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Hormones and Behavior

Pheromone receptors in mammals

Ivan Rodriguez*

Department of Zoology and Animal Biology and NCCR Frontiers in Genetics, University of Geneva, Geneva, Switzerland

Received 4 December 2003; revised 16 March 2004; accepted 18 March 2004

Abstract

In most mammals, pheromone perception mediates intraspecies interactions related to reproduction, such as mate recognition, intermale

aggressive behaviors, or exchanges between females and their offspring. Recent molecular findings, particularly the identification of two

large pheromone receptor gene superfamilies, provide today invaluable tools to better understand the way mammals make sense of

pheromonal information.

D 2004 Published by Elsevier Inc.

Keywords: Mammals; Pheromones; Reproduction

Introduction

All living species have evolved various communication

strategies, many of them involving the use of chemosensory

organs. Thus, mammals possess an extremely sophisticated

tool, the olfactory system, which allows them to probe the

outside world in a single whiff.

Since the identification of the vomeronasal organ

(VNO) in 1813 by the anatomist Ludwig Jacobson

(Jacobson et al., 1998), it has been known that the

mammalian olfactory system is physically divided into

two parts: the main olfactory and the vomeronasal systems.

However, it took scientists over a century to realize that the

VNO, a structure that had been considered as a secondary

and somehow useless subpart of the main olfactory system,

was not only physically distinct from this latter, but also

differed functionally. This major step in the recognition of

the role played by this organ was achieved when it became

apparent that its removal did not perturb the ability of most

0018-506X/$ - see front matter D 2004 Published by Elsevier Inc.

doi:10.1016/j.yhbeh.2004.03.014

* Department of Zoology and Animal Biology and NCCR Frontiers in

Genetics, University of Geneva, Quai Ernest Ansermet 30,1211 Geneva 4,

Switzerland.

E-mail address: Ivan.Rodriguez@zoo.unige.ch.

animals to perceive smells, but dramatically impaired their

ability to interact with their congeners. Thus, this small

structure, found in many vertebrates and in almost all

terrestrial mammals, was mainly responsible for the

detection of pheromones. These latter, which are surpri-

singly still very poorly defined in mammals, are released in

the outside world in bodily fluids such as urine, or

produced and kept on the emitter’s body, e.g., in sweat

or saliva; they are then perceived by another member of

the same species, and provide the recipient information

such as sexual or social status of the emitter. Thus, long-

lasting effects such as an advanced onset of puberty or the

termination of a pregnancy, or more short-term behaviors

such as copulation, protection of the pups in lactating

females, or intermale aggression, are mediated by pher-

omones (Doving and Trotier, 1998; Halpern, 1987;

Johnston, 1998; Keverne, 1999). The ability to detect

and make sense of these particular molecules is therefore

necessary, if not for the survival of the individual, at least

for the survival of the species.

The organ of Jacobson, a bilateral tubular structure

(Fig. 1), is located at the base of the nasal septum and opens

via a small duct into the nasal cavity, the mouth, or both,

depending on the species. It is filled with a few hundred

thousand bipolar sensory neurons, each projecting a single

46 (2004) 219–230

Fig. 1. The vomeronasal neurosensory epithelium and its projections.

Bottom: Coronal view of a VNO. Neurons in the apical sensory layer

(orange) express V1r receptors while basal sensory neurons (green) express

V2r receptors. This differential expression correlates with the expression of

the G protein subunits ai2 and ao, respectively. Each VSN projects a single

axon toward the accessory olfactory bulb (top). Top: Dorsal view of the

accessory bulb. Axonal projections from V1r- and V2r-expressing VSNs

coalesce into multiple glomeruli in the rostral (top of drawing, orange) and

caudal (bottom of drawing, green) accessory bulbs, respectively.

Fig. 2. Top: Phylogenetic tree corresponding to the complete mouse V1r

repertoire. The 12 V1r families (V1ra-l) are clearly distinct. Each terminal

branch represents a single V1r gene (adapted from Rodriguez et al., 2002).

Bottom: Monoallelic expression of V1r genes. The picture represents a

coronal section through the vomeronasal neuroepithelium of a compound

heterozygote knock-in mouse in which VSNs expressing the paternal and

maternal alleles of the V1rb2 gene appear green and red fluorescent,

respectively. No co-expression of the two parental alleles is observed

(adapted from Rodriguez et al., 1999).

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230220

axon toward a specialized part of the olfactory bulb, in an area

inadequately called (because the name was coined before the

specific role of the VNO became apparent) the accessory

olfactory bulb. The dendritic endings of these neurons float

into a mucus that is in contact with the outside world and that

can be actively expelled from or sucked into the VNO.

Pheromones, previously dissolved into this mucus, are then

recognized by specific vomeronasal sensory neurons (VSNs),

an interaction that initiates a still ill-defined transduction

cascade, and culminates in the generation of action potentials

that are transmitted along the axonal projections toward the

accessory olfactory bulb. At this first relay level, secondary

neurons form synapses with the axonal projections of the

sensory neurons and transmit the information to parts of the

brain’s limbic system, the amygdala and hypothalamus,

bypassing higher cognitive centers. The resulting signals

then affect the endocrine status of the recipient, usually

through modulation of luteinizing hormone or prolactin

(Halpern, 1987).

How are molecules recognized by olfactory sensory

neurons? The olfactory system faces a task quite com-

parable to the one of the immune system, as it also has to

make sense of thousands of unknown external molecules.

The immune system got around this problem by using a

combinatorial strategy allowing a few genes to recombine,

a gene rearrangement resulting in an enormous diversity of

receptors. Multiple mechanisms were proposed for olfac-

tory recognition during the second part of the 20th century,

some of which involved very complex and supposedly

unusual physical properties of olfactory sensory neurons.

Most of these explanations appear today highly improb-

able, if not simply wrong. The major blow to these

hypotheses was given by the identification of a unique tool

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230 221

possessed by the mammalian main olfactory system to

identify chemicals: a gigantic and amazingly diverse

repertoire of genes encoding for odorant receptors (Buck

and Axel, 1991). These genes number indeed over 1000 in

rodents and represent the largest protein superfamily in

mammalian genomes.

Odorant receptors are, however, not expressed in the

vomeronasal organ; which other olfactory receptor type

then allow VSNs to recognize pheromones? The discovery

by Catherine Dulac and Richard Axel of a large receptor

superfamily exclusively expressed by VSNs gave a first

answer in 1995 (Dulac and Axel, 1995). The identification

of a second vomeronasal-specific receptor superfamily

quickly followed this major breakthrough. This review

summarizes the progresses on vomeronasal receptors over

the years that followed the identification of the first

receptor family.

The concept of pheromone

It may appear singular to discuss the concept of

pheromone, which most of us would think is well defined.

This is, however, not the case. Because this review focuses

on pheromone receptors, a short history of our under-

standing of this concept may be useful.

The term bpheromoneQ was initially coined by Karlson

and Luscher (1959) over 40 years ago. It was designed to

describe biologically active substances produced by an

insect, which, when perceived by another member of the

emitter’s species, is able to induce a specific reaction in the

recipient such as a particular developmental process or a

given behavior. Some time later, pheromones were classi-

fied into multiple categories, allowing for example to

distinguish those eliciting immediate behavioral responses,

or those airborne, or even those that are consciously

perceived, from the others.

Today, the term pheromone is widely used by people

working in very different fields ranging from psychology

to electrophysiology, some studying life forms as different

as yeasts, butterflies, or ferrets. As a result, the same term

is now often used to describe very different types of

chemosensory stimuli. Thus, molecules detrimental to the

emitter, or particular complexes of chemicals used for

interspecies communication, or even synthetic compounds

with no natural counterparts, are often called pheromones.

This expansion of the use of a previously quite restricted

term has bothered and has been discussed by many. A

recent review proposes a definition that has the quality of

not being too limited, and that has the advantage of not

relying on the chemical nature of the molecules: Pher-

omones are produced by individuals of a given species and

are defined in the context of mutually beneficial commu-

nication between members of this species (Meredith,

2001). Although a distinction between mutually and

single-sided beneficial information would be of great

interest, benefits may not be immediately apparent in

many cases or, even worse (because of our different

understandings of a benefit), may be subject to debate. A

slight extension of the previously mentioned definition of

pheromonal interaction will therefore be used here, which

includes chemosensory-mediated information not necessa-

rily mutually beneficial. The somewhat arbitrary but

widely adopted limit of pheromone communication to

intraspecies interactions will be kept because it makes the

concept easier to use.

Identification of the first vomeronasal receptor genes

The identification of odorant receptors by Buck and

Axel in 1991 was expected to be very soon followed by

the identification of vomeronasal receptors. The discovery

of the odorant receptor gene superfamily was based on the

assumption that it consisted of a novel and large family,

and that the proteins encoded by these genes were likely to

be seven transmembrane proteins. The strategy used to

clone the first odorant receptor was based on the fact that a

very large seven transmembrane receptor family (called

rhodopsin-like), from which odorant receptors were possi-

bly members, possessed various amino acid segments

common to all its members. The use of degenerate primers

corresponding to these segments thus allowed the cloning

of the first members of the odorant receptor gene super-

family (Buck and Axel, 1991).

Similar assumptions were applied to the putative

vomeronasal receptor repertoire. Against all expectations,

no vomeronasal receptor gene could be obtained with the

homology-based approach used for the identification of

odorant receptors. Facing this problem, Dulac and Axel

opted for a unique molecular strategy, based on another

assumption: Different populations of VSNs should express

different receptors. The main idea consisted in the

subtraction of messenger RNAs corresponding to these

different populations, hoping that one of the differences

between these sensory neurons would consist in the long-

sought chemosensory receptors. The tour de force involved

the generation of single-cell libraries containing the tran-

scriptomes of a few randomly chosen rat single VSNs, and

the subtraction of these transcriptomes. Following this

approach, Dulac and Axel reported the identification of the

first vomeronasal receptors, termed V1rs, in 1995 (Dulac

and Axel, 1995).

V1r receptors are, as was foreseen, numerous. At the time

of the initial identification of the rat sequences, the size of the

repertoire was assumed, based on low-stringency hybrid-

izations of genomic DNA, to contain around 40 members.

These receptors are, again as foreseen, putative seven

transmembrane receptors. Consistent with the failure of the

initial homology-based approach, V1rs share no sequence

homology with receptors from the rhodopsin family. Less

expectedly, this observation extends to a lack of homology to

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230222

any other known protein, except a weak relationship with a

family of taste receptors called T2R, whose members mediate

bitter tasting.

A very diverse superfamily

The complete picture of the mouse V1r superfamily

repertoire took a few years to emerge. Following the initial

identification of the rat V1r sequences, three quite distant

V1r families were identified in the mouse (Del Punta et al.,

2000; Pantages and Dulac, 2000). At the time, the very

limited homology of one of these families with the other

two even suggested that it could represent a totally new

class of receptors (Pantages and Dulac, 2000). The

sequencing of the complete mouse genome by Celera

(and later by public efforts) then allowed a global view of

a V1r gene repertoire (Rodriguez et al., 2002; Zhang et al.,

2004). About 150 V1r receptors are distributed among 12

very divergent families (Fig. 2); families share from 15%

to 40% of their amino acid residues between each other,

with intrafamily identities varying between 40% and 99%.

A high sequence variability is found in transmembrane

domain 2, in intracellular loop 1, and in extracellular loops

2 and 3, while transmembrane domain 3 is highly

conserved. A few amino acid residues mostly located

between transmembrane domains 5 and 6, and a potential

glycosylation in extracellular loop 2, are common to

virtually all members of the 12 V1r families, while specific

amino acid signatures are characteristic of each of the 12

families (Rodriguez et al., 2002).

In accordance with these previous observations, analysis

of substitutions in coding regions of the duplicated V1rs

(such analysis indicates the nature of the selective forces

that have shaped the evolution of a gene) reveals that V1rs

have been subject to selection both against and for change

in specific domains. In mouse V1rs, negative selection

(selection against change) is observed for example in the

third transmembrane domain [average ratio of nonsynon-

ymous (Ka) vs. synonymous (Ks) substitutions of 0.29],

and a Ka/Ks higher than 1 (selection for change, positive

selection) is found for the loop between the first two

transmembrane domains (Lane et al., 2002). Other evi-

dences for positive selection acting on the V1r repertoire

have been reported, including in nonrodent species (Mundy

and Cook, 2003; Zhang et al., 2004). Positive selection is

mostly observed in genes evolving rapidly, such as those

involved in reproduction and in the immune system

(Hughes et al., 1990; Pamilo and O’Neill, 1997; Wyckoff

et al., 2000).

Diversity of the mouse V1r repertoire is not limited to

intergenic variations. Indeed, allelic polymorphisms, which

can generate different products of the same gene, appear to

be quite frequent in mouse V1r genes. After an initial report

of intraspecies polymorphisms in a single V1r (Rodriguez

et al., 1999), a thorough estimation of such polymorphisms

between V1r repertoires of different mouse strains indicated

an average of 1.28 single nucleotide polymorphisms per

1000 base pairs in V1r coding regions (to be compared to a

mean of 0.82 for mouse genes) (Zhang et al., 2004). Such

variations at the nucleic acid level may or may not lead to a

modification of the corresponding protein. In vertebrate

genes, most polymorphic codons do not lead to a

modification of the corresponding amino acids. Interest-

ingly, this is not what is observed in V1rs, where the

fraction of polymorphisms leading to amino acid changes is

larger than the one leading to a conservation of the amino

acid change (Zhang et al., in press). The intragenic genomic

variability of V1rs in a given species is therefore also

reflected at the protein level.

Genomic organization of the V1rs

Genomic organization of the V1r repertoire has mostly

been investigated in the mouse (Rodriguez et al., 2002;

Zhang et al., 2004). Similar to what is known for odorant

receptor genes, the V1r repertoire consists of small

multiexonic genes, each possessing a coding sequence of

about 900 base pairs entirely included in a single exon.

While most of the 150 mouse V1r genes are found on

chromosomes 6, 7, and 13, they are also present on other

autosomes (Rodriguez et al., 2002; Zhang et al., 2004). A

single V1r gene from the V1rb family is also found on the

X chromosome (Del Punta et al., 2000). A large proportion

of V1r genes is still not assigned to any chromosome,

probably because of the very high content of repetitive

elements surrounding each V1r gene unit, which renders

genomic mapping difficult. V1r gene families are usually

clustered and include both intact and pseudogenic sequen-

ces, with a mean intergenic distance of less than 20

kilobases. In these clusters, transcriptional orientation of

each V1r gene is apparently unrelated to the orientation of

its flanking V1r genes. Very rare non-V1r genes are present

in the clusters, which instead, as previously mentioned, are

densely populated with repetitive elements, mostly mem-

bers of the Line 1 repeat family. A single 600-kilobase

cluster on chromosome 6 has been thoroughly investigated,

and contains 23 genes and pseudogenes from the V1ra and

V1rb families, among which 16 appear intact (Del Punta et

al., 2000; Lane et al., 2002). Exceptional noncoding

homology, possibly related to transcription regulatory

regions, is observed across members of this cluster (Lane

et al., 2002). This homology is surprisingly not shared by

V1r sequences of other clusters, suggesting some kind of

locus-specific transcriptional regulation.

Different species, different V1r repertoires

A look at V1r molecular trees suggests that expansion

of many families of the V1r repertoire occurred in bursts

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230 223

(Fig. 2) (Lane et al., 2002; Rodriguez and Mombaerts,

2002; Rodriguez et al., 2002). The analysis of substitution

rates of duplicated blocks in three different mouse clusters

(containing 73 V1r genes and therefore only a fraction of

the entire mouse repertoire) indicates that the majority of

the expansion of these clusters began between 22 and 32

million years ago through large local duplications from a

few ancestral genes, around the time of mouse–rat

speciation (Lane et al., 2002).

Comparisons of the complete V1r repertoire of different

species, including mouse, rat, dog, and human, indicates

that mammalian species differ in the size and composition

of their V1r repertoires (Capello and Rodriguez, unpub-

lished data; Lane et al., 2004; Rodriguez and Mombaerts,

2002). A partial overlap is indeed observed, not at the level

of orthologous genes (the concept of orthology is here

often meaningless), but rather at the level of V1r families.

Species-specific families are also found, and may for

example be present in two species, but not in a third

species. Because the rate of emergence of new V1r

receptor genes is apparently very rapid and the sequences

quite divergent between species, and because the corre-

sponding receptors are involved in the regulation of

reproductive and other social behaviors, an attractive

hypothesis would be to consider V1rs as instrumental or

even at the origin of some speciation events in vertebrates;

they could thus contribute to premating reproductive

isolation. A related situation is found in an Ostrinia

species (a moth), where changes in pheromone signaling

and perception appear to have allowed for such isolation

and possibly speciation (Roelofs et al., 2002).

V1rs in primates

During the evolution of higher primates, the VNO

decreased in size. Thus, New World monkeys possess a

quite small organ, while most catarrhine primates (a group

that includes apes and Old World Monkeys), despite the

development of a vomeronasal system during embryo-

genesis, exhibit a vestigial VNO after birth, very likely

without function (Hunter et al., 1984; Maier, 1997;

Meredith, 2001), although this is still debated. In favor

of this absence of vomeronasal function, an ion channel,

the transient potential cation Trpc2 channel, which is likely

involved in vomeronasal transduction (Leypold et al.,

2002; Liman and Innan, 2003; Liman et al., 1999; Stowers

et al., 2002; Zhang and Webb, 2003), is a pseudogene in

many catarrhine primates (Liman and Innan, 2003; Zhang

and Webb, 2003). Moreover, numerous V1r pseudogenes

have been identified in catarrhines (Giorgi and Rouquier,

2002; Giorgi et al., 2000; Kouros-Mehr et al., 2001; Zhang

and Webb, 2003). This observation is, however, also true

in New World Monkeys and even in rodents. It appears

that vision-based signaling, especially because trichromatic

vision emerged (which occurred in the common ancestor of

hominoids and Old World Monkeys after they were

separated from New World Monkeys), has partly replaced

VNO-mediated social interactions in catarrhines, including

humans (Liman and Innan, 2003; Zhang and Webb, 2003).

In this light, it may appear surprising that many of these

latter species still exhibit unambiguous pheromone-induced

responses. An explanation for such responses without

functional VNO could be that, similar to what is observed

in some mammals such as rabbits, pigs, or ewes (Cohen-

Tannoudji et al., 1989; Dorries et al., 1997; Hudson and

Distel, 1986), the main olfactory system is able to mediate

pheromone perception in these primates. If true, still

undefined receptors, odorant receptors, or vomeronasal-

type receptors could be involved in this process. These

latter indeed appear to possess unique characteristics

because VSNs expressing them are apparently very

narrowly tuned, extremely sensitive to low agonist

concentrations, and do not adapt under prolonged stimulus

exposure (Holy et al., 2000; Leinders-Zufall et al., 2000;

Zufall et al., 2002).

Data on V1r gene evolution in primates are still rather

scarce, but some interesting observations have been made.

In humans, among a very large set of pseudogenes (Giorgi

et al., 2000; Kouros-Mehr et al., 2001), five V1r genes,

termed V1RL1 to 5 (for V1r-like), appear to be intact in

many individuals (Rodriguez and Mombaerts, 2002;

Rodriguez et al., 2000; Zhang and Webb, 2003). However,

two of them, V1RL1 and 5, show signs of pseudogeniza-

tion (Zhang and Webb, 2003). Interestingly, mature tran-

scripts of V1RL1 (the only one of the five V1RL genes

tested), are expressed in the human olfactory mucosa

(Rodriguez et al., 2000).

These human V1r sequences (or a fraction of those)

could represent intact and functional V1r genes, or

alternatively relics of an ongoing pseudogenization proc-

ess. In favor of a pressure to maintain a specific V1RL

receptor, 14 of the 15 residues most conserved in rodent

V1r sequences, in addition to a conserved potential

glycosylation site in extracellular loop 2, are present in

V1RL1. Contradictory data have, however, been published

(Zhang and Webb, 2003) based on the probability that, by

chance, a few human V1r receptor coding sequences may

have escaped pseudogenization. Satisfactory statistical

evaluation of such possibility requires the number of

functional V1r genes at the time the functional constraint

potentially disappeared, a number that we still ignore but

that may be particularly low (Capello, Pfister, and

Rodriguez, unpublished). In addition, evaluation of the

probability to have maintained expression of the V1RL1

gene without selection pressure is impossible today, as we

still have to identify a single V1r promoter. Definitive

proof for a chemosensory role played by human V1RLs

may take a long time, and will require the identification of

potential ligands, and likely the evaluation of mutant

individuals; similarly, a demonstration of their existence as

simple evolutionary remnants will be extremely difficult.

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230224

The presumed ortholog of V1RL1 has been sequenced

from New World Monkeys (a large sample including

common marmoset, pygmy marmoset, Goeldi’s monkey,

night monkey, golden-headed lion tamarin, saddle-backed

tamarin, white-fronted capuchin, mantled howler, Bolivian

red howler, and red howler), and apes (common chimpanzee,

western lowland gorilla, orangutan, and lar gibbon) (Mundy

and Cook, 2003). In eight of these species, V1RL1 is a

pseudogene, and phylogenetic analyses indicate that the

corresponding pseudogenization events occurred independ-

ently in the different lineages. The relative rates of

synonymous and nonsynonymous substitutions were eval-

uated for these presumed orthologs. Among lineages that

maintained a complete V1RL1 open reading frame, sites

under purifying selection were identified, while a few others

were found under strong positive selection. Such evolu-

tionary pressures, which are very good evidences for a

function of this receptor in these diverse species, do not,

however, link the expression of V1RL1 to pheromone

detection.

A second vomeronasal receptor family

In 1997, three groups independently identified a novel

seven transmembrane superfamily, V2r, exclusively

expressed in the basal layer of the vomeronasal neuro-

epithelium. Two approaches were used, one involving a

subtractive strategy similar to the one successfully used for

the identification of V1rs, and another involving the

amplification of transcripts with degenerate primers (Her-

rada and Dulac, 1997; Matsunami and Buck, 1997; Ryba

and Tirindelli, 1997).

In contrast to V1rs, V2rs possess a long extracellular N-

terminus, suggesting a different mode of ligand recog-

nition. They are related to Ca2+-sensing and metabotropic

glutamate receptors, and to the taste receptors that mediate

sweet perception. V2r genes number about 100 to 140 in

rodents, are alternatively spliced, and are organized in

clusters. In addition, similar to what is observed (but to a

lesser degree) in the mouse V1r repertoire, a very large

part of the rodent V2r repertoire is composed of

pseudogenes (Herrada and Dulac, 1997; Matsunami and

Buck, 1997; Ryba and Tirindelli, 1997).

Large-scale sequence information concerning the V2r

superfamily is very scarce, even in the mouse. This is mainly

because the extraction of V2r coding sequences from

genomic databases is more complex than for odorant and

V1r receptor genes, because V2rs possessmultiexonic coding

sequences, a unique feature among olfactory receptor genes.

Does size matter?

Why does a mouse need over 250 different vomeronasal

receptors? Only a very limited number of rodent behaviors

or endocrine modifications are known to be pheromone-

mediated and to be at least partially dependent on a

functional vomeronasal system. These include, for exam-

ple, the return to estrus of newly mated female mice when

exposed to strange males before embryo implantation

(Bellringer et al., 1980), or the induction of estrus in

house grouped females made anestrus by group housing. A

few molecules, mostly produced in the urine, such as

E,a-farnesene, 6-hydroxy-6-methyl-3-heptanone, (s)-2-sec-

buty-4,5-dihydrothiazole, or dehydro-exo-brevicomin, are

known to be able to trigger some of these events (Jemiolo et

al., 1986; Novotny, 2003; Novotny et al., 1986, 1999;

Schwende et al., 1984). Despite our surely still very partial

view of the mammalian pheromone interactions complexity,

such a massive number of receptors whose unique role would

essentially be to mediate pheromone detection is quite

puzzling.

One explanation could simply be the following: in

insects, for example, pheromones are often found in

blends; by extension, such mixes involving different

compounds may be used as chemical signals in mammals.

Multiple specific receptors could be specialized in the

detection of each of these compounds, and the activation of

all these receptors would be necessary to induce a specific

behavior. A large number of receptors could therefore be

necessary to extract information from complex signals.

However, it appears that in mammals, single molecules are

able to elicit responses apparently similar to the ones

produced by natural pheromone-containing fluids, such as

2-methylbut-2-enal that allows nipple location in rabbits

(Schaal et al., 2003).

Another nonexclusive potential role played by the large

vomeronasal receptor repertoire could reside in a VNO-

mediated perception of not only pheromones, but of the

largest group of semiochemicals. These represent mole-

cules from which animals extract information from their

environment, such as chemicals produced by surrounding

plants or animals from other species. Indications that in

rodents the VNO may be able to perceive odorant

molecules not considered as pheromones have been

published (Holy et al., 2000; Sam et al., 2001; Trinh and

Storm, 2003), although other data question the ability of

the vomeronasal system to respond to such volatile

molecules (Luo et al., 2003).

An extension of this idea, which involves potential

multiple roles played by V1rs, would expect these

receptors to be expressed outside the vomeronasal neuro-

epithelium. This is in fact the case, as few V1r transcripts

have been detected in the main olfactory epithelium of

humans and goats (Hagino-Yamagishi et al., 2001;

Rodriguez et al., 2000). Their role as chemodetectors in

this tissue is still unclear, but could be related to the ability

of many mammals, as previously mentioned, to respond to

pheromones independently of a functional VNO. More

astonishing is the identification of mRNAs corresponding

to nine different V1rs, called TV1rs, in male rodents

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230 225

developing germ cells (Tatsura et al., 2001). These tran-

scripts are apparently expressed by subsets of spermatids,

but no specific role has yet been attributed to these

receptors. Obviously, in addition to this quite unexpected

V1r-expressing tissue, transient V1r transcription in other

cell types may have escaped our attention.

An alternative explanation would consider the rodent

vomeronasal receptor repertoire as the result of an over-

expansion of gene families, with no specific evolutionary

pressure on any given member of the repertoire (because

many V1rs from a given family are very similar and

potentially recognize the same molecules), but rather with

a pressure on the existence of the different families. This

view would explain why such a large proportion of V1rs

(over 30%) consists of pseudogenes in rodents (Rodriguez

et al., 2002; Zhang et al., 2004).

Expression of vomeronasal receptors

Vomeronasal receptor genes are essentially expressed by

VSNs, and their products are believed to be carried toward

the dendritic endings of these latter such that they contact

the outside world.

The vomeronasal neuroepithelium is a pseudostratified

epithelium that constantly renews itself during adult life.

VSNs located basally or more apically in the neuroepithe-

lium represent fully mature and functional cells, a situation

which contrasts with the one observed in the main olfactory

neuroepithelium where basal neurons are immature. In the

VNO, the main stem cell population is located mostly

laterally (and not basally), at the boundary between the

sensory and the nonsensory epithelia. Molecular markers

allow a distinction between sensory neurons in the apical or

basal zones of the vomeronasal neuroepithelium. Thus,

subunits of a protein (a G protein), thought to directly

interact with vomeronasal receptors and to be involved in the

vomeronasal receptor-induced signal transduction cascade,

are differentially expressed in these two layers: The G

subunits ai2 and ao are, respectively, expressed in the apical

and the basal vomeronasal sensory layers (Fig. 1).

V1rs

V1r expression has been reported in multiple terrestrial

mammals. In rodents, neurons expressing specific V1rs are

exclusively located in the apical part of the neuroepithe-

lium (Fig. 1) (Saito et al., 1998). In situ hybridizations on

vomeronasal sensory epithelia indicates that only a fraction

of these VSNs expresses a given V1r receptor (Dulac and

Axel, 1995). The distribution of VSNs expressing a given

V1r is punctate, and apparently random; the use of

different combinations of probes and the generation of

single cell libraries suggest that each apical sensory neuron

expresses a single V1r gene (Dulac and Axel, 1995), but

definitive demonstration of this monogenic paradigm is

still lacking. Use of transgenic mice with both parental

alleles of a specific V1r gene (V1rb2) targeted with

different markers shows that VSNs not only express a

single (or a few) V1r gene(s), but also express each V1r

from a single parental allele (Fig. 2; Rodriguez et al.,

1999). Thus, an adult mouse probably expresses each V1r

from only about 500 to 1000 sensory neurons, with an

equal probability to express it from the maternal or the

paternal alleles.

V2rs

V2rs are likely to be present in most vertebrates; they

have indeed been identified in rat and mouse, and V2r-

related transcripts have also been identified in the

olfactory epithelium of goldfish and fugu (Cao et al.,

1998; Naito et al., 1998; Speca et al., 1999). In mammals,

and in contrast to what is observed for V1r expression,

V2r transcription is restricted to Gao-expressing sensory

neurons in the basal layer of the vomeronasal neuro-

epithelium (Fig. 1; Matsunami and Buck, 1997; Ryba and

Tirindelli, 1997), with the single exception of the V2r

gene Go-VN2, which is apically expressed in male rats

and in more basal VSN populations in females (Herrada

and Dulac, 1997). This latter example represents the only

sexually dimorphic vomeronasal receptor expression

reported today, and has not been shown in the mouse yet.

Expression of most V2rs is punctate and reminiscent of

what is observed in the apical sensory layer for V1rs.

Indirect evidence for monoallelic expression of one

member of the superfamily, the V2r1b gene, has been

obtained (Del Punta et al., 2002b). However, contrary to

what is known for V1rs, co-expression of different V2r

genes in a single neuron does occur in rodents (although

only a few are likely to be coexpressed). Staining of rat or

mouse vomeronasal epithelium with V2r2-specific anti-

bodies reveals a co-expression of this receptor (or members

of its subfamily) in basal VSNs expressing other V2rs

(Martini et al., 2001). A similar situation may also be true

in the olfactory system of goldfish, where two V2r-related

receptors, specifically 5.3 and 5.24, are broadly expressed,

while others are restricted to very limited sensory

population (Speca et al., 1999). Interestingly, both mouse

and goldfish widely expressed V2r-like receptors are

particularly divergent in sequence when compared to their

respective V2r repertoires; this observation may, however,

simply reflect our still incomplete deciphering of these two

latter repertoires.

Consistent with a role of vomeronasal receptors in adult

interactions, the onset of expression of V1rs and V2rs in

rodents begins late during embryogenesis, or even after

birth. Consequently, the spatial organization of the vomer-

onasal neuroepithelium emerges only during the postnatal

period and reaches its definitive pattern at sexual maturity,

in contrast with the well-defined odorant receptor zones

present early after birth in the main olfactory epithelium.

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230226

Targeting of the vomeronasal receptors to the dendrites

The notion that seven transmembrane receptors are not

transported alone to the cellular membranes and that

companion molecules are required for their expression is

not new. For example, g-aminobutyric acid B (GABAB)

receptors require dimerization for surface expression, and

receptor activity modifying proteins (RAMPs) regulate the

transport and ligand specificity of the calcitonin-receptor-like

receptor.

Although it has been shown that V1r receptors are

present in vomeronasal dendritic knobs (Takigami et al.,

1999), not much is known about the way V1rs mature and

reach these specialized structures.

A recent report sheds some light on the way V2r receptors

are processed. A very unexpected partner was found to be

necessary for the correct addressing of V2rs to the VSN

dendritic endings (Loconto et al., 2003). Members of two

families of major histocompatibility complex class Ib

molecules, the M1 and M10 families that comprise nine

members, were found to be expressed in the vomeronasal

sensory epithelium, specifically in the basal, V2r-expressing

zone (Ishii et al., 2003; Loconto et al., 2003). The function of

class Ib molecules, which are similar to class Ia molecules

usually associated with h2-microglobulin, is not fully

understood, but some of these proteins have demonstrated

the capacity of presenting antigenic peptides. At the time of

their identification as part of the vomeronasal transcriptome,

the M1-10 genes had been known for some time, but no

tissue was known to express them. In situ hybridizations with

probes recognizing the M1-10 transcripts indicates that these

genes are expressed exclusively in the VNO, the first signals

being detectable only after birth. They are found differ-

entially expressed in subsets of VSNs, with more than one M

gene being sometimes expressed per neuron. Data suggest

that basal VSNs express nonrandom combinations of M10

and V2r receptors, and that a precise correlation between the

expression of both gene families may exist (Ishii et al., 2003;

Loconto et al., 2003).

Immunocytochemical and biochemical analysis indicate

that molecular complexes including M10s, V2rs, and the

molecule commonly associated with MHCs, h2-micro-

globulin, are present in VSN dendrites. Moreover, and in

agreement with this finding, the presence of M10 receptors

and the expression of h2-microglobulin appear to be

necessary for a correct traffic of V2rs to the cell surface.

Consistent with a role played by h2-microglobulin in the

elaboration of a functional vomeronasal system, h2-micro-

globulin null mice exhibit abnormalities in the dendritic

expression of a V2r receptor, together with a lack of

aggressive behavior toward other males (Loconto et al.,

2003). Because h2-microglobulin expression is not limited

to the olfactory system, this last and striking observation

should, however, be evaluated with care.

It appears that in mice, and likely also in other mammals,

major histocompatibility complex (MHC) genes influence

mating preferences, such that conspecifics possessing

different MHC groups are most attracted to each other

(Jordan and Bruford, 1998). A relation between this

observation and the expression of MHC genes in the

VNO is an attractive possibility. However, a recent report

casts doubts on the role played by the VNO in the MHC

determination of conspecifics, as mice lacking such struc-

ture are still able to identify MHC-determined odor types

(Wysocki et al., in press). This last report reminds us that the

simple idea of a quite strict functional dichotomy between a

main olfactory system limited to odor perception and a

vomeronasal system restricted to pheromones detection is

contradicted by many observations.

Vomeronasal receptors as pheromone receptors

Until very recently, the only direct evidence linking V1r

receptors to pheromone detection was their selective

expression in VSNs. As no functional data for a role of

these seven transmembrane proteins in pheromone response

was available, they were called bcandidate pheromone

receptorsQ. An initial report pointing to a potential chemo-

sensory role played by a V1r receptor indicated that a

chimeric V1r receptor (a fusion between a portion of a V1r

and influenza hemagglutinin), expressed at nonphysiolog-

ical levels, in vitro, could provide a response to urine

(Hagino-Yamagishi et al., 2001). Two reports then allowed

the removal of the Tcandidater qualifier used to refer to V1r

pheromone receptors.

The first approach took advantage of the family-specific

organization in clusters of V1rs. A genomic region of about

600 kilobases, containing almost all members of the V1ra

and V1rb families (representing 12% of the V1r repertoire),

was deleted by chromosome engineering in the mouse (Del

Punta et al., 2002a). The behavior of these animals was

studied in detail. The resulting mice appeared normal and

fertile, and performed equally than control animals in

multiple VNO-dependent behaviors, including for males,

female-induced 70-kHz ultrasound vocalizations, and male-

induced aggression. Mutant females exhibited normal

estrous cycling, and an unaltered ability to retrieve their

pups when these latter were dispersed in the cage. However,

two VNO-dependent behaviors were found to be deficient in

the mutant animals. First, lactating females, which should be

naturally aggressive toward intruders invading their nest

area, exhibited a significant reduction of all measures of

such aggressivity (latency to attack, number of attacks,

number of fights, and number of tail rattles). Second, male

sexual behavior toward females (but also toward males) was

decreased in mutants animals; moreover, the gain of sexual

activity, which is usually observed after experience, was

absent in the deletion animals.

In addition to these behavioral observations, electro-

physiological responses of VSNs to three specific pher-

omones were abolished in the mutants. Thus, despite a

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230 227

relatively limited deletion in terms of number of V1r genes

removed, both genders of the mutant mice displayed

deficits in the detection of particular molecules, and very

selectively, an impairment (although partial) of a subset of

VNO-dependent behaviors. These results indicate an

apparent low level of redundancy within the V1r repertoire,

unlike what is believed to be the case within the odorant

receptor repertoire; such functional specialization is con-

sistent with the phylogenetic isolation of the different V1r

families. It should, however, not be assumed that these

results demonstrate that the activation of a given V1r

family corresponds to a specific behavior, because as

previously mentioned, pheromonal signals may consist of

blends of compounds; indeed, the deletion of a part of the

receptor repertoire may lead to an altered representation of

the blend and a corresponding affected behavior.

A second approach, which led to the identification of the

first pheromone–receptor pair in mammals, used mouse

mutant lines with targeted V1r alleles, allowing the visual-

ization of VSNs expressing these alleles. A direct correla-

tion was established between the expression of the

vomeronasal receptor V1rb2 and the response of a VSN

to minute amounts of the mouse pheromonal compound 2-

heptanone; moreover, this response was shown to be

dependent on the expression of the V1rb2 receptor (Boschat

et al., 2002). In the same experiment, 2-heptanone-related

compounds were shown to be unable to trigger a reaction in

V1rb2-expressing sensory neurons, arguing in favor of a

strong selectivity of this receptor, and consistent with

previous observations showing a narrow tuning of VSNs

to single ligands (Zufall et al., 2002).

We thus dispose today of convincing evidence support-

ing a role of V1rs as pheromone receptors.

There is still no strong data supporting the role played

by V2rs as pheromone receptors in mammals. The

evidence indeed mostly relies on the specific expression

of V2rs in VSNs, and on sequence homologies shared

between V2rs and other membrane receptors. However, in

support of a chemosensory role played by members of this

superfamily, a specific V2r-like receptor, receptor 5.24,

expressed in the goldfish olfactory rosette has been shown

to be preferentially tuned (Kd = 100 nM) to recognize

arginine and lysine (Speca et al., 1999), compounds which

for a fish mean feeding cues.

V1rs as axon guidance molecules

Each VSN expressing a given V1r projects a single

axon toward the rostral part of the accessory olfactory

bulb. Gene targeting approaches in the mouse allowed the

visualization of VSNs expressing a specific receptor along

with their axonal projections, and therefore the entire

topographical map in the accessory olfactory bulb corre-

sponding to this receptor. These analyses revealed that (1)

axons from VSNs expressing the same receptor coalesce

into 10–30 spherical neuropil-rich structures, called glo-

meruli, which are usually exclusively innervated by fibers

with the same specificity and (2) that the capacity to

converge and to form these glomeruli depends on the

ability to express the chosen V1r gene (Belluscio et al.,

1999; Rodriguez et al., 1999). Thus, multiple topographical

maps, each corresponding to the projections of VSNs

expressing one of the 150 V1r receptors, are present in the

accessory olfactory bulb, and are dependent on the

expression of the V1rs.

These studies showed that VSN fibers expressing a

truncated V1r gene and unable to express the correspond-

ing receptor no longer converge in the accessory olfactory

bulb, but instead penetrate glomeruli corresponding to

fibers expressing other V1rs. This phenotype could be

explained either by a missing information concerning the

target, and the resulting Vloss of directionJ from the

fibers expressing the deleted allele. Another view would be

to consider the VmistargetedJ fibers as axonal projec-

tions from VSNs expressing a novel and random V1r, as

the result of an inability of the deleted allele to prevent the

expression of other V1rs. This latter hypothesis would be

consistent with a recently identified role played by odorant

receptors (Serizawa et al., 2003). Both explanations

involve the V1r as a main key to the formation of the

bulbar map. This role may be direct (the V1rs could be

expressed in growth cones of VSNs and be directly

involved in sensing guidance cues, but no data supports

this yet), or indirect (the V1rs could be involved in some

dendrite-initiated activity-dependent guidance process for

example). This latter possibility may, however, appear

unlikely because mouse mutants lacking a key element of

the putative V1r-dependent transduction cascade such as

the Trpc2 channel (Liman et al., 1999) exhibit unaltered

vomeronasal projection maps in the accessory bulb

(although a decrease in the number of VSN axonal

projections is observed) (Norlin et al., 2003; Stowers

et al., 2002).

Similar to what is observed for VSNs expressing V1rs,

those expressing V2rs also send axons toward the

accessory bulb; these projections are, however, restricted

to its caudal part (Fig. 1). VSNs expressing the V2r1b

receptor converge toward 6–10 glomeruli, distributed in

globally conserved areas of the accessory olfactory bulb,

and innervated exclusively by neurons expressing V2r1b

(Del Punta et al., 2002b). We still ignore if the expression

of the V2r1b receptor is required for the elaboration of

the olfactory topographical map, but because both odorant

and V1r receptors play such a remarkable role, it would

not be surprising for V2r receptors to similarly instruct

axons.

It has been shown that secondary neurons in the

accessory olfactory bulb, the mitral cells, which send

multiple dendrites toward different glomeruli, project to

glomeruli innervated by axons of sensory neurons express-

ing the same V1r or V2r receptor, thus resolving the

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230228

divergent (or partially convergent) pattern of projections in

the accessory olfactory bulb by dendritic convergence (Del

Punta et al., 2002b). It is not known if the identity of the

V1r or V2r receptor corresponding to the linked glomeruli

is involved in the mechanism leading to this secondary

convergence, but in the light of the instructive role these

receptors play in the formation of the glomeruli, their

potential involvement should not be disregarded.

Possibly related to the axon guidance role of the odorant

receptors, the V1rs expressed in germ cells have been

proposed to play a role in the chemosensory guidance of

sperm (Tatsura et al., 2001). Odorant receptors have

similarly been identified in sperm, and evidence for odorant

receptor-mediated guidance in mature germ cells has been

obtained (Spehr et al., 2003).

One VNO and two vomeronasal receptor families

Multiple molecular and electrophysiological evidences

point to a distinctive role played by VSNs in the basal or

apical compartments of the VNO. These include the

differential expression of the two chemosensory receptor

families and associated molecules, the separate axonal

projections emanating from VSNs expressing these two

receptor types, distinct electrical properties between V1r-

and V2r-expressing VSNs, and also evidences that VSNs

from the apical zone respond to different types of stimuli

than those in the basal zone (Fieni et al., 2003; Krieger et

al., 1999; Kumar et al., 1999; Sugai et al., 2000).

Moreover, mutant mice with deficiencies affecting differ-

ent portions of the VSN pool exhibit quite specific

behavioral alterations, although some of them overlap.

These include impairment of male–male aggressivity (h2-microglobulin null) (Loconto et al., 2003), and decreased

attacks toward intruders by lactating females (V1ra/b

deletion and Gai2 mutants) (Del Punta et al., 2002a;

Norlin et al., 2003).

A mouse mutant line lacking the ion channel Trpc2 is of

particular interest. Trpc2 is believed to be part of the

vomeronasal signal transduction cascade, a cascade that is

still not fully understood today. The channel deletion in the

mouse results in VSNs strongly impaired in their ability to

respond to pheromones (Leypold et al., 2002; Stowers et al.,

2002), and thus offers a unique tool to investigate the

behavioral consequences of a genetic lesion of the vomer-

onasal system. Gonadal hormone levels and most olfactory-

mediated behaviors of the mutant mice appear normal,

including, surprisingly, male–female courting and mating.

Even less expected is the behavior of mutant males, which

instead of displaying aggression toward male intruders,

initiate courtship: Males lacking the channel appear to be

unable to discriminate between genders, with no improve-

ment of this deficit after sexual experience. The behavior of

the Trpc2 null animals is not impaired in the sense that the

mutant mice are still able to fight and mate, but these

behaviors occur during inappropriate situations, as a result of

gender blindness.

These results are interesting to be compared with

previous and numerous studies that involve the physical

removal of the VNO in adult rodents, and that show

resulting impairments in aggressive and sexual behaviors

(Labov and Wysocki, 1989; Maruniak et al., 1986; Wysocki

and Lepri, 1991; Wysocki et al., 1983). In these studies, no

gender discrimination deficiency was reported, but such

phenotype may have been overlooked because it was likely

not tested. These apparently conflicting observations may

have other explanations, because both approaches are quite

different. First, in a conventional Trpc2 knockout, mice

should not experience any VNO-perceived stimulus, while

in the approach involving the physical extirpation of the

VNO (and in addition to the traumatic experience that is

difficult to control), experience may already have taken

place at the time of the surgical procedure. Second, it

appears that a residual electrical activity may still be present

in the VNO of the genetically engineered mice (Leypold et

al., 2002), possibly reflecting a Trpc2-independent sensory

population. Third, in the genetically lesioned animals, the

lack of electrical activity in the VNO during embryogenesis

and early postnatal life could impair developmental pro-

cesses, such as the migration of gonadotropin-releasing

hormone neurons to the forebrain via vomeronasal axons.

This veil of uncertainty will be lifted by the generation of

conditional Trpc2 mutant mice, which will allow at will, at

any time during the life of an animal, the genetic ablation of

the function of the VSNs expressing the channel.

Moreover, similar genetic tools will permit to answer the

question of the respective roles played by the V1r- and V2r-

expressing VSN populations. Indeed, selective genetic

ablation of the V1r- or V2r-expressing VSNs (which express

the Gai2 or Goa subunits, respectively) using a conditional

tissue-specific recombinase-mediated knockout strategy is a

quite easy task, and will allow unambiguous attribution of

the respective roles played by the two receptor families in

vomeronasal-dependent behaviors.

In less than 10 years, our molecular understanding of

mammalian pheromone perception has dramatically

changed. Two very large, monoallelically expressed seven

transmembrane receptor gene superfamilies are likely to be

responsible for the detection of pheromones by the

mammalian VNO; their corresponding products play a

surprising dual role as they also appear to be involved in

axonal pathfinding processes in the vomeronasal system.

Novel and major questions arise from these remarkable

capacities. Which mechanism is used by a VSN to choose a

single vomeronasal receptor gene to be expressed, more-

over, from a single allele? How do vomeronasal chemo-

receptors instruct axons to find their targets? Which (if any)

role does the V1r repertoire rapid evolution play in

speciation? We have at our disposal the molecular frame-

work to tackle these novel questions, and their elucidation

will surely bring new surprises, and questions.

I. Rodriguez / Hormones and Behavior 46 (2004) 219–230 229

Acknowledgments

I wish to thank Pierre Vassalli and the members of my

laboratory for helpful comments on the text, and the Swiss

National Science Foundation for its financial support.

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