GENERAL AND COMPARATIVE

ENDOCRINOLOGY

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 135 (2004) 1–16

Minireview

Evolutionary aspects of GnRHs, GnRH neuronal systemsand GnRH receptors in teleost fish

Christ�eele Lethimonier,a Thierry Madigou,a Jos�ee-Antonio Mu~nnoz-Cueto,b

Jean-Jacques Lareyre,c and Olivier Kaha,*

a Endocrinologie Mol�eeculaire de la Reproduction, UMR CNRS 6026, 35042 Rennes cedex, Franceb Departamento de Biologia, Facultad de Ciencias del Mar y Ambientales, Puerto Real, Cadiz, Spain

c INRA SCRIBE Universit�ee de Rennes 1, Campus de Beaulieu, Rennes, France

Accepted 14 October 2003

Abstract

Gonadotrophin-releasing hormone (GnRH) was originally believed to be released by a unique set of hypophysiotrophic neurons

to stimulate the release of gonadotrophins from the pituitary, therefore acting as a major initiator of the hormonal cascade con-

trolling the reproductive axis. However, it now appears that each vertebrate species expresses two or three GnRH forms in multiple

tissues and that GnRHs exert pleiotropic actions via several classes of receptors. This new vision of the GnRH systems arose

progressively from numerous comparative studies in all vertebrate classes, but fish in general, and teleosts in particular, have often

plaid a leading part in changing established concepts. To date fish still appear as attractive models to decipher the evolutionary

mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest variety of GnRH variants,

but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptors. This paper intends to

summarize the current situation with special emphasis on interspecies comparisons which provide insights into the possible evo-

lutionary mechanisms leading to the diversification of GnRH functions.

� 2003 Elsevier Inc. All rights reserved.

Keywords: GnRH; GnRH receptors; Genome analysis; Hypothalamus; Pituitary; Reproduction; Teleost

1. Introduction

Since the pioneer studies of Breton and colleaguesshowing that a hypothalamic factor stimulates the re-

lease of pituitary gonadotropic hormone in carp (Breton

et al., 1971) and the characterization of the first fish

GnRH, salmon GnRH (Sherwood et al., 1983), research

on GnRH in teleost fish has focused increasing attention.

This is not only due to the important potential applica-

tions of GnRH in fish farming (Zohar and Mylonas,

2001), but also to the fact that teleost fish have turnedout to be of special interest to understand the mecha-

nisms underlying the evolution of GnRH genes in ver-

tebrates. In parallel, following the early work of Habibi

et al. (1987) on the binding properties of GnRH in the

* Corresponding author. Fax: +33-2-23-23-67-94.

E-mail address: olivier.kah@univ-rennes1.fr (O. Kah).

0016-6480/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2003.10.007

goldfish pituitary and the cloning of the first fish GnRH

receptor (GnRH-R) in catfish (Tensen et al., 1997), a

growing number of studies are devoted to fish GnRH-R.These studies have largely contributed to the discovery

that there are several GnRH-R subtypes with differential

structural characteristics and expression sites.

This article intends to review recent data on the

evolution of GnRH forms and neuronal circuits and the

presence of multiple GnRH-R subtypes in teleosts with

special emphasis on evolutionary aspects.

2. Gonadotrophin-releasing hormones

2.1. Eight GnRH variants in teleost fish

The number of GnRH family members in vertebrates

has rapidly increased over the last decade, now reaching

a total of 14 variants, however unique GnRH forms

2 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

have also been found in prochordates (Adams et al.,2003) and in invertebrates (Iwakoshi et al., 2002). As

GnRH-like peptides have been found in cnidaria con-

sidered as the first animals with neurons and true syn-

apses (Anctil, 2000), it is possible that the origin of the

GnRH family goes back around 600 millions years.

Among vertebrates, teleost fish represent the group ex-

hibiting the highest number of GnRH variants. Fol-

lowing the identification of salmon GnRH (sGnRH;Sherwood et al., 1983), 7 other GnRH forms have been

purified in teleosts by high performance liquid chro-

matography (HPLC) and sequencing of GnRH-immu-

noreactive peaks (Table 1). These variants, usually

named after the first species in which they have been

characterized, are presented in Table 1. Apart from

mammalian GnRH (mGnRH; Matsuo et al., 1971) and

chicken GnRH-II (cGnRH-II; Miyamoto et al., 1984),identified in fish for the first time in eel (King et al.,

1990) and goldfish (Yu et al., 1988), respectively, the 6

other forms are specific of the fish lineage. These forms

are catfish GnRH (cfGnRH; Bogerd et al., 1992;

Ngamvongchon et al., 1992), sea bream GnRH

(sbGnRH; Powell et al., 1994), herringGnRH (hgGnRH;

Carolsfeld et al., 2000), pejerey GnRH (pjGnRH; Mon-

taner et al., 2001; also calledmedakaGnRH:Okubo et al.,2000a) and whitefish GnRH (wfGnRH; Adams

et al., 2002).

As now shown in all vertebrate classes, the brain of

teleosts contains at least two GnRH variants, but there

is a growing number of species in which three GnRH

variants have been found (Table 2). Until now, all te-

leosts examined express cGnRH-II, which has also been

characterized in primitive bony fish (Lepretre et al.,1993; Sherwood et al., 1991) and tetrapods. Thus, this

form appears to be highly conserved in both sarco-

pterygii and actinopterygii. With the notable exceptions

Table 1

Amino acid sequence of the 14 GnRH variants identified in vertebrates

Teleosts

Mammalian GnRH pGlu His Trp Ser Tyr Gl

Chicken GnRH-II — — — — His —

Pejerey GnRH — — — — Phe —

Seabream — — — — — —

Salmon GnRH — — — — — —

Catfish GnRH — — — — His —

Herring GnRH — — — — His —

Whitefish GnRH — — — — — —

Other vertebrates

Mammalian GnRH pGlu His Trp Ser Tyr Gl

Frog GnRH — — — — — —

Lamprey GnRH-I — — Tyr — Leu Gl

Lamprey GnRH-III — — — — His As

Dogfish GnRH — — — — His —

Guinea pig GnRH — Tyr — — — —

Chicken GnRH-I — — — — — —

Eight of these variants are found in teleost fish.

of the butterfly fish (osteoglossiforme; O�neill et al.,1998), European eel (anguilliforme; King et al., 1990),

and catfish (siluriforme; Ngamvongchon et al., 1992),

salmon GnRH has been detected in all teleost species

from primitive fish, such as osteoglossiformes (Okubo

and Aida, 2001; O�neill et al., 1998), to the acanth-

opterygii. In species expressing three GnRH forms, the

third variant appears less conserved and can be either

hgGnRH, wfGnRH, pjGnRH or sbGnRH.Table 2 summarizes the distribution of these 8 forms

in the different orders of teleosts fish and shows that it is

difficult to draw any clear conclusion regarding the

correlation between the presence of either two or three

variants and the position of a given species in the teleost

lineage. Indeed, although it was originally believed that

the presence of 3 GnRH variants in the same species was

a characteristic of evolved fish, three GnRH forms havebeen found in the Pacific herring (Clupea harengus

pallasi), a rather primitive teleost, in which a new form,

hgGnRH, was found in addition to sGnRH and

cGnRH-II (Carolsfeld et al., 2000). Another interesting

example is the whitefish (Coregonus clupeaformis), a

basal salmonid which also expresses 3 forms including

the unique whitefish GnRH variant (Adams et al.,

2002). According to these authors, this third form wouldhave emerged after a genome duplication, but was lost

later in salmonids, such as the Rainbow trout or the

Masu salmon, due to chromosomal rearrangements

(Adams et al., 2002). It is however interesting to men-

tion that species belonging to several orders of

Acanthopterygii have all been shown to express three

GnRH variants (see Somoza et al., 2002), including an

hypophysiotrophic form with a serine in position 8(pjGnRH or sbGnRH). Atheriniformes, synbranchi-

formes, beloniformes and cyprinodontiformes, some-

times grouped under the taxon Smegmamorpha, and

y Leu Arg Pro Gly-NH2 Matsuo et al. (1971)

Trp Tyr — — Yu et al. (1988)

— Ser — — Montaner et al. (2001)

— Ser — — Powell et al. (1994)

Trp Leu — — Sherwood et al. (1983)

— Asn — — Bogerd et al. (1992)

— Ser — — Carolsfeld et al. (2000)

Met Asn — — Adams et al. (2002)

y Leu Arg Pro Gly-NH2 King et al. (1990)

— Trp — — Yoo et al. (2000)

u Trp Lys — — Sower et al. (1993)

p Trp Lys — — Sower et al. (1993)

Trp Leu — — Lovejoy et al. (1992)

Val — — — Jimenez-Linan et al.

(1997)

— Gln — — Miyamoto et al. (1982)

Table 2

Distribution of GnRH variants in the brain of teleost fish

Tel POA MT Reference

Anguilliformes

Eel Anguilla anguilla m m cII Montero et al. (1994)

Pantodonidae

Butterfly fish Pantodon buchholzi m? m? cII? O�neill et al. (1998)Osteoglossiformes

Arowana Scleropages jardini s? s? cII? Okubo et al. (2001)

Clupeiformes

Herring Clupea harangus s? hg? cII? Carolsfeld et al. (2000)

Cypriniformes

Goldfish Carassius auratus s s cII Yu et al. (1988)

Zebrafish Danio rerio s s cII Powell et al. (1996)

Siluriformes

African catfish Clarias gariepinus cf cf cII Zandbergen et al. (1995)

Characiformes

Pacu Piaractus mesopotamicus s sb cII Powell et al. (1997)

Salmoniformes

Whitefish Coregonus clupeaformis s? wf? cII? Adams et al. (2000)

Rainbow trout Oncorhynchus mykiss s s cII Okuzawa et al. (1990)

Masu salmon Oncorhynchus masou s s cII Amano et al. (1991)

Atheriniformes

Pejerey Odonthestes bonariensis s pj cII Montaner et al. (2001)

Synbranchiformes

Swamp eel Synbranchus marmoratus s? pj? cII? Somoza et al. (2002)

Beloniformes

Medaka Oryzias latipes s pj cII Okubo et al. (2000a,b)

Cyprinodontiformes

Latyfish Xiphophorus maculatus s? pj? cII? Somoza et al. (2002)

Scorpaeniformes

Grass rockfish Sebastes rastrelliger s? sb? cII Powell et al. (1996)

Perciformes

African cichlid Haplochromis burtoni s sb cII White et al. (1995)

Seabream Sparus aurata s sb cII Gothilf et al. (1996)

Striped bass Morone saxatilis s sb cII Chow et al. (1998)

Sea bass Dicentrarchus labrax s sb cII Gonzalez-Martinez et al. (2001)

Red seabream Pagrus major s sb cII Senthilkumaran et al. (1999)

Tilapia Oreochromis mossambicus

Pleuronectiformes

Barfin flounder Verasper moseri s sb cII Amano et al. (2002)

Turbot Scophthalmus maximus s sb cII Andersson et al. (2001)

Tetraodontiformes

Torafugu Fugu rubripes s? sb? cII? Aparicio et al. (2002); this study

For clarity purpose, three main populations (olfactory bulbs+ telencephalon: Tel, preoptic area: POA and midbrain tegmentum: MT) have been

considered. Question marks refer to studies in which the distribution has not been formally established from neuroanatomical studies, but can be

predicted on the basis of comparison with other species and phylogenetical analysis.

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 3

Scorpaeniformes were found to have pjGnRH (Somoza

et al., 2002), whereas perciformes and pleuronectiformes

are until now characterized by the presence of sbGnRH.

To date, no GnRH has been purified in tetraodonti-formes, but a BLAST search performed on the torafugu

genome (http://www.fugu-sg.org/) indicates the presence

of sGnRH (SINFRUG00000081202; Scaffold 7),

sbGnRH (SINFRUP00000080578; Scaffold 137), and

cGnRH-II (SINFRUG00000062489; Scaffold 119).

2.2. Phylogenetic analysis of preproGnRH

Considering the number of GnRH variants in fish

and other vertebrates, one of the major issues concerns

the phylogenetic relationships between these different

variants. Because GnRHs are short peptides, it is thus

necessary to consider the sequence of their precursor,

the preproGnRH. All GnRHs are issued from a largeprecursor which includes a signal peptide (around 20–25

residues), the biologically active GnRH sequence, a

processing tripeptide (Gly–Lys–Arg) and the GnRH-

associated peptide (GAP; around 40–50 residues). The

cDNAs corresponding to the two or three GnRH vari-

ants have been cloned in a number of species allowing

sequence comparisons to be made. Such an analysis was

performed by Okubo et al. (2000a), who cloned twocDNAs corresponding to sGnRH and cGnRH-II in the

arowana (Scleropages jardini), belonging to the order

4 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

osteoglossiforme, one of the most primitive teleost or-ders. This analysis using the neighbor-joining method,

clearly shows three branches, two of which encompass-

ing sequences corresponding to a unique GnRH variant.

One includes all precursors of cGnRH-II, while another

contains all sequences corresponding to preprosGnRH.

The third branch includes sequences encoding eel

mGnRH, medaka pjGnRH, perciform sbGnRH, and

catfish cfGnRH (Okubo et al., 2001), all known forcorresponding to the main hypophysiotrophic form in

these species (see below). An interesting observation

resulting from such analyses is that each branch includes

species representative of basal, intermediate, and mod-

ern teleosts, which would indicate that these three

branches are ancient and evolved before the diversifi-

cation of teleosts. Unfortunately, the cDNA sequences

encoding wfGnRH and hgGnRH are not presentlyavailable.

When sequences encoding preproGnRHs from other

vertebrate classes are included in the analysis, there are

again three main branches (Fig. 1), as already shown

by White et al. (1998) who proposed to name them

type 1, 2, and 3. One contains hypophysiotrophic

variants, such as frog GnRH, guinea pig GnRH,

mammalian mGnRH in addition to a number of fishhypophysiotrophic variants (type 1). Another branch

clusters all cGnRH-II from mammals and fish (type 2),

and a third one includes only the fish preprosGnRH

(type 3). It is interesting to mention that lamprey

GnRH-I shows higher identity to prepro chicken

GnRH-II suggesting that they share a common origin.

As already stated by White et al. (1998), each of these

three branches correspond to forms with distinct ex-pression patterns and probably different biological

properties. Further evidence that type 1 and type 2 fish

GnRH genes are orthologs to human mGnRH and

cGnRH-II, respectively, was obtained from character-

ization of GnRH loci in the medaka and human ge-

nomes (Okubo et al., 2002). The fact that branches 1

and 2 contain sequences from both fish and land ver-

tebrates indicate that these branches are ancient andemerged before the divergence of these groups. Branch

3 includes only sGnRH fish sequences and this could

mean either that the corresponding gene has been lost

in land vertebrates or remains to be found. Alterna-

tively, it is possible that the gene duplication that gave

birth to this branch occurred within the fish lineage,

but in this case one would expect this branch to cluster

with one of the two other fish branches (White et al.,1998). Based on this analysis, it is surprising that no

type 1 GnRH sequence has been found in cyprinifor-

mes (ostariophysii) or some salmoniformes (protac-

anthopterygii). The fact that the whitefish, a basal

salmonid has three forms tends to indicate that indeed

some salmonids have lost one gene, or that it remains

to be found (Adams et al., 2002).

2.3. Organization of GnRH systems in the brain of teleost

fish

Another mean to find relationships between GnRH

variants is to look at their sites of expression as an in-

dication of their potential function. The assumption is

that ortholog genes will share common expression pat-

terns. A considerable amount of work has been devoted

to the identification and localization of GnRH-ex-pressing neurons in the brain of fish using immunohis-

tochemistry or, more recently, in situ hybridization.

Early studies in goldfish, using sGnRH antibodies, had

shown that GnRH neurons are distributed in the ante-

rior ventral brain from the olfactory nerves to the me-

diobasal hypothalamus (Kah et al., 1986). Small cell

clusters or isolated neurons were found in the olfactory

nerves and bulbs, ventral telencephalon, preoptic areaalong a tract of GnRH-immunoreactive fibers running

to the anterior pituitary. A small number of isolated

neurons were also found along these fibers in the me-

diobasal hypothalamus. An additional population of

large sGnRH-immunoreactive neurons was also identi-

fied in the synencephalon and immunoreactive fibers

were widely present in many brain regions including in

the spinal cord (Kah et al., 1986). Such a pattern oforganization was then found with minor variations in

many, if not all, teleost species studied, and three main

groups of GnRH neurons are usually recognized, al-

though this is a simplified view of a more complex sit-

uation: a clearly identified population of large GnRH

neurons in the tegmentum of the midbrain (MT), an

anterior telencephalic population including the terminal

nerve associated neurons (Tel), and a third populationin the caudal telencephalon-preoptic region (POA).

What has emerged from recent studies is that these

different populations express two or three GnRH vari-

ants depending on the species (Table 2 and Fig. 2).

2.3.1. Two GnRH variants

The presence of two GnRH variants in the brain of

a single teleost was first demonstrated in goldfish byYu et al. (1988) who, using HPLC and radio-

immunoassays on microdissected brain regions, showed

that the anterior brain contained more sGnRH and the

posterior brain more cGnRH-II. The first species in

which the differential distribution of two GnRH vari-

ants was demonstrated by means of immunohisto-

chemistry using specific antibodies to sGnRH and

cGnRH-II was the Masu salmon (Amano et al., 1991).It was found that neurons of the anterior ventral brain

(Tel + POA) were immunoreactive to sGnRH whereas

the large cell bodies of the synencephalon were positive

with antibodies against cGnRH-II (Fig. 2). This latter

cGnRH-II population was subsequently described in

many other teleost species (see Table 2) and other

vertebrate classes.

Fig. 1. Phylogenetic tree for preproGnRHs in vertebrates showing three main branches in GnRH evolution; one clustering all sGnRH from fish (3),

another including cGnRH-II from fish and mammals (2), and the third one with hypophysiotrophic forms GnRH forms from fish amphibian and

mammals (1). The tree was generated using clustalW based on the neighbor-joining method. The scale bar corresponds to estimated evolutionary

distance units. Sequence access numbers: African catfish (Clarias gariepinus): cf GnRH1 (X78049), cfGnRH2 (X78048), cGnRH-II (X78047);

arowana (Scleropages jardinii): sGnRH (AB047325), cGnRH-II (AB047326); cichlid (Haplochromis burtoni): sbGnRH (U31865), cGnRH-II

(L27435), sGnRH: (S63657); eel (Anguilla japonica) mGnRH (AB026989), cGnRHII (AB026990); frog (Rana dybowskii) GnRH (AF139911);

goldfish (Carassius auratus): sGnRH1 (AB017271), sGnRH2 (AB017272), cGnRH-II1 (U30386), cGnRH-II2 (U40567); guinea pig (Cavia porcellus)

gpGnRH (AF426176); human (Homo sapiens) mGnRH: (X01059), cGnRH-II: (AF036329); lamprey (Petromizon marinus) lGnRH (AF144479);

medaka (Oryzias latipes): sGnRH (AB041335), cGnRH-II (AB041334), mdGnRH (AB041334); musk shrew (Suncus murinus) cGnRH-II

(AF107315); rat (Rattus norvegicus) mGnRH (M15527); rhesus monkey (Macaca mulatta) cGnRH-II (AF097356); salmon (Oncorhynchus nerka)

sGnRH 1 (D31868), sGnRH 2 (D31869); sea bass (Diecentrarchus labrax): sbGnRH (AF224279), cGnRH-II (AF224281), sGnRH (AF224280);

seabream (Sparus aurata): sbGnRH, (U30320), sGnRH, (U30311), cGnRH-II, (U30325); tree shrew (Tupaia glis belangeri) mGnRH (U63326),

cGnRH-II (U63327); zebrafish (Danio rerio) sGnRH (AJ304429).

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 5

This pattern of organization is found in other species

expressing two GnRH variants, such as the African

catfish (Zandbergen et al., 1995) and the European eel

(Montero et al., 1994), although in these species anterior

neurons (Tel and POA), express cfGnRH and mGnRH,

respectively (Fig. 2). However, although the GnRH

Fig. 2. The main types of organization of the GnRH systems in teleosts. The eel illustrates the situation in which a type 1 GnRH is found together

with a type 2 GnRH (adapted from Montero et al., 1994). This situation is also found in catfish (Dubois et al., 2001). The goldfish reflects a unique

situation where a type 3 and a type 2 forms are found in neurons of the anteroventral brain (adapted from Kim et al., 1995). The Masu salmon is an

example where only a type 3 GnRH is expressed in neurons of the anterior ventral brain (adapted from Amano et al., 1991). The European sea bass is

an example of a species with three GnRH variants (types 1, 2, and 3). In this case, the distribution of sGnRH and sbGnRH neurons overlap in the

forebrain (adapted from Gonzalez-Martinez et al., 2002a). The relative abundance of pituitary GnRH fibers is indicated.

6 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

forms detected in Tel and POA neurons in catfish or eelbelong to branch 1 of the phylogenetical tree, the one

expressed in these cell populations in salmonids is found

in branch 3. If some salmonids have lost the type 1 gene,

it is then possible that a type 3 GnRH was recruited to

fulfill the hypophysiotropic roles. In the African catfish,

based on differences in temporal, spatial, and morpho-

logic appearance, two distinct cfGnRH populations

were identified in the ventral forebrain: a populationinnervating the pituitary (ventral forebrain system) and

a so-called terminal nerve (TN) population. DiI tracing

studies revealed that the TN population has no neuronal

connections with the pituitary (Dubois et al., 2001), as

already demonstrated by Oka and Matsushima (1993) in

the dwarf gourami.

A unique situation has been reported in the goldfish

(Fig. 2) known to express sGnRH and cGnRH-II (Yuet al., 1988). Indeed, both sGnRH and cGnRH-II-im-

munoreactive neurons are found in the anteroventral

brain (Kim et al., 1995), a result in agreement with

studies based on preproGnRH mRNA detection (Lin

and Peter, 1997). To date the goldfish remains the only

teleost species with cGnRH-II expression outside the

midbrain tegmentum.

2.3.2. Three GnRH variants

The first species in which three GnRH variants,

sGnRH, sbGnRH and cGnRH-II, were characterized is

the gilthead seabream (Powell et al., 1994). Subsequent

studies in other perciformes and pleuronectiformesshowed that these three forms have differential expres-

sion patterns with the newly discovered sbGnRH being

expressed mainly in the POA cells, sGnRH mostly in the

olfactory bulbs, and cGnRH-II in the midbrain teg-

mentum (Amano et al., 2002; Andersson et al., 2001;

Gonzalez-Martinez et al., 2001, 2002a; Gothilf et al.,

1996; Senthilkumaran et al., 1999; White et al., 1995).

The organization of three GnRH systems was studied indetails in the European sea bass by in situ hybridization

and immunohistochemistry with highly specific anti-

bodies against sea bass recombinant GAPs (Gonzalez-

Martinez et al., 2001, 2002a; Zmora et al., 2002). The

salmon GAP immunostaining was mostly detected in

terminal nerve neurons, but also in ventral telencephalic

and preoptic perikarya (Fig. 2). Salmon GAP-immu-

noreactive (ir) fibers were observed mainly in the fore-brain, although sGAP-ir projections were also evident in

the optic tectum, mesencephalic tegmentum, and ventral

rhombencephalon. The pituitary receives a small num-

ber of sGAP-ir fibers. The seabream GAP-ir cells were

mainly detected in the preoptic area. Nevertheless,

sbGAP-ir neurons were also found in olfactory bulbs,

ventral telencephalon, and ventrolateral hypothalamus.

The sbGAP-ir fibers were only observed in the ventralforebrain and massively innervate the pituitary gland

only. Finally, chicken-II GAP immunoreactivity was

only detected in large synencephalic cells, which are the

origin of a profuse innervation reaching the telenceph-

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 7

alon, preoptic area, hypothalamus, thalamus, pretec-tum, posterior tuberculum, mesencephalic tectum and

tegmentum, cerebellum, and rhombencephalon. How-

ever, no cIIGAP-ir fibers were detected in the hypoph-

ysis. These results showed the overlapping of sGAP- and

sbGAP-expressing cells in the forebrain of the sea bass,

and provided unambiguous information on the distri-

bution of projections of the three different GnRH forms

expressed in the brain of a single species (Gonzalez-Martinez et al., 2001, 2002a; Zmora et al., 2002).

2.4. Embryonic origins of the brain GnRH systems in

teleosts

If it seems clear that mesencephalic cGnRH-II neu-

rons differentiate early from a midbrain germinative

primordium and GnRH neurons of the terminal nervemigrate from the olfactory region, there is currently a

debate concerning the origin of the preoptic GnRH

system in species expressing three GnRH forms. Some

studies indicate that preoptic sbGnRH neurons also

originate from the olfactory region and migrate to their

final position (Gonzalez-Martinez et al., 2002b; White

and Fernald, 1998). This assumption is based on the fact

that sbGnRH neurons are first detectable in the olfac-tory region and then are found more caudally as

development proceeds. Based on the fact that sbGnRH-

expressing cells are first visible in their final position in

the POA, other groups claim that sbGnRH neurons

originate from the anteroventral preoptic area (Parhar,

2002). Given that sbGnRH is the ortholog of the genes

encoding the tetrapod GnRH forms expressed in hyp-

ophysiotrophic neurons, which are well known for dif-ferentiating from the olfactory region, it is very unlikely

that this second hypothesis is viable, unless it is shown

that proliferation markers are expressed in preoptic

GnRH neurons. Of particular interest are data obtained

in the African catfish showing that the cfGnRH TN and

the POA neurons, both originate from the olfactory

region, but at two different periods of development

(Dubois et al., 2001, 2002).In summary, based on the structural identities of

preproGnRHs and their sites of expression, there seems

to be three paralog genes in teleosts, but it appears that

some families have lost one of them (type 3 in eels and

catfish, type 1 in salmonids and cyprinids). However,

based on the very similar overall pattern of distribution

of the GnRH-expressing neurons, one can suggest that

another gene has been recruted to fulfill the roles of themissing form. In this respect, it is interesting to note that

in some perciformes sGnRH neurons send projections

to the pituitary (Gonzalez-Martinez et al., 2002a), in-

dicating that these neurons have retained the capacity to

fullfil hypophysiotropic functions. If it seems clear that

type 1 GnRH is involved in the regulation of pituitary

functions, the precise roles of the type 2 (see Millar, 2002

for review) and type 3 (see Oka, 2002 for review)GnRHs still remain highly enigmatic. Recent data in

tunicates indicate that 2 genes were already present in

protochordates, with characteristics similar to the type 1

and type 2 human genes (Adams et al., 2003). Finally,

GnRH peptides and mRNAs have been found in the

gonads where they may be involved in steroidogenesis,

reinititation of meiosis in females and germ cell prolif-

eration in males (see Pati and Habibi, 2002).

3. Teleost GnRH receptors

The first molecular characterization of a full-length

piscine GnRH-R was described in 1997 in African cat-

fish (Tensen et al., 1997). Since then, cDNA encoding

GnRH-R have been obtained in various teleost speciesincluding goldfish (two receptors; Illing et al., 1999),

rainbow trout (Madigou et al., 2000), African catfish

(two receptors; Bogerd et al., 2002), medaka (two

receptors; Okubo et al., 2001), striped bass (Mo-

rone saxatilis; Alok et al., 2000), amberjack (Seriola

dumerilii; GenBank AJ130876), African cichlid (two

receptors; Robison et al., 2001), European sea bass

(DLA419594), and Japanese eel (Okubo et al., 2000b).Information obtained about these receptors is summa-

rized in Table 3.

3.1. Piscine GnRH-R structure

Analysis of the primary amino acids sequence of fish

GnRH receptors indicates that they belong to the G

protein coupled receptors family (GPCRs). A recentphylogenetic analysis of the GPCRs shows that they

segregate into five subfamilies tentatively termed gluta-

mate, secretin, adhesion, frizzled/TAS2, and rhodopsin

subfamilies (Fredriksson et al., 2003). This latter sub-

family is itself subdivided into four subgroups (a; b; c; d)and many of the peptide hormone receptors, including

the GnRH-R, belong to the beta subgroup. Members of

the GPCRs show three main functional domains thatincludes an N-terminal extracellular domain (30–40 aa),

a large transmembrane domain (280–290 aa), and a

short C-terminal cytoplasmic domain (30–50 aa). The

transmembrane domain is constituted of seven highly

conserved transmembrane alpha helix (TM) that are

required to anchor the receptor to the cell membrane. In

contrast to their mammalian counterparts, fish GnRH-

R show an Asp residue in transmembrane domain 2 thatis conserved among members of the GPCRs family. This

Asp residue, that is converted to an Asn residue in

mammalian GnRH receptors, is required for piscine

receptor function (Blomenr€oohr et al., 2002). Intrigu-

ingly, opposite to other GPCRs, mammalian and fish

GnRH-R have a conserved Asp residue in the trans-

membrane helix 7, but conversion of this residue to an

Table

3

Pharm

acologicalcharacterization,tissuedistribution,andclassificationofknownpiscineGnRH-R

Reference

Receptor

abbreviation

GnRH-R

type

Ligand

selectivity

Tissue

distribution

Anguilliform

es

Eel

Okuboet

al.(2000a,b)

IIND

Pituitary

>brain>testis>eye>olfactory

epithelium

Cypriniform

es

Goldfish

Illinget

al.(1999)

GfA

IIGfA

*:cG

nRH-II>sG

nRH>mGnRH>sbGnRH

Brain

(butonly

GfA

isexpressed

in

ventraltelencephalon)>pituitary

(proxi-

malpars

distalis)>ovary

(interstitialcells

andtheca-granulosa

celllayers)¼liver

GfB

GfB:cG

nRH-II>sG

nRH>mGnRH>sbGnRH

Beloniform

es

Medaka

Okuboet

al.(2001)

GnRH-R

1I

GnRH-R

1:cG

n-

RHI>

¼sG

nRH¼mGnRH¼mdGnRH

ND

GnRH-R

2II

GnRH-R

2*:cG

n-

RHII>sG

nRH>mGnRH>mdGnRH

ND

Siluriform

es

Catfish

Tensenet

al.(1997)

cfGnRH-R

1II

cGnRH-II>cfGnRH

Pituitary

>brain>cerebellum>testis

Bogerdet

al.(2002)

cfGnRH-R

2II

cGnRH-II>cfGnRH¼mGnRH

Brain>ovary

>heart>

testis>cerebel-

lum>pituitary

Salm

oniform

es

Rainbow

trout

Madigouet

al.(2000)

rtGnRH-R

IIND

Brain>testis>ovary

>retina>pituitary

Perciform

es

Sea

bass

Gonzalez-Martinez

etal.

(2002c)

IND

Pituitary

>brain

Seriola

dumerilli

AJ130876

IND

ND

Striped

bass

Aloket

al.(2000)

stbGnRH-R

IcG

nRH-II>mGnRHa>

¼sG

nRH>sbGnRH

Pituitary

>brain>ovary

Astatotilapia

Robisonet

al.(2001)

GnRH-R

1II

cGnRH-II>sG

nRH¼

mGnRH>sbGnRH

Brain>testis>kidney

>

retina>muscle>pituitary

GnRH-R

2I

ND

ND

Asterisk(*)indicatesreceptorsubtypeshowingahigher

cGnRH-IIsensitivityin

thespeciesofinterest.GnRH-R

typerefers

tothephylogeneticalanalysisperform

edin

Fig.4.

8 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 9

Asn does not change receptor function in transfectedcells. The regions between the alpha helices form intra-

cellular or extracellular loops and have been involved in

signal transduction and ligand selectivity. For instance,

the motif DRXXXI/V located at the cytoplasmic end of

the third TM is involved in signal transduction and re-

ceptor activation. In addition, putative ligand contact

sites are evolutionary well conserved. The N-terminal

domain is less conserved, but contains glycosylationsites that may be required for GnRH-R expression. Fi-

nally, the most striking change in receptor structure

between piscine and mammalian receptors is the pres-

ence of a C-terminal cytoplasmic tail. This tail is re-

quired for piscine GnRH-R function (Blomenr€oohr et al.,2002) and has been involved in the desensitization of a

chimeric receptor by decreasing the rate of internaliza-

tion (Lin et al., 1998).

3.2. Two main GnRH-R types with subtypes

To determine the number of sequences encoding

GnRH receptors in teleosts, we have carried out an in

silico analysis on the whole genome of the tetraodonti-

forme Takifugu rubripes (Aparicio et al., 2002) and

partial zebrafish genome draft (Sanger Institute; ftp://ftp.sanger.ac.uk/pub/yyy). In the fugu, five different loci

showing open reading frames (termed GnRH-R1 to

GnRH-R5) encoding putative GnRH-R were identified

(Fig. 3). In the zebrafish, four open reading frames cor-

responding to putative GnRH-R were retrieved and

termed GnRH-R1 to GnRH-R4 (Fig. 3). Phylogenetical

analysis of these sequences and other known teleost

GnRH-R indicates the presence of two main types,termed type I and type II (Fig. 4). According to this

analysis, 3 of the putative fugu receptors (GnRH-R1,

GnRH-R2, and GnRH-R3) would belong to type I

whereas GnRH-R4 and GnRH-R5 are found with the

type II receptors. Two of the zebrafish sequences cluster

with type I receptors, while the other two are found

among type II. It is interesting to mention that the two

GnRH-R cloned in goldfish and catfish cluster in thesame branch (type II), and thus would appear as sub-

types of type II. In contrast, the two receptors identified

in medaka and African cichlid, two Acanthopterygii, are

found in the two main branches. In addition, a partial

sequence of a European eel GnRH-R (T. Madigou et al.,

unpublished) corresponding to a type I appears different

from that of the Japanese eel type II previously published

(Okubo et al., 2000b). Examination of the sequences oftype I and type II receptors revealed the presence of di-

vergent motifs, notably in transmembrane domains

(TM) 3 and 7 (Fig. 5). Indeed, all type I receptors have a

C/GAFVT motif in TM 3 whereas type II exhibit a

SAFIL type motif. All type I receptors have a DLE-

GKVSHSLTH like sequence at the beginning of TM7,

while type II have a VTPEYmotif at this site. In order to

see if those two types also exist in salmoniformes, wedecided to use primers specific to these motifs in order to

clone a type I GnRH-R in the rainbow trout, a species in

which a type II receptor was already characterized

(Madigou et al., 2000). Accordingly, a type I GnRH-R

sequence could be obtained (Fig. 4), whereas a sequence

corresponding to a type II receptor could be retrieved

using the same strategy in the European sea bass, in

which a type I receptor was already cloned(DLA419594). Thus, it appears that, within the teleost

lineage, two main types of GnRH-R could exist each of

which may include 2 or 3 subtypes as evidenced by the

fugu, the zebrafish, the goldfish or the catfish. At the

moment, examples of species exhibiting those two types

belong to the orders anguilliforme, cypriniforme, sal-

moniforme, beloniforme, perciforme, and tetraodonti-

forme showing a widespread distribution of these twoGnRH-R types. The presence of at least two GnRH-R

types, with possible subtypes, is not restricted to fish

since two GnRH-R have been characterized in mammals

(Millar, 2002) and three in bullfrog (Wang et al., 2001).

The existence of different types and subtypes of

GnRH-R in a single fish species raises the question of

whether these receptors have redundant or distinct

functions. To address this important issue, it is necessaryto combine the information on the gene structure and

regulation, ligand specificity and selectivity, and tissue/

cellular distribution of the different GnRH-R types and

subtypes.

3.3. Genes encoding distinct GnRH-R types show different

genomic organization

The genomic organization of GnRH-R genes has

been determined in medaka (Okubo et al., 2001, 2002),

eel (Okubo et al., 2000b), and in trout (Madigou et al.,

2000; Madigou et al., 2002). The genomic structure of

the fugu, zebrafish, and known GnRH-R genes was

compared with respect to their membership to each

clade (Fig. 6). The exon size and phase is well conserved

within each clade except for the first and last exon due tountranslated regions. Genes encoding Type I GnRH

receptors consist in 3 exons separated by two introns.

The 50 flanking region shows the presence of TATA and

CAAT boxes. This structure has been evolutionary

conserved in human type II GnRH receptor (Neill,

2002). The structure of the genes encoding piscine type

II GnRH-R is more complex. In trout, the use of an

alternative promoter and splicing leads to a gene struc-ture consisting in either three or four exons encoding a

receptor with a classic or shorter N-terminal end, re-

spectively (Madigou et al., 2002). In fugu, we also de-

termined that GnRH-R4 and GnRH-R5 genes were

composed of at least three exons similar to the trout and

eel genes. The medaka type II GnRH-R gene contains

four exons. Interestingly, in the second exon, there is an

Fig. 3. Several genes encoding putative GnRH receptors are present on the Fugu rubripes and zebrafish genomes. Using trout GnRH receptor

(GnRH-R) amino acids sequence as template an in silico search for orthologous genes has been carried out on the recently released F. rubripes

(Aparicio et al., 2002) and zebrafish genome draft using the links http://bahama.jgi-psf.org/fugu/bin/blast.fugu.cgi and http://www.ensembl.

org/Danio_rerio/. Five open reading frames present on the takifugu genome, numbered GnRH-R1 (scaffold 10498), GnRH-R2 (scaffold 1609),

GnRH-R3 (scaffold 4966), GnRH-R4 (scaffold 468), and GnRH-R5 (scaffold 4098), showed 58, 58, 61, 71, and 75% homology to trout GnRH-R,

respectively. Four open reading frame, numbered from GnRH-R1 to GnRH-R4 were identified on the zebrafish genome draft. Multiple amino acids

sequences alignment was carried out using the clustalW algorithm of the BioEdit shareware (Hall, 2002). Black bars indicate putative transmembrane

domains.

10 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

in frame ATG that corresponds to the putative trout

translation initiation codon ATG-2 found in alterna-

tively spliced messengers (Madigou et al., 2002). The

location of this ATG is also conserved in fugu GnRH-

R4 and GnRH-R5 genes as well as in the eel gene.

However, the use of an alternative splicing in medaka,eel, and fugu type II GnRH-R genes that could result in

a shorter GnRH-R remains to be demonstrated. Finally,

the analysis of GnRH-R transcripts in astatotilapia

suggests a more complex gene structure with five exons

(Robison et al., 2001). In conclusion, type I and type II

GnRH-R gene structures have striking differences in

exon number, exon size, and promoter components.

This observation strengthens the data obtained from the

phylogenetic data presented above which needs to be

completed by gene cluster analyses.

3.4. Ligand for GnRH receptors

The presence of different types and subtypes of

GnRH-R in a single fish species raises the question

whether the different GnRH-related peptides have sim-

Fig. 4. Phylogenic analysis of teleost GnRH-R. The phylogenic analysis

was carried out as described in Fig. 2. Only the region spreading from

TM5 to TM6 was considered in the study. Note that piscine GnRH-R

segregate (arrow) into two main phyla, termed type I and type II

GnRH-R. Sequences Accession number are eel (GnRH-R1: AB041327;

GnRH-R2: Madigou et al., unpublished), sea bass (GnRH-R1:

AJ419594; GnRH-R2: Lethimonier et al., unpublished), trout

(rtGnRH-R1: AJ272116; rtGnRH-R2: Lethimonier et al., unpub-

lished), catfish (GnRH-R1: X97497; GnRH-R2: AF329894), seriola

dumerilii GnRH-R (AJ130876), striped sea bass GnRH-R (AF218841),

Astatotilapia (AY028476), goldfish (gfGnRH-RA: AF121845; GnRH-

RB: AF121846), medaka (mdGnRH-R1: AB057677; mdGnRH-R2:

AB057676), and human GnRH-R2 (NM_057163).

Fig. 5. Type I and type II fish GnRH-Rs show different motifs in transmemb

an in silico analysis of the takifugu (fgGnRH-R) or zebrafish genome drafts.

sea bass (GnRH-R1: AJ419594), trout GnRH-R, catfish (GnRH-R1: X97

striped sea-bass GnRH-R (AF218841), Astatotilapia (AY028476), goldfish

GnRH-R2: AB057676).

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 11

ilar potency to activate these different GnRH-R. Apharmacological characterization of the GnRH-R types

was performed in different species including catfish

(Bogerd et al., 2002; Tensen et al., 1997), goldfish (Illing

et al., 1999), astatotilapia (Robison et al., 2001), striped

bass (Alok et al., 2000), and medaka (Okubo et al.,

2001). Binding of GnRH to GnRH-R results in the

activation of signal transduction mechanisms involving

G-proteins and membrane bound phospholipases (re-view Klausen et al., 2002). This leads to an increase in

cytosolic diacylglycerol (DAG) and inositol 1,4,5-

triphosphate (IP3) that, in turn, releases calcium from

intracellular stores. Moreover, GnRH-induced response

may also involves other signaling pathways leading to

production of cyclic adenosine monophosphate (cAMP)

and arachidonic acid (Bogerd et al., 2002; Pati and

Habibi, 2002).Only a few binding studies have been carried out on

fish GnRH-Rs (Bogerd et al., 2002; Robison et al., 2001)

and demonstrated that GnRH-Rs bind with different

affinities to different GnRH variants. Direct binding

studies (Bogerd et al., 2002) showed that cGnRH-II

binds to catfish type II GnRH-R with a much higher

affinity compared to other GnRH forms (Ka ¼ 2:18(cfGnRH-R1) to 4.3 nM (cfGnRH-R2) for cGnRH-IIversus Ka ¼ 1 (cfGnRH-R1) to 10 lM (cfGnRH-R2) for

cfGnRH). Competitive binding on type II astatotilapia

GnRH-R1 (Robison et al., 2001) showed similar results

since its binds cGnRH-II (EC50 ¼ 14:2 nM) with a much

rane domains 3 and 7. Asteriks (*) indicate sequences determined from

Eel (GnRH-R1: AB041327; GnRH-R2: Madigou et al., unpublished),

497; GnRH-R2: AF329894), seriola dumerilii GnRH-R (AJ130876),

(GfA: AF121845; GfB: AF121846), medaka (GnRH-R1: AB057677;

Fig. 6. Analysis of the structures of known GnRH-R genes. The in silico search for genes encoding putative fugu or zebrafish GnRH-R has been

carried out as described in Fig. 1 using tblastn algorithm and ORF finder. Our analysis indicates that genes encoding type I GnRH-R have a CAAT

and TATA box in the promoter region and are constituted of three exons interrupted by two introns. All exon/intron boundaries are conserved. The

size of exon 2 (205 nucleotides) is also conserved. Genes corresponding to type II GnRH-R are TATA-box less and constituted of at least three exons.

However, in trout, an alternative promoter usage and splicing has been described leading to an additive upstream exon (Madigou et al., 2002). This

leads to the use of another translation initiation codon (as shown with asterisks) that is conserved in all genes studied. Grey squares represent

transmembrane domains, black boxes: non-coding region; white boxes: coding region. Exon and intron sizes are indicated in bp.

12 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

higher affinity than sGnRH (EC50 ¼ 2:5lM) or

sbGnRH (EC50 ¼ 30lM). In addition, in all studies

carried out in transfected cells, both receptors are acti-

vated by different GnRH peptides. However, both re-

ceptors have a clear preference in terms of sensitivity for

cGnRH-II, followed by sGnRH and a third endogenousGnRH forms (species specific) when identified (Table 4).

Table 4

Biopotency of the different GnRH forms relative to mGnRH to stimulate IP

R types cGnRH-II sGnR

Medaka R1 Type 1 33 5.3

Medaka R2 Type II 1174 4.8

Goldfish GfA Type II 7000 45.6

Goldfish GfB Type II 838 51

Astatotilapia Type II 2000 2

Catfish GnRH-R1 Type II 1199 ND

Catfish GnRH-R2 Type II 6446 ND

ND: not determined.

Nevertheless, the different types of medaka GnRH-R

show different selectivity for GnRH variants (Okubo

et al., 2001). Medaka GnRH-R2 is particularly sensitive

to cGnRH-II whereas medaka GnRH-R1 shows no

clear preference for any of the GnRH tested. On the

other hand, catfish GnRH-Rs that belong to the samepiscine type II GnRH-R branch show no obvious

production in transfected cells

H Third GnRH form

0.023 (mdGnRH) Okubo et al. (2001)

0.316 (mdGnRH) Okubo et al. (2001)

0.3 (sbGnRH) Illing et al. (1999)

0.28 (sbGnRH) Illing et al. (1999)

0.1 (sbGnRH-R) Robison et al. (2001)

1.94 (cfGnRH) Bogerd et al. (2002)

6.8 (cfGnRH) Bogerd et al. (2002)

C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16 13

difference in ligand selectivity (Bogerd et al., 2002).However, in goldfish, GnRH-R subtypes GfA and GfB

have different ligand selectivity (Illing et al., 1999). In

conclusion, the pharmacological characterization of the

different GnRH-R types in a single fish species suggests

that they may have different ligand selectivity, but

whether two subtypes belonging to the same type share

similar ligand-induced potency remains unclear. The fish

lineage-specific gene duplication events may have led todifferent ligand selectivity for GnRH-R subtypes ob-

served in different species.

3.5. Tissue distribution of the GnRH receptors

Altogether GnRH-R genes are expressed widely, but

mostly in reproductive tissues (Table 3). However, dif-

ferences in tissue and/or cellular distribution have beendescribed for each GnRH-R types and subtypes identi-

fied in a single fish species. Sea bass and striped bass

GnRH-R genes belonging to type I are highly expressed

in the pituitary and to a lesser extent in brain and ovary.

In addition, pituitary GnRH-R type I gene expression

increases as sea bass and striped bass sexually mature

(Alok et al., 2000; Gonzalez-Martinez et al., 2002c). In

contrast, the trout GnRH-R (type II) is very poorlyexpressed in the pituitary, but was found in the brain

and the gonads (Madigou et al., 2000, 2002). The tissue

distribution of GnRH-R belonging to type II shows a

more complex picture. Certain fish type II receptors (i.e.,

goldfish GfA, trout rtGnRH-R, catfish cfGnRH-R2,

and astatotilapia GnRH-R1) have been found mainly

expressed in different brain regions (optic tectum, hy-

pothalamus, telencephalon, cerebellum) and moderatelyin pituitary, testis, and retina. In the goldfish, GfA was

shown to have a more widespread distribution than GfB

(Peter et al., 2003). However, although expressed in

brain, other subtypes belonging to this branch are

mostly expressed in pituitary as shown in catfish

(cfGnRH-R1; Bogerd et al., 2002), and eel (Okubo

et al., 2000b). In the goldfish, there is indication that

GfA and GfB could be expressed in gonadotrophs and,to a lesser extent in somatotrophs (Illing et al., 1999). In

the pituitary of tilapia, immunohistochemical studies

strongly suggest that two GnRH-R subtypes, belonging

to type II clade and termed 1A and 1B, show different

spatio-temporal expression patterns in LH containing or

prolactin producing cells, respectively (Parhar et al.,

2002). In addition, the use of a third antibody raised

against a type I piscine GnRH-R (striped bass) indicatesthat this GnRH-R type could be restricted to GH-im-

munoreactive cells.

Finally, there is need for more accurate information

on the expression of GnRH-R types and subtypes in

gonads. In goldfish ovary, GnRH-R (GfA) expression

appears to be restricted to intersticial and theca-granu-

losa cell layers (Illing et al., 1999).

In summary, there is now growing evidence that twodistinct GnRH-R types showing structural differences

and different tissue distribution are expressed in a single

fish species as observed in other vertebrate groups.

However, the evolutionary relationships between mam-

malian and fish GnRH-R remains unclear, mainly be-

cause of diverging sequences. One can expect that

comparison of large genomic regions harboring each

receptor type in fish and mammals will provide newinsights regarding evolution of the GnRH-R family.

In addition, a subsequent amplification of the GnRH-

R genes likely occurred in certain fish lineages. The re-

tention of distinct genes encoding GnRH-R strongly

suggests that they have gained non-redundant functions.

The functional characterization of fish GnRH-R will

require to address and combine different issues including

ligand selectivity, regulation, and spatio-temporal ex-pression patterns of each GnRH-R types and subtypes

within a single fish species. The use of technologies al-

lowing to switch off gene expression either in vivo or in

vitro would also bring new insights on type- and/or

subtype-specific function.

4. Conclusion

There is now evidence for a complex interplay be-

tween several GnRH-like peptides and different GnRH-

R types and subtypes within a single fish species but,

because of the long evolutionary history and the great

diversity of teleost fish, it is difficult at the present stage

to draw strait conclusions with regards to co-evolution

of GnRH peptides and GnRH receptors. Expansion ofgene families in euteleost fish is well documented, but

the mechanisms that generated these events are unclear.

One hypothesis is that an ancient genome duplication

occurred first during the evolution of ray-finned fishes

(Acanthopterygii) followed by subsequent fish lineage-

specific tetraploidization and gene loss events. However,

the importance of the ancestral whole genome duplica-

tion event in the abundance of duplicated genes in fishremains under debate (Taylor et al., 2001). Gene du-

plication is an important strength that triggers genome

and biological evolution. It is believed to provide op-

portunities for evolutionary novelties (Ohno, 1970). One

copy of the duplicated gene may accumulate degenera-

tive mutations leading to acquisition of new gene func-

tions. However, although fish genes seem to accumulate

substitutions at higher rate than mammalian genes(Robinson-Rechavi and Laudet, 2001), this process

known as neo-functionalization is predicted to be a rare

event (Lynch and Conery, 2000). Most of the duplicated

genes are predicted to be silenced or lost in mammals

and in fish (Bailey et al., 1978; Li, 1980; Lynch and

Conery, 2000). Another outcome of gene duplication

may explain retention and functional divergence of the

14 C. Lethimonier et al. / General and Comparative Endocrinology 135 (2004) 1–16

duplicated genes. Degenerative mutations can alter thespatial and/or temporal gene expression pattern and/or

regulation mechanisms of both gene copies so that they

are required to assume the biological function of the

single-copy ancestral gene. This sub-functionalization

process increases the likelihood of duplicated gene re-

tention, but also constitutes a resource for future de-

generative mutations that will lead ultimately to a novel

protein function. Although functional analyses remainto be carried out in fish to determine whether the dif-

ferent GnRH peptides on one hand and cognate recep-

tors on the other hand have redundant biological

function, the emergence of new GnRH systems showing

distinct spatial and temporal expression pattern suggests

that GnRH peptides may have pleiotropic function in

the reproductive physiology of the fish.

Acknowledgments

This work was supported by the European Union

Q5RS-2002-01801 PUBERTIMING.

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