Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family
et al. (2012) Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the
Evolution of This Receptor Family. PLoS ONE 7(11): e48931. doi:10.1371/journal.pone.0048931
Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family
Je re my Pasquier 0
Anne-Gae lle Lafont 0
Shan-Ru Jeng 0
Marina Morini 0
Ron Dirks 0
Guido van den Thillart 0
Jonna Tomkiewicz 0
Herve Tostivint 0
Ching-Fong Chang 0
Karine Rousseau 0
Sylvie Dufour 0
Hubert Vaudry, University of Rouen, France
0 1 Muse um National d'Histoire Naturelle, Research Unit BOREA, Biology of Aquatic Organisms and Ecosystems, CNRS 7208- IRD207- UPMC, Paris, France, 2 National Kaohsiung Marine University, Department of Aquaculture, Kaohsiung, Taiwan, 3 Leiden University, ZF-screens B.V. and Institute of Biology, Leiden, The Netherlands, 4 Technical University of Denmark, National Institute of Aquatic Resources , Charlottenlund, Denmark , 5 Muse um National d'Histoire Naturelle, UMR 7221 CNRS/MNHN Evolution des Re gulations Endocriniennes, Paris, France, 6 National Taiwan Ocean University, Department of Aquaculture and Center of Excellence for Marine Bioenvironment and Biotechnology , Keelung , Taiwan
Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current teleosts.
Funding: JP is a recipient of a PhD fellowship from the Ministry of Research and Education. This work was supported by grants from the National Research
Agency, PUBERTEEL number ANR-08-BLAN-0173 to SRJ, CFC, KR and SD, and from the European Community, 7th Framework Programme, PRO-EEL number
245257 to AGL, RD, GT, JT and SD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
In 1999, a novel G protein-coupled receptor named GPR54 was
cloned from the rat brain . GPR54 ligands were later shown to
be kisspeptins, previously described as metastasis suppressors,
encoded by Kiss1 gene . As soon as this link between kisspeptins
and GPR54 was unveiled, major discoveries in the field of
reproductive endocrinology were realised. Kisspeptin and its
receptor (GPR54/Kissr) emerged as major upstream regulators of
the gonadotropic axis in mammals, by their key roles in the control
of GnRH, mediation of steroid feedbacks, as well as initiation of
In mammalian species, a single gene, named Kiss1r, encodes for
the kisspeptin receptor. To date the only exception is the platypus
(Ornithorhynchus anatinus), a non-placental mammal, in which two
receptors are present . Contrasting situations are found in other
tetrapods, as shown by a lack of Kissr in birds and up to three Kissr
paralogous genes in an amphibian species, the xenopus (Xenopus
tropicalis) . In teleosts, at least one Kissr is present in all species
investigated so far (for review ). A second Kissr gene could be
evidenced in some species including zebrafish (Danio rerio) ,
goldfish (Carassius auratus) , medaka (Oryzias latipes) , and
striped bass (Morone saxatilis) . However, this second paralog is
lacking in the genomes of other teleosts, such as fugu (Takifugu
niphobles), tetraodon (Tetraodon nigroviridis) and stickleback
(Gasterosteus aculeatus). Concerning cyclostomes, we recently identified one
Kissr gene in the sea lamprey (Petromyzon marinus) genome . The
homology relationships between the various Kissr and the
evolutionary events that led to such diversity are still ambiguous.
Recently the genomes of three osteichthyan species of particular
phylogenetic interest have been published: the coelacanth,
Latimeria chalumnae, a representative of early sarcopterygians
(coelacanth genome project, Broad Institute), the spotted gar,
Lepisosteus oculatus, a non-teleost actinopterygian , and the
European eel, Anguilla anguilla, a member of an early group of
teleosts (elopomorphs) . In the present study, we investigated
the presence of Kissr in the genome of those relevant species.
We previously initiated the study of Kissr in the eel . Due to its
phylogenetical position, the eel may provide insights into ancestral
regulatory functions in teleosts , the largest group of
vertebrates. Furthermore, its striking biological cycle, with a
blockade of sexual maturation as long as the reproductive oceanic
migration is not performed, makes the eel a powerful model to
investigate neuroendocrine mechanisms of puberty . We
formerly cloned the cDNA of one Kissr from the European eel
In the present study, we report the occurrence of two additional
Kissr in the eel, providing the first evidence for three Kissr in a
teleost species. We also identified four Kissr in both coelacanth and
spotted gar genomes, providing the first evidence for the presence
of four Kissr in vertebrate species. Phylogenetic and syntenic
analyses allowed us to assess the existence of four Kissr paralogs in
osteichthyans and to raise new hypotheses on the origin and
evolutionary history of vertebrate Kissr family. These data also let
us propose a clarified Kissr nomenclature, based on these four
paralogons. Finally, in order to get some insights into the potential
process driving the conservation or the loss of multiple Kissr, we
focused on the analyses of their tissue distributions and regulations
during experimental maturation in the eel.
Materials and Methods
European eels (Anguilla anguilla) were at the prepubertal silver
stage, which corresponds to the last continental phase of the eel life
cycle, preceding the oceanic reproductive migration. Cloning and
tissue distribution were performed using female and male eels
purchased from Rungis International Market (Rungis, France) and
transferred to MNHN, France. Eel manipulations were performed
according to the guidelines of the French Ministry of Agriculture
and Fisheries; Veterinary Department for Animal Health and
Protection, under the supervision of authorised investigators
(agreement NuI-75UPMC-F1-07). Experimental gonadal
maturation was performed on farmed female eels at the DTU Aqua
research facility at Lyksvad Fishfarm, Vamdrup, Denmark. Eel
manipulations and experimental maturation were performed
according to the guidelines of Danish Ministry of Food,
Agriculture and Fisheries; Danish Veterinary and Food
Administration (Approval reference for maturation experimentations:
2010/561-1783 artificial reproduction of European eel). Pain,
suffering and stress were attempted minimized during transport
and rearing throughout the experiments. All eels were anesthetized
using benzocaine before sacrifice and whenever needed in relation
Identification of Kissr sequences
European eel genome database analysis. The TBLASTN
algorithm of the CLC DNA Workbench software (CLC bio,
Aarhus, Denmark) was used to retrieve the genomic sequences of
three Kissr (named Kissr-1, Kissr-2 and Kissr-3) from the European
eel genome database . The peptidic sequences of the
previously characterized eel Kissr (named here Kissr-2) , of
the two zebrafish Kissr, and of the three xenopus Kissr were used as
Coelacanth and spotted gar genomic database
analyses. The TBLASTN algorithm (search sensitivity: near
exact matches (short)) of the e!ENSEMBL website was used to
retrieve the genomic sequences of four coelacanth Kissr and four
spotted gar Kissr from their genomic databases, respectively. The
peptidic sequences of the three eel Kissr identified in the present
study, of the three xenopus Kissr and of the two predicted platypus
Kissr were used as queries.
The exons and splicing junctions were predicted using the
empirical nucleotidic splicing signatures, i.e. intron begins with
GT and ends with AG. The 7 transmembrane domains were
determined using TMHMM software (TMHMM Server v. 2.0).
Amino-acid sequences of 51 known or predicted Kissr were first
aligned using ClustalW , then manually adjusted. The JTT
(Jones, Taylor and Thornton) protein substitution matrix of the
resulting alignment was determined using ProTest software .
Phylogenetic analysis of the Kissr sequence alignment was
performed using the maximum likelihood method (RaxML
software ), with 1,000 bootstrap replicates.
Identity and similarity percentages between two sequences were
calculated using EMBOSS Matcher.
The synteny analyses of the eel Kissr-1, Kissr-2, Kissr-3 genomic
regions were manually performed using CLC DNA Workbench 6
software and the European eel genome database. The analyses of
the neighbouring genes of the four predicted spotted gar Kissr were
performed using the preliminary gene annotation of the genome
assembly LepOcu1 generated by Ensembl release 67. Synteny
maps for the genomic neighbourhoods of the Kissr genes in human,
platypus, lizard (Anolis carolinensis), xenopus, zebrafish, medaka,
stickleback, tetraodon and coelacanth ( as well as of the
corresponding region in chicken (Gallus gallus) were performed
using the PhyloView of Genomicus v67.01 web site . The
analysis of the neighbouring genes of the four predicted coelacanth
Kissr paralogs was completed by the automatic and manual gene
annotation of the coelacanth genome.
Cloning of the full-length cDNAs encoding eel Kissr-1 and
Total RNA from eel brain (di-/mes-encephalon) was extracted
using Trizol reagent and reverse-transcripted as previously
described . 59 and 39 UTR genomic sequences of predicted
Kissr-1 and Kissr-3 were used to design specific primers (Table S1)
in order to amplify by PCR their full coding sequences (CDS).
Standard PCRs were performed as follows: an initial step of
polymerase activation for 3 min at 94uC; then 40 cycles with 30 s
at 94uC for denaturing, 30 s at various temperatures (54uC for
Kissr-1 and 66uC for Kissr-3) for annealing, 1 min 30 s at 72uC for
primer extension, and a single final extension step of 5 min at
72uC. PCR products were purified with the QUIAquick PCR
Purification Kit (Qiagen, Hilden, Germany) and inserted in a
pCRTM4-TOPOH TA vector provided by the TOPOH TA
CloningH Kit (Invitrogen). The vectors were then transfected in
One ShotH TOP10 Chemically Competent E. coli (Invitrogen).
After the bacteria containing a vector with insert had grown in
miniprep cultures, vectors were extracted and purified using
QUIAquick Spin Miniprep Kit (Qiagen, Hilden, Germany). Their
inserts were then sequenced at GATC biotech Ltd (Konstanz,
Germany). The obtained sequences were submitted to EMBL.
Kissr tissue distribution in the eel
Various tissues were collected from eight freshwater female
silver European eels to investigate the distribution of Kissr-1, Kissr-2
and Kissr-3 expressions, using qPCR. Eels were sacrificed by
decapitation. The following organs were sampled, stored in
RNAlater (Ambion-Inc, Austin, TX, USA) and kept frozen at
220uC until RNA extraction: brain, pituitary, ovary, muscle, eye,
liver, adipose tissue, kidney, intestine and spleen. The brain was
dissected into five parts : olfactory bulbs, telencephalon, di-/
mes-encephalon, cerebellum and medulla oblongata. In addition, testes
from eight freshwater male silver European eels were also
Eel experimental maturation
Farmed female silver European eels transferred to seawater
(36%) received weekly injections of salmon pituitary extract for
four months to induce vitellogenesis followed by one
dihydroxyprogesterone injection to induce final oocyte maturation and
ovulation, according to . Analyzes were performed on nine
matured eels and seven controls. After sacrifice, anterior brain
divided in two parts (olfactory bulbs/telencephalon;
di-/mesencephalon), pituitary and ovary were dissected and stored in
RNAlater and kept frozen at 220uC until RNA extraction.
Quantitative real-time PCR (qPCR)
Eel Kissr-1, Kissr-2 and Kissr-3 specific primers (Table S1) were
designed based on the full-length European eel CDS sequences
cloned in this study (Kissr-1 and Kissr-3) and previously  (Kissr-2),
using Primer3 Software (Whitehead Institute/Massachusetts
Institute of Technology, Boston, MA). The Kissr-3 qPCR primers were
designed in exon-3, to quantify the expression level of the three
Kissr-3 mRNA splicing isoforms at once. To optimize the assays,
different annealing temperatures were tested according to the
melting temperature (Tm) of primers. To assess their specificity,
amplification products were sequenced at GATC Biotech Ltd.
Primers for European eel LHb, mGnRH and reference gene b-actin
were as previously designed [18,20] (Table S1). All primers were
purchased from Eurofins (Ebersberg, Germany).
Quantitative assays of eel Kissr-1, Kissr-2, Kissr-3, mGnRH, LH-b
and b-actin mRNAs were performed using the LightCyclerH
System (Roche, Ltd. Basel, Switzerland) with SYBER Green I
sequence-unspecific detection as previously described [18,20,21].
The qPCR primers are listed in Table S1. The qPCRs were
prepared with 4 ml of diluted cDNA template, 2 ml of PCR grade
water, 2 ml of SYBR Green master mix and 1 ml of each forward
and reverse primer (0.5 pmole each at final concentration). The
qPCRs were performed as follows: an initial step of polymerase
activation for 10 min at 95uC; then 41 cycles with 15 s at 95uC for
denaturing, 5 s at 60uC (Kissr-1, Kissr-2, mGnRH, LH-b and b-actin)
or 62uC (Kissr-3) for annealing, 10 s at 72uC for primer extension,
5 s at 83uC to avoid measurement of non-specific annealing; and a
single final extension step of 5 min at 72uC. Each qPCR run
contained a non-template control (cDNA was substituted by water)
for each primer pairs to confirm that reagents were not
contaminated. The efficiency of all primers was tested and the
specificity of each reaction was assessed by melting curve analysis
to ensure the presence of only one product, and by sequencing.
Individual tissue samples were then analyzed in duplicate by
qPCR. Serial dilutions of cDNA pool of brain tissues were run in
duplicate and used as a common standard curve and also included
in each run as a calibrator. Normalisation of data was performed
using total RNA content for the tissue distribution samples, and
using b-actin mRNA level for experimental maturation samples.
Results and Discussion
Characterization of three European eel Kissr
Genomic prediction of three eel Kissr genes. We used the
deduced peptidic sequence from the previously cloned European
eel Kissr cDNA  (considered as Kissr-2 in the present study) as a
query to retrieve Kissr sequences in the European eel genome.
TBLASTN results revealed the presence of three different Kissr
genes, called Kissr-1, -2, -3, each made of 5 exons and 4 introns
(Fig. S1), that constitutes the conserved structure of Kissr genes.
The lengths of exon-2, exon-3 and exon-4 are the same for all
three genes with 125 bp, 136 bp and 239 bp, respectively. The
exon-1 of Kissr-1, Kissr-2 and Kissr-3 is 235 bp, 214 bp and 229 bp,
respectively. The exon-5 is 348 bp, 372 bp and 351 bp,
respectively. This leads to predicted Kissr-1, Kissr-2 and Kissr-3 CDS of
1083 bp, 1086 bp and 1080 bp, respectively.
Cloning of eel Kissr cDNAs. Using European eel specific
Kissr-1 primers, PCRs performed on brain cDNAs led to a single
product. Its sequence (EMBL: HE802271) encompassed partial 59
and 39 UTRs of 199 bp and 102 bp, respectively, and a full CDS
of 1083 bp. Once translated, the cloned Kissr-1 CDS gives a
360aa receptor, exhibiting the seven transmembrane domains (TMD)
characteristic of the GPCR family (Fig. 1A).
The previously cloned eel Kissr cDNA  is named Kissr-2 in the
Using European eel specific Kissr-3 primers, PCRs performed
on brain cDNAs led to the isolation of three different products
(EMBL: HE802272, HE802273 and HE802274). Their sequence
revealed three Kissr-3 isoforms named here Kissr-3_v1 (Fig. 1B),
Kissr-3_v2 and Kissr-3_v3 (Fig. S2). The partial 59 and 39 UTR are
167 and 147 bp for all three isoforms. The Kissr-3_v1 isoform
corresponds to the 5 predicted exons from the eel genome. Once
translated, it gives a 359-aa receptor exhibiting the seven
conserved TMD (Fig. 1B). The Kissr-3_v2 CDS corresponds to
the 5 predicted exons, minus the 76 first nucleotides of the exon-2
that are missing (Fig. S2A). The loss of these 76 nucleotides results
in a shift of the Kissr-3 reading frame and the occurrence of a first
premature termination codon at position 280 pb. Once translated,
it gives a 93-aa protein. This shorter protein exhibits a single TMD
encoded by exon-1. The Kissr-3_v3 CDS corresponds to exon-1,
exon-3, exon-4 and exon-5, while exon-2 is completely missing
(Fig. S2B). The loss of exon-2 results in a shift of the Kissr-3 reading
frame without the occurrence of any premature termination
codon. Once translated, it gives a 361-aa protein, which exhibits
only one TMD encoded by exon-1. The presence of the three
Kissr-1, -2, and -3 transcripts and of the three Kissr-3 mRNA
isoforms was also observed in Japanese eel (Anguilla japonica) brain
cDNAs (data not shown).
To date, only one or two Kissr genes had been found in the
genome of teleosts (reviewed by ), so the eels provide the first
evidence of the presence of three Kissr genes in teleosts. Until now
three Kissr genes had been found only in an amphibian species,
In addition to this Kissr gene diversity, European and Japanese
eels present three Kissr-3 mRNA isoforms revealing similar
alternative splicing in both species. The existence of Kissr mRNA
splicing isoforms has been described in two other teleosts, the sole
(Solea senegalensis)  and the zebrafish . In the sole, two
isoforms were identified for the single Kissr (Kissr-2 type) gene
Figure 1. Molecular cloning of eel Kissr-1 and Kissr-3_v1. Nucleotide and deduced amino-acid sequence of the cDNA encoding the eel Kissr-1
(A) and Kissr-3_v1 (B). Nucleotides (top) are numbered from 59 to 39. The amino-acid residues (bottom) are numbered beginning with the first
methionine residue in the ORF. The asterisk (*) indicates the stop codon. The predicted seven transmembrane domains (TMD) are underlined and the
cysteines involved in a disulphide bridge are shaded in grey.
present in this species and they differ by the retention of intron III,
while in the eel they differ by partial or complete deletion of
exon2 in Kissr-3 . In the zebrafish, five isoforms were identified for
Kiss1rb (Kissr-3 type), while no splicing isoform was observed
concerning Kiss1ra (Kissr-2 type). Among the five zebrafish Kiss1rb
isoforms, one of them, called KRBDP1, resulted from the deletion
of exon-2 and corresponds to eel Kissr-3_v3 . Even though the
existence of other Kissr mRNA isoforms in the eel cannot be
excluded, the three genes and several isoforms described here
highlight the Kissr molecular diversity in basal teleosts. They also
could reflect an ancestral post-transcriptional regulatory process of
Genomic prediction of four coelacanth and four spotted
gar Kissr genes
To further assess the Kissr diversity in vertebrates, we
investigated the presence of these genes in the genomes of the
spotted gar, a non-teleost actinopterygian, and the coelacanth, a
basal sarcopterygian, two species of relevant phylogenetical
positions. We performed a TBLASTN in both genomes (Ensembl)
using the three European eel, the two zebrafish and the three
xenopus Kissr proteins as queries. The TBLASTN results revealed
the existence of four Kissr genes in both coelacanth and spotted gar
genomes, named here Kissr-1, Kissr-2, Kissr-3 and Kissr-4.
Four Kissr genes in the coelacanth genome. The four
predicted Kissr genes of coelacanth are made of 5 exons and 4
introns. The predicted CDS and the exon-exon junctions of each
putative transcript are shown in Fig. S3. Once translated, the
putative transcripts lead to four predicted proteins, i.e. coelacanth
Kissr-1, Kissr-2, Kissr-3 and Kissr-4, of 368-aa, 367-aa, 377-aa
and 367-aa, respectively (Fig. S3).
Four Kissr genes in the spotted gar genome. The four
predicted Kissr genes of spotted gar are also made of 5 exons and 4
introns. Although Kissr-1 exon-4 and Kissr-4 exon-5 are partial due
to the current status of the genome, the predicted CDS and the
exon-exon junctions of each putative transcript are shown in Fig.
S4. Once translated, the putative transcripts lead to four predicted
proteins, i.e. spotted gar Kissr-1 to 4 of 318-aa, 362-aa, 382-aa,
and 385-aa, respectively (Fig. S4).
In both coelacanth and spotted gar, all predicted Kissr proteins
present the typical seven TMD of the GPCR family (Fig. S3 and
These new findings evidence for the first time the potential
existence of four Kissr paralogs in vertebrate species,
representatives of the sarcopterygian (coelacanth) and the actinopterygian
(spotted gar) lineages.
Phylogenetic analysis of the Kissr family
Other genomic database analyses. In addition to the
characterisation and/or the prediction of European eel,
coelacanth and spotted gar Kissr, we used the ENSEMBL Genome
Browser to retrieve the sequences of two Kissr in the medaka
genome (GPR54-1, chromosome 17 and GPR54-2, chromosome
9; ), and to search for new candidate Kissr genes in the genomes
of other vertebrates. We found one putative Kissr gene, named
here Kissr-2, in several teleost genomes, stickleback (chromosome
group III), tetraodon (chromosome 15-random) and cod (Gadus
morhua; scaffold 377), one Kissr in a cyclostome genome, sea
lamprey (scaffold GL478157; ), and one in the lizard genome
Phylogenetic analysis. Based on an alignment of 51 Kissr
peptidic sequences (Fig. S5), and assuming ambulacrarian (acorn
worm, Saccoglossus kowalevskii, and purple sea urchin,
Strongylocentrotus purpuratus (Table S2)) Kissr as outgroup, a phylogenetic tree
was generated using the Maximum Likelihood method (the list of
sequences and accession numbers is provided in Table S2). As
shown in Fig. 2, it clusteres the vertebrate Kissr into four main
clades, which are supported by significant bootstrap values (99, 90,
73, and 90%, respectively). Based on this analysis and the
determination of those four clades, we propose the following new
nomenclature of the different Kissr.
The first clade mainly encompasses sarcopterygian Kissr
including all eutherian Kissr, a metatherian Kissr (Monodelphis
domestica; opossum GPR54), a prototherian Kissr (platypus
GPR54a), a xenopus Kissr (GPR54-1a) and coelacanth Kissr-1.
In addition, two actinopterygian Kissr, spotted gar and European
eel Kissr-1, branch together at the base of this clade. This is the
first evidence of the presence of an ortholog to eutherian Kissr in
actinopterygians. Acknowledging the presence of eutherian
(including human) Kissr, which have been the first to be
discovered, this clade was named Kissr-1 clade. The eel is up to
now the only teleost presenting an ortholog (eel Kissr-1) to
The second clade, named Kissr-2, clusters mainly
actinopterygian Kissr, i.e. spotted gar Kissr-2 and most of the previously
described teleost Kissr including zebrafish Kiss1ra, goldfish
GPR54a, medaka GPR54-1 and European eel Kissr-2. This clade
also clusters together three sequences from sarcopterygian species,
xenopus GPR54-2, bullfrog (Rana catesbeiana) GPR54 and
The third clade, named Kissr-3, clusters two sarcopterygian
Kissr (xenopus GPR54-1b and coelacanth Kissr-3) and some
actinopterygian Kissr (spotted gar Kissr-3 and a few teleost Kissr:
zebrafish Kiss1rb, goldfish GPR54b, medaka GPR54-2 and
European eel Kissr-3).
The fourth clade, named Kissr-4, clusters three sarcopterygian
Kissr (platypus GPR54b, lizard Kissr and coelacanth Kissr-4) with
one actinopterygian Kissr, the spotted gar Kissr-4.
This phylogenetic analysis suggests the existence of four distinct
paralogous Kissr in osteichthyans. Furthermore, it shows that each
sarcopterygian Kissr is orthologous to actinopterygian Kissr. This
is specially highlighted by the relationship between the four
coelacanth and spotted gar Kissr.
Syntenic analysis of Kissr genes
In order to test the results obtained with the phylogenetic
analysis, we performed a syntenic analysis of the Kissr
neighbouring genes, an approach which is applied to determine gene
orthology relationships as well as the origin and evolutionary
history of gene families. For this analysis (Fig. 3), we considered the
following vertebrate representatives: mammals (eutherian: human
and prototherian: platypus), bird (chicken), squamate (lizard),
amphibian (xenopus), basal sarcopterygian (coelacanth),
nonteleost actinopterygian (spotted gar) and teleosts (zebrafish,
medaka, stickleback, tetraodon and European eel). As already
reported (for review: [5,2426]), genomic synteny analysis shows
that birds do not possess any Kissr gene.
The mammalian, amphibian, coelacanth, spotted gar and
European eel genes from Kissr-1 clade are positioned in genomic
regions containing common loci, including PALM, PTBP1, LPPR3,
MED16, ARID3A, WDR18, GRIN3B, C19orf6, GADD45B and
DIRAS1, exhibiting well conserved synteny (Fig. 3A). This supports
the orthology of the Kissr-1 genes. Syntenic analysis suggests that
the other teleost genomes do not contain any Kissr-1 gene, though
the above-mentioned neighbouring genes are present in the
corresponding genomic regions (Fig. 3A). The eel currently
provides a unique example of Kissr-1 ortholog in teleosts.
The amphibian, coelacanth, spotted gar and teleost genes from
Kissr-2 clade are positioned in genomic regions containing
common loci, including PTPRF, KDM4A, ST3GAL3, DPH2,
ATPV60B, B4GALT2, CCDC24, KIF2C, PTCH2, EIF2B3,
ZCCHC11, GLIS1, GADD45A, DIRAS3, LPAR3, ZNF644, PTBP2
and PALMD, exhibiting well conserved synteny (Fig. 3B). This
supports the orthology of the Kissr-2 genes. European eel Kissr-2
orthology is only supported by the presence of partial GLIS1 due to
the small size of the scaffold. Syntenic analysis suggests that
squamate (lizard) and mammalian genomes do not contain any
Kissr-2 gene, though the above-mentioned neighbouring genes are
present in the corresponding genome regions (Fig. 3B).
The amphibian, teleost (zebrafish, medaka, European eel) and
spotted gar Kissr-3 genes are positioned in genomic regions
containing common loci including PSAT1, ISCA1, ZCCHC6,
GADD45G, DIRAS2, PALM2, LPAR1 and PTBP3, exhibiting
conserved synteny (Fig. 3C). This supports the orthology of the
Kissr-3 genes. Syntenic analysis suggests that the other teleost and
tetrapod genomes do not contain any Kissr-3 gene, though the
above-mentioned neighbouring genes are present in the
corresponding genome regions. The coelacanth predicted Kissr-3 is split
into the scaffolds JH131603.1 and JH131921.1, which are too
small to contain any other gene (Fig. 3C).
Platypus and lizard Kissr-4 gene neighbouring regions present
only RETN gene in common, due to the small size of the platypus
scaffold (Fig. 3D). Assemblage of three scaffolds (JH133705.1,
JH132986.1, and JH127844) from coelacanth genome allowed us
to reveal a Kissr-4 neighbouring region comprising LPAR2,
STXBP2 and EPHX3 genes, with STXBP2 and EPHX3, being
also present in lizard and spotted gar Kissr-4 region (Fig. 3D).
These data suggest that Kissr-4 genes can be considered as
orthologous. Syntenic analysis also suggests that the genomes from
placental mammals, amphibian and teleosts do not contain any
Kissr-4 gene, though they present in a common region some
conserved genes including CAMSAP3, XAB2, STXBP2, RETN,
RAB3D, PALM3, NOTCH3, EPHX3, WIZ and LPAR2 (Fig. 3D).
This syntenic analysis of Kissr genes delineated four different
conserved genomic regions among osteichthyans. For each
conserved genomic region, Kissr genes from various species
clustered in the corresponding phylogenetic Kissr clade. Thus,
the results of the syntenic analysis fully validated the orthology
relationships of the phylogenetic analysis and further supported
our proposal for a new nomenclature.
Evolutionary history of Kissr family
Origin of the four Kissr present in basal
osteichthyans. So far, the presence of Kissr has been
characterised in two non-vertebrate species, the purple sea urchin and
the acorn worm (Table S2). This revealed the existence of at least
one ancestral Kissr before the vertebrate emergence. The
predictions of four Kissr in the genomes of a basal sarcopterygian,
the coelacanth, and of an actinopterygian, the spotted gar,
together with the results of both phylogenetic and syntenic
analyses, enabled us to hypothesise the existence of at least four
Kissr paralogous genes in the common osteichthyan ancestor of the
sarcopterygian and actinopterygian lineages. A remaining question
was to infer whether these four genes resulted from the
duplications of one or multiple ancestral Kissr genes.
It is currently admitted that two rounds of whole genome
duplication (1R and 2R) occurred in the early vertebrate
evolutionary history, resulting in four-fold replicates of the
ancestral genome. Even though numerous genomic
rearrangements and loss events occurred during the vertebrate radiation, the
vestiges of these two successive genome duplications can be
revealed in the current vertebrate species by the existence of
numerous four-fold repeated regions (tetra-paralogon) carrying
paralogous genes .
Our syntenic analysis demonstrated that the current
osteichthyan Kissr genes are localised in four different genomic regions. In
the Fig. 3, in addition to the Kissr genes, we could reveal paralogs
from eight gene families present among the four syntenic regions
of the osteichthyan representative species. The eight considered
families are PALM (4 paralogs), PTBP (3 paralogs), GRIN3 (2
paralogs), GADD45 (3 paralogs), DIRAS (3 paralogs), ZCCHC (2
paralogs), LPAR (3 paralogs) and ZNF644/WIZ (2 paralogs). Fig. 4
focuses on the comparison of these four conserved Kissr syntenic
regions in one sarcopterygian, the human, and one
actinopterygian, the spotted gar, two species chosen for their genomic
assembly in chromosomes and their phylogenetic positions among
osteichthyans. Fig. 4 shows that the members of these eight gene
families delineate a tetra-paralogon with the four conserved
genomic regions of both human and spotted gar. This syntenic
observation strongly suggests that the four Kissr resulted from
enbloc duplications of a unique ancestral genomic region.
Recently, two studies proposed reconstructions of the genomes
of vertebrate and chordate ancestors. In the first study, ten
protochromosomes of the ancestral vertebrate karyotype and their
linkage to the corresponding tetra-paralogons in the human
genome were hypothesised . The second study proposed a
reconstruction into seventeen proto-chromosomes of the last
chordate ancestor genome and their linkage to the human
tetraparalogons . Considering these two studies, together with our
localisation of the four Kissr syntenic regions in the human
genome, we can hypothesise that the corresponding
tetraparalogons resulted from the duplications of one single region
localised on the proto-chromosome-A of the vertebrate ancestor
and on the proto-chromosome-1 of the last chordate ancestor.
From these analyses, we can infer that the four Kissr paralogons
may have resulted from the two successive rounds of whole
genome duplication (1R and 2R) that occurred in basal vertebrates
To date, the exact timing of the 2R occurrence is uncertain and
the impact of this event on the cyclostomes is still debated [30,31].
In our study, neither phylogenetic nor syntenic analyses enabled us
to specifically relate the sea lamprey Kissr to one of the four
osteichthyan Kissr. The currently available data also led to a
polytomy of the four Kissr clades and did not cluster them into two
major clades, each of them divided in two sub-clades, which would
have reflected the successive 1R and 2R. In the future, increasing
number of characterised Kissr sequences, especially from Kissr-3
and Kissr-4 clades, may bring sufficient new information to resolve
this polytomy. Though kisspeptin genes have been characterised in
chondrichthyans, it could be of particular interest to investigate the
presence of its receptor in this lineage to further assess Kissr history
in early gnathostomes.
A subsequent history of losses. Our study suggests that
four Kissr paralogs would have been present in ancestral
gnathostomes, resulting from 1R and 2R. It also shows that these
four paralogons are still present in two early emerged
osteichthyans, a sarcopterygian, the coelacanth and an actinopterygian, the
spotted gar. All other vertebrate species investigated so far possess
less Kissr genes (from 3 to none) indicating multiple events of Kissr
losses in both the sarcopterygian and actinopterygian lineages
In tetrapods (sarcopterygians), Kissr-4 would have been lost in
amphibians, Kissr-1, -2 and -3 being present in the xenopus, while
in amniotes the losses would have first concerned Kissr-2 and
Kissr3, Kissr-1 and Kissr-4 being present in a prototherian mammal
(Fig. 5). Further alternative losses occurred in amniotes, with only
Kissr-1 remaining in eutherian mammals, but only Kissr-4 in
squamates (lizard) (Fig 5). Finally, an additional loss would have
led to the complete absence of Kissr in birds.
In teleosts (actinopterygians), a third round of whole genome
duplication (3R) is supposed to have occurred specifically in the
early history of this group. The 3R is usually considered as one of
the main factors that drove the large radiation and adaptative
success of the teleost lineage [11,32]. As four kissr were present in
basal actinopterygians, the teleost-specific 3R implied the potential
existence of up to eight Kissr genes in the early teleost history.
However, our results show that the largest number of Kissr
exhibited by current teleosts is three in the eel, and that each of
them is orthologous to one of the coelacanth and spotted gar Kissr.
This indicates that 3R did not impact the number of Kissr in
teleosts, suggesting an early loss of teleost-specific duplicated Kissr
genes, before the emergence of the elopomorphs (Fig. 5). Apart
from the eel, only one or two Kissr genes have been described so far
in teleosts, indicating additional loss events after the emergence of
the elopomorphs (Fig. 5).
The occurrence of these many independent loss events may
have led to the current situation of Kissr in the various vertebrate
lineages. Indeed, some species seem to be more conservative than
others, and it is of particular interest to clarify what may have
driven the conservation or the loss of Kissr.
Conservation of multiple Kissr: the example of the eel
As shown by the present study, the eel is one of the most
conservative species among current vertebrates, as three different
Kissr have been retained. Conservation of multiple Kissr may reflect
evolutionary processes such as neo- or sub-functionalisation. The
comparative analyses of eel Kissr peptidic sequences and eel Kissr
tissue distribution and regulation may constitute the first steps in
the understanding of such processes.
Comparison of eel Kissr peptidic sequences. The
analysis of the peptidic sequences deduced from the three cloned eel
Kissr cDNAs revealed a conserved disulfide bridge between
cysteines at positions 112/192 for Kissr-1, 105/185 for Kissr-2
and 110/190 for Kissr-3. The seven TMD of each receptor
comprise 23 aa, except for TMD3 of Kissr-1 and Kissr-2 which
comprise 18 aa and 19 aa, respectively. The pairwise comparison
of the three peptidic sequences revealed 60.5% identity for
Kissr1/Kissr-2, 63.3% for Kissr-1/Kissr-3, and 63.4% for Kissr-2/
Kissr-3. These low identities are mostly due to differences between
the N-terminal extracellular domains (28.6% to 40.5% identity)
and between the C-terminal intracellular domains (43.6% to
58.3% identity) (Fig. S6). Differences within the N-terminal
domains could reflect variations in ligand binding properties,
while those in the C-terminal domain may correspond to
differences in G protein association properties and activation of
intracellular signalling pathways. Future studies, including
recombinant receptors and characterization of endogenous ligands, will
aim at further investigating the potential differences in the
structure/function of three eel Kissr.
Differential tissue distribution of the three eel
Kissr. Specific qPCR were developped for each eel Kissr and
applied to the analysis of their respective tissue distribution (Fig. 6).
Kissr-1 mRNA was expressed in all parts of the brain and in the
pituitary. Its highest expression level was found in the gonads, in
both ovary and testis. Low Kissr-1 mRNA levels were measured in
muscle, retina and fat tissue, while its expression was close to the
limit of detection in the other peripheral tissues investigated (liver,
kidney, intestine, and spleen).
Kissr-2 mRNA was also expressed in the brain and pituitary,
with the highest transcript levels in the telencephalic and
di-/mesencephalic areas, in agreement with our previous study . A low
expression of Kissr-2 mRNA was measured in the testis, while its
expression in the ovary and all the other peripheral organs was
close to the limit of detection.
Kissr-3 mRNA was highly expressed in the di-/mes-encephalic
area and to a much lesser extent in the other parts of the brain.
Transcript level was close to the limit of detection in the pituitary.
Low expression of Kissr-3 mRNA was measured in the gonads,
ovary and testis, as well as in the muscle, while its expression in the
other peripheral organs was close to the limit of detection.
Figure 5. Current status and proposed evolutionary history of Kissr family. This representation is based on the phylogenetic and synteny
analyses. The names of the main phyla are given on the corresponding branches. The names of the current representative species of each phylum are
given at the end of the final branches, together with the symbol of the Kissr gene they possess. This hypothesis assumes the presence of the four Kissr
paralogs in the osteichthyan lineage resulting from the two rounds of vertebrate whole genome duplication. Multiple subsequent Kissr gene loss
events are indicated in the actinopterygian and sarcopterygian lineages.
The three eel Kissr thus appear mainly expressed in the eel
Brain-Pituitary-Gonad (BPG) axis, which highlights the potential
involvement of the kisspeptin system in the eel reproductive
function. Considering the phylogenetic position of the eel, this
tissue distribution could reflect an ancestral role of the kisspeptin
system in reproduction, which would have been largely conserved
across vertebrate evolution. The three Kissr transcripts are all
highly expressed in the eel brain, but with various relative levels
according to brain regions. Both Kissr-1 and Kissr-2 are expressed
in the pituitary, where they may mediate direct kisspeptin effects as
previously investigated . Kissr-1 is also highly expressed in both
the ovary and testis. Thus, the three eel Kissr present a differential
distribution among BPG axis that suggests differential putative
roles in the control of eel reproduction and implies a potential
subfunctionalisation of the three receptors.
In the other teleosts, only Kissr-2 and for some species Kissr-3
have been described so far. As in the eel, they are expressed in the
BPG axis (grey mullet, Mugil cephalus ; zebrafish [6,34]; fathead
minnow, Pimephales promelas ; tilapia, Oreochromis niloticus
[36,37]; goldfish ; fugu ). In xenopus, which presents the
three orthologs of eel Kissr , all three receptors are expressed in
the brain, while only GPR54-1a (kissr-1) and GPR54-2 (kissr-2)
mRNA are detected in the pituitary, similarly to the eel. In
placental mammals, only Kissr-1 ortholog is present and expressed
in the brain, pituitary (human [2,39]; mouse ) and ovary (rat
; human and marmoset ), where a role in local control of
ovarian functions has been suggested.
Differential regulation of the three eel Kissr by
experimental maturation. Gonadal development in the silver
eel is blocked at a pre-pubertal stage due to a deficient production
of pituitary gonadotropins, which results from a dual brain
blockade: lack of stimulation by GnRH and direct inhibition by
dopamine (for review: ). Experimental gonadal maturation
was induced in female silver eels according to classical hormonal
treatments . The expressions of the three Kissr were analyzed
by qPCR in the BPG axis, which are the major sites of Kissr
expression in the eel.
In the brain (Fig. 7), Kissr-1 transcript level was significantly
upregulated in matured eels as compared to control ones (x3.73,
P,0.0001 in olfactory bulbs/telencephalon and x7.15, P,0.0001
in di-/mes-encephalon), while no changes were recorded for
Kissr2 nor Kissr-3. In the pituitary (Fig. 7), Kissr-1 and Kissr-2 transcript
levels were significantly down-regulated in matured eels compared
to controls (x0.42; P,0.05 and x0.14; P,0.01, respectively), while
Kissr-3 remained at the limit of detection. In the ovary (Fig. 7),
Kissr-3 transcript level was significantly down-regulated in matured
eels (x 0.39; P = 0.01), while Kissr-1 transcript levels did no change
significantly and Kissr-2 remained at the limit of detection.
We also analyzed by qPCR the expression of brain mGnRH and
pituitary LH. Significant increases in mRNA levels for brain
mGnRH (x5.6; P,0.001) and pituitary LH (x206; P,0.0001) were
measured in matured eels, as compared to control eels (data not
shown) in agreement with our previous studies [20,44,45].
A parallel study was performed on the regulation of the three
Kissr transcript levels in experimentally matured Japanese female
eels. Eels were treated according to Jeng et al. . As for the
European eel, a differential regulation was evidenced. This was
shown by selective increase in brain Kissr-1, decreases in pituitary
Kissr-1 and Kissr-2 and decrease in ovarian Kissr-3 transcripts, in
matured eels as compared to controls (data not shown).
Those results clearly evidence a differential regulation of the
three eel Kissr, with receptor- and tissue- specific variations. The
selective up-regulation of Kissr-1 expression in the brain suggests
that Kissr-1 may contribute to enhance kisspeptin stimulatory
control of GnRH at puberty in the eel. Similarly, brain Kissr-1
expression increases at the onset of puberty in mammals (rodent
; monkey ). This role may have shifted to Kissr-2 in other
teleosts, which lack Kissr-1 ortholog. For instance, parallel
variations in the brain expressions of Kissr-2 ortholog and GnRH
were observed during sexual maturation/puberty in several
teleosts (cobia, Rachycentron canadum ; grey mullet ; fathead
minnow ; Nile tilapia ). We recently revealed an
unexpected inhibition by kisspeptins of LHb expression in eel
pituitary cells in vitro, providing the first evidence of an inhibitory
role of kisspeptin on gonadotropic function . In the present
study, we observed a down-regulation of eel Kissr-1 and Kissr-2 in
the pituitary, while LHb expression largely increased. This suggests
that the inhibitory control of kisspeptin on LHb expression could
be removed during the sexual maturation by the down regulation
of its receptors in the pituitary. Kissr-3 was down-regulated in the
ovary implying a potential involvement of this receptor in ovarian
function. Future studies should aim at clarifying the role of
kisspeptin system at gonadal level in teleosts.
The regulation of the expression of the three Kissr in the BPG
tissues reinforces the hypothesis of a conserved ancestral role in
vertebrate reproductive function. In addition to their differential
distribution in the BPG axis, their differential regulations further
strengthen the potential sub-functionalisation of the three Kissr
paralogs for the control of eel reproductive function.
Our previous studies, including castration experiments and
steroid treatments, demonstrated that the activation of mGnRH
and LH in matured eel result from a positive feedback by sex
steroids [44,45,50,51]. Increasing data support a role of brain
kisspeptin system in the mediation of steroid feedbacks on GnRH
neurons in mammals (for review: [52,53]) and recently in teleosts
(medaka ; goldfish ; zebrafish ). Future studies will aim
at investigating the role of sex steroids in the differential regulation
of Kissr paralogs in the eel.
In conclusion, our study provides the first evidence of multiple
Kissr paralogs in basal osteichthyans, with the cloning of three Kissr
in a basal teleost, the eel, and the prediction of four Kissr in a
nonteleost actinopterygian, the spotted gar, and four Kissr in a basal
sarcopterygian, the coelacanth. Phylogenetic and syntenic analyses
support the existence of four Kissr paralogs in osteichthyans,
leading to the proposal of a new, simplified classification and
nomenclature (Kissr-1 to 4). The four Kissr paralogs may have
arisen during the two rounds of whole genome duplication (1R
and 2R) in early vertebrates, followed by multiple gene loss events
in the various groups of actinopterygian and sarcopterygian
lineages. In particular, no impact of teleost-specific 3R can be
recorded on the number of Kissr paralogs in current teleosts.
Subfunctionalisation of the three eel Kissr, as shown by differences in
their sequences, tissue distributions and regulations during sexual
maturation, may have represented significant evolutionary
constraints for the conservation of multiple Kissr paralogs in this
Figure S1 Three eel Kissr gene sequences. Genomic sequences of
the eel Kissr-1 extracted from the scaffold 90.1 (A), Kissr-2
extracted from the scaffold 3158.1 (B) and Kissr-3 extracted from
the scaffold 1832.1 (C) (European eel genome ). Nucleotides
are numbered from 59 to 39. The five exons of each gene are
shaded in grey.
Figure S2 Molecular cloning of eel Kissr-3 splicing variants
Kissr3_v2 and Kissr-3_v3. Nucleotide and deduced amino-acid sequence
of the cDNAs encoding the eel Kissr-3_v2 (A) and Kissr-3_v3 (B).
Nucleotides (top) are numbered from 59 to 39. The amino-acid
residues (bottom) are numbered beginning with the first
methionine residue in the ORF. The asterisk (*) indicates the stop codon.
The predicted transmembrane domains (TMD) are underlined.
Figure S3 Prediction of four Kissr CDS from the coelacanth
genome. Nucleotide and deduced amino-acid sequences of the
CDS encoding the coelacanth Kissr-1 (A), Kissr-2 (B), Kissr-3 (C)
and Kissr-4 (D). Nucleotides (top) are numbered from 59 to 39. The
amino-acid residues (bottom) are numbered beginning with the
first methionine residue in the ORF. The asterisk (*) indicates the
stop codon. The predicted transmembrane domains (TMD) are
underlined. The exon-exon junctions are represented by two
nucleotides coloured in red.
Figure S4 Prediction of four Kissr CDS from the spotted gar
genome. Nucleotide and deduced amino-acid sequences of the
CDS encoding the spotted gar Kissr-1 (A), Kissr-2 (B), Kissr-3 (C)
and Kissr-4 (D). Nucleotides (top) are numbered from 59 to 39. The
amino-acid residues (bottom) are numbered beginning with the
Figure S5 Alignment of the amino-acid sequences of 51 Kissr
used for the phylogenetic analysis. The amino-acid sequences were
aligned by ClustalW and manually adjusted. The identical
aminoacid residues between sequences are shaded in black and the
similar (with similar physico-chemical properties) amino-acid
residues are shaded in grey. Sequence references are listed in
Figure S6 Alignment of the deduced amino-acid sequences of
the three eel Kissr. The entire amino-acid sequences were aligned
by ClustalW and manually adjusted. The identical amino-acid
residues between the three sequences are shaded in black and the
similar (with similar physico-chemical properties) amino-acid
residues are shaded in grey.
Table S1 European eel gene specific primers. Specific primers (F
for Forward and R for Reverse) were designed for PCR and qPCR
amplifications. Kissr, kisspeptin receptor; LHb, Luteinizing
Hormone b subunit; mGnRH, mammalian Gonadotrophin Releasing
We thank Dr. B. Querat (CNRS, Paris, France) for his helpful advices and
discussions concerning phylogeny and synteny. We are grateful to Dr C.
Henkel (ZF-screen, Leiden, Netherlands) for his work on the European eel
genome, P. Lauesen (Billund Aquaculture Service, Billund, Denmark), M.
Kruger-Johnsen (DTU aqua, Copenhagen, Denmark) and C. Graver
(Danish Eel Farmers Association, Ribe, Denmark) for performing eel
maturation experiment, and S. Baloche (CNRS, Paris, France) for her
technical assistance. Special thanks to L. Hardman and C. Atkinson
(European Programme Erasmus, Keele University, Keele, UK/MNHN,
Paris, France) for English correction. We also thank Eric Ryckelynck and
his team from Nodaiwa (Paris, France) for their kind cooperation.
Conceived and designed the experiments: SD CFC KR SRJ. Performed
the experiments: JP SRJ MM JT. Analyzed the data: JP AGL SRJ HT.
Contributed reagents/materials/analysis tools: RD GvdT. Wrote the
paper: JP AGL KR SD. Provided comments on the manuscript: JP AGL
SRJ MM RD GvdT JT HT CFC KR SD.
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