regulation of gonadotropin subunit genes in tilapia

Ž .Comparative Biochemistry and Physiology Part B 129 2001 489�502

Review

Regulation of gonadotropin subunit genes in tilapia

Z. Yarona,�, G. Gur a, P. Melameda,1, H. Rosenfeldb, B. Levavi-Sivanc, A.Elizur b,d

aDepartment of Zoology, Tel-A�i� Uni�ersity, Tel A�i� 69978, IsraelbNational Center of Mariculture, IOLR, Eilat 88112, Israel

cDepartment of Animal Sciences, Faculty of Agriculture, The Hebrew Uni�ersity, Reho�ot 76100, IsraeldBribie Island Aquaculture Research Centre, Department of Primary Industries, Bribie Island, Queensland 4507, Australia

Received 4 August 2000; received in revised form 29 January 2001; accepted 10 February 2001

Abstract

Ž . ŽA steroidogenic tilapia gonadotropin taGtH�LH was purified from pituitaries of hybrid tilapia Oreochromis.niloticus�O. aureus and a homologous RIA was established. This RIA enabled the study of the endocrine regulation of

GtH release, the transduction pathways involved in its secretion and its profile during the spawning cycle. Discrepanciesbetween steroid and taGtH peaks during the cycle led to the conclusion that an additional gonadotropin similar tosalmonid FSH operates early in the cycle. In order to identify this hormone and to study the endocrine control of

Ž .synthesis of all gonadotropin GtH subunits, a molecular approach was taken. The cDNA sequences and the entiregene sequences encoding the FSH� and LH� subunits, as well as an incomplete sequence of the glycoprotein hormone

Ž . Ž .� subunit GP� , were cloned. Salmon gonadotropin-releasing hormone sGnRH elevated mRNA steady-state levels ofŽ .all three GtH subunits in cultured pituitary cells. Pituitary adenylate cyclase-activating polypeptide PACAP and

Ž .neuropeptide Y NPY also stimulated the expression of these subunits and potentiated the effect of GnRH, except thatNPY did not affect FSH�. The GnRH and NPY effects were found to be mediated mainly through protein kinase CŽ . Ž . Ž .PKC , while protein kinase A PKA cascade was involved to a lesser extent. Mitogen-activated protein kinase MAPK

Ž . Ž .cascade takes part in mediating GnRH effects, possibly via PKC. Testosterone T and estradiol E2 , but notŽ .11-ketotestosterone KT , are able to elevate GP� and LH� mRNAs in pituitary cells of early maturing or regressing

males. Low levels of T exposure are associated with elevated FSH� mRNA in cells of mature fish, while higher levelssuppress it, but elevate LH� mRNA. In vivo observations also showed the association of low T levels with increasedFSH� mRNA and high T levels with elevated LH� mRNA. In accordance with these findings, analysis of LH� and

Ž .FSH� 5� gene-flanking regions revealed on both gene promoters a GtH-specific element GSE , half site estrogenŽ . Ž .response elements ERE , cAMP response element CRE and AP1. In vitro experiments showed that recombinant

� Corresponding author. Norman and Rose Lederer Chair of Experimental Biology, Tel-Aviv University. Fax: �972-3-640-9403.Ž .E-mail address: [email protected] Z. Yaron .

1Present address: Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore.

1096-4959�01�$ - see front matter � 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 6 - 4 9 5 9 0 1 0 0 3 4 5 - 1

( )Z. Yaron et al. � Comparati�e Biochemistry and Physiology Part B 129 2001 489�502490

human activin-A leads to higher levels of GP� , FSH� and LH� mRNAs in pituitary cell culture. Porcine inhibinŽ .marginally decreased the mRNA levels of GP� and FSH�, but at a low level 1 ng�ml it stimulated that of LH�. These

results shed some light on certain hypothalamic and gonadal hormones regulating the expression of GtH subunit genesin tilapia. In addition, they provide evidence for their differential regulation, and insight into their mode of action.� 2001 Elsevier Science Inc. All rights reserved.

Ž . Ž .Keywords: Activin; Estradiol; Follicle-stimulating hormone FSH ; Gonadotropin-releasing hormone GnRH ; Inhibin; LuteinizingŽ . Ž . Ž .hormone LH ; Mitogen-activated protein kinase MAPK ; mRNA; Neuropeptide Y NPY ; Oreochromis; Pituitary adenylate

Ž .cyclase-activating polypeptide PACAP ; Pituitary; Promoter; Testosterone

1. The search for tilapia gonadotropins

The early search for the gonadotropic hor-Žmones in tilapia hybrid Oreochromis niloticus�

.O. aureus emanated from a study on in vitrosecretion of estradiol by ovarian fragments. Thestudy provided the protocol for optimal secretionin response to gonadotropic stimulation, whichwas consequently utilized as a sensitive bioassay

Žfor gonadotropins Bogomolnaya and Yaron,.1984; Yaron et al., 1985 . Using the above bioas-

Žsay and classic chromatographic procedures af-finity chromatography on conconavalin ASepharose, ion exchange chromatography onDEAE�Trisacryl, and gel filtration on Ultrogel

. Ž .AcA54 a steroidogenic gonadotropin taGTHŽ .was isolated Bogomolnaya et al., 1989 . The ra-

Ž .dioimmunoassay RIA for tilapia steroidogenicŽ .gonadotropin taGtH facilitated research on go-

nadotropic activity in the fish under natural con-ditions or experimental manipulations.

Tilapias are group-synchronous spawners, ex-hibiting several spawning cycles during the breed-ing season. Under conditions prevailing in Israeliaquaculture, each spawning cycle in Oreochromis

Žniloticus lasts for approximately 28 days Roth-.bard et al., 1983 . A study on the hormonal profile

during such a cycle showed an increase in testos-Ž . Ž .terone T and estradiol E2 levels in mid-cycle,

well before any increase could be noted in taGtHlevel, which only occurred around ovulation andactual spawning. Such an increase would be anal-

Žogous to that of salmonid gonadotropin II GtH.II�LH , and the question arose as to the iden-

tity of a hormone that stimulated the steroidincrease at earlier phases of the cycle. Such ahormone would be physiologically similar to go-

Ž .nadotropin I GtH I�FSH in the salmonidŽ .model Rothbard et al., 1991, 1997 . In order to

identify this hormone and to study the endocrinecontrol of synthesis of all GtH subunits, a molec-ular approach was taken.

2. Isolation and cloning of tilapia gonadotropinsubunit cDNAs and genes

The cDNAs encoding the FSH�, LH� andŽ .glycoprotein hormone GP � subunits were am-

plified from pituitary RNA using RACE-PCR.The amplified products were cloned and se-quenced confirming the identity of each subunit.Comparisons of the amino acid sequences oftilapia gonadotropin subunits with those of otherfish revealed that the LH� and �-subunit have a

Žhigh degree of sequence identity av. 85 and 78%,.respectively , while the FSH� is much more di-

vergent, and has relatively low sequence identityŽ .av. 52%; Rosenfeld et al., 1997 . The sequencesencoding the mature FSH�, LH� and GP� wereused as probes to study the temporal and spatialexpression of the respective genes, as described inthe following sections.

The full sequences of the tilapia FSH� andLH� genes were isolated from the tilapiaŽ . ŽOreochromis mossambicus genomic library akind gift from Dr Swennen and Dr Rentier-

.Delrue, Liege . The genomic organization of both`genes conforms with the pattern characterizing allgonadotropins studied so far in vertebrates, i.e.

Žthree exons, interrupted by two introns Rosen-.feld et al., 1997 . The first intron is positioned at

the signal peptide in the LH� gene, or at the 5�Žuntranslated region in that of FSH� Rosenfeld

.et al., in press . The second intron is located atthe highly conserved position of three amino acidsdownstream from the fifth cysteine. The exon�intron boundaries were defined by comparing thegene sequences with the corresponding full-length

Ž .complementary DNA cDNA clones obtained inthis study by RACE-PCR. Of particular note arethe markedly large introns of both genes.

Southern blot analysis of tilapia genomic DNArevealed that the LH� and FSH� genes are eachpresent in a single copy. These results differ from

( )Z. Yaron et al. � Comparati�e Biochemistry and Physiology Part B 129 2001 489�502 491

preliminary findings in the chinook salmonŽ .Oncorhynchus tshawytscha , in which the possiblepresence of multiple copies of the FSH� gene has

Ž .been indicated Xiong et al., 1994b , and from thegoldfish, in which two distinct genes encodingFSH� are present and expressed in the pituitaryŽ .Sohn et al., 1998a . The multiplicity of the FSH�gene in these species may be a result of theirtetraploid genomes.

3. The FSH� and LH� 5� promoter region andputative response elements

Sequence analysis of the tilapia FSH� and LH�Ž5� flanking regions approx. 1.7 and 1.2 kb, re-

.spectively revealed potential CCAAT and TATApromoter proximal elements. In addition, theyinclude several sequences of cis-acting motifs,which have been demonstrated to be essential forinducible and tissue-specific transcriptional regu-lation of other glycoprotein hormone genes.Among these upstream regulatory sequences are:

Ž .the gonadotrope-specific element GSE ; half sites1Ž .of estrogen response elements ERE ; cAMP2

Ž .response element CRE ; and AP1, the recogni-tion site for the Fos and Jun transcriptional fac-tors. Comparison of the FSH� and LH� 5�flanking regions of tilapia with the available coun-terparts from other teleosts, namely common carp

Ž .LH� Chang et al., 1992 , chinook salmon LH�Ž .Xiong et al., 1994a , and goldfish FSH� and

Ž .LH� Sohn et al., 1998a, 1999, respectively , re-vealed that the promoters from all species share anumber of putative elements; however, their in-ternal organization differs. Unlike the chinooksalmon LH� promoter, which contains a fullfunctional palindromic ERE, as well as several

Ž .half ERE Xiong et al., 1994a; Liu et al., 1995 ,only half ERE motifs were identified in tilapia,carp and goldfish promoters. Moreover, it is inter-

Ž .esting to note the absence of androgen AREand GnRH response elements in both the tilapiaFSH� and LH� gene promoter regions. Thiscomparison also revealed that the promoter re-gion of both tilapia genes does not contain acanonical TATA sequence in the appropriate lo-cation, which is approximately 30 bp upstream ofthe transcription start site. The first TATA box inthe tilapia FSH� gene is located 97 bp upstream

Ž .of the CAP site Rosenfeld et al., in press , andŽ .even further upstream �228 in the LH� gene.

4. Functional analysis of the FSH� promoter

The isolated tilapia FSH� promoter and poten-tial regulatory regions were fused to the coding

Žregion of the firefly luciferase gene Rosenfeld et.al., in press . These gene constructs were trans-

fected into tilapia pituitary cell cultures. An effi-cient expression directed by the tilapia FSH�promoter was obtained in cultured pituitary cellsunder basal conditions. Exposing the transfectedcells to GnRH resulted in a dose-dependent in-crease in luciferase activity, while exposure to

Žgraded doses of E2 suppressed this activity Ro-.senfeld et al., in press . These results are in line

with the FSH� response in non-transfected pitu-itary cells, which showed a positive response toGnRH and a negative response to high levels of T

Ž .or E2 Melamed et al., 2000 .Sequential deletion analysis of the tilapia FSH�

5� flanking regions showed that a construct con-Ž .taining 1211 bp of the promoter �1211 LUC,

which includes the putative CRE sequence, wasŽ .expressed at much higher levels four-fold than

Ž .the longest construct prepared, �1700 LUC. TheŽ .high expression levels observed with �1211 LUC

were dramatically reduced for the consecutive,Ž .�1389 LUC construct, suggesting the presenceof both positive and negative regulatory se-quences, which may be related to the cAMP

Ž .response element CRE within the promoter re-gion examined. These results are in agreement

Ž .with the findings described below Section 6.3that the expression of FSH� is mediated by thecAMP�PKA cascade.

5. Cellular localization of � subunit messenger( )ribonucleic acids mRNAs

In situ hybridization using the cDNA probesfor the GtH � subunits revealed that cells ex-pressing FSH� mRNA are situated adjacent tothe palisade of the somatotrophs that surroundthe nerve fibers ramifying within the proximal

Ž .pars distalis PPD . Cells exhibiting LH� mRNAare more numerous and occupy a more periph-

Žeral position of the PPD Melamed et al., 1998;.Fig. 1 . The area occupied by the latter cells in

pituitary sections overlaps that of cells reactingpositively to the polyclonal anti-taGtH serum.


Ž . Ž .Fig. 1. a Localization of FSH� and b LH� mRNAs in theproximal pars distalis of the pituitary of male tilapia hybridŽ .Oreochromis niloticus�O. aureus . In situ hybridization was

Ž .carried out using digoxygenin DIG -labeled cDNA probes asŽ .in Melamed et al. 1998 . FSH, FSH�; LH, LH�; N, ramifica-

tion of nerve fibers; S, somatotrophs. Scale, 200 �m.

6. The effect of gonadotropin releasing-hormone( )GnRH , pituitary adenylate cyclase-activating

( ) ( )polypeptide PACAP and neuropeptide Y NPY ,and their signal transduction pathways

6.1. LH release and its signal transduction cascades

As in other vertebrates, including fish, GnRHwas found to stimulate LH release in tilapia hy-brids. The in vivo effect of a superactive salmon

Ž .GnRH analog sGnRHa is exerted within 10�15min, reaching a peak after 3�6 h. The height ofthe peak is positively dependent on the tempera-

Ž .ture Gissis et al., 1991 . Studies using perifusionŽexperiments Levavi-Sivan and Yaron, 1989;

.Yaron and Levavi-Sivan, 1990 or static culture ofŽdispersed pituitary cells Levavi-Sivan and Yaron,

.1992, 1993; Levavi-Sivan et al., 1995 demon-

strated certain steps in the transduction of GnRHsignal. These include Ca2� influx and itsmobilization from intracellular sources, phospho-

Ž .lipase C PLC , known to stimulate phosphoinosi-tide turnover, resulting in the formation of inosi-

Ž . Ž .tol trisphosphate IP and diacylglycerol DAG ,3Ž .and protein kinase C PKC . Phospholipase A 2

and arachidonic acid were also found to be asso-ciated with taGtH release from the pituitary of

Ž .tilapia Yaron and Levavi-Sivan, 1990 .ŽIn contrast to the situation in goldfish Jobin et

.al., 1996 , transduction of the GnRH signal intilapia also involves a cAMP�protein kinase AŽ .PKA pathway as an additional and parallel

Žtransduction cascade Levavi-Sivan and Yaron,.1992 . However, the exact interaction or cross-talk

between PKA and other transduction cascades intilapia needs further investigation.

6.2. GnRH effects on GtH subunit mRNAs

A direct effect of sGnRH on LH� mRNA wasfirst demonstrated in primary culture of tilapia

Ž .hybrids Oreochromis niloticus�O. aureus pitu-itary cells. A time�response study indicated aninitial increase in the transcript 12 h after expo-

Ž .sure to the neuropeptide 10 nM , and furtherŽelevation occurred after 18 and 24 h Melamed et

.al., 1996 . In a subsequent study in tilapia, thesame concentration of sGnRH led, after 24 h, toa 3.7-fold increase in GP� mRNA, 2.7-fold inLH� mRNA and only 1.7-fold in that of FSH�Ž .Gur et al., 2000 .

These results are in line with the finding in theŽ .striped bass Morone saxatilis , in which the re-

sponses of GP� and LH� mRNAs of maturingmales to GnRH implantation were higher than

Ž .that of FSH Hassin et al., 1998 . Similarly, im-planting GnRHa in vitellogenic homing sockeyesalmon resulted in increased mRNA levels of

ŽGP� and LH�, but not those of FSH� Kitahashi.et al., 1998 . Apparently opposite results were

obtained in pituitary cell primary culture of 2-Ž .year-old coho salmon Oncorhynchus kisutch ,

Ž .where sGnRH 1 and 100 nM resulted in in-Žcreased levels of the two GP� subunits � and1

.� and FSH� mRNAs, but not in that of LH�2Ž .Dickey and Swanson, 2000 . It was assumed thatthe donor fish were at a stage too early for LHproduction, which normally appears only whenfish approach spawning.


FSH� and LH� mRNAs in common carpŽ .Cyprinus carpio were stimulated differentially bysGnRHa, the effect being dependent on the re-productive phase. In vitellogenic females, mRNAlevel of both FSH� and LH� increased 6 h after

Ž .a single injection of the peptide 10 �g�kg , withthe increase in FSH� being higher than that ofLH�. However, in post-vitellogenic females, onlyLH� mRNA increased in response to GnRHaŽ .Kandel-Kfir et al., 2000 . Different potencies forsGnRH and chicken GnRH II were reported ingoldfish, but FSH� mRNA was not included in

Ž .the report Khakoo et al., 1994 .Information on additional fish and stages of

reproduction is still needed. However, it wouldappear that the type of the GtH subunit genepreferentially expressed in response to GnRH isdependent on the reproductive phase. How thisinformation is relayed to the gonadotrophs is animportant topic for future studies, with the tem-poral and spatial distribution of GnRH receptorbeing a possible candidate for such a differentialresponse.

6.3. Effects and signal transduction of PACAP andNPY

Although ample information is available on thehypothalamic regulation of LH release in fish and

Žthe transduction of GnRH signal reviewed byChang and Jobin, 1994; Van Der Kraak et al.,

.1998 , much less is known on the regulation ofGtH subunit genes by other hypothalamic pep-tides. The studies in tilapia were undertaken inorder to examine the effect of PACAP and NPYon GtH subunit mRNA levels as a proximateparameter for hormone production. Pituitary cells

�from maturing male tilapia hybrids Oreochromisniloticus�O. aureus; body weight, 30�51 g; gona-

Ž . �dosomatic index GSI , 0.27�0.04 were culturedfor 4 days and then exposed for 24 h to sGnRH

Ž . Ž .10 nM , PACAP 38 0.001�0.1 nM or NPYŽ .0.01�1 nM , either alone or with sGnRH. Theinvolvement of PKA and PKC in the signal trans-duction of the peptides was examined by exposing

Ž .the cells to either PKA inhibitor H89 or to theŽ .PKC inhibitor GF109203X; GF given 15 min

prior to the stimulants.Exposure to sGnRH increased the transcript

levels of all GtH subunits, with FSH� being leastaffected. However, the GnRH effect seems to betransduced differently on each subunit. While thesGnRH-dependent increase in GP� and LH�mRNA could be reduced by PKC inhibition, thatof FSH� was attenuated only when PKA was

Žinhibited Fig. 2 illustrates the effect on FSH�mRNA; see Table 1 for summary of results; Gur

.et al., 2000 .PACAP 38, which acts as growth hormone-

Žreleasing hormone in fish Parker et al., 1997;.Montero et al., 1998 , was found to increase taGtH

release in tilapia hybrids. PACAP also led toincrease in mRNA levels of all GtH subunitmRNAs. As the inhibitors of both PKA and PKCsuppressed the stimulatory effect of PACAP onmRNA levels of the three subunits, it was as-sumed that both PKA and PKC pathways are

Ž .involved in this stimulation Gur et al., 2000 .This assumption is in agreement with the findingof a putative cAMP response element, and AP1response element, in the 5� untranslated region of

Žboth FSH� and LH� genes of tilapia Elizur et.al., 2000 . Exposing trout pituitary cells tran-

siently transfected with salmon GP��CATchimeric plasmids to 8-Br-cAMP stimulated theiractivity, also suggesting cAMP�PKA involvementin GP� gene transcription in the chinook salmonŽ .Suzuki et al., 1995 . In mammals, PACAP wasobserved to stimulate LH release by accumula-tion of intracellular cAMP in the gonadotrophs,but the release was also found to be dependent

Ž .on the PKC activation reviewed by Evans, 1999 .

Table 1aSummary of signal transduction pathways

LH LH� FSH� GP�

release transcription transcription transcription

GnRH PKA, PKC PKC PKA PKA, PKCPACAP38 PKA, PKC PKA, PKC PKA, PKC PKA, PKCNPY PKC PKA, PKC No effect PKA, PKC

a Summary of signal transduction pathways of GnRH, PACAP38 and NPY on GtH release from pituitary cell culture of tilapia andGtH subunit genes transcription. Principal pathways in bold letters.


Fig. 2. FSH� mRNA levels in pituitary cells of maturing maleŽhybrid tilapia Oreochromis niloticus�O. aureus; body weight,

. Ž30�51 g; GSI, 0.27�0.04 exposed in culture to GnRH Gn,. Ž . Ž .10 nM , PACAP38 PA, 0.01 nM or NPY NP, 0.1 nM in the

Ž .absence or presence of 100 nM of the PKA inhibitor H89 HŽ .or the PKC inhibitor GF109203X GF . Mean�SEM, n�

3�6. Means marked by the same letter do not differ from eachŽ .other P�0.05 .

Similar to the situation in mammals, it is possiblethat the activation by PACAP in tilapia involvestwo intracellular pathways that have been tradi-tionally considered distinct. The possible exis-tence of cross-talk between these pathways has

Žrecently become evident e.g. Han and Conn,.1999 .

Exposure to NPY was followed by increasedmRNA levels of GP� and LH�. The effects onLH release were mediated through the PKCpathway, whereas the regulation of LH� tran-script was found to be via PKA. It is significant tonote that NPY had no effect on FSH� mRNAŽ .Fig. 2; Table 1; Gur et al., 2000 .

In addition to the direct modulatory activity ofPACAP and NPY on gonadotropin secretion andsubunit gene expression, these peptides werefound to augment GP� mRNA levels in responseto GnRH when applied directly to the cultured

Ž .cells Gur et al., 2000 . In goldfish implanted invivo with E2 and T, NPY significantly stimulatedthe release of GnRH from hypothalamic slicesand was considered to be one of the mechanismsmediating the feedback of these steroids in the

Ž .pituitary Peng et al., 1993 . Similarly, NPY wasfound to stimulate the in vitro release of seabreamGnRH in the preoptic area anterior hypothala-mus, and also from the pituitary of the red

Žseabream, Pagrus major Senthilkumaran et al.,.2000 . These results corroborate earlier findings

in ovulated rainbow trout, in which NPY-stimu-lated GtH release was reduced by GnRH antago-

Ž .nist Breton et al., 1990 . However, this route ofaction cannot explain the foregoing effects ofNPY in pituitary cells of tilapia. This is becauseGnRH by itself increased the mRNA levels of allGtH subunits, whereas NPY lacked any effect onFSH� mRNA. If NPY had acted on the pituitarycells only indirectly, by increasing the release ofGnRH from pituitary cells, it would have alsoincreased the level of FSH� mRNA. As suchaction did not occur, it is concluded that NPY intilapia can exert its effect directly at the level ofthe gonadotrophs.

7. The stabilizing effects of cAMP on LH� mRNA

In order to reveal possible ways by which thecAMP�PKA pathway affects the LH� steady-state mRNA level, pituitary cells in culture wereexposed for 24 h to either forskolin or the PKC

Žstimulator TPA 1-O-tetradecanoyl phorbol-13-.acetate . Actinomycin D was then added to these

and to control cells to block further transcription,and the level of remaining LH� mRNA was

Ž .determined at intervals Melamed et al., 1996 .The rate of mRNA degradation in cells exposedto forskolin was considerably lower than in con-

Ž 1trol or TPA-treated cells apparent T of �14 vs.2

.�8.4 h . The mechanism by which mRNA isstabilized has been reported in rat pituitary cells,where forskolin increased the size of the LH�transcripts, reflecting changes in the length of the

Ž .poly A tail which is associated with increasedŽtranscript stability Weiss et al., 1992; Ishizaka et

.al., 1993 .

( )8. Mitogen-activated protein kinase MAPK —ERK1��2

Ž .G-protein-coupled receptors GPCRs , such asthat of GnRH, are known to convey the extracel-lular signal by a set of sequentially activatedprotein kinases, which are collectively known as


Ž .the mitogen-activated protein kinase MAPKcascades. The GPCRs activate the MAPK cas-cade via mechanisms involving PKC-dependent or-independent pathways, by the �� subunits ofG-proteins and by various potential sites of acti-

Žvation, such as Ras and Raf-1 Seger and Krebs,.1995 . Since MAPK is translocated to the nu-

cleus, stimulation of MAPK leads to the activa-Ž .tion of transcription factors e.g. Elk and c-fos ,

initiating cellular responses, such as growth anddifferentiation. In mammals, GnRH stimulates

ŽMAPK also known as extracellular signal-regu-.lated kinases, ERK1�2; p42�44 MAPK in pitu-

itary cells and, in turn, is involved in gene expres-Žsion of the gonadotropin �-subunit Roberson et

.al., 1995; reviewed by Naor et al., 2000 . Fol-lowing this paradigm, it was hypothesized that theeffects of GnRH on GtH subunit gene expressionin tilapia also involve the MAPK cascade. To testthis hypothesis, pituitary cells were exposed tosGnRH with or without the PKC inhibitorŽ . Ž .GF109203X; GF or the MEK MAPK kinase

Ž .inhibitor PD-098059; PD given 15 min beforesGnRH. Cell protein extract was immunoblotted

Žusing antisera for mammalian active phosphory-. Žlated ERK 42�44 and general unphosphory-. Ž .lated ERKs Gur et al., in press .

Exposure to sGnRH resulted in a dose-relatedincrease in active ERK1 and ERK 2, while GFand PD suppressed ERK1 dose-dependently.Moreover, inhibiting MEK by PD reduced, notonly ERK1�2, but also the mRNA levels of GP�and LH�. However, mRNA levels of FSH� wereunaffected. These results indicate the involve-ment of the MAPK cascade in the transduction ofGnRH signal leading to GP� and LH� geneexpression, and that this activation is PKC-depen-

Ž .dent Gur et al., in press . The lack of MAPKinvolvement in FSH� transcription is in line withthe results of its promoter analysis, highlightingthe role of the cAMP�PKA cascade in its regula-

Ž .tion see Section 4; Rosenfeld et al., in press .

9. Sex steroids and GtH subunit transcripts

9.1. Studies in tilapia

Information on the impact of sex steroids onGtH subunit mRNA in tilapia is derived from two

Ž .sources: a from observations that fish at differ-ent stages of gonadal development, exhibiting

varying levels of circulating sex steroids, havedifferent levels of FSH� and LH� mRNA in the

Ž .pituitary; and b from in vitro experiments onexposure of cultured pituitary cells to sex steroids.

9.1.1. FSH� and LH� mRNA in fish with �ariousle�els of circulating sex steroids

ŽMale hybrid tilapia Oreochromis niloticus�O..aureus , collected from local farms and reared in

tanks in a greenhouse under natural photoperiod,were examined either during the breeding seasonŽ .June�September, water temperature 22�28�C ,

Žor during the reproductive resting phase late.November�January, water temperature 16�20�C .

Fish were weighed, blood sampled, gonads wereexcised and weighed, and pituitaries were takenfor determination of GtH� subunit mRNA levelsby slot-blot hybridization.

High levels of FSH� mRNA were observedonly in fish with relatively low levels of sex steroidsŽ .�1 ng�ml T in males or E2 in females . In fishwith higher or lower steroid levels, the quantitiesof FSH� mRNA were low. In fish with highersteroid levels, LH� transcript levels were alsohigh. In regressed male fish, a positive linearcorrelation was observed between the quantitiesof LH� mRNA in the pituitary and circulating T

Ž .levels Melamed et al., 2000 .

9.1.2. In �itro effect of T and E2 on gonadotropinsubunit mRNAs

In order to reveal whether the steroids affectGtH subunit gene transcription directly at thepituitary level, cells taken from fish at variousstages of their reproductive phase were dispersedand cultured. On the third day of culture, T or E2were added to the medium for 48 h, after whichthe RNA was extracted from the cells and hy-bridized.

ŽIn pituitary cells from immature males GSI�.0.1�0.02 , the FSH� transcript increased in re-

sponse to 10 nM T given for 36�48 h. In cellsŽ .from maturing males GSI�0.56�0.1 , only ex-

Ž .posure to low doses of T 10�100 pM caused anŽincrease in FSH� mRNA levels Melamed et al.,

.1997 . In fish at the end of the spawning seasonŽ .GSI�0.42�0.07 , levels of the FSH� mRNAdecreased following exposure to higher doses of

Žthe steroids T at 10 and 100 nM and E2 at 1�100. Ž .nM . The addition of T or E2 10 pM�100 nM

did not result in any change in LH� mRNAŽ .Melamed et al., 2000 . Cultured pituitary cells


Ž .Fig. 3. Schematic presentation of testosterone 10 nM effecton mRNA levels of FSH� and LH� subunits at various stagesof gonadal development in tilapia.

Ž .taken from regressed fish GSI�0.125�0.02Ž .and exposed to T 0.1�10 nM showed a dose-

dependent increase in the mRNA of LH� levelŽ .up to 450% . However, no change was observed

Žin the levels of FSH� mRNA summarized in Fig..3 . Exposure of the cells to 11-ketotestosteroneŽ .KT at the same concentrations was not followedby any change in the mRNA levels, either of

Ž .FSH� or of LH� Melamed et al., 1998 .To study the effect of T on GP� subunit,

pituitary cells were taken from immature maleŽ .hybrid tilapia GSI�0.18�0.05 or mature males

Žat the beginning of the reproductive season GSI.�0.33�0.05 and cultured for 3 days. They were

Ž .then exposed to graded doses of T 0.01�100 nM .GP� mRNA levels increased only in cells takenfrom the fish with the more developed gonadsŽ .Gur et al., 1999 .

The results presented indicate that T has aneffect similar to that of E2 on the transcription ofGtH subunits, and that the non-aromatizable an-drogen lacks such an effect. This implies that theeffect of T in tilapia pituitary is exerted via itsaromatization to E2. When dispersed pituitarycells were separated on a density gradient,aromatase activity was localized to the go-nadotropin-enriched fraction and was not pres-ent in the fraction containing the somatotrophsŽ .Melamed et al., 1999 .

9.2. The situation in other fish

9.2.1. LH�Testosterone, other aromatizable androgens, or

estradiol, increase the mRNA levels of LH� inŽgoldfish Khakoo et al., 1994; Huggard et al.,

.1996; Sohn et al., 1998b; Kobayashi et al., 2000 ,

Ž .coho salmon Dickey and Swanson, 1998 andŽ .European eel Querat et al., 1991 . Similarly, LH´

pituitary content increased following treatmentŽ .with T or E2 in rainbow trout Breton et al., 1997

Žand mature parr Atlantic salmon Antonopoulou.et al., 1999 . LH� transcript and LH de novo

synthesis were elevated by exposure of AfricanŽcatfish pituitary cells to T or E2 Rebers et al.,

.2000 .

9.2.2. FSH�Similar to the situation in tilapia, transcript

levels of FSH were reduced by T or E2 treatmentŽ .in coho salmon Dickey and Swanson, 1998 and

in juvenile goldfish fed steroid-containing pelletsŽ .Kobayashi et al., 2000; Sohn et al., 2000 . FSHpituitary content declined 30 days after implanta-

Ž .tion of E2 in trout Breton et al., 1997 . Remov-ing the source of sex steroids by castration re-sulted in a dramatic increase in pituitary FSHcontent in mature parr Atlantic salmon in summerŽ .Antonopoulou et al., 1999 , indicating a negative

Ž .feedback of gonadal hormone s on FSH. Theelevation of FSH� mRNA in castrated goldfish

Žcould be reversed by T, E2 or KT Kobayashi et.al., 2000 .

9.2.3. GP�Chronic in vivo treatment with T or E2 was

followed by an increase in GP� mRNA in Euro-Ž .pean eel Counis et al., 1987; Querat et al., 1991 .´

However, addition of T or E2 to eel pituitary cellculture for 24 or 72 h, or even 13 days, failed toincrease GP� mRNA. These results are in con-trast with the increased transcript levels in Africancatfish pituitary cell culture exposed to T for 48 h,with such an increase being abated by the aro-

Ž .matase inhibitor, ATD Rebers et al., 2000 . Thesituation in goldfish is controversial. In sexuallyimmature fish, T or 11-hydroxyandrosterone atphysiological doses increased GP� mRNA, whilehigh, non-physiological doses of both androgensgiven in vivo was not stimulatory, or even sup-pressed the transcript levels. However, when suchhigh doses were given to mature fish, the GP�transcript did increase. T was also effective whengiven continuously to pituitary fragments in peri-

Ž .fusion Huggard et al., 1996 . It should be noted,however, that such perifused pituitary fragmentscontain nerve fibers carrying hypothalamic hor-mones, which may be sensitive to the presence ofthe steroids. In another study on adult goldfish


Žimplanted for 2 weeks with T, E2 or KT Sohn et.al., 1998b , or in juvenile goldfish fed with these

steroids, no change could be noted in the level ofŽ .GP� transcript Kobayashi et al., 2000 .

9.3. Is the effect androgenic or estrogenic?

In most fish species, the effect of sex steroidson GtH subunit gene expression or on GtH con-tent is attributed to estradiol, since the ad-ministration of non-aromatizable androgens orabating aromatase activity abolished the effectŽ .Antonopoulou et al., 1999; Rebers et al., 2000 .An exception is the situation in the European eel,in which T elevated LH� mRNA, but not that ofGP� , and also stimulated LH production. Similareffects could also be induced by non-aromatizableandrogens, but not by estradiol, indicating an

Žandrogen-specific effect in this fish Huang et al.,.1997 . There is no agreement about the situation

in goldfish, as the non-aromatizable androgen11-hydroxyandrosterone was effective in increas-

Žing both GP� and LH� mRNAs Huggard et al.,.1996 . In another study, KT failed to increase the

levels of these transcripts, whereas T and E2 wereŽeffective Sohn et al., 1998b; Kobayashi et al.,

.2000 .

The foregoing account and the review by Du-Ž .four et al. 2000 show that a considerable volume

of information has been accumulated on the sti-mulatory effect of T and E2 on LH� mRNA andon LH pituitary content and release. Most sourcesreport the results of steroid treatment in vivo;however, such a route of administration cannotaccurately target the site of steroid effect or itsmode of action. Therefore, reports on steroideffect on GtH content or mRNA achieved in vivomay not match, or may even contradict results

Žobtained in vitro see Huang et al., 1997; Rebers.et al., 1997, 2000 . Specifically, these studies can-

not distinguish between direct effects of thesteroids on the pituitary and possible indirect

Žeffects mediated by hypothalamic agents re-.viewed by Van Der Kraak et al. 1998 . For exam-

ple, E2 alone, or in combination with androgens,can elevate brain and pituitary mGnRH levels,which will also lead to an increase in pituitary

Ž .GtH content Montero et al., 1995 . Furthermore,sex steroids are able to stimulate the release ofother hypophysiotropic hormones, such as NPY,which may exert their own effect on the pituitary

Ž .or on GnRH release Peng et al., 1993 . T and E2can potentiate the in vitro stimulation of LH

Ž .release and FSH to some extent by gamma-

ŽFig. 4. mRNA levels of GP� , FSH� and LH� in pituitary cells of hybrid male tilapia Oreochromis niloticus�O. aureus; body weight,. Ž . Ž . Ž .56�120 g; GSI, 0.13�0.01 exposed in culture to: a recombinant human activin A; and b porcine inhibin. Mean�SEM n�3�4 .

Unmarked bars indicate SEM too small to be presented graphically. Means marked by the same letter do not differ from each otherŽ .P�0.05 .


Ž . Ž .aminobutyric acid GABA Mananos et al., 1999 ,˜ ´although it is not known yet whether such effectsalso involve increased subunit gene transcription.

10. Inhibin and activin effect on GtH subunitmRNAs

Preliminary results in male tilapia hybridsŽ .Oreochromis niloticus�O. aureus have shownthat recombinant human activin A added to cul-tured pituitary cells at 0.5�20 ng�ml for 48 h ledto an increase in all gonadotropin subunitmRNAs. FSH� mRNA increased by more than24-fold at the maximal concentration of 20 ng�ml,while that of LH� increased by up to 12-fold. Theleast affected was GP� mRNA, which increased

Ž .by only 2.5-fold Fig. 4a .Only in one respect do these results agree with

those reported in goldfish, where the homologousrecombinant activin B given to culture of pitu-itary cells led to an increased level of FSH�

Ž .mRNA Yam et al., 1999 . In contrast to thesuppressive effect of activin B on LH� mRNAlevel in goldfish, in tilapia there was an increasein LH� transcript after 48-h exposure to humanactivin A, although utilized at lower concentra-tions compared to goldfish. This discrepancy can

Žbe attributed to the different type of activin A or.B used in these studies, to the heterologous

Ž .source of the peptide human used in tilapia; orto an inherent difference between the fish species.Pituitary cells of coho salmon also responded torecombinant human activin A by an increase in

Ž .mRNA of both GP� 1 and 2 and FSH�, with noŽ .effect on LH� Davies et al., 2000 . However, the

donor fish in this study were immature malesŽ .GSI 0.04 , a stage at which LH� mRNA did not

Žincrease, even in response to GnRH Dickey and.Swanson, 2000 . It should be noted that porcine

inhibin A and activin A both stimulate the releaseof LH from cultured pituitary cells of goldfishŽ .Ge et al., 1992 .

A 48-h exposure of cultured pituitary cells ofŽ .tilapia hybrids to porcine inhibin 0.5�20 ng�ml

resulted only in a marginal and non-significantdecrease in the mRNA levels of GP� and FSH�.The response of LH� was more complicated: atinhibin concentrations of 0.5 and 1 ng�ml therewas a dose-dependent increase of up to five-foldin the transcript, whereas at higher concentra-tions of the peptide, the stimulatory effect was

Ž .reduced considerably Fig. 4b . These results bearsome similarity to the situation in rainbow trout,in which recombinant human inhibin was foundto inhibit FSH release, but to stimulate LH re-

Žlease from cultured pituitary cells Chyb and Bre-.ton, 2000 . Due to the foregoing contradictory

results, it would appear that more information isrequired on the effect of these gonadal peptideson gonadotropin subunit genes, in respect to go-nadal stage, before any solid conclusion can bereached.

11. Conclusions

There is a clear indication in the foregoingparagraphs that the response of the pituitary go-nadotrophs to hypothalamic or gonadal hormonesis dependent on the maturation stage of thetreated fish or the donor fish. Our attempt tosummarize the results published so far was ham-pered by inaccurate descriptions of the gonadalstate. GSI can provide only an approximate esti-mate of the real situation of the gonads, but notof the receptor abundance of the stimulating hor-mone. Furthermore, comparison between gonadalstate in males and females is questionable, whichcould well be one source of discrepancy betweensimilar experiments. Another source of differencederives from results achieved in vivo or in vitro.Indeed, experiments on isolated cells suffer fromthe artificial environment in which the cells arecultured. However, such experiments have theadvantage of examining the direct effect of theadministered hormone with minimal interventionof other factors.

In spite of the above, the data presented in thisreview indicate that expression of each go-nadotropin subunit responds differently to theadministered hormones. In general, the responsesof LH� and GP� are similar, whereas that ofFSH� differs. It is suggested that transduction ofthe GnRH signal, leading to the increased expres-sion of LH� and GP� , is mainly mediated throughPKC and MAPK cascades, whereas that of FSH�is through cAMP�PKA. The results of the FSH�promoter analysis in tilapia corroborate this sug-gestion.

NPY stimulates the transcription of LH� andGP� , but has no effect on that of FSH�. A hightranscript level of FSH� is associated with rela-tively low testosterone or estradiol levels, while a


high transcript level of LH� is associated withhigh levels of these steroids.

Still more information is required on the regu-lation of fish gonadotropin subunit genes and theactual levels of the two gonadotropins in thecirculation of fish. We hope that the above ac-count will stimulate research into this importantfield of comparative endocrinology.

Acknowledgements

The article is based on research projects sup-ported by research grants from the Israeli Min-istry of Science and Technology to A.E. and Z.Y.,and from the Israel Science Foundation to Z.Y.and A.E. We thank Mr A. Gissis and KibbutzHa’Maapil hatchery for the supply of fish, MsMichal Ofir, Ms Sara Kinnamon, Ms M. Kandel-Kfir, Mr Y. Cohen, Ms Yael Zilberstein and MsHelena Safarian for their help. We thank Ms NilliZmora and Ms Iris Meiri for their valuable con-tribution towards the molecular cloning of theGtH subunit cDNAs, and Mr Itai Ivri and MrBarak Yarden for the maintenance of tilapiastocks. Our thanks extend to Professor Z. Naorand Mr D. Bonfil for advice and to Ms Naomi Pazfor reading the manuscript.

References

Antonopoulou, E., Swanson, P., Mayer, I., Borg, B.,1999. Feedback control of gonadotropins in Atlanticsalmon, Salmo salar, male parr � II. Aromataseinhibitor and androgen effects. Gen. Comp. En-docrinol. 114, 142�150.

Bogomolnaya, A., Yaron, Z., Hilge, V., Graesslin, D.,Lichtenberg, V., Abraham, M., 1989. Isolation andradioimmunoassay of a steroidogenic gonadotropinof tilapia. Isr. J. Aquaculture 41, 123�136.

Bogomolnaya, A., Yaron, Z., 1984. Stimulation in vitroof estradiol secretion by the ovary of the cichlid fish,Sarotherodon aureus. Gen. Comp. Endocrinol. 53,187�196.

Breton, B., Mikolajczyk, T., Weil, C., Danger, J.M.,Vaudry, H., 1990. Studies on the mode of action of

Ž .neuropeptide Y NPY on maturational go-Ž .nadotropin GtH secretion from perifused rainbow

trout pituitary glands. Fish Physiol. Biochem. 8,339�346.

Breton, B., Sambroni, E., Govoroun, M., Weil, C., 1997.Effects of steroids on GtH I and GtH II secretionand pituitary concentration in the immature rainbow

trout Oncorhynchus mykiss. C. R. Acad. Sci. Ser.III-Vie 320, 783�789.

Chang, J.P., Jobin, R.M., 1994. Regulation of go-nadotropin release in vertebrates: a comparison ofGnRH mechanisms of action. In: Davey, K.G., Pe-

Ž .ter, R.E., Tobe, S.S. Eds. , Perspectives in Compara-tive Endocrinology. National Research Council ofCanada, Ottawa, pp. 41�51.

Chang, Y.S., Huang, F.L., Lo, T.B., 1992. Isolation andsequence analysis of carp gonadotropin �-subunitgene. Mol. Mar. Biol. Biotechnol. 1, 97�105.

Chyb, J., Breton, B., 2000. Effects of recombinant hu-man inhibin on GTH I and GTH II secretion fromdispersed pituitary cells of female rainbow troutduring the reproductive cycle. In: Norberg, B.,Kjesbu, O.S., Taranger, G.L, Andersson, E., Ste-

Ž .fansson, S.O. Eds. , Proceedings of the 6th Interna-tional Symposium on Reproductive Physiology ofFish, Bergen, 1999. University of Bergen, Bergen,pp. 472�474.

Counis, R., Dufour, S., Ribot, G., Querat, B., Fontaine,´Y-A., Jutisz, M., 1987. Estradiol has inverse effectson pituitary glycoprotein hormone �-subunitmessenger ribonucleic acid in the immature Euro-pean eel and gonadectomized rat. Endocrinology121, 1178�1184.

Davies, B., Dickey, J., Swanson, P., 2000. Regulation ofŽ . Ž .FSH GTH I and LH GTH II in coho salmon

Ž .Oncorhynchus kisutch : the action of recombinanthuman activin and a salmon testis extract. In: Nor-berg, B., Kjesbu, O.S., Taranger, G.L, Andersson, E.,

Ž .Stefansson, S.O. Eds. , Proceedings of the 6th Inter-national Symposium on Reproductive Physiology ofFish, Bergen, 1999. University of Bergen, Bergen, p.486.

Dickey, J.T., Swanson, P., 1998. Effects of sex steroidsŽ .on gonadotropin FSH and LH regulation in coho

Ž .salmon Oncorhynchus kisutch . J. Mol. Endocrinol.21, 291�306.

Dickey, J.T., Swanson, P., 2000. Effects of salmon go-nadotropin-releasing hormone on follicle stimulatinghormone secretion and subunit gene expression in

Ž .coho salmon Oncorhynchus kisutch . Gen. Comp.Endocrinol. 118, 436�449.

Dufour, S., Huang, Y.S., Rousseau, K. et al., 2000.Puberty in teleosts: new insights into the role ofperipheral signals in the stimulation of pituitary go-nadotropins. In: Norberg, B., Kjesbu, O.S., Taranger,

Ž .G.L, Andersson, E., Stefansson, S.O. Eds. ,Proceedings of the 6th International Symposium onReproductive Physiology of Fish, Bergen, 1999. Uni-versity of Bergen, Bergen, pp. 455�461.

Elizur, A., Zmora, N., Meiri, I. et al., 2000. Go-nadotropins � from genes to recombinant proteins.In: Norberg, B., Kjesbu, O.S., Taranger, G.L, Ander-

Ž .sson, E., Stefansson, S.O. Eds. , Proceedings of the


6th International Symposium on Reproductive Phys-iology of Fish, Bergen, 1999. University of Bergen,Bergen, pp. 462�465.

Evans, J.J., 1999. Modulation of gonadotropin levels bypeptides acting at the anterior pituitary gland. En-docrine Rev. 20, 46�67.

Ge, W., Chang, J.P., Peter, R.E., Vaughan, J., Rivier, J.,Vale, W., 1992. Effects of porcine follicular fluid,inhibin A and activin A on goldfish gonadotropinrelease in vitro. Endocrinology 131, 1922�1929.

Gissis, A., Levavi-Sivan, B., Rubin-Kedem, H., Ofir, M.,Yaron, Z., 1991. The effect of gonadotropin-releas-ing hormone superactive analog and dopamine an-tagonists on gonadotropin level and ovulation intilapia hybrids. Isr. J. Aquacult.-Bamidgeh 43,123�136.

Gur, G., Melamed, P., Zilberstein, Y. et al., 1999.Effects of GnRH and steroids on the transcriptionof the tilapia glycoprotein hormone � subunit gene.In: Roubos, E.W., Wendelaar Bonga, S.E., Vaudry,

Ž .H., De Loof, A. Eds. , Recent Developments inComparative Endocrinology and Neurobiology.Shaker Publishers, Maastricht, pp. 29�30.

Gur, G., Melamed, P., Rosenfeld, H., Elizur, A., Yaron,Z., 2000. Mechanisms involved in the effect ofGnRH, PACAP and NPY on gonadotropin subunitmRNAs in tilapia pituitary cells. In: Norberg, B.,Kjesbu, O.S., Taranger, G.L, Andersson, E., Ste-


Gur, G., Bonfil, D., Safarian, H., Naor, Z., Yaron, Z.GnRH receptor signaling in tilapia pituitary cells:

Ž .role of mitogen-activated protein kinase MAPK .Comp. Biochem. Physiol., in press.

Han, X-B., Conn, P.M., 1999. The role of proteinkinase A and C pathways in the regulation of mito-gen-activated protein kinase activation in responseto gonadotropin-releasing hormone receptor activa-tion. Endocrinology 140, 2241�2251.

Hassin, S., Gothilf, Y., Blaise, O., Zohar, Y., 1998.Gonadotropin-I and -II subunit gene expression of

Ž .male striped bass Morone saxatilis after go-nadotropin-releasing hormone analogue injection:quantitation using an optimized ribonuclease protec-tion assay. Biol. Reprod. 58, 1233�1240.

Huang, Y.S., Schmitz, M., Lebelle, N., Chang, C.F.,Querat, B., Dufour, S., 1997. Androgens stimulate´gonadotropin-II� subunit in eel pituitary cells invitro. Mol. Cell. Endocrinol. 131, 157�166.

Huggard, D., Khakoo, Z., Kassam, G., Mahmoud, S.S.,Habibi, H.R., 1996. Effect of testosterone on matu-rational gonadotropin subunit messenger ribonucleicacid levels in the goldfish pituitary. Biol. Reprod. 54,1184�1191.

Ishizaka, K., Tsujii, T., Winters, S.J., 1993. Evidence fora role for the cyclic adenosine 3�,5�-monophos-phate�protein kinase-A pathway in regulation of thegonadotropin subunit messenger ribonucleic acids.Endocrinology 133, 2040�2048.

Jobin, R.M., Van Goor, F., Neumann, C.M., Chang,J.P., 1996. Interaction between signaling pathways inmediating GnRH-stimulated GTH release fromgoldfish pituitary cells: protein kinase C, but notcyclic AMP, is an important mediator of GnRH-stimulated gonadotropin secretion in goldfish. Gen.Comp. Endocrinol. 102, 327�341.

Kandel-Kfir, M., Gur, G., Melamed, P. et al., 2000.FSH� and LH� mRNAs in maturing and mature

Ž .common carp Cyprinus carpio : their response toGnRH. In: Norberg, B., Kjesbu, O.S., Taranger, G.L,

Ž .Andersson, E., Stefansson, S.O. Eds. , Proceedingsof the 6th International Symposium on ReproductivePhysiology of Fish, Bergen, 1999. University ofBergen, Bergen, p. 483.

Khakoo, Z., Bhatia, A., Gedamo, L., Habibi, H.R.,1994. Functional specificity for salmon go-

Ž .nadotropin-releasing hormone GnRH and chickenGnRH II coupled to gonadotropin release and subu-nit messenger ribonucleic acid level in the goldfishpituitary. Endocrinology 134, 838�847.

Kitahashi, T., Alok, D., Ando, H. et al., 1998. GnRHanalog stimulates gonadotropin II gene expression inmaturing sockeye salmon. Zool. Sci. 15, 761�765.

Kobayashi, M., Sohn, Y.C., Yoshiura, Y., Aida, K.,2000. Effects of sex steroids on the mRNA levels ofgonadotropin subunits in juvenile and ovariec-tomized goldfish Carassius auratus. Fish. Science 66,223�231.

Levavi-Sivan, B., Yaron, Z., 1989. Gonadotropin secre-tion from perifused tilapia pituitary in relation togonadotropin-releasing hormone, extracellular cal-cium, and activation of protein kinase C. Gen. Comp.Endocrinol. 75, 187�194.

Levavi-Sivan, B., Yaron, Z., 1992. Involvement of cyclicadenosine monophosphate in the stimulation of go-nadotropin secretion from the pituitary of a teleostfish, tilapia. Mol. Cell. Endocrinol. 85, 175�192.

Levavi-Sivan, B., Yaron, Z., 1993. Intracellular media-tion of GnRH action on GTH release in tilapia. FishPhysiol. Biochem. 11, 51�59.

Levavi-Sivan, B., Ofir, M., Yaron, Z., 1995. Possiblesites of dopaminergic inhibition of gonadotropin re-lease from the pituitary of a teleost fish, tilapia. Mol.Cell. Endocrinol. 109, 87�95.

Liu, D., Xiong, F., Hew, C.L., 1995. Functional analysisof estrogen responsive elements in chinook salmonŽ .Oncorhynchus tschawytscha gonadotropin II� sub-unit gene. Endocrinology 136, 3486�3493.

Mananos, E.L., Anglade, I., Chyb, J., Saligaut, C., Bre-˜ ´ton, B., Kah, O., 1999. Involvement of gamma-


aminobutyric acid in the control of GtH-1 and GtH-2secretion in male and female rainbow trout. Neu-roendocrinology 69, 269�280.

Melamed, P., Gur, G., Elizur, A., Rosenfeld, H., Ren-tier-Delrue, F., Yaron, Z., 1996. Differential effectsof gonadotropin-releasing hormone, dopamine andsomatostatin and their second messengers on themRNA levels of gonadotropin II� subunit andgrowth hormone in the teleost fish, tilapia. Neuroen-docrinology 64, 320�328.

Melamed, P., Gur, G., Rosenfeld, H., Elizur, A., Yaron,Z., 1997. The mRNA levels of GtH I�, GtH II� andGH in relation to testicular development and testos-terone treatment in pituitary cells of male tilapia.Fish Physiol. Biochem. 17, 93�98.

Melamed, P., Rosenfeld, H., Elizur, A., Yaron, Z.,1998. Endocrine regulation of gonadotropin andgrowth hormone gene transcription in fish. Comp.Biochem. Physiol. 119C, 325�338.

Melamed, P., Gur, G., Rosenfeld, H., Elizur, A., Yaron,Z., 1999. Possible interactions between go-nadotrophs and somatotrophs in the pituitary oftilapia: apparent roles for insulin-like growth factor Iand estradiol. Endocrinology 140, 1183�1191.

Melamed, P., Gur, G., Rosenfeld, H., Elizur, A., Schulz,R.W., Yaron, Z., 2000. Reproductive development of

Žmale and female tilapia hybrids Oreochromis niloti-.cus�O. aureus and changes in mRNA levels of

Ž .gonadotropin GtH I� and II� subunits. J. Exp.Zool. 286, 64�75.

Montero, M., Lebelle, N., King, J.A., Millar, R.P., Du-four, S., 1995. Differential regulation of the 2 forms

Žof gonadotropin-releasing hormone mGnRH and.cGnRH-II by sex steroids in the European femaleŽ .silver eel Anguilla anguilla . Neuroendocrinology 61,

525�535.Montero, M., Yon, L., Rousseau, K. et al., 1998. Dis-

tribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguillaanguilla. Endocrinology 139, 4300�4310.

Naor, Z., Outhiriaradjou, B., Seger, R., 2000. Activa-tion of MAPK cascade by G-protein-coupled recep-tors: the case of gonadotropin-releasing hormonereceptor. TEM 11, 91�99.

Parker, D.B., Power, M.E., Swanson, P., Rivier, J.,Sherwood, N.M., 1997. Exon skipping in the geneencoding pituitary adenylate cyclase-activating po-lypeptide in salmon alters the expression of twohormones that stimulate growth hormone release.Endocrinology 138, 414�423.

Peng, C., Trudeau, V.L., Peter, R.E., 1993. Seasonalvariation of neuropeptide-Y actions on growth-hormone and gonadotropin-II secretion in the gold-fish-effects of sex steroids. J. Neuroendocrinology 5,273�280.

Querat, B., Hardy, A., Fontaine, Y.A., 1991. Regulation´of type II gonadotrophin � and � subunit mRNAsby oestradiol and testosterone in the European eel.J. Mol. Endocrinol. 7, 81�86.

Rebers, F.E.M., Tensen, C.P., Schulz, R.W., Goos,H.J.Th., Bogerd, J., 1997. Modulation of glycoproteinhormone �- and gonadotropin II�-subunit mRNAlevels in the pituitary of mature male African catfish,Clarias gariepinus. Fish Physiol. Biochem. 17, 99�108.

Rebers, F.E.M., Hassing, G.A.M., Zandbergen, M.A.,Goos, H.J.Th., Schulz, R.W., 2000. Regulation ofsteady-state luteinizing hormone messenger ribonu-cleic acid levels, de novo synthesis, and release bysex steroids in primary pituitary cell cultures of maleAfrican catfish, Clarias gariepinus. Biol. Reprod. 62,864�872.

Roberson, M.S., Misra-Press, A., Laurance, M.E., Stork,P.J., Maurer, R.A., 1995. A role for mitogen-activatedprotein kinase in mediating activation of the gly-coprotein hormone �-subunit promoter by go-nadotropin-releasing hormone. Mol. Cell. Biol. 15,3531�3539.

Rosenfeld, H., Levavi-Sivan, B., Melamed, P., Yaron,Z., Elizur, A., 1997. The GtH � subunits of tilapia:gene cloning and expression. Fish Physiol. Biochem.17, 85�92.

Rosenfeld, H., Levavi-Sivan, B., Gur, G., et al. Charac-terization of the tilapia FSH� gene and analysis ofits 5� flanking region. Comp. Biochem. Physiol., inpress.

Rothbard, S., Levavi-Sivan, B., Yaron, Z., 1997. Breed-ing behavior and the associated taGtH and steroidlevels in female Nile tilapia, Oreochromis niloticus.Pol. Arch. Hydrobiol. 44, 83�92.

Rothbard, S., Ofir, M., Levavi-Sivan, B., Yaron, Z.,1991. Hormonal profile associated with breeding be-havior in Oreochromis niloticus. In: Scott, A.P.,

Ž .Sumpter, J.P., Kime, D.E., Rolfe, M.S. Eds. ,Proceedings of the 4th International Symposium onthe Reproductive Physiology of Fish, FishSymp91,

Ž .Sheffield p. 206 .Rothbard, S., Solnik, E., Shabbath, S., Amado, R.,

Grabie, I., 1983. The technology of mass productionof hormonally sex-inversed all-male tilapias. In:

Ž .Fishelson, L., Yaron, Z. Eds. , Proceedings of Inter-national Symposium on Tilapia in Aquaculture. Tel-Aviv University, Tel Aviv, pp. 425�434.

Seger, R., Krebs, E.G., 1995. The MAPK signalingcascade. FASEB J. 9, 726�735.

Senthilkumaran, B., Okuzawa, K., Gen, K., Nagahama,Y., Kagawa, H., 2000. Neuropeptide Y stimulates invitro seabream GnRH release from preoptic ante-rior hypothalamus and pituitary of pre-pubertal redseabream. In: Norberg, B., Kjesbu, O.S., Taranger,

Ž .G.L, Andersson, E., Stefansson, S.O. Eds. ,Proceedings of the 6th International Symposium on


Reproductive Physiology of Fish, Bergen, 1999. Uni-Ž .versity of Bergen, Bergen p. 62 .

Sohn, Y.C., Suetake, H., Yoshiura, Y., Kobayashi, M.,Aida, K., 1998a. Structural and expression analysis of

Žgonadotropin I� subunit genes in goldfish Carassius.auratus . Gene 222, 257�267.

Sohn, Y.C., Yoshiura, Y., Kobayashi, M., Aida, K.,1998b. Effect of sex steroids on the mRNA levels ofgonadotropin I and II subunits in the goldfish Caras-sius auratus. Fish. Sci. 64, 715�721.

Sohn, Y.C., Yoshiura, Y., Suetake, H., Kobayashi, M.,Aida, K., 1999. Nucleotide sequence of gonadotropinII� subunit gene in goldfish. Fish. Sci. 65, 800�801.

Sohn, Y.C., Kobayashi, M., Aida, K., 2000. DifferentialŽexpression and structure of goldfish FSH� GTH

. Ž .I� and LH� GTH II� genes. In: Norberg, B.,Kjesbu, O.S., Taranger, G.L, Andersson, E., Ste-


Suzuki, K., Liu, D., Hew, C.L., 1995. A gene encodingŽ .chinook salmon Oncorhynchus tschawitscha go-

nadotropin � subunit: gene structure and promoteranalysis in primary pituitary cells. Mol. Mar. Biol.Biotechnol. 4, 9�10.

Van Der Kraak, G., Chang, J.P., Janz, D.M., 1998.Ž .Reproduction. In: Evans, D.H. Ed. , The Physiology

of Fishes. CRC Press, pp. 465�488.Weiss, J., Crowley, W.F., Jameson, L., 1992. Pulsatile

gonadotropin-releasing hormone modifies poly-adenylation of gonadotropin subunit messenger ri-bonucleic acids. Endocrinology 130, 415�420.

Xiong, F., Liu, D., Elsholtz, H.P., Hew, C.L., 1994a.The chinook salmon gonadotropin II� subunit genecontains a strong minimal promoter with a proximalnegative element. Mol. Endocrinol. 8, 771�781.

Xiong, F., Liu, D., Le Drean, Y., Elsholtz, H.P., Hew,´C.L., 1994b. Differential recruitment of steroid-hormone response elements may dictate the expres-sion of the pituitary gonadotropin II� subunit geneduring salmon maturation. Mol. Endocrinol. 8,782�793.

Yam, K.M., Yoshiura, Y., Kobayashi, M., Ge, W., 1999.Recombinant goldfish activin B stimulates go-nadotropin-I� but inhibits gonadotropin-II� expres-sion in the goldfish, Carassius auratus. Gen. Comp.Endocrinol. 116, 81�89.

Yaron, Z., Bogomolnaya, A., Donaldson, E.M., 1985.Stimulation of estradiol secretion by the ovary ofSarotherodon aureus as a bioassay for fish go-

Ž .nadotropin. In: Lofts, B., Holmes, W.N. Eds. , Cur-rent Trends in Comparative Endocrinology. HongKong University Press, Hong Kong, pp. 225�228.

Yaron, Z., Levavi-Sivan, B., 1990. Intracellular eventsassociated with GnRH and dopamine effects on GTHsecretion in tilapia. In: Epple, A., Scanes, C.G.,

Ž .Stetson, M.H. Eds. , Progress in Comparative En-docrinology. Progress in Clinical and Biological Re-search, 342. Wiley-Liss, New York, pp. 409�414.

regulation of gonadotropin subunit genes in tilapia

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