Chem. Senses 27: 529-537,
2002
© Oxford University Press 2002
Identification of V1R-like Putative Pheromone Receptor Sequences in Non-human Primates. Characterization of V1R Pseudogenes in Marmoset, a Primate Species that Possesses an Intact Vomeronasal Organ
IGH, CNRS UPR 1142, Montpellier, France
Correspondence to be sent to: Sylvie Rouquier, IGH, CNRS UPR 1142, rue de la Cardonille, 34396 Montpellier cédex 5, France. e-mail: rouquier{at}igh.cnrs.fr
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Abstract |
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The vomeronasal organ (VNO) is responsible in terrestrial vertebrates for the sensory perception of some pheromones, chemicals that elicit characteristic behaviors among individuals of the same species. Two multigene families (V1R, V2R) that encode proteins with seven putative transmembrane domains that are expressed selectively in different neuron subsets of the VNO have been described in rodents. Pheromone-induced behaviors and a functional VNO have been described in a number of mammals, but this sensory organ seems absent in adult catarrhines and apes, including humans. Until now, only pseudogenes have been isolated in humans, except one putative V1R (hV1RL1) sequence expressed in the main olfactory epithelium. We sought to isolate V1R-like genes in a New World monkey species, the marmoset Callithrix jacchus, that possesses an intact VNO and for which pheromone-induced behavior has been well documented. Using library screening approaches, we have identified five different sequences that exhibit characteristic features of V1R sequences, but that are non-functional pseudogenes. In an attempt to sort out functional V1R genes, we next cloned by polymerase chain reaction (PCR) the primate orthologues of hV1RL1. This approach was successful for gorilla, chimpanzee and orangutan, but not for the other species, including marmoset, probably because these species are too divergent from humans. Chimpanzee and orangutan V1RL1 genes are pseudogenes, whereas the gorilla counterpart is potentially functional. These observations raise the possibility that the V1R family has evolved in such a manner in mammals that every species that relies on a VNO-mediated sensory function possesses its own set of functional vomeronasal genes.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chemoreception is an ancient sense system that enables an organism to detect chemicals in its environment. Being thus able to sense molecules in their environment allows organisms to alter their behavior accordingly. Chemosensation in vertebrates is achieved by at least two distinct nasal organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) (Bargmann, 1997
). Sensory
neurons of the MOE detect volatile odorants and are therefore responsible for
the perception of smell. Those of the VNO are believed to respond mainly to
pheromones, eliciting a characteristic array of innate reproductive and social
behaviors, along with neuroendocrine responses. However, only a few pheromones
are known to stimulate vomeronasal sensory neurons and some pheromones appear
to be detected by the main olfactory system.
In mammals, the olfactory receptors (ORs) of the MOE are encoded by a large
family of 
1000 genes. They belong to the superfamily of
seven-transmembrane-domain (7TM) G-protein-coupled receptors (GPCR)
(Mombaerts, 1999a). In
rodents, differential screenings of cDNA libraries have led to the isolation
of two large independent superfamilies of putative pheromone receptor genes
(VRs), the V1Rs and V2Rs (Dulac and Axel,
1995; Herrada and Dulac,
1997; Matsunami and Buck,
1997; Ryba and Tirindelli,
1997). V1Rs are restricted to G
i2-expressing neurons, and
V2Rs are restricted to G0-expressing neurons in the VNO. They both
constitute novel families of 7TM receptors with no sequence similarity with
ORs.
During the course of this work, a third family of pheromone receptors has
been reported as the V3R family (Pantages
and Dulac, 2000), but recent complementary data indicate that V3R
correspond to one of the 12 V1R families
(Rodriguez et al.,
2002). In mouse, the V1R family comprises 300 receptor genes,
distributed in 12 families, with pseudogenes accounting for 53% of the
total. Indeed, besides the four gene families that were first described (V1ra,
V1rb, V1rc and V3R) (Dulac and Axel,
1995; Del Punta et
al., 2000; Dulac,
2000; Pantages and Dulac,
2000), eight new families have been characterized by database
mining of the mouse genome, revealing a large sequence diversity among the
V1Rs (Rodriguez et al.,
2002). V2Rs contain a large extracellular N-terminal domain and
share similarity to extracellular calcium-sensing receptors and metabotropic
glutamate receptors. The rodent genome is estimated to contain 100
V2R-like genes, including a large number of pseudogenes
(Dulac, 2000).
In mammals, nerve fibers from the VNO converge to the accessory olfactory
bulb (AOB), that projects to areas of the brain that are involved in hormonal
and reproductive functions. Although the existence of a functional VNO is
still debated (Meredith,
2001), a study reporting anatomical, histological and
immunohistochemical data indicates that in human adults, the vomeronasal
structure is a remnant of the VNO and probably cannot function as a sensory
organ (Trotier et al.,
2000). Indeed, it appears that a VNO-like structure is present
during early human embryogenesis and regresses after birth to become vestigial
in adults (Rodriguez et al.,
2000). Furthermore, there is no AOB in humans. Although different
studies argue in favor of a pheromone-like communication in human, as for
example the synchronization of menstrual cycles among women living together
(Stern and McClintock, 1998),
the proof that this is mediated by the VNO is lacking. In other catarrhines
the VNO is also vestigial, with no obvious thick sensory epithelium, while it
is present in New World monkey platyrrhines
(Stoddart, 1980;
Taniguchi et al.,
1992).
Based on known and potentially functional rodent genes, genomic and
bioinformatics approaches were used to explore the possibility that humans
possess functional V1R-like sequences; only pseudogenes were found
(Giorgi et al.,
2000). Nevertheless, a single V1R/V3R (V1RL1) sequence with an
open-reading frame and expressed in the olfactory mucosa has been recently
isolated (Pantages and Dulac,
2000; Rodriguez et
al., 2000). On the other hand, despite the mouse V1R
repertoire recently having been characterized by database mining, no data are
available in other species due to the difficulty of cloning these receptors by
conventional approaches. To shed light on the evolution of the V1R family, we
chose to investigate the genome of a primate species that possesses an intact
VNO, i.e. the marmoset (Callithrix jacchus, New World monkey), in an
attempt to isolate potentially functional V1R genes. Because many primates are
endangered species from which it is nearly impossible to obtain VNO tissue
suitable for RNA isolation, we used a genomic approach. The absence of introns
in the coding region of V1Rs (Dulac and
Axel, 1995) permits the determination of the coding sequence
directly from genomic clones. In addition, we sought to clone the primate
orthologues of the human V1RL1 gene, the only potentially functional primate
V1R gene described to date.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Probes
Human V1R clones Ph2 and PhB4C5 (Giorgi
et al., 2000) were amplified by polymerase chain reaction
(PCR) using internal V1R-sequence primers. Twenty-five-microliter PCRs were
performed using 10 ng of recombinant plasmid DNA and primer sets CL2-1F
(5'-GAT GAG GGA CTC TCC ATC TGC-3') and CL2-2R (5'-AAG GGC
TCA CAG TGG CAT AGG) for clone Ph2 and BC5'F (5'-TAT TTG CAC AAG
GTG ATG AGG) and BCR (5'-GAA AGC ACA CAG TTC TTG GC-3') for clone
PhB4C5. An initial denaturation step at 94°C for 2 min was followed by 30
cycles (94°C for 15 s, 66°C for Ph2 or 55°C for PhB4C5 for 30s,
72°C for 45 s) and a final extension at 72°C for 10 min. The rat VN1
receptor sequence (Dulac and Axel,
1995) was cloned by PCR using 100 ng of genomic rat DNA and primer
sets rVN1-F (5'-AGA ACA GCA GAC TCT ACA CTG-3') and rVN1-R
(5'-ACA TGG ACC TCA AAG AGT TAA C-3'). Cycling conditions were as
indicated above, except that the annealing temperature was 58°C and the
duration of the extension step 90 s. The marmoset probe was PCR-generated from
subclone Ms7E9 (issued from cos Ms7) containing the V1R sequence (see Results)
with primers Ms7E9-2F (5'-GAA TAA CTT CAT CTG TCA GGC-3') and
Ms7E9-2R (5'-ATG TAT GCA CTG AGA GCA GC-3'), using an annealing
temperature of 58°C and an extension step of 45 s. PCR products were then
purified by electrophoresis on low-melting-point agarose and directly
radiolabeled by random hexamer priming
(Feinberg and Vogelstein, 1983)
using [-32P]dCTP.
Cosmid library screening
A C. jacchus cosmid library gridded on high density filters
(marmoset genome coverage of 1.5x) was purchased from the Resource
Center of the German Human Genome Project (RZPD, Berlin, Germany).
The different screenings were performed separately at low stringency with human, rat and marmoset V1R clones as probes. Filter hybridization was carried out overnight at 55°C in 6x SSC, 5x Denhardt's, 0.5% SDS and 100 µg/ml sonicated herring sperm DNA. Following the hybridization, filters were washed twice in 2x SSC and 0.1% SDS at 55°C. Positive clones were identified by autoradiography after overnight or longer exposure at -80°C.
Cosmid clone analysis
Cosmid DNAs were isolated by alkaline lysis and purification using a
High-Pure Plasmid Isolation kit (Roche Diagnostic) or Qiagen tip- 100 (Qiagen)
columns, following the procedure recommended by the manufacturers. DNAs were
digested with various restriction enzymes in buffers supplied by the
manufacturer (New England Biolabs). The restriction digests were analysed by
electrophoresis on 0.8% agarose gels. The gels were alkali blotted as
described previously (Gemmill et
al., 1987) onto nylon membranes (Hybond N+; Amersham) and
used for hybridization as described above. Because of their extreme
instability, some cosmid DNAs were rescued by in vitro packaging and
transfected in the bacterial host DH5MCR
(Yokobata et al.,
1990) using Gigapack III Gold Packaging extract from Stratagene
and following the instructions of the manufacturer.
PCR with degenerate primers
Degenerate PCR primers were designed in the most conserved regions of the
VIR receptor sequences isolated from rat
(Dulac and Axel, 1995) and from
human (Giorgi et al.,
2000). VIR-2F is located in the fourth transmembrane domain
[5'-TC(A/T) ATT TGT C(C/T)T T(C/T)A (A/G) (C/T)A GT(G/T) (A/G)C] and
VIR-2R is located between transmembrane domains V and VI [5'-T(C/T)
(C/T) (A/C/T) (C/T)A (C/T)A(C/T) GCT (C/T)GT (C/T)GT GCT GTG-3']. These
primers were used in PCR assays to isolate V1R-like sequences from cosmids
issued from library screenings and confirmed as positives by Southern-blot
hybridization. The cycling conditions consisted of an initial denaturation at
94°C for 2 min, followed by 30 cycles (94°C for 30 s, 50°C for 40
s, 72°C for 45 s) and a final extension step at 72°C for 10 min. PCR
products were subcloned in a PCR cloning system (TA cloning kit; Invitrogen).
Recombinant clones were analysed by PCR and sequenced using M13-vector primers
and dye-labelled dideoxy terminator chemistry.
Sequencing of the V1R fragments
Directional sequencing with custom-made primers was performed directly on cosmid DNA that yielded a positive V1R band with degenerate primers V1R-2F/2R, following the instructions of the manufacturer.
Cosmids that did not amplify with the V1R primers, but that produced a positive signal by Southern-blot hybridization, were analyzed using conventional methods. Fragments containing putative V1R sequences were subcloned into pBluescript KS-(Stratagene). Sequencing was carried out by a combination of vector and custom-made primers using dye-labelled dideoxy terminator chemistry.
Multiple alignment was performed using the PileUp program from the GCG
package (University of Wisconsin) and the CLUSTAL W v. 1.8 software package
(Thompson et al.,
1994). The percentage of identity for pairwise comparisons was
calculated by using the GAP program (Wisconsin Package v. 8, GCG).
Analysis of cos Ms7 subclone
PCR primers surrounding the junction between the V1R sequence and the non-V1R sequence were designed: Ms7E9-C1F (5'-CTT GCA GGA GGT AAA TAA TTG-3') and Ms7E9-C1R (5'-ATT ACA CTC AGG ATG CAG AC-3'). PCR reaction was performed as indicated above, with an annealing temperature of 55°C and an extension step of 45 s.
PCR cloning of primate orthologues of the hV1RL1 gene
PCR primers (PM1F, 5'-ATG GTT GGA GAC ACA TTA AAA C-3' and PM1R, 5'-TCA TGG CAT GAC AAC CAG ATT AG-3') spanning both ends of human V1RL1 (GenBank AF255342) were used to amplify genomic DNA from different primate species kindly provided by Dr A. Blancher: human (Homo sapiens); chimpanzee (Pan troglodytes); gorilla (Gorilla gorilla); orangutan (Pongo pygmaeus); gibbon (Hylobates lar); macaque (Macaca sylvanus); baboon (Papio papio); marmoset (Callithrix jacchus); squirrel-monkey (Saimiri boliviensis); and lemur (Eulemur fulvus). The cycling conditions consisted of an initial denaturation at 94°C for 2 min, followed by 30 cycles (94°C for 15 s, 55°C for 30 s, 72°C for 90 s) and a final extension step at 72°C for 10 min. PCR products were subcloned and recombinant clones were analysed as described above.
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Results |
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Screening of a marmoset cosmid library with human probes
Given the phylogenetic proximity of marmoset and human, we first screened
at low stringency a C. jacchus cosmid library with probes
corresponding to two human V1R coding regions (Ph2 and PhB4C5) belonging to
two different subfamilies (Giorgi et
al., 2000). Nineteen positive clones that produced medium or
weak signals were isolated. Out of them, nine clones were confirmed by
hybridization of a Southern blot of the EcoRI-digested DNAs with the mixed
probe used for the library screening. The cosmids were analyzed by
restriction-digest fingerprinting and were grouped on the basis of the
similarity of their restriction pattern (not shown). Duplicate clones were
eliminated and analysis was pursued on five clones: Ms7 (ICRF MPMGc
160-K02359Q3); Ms8 (ICRF MPMGc160E14338Q3); Ms11 (ICRF MPMGc160I04322Q3); Ms18
(ICRF MPMGc160H0890Q3); and Ms19 (ICRF MPMGc160N01207Q3). Ms7 was positive
with both probes Ph2 and PhB4C5; Ms8 was positive only with Ph2; Ms11, Ms18
and Ms19 were positive only with PhB4C5.
Analysis of the positive clones
All these cosmid clones showed an extreme instability and were deleting. We
rescued them by in vitro packaging and transfection into the
bacterial host Escherichia coli DH5-MCR. Fragments testing
positive with the human V1R probes were subcloned and sequenced. To our
surprise, except Ms7 that contained a V1R-like sequence, all the remaining
clones contained the calossin gene (GenBank Y17920), a calmodulin-binding
protein of unknown function conserved throughout animal phylogeny. Pairwise
comparisons between nucleotide sequences of the calossin gene and the human
V1R probes did not reveal any stretch of identity or common repetitive
sequences that could explain this result. Analysis of the Ms7E9 subclone
revealed 67.8 and 70.1% nucleotide sequence identity (NSI) with human V1R
genes Ph2 and PhB4C5, respectively, and 55.6% NSI with VN6, the most
homologous rat V1R gene (Dulac and Axel,
1995). During the course of this work, other workers
(Del Punta et al.,
2000) determined the sequences of the coding region of 25
potentially functional mouse V1R genes and seven pseudogenes. These sequences
are distributed into three distinct groups—a, b, and c—with group
c defining a novel group of V1R genes. In addition to V3Rs (V1rd), eight
supplementary families have been identified very recently
(Rodriguez et al.,
2002). The Ms7E9 sequence presents 66.9% NSI with the closest V1Rc
sequence (V1rc3), while the closest homologues in groups a and b are V1Ra2
(57% NSI) and V1Rb3 (56.5% NSI).
These results clearly indicate that Ms7 belongs to this new group c (Figure 1). However, the homology with known V1R sequences starts at the end of TMII, the 5' end containing numerous stop codons indicating that this gene is either a truncated V1R-sequence or has accumulated so many mutations in the 5' moiety that the amino-acid identity with V1Rs is not evident anymore. To verify that this sequence was a faithful representation of the marmoset genome and not an artefact due to the deletion of the cosmid, we designed PCR primers surrounding the junction between the V1R sequence and the unknown genomic sequence. The PCR reactions were carried out on marmoset genomic DNA, cosmid DNA and plasmid subclone DNA. In the three cases, the same band at the expected size of 250 bp was obtained, confirming that this sequence is a true pseudogene, not a cloning artefact and therefore does not code for a full-length V1R protein (not shown).
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Screening of the marmoset library with rat probes
We next decided to screen the cosmid library at low stringency with a rat coding V1R sequence (VN1). Indeed, given the result of the first library screening, we thought that even though marmoset is phylogenetically closer to human than rat, functional V1R sequences could be closer to potentially functional rat sequences than to human V1R pseudogenes. This approach was unsuccessful. Twenty-four weakly positive clones were selected, but were not confirmed to contain V1R sequences by Southern-blot hybridization.
Screening of the marmoset library with the marmoset Ms7E9 clone
We performed a last screening at low stringency with the Ms7E9 V1R-like sequence. Four calossin-containing clones were again isolated. Twelve other clones yielded a positive signal ranging from weak to strong and were analyzed by Southern blot (Figure 2). Out of them, one clone did not grow, four produced a weak signal (MsM2, ICRF MPMGc160P0934; MsM4, ICRF MPMGc160M09317; MsM10, ICRF MPMGc160G16333Q2; MsM11, ICRF MPMGc160M1118Q2) and four clones gave a medium to strong signal (MsM5, ICRF MPMGc160O2195Q2; MsM6, ICRF MPMGc160O19149Q2; MsM9, ICRF MPMGc160B15280Q2; MsM12, ICRF MPMGc160N02356Q2) comparable to the positive control Ms7. Clones MsM1, MsM7 and MsM8 were negative. Clones MsM2 and MsM10 were further shown to contain repeats but no V1R-like sequences.
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Analysis of the marmoset V1R-like clones
In parallel, degenerate PCR primers chosen in conserved domains of rat and
human V1Rs were tested on these cosmids as well as on the cosmids isolated
during the two other screening steps. Primer pair V1R-2F/2R was able to
amplify products only from cosmids Ms7, MsM4, MsM5, MsM6, MsM9, MsM11 and
MsM12 (not shown). These PCR products were subcloned and sequenced. Sequences
obtained from clones M5 and M12 were identical, as well as those from MsM6 and
MsM9. We established the full-length sequences of all these clones, except
MsM4 (for which 16 amino acids are missing on the 5' end), by
primer-walking on cosmid DNA. Ms7 presents 94 and 93.7% NSI with MsM5/M12 and
MsM6/M9, respectively. Furthermore, as observed in Ms7, the homology of
MsM5/M12 and MsM6/M9 with rodent V1R sequences starts at the beginning of
domain II, with a very diverged 5' end.
MsM4 and MsM11 present, respectively, 73.9 and 48.7% NSI with Ms7. When
compared to groups a, b and c (Figure
1), MsM4 clearly belongs to group c (68.7% NSI with V1Rc3), while
MsM11 presents an average NSI of 45% with the sequences of the three
groups (44% with V1ra1, 41.4% with V1Rb1 and 49.4% with V1Rc3). When compared
to human sequences, MsM4 and MsM11 present, respectively, 69 and 49% NSI with
Ph2 and 73 and 44.9% NSI with PhB4C5. As in the case of Ms7, the four
additional V1R-like sequences obtained from these six cosmid clones contained
stop codons and/or frameshifts, indicating that they correspond to
pseudogenes.
Although the proteins deduced from these marmoset V1R-like sequences are
similar to V1Rs, the inferred amino-acid sequences were uncertain in a few
regions of some clones due to many frameshift-causing indels
(insertion/deletion events). We therefore present in
Figure 1 the relationships
among these sequences in the form of a phylogenetic tree based on the
nucleotide sequences. The tree includes the seven rat V1R sequences
(Dulac and Axel, 1995), the
mouse V1R genes and pseudogenes belonging to the three different groups a, b
and c recently characterized (Del Punta
et al., 2000) and the seven human pseudogenes that we
previously described (Giorgi et
al., 2000). Except for PhA4 and MsM11 that are more distantly
related, marmoset and human V1R-like sequences belong to group c initially
defined by mouse V1R sequences, but form a sub-group distinct from rodent
group c sequences. MsM4 groups with PhH8 and PhD1 that localize human
chromosome 7, and sequences Ms7E9, MsM5/M12 and MsM6/M9 form another
sub-group. MsM11 may constitutes a new group that is not a, b or c.
The marmoset sequences show a NSI ranging from 44.5 to 94%, with M11 being
the most divergent, leading in all cases to an amino-acid sequence identity
(ASI) > 40%. One classification proposed for multigene families
(Nebert et al., 1991;
Ben-Arie et al., 1994)
defines 40% ASI as a threshold for belonging to the same family. According to
this definition, all marmoset sequences are members of the same family. Except
for MsM11, the group of marmoset sequences is very close to the mouse group c
sequences that are considered as a new sub-group
(Del Punta et al.,
2000). Families are then subdivided in sub-families (ASI >
60%). Accordingly, only M11 belongs to a distinct sub-family with an NSI with
the other V1R genes ranging from 44 to 48%. As shown in
Figure 3, assembling of the
marmoset protein sequences by correcting numerous frameshifts revealed that
they contain the amino-acid residues described previously
(Del Punta et al.,
2000) that are specific to the 7TM-GPCR superfamily (N28, C88,
C176 and F254) and the V1R family (G24, L31, R99, L109, K128, N163 and
L250).
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Characterization of primate orthologues of the hV1RL1 gene
In an attempt to characterize potentially functional V1R genes in primates,
we took advantage of the ORF (open reading frame)-containing human gene hV1RL1
(GenBank AF255342) to characterize by PCR the orthologues in various species.
Human hV1RL1 is the only gene reported to date to encode a potentially
functional VR-type receptor in primates. PCR products were obtained with DNA
from human, chimpanzee, gorilla and orangutan, but not from the other species
(gibbon, macaque, baboon, marmoset, squirrel-monkey and lemur), suggesting
that these latter sequences are too divergent from their human counterpart.
The human sequence was 100% identical to the published hV1RL1 sequence
(Pantages and Dulac, 2000;
Rodriguez et al.,
2000). Protein sequence comparison
(Figure 4) indicates that
human, chimpanzee and gorilla share 97% of amino-acid sequence identity
(ASI), whereas orangutan, that is more distantly related on the evolutionary
tree, shares 92% ASI with the three other species. Surprisingly, only
human and gorilla contain an ORF. Chimpanzee and orangutan contain obvious
pseudogenes due to frameshift mutations interrupting the ORF. The orangutan
sequence contains five frameshifts and the chimpanzee sequence contains one
frameshift associated with a motif change in conserved transmembrane domain
TM7 (CIH instead of VSL in the other species). In addition, the four species
present 38 different amino-acid substitutions, a number of which are
non-synonymous (Figure 4).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The VNO is a chemosensory organ located at the base of the nasal cavity. It is distinct from the olfactory epithelium and is present in most amphibians, reptiles and non-primate mammals, but is absent in birds, adult catarrhine monkeys and apes (Stoddart, 1980
). It plays an important role in intraspecific chemical
(pheromone) communication (Halpern,
1987; Keverne,
1999; Holy et al.,
2000; Leinders-Zufall et
al., 2000). Two main families of putative pheromone receptors
expressed in rodent VNO have been described, i.e. V1R/V3R and V2R, each
comprising 300 and 100-150 members, respectively. Pseudogenes have been
reported for the mouse V1R (50%) and V2R (66%) families. V1R sequences
have been cloned in human in three separate studies. In the three analyses,
the human V1R-like family appears to be much smaller than the rodent family
since only seven, eight and 34 different sequences, respectively, were
characterized (Dulac and Axel,
1995; Giorgi et al.,
2000; Kouros-Mehr et
al., 2001). So far, only human V1R pseudogenes have been
cloned. However, one potentially functional human sequence, V1RL1 (GenBank
AF255342) described (Pantages and Dulac,
2000; Rodriguez et
al., 2000) and expressed in the MOE has been isolated. This
is the only example of a potentially functional primate VR gene reported to
date.
Platyrrhines are the last primates to diverge from the human evolutionary
lineage that have the VNO morphology of most other mammals. We thought that a
primate species with an intact and functional VNO would retain a high
proportion of functional V1R genes, as in rodents. We therefore sought to
isolate putative marmoset V1R homologs to approach the question of the
relationship between an intact VNO and potentially functional V1R sequences.
As a result, we have only identified five different marmoset V1R-like
pseudogenes using an approach based on genomic library screenings at low
stringency with V1R probes derived either from humans or rodents. These
results are similar to those obtained in humans using rodent probes, i.e. a
small number of different clones containing only V1R pseudogenes were isolated
(Giorgi et al.,
2000). Marmosets possess an intact VNO and the role of olfactory
communication in social behaviors such as recognition of group members
(Epple et al., 1993)
and reproductive status (Smith and Abbott,
1998) has been documented. Despite these observations, our
approach did not allow us to isolate functional marmoset V1R genes. Similarly,
an approach conducted in pig, another species with an intact VNO and whose
behavior related to pheromones is well established, resulted in the
identification of pseudogenes only (S. Rouquier et al., unpublished
results).
Nevertheless, the release of the ORF-containing hV1RL1 gene
(Rodriguez et al.,
2000) allowed us to isolate by PCR the orthologues of this gene in
different primate species, in an attempt to pull out potentially functional
primate genes. Among the different species tested and in addition to humans,
we were able to amplify only chimpanzee, gorilla and orangutan sequences, the
other species, including marmoset, presumably being too divergent. Similarly,
a pig library screening using human V1RL1 as a probe did not allow us to
select for a putative pheromone receptor gene (S. Rouquier et al.,
unpublished results). Chimpanzee and orangutan V1RL1 genes are pseudogenes
and, despite the fact that human and gorilla contain an ORF, these
observations raise the question of whether the gene is functional in these two
last species. Indeed, from an evolutionary point of view, knowing the
regression of the olfactory structures (VNO, MOE) and the increasing
pseudogenization level of the genes involved in the corresponding chemosensory
functions from mouse to humans, and even more precisely from New World monkeys
to hominoids (Rouquier et al.,
1998a; Giorgi et al.,
2000; Kouros-Mehr et
al., 2001), the notion that the human V1RL1 gene became
functional again, or retained a function, while it is inactivated in
chimpanzees and orangutans is questionable. Alternatively, due to its
expression pattern (olfactory mucosa), it is possible that V1RL1 was primarily
a VNO receptor which evolved into an odorant receptor with pheromone-like
ligand properties (Kouros-Mehr et
al., 2001). This question will be ultimately addressed by a
functional test.
Our results lead us to hypothesize that during evolution, the V1R gene
repertoire diversified to generate different sets of functional genes that are
specific of each species. Indeed, using classical heterologous screening
strategies, we were able to select only for vestigial genes, whereas the
presumed functional pheromone receptor genes that are specific for a given
species may be too divergent to be isolated by these approaches. These
observations suggest that the species-specific nature of pheromone
communication makes this system quite different from regular olfaction, since
functional olfactory receptor genes are quite conserved in vertebrates,
especially in mammals, and can be isolated from one species to another using
various strategies (Mombaerts,
1999b). This hypothesis is reinforced by data published while this
work was under submission (Lane et
al., 2002). The authors performed a sequence analysis of
mouse V1R gene clusters and were unable to identify the human orthologues,
suggesting that these genes have been lost during primate evolution. In the
absence of extensive genome sequence data, it is likely that cDNA approaches
using complete VNO or single-cell PCR would be more appropriate to
characterize putative pheromone receptor genes of a particular species.
The results reported here indicate that V1R genes with sequences likely to
code for functional proteins are weakly homologous between species and
therefore suggest alternative approaches for their identification. Indeed, the
characterization of the V1R repertoire is rather slow and very few papers have
been published for other species than rodents. On the other hand, the
identification of a human V1R/V3R ORF that is expressed in the MOE suggests
that the VNO should not be considered as the exclusive site for VR type
receptors, as previously described in goldfish
(Cao et al.,
1998).
Overall, these observations lead to several reflections on the evolution of chemical communication which are not mutually exclusive, as follows.
- The pseudogenization process parallels the regression of the structures
(VNO, AOB, MOE) devoted to chemical communication and the need for this
function (Giorgi et al.,
2000; Rouquier et
al., 2000; Kouros-Mehr
et al., 2001), i.e. in absence of selective pressure
there would be a parallel degeneration of organs and genes, meaning that
primates (at least hominoids) would not extensively use chemicals to
communicate as rodents do.
- One may wonder why so few VR genes are identified by using heterologous
(rodent) probes in primates. Two explanations are possible. Either numerous
genes were deleted over time, or they evolved rapidly in a species-specific
fashion leading to a loss of the homology necessary to ensure a specific
hybridization.
- In animals that possess a VNO and for which chemical communication via
pheromones has been well established (pig, marmoset), we see the same type of
results, i.e. very few genes were identified using heterologous probes and all
of them were pseudogenes. Starting from the hypothesis that these species have
functional VR genes (GPCR), we may hypothesize that, during evolution, each
species acquired its own set of functional VR genes and that each set does not
overlap with those of other species, except perhaps in the case of very close
species such as rat and mouse, or human and chimpanzee or gorilla. This
situation evokes the case of olfactory receptors of vertebrates and insects,
i.e. vertebrate sequences could not be used to pull out insect olfactory
receptor genes.
- Alternatively, primates or any species communicating via pheromones might
use receptors radically different from those used as probes in this study.
- Finally, it is also possible that in absence of a functional VNO, other
genes different from the VR genes and expressed in other tissues may function
as pheromone receptor genes. Olfactory receptors could play this role. For
example, gene OR912-93 which is potentially functional in chimpanzee,
orangutan and gibbon (Rouquier et
al., 1998b), binds the odorant 2-heptanone
(Gaillard et al.,
2002), a chemical which displays pheromone activity in mouse
(Leinders-Zufall et al.,
2000). However, it should be demonstrated that the signal
transmission in the nervous system is in accordance with a pheromone stimulus
inducing modifications of the physiology and/or behavior.
In most species that rely on pheromone communication, the receptors responsible for the function remain to be discovered. The study of the evolution of the VR genes in primates is an important step in discovering whether humans possess functional VR genes.
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Acknowledgments |
|---|
We are grateful to S. Dubel for critical reading of the manuscript, and to Prof. A. Blancher for providing primate DNAs.
|
Notes |
|---|
Note
Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession Nos AF397897-AF397901 and AF426106-AF426108
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References |
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