warby C, Sherwood NM. Primaryand interassay coefficients of variations for the RIA for cGnRH-II were 6.87structure of solitary form of gonadotropin-releasing hormone (GnRH)(n=7) and 4.81% (n=5), respectively. We already described the sensitivityin cichlid pituitary; three forms of GnRH in brain of cichlid andand intra- and interassay coefficients of variations of the RIA for sGnRHpumpkinseed fish. Reg Pept 1995; 57: 43–53.elsewhere (12).

12 Okuzawa K, Amano M, Kobayashi M, Aida K, Hanyu I, Hasegawa Y,Statistical analysis Miyamoto K. Differences in salmon GnRH and chicken GnRH-II

contents in discrete brain areas of male and female rainbow troutAll data were expressed as a mean±SEM. Parallelism of displacement curvesaccording to age and stage of maturity. Gen Comp Endocr 1990;for brain and pituitary extracts to each standard curve was tested by anova.80: 116–126.Seasonal variations in levels of the three forms of GnRH in the brain and

13 Kobayashi M, Amano M, Hasegawa Y, Okuzawa K, Aida K. Effects ofpituitary of red seabream were examined by one-way anova followed byolfactory tract section on brain GnRH distribution, plasma gonadotropinDuncan’s multiple-range test, and the GSIs were analysed by the Kruskal–levels, and gonadal stage in goldfish. Zool Sci 1992; 9: 765–773.Wallis test.

14 Gothilf Y, Munoz-Cueto JA, Sagrillo CA, Selmanoff M, Chen TT,Kah O, Elizur A, Zohar Y. Three forms of gonadotropin-releasinghormone in a perciform fish (Sparus aurata): complementary deoxyribo-Acknowledgementsnucleic acid characterization and brain localization. Biol Reprod 1996;55: 636–645.This study was supported in part by a grant-in-aid from the Ministry of

15 Oka Y. GnRH neuronal system of fish brain as a model system for theAgriculture, Forestry and Fisheries (BDP 98-IV-2-4). B. Senthilkumaran andstudy of peptidergic neuromodulation. In: Parhar IS, Sakuma Y, eds.K. Gen are grateful to the Science and Technology Agency (STA) of JapanGnRH Neurons Gene to Behavior. Tokyo: Brain Shuppan, 1997: 245–276.for fellowships.

16 Gentile F, Lira O, Marcano-de Cotte D. Relationship between braingonadotrophin-releasing hormone (GnRH) and seasonal reproductive

Accepted 27 July 1998 cycle of ‘caribe colorado’, Pygocentrus notatus. Gen Comp Endocr 1986;64: 239–245.

17 Amano M, Aida K, Okumoto N, Hasegawa Y. Changes in salmonReferences GnRH and chicken GnRH-II contents in the brain and pituitary, and

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2 Sherwood NM, von Schalburg K, Lescheid DW. Origin and evolution Biochem 1993; 11: 233–240.of GnRH in vertebrates and invertebrates. In: Parhar IS, Sakuma Y, 19 Yu KL, Nahorniak CS, Peter RE, Corrigan A, Rivier JE, Vale WW.eds. GnRH Neurons Gene to Behavior. Tokyo: Brain Shuppan, 1997: 3–25. Brain distribution of radioimmunoassayable gonadotropin-releasing hor-

3 Amano M, Urano A, Aida K. Distribution and function of gonadotropin- mone in female goldfish: seasonal variation and periovulatory changes.releasing hormone (GnRH) in the teleost brain. Zool Sci 1997; 14: 1–11. Gen Comp Endocr 1987; 67: 234–246.

4 Powell JFF, Zohar Y, Elizur A, Park M, Fischer WH, Craig AG, Rivier 20 Kobayashi M, Amano M, Kim MH, Furukawa K, Hasegawa Y, Aida K.JE, Lovejoy DA, Sherwood NM. Three forms of gonadotropin-releasing Gonadotropin-releasing hormones of terminal nerve origin are nothormone characterized from brains of one species. Proc Nat Acad Sci essential to ovarian development and ovulation in goldfish. Gen CompUSA 1994; 91: 12081–12085. Endocr 1994; 95: 192–200.

5 White SA, Kasten TL, Bond CT, Adelman JP, Fernald RD. Three 21 Parry DM, Goldsmith AR, Millar RP, Glennie LM. Immuno-gonadotropin-releasing hormone genes in one organism suggest novel cytochemical localization of GnRH precursor in the hypothalamus ofroles for an ancient peptide. Proc Nat Acad Sci USA 1995; 92: 8363–8367. European starlings during sexual maturation and photorefractoriness

6 Powell JFF, Krueckl SL, Collins PM, Sherwood NM. Molecular forms J Neuroendocr 1997; 9: 235–243.of GnRH in three model fishes: rockfish, medaka and zebrafish. J Endocr 22 Kim MH, Oka Y, Amano M, Kobayashi M, Okuzawa K, Hasegawa Y,1996; 150: 17–23. Kawashima S, Suzuki Y, Aida K. Immunocytochemical localization of

7 Powell JFF, Standen EM, Carolsfeld J, Borella MI, Gazola R, Fischer sGnRH and cGnRH-II in the brain of goldfish, Carassius auratus.J Comp Neurol 1995; 356: 72–82.WH, Park M, Craig AG, Warby CM, Rivier JE, Val-Sella MV, Sherwood

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