www.elsevier.com/locate/gene

Gene 316 (2003) 157–165

Identification and characterization of the reptilian GnRH-II gene

in the leopard gecko, Eublepharis macularius, and its

evolutionary considerations$

Tadahiro Ikemoto*, Min Kyun Park

Laboratory of Endocrinology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo,

7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

Received 7 March 2003; received in revised form 2 June 2003; accepted 17 June 2003

Received by T. Gojobori

Abstract

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid

sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of

all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved

without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized

the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons

and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor

cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding

polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This

widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus–

pituitary–gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the

leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These

results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with

amniote evolution.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Chicken GnRH-II; Leopard gecko (Eublepharis macularius); Precursor sequence; Genomic structure; Spatial expression pattern; Phylogeny and

evolution

1. Introduction decapeptide, gonadotropin-releasing hormone, which stim-

GnRH plays a pivotal role in the regulation of reproduc-

tion. GnRH was originally identified as a hypothalamic

0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0378-1119(03)00758-3

Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA;

cGnRH-II, chicken GnRH-II; GAP, GnRH-associated peptide; GnRH,

gonadotropin-releasing hormone; GnRH-II, gene encoding GnRH-II; kb,

kilobase(s) or 1000 bp; NUP, Nested Universal Primer A; ORF, open

reading frame; PCR, polymerase chain reaction; RACE, rapid amplification

of cDNA ends; RT, reverse transcription; TAE, Tris-acetate-EDTA; UPM,

Universal Primer A Mix; UTR, untranslated region(s).$ The nucleotide sequence reported in this paper has been deposited in

the DDBJ/EMBL/GenBank with the accession number AB104485.

* Corresponding author. Tel./fax: +81-3-5841-4439.

E-mail address: tikemoto@infoseek.jp (T. Ikemoto).

ulates pituitary gonadotropes to synthesize and release

gonadotropins. Since the structure of the decapeptide was

determined from porcine and ovine brains (Matsuo et al.,

1971; Burgus et al., 1972), more than 20 GnRH molecules

or GnRH-like sequences have been identified thus far from

various vertebrates and some invertebrates (Adams et al.,

2003). With a given species, it is generally found that

multiple GnRH isoforms and multiple types of GnRH

receptor are distributed in a wide range of tissues. Thus,

GnRH is thought to have diverse physiological functions in

addition to gonadotropin secretion (Millar, 2003).

Of all the structural variants of GnRH, chicken GnRH-II

(cGnRH-II; first isolated from chicken brain by Miyamoto

et al., 1984) has been found to be universally present in and

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165158

uniquely conserved among vertebrates without any se-

quence substitutions (Millar and King, 1987). Chicken

GnRH-II appears in all classes of jawed vertebrates, reveal-

ing that it has remained unchanged for at least 500 million

years. This remarkable conservation suggests that it has

been highly constrained throughout evolution, apparently

indicating an important conserved role. This GnRH form

has been renamed GnRH-II (Sealfon et al., 1997).

It is generally accepted that different forms of GnRH

have arisen through gene duplications from a single ances-

tral GnRH form whose origin predates vertebrates (Millar

and King, 1987; Sherwood et al., 1993; Fernald and White,

1999). Indeed, GnRH-like immunoreactivities were reported

in several invertebrates (Tsutsui et al., 1998; Rastogi et al.,

2002), and several GnRH peptides were isolated from two

tunicates and an octopus (Craig et al., 1997; Di Fiore et al.,

2000; Iwakoshi et al., 2002). For analyses of phylogeny and

evolution based on a particular peptide, including GnRH,

the limited primary amino acid sequence of the low molec-

ular weight mature polypeptide is not useful for constructing

molecular phylogenetic trees (Dores et al., 1996). Rather,

precursor sequences from various species are needed to

achieve an understanding of GnRH evolution.

After the discovery and sequencing of the complemen-

tary DNA (cDNA) encoding the GnRH precursor in humans

(Seeburg and Adelman, 1984), a large body of GnRH

precursor sequence data from various species has been

accumulated. Precursor cDNAs for the remarkably con-

served GnRH-II have also been cloned from fishes, amphib-

ians and mammals (for details, see Section 2.7); however,

no sequence data have been reported from reptiles and birds.

Reptiles show some features of both higher and lower

vertebrates. They, together with mammals and birds, are

amniotes. On the other hand, extant reptiles share with

fishes and amphibians the characteristic of being ectother-

mic. Altogether, reptiles are important animals that may be

considered a crucial evolutionary bridge among vertebrates,

and would be absolutely indispensable for clarifying the

evolution of GnRH. The same is also true of GnRH-II,

because its mature peptide sequence remains unchanged but

its precursor sequence is remarkably divergent between

amphibians and mammals (Wang et al., 2001).

These considerations prompted us to search for the

GnRH-II precursor cDNA in reptiles. The existence of

GnRH-II in reptiles was verified by Powell et al. (1986).

Subsequently, this was confirmed by its sequencing in the

American alligator (Lovejoy et al., 1991). Here we describe

the first molecular cloning and characterization of the full-

length GnRH-II precursor cDNA from a reptilian species,

leopard gecko Eublepharis macularius. The physiology and

behavior of this reptile have been vigorously investigated

(Crews et al., 1998). Its ease of breeding and early matura-

tion also make this species a good experimental model. We

also determined the genomic structure of the GnRH-II gene.

In addition, to identify the sites where GnRH-II decapeptide

might be synthesized and to seek clues to its potential

function, the spatial expression pattern of the GnRH-II

precursor was examined by the reverse transcription–poly-

merase chain reaction (RT–PCR)-sequencing method. Fi-

nally, we constructed a molecular phylogeny of the

evolution of GnRH-II, incorporating our new sequence data

from the leopard gecko.

2. Materials and methods

2.1. Animals

Animals were treated according to the guideline of the

Bioscience Committee at the University of Tokyo. The

leopard geckos (E. macularius) were bred over several

generations in groups of one male and at least two females

in the laboratory. Fertile eggs were placed in cases filled

with moist vermiculite at a constant incubation temperature

of 29.0 jC, which produces a female-biased sex ratio

(Crews et al., 1998). One-year-old, fertile male (body

weight 80–100 g) and female (60–80 g) leopard geckos

were maintained under the conditions of 29.0 jC and a long

day photoperiod (14 h light and 10 h dark). The animals

were provided crickets and water ad libitum. Reproductive

fertility was confirmed by egg-laying and at least one

vitellogenic follicle in females in each group. Animals were

anesthetized with sodium pentobarbital (25 Ag/g) and killed

by rapid decapitation, followed by complete bloodletting.

Tissues and organs were immediately dissected and frozen

in liquid nitrogen, and stored at � 80 jC until use.

2.2. RNA preparation and cDNA synthesis

Total RNA was extracted using ISOGEN (NIPPON

GENE, Tokyo, Japan). Male whole brain cDNA used for

rapid amplification of cDNA ends (RACE; see Section 2.3)

was synthesized from 1 Ag of total RNA using a SMART

RACE cDNA Amplification Kit (BD Biosciences Clontech,

Palo Alto, CA), according to the manufacturer’s instructions.

The cDNAs used as templates for RT–PCRwere synthesized

from 3 Ag of denatured total RNA using 5 AM oligo(dT)

primer and 100 units of M-MLV Reverse Transcriptase

(Promega, Madison, WI) in a 20-Al reaction volume with

incubation at 38 jC for 30 min followed by 42 jC for 1.5 h.

2.3. Molecular cloning of the GnRH-II precursor cDNA by

RACE and RT–PCR

RACE was carried out to obtain a partial GnRH-II

precursor cDNA from male whole brain cDNA using four

degenerate primers (Table 1), Universal Primer A Mix

(UPM) and Nested Universal Primer A (NUP) (Clontech).

All of the following PCR amplifications were performed in a

20-Al reaction mixture containing each primer at 1 AM, 0.25

unit of TaKaRa Ex Taq (TaKaRa, Shiga, Japan), each dNTP

at 250 AM and Ex Taq Buffer (TaKaRa). The primary PCRs

Table 1

Oligonucleotide primers used for RACE, RT–PCR and genomic structure determination

Name Nucleotide sequencea Usage and location

01SE 5V-CAYTGGTCNCAYGGNTGGTA-3V for 3V-RACEnest02SE 5V-TGGTCNCAYGGNTGGTAYCC-3V for nested 3V-RACE03AS 5V-GGRTACCANCCRTGNGACCA-3V for 5V-RACEnest04AS 5V-TACCANCCRTGNGACCARTG-3V for nested 5V-RACEGSP-01SE 5V-GAGGCAGAAGAGCCAAGAGGTGAGG-3V for RT–PCR (in the 5V-UTR)GSP-02AS 5V-CAGACGTGAGTGAACACAGCAAGTC-3V for RT–PCR (in the 3V-UTR)EX1-12SE 5V-NNNNNTGGGATTTTCGGAGG-3V for intron A (in exon 1)

EX2-21SE 5V-GCATAATGATCATCGCCACTATCC-3V for intron B (in exon 2)

EX2-22AS 5V-GTGGCGATGATCATTATGCAGAGG-3V for intron A (in exon 2)

EX3-31SE 5V-GCTATGTGACGGGGACGACTGCAC-3V for intron C (in exon 3)

EX3-32AS 5V-GTGCAGTCGTCCCCGTCACATAGC-3V for intron B (in exon 3)

INT2-21AS 5V-GATTCGAACCATCGGTAAAGAAGTAC-3V for intron A (in intron B)

a Abbreviations for degenerate nucleotides: Y, C or T; R, G or A. N represents all four nucleotides.

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165 159

were performed with 01SE for 3V-RACE or 03AS for 5V-RACE in combination with UPM. The reaction conditions

for the primary PCR were as follows: 94 jC for 5 min, 5

cycles of 94 jC for 40 s, 67 jC for 30 s, 72 jC for 2 min, 7

cycles of 94 jC for 40 s, 65 jC for 30 s, 72 jC for 2 min, 10

cycles of 94 jC for 40 s, 63 jC for 30 s, 72 jC for 2 min, and

72 jC for 7 min. The 40-fold-diluted primary PCR solutions

served as templates for the nested PCRs with nest02SE for

3V-RACE or nest04AS for 5V-RACE in combination with

NUP. The reaction conditions for nested PCR were as

follows: 94 jC for 5 min, 5 cycles of 94 jC for 40 s, 67

jC for 30 s, 72 jC for 2 min, 15 cycles of 94 jC for 40 s, 64.5

jC for 30 s, 72 jC for 2 min, 20 cycles of 94 jC for 40 s, 62

jC for 25 s, 72 jC for 2 min, and 72 jC for 7 min. The

amplified products were separated by electrophoresis in a

1.5% Tris-acetate-EDTA (TAE) agarose gel and visualized

using ethidium bromide staining and a UV transilluminator.

DNA fragments were extracted using a QIAquick Gel

Extraction Kit (QIAGEN K.K., Tokyo, Japan) and directly

sequenced. These analyses were repeated independently

three times to avoid any PCR amplification errors.

After determination of the sequences of the 3V- and 5V-ends of the GnRH-II precursor cDNA, amplification of

cDNA fragments including the entire open reading frame

(ORF) was carried out to determine the complete sequence.

This amplification was performed with gene-specific pri-

mers, GSP-01SE complementary to the 5V-untranslatedregion (5V-UTR) and GSP-02AS complementary to the 3V-UTR (Table 1). The reaction conditions were as follows: 94

jC for 5 min, 30 cycles of 94 jC for 40 s, 65 jC for 30 s,

72 jC for 40 s, and 72 jC for 5 min. The PCR products

were sequenced as described above. This sequence deter-

mination was repeated using cDNAs derived from whole

brain, heart and ovary to rule out any possible errors

introduced during the procedure.

2.4. Comparison of the amino acid sequences of various

GnRH-II precursors

The CLUSTAL X program (version 1.81) (Thompson et

al., 1997) was used with default settings to align the

deduced amino acid sequences of the GnRH-II precursor

of the leopard gecko and other representative species with

each other. The amino acid identities were calculated

between entire ORFs, signal peptides, amidation/proteolytic

processing signals and GnRH-associated peptides (GAPs),

respectively, using the GeneDoc software (version 2.6.002)

(Nicholas and Nicholas, 1997). The amino acid similarity

score, based on the number of similar residues or conser-

vative substitutions, was also calculated between signal

peptides by GeneDoc.

2.5. Genomic structure of the GnRH-II gene

Genomic DNA was extracted from the liver using ISO-

GEN (NIPPON GENE). The GnRH-II gene was cloned by

PCR amplification using several primer pairs (Table 1, Fig.

3A). The reaction conditions were as follows: 94 jC for 5

min, 40 cycles of 94.3 jC for 45 s, 65 jC for 30 s, 72 jC for

2 min, and 72 jC for 7 min. The PCR products were

sequenced as described above. Sequencing procedure was

repeated independently three times.

2.6. Expression analysis

To identify sites where GnRH-II decapeptide might be

synthesized and obtain clues about its potential function, the

spatial expression pattern of the GnRH-II precursor was

examined by the RT–PCR-sequencing method. One micro-

liter of each five-fold-diluted RT product was amplified with

GSP-01SE and GSP-02AS. These primers, specific to the

GnRH-II precursor, were designed to amplify the entire

ORF and span all exon–intron boundaries, so any products

from possible genomic contamination could be eliminated.

For negative controls, PCR amplifications were also con-

ducted using each RNA sample without the RT reaction.

The reaction conditions were as follows: 94 jC for 5 min,

45 cycles of 94 jC for 40 s, 67 jC for 30 s, 72 jC for 40 s,

and 72 jC for 5 min. The PCR products were analyzed by

electrophoresis on a 1.2% TAE agarose gel. Each DNA

fragment was extracted from the gel and sequenced directly

to confirm that it was derived from the GnRH-II precursor

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165160

mRNA. Some of these fragments required reamplifications.

This procedure was repeated independently at least two

times.

2.7. Molecular phylogenetic analysis

The nucleotide sequences of the entire ORF and the

deduced amino acid sequences of the GnRH-II precursors in

the leopard gecko and all reported species were aligned with

each other using CLUSTAL X with default settings, fol-

lowed by some minor revisions to correct obvious mistakes.

The alignments of both nucleotide and amino acid sequen-

ces were used to generate phylogenetic trees. Phylogenetic

trees were constructed using two methods: the neighbor-

joining method (Saitou and Nei, 1987) with the Tamura 3-

parameter model (Tamura, 1992) for nucleotide sequences

and with default settings for amino acid sequences in the

Mega software (version 2.1) (Kumar et al., 2001), and the

maximum likelihood method with default settings for nu-

cleotide and amino acid sequences in the PHYLIP software

(version 3.6a3) (Felsenstein, 2002). Octopus GnRH (Iwa-

koshi et al., 2002) was used as an outgroup. Bootstrap

values were calculated with 1000 replications to estimate the

robustness of internal branches. Full species names and

GenBank accession numbers of all the reported GnRH-II

precursor sequences are as follows: Anguilla japonica

(Japanese eel), AB026990; Astatotilapia burtoni (Burton’s

mouth-brooder), L27435; Cyprinus carpio (common carp),

AY189961; Carassius auratus (goldfish), (a) U30386, (b)

U40567; Clarias gariepinus (North African catfish),

X78047; Danio rerio (zebrafish), AF511531; Dicentrarchus

labrax (European sea bass), AF224281; Homo sapiens

(human), NM_001501; Macaca mulatta (rhesus monkey),

AF097356; Morone saxatilis (striped sea-bass), AF056313;

Oncorhynchus mykiss (rainbow trout), AF125973; Oreo-

Fig. 1. Nucleotide and deduced amino acid sequence of the cDNA encoding the Gn

5Vto 3V, beginning with the initiator codon (ATG) in the coding region. The ORF i

case letters. Amino acid residues (bottom) are numbered beginning with the first M

decapeptide sequence is depicted in the open box. Sequences for the signal peptide

(Gly-Lys-Arg) is shown by the dotted line. Arrowheads indicate the splice sites. T

sequence has been deposited in the DDBJ/EMBL/GenBank under the accession

chromis niloticus (Nile tilapia), AB101666; Oryzias latipes

(Japanese medaka), AB041330; Rana catesbeiana (bull-

frog), AF186096; Rutilus rutilus (roach), U60668; Sclero-

pages jardinii (Australian bonytongue), AB047326; Sparus

aurata (gilthead seabream), U30325; Suncus murinus

(house shrew), AF107315; Trichosurus vulpecula (silver-

gray brushtail possum), AF193516; Tupaia glis belangeri

(common tree shrew), U63327; Typhlonectes natans (Rio

Cauca caecilian), AF167558; Verasper moseri (barfin floun-

der), AB066359; Octopus vulgaris (common octopus),

AB037165.

3. Results and discussion

3.1. Molecular cloning of the GnRH-II precursor cDNA in

the leopard gecko

The full-length GnRH-II precursor cDNA was isolated

from the leopard gecko whole brain by RACE and RT–PCR

(Fig. 1). The nucleotide sequence was determined by

independent amplifications using different organs (whole

brain, heart and ovary), at least three times each. The 597-

base pair (bp) cDNA consisted of a 5V-UTR of 71 nucleotides,

an ORF of 258 nucleotides encoding a prepro-GnRH-II of 86

amino acid residues, a stop codon (TGA) and a 3V-UTR of 265

nucleotides. The initiation site for translation would be the

ATG at positions + 1 to + 3 (Fig. 1), and was generally in

agreement with the initiation sites of all the other species

analyzed (Fig. 2A). Moreover, the CACCATGG sequence at

positions � 4 to + 4 is the typical consensus sequence for

translation initiation sites in eukaryotic mRNAs (Kozak,

1987). Although there is an additional in-frame ATG at

positions + 40 to + 42, CATAATGA at positions + 36 to

+ 43 is less similar to the Kozak consensus sequence. No

RH-II precursor in the leopard gecko. Nucleotides (top) are numbered from

s indicated by capital letters, and the 5V- and 3V-UTRs are indicated by lower

et residue in the ORF. The asterisk indicates the stop codon. The GnRH-II

and GAP are underlined. The amidation/proteolytic processing (A/P) signal

he polyadenylation signal (AATAAA) is double-underlined. The nucleotide

number AB104485.

Fig. 2. Comparison of the prepro-GnRH-II polypeptide of the leopard gecko and other representative species. (A) Alignment of the amino acid sequences of

various GnRH-II precursors. The entire ORF amino acid sequences were aligned with each other using the CLUSTAL X program and displayed using the

GeneDoc software. Hyphens were inserted for optimal alignment. The prepro-GnRH-II polypeptide is composed of a signal peptide, the GnRH-II decapeptide,

an amidation/proteolytic processing (A/P) signal and a GAP. Shaded areas indicate the identical residues with those of the leopard gecko. Heavily shaded areas

are conserved throughout all used species. Numbers on the left and right indicate the amino acid positions. For species names and GenBank accession numbers,

see Section 2.7. sg brushtail possum, silver-gray brushtail possum. (B) Amino acid comparison between prepro-GnRH-II of the leopard gecko and those of

other representative species. Each score in parenthesis shows the percentage of amino acid similarity, and the other values are the percentages of amino acid

identity. The similarity score is based on the number of similar residues or conservative substitutions. These scores were obtained using GeneDoc.

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165 161

other possible ATG translation initiation site was found in the

upstream of this ORF. The 3V-UTR contained a canonical

polyadenylation signal (AATAAA) slightly upstream of the

poly(A) tail (Proudfoot and Brownlee, 1976). The nucleotide

sequence of the GnRH-II precursor cDNA has been deposited

in the DDBJ/EMBL/GenBank under the accession number

AB104485.

The leopard gecko prepro-GnRH-II has a structure char-

acteristic of prepro-GnRH polypeptides: a signal peptide (24

residues), a GnRH decapeptide (GnRH-II), an amidation/

proteolytic processing signal (Gly-Lys-Arg) and a GAP (49

residues) (Fig. 1). The signal peptide includes a high

proportion of hydrophobic amino acids (16 out of 24

residues), which is generally common for signal peptides

of prepro-GnRH polypeptides in other species (Bogerd et

al., 1994; Wang et al., 2001). The Gly-Lys-Arg sequence

that followed the GnRH-II decapeptide is identical to those

of all the reported prepro-GnRH polypeptides; the glycine

residue is the standard donor of the amino group for

carboxy-terminal amidation and the Lys-Arg functions in

proteolytic processing, as is also true for many neuroendo-

crine peptide precursors (Douglass et al., 1984).

3.2. Comparison of the amino acid sequences of various

GnRH-II precursors

The alignment of the deduced prepro-GnRH-II polypep-

tides of the leopard gecko and other representative species is

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165162

shown in Fig. 2A. For the entire ORF, the leopard gecko

prepro-GnRH-II had the highest identity (62%) with the Rio

Cauca caecilian prepro-GnRH-II (Fig. 2B). The leopard

gecko prepro-GnRH-II also showed relatively high identi-

ties (48–62%) with its counterparts in nonmammalian

species, whereas it exhibited low identities ( < 30%) with

its counterparts in mammalian species except the silver-gray

brushtail possum (38%). This tendency was also observed

for the comparison of the GAP, but the GAP sequences were

remarkably divergent, especially in length, between mam-

malian and nonmammalian species (Fig. 2A). The signal

peptide generally showed low identities ( < 40%), but

exhibited higher similarities (50–66%), which was mainly

due to the common feature of the prepro-GnRH polypeptide

of having a high proportion of hydrophobic amino acids in

the signal peptide.

3.3. Genomic structure of the GnRH-II gene

Sequencing of the PCR-amplified leopard gecko GnRH-

II gene revealed that it was composed of four exons

(designated exons 1, 2, 3 and 4) separated by three introns

(designated introns A, B and C) at similar positions as found

in other GnRH genes (Fig. 3A). The exon–intron bound-

aries were determined by sequence comparison with the

GnRH-II precursor cDNA. The three intron sites are shown

in Fig. 1. All of three 5V- and 3V-splice sites correspond to theconsensus sequences: 5V-GT (donor) and 3V-AG (acceptor)

intron splice sites (Mount, 1982) (Fig. 3B). All three introns

contains a pyrimidine-rich region for mRNA splicing im-

mediately upstream of the 3V-splice sites (Mount, 1982) (Fig.

3B). The three introns are located in the 5V-UTR, in the

Fig. 3. Schematic diagram of the GnRH-II gene in the leopard gecko. (A) Genomi

lines indicate exons and introns, respectively. Arrows indicate the locations of ol

proteolytic processing signal. (B) Sequences of exon– intron boundaries of the Gn

introns are by lower case letters. Exon– intron boundaries are depicted by slashe

contain pyrimidine-rich region (underlined) immediately upstream of the 3V-splice

middle of the GAP, and shortly upstream of the 3V-UTR,respectively. Exon 1 contains a part of the 5V-UTR. Exon 2

encodes the rest of the 5V-UTR, the signal peptide, the

GnRH-II decapeptide, the amidation/proteolytic processing

signal and the N-terminus of the GAP. Exon 3 encodes the

central portion of the GAP. Exon 4 encodes the C-terminus

of the GAP along with the 3V-UTR. It is widely recognized

that the structure of all known vertebrate GnRH genes is

conserved and is composed of four exons and three introns

(Farahmand et al., 2003). The lengths of the introns in all of

the GnRH-II genes reported to date vary between 82

(Japanese medaka intron B) and 845 bp (human intron C)

(White et al., 1998; Okubo et al., 2002). The length of intron

B (91 bp) is relatively short, while those of introns A and C

(about 2 and 4 kilobase (kb), respectively) are much longer

than those found in other species.

3.4. Expression of the GnRH-II precursor mRNA in the

leopard gecko

Single RT–PCR products of the expected size (414 bp)

were obtained from more than half of the tissues and organs

examined (Fig. 4). The authenticity of the RT–PCR prod-

ucts was confirmed by direct sequencing. The possibility of

genomic contamination was eliminated by the observation

of amplifications spanning all exon–intron boundaries. No

products were detected from any negative controls without

the RT reaction. This widespread expression of the GnRH-II

precursor mRNA is consistent with the previous findings in

several species. The presence of the GnRH-II precursor

mRNA in gonads has been reported in the goldfish (Yu et

al., 1998), Burton’s mouth-brooder (White and Fernald,

c structure of the GnRH-II gene in the leopard gecko. Boxes and horizontal

igonucleotide primers to amplify the gene partially. A/P signal, amidation/

RH-II gene in the leopard gecko. Exons are indicated by capital letters, and

s. All of three introns have the consensus 5V-GT and 3V-AG sequences and

sites.

Fig. 4. Expression of the GnRH-II precursor mRNA in the leopard gecko. RT products were PCR-amplified to span all exon– intron boundaries and were

analyzed by electrophoresis on a 1.2% TAE agarose gel with ethidium bromide staining. The authenticity of the RT–PCR products was confirmed by direct

sequencing of each band of the expected size (414 bp; indicated by an arrow). The lane of RT-minus represents the negative control using male whole brain

RNA sample without the RT reaction. M, molecular marker; � , under detection level.

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165 163

1998), Japanese eel (Okubo et al., 1999), rainbow trout (von

Schalburg et al., 1999), North African catfish (Bogerd et al.,

2002) and humans (Millar, 2003). In the North African

catfish, ubiquitous expression of the GnRH-II precursor

mRNA was observed in all tissues and organs tested

(Bogerd et al., 2002). The hemipenis is the male copulatory

organ, and is one of the major features distinguishing the

Fig. 5. Unrooted neighbor-joining phylogenetic tree of the GnRH-II precursors. The

parameter model for entire ORF nucleotide sequences using the Mega software. Oc

are indicated for all nodes on the tree. The scale bar beneath the tree corresponds

accession numbers, see Section 2.7.

both sexes of the leopard gecko and some other reptiles.

This is the first report to the best of our knowledge on the

expression of the GnRH precursor mRNA in the male

copulatory organ, the hemipenis, and its counterpart in

female, the hemiclitoris.

The roles of GnRH outside the hypothalamus–pituitary–

gonadal axis remain largely unknown. Although the expres-

tree was constructed using the neighbor-joining method with the Tamura 3-

topus GnRH was used as an outgroup. Bootstrap values of 1000 resamplings

to estimated evolutionary distance units. For species names and GenBank

T. Ikemoto, M.K. Park / Gene 316 (2003) 157–165164

sion of the GnRH-II precursor mRNA does not ensure its

translation into a functional GnRH-II decapeptide, the

present results support the notion that GnRH-II may act as

a neurotransmitter and/or a neuromodulator in the brain and

an autocrine and/or a paracrine hormone outside the brain.

Verifying this, however, will require further studies. Under-

standing the roles of the widespread GnRH-II expression

will require detailed knowledge of its specific sites of action

and the distribution of its receptor.

3.5. Molecular phylogenetic analysis

Molecular phylogenetic trees of the GnRH-II precursors

were constructed using the methods of neighbor-joining and

maximum likelihood for nucleotide and amino acid sequen-

ces. Each method yielded essentially the same tree topology.

Fig. 5 shows an unrooted neighbor-joining tree based on the

entire ORF nucleotide sequences. Octopus GnRH used as an

outgroup indicates the putative position of the root. The

robustness of internal branches was estimated by 1000

bootstrap resamplings. As inferred from the results of the

sequence comparison, there were two major clusters: mam-

malian species (except for a marsupial mammal, the silver-

gray brushtail possum), and the other species. The leopard

gecko has a similar precursor sequence with those of fishes

and amphibians, while eutherian mammals have distinct

sequences, especially in the GAP region. Besides, some

mammalian species are thought not to have a GnRH-II

decapeptide (Millar, 2003). Thus the precursor sequence

of the evolutionarily conserved GnRH-II may have become

somewhat divergent during amniote evolution.

3.6. Conclusions

We have identified and characterized the full-length

cDNA encoding the GnRH-II precursor and determined its

genomic structure in the leopard gecko, E. macularius. This

is the first report of such an analysis in reptiles. The deduced

prepro-GnRH-II polypeptide had the highest identities with

the corresponding polypeptides of amphibians.

Expression of the GnRH-II precursor was detected in

more than half of the tissues and organs examined by the

RT–PCR-sequencing method. This widespread expression

is consistent with the previous findings in several species,

though the role of this decapeptide outside the hypothala-

mus–pituitary–gonadal axis remains largely unknown.

Molecular phylogenetic analysis combined with se-

quence comparison showed that the leopard gecko is more

similar to fishes and amphibians than to eutherian mammals

with respect to the GnRH-II precursor sequence. These

results strongly suggest that the divergence of the GnRH-

II precursor sequences seen in eutherian mammals may have

occurred along with amniote evolution. Further character-

ization of the precursor sequence of GnRH, including

GnRH-II, will provide strong insights into the phylogeny

and evolution of GnRH.

Acknowledgements

The authors would like to thank the editor and reviewers

for their helpful comments.

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