Physiology & Behavior 77 (2002) 167 – 176

Attractive properties of sexual pheromones in mice:

Innate or learned?
Jose Moncho-Bogania,1, Enrique Lanuzab,1, Adoración Hernándeza,
Amparo Novejarquea, Fernando Martı́nez-Garcı́aa,*
a
Departament de Biologia Animal (Unitat de Morfologia Microscòpica), Facultat de Ciències Biològiques, Universitat de València, C. Dr. Moliner 50,
ES-46100 Burjassot, València, Spain
b
Departament de Biologia Cel
lular, Facultat de Ciències Biològiques, Universitat de València, C. Dr. Moliner 50, ES-46100 Burjassot, València, Spain
Received 8 March 2002; received in revised form 22 April 2002; accepted 26 June 2002

Abstract

It is generally assumed that chemical signals (sexual pheromones) constitute the primary stimulus for sexual attraction in many mammals.
However, it is unclear whether these pheromones are volatile or nonvolatile and which sensory systems are involved in their detection
(vomeronasal and/or olfactory). Moreover, it has been demonstrated that experience influences the behavioral response to sexual pheromones
and the sensory systems implicated. In order to clarify this issue, the attractive properties of volatile and nonvolatile components of the male-
soiled bedding have been analyzed in female mice that had no previous experience with adult male-derived chemical signals (chemically
naı̈ve females) using two-choice preference tests. The results indicate that some nonvolatile male-derived substances exert an innate attraction
to females, but volatiles derived from male-soiled bedding do not attract chemically naı̈ve females. Therefore, the primary attractive sexual
pheromone includes a nonvolatile compound (e.g. major urinary proteins, MUPs). On the other hand, male-derived volatiles become
attractive to females because of repeated exposure to male-soiled bedding. This represents a Pavlovian-like associative learning in which
previously neutral volatiles (very likely odorants) acquire attractive properties by association with the nonvolatile, innately attractive
pheromone(s). These findings indicate that not only the sexual but also the ‘chemical’ experience (previous experience with sexual
pheromones) has to be taken into account to interpret the role of chemicals as releaser or primer pheromones. The sensory systems involved
in the detection of these stimuli and the neural basis of the odor – pheromone association are discussed.
D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Pheromones; Vomeronasal system; Olfactory system; Sexual behavior; Emotional learning

1. Introduction the amygdala [4,5]. Despite these solid anatomical data,

Vertebrates possess two nasal chemosensory organs, the
olfactory and vomeronasal epithelia. The olfactory epithe-
lium detects a myriad of air-borne (volatile) substances [1],
whereas the vomeronasal organ (VNO) seems primarily
specialized in detecting pheromones, which are important
for intraspecies communication [2,3]. Olfactory and vomer-
onasal stimuli are processed through two parallel neuro-
anatomical pathways arising from the main and accessory
olfactory bulbs, respectively, which converge at the level of
* Corresponding author. Tel.: +34-96-398-3225; fax: +34-96-386-
4372.
E-mail address: fernando.mtnez-garcia@uv.es (F. Martı́nez-Garcı́a).
1
These authors contributed equally to this work.
current ideas on the functional neuroanatomy of the amyg-
dala largely neglect its functions in chemosensory process-
ing [6]. Indeed, most of the functional studies on the
amygdala are focused on its well-known role in the emo-
tional evaluation of incoming stimuli by means of their
association with previously fear-arousing (aversive) or
rewarding (attractive) stimuli, thus resulting in aversive or
appetitive emotional learning [7 –9]. A similar role of the
amygdala in emotional evaluation of chemosensory stimuli
is suggested by studies of the neuronal response to olfactory
stimuli by the basolateral amygdala [10,11].
As pointed out by Beauchamp et al. [12,13], at least some
VNO-detected stimuli appear intrinsically rewarding [2,14].
In fact, most of the stimuli supposedly detected by the VNO,
such as pheromones or chemical signals from usual predators

0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 8 4 2 - 9
168 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
[15] or preys [16], are likely to have an intrinsic emotional
value. Thus, a possible role of the amygdala might be to allow
emotional tagging of odors by means of their association with
pheromones or other VNO-dependent stimuli with an innate
emotional significance.
Our group has started a research project to test the
involvement of the amygdala in these kinds of associations.
As a first step towards this goal, we have tested whether
sexual pheromones are intrinsically attractive to adult mice.
To do so, we have studied if ‘chemically naı̈ve’ (without
previous experience with chemical signals of adult conspe-
cifics of the other gender), sexually mature mice show any
attraction to soiled bedding from adults of the other gender.
Since males are, unavoidably, in contact with an adult
female (their mother), we decided to use females as experi-
mental animals.
Male-soiled bedding contains both volatile (putative
odorants) and nonvolatile compounds (e.g. major urinary
proteins, MUPs [17]) that are known to be important in
intraspecific communication [18 –23], whose role in the
attractive properties of the male-soiled bedding are not clear
yet. To tackle this issue, we have analyzed the chemo-
investigatory behavior of chemically naı̈ve female mice
towards male-soiled bedding under two different experi-
mental conditions: (a) when contact with the male-soiled
bedding was allowed, so that the female got access to every
component of the soiled bedding, and (b) when females
were allowed only to investigate volatile compounds eman-
ating from the male-soiled bedding.
Finally, we have studied the influence that repeated
experience with male-soiled bedding has on the behavioral
response that females show towards it. In particular, we test
whether, as suggested by Beauchamp et al. [12,13], odorants
(probably detected by the main olfactory system) can
acquired pheromonal properties by association with non-
volatile pheromones (probably detected by the accessory
olfactory system).
2. Materials and methods
2.1. Animals
The present studies were performed using female mice of
the CD-1 strain (Harlam, Barcelona, Spain) that were treated
throughout according to the EEC guidelines for European
Communities Council Directives of 24th November 1986
(86/609/EEC). To ensure that the females used in these
experiments are ‘chemically naı̈ve,’ that is to say, they had
never been exposed to chemical signals from sexually
mature males, pregnant females were housed in a clean
room without males. Nineteen days after delivery (early
before puberty), pups were sexed, males were removed, and
their female siblings were brought to a clean room in
complete absence of adult male chemical signals where
they were kept in groups of five per cage until they were
used for the behavioral experiments, at the age of 9 weeks.
Thirty females reared in these conditions were used.
2.2. Preference tests
Before the start of the experiments, animals were ha-
bituated to handling and to the test cage for 10 min daily
during 1 week, at the same time of the day in which the
preference tests were going to be performed (between 9 and
13 h). Preference tests were undertaken in plastic cages of
25  50  30 cm in size. Test cages were divided into two
compartments of equal size (left side or Compartment 1; right
side or Compartment 2) communicated by a door of 6.5  6.5
cm through which the animals could move freely. Each
compartment contained 40 g of either male- or female-soiled
bedding, depending on the experiment (see below). The
female-soiled bedding was collected from several housing
cages of the experimental females after 1 week of use. The
male-soiled bedding was collected from several cages con-
taining two to five adult males for a week. To ensure a homo-
geneous composition throughout the experiments, male-
soiled bedding from several cages was thoroughly mixed
and stored in the freezer until the day of the experiment.
For the preference tests, female mice were released in
Compartment 1 (left side) of the test cage, the experimenter
left the room, and the behavior was videotaped for 5
(Experiments 1 and 3) or 10 min (Experiment 2). The time
the animal’s snout was in Compartment 2 (right side of the
cage) during the test was measured by a person who was
blind to the experimental conditions. Since the test cage has
only two compartments (1 and 2), the time an animal spends
in a given compartment equals the total time minus the time
spent in the other compartment. Results are presented in
seconds, as the average of the time spent in a given
compartment ± the standard error (S.E.).
2.2.1. Experiment 1
Analysis of female preference for male-soiled bedding
with direct access to the bedding. A group of chemically
naı̈ve females (n = 10) was used in this experiment. The
estrous status of these females was assessed by means of a
standard vaginal cytology just before the experiment.
Females were used when leukocytes and abundant nucleated
epithelial cells, but no cornified cells, were observed.
Therefore, the females used in this experiment were not in
estrus, but very likely in diestrus.
These females were first tested in the control condition
(female- vs. female-soiled bedding preference test; F/F test).
The next day, the same females were tested in the female-
vs. male-soiled bedding preference test (F/M test). For this
test, in five randomly chosen females, the male-soiled
bedding was located in Compartment 1, whereas in the
remaining ones it was placed in Compartment 2. This
allowed discarding any influence of the compartment in
which the male-soiled bedding was located on the pref-
erence shown for male- vs. female-soiled bedding (see
 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
Statistical analysis). Consequently, for Experiments 2 and 3,
the male-soiled bedding was placed in Compartment 2 in all
the female- vs. male-soiled bedding preference tests.
2.2.2. Experiment 2
Analysis of the time course of the female preference for
male-soiled bedding. In this experiment, a group of chem-
ically naı̈ve females (n = 10) was tested in the control
condition (F/F test) during 10 min, and the next day the
same females were tested in the female- vs. male-soiled
bedding preference test (F/M test) during 10 min. The aim
of this experiment was to study the possible variations with
time of the preference of females for male-soiled bedding
(as observed in Experiment 1). More specifically, we studied
if the response of females to male-soiled bedding shows
habituation.
2.2.3. Experiment 3
Analysis of the female preference for male-soiled bed-
ding without direct access to the bedding before and after
experience with adult male chemical signals. In this experi-
ment two different kinds of tests were performed. In some
cases, the animals were allowed to contact the bedding, thus
having access to both volatile and nonvolatile components
contained in it. In other cases, a plastic platform with evenly
distributed (every 3.5 cm) small holes (6 mm in diameter)
separated the animals from the soiled bedding some 4.5 cm,
so that animals had access only to air-borne volatile sub-
stances emanating from the soiled bedding.
As outlined in Fig. 1, Experiment 3 lasted 9 days. On
Day 1, a group of chemically naı̈ve females (n = 10) was
tested in the control situation (female- vs. female-soiled
bedding) but on a platform that prevented direct access to
the bedding (F/Fp test). On Day 2, the same females were
Fig. 1. Outline of the design of Experiment 3.
169
tested in the control situation (F/F) with direct access to the
bedding. On Day 3, females were tested in the female- vs.
male-soiled bedding preference test on the platform, there-
fore without direct access to the bedding (F/Mp test). On
Day 4, the females were tested in the female- vs. male-soiled
bedding preference test with direct access to the bedding (F/
M test). During the next 4 days, the females were exposed
daily for 10 min to male-soiled bedding in a regular housing
cage (‘experience’). This exposure took place in a different
room from the testing room, to avoid any contextual
association. On Day 9, the females were tested again in
the female- vs. male-soiled bedding preference test on the
platform, to observe the possible effects of experience on
that test (F/Mpe).
2.3. Statistical analysis
Data derived from all the tests were statistically analyzed
using the SPSS software package (version 10.0). For
Experiment 1, a Student’s t-test for independent samples
reveals that the compartment in which the male-soiled
bedding was placed has no effect on the time females spent
in it (male-soiled bedding in Compartment 1, n = 5,
185.8 ± 4.2 s; male-soiled bedding in Compartment 2,
n = 5, 184.6 ± 3.8 s, t = 0.142, P > .45). Therefore, the data
of all 10 females were pooled and used to compare the time
spent in the compartment containing the male-soiled bed-
ding (either 1 or 2) by the females in the F/M test with the
time spent in the same compartment in the control situation
(F/F test), using a Student’s t-test for paired samples.
The data obtained from the 10 min measured in Experi-
ment 2 were divided into two 5-min periods (0 –5 min and
5 –10 min). The mean times the females spent in Compart-
ment 2 during both periods were compared using a standard
170 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176

analysis of variance (ANOVA). In our design, we have
repeated measures for each animal under two within-subject
factors, namely absence (F/F) and presence (F/M) of male-
soiled bedding in Compartment 2.
Concerning Experiment 3, a Kolmogorov – Smirnov test
of normality with Lilliefors’ correction applied to the pooled
data of all the preference tests of this experiment showed
that the time spent by the females in Compartment 2 follows
a normal distribution. Therefore, a one-way ANOVA was
applied to test the influence of the test (fixed factor), the
animal (random factor), and the interaction between test and
animal on the time spent in Compartment 2 by the females.
A post hoc Student–Newman –Keuls test allowed further
assessment of the presence of heterogeneities and homoge-
neities in the data derived from the five tests under scrutiny.
3. Results
3.1. Experiment 1
Chemically naı̈ve females showed a significant pref-
erence (t = 6.366, P < .001) for the compartment with
male-soiled bedding as compared with controls (Fig. 2).
Thus, in the F/M test, females spent nearly 62% of the
duration of the test in the compartment having male-soiled
bedding (185.2 ± 7.4 s), whereas in the control situation (F/F
test), females spent about half of the time in the corres-
ponding compartment (150.8 ± 4.2 s).
3.2. Experiment 2
This experiment was designed to test whether or not the
attraction that chemically naı̈ve females show towards male-
soiled bedding (see Experiment 1) undergoes habituation.
The results of the ANOVA analysis indicate that only the
experimental condition (presence or absence of male-soiled
bedding) has a significant influence on the time the females
spent in Compartment 2 [ F(1,9) = 9.096, P=.015]. In fact,
neither the period of time [ F(1,9) = 0.522, P=.488] nor the
Experimental Condition  Period of Time interaction
[ F(1,9) = 0.05, P=.946] showed significant influence on
the behavior observed. These data indicate that the pref-
erence for the compartment with the male-soiled bedding
does not change with time so that there is not habituation in
the period of time investigated.

Fig. 2. Experiment 1: Female mice that have never been exposed to adult male chemical signals show a strong preference for the male-soiled bedding. Bars
represent the average time ( ± S.E.) spent by the females in the compartment with male-soiled bedding (either Compartment 1 or 2; see Methods) during the
female- vs. male-soiled bedding preference test (F/M) and the time spent in the same compartment in the control condition (F/F: female-soiled bedding in both
compartments). * P < .01 resulting from the application of a Student’s t-test for paired samples.
 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176 171

Fig. 3. Experiment 3: The preference that female mice show for volatiles emanating from the male-soiled bedding is dependent on previous experience with
male sexual pheromones. Bars represent the time spent in Compartment 2 by the same 10 females in five consecutive tests. F/Fp: Control (female- vs. female-
soiled bedding) with a platform interposed between the animal and the bedding. F/F: Control test with female-soiled bedding in both compartments. F/Mp:
Female- vs. male-soiled bedding preference test in which a platform separated the animals from the bedding. F/M: Female- vs. male-soiled bedding preference
test with direct access of the animals to the bedding. F/Mpe: Female- vs. male-soiled bedding preference test with the platform interposed after repeated
exposure of the females to male-soiled bedding (‘experience’).

3.3. Experiment 3 tests) and the F/Mp test. In their first exposure to volatiles
emanating from the male-soiled bedding, chemically naı̈ve
The third experiment was designed to address two females do not show any preference for these volatiles as
questions. Firstly, to discriminate whether the preference compared with the control situation. However, females with
for the male bedding obtained in Experiment 1 is due to experience (repeated exposure to male-soiled bedding
volatile or to nonvolatile chemicals present in the bedding. allowing a direct contact with it) show a response to the
Secondly, the results of this experiment allow studying the volatiles similar to the one they show if direct contact with
effects of previous experience with adult male-derived the bedding is allowed. Therefore, experience changes the
chemicals on the preference of females to volatiles from response to male-derived volatiles.
adult male-soiled bedding.
Fig. 3 shows the time spent in Compartment 2 by the
females in the five consecutive tests that constitute this Table 1
experiment: F/Fp, F/F, F/Mp, F/M, and F/Mpe. The Summary (as obtained with SPSS software) of the results of the Student –
ANOVA indicates a significant effect of the factors ‘test’ Newman – Keuls post hoc analysis of the data from Experiment 3 (common
[ F(4,36) = 7.644, P < .001] and ‘animal’ [ F(9,36) = 2.485, level of significance a=.05)
P=.025], as well as of their interaction [ F(1,9) = 2498.991, Test n Subgroup
P < .001] on the time females spent in Compartment 2. 1 2
A post hoc Student – Newman– Keuls test was applied to F/Fp 10 145.00
explore the differences between tests, with a common level F/F 10 147.80
of significance (a=.05). The results indicate that there are F/Mp 10 151.80
two sets of homogeneous means (see Table 1). On the one F/Mpe 10 167.80
F/M 10 172.30
hand, there are no significant differences between the F/M Significance .533 .480
and the F/Mpe tests or among the F/F, F/Fp, and F/Mp tests.
The results indicate that there are two sets of homogeneous means
On the other hand, the females spent significantly more time (Subgroups 1 and 2). Tests are ordered according to their average. The
in Compartment 2 in the F/M and F/Mpe tests than in the significance refers to the comparison between the two most distant means
two control tests (with and without platform; F/F and F/Fp within the subgroup.
172 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
4. Discussion
Our results indicate that some of the substances con-
tained in the male-soiled bedding are attractive to females,
since they spend more time investigating the compartment
of the test cage containing male-soiled bedding than the one
that contains female-soiled bedding. These attractive prop-
erties of the male-soiled bedding to females are not learned,
since chemically naı̈ve females show preference for the
male-soiled bedding in their first exposure to it. In fact,
the chemically naı̈ve females used in this work had been in
contact with their prepubertal male siblings up to weaning,
at 19 days of age. Puberty induces important changes in the
composition of the male mice chemical signals [24], so that
prepubertal male-derived chemical signals do not elicit
neuroendocrine responses (Vanderbergh effect [25]). More-
over, Hayashi and Kimura [26] demonstrated that females
preferred the odor of normal males to odors of castrated
males whether or not they were reared with their litter
brothers before weaning. Therefore, it is very unlikely that
chemosensory experience of the females before weaning
accounts for the preference of adult females for adult male
chemical signals, so that this preference seems to be innate.
This preference of chemically naı̈ve females for the male-
soiled bedding is not just due to novelty but reflects actual
attractive properties of some of the substances contained in
the male-soiled bedding. This conclusion is based on two
observations. First, the preference for the male-soiled bed-
ding does not show habituation in a 10-min period (Experi-
ment 2) as would be expected if female mice were just
investigating a new stimulus. Second, females do not show
preference for the volatiles that originated from the male-
soiled bedding the first time they are exposed to them, when
a novelty effect is to be expected, but are attracted to the
volatiles after experience, when the stimuli do not represent
a novelty anymore (Experiment 3). In addition, the pref-
erence of female mice for male urine has been demonstrated
in a number of previous works in different laboratories
using experienced animals [19,26,27], in which novelty
effect is not present.
An important point of our results is that a platform
separating the females 4.5 cm from the bedding suppresses
the preference for the male-soiled bedding in chemically
naı̈ve animals (Fig. 2). This indicates that just a few
centimeters away from the bedding, the concentration of
the pheromone(s) responsible for the attraction is not high
enough as to provoke attraction. Therefore, our results
strongly suggest that male mice produce some nonvolatile
sexual pheromone(s) that are innately attractive to females.
Another important conclusion that can be inferred from
our results is that the experience with male chemical signals
changes the way in which females detect and respond to
sexual pheromones. Apparently, volatile substances eman-
ating from the male-soiled bedding that do not attract
chemically naı̈ve females become attractive to the same
animals after repeated contact with male-soiled bedding.
The most likely explanation for this change in the response
to the volatiles is, in our view, that this is a case of simple
emotional learning in which a previously neutral stimulus
(volatiles derived from male’s excretions and/or secretions)
becomes attractive by association with an innately attractive
one (some nonvolatile components of the soiled bedding).
This interpretation is in agreement with the hypothesis of
the reinforcing value of urine-contained nonvolatile pher-
omones previously suggested to explain the extinction of the
response of male guinea pigs to female urine odor following
VNO removal [12,13] or after preventing access to the urine
nonvolatiles by placing it under a wire mesh screen [28].
Alternative explanations, such as a preference for the urine
volatiles developed after the first presentation (an effect
similar to the mere exposure effect), are extremely unlikely
because in the F/Mpe test, the females are making a choice
between a compartment with female bedding (which is
familiar and probably makes them feel comfortable and
safe) and male-soiled bedding.
4.1. Identity of the primary attractive male sexual
pheromone
The most likely candidates for the nonvolatile, innately
attractive pheromones contained in male-soiled bedding are
the MUPs or complexes of MUPs and urine-borne volatiles
(see below). Mucignat-Caretta et al. [29] demonstrated that
the MUPs were attractive to adult females and aversive for
prepubertal ones, thus reproducing the natural response of
females to male chemical signals [27]. Like other lipocalins,
MUPs bind small odorants with a medium affinity in the
104- to 105-M 1 range [30] and in fact, odorants and MUPs
co-purify [31]. For this reason, MUPs are usually seen as a
simple reservoir for storage and slow release of volatiles
from urine [17], and Mucignat-Caretta et al. [29] attribute
the attractive properties of MUPs to their associated odor-
ants. However, the content of MUPs in male rodents is not
only quantitatively but also qualitatively different to that of
females [32], and MUPs of the wild house mice exhibit a
very high level of polymorphism [33]. Such a molecular
diversity is not expected for a simple pheromone carrier
[23].
This view contradicts previous observations that attribute
this attraction to urine-borne volatiles such as (E)-b-farne-
sene and (E,E)-a-farnesene (farnesenes) [19,34], 2,3-dehy-
dro-exo-brevicomin (brevicomin), and 2-sec-butyl-4,5-
dihydro-thiazole (thiazole) [18]. If these highly volatile
substances were innately attractive, our chemically naı̈ve
females would have shown preference for the male-soiled
bedding even through the platform. Using a two-choice test,
Jemiolo et al. [19] reported that sexually experienced
females preferred synthetic farnesenes (against water) at
concentrations similar to those in male urine, and these data
led the authors to conclude that farnesenes are the compo-
nents of male urine that are attractive to female mice.
However, when tested in the same way, virgin females only
 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
showed preferences for farnesenes at concentrations much
higher (50 – 100 times) than their content in adult male urine
but not at natural concentrations. This indicates that, accord-
ing to our results, farnesenes are not the primary attractive
male pheromones but acquire their attractiveness after
sexual (but maybe also chemical) experience. This view is
supported by previous observations [35] indicating that the
preference of adult female mice for preputial-derived odor-
ants (such as farnesenes) is the result of an imprinting-like
learning during the first weeks of their life.
The attractiveness of brevicomin and thiazole (already
present in bladder urine [36]) was studied also by Jemiolo et
al. [18] in virgin females. Their results indicate that both
compounds spiked together onto urine of castrated male
mice (at natural concentrations, about 1.3 ppm) mimic the
attractive properties of urine of intact males. In fact, virgin
females preferred to sniff urine of castrated males supple-
mented with the mixture of brevicomin and thiazole to urine
of castrated males alone. However, these results are in
conflict with those of the above-cited paper by the same
group [19] indicating that, in the same experimental con-
ditions, virgin females do not prefer bladder urine from
intact males to water (see Test 3 in their Table 1). Our
findings might help to explain these conflicting results by
considering the chemical experience of the females. Appa-
rently, the virgin females employed by Jemiolo et al. [18]
were not chemically naı̈ve so that their responses to brevi-
comin and thiazole might be a consequence of their previous
experience. On the contrary, the virgin female mice used by
Jemiolo et al. [19] to test attraction to farnesenes seem to
have not been exposed to male chemical signals. This fact
would explain both their inability to respond to farnesenes at
natural concentrations and their lack of preference for
bladder male urine against water in a situation in which
contact with urine is not allowed.
These data suggest that the primary (innate) attractive
male sexual pheromone might be a complex of urine-borne
volatiles and nonvolatiles, in line with recent data obtained
in rats [37]. This would explain why chemically naı̈ve
animals respond to the volatiles contained in urine when
presented at very high concentrations (50 – 100 times their
content in normal male urine [19]) but not at natural
concentrations (our results [19]). However, they respond
to the volatiles at natural concentrations if access to the
nonvolatiles contained in urine is allowed (our results).
Evidence supporting a cooperative action of volatiles and
nonvolatile chemicals as releaser pheromones comes from
recent experiments [22], in which the behavior of scent
marking in male mice towards fractions of conspecific male
mice urine (countermarking) were studied. Their results
demonstrate that countermarking is stimulated by the frac-
tion containing proteins associated to volatiles (mainly
brevicomin and thiazole) but not by fractions containing
only volatiles. In fact, it has been recently demonstrated [23]
that MUPs play a role in individual recognition among male
mice and the same can be true for females.
173
Apparently, all the responses elicited by urine-borne
proteins are dependent on the VNO [17], thus fitting the
generally accepted view that the VNO, which is isolated
from the airstream and requires active mechanisms for
introduction of the stimulus, is harnessed to detect non-
volatile, water-soluble molecules [3,38]. In fact, diluted
urine specifically stimulates VNO neurons and some of
the VNO neurons respond in a differential way to either
male or female urine [39]. Moreover, the use of both
electrophysiological methods and optical imaging [40,41]
has demonstrated that VNO neurons respond in a very
sensitive and specific way to volatile substances contained
in male urine such as farnesenes, heptanones, brevicomin,
dimethylpirazine, and thiazole.
Although the response of VNO neurons to MUPs has
never been analyzed, the expression of the immediate early
genes (c-fos and egr-1) in the accessory olfactory bulb
(which is the target for VNO neurons) strongly suggests
that MUPs detected at the level of the VNO are important
for the biological response(s) of females to male urine. First,
a mixture of brevicomin and thiazole combined with MUPs
is extremely effective in inducing c-fos mRNA expression in
the VNO whereas the volatiles alone are not [42]. Second,
both male-derived urine-borne volatiles and stripped MUPs
elicit the expression of egr-1 in mitral cells of the accessory
olfactory bulb, but the population of mitral cells expressing
egr-1 in response to MUPs was different to the one elicited
by the volatiles brevicomin and thiazole [43].
Although the role of the VNO in female mice sexual
behavior has been largely neglected, lesions of the VNO in
females of other rodent species severely impair their behav-
ioral responses to males and their chemical signals. For
instance, VNO lesions significantly disrupt lordosis behav-
ior in female prairie voles [44] and significantly diminish
both the interest of female hamsters towards male flank
gland secretion and scent (flank and vaginal) marking
behavior to conspecific odors [45]. Lesions of the VNO
apparently impair the ability of female hamsters to express
sex preference through specialized species-specific behav-
iors such as scent marking [45] or ultrasound vocalizations
[46]. Furthermore, using genetically modified mice whose
VNO has been shown to be nonfunctional, it has been
recently demonstrated that the VNO is essential for sex
discrimination in male mice [47], and therefore it is likely to
play the same role in females.
4.2. The acquired attraction to male-derived volatiles:
association pheromones– odors
Urine-borne volatiles do not elicit attraction in chem-
ically naı̈ve females but they become attractive to chem-
ically experienced females (see Results). This represents a
case of associative learning that occurred during repeated
exposure of the females to both volatile and nonvolatile
components in the male-soiled bedding. As discussed
above, the unconditioned stimulus seems to be the complex
174 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
of MUPs and associated volatiles detected by the VNO,
which seems to be innately attractive. The conditioned
stimulus consists of volatiles, probably detected by the
olfactory epithelium.
It is very likely that the same volatiles that, in association
with MUPs, are detected by the VNO [40 – 43] and elicit
innate attraction, can be also detected by the olfactory
epithelium. In fact, the fraction of urine containing the
MUPs plus associated volatiles shows a weak ‘mousey’
scent [22,31] whereas the fractions rich in free brevicomin
and thiazole are reported to display an acrid ‘mousey’ odor
to the human nose. Moreover, Guo et al. [42] reported an
increase in the expression of c-fos mRNA elicited by both
complete male-soiled bedding and brevicomin + thiazo-
le + MUPs not only in the accessory olfactory bulb but also
in the main olfactory bulb. The expression of c-fos in the
main olfactory bulb is present even in females whose VNO
was removed, thus indicating that the olfactory epithelium
also detects the MUP-associated volatiles contained in male
urine.
This raises the possibility that the associative learning
during repeated exposure of females to male-soiled bedding
consisted of a VNO – olfactory association. By means of a
process of Pavlovian conditioning, there would be a transfer
of the attractive properties of VNO-detected nonvolatile
pheromones (likely the complex of MUPs and odorants)
to the urine-borne volatiles detected by the olfactory epi-
thelium, in a case of emotional tagging of odors. This would
explain why solutions of synthetic brevicomin, thiazole, and
farnesenes detected in a context in which direct contact with
the solution is avoided [18,19] are attractive to chemically
(or even sexually) experienced females.
Beauchamp et al. [12,13] put forward a hypothesis
similar to ours to explain the effects of VNO removal on
the chemoinvestigatory behavior of male guinea pigs
towards female urine. Removal of the VNO of male guinea
pigs results in an extinction-like decrease of the chemo-
investigatory behavior (head bobbing) towards female urine,
thus suggesting that the VNO-mediated detection of sexual
pheromones is attractive and reinforcing to males, whereas
the attractive properties of volatiles detected by the olfactory
epithelium are learned by association with VNO-detected
pheromones. Other cases of conditioned behavioral
responses to odors have been reported in rodents. For
instance, Nyby et al. [48] reported that, after encountering
females odorized with perfume, male mice emit ultrasound
courtship vocalizations to the perfume itself. Prepubertal
experience can also influence the sexual response to odors
in the adult life [26,35,49].
Studies performed in female pigs indicate that the olfact-
ory epithelium is involved in the behavioral response to the
male pheromone androsterone, since the response of ani-
mals whose vomeronasal duct was blocked did not differ
from that of controls [50]. Although the authors discuss the
possibility that their results are due to the sexual experience
of the females, our findings raise the possibility that the
response observed in VNO-blocked females be the result of
vomeronasal – olfactory associative learning. In fact, all the
female subjects of that study were housed with boars so that
they had previous experience with male sexual pheromones
allowing such an association to occur.
Some of the neuroendocrine responses elicited by pher-
omones might undergo a similar process of Pavlovian
conditioning by association of the actual pheromone with
odorants. This would explain the results reported by
Mucignat-Caretta et al. [20] indicating that female mice
reared with both parents or just sprayed with adult male
urine prior to weaning show enhanced puberty acceleration
in response to adult male urine after weaning. A possible
explanation for these results is that, due to a Pavlovian
association prior to weaning, prepubertal females respond
both to the actual pheromone (VNO-mediated uncon-
ditioned response) and to odorants of the male urine
(olfactory epithelium-mediated conditioned response) thus
resulting in a more robust neuroendocrine response. Our
results also raise the possibility that the neuroendocrine
response of ewes to male-derived chemicals detected by
the olfactory epithelium [51] is a consequence of previous
experience allowing pheromone– odorants association.
As a conclusion, one of the main outcomes of our work is
that previous experience with sexual pheromones has to be
taken into account to interpret the role of chemicals as
releaser or primer pheromones. As put forward by Beau-
champ et al. [52] and further discussed by Nyby et al. [48],
since learning might confer ‘pheromonal’ properties to
virtually every odorant, the classical concept of pheromone
cannot be easily applied to the mammalian intersexual
communication. Therefore, it seems appropriate to restrict
the concept of pheromone to those substances that have
innate biological value for intraspecies communication.
Another interesting question raised by our results is
where in the brain would such an odorant – pheromone
association occur. Olfactory and vomeronasal stimuli relay
in the main and accessory olfactory bulbs, respectively. The
main olfactory bulbs project not only to the anterior olfact-
ory nucleus and olfactory tubercle but also to portions of the
paleocortex that include the prepiriform, piriform, and
entorhinal cortices, as well as the anterior and posterolateral
regions of the cortical amygdala and the amygdalo-piriform
transition. On the other hand, the accessory olfactory bulbs
project to the posteromedial cortical amygdala, the medial
amygdala, and portions of the bed nucleus of the stria
terminalis [4]. Although these secondary olfactory and
vomeronasal projections appear quite segregated, physio-
logical data indicate that convergence of both kinds of
stimuli occur at the level of the medial amygdala [53].
On the other hand, the use of anterograde neuroanatom-
ical tracers indicates that both secondary vomeronasal and
olfactory centers (the medial amygdala [54], the piriform
cortex [55], and the amygdalo-piriform transition [56])
project to the basolateral division of the amygdala, a
convergence that is facilitated by the complex set of intrinsic
 J. Moncho-Bogani et al. / Physiology & Behavior 77 (2002) 167–176
connections within the basolateral amygdala [57]. These
data suggest that the basolateral amygdala, which is known
to be involved in the emotional tagging of olfactory stimuli
by means of Pavlovian conditioning using a diversity of
unconditioned stimuli [10,11,58], might also be involved in
the emotional labeling of odorants by association with
VNO-detected pheromonal stimuli. The experimental setup
we have designed seems appropriate to test this hypothesis
using current methods for research in functional neuro-
anatomy.
Acknowledgements
We thank A. Lázaro and M. Ferrer for technical
assistance, Dr. G. Ayala for statistical advice, and Drs.
D.C. Davies, I. González, M.J. Lorente, and A. Martı́nez-
Marcos for discussion. This work was supported by the
Valencian Government (GV00-161-05) and the Spanish
Ministry of Science and Technology and European Funds
for Regional Development (FEDER/BFI2001-3535).
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