Chem. Senses 27: 207-213,
2002
© Oxford University Press 2002
A Putative Pheromone Receptor Gene Is Expressed in Two Distinct Olfactory Organs in Goats
1 Department of Ultrastructural Research, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan 2 Laboratory of Veterinary Ethology, Department of Animal Resource Science, University of Tokyo, 1 - 1 - 1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 3 Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan 4 CREST (Core Research for Evolutional Science and Technology)
Correspondence to be sent to: Kimiko Hagino-Yamagishi, Department of Ultrastructural Research, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. e-mail: kyamagis{at}rinshoken.or.jp
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Abstract |
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Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Mammals have two distinct olfactory systems (Eisthen, 1992
). Volatile
odorants are detected by the sensory neurons in the olfactory epithelium (OE),
and these chemical signals are transmitted to the main olfactory bulb (MOB)
(Kosel et al., 1981).
On the other hand, pheromone molecules, which elicit innate reproductive and
social behavior within a species, are detected by the sensory neurons in the
vomeronasal organ (VNO), and pheromonal information is transmitted to the
accessory olfactory bulb (AOB) (Halpern,
1987).
Three families of receptor genes (V1R, V2R and V3R genes) have been
expressed specifically in the vomeronasal neurons
(Dulac and Axel, 1995;
Herrada and Dulac, 1997;
Matsunami and Buck, 1997;
Ryba and Tirindelli, 1997;
Pantages and Dulac, 2000).
Each receptor gene is expressed in small subsets of vomeronasal neurons, but
there is no sequence homology between V1R and V2R genes. V3R genes are
distantly related to V1R genes (Pantages
and Dulac, 2000). These findings suggest that these genes encode
pheromone receptors, although there is no direct evidence that these receptors
recognize pheromone molecules.
Recently, Leinders-Zufall et al.
(Leinders-Zufall et al.,
2000) showed physiological responses in mouse vomeronasal neurons
to substances reported to be pheromones in that species. Their data implied
that the responsive neurons seemed to be mostly expressing V1Rs. However, the
expression of the V1R family genes in VNO has been reported in only the rodent
thus far (Dulac and Axel, 1995;
Saito et al., 1998).
Thus, we attempted to identify the V1R family genes and analyze the expression
of these genes in goat, in which activation of the pheromone-induced
neuroendocrine secretion has been well documented
(Chemineau, 1987). We found
that one of the genes was expressed not only in the sensory neurons of VNO but
also in those of OE, implying that two distinct olfactory systems are involved
in goat pheromone sensing.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Histology
VNOs were dissected from adult goats and mice [Crj: CD-1 (ICR)]. Cryostat sections of 10 µm thickness were stained with Mayer's hematoxylin. To estimate the volumes of VNOs and neuroepithelia in goats and mice, we assumed that the VNOs had a columnar structure. The volumes were calculated as follows: the cross-sectional area x rostral—caudal length of the VNOs. The cross-sectional area was measured using NIH image software (available on the Internet at http://rsb.info.nih.gov/nih-image/ ).
Cloning of goat V1R homologue genes
To isolate goat V1R homologue genes, polymerase chain reaction (PCR) was
performed using degenerate primers derived from the conserved regions of gene
families of rat and mouse V1Rs, with goat genomic DNA as the template
(Dulac and Axel, 1995;
Saito et al., 1998).
The degenerate primers used were S1
(5'-CACIIGAAIYARIYCYRRYAYYTTCA-3'), S2
(5'-CTCARYSYYARAARMTCCTGTTT-3'), AS1
(5'-AWACAAAKAARSTCAYDAGC-3') and AS2
(5'-ACARAARGRCTKAYDGTRGCATA-3'), which corresponded to the amino
acid sequences of H(R/K) (K/N)Q(T/I/A)(R/Q)HL(Q/H), LSP(R/K)(S/K/N)SCL,
LM(S/T)(F/L)FV and YAT(V/L)S(P/L)(F/S)V, respectively.
To obtain the entire coding sequences of goat V1R homologue genes, the
inverse PCR method was used (Triglia
et al., 1988). The specific PCR primers used for the
first PCR were 5'-ACCCAGACCATTCTATTCCTC-3' and
5'-TTCTCAAGAATGTTGTGGGATTA-3' for gV1R1, and
5'-GAAGAAAGGACCACTGAGACC-3' and
5'-GACCACTGAGACCGTCCTGC-3' for gV1R2. PCR primers used for the
second PCR were 5'-ATACCTAAGGAAGTGAATCACG-3' and
5'-GGAACCATTGATGAACATGTTG-3' for gV1R1, and
5'-GGATGCTTTTGGAGAAAGCTT-3' and
5'-TTTGGAGAAAGCTTGGTGCTG-3' for gV1R2. The generated PCR products
were cloned and sequenced.
Southern hybridization of reverse transcription—PCR (RT-PCR) products
Total RNAs were extracted from various tissues using ISOGEN (Nippon gene), and were treated with DNase I. The poly(A)+ RNAs were purified using an Oligotex-MAG mRNA purification kit (Takara). The cDNAs were synthesized from these poly(A)+ RNAs by AMV RT-XL (Takara), and were used as templates for RT—PCR. The specific PCR primers used were 5'-TAATCCACATAGTCATGCCTA-3' and 5'-TTCAGTACTGATTAGCACCA-3' for gV1R1, and 5'-TAATCCACATAGTCATGCTA-3' and 5'-TAATACGACTCACTATAGGG-3' for gV1R2.
To analyze the expression of V1R family genes in mouse VNO and OE,
RT—PCR was performed. The specific primers used were mouse V1R-S primer
(5'-TAGAAGCTCCTGTTTARCAA-3') and mouse V1R-AS primer
(5'-GAAGATGCYGGGYCTGWTT-3'), which corresponded to the amino acid
sequences of RSSCL(A/T) and (K/N)Q(T/A)(Q/R)HL, respectively. The PCR products
were electrophoresed, blotted and hybridized in 4x sodium
citrate/chloride buffer (SSC) containing 50% formamide, 50 mM phosphate buffer
(PB) pH 7.5, 10x Denhardt's solution and 0.1% sodium dodecyl sulfate
(SDS) at 42°C. The filters were washed in 0.2x SSC containing 0.1%
SDS at 65°C. In some cases, the filters were hybridized under relaxed
conditions in 4x SSC containing 25% formamide, 50 mM PB, pH 7.5,
10x Denhardt's solution and 0.1% SDS at 42°C, followed by relaxed
washing with 2x SSC containing 0.1% SDS at 42°C. The probes used for
hybridization were derived from the coding sequences of gV1R1 and gV1R2 genes,
and the entire coding sequence of the mouse V1R gene (clone 2-4)
(Hagino-Yamagishi et al.,
2001), respectively.
The specific primers used for the detection of the olfactory marker protein (OMP) gene were 5'-ACRGGCACCTCGCAGAACTG-3' and 5'-CCAAAGGTGAYGAGGAARTACAT-3', and those for the detection of ß-actin were 5'-CAATGGATCCGGTATGTG-3' and 5'-CGTTGTAGAAGGTGTGATGCC-3'.
In situ hybridization
VNOs and OEs of male goats were collected and fixed in Bouine's solution
(Sigma). The OE was decalcified in 10% ethylenediamine tetraacetic acid (EDTA)
for 2 days. These fixed tissues were processed through a standard paraffin
embedding protocol. Sections were cut into 15 µm thickness and attached to
silanized slides. A riboprobe was prepared and labeled with digoxigenin as
described previously (Schaeren-Wiemers and
Gerfin-Moser, 1993). The 710 bp DNA fragment of gV1R1 was used for
the preparation of the riboprobe. In situ hybridization procedures
were essentially the same as those described previously
(Schaeren-Wiemers and Gerfin-Moser,
1993). Hybridization was carried out in a hybridization solution
containing 50% formaldehyde, 10 mM Tris—HCl pH 7.6, 200 µg/ml tRNA,
1x Denhardt's solution, 600 mM NaCl, 0.25% SDS and 1 mM EDTA pH 8.0 at
55°C overnight. Hybridized RNAs were detected with an alkaline
phosphatase-conjugated anti-DIG Fab fragment antibody (Roche), and signals
were visualized using 4-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl-phosphate (Roche) as chromogenic substrates.
Southern hybridization of goat genomic DNA
Ten micrograms of goat genomic DNA, which was digested with EcoRI, BamHI and HindIII, was blotted and hybridized with probes specific for gV1R1 and gV1R2 genes. Hybridization was carried out under the relaxed conditions described above.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Goat vomeronasal organ
Goat VNO is a columnar structure located at the base of the nasal septum.
One feature of goat VNO is that the neuroepithelial layer (indicated by `n' in
the figures) is thinner than that in mouse VNO
(Figure 1A,B). The ratio of the
volume of the neuroepithelial layer to that of the whole VNO was 
2% in
the goat and 25% in the mouse. These results show that the ratio of the volume
of the neuroepithelial layer to that of the whole VNO in the goat is 8%
of that in the mouse.
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Cloning of goat V1R genes
We identified two V1R homologue genes (gV1R1 and gV1R2 genes) from goat
genomic DNA using degenerate primers derived from highly conserved regions in
rat and mouse V1R family genes (Dulac and
Axel, 1995; Saito et
al., 1998). The deduced amino acid sequences of the gV1R gene
show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R
family genes, respectively (Dulac and Axel,
1995; Pantages and Dulac,
2000), suggesting that the newly isolated goat VNO receptor genes
are members of the V1R gene family rather than the V3R gene family. The gV1R1
gene has an open reading frame that encodes a polypeptide of 309 amino acids
(Figure 2A). On the other hand,
the gV1R2 gene has multiple frameshifts and stop codons in its coding sequence
(Xs in Figure 2A), thereby
indicating that gV1R2 does not function as a receptor protein.
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Hybridization analysis of goat genomic DNA shows that even under relaxed
conditions, only a single band can be detected using probes specific for gV1R1
and gV1R2 genes (Figure 2B).
This result is inconsistent with the previous notion that multiple bands can
be detected when genomic DNAs from the rodent are hybridized with probes
specific for V1R family genes under relaxed conditions
(Dulac and Axel, 1995;
Saito et al., 1998).
Our data imply that the size of the V1R gene family in the goat is relatively
smaller than that in the rodent.
Expression of gV1R1 and gV1R2 genes
As our first trial, we examined the expression of V1R homologue genes in goat VNO by Northern hybridization analysis, but could not obtain positive signals (data not shown). We assumed that the relative amount of gV1R-expressing cells in the entire VNO preparation was too low to allow detection of gV1R transcripts, because the ratio of the volume of the neuroepithelial layer to that of the whole VNO in the goat was only 8% of that in the mouse (Figure 1). Thus, we adopted a more sensitive method, the Southern hybridization analysis of RT-PCR products, and found that the gV1R1 gene was expressed not only in VNO and AOB, but also in OE and MOB (Figure 3A, top panel). It should be noted that we obtained OE RNA from the lateral side of OE to eliminate possible contamination by vomeronasal neurons that project their axons along the nasal septum to the AOB. The gV1R1 gene was also expressed in the testis but not in other tissues (Figure 3A, top panel). The same results were obtained from four independent experiments (one male and three female goats) (data not shown).
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To examine whether the expression of putative pheromone receptors in OE is
a common feature in mammals, we analyzed the expression of V1R genes in mouse
OE by Southern hybridization analysis of RT-PCR products. The lateral side of
OE was carefully excised from 10 male and 10 female mice, and mRNA was
purified from these OE. Using this mRNA as a template, RT-PCR was performed
using specific primers derived from the conserved region of V1R family genes.
Note that we detected at least 13 V1R family genes in the mouse using these
primers (data not shown). The PCR products were blotted and hybridized with
the probe that encoded the entire coding region of mouse V1R clone 2-4
(Hagino-Yamagishi et al.,
2001) under relaxed conditions. As shown in
Figure 3B, V1R family genes
were not expressed in OE in both male and female mice, although they were
clearly expressed in VNOs. These results strongly suggest that mouse V1R genes
are expressed only in VNO and not in OE, and the expression of the putative
pheromone receptor gene in OE is characteristic of the goat and not of the
mouse.
Then, the type of gV1R1-expressing cells in OE and VNO was determined by in situ hybridization analysis (Figure 4). The gV1R1-expressing cells were detected in the neuroepithelial layer (indicated by `n') in both VNO and OE (Figure 4A,C) using an anti-sense riboprobe, but no signal was detected using the sense probe (Figure 4B,D). These results strongly suggest that the gV1R1-expressing cells in the VNO and OE are vomeronasal and olfactory sensory neurons, respectively.
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In addition, the distribution of the gV1R1-expressing cells throughout the neuroepithelial layer of VNO, and a distinct spatial segregated pattern of the gV1R1-expressing cells, which was clearly detected in rodents, were not observed (data not shown).
The gV1R2 gene was not expressed in any tissues except the testis, suggesting that it is not expressed and does not function in the vomeronasal system (Figure 3A, bottom panel).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We identified homologue genes of the rodent putative pheromone receptor in goat (gV1Rs), and found that the gV1R1 transcripts were expressed not only in sensory neurons of goat VNO but also in those of OE (Figure 3A, top panel; Figure 4A, C). The expression of the same receptor in the VNO and OE implied that the goat recognizes and processes ligand information through the two distinct olfactory systems.
The expression of the V1R homologue gene in OE was recently reported in
human (Rodriguez et al.,
2000), despite the fact that adult human does not have a
functional vomeronasal system similar to that in rodents and even-toed
ungulates (Stensaas et al.,
1991). Thus, the expression of V1R homologue genes in OE is not
restricted in even-toed ungulates. In addition, previous reports showed that
the rabbit and the pig with surgically removed or impaired VNOs still
exhibited pheromone-induced stereotype behavior such as nipple-search behavior
or reproductive behavior (Hudson and
Distel, 1986; Dorries et
al., 1997), implying that pheromones could be detected not
only by VNO but also by OE in these animals. These data support the notion
that pheromone detection by the main olfactory system may play an important
part in many mammalian species. The gene expression of the putative pheromone
receptor in sensory neurons of goat VNO and OE shows the possible involvement
of the two distinct sensory organs in mammalian pheromone detection.
Morphological observation of goat VNO revealed that the ratio of the volume of the neuroepithelial layer to that of the whole VNO in the goat is only 8% of that in the mouse (Figure 1), and the size of goat V1R gene family seems to be smaller than that of rat and mouse V1R gene families (Figure 2B). In addition, we have cloned eight genes of goat V2R homologues thus far, and found that these genes have multiple frameshifts and/or termination codons within their coding sequences (data not shown). Thus, the majority of goat V2R gene products may not function as receptors. Taken together, it seems that the ability of pheromone detection by VNO is more inefficient in goats than in rodents. The expression of the putative pheromone receptor gene in goat OE may compensate for the pheromone sensing function by VNO.
In rodents, olfactory neurons project their axons to MOB
(Kosel et al., 1981),
whereas vomeronasal neurons project their axons to AOB
(Halpern, 1987). These two
olfactory systems seem to be clearly separated from each other. However in the
goat, the gV1R1 transcripts are expressed not only in AOB but also in MOB
(Figure 3A, top panel).
Although we cannot completely rule out the possibility of trace amounts of
contamination of AOB in the MOB sample, our results showed the possibility
that the axons of gV1R1-expressing sensory neurons of VNO and/or OE project to
MOB. Another possibility is that gV1R1 transcripts were expressed in cells of
AOB/MOB themselves. To examine these possibilities, determination of the cell
type that expresses the gV1R1 transcripts in the olfactory bulb is
required.
Both gV1R1 and gV1R2 transcripts are expressed in the testis
(Figure 3A), although gV1R2 is
not a functional receptor (Figure
2A). Mouse V1Rs are also expressed in both VNO and the testis, and
it has been argued that they are involved in sperm chemotaxis
(Tatsura et al.,
2001). However, the biological significance of the expression
remains unclear.
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Acknowledgments |
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We would like to thank Dr Toru Sawazaki for providing Shiba goats from the closed colony of the University of Tokyo. We would also like to thank Dr Eri Iwata and Mr. Soichiro Matsuse for their help in preparing goat tissues. This study was supported by CREST of the Japan Science and Technology Corporation.
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Accepted November 8, 2001
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