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Comparison of Urinary Scents of Two Related Mouse
Species, Mus spicilegus and Mus domesticus
Helena A. Soini &Donald Wiesler &Sachiko Koyama &
Christophe Féron &Claude Baudoin &Milos V. Novotny
Received: 13 January 2009 /Revised: 11 March 2009 /Accepted: 1 April 2009
#Springer Science + Business Media, LLC 2009
Abstract Whereas the house mouse (Mus domesticus) has
been studied extensively in terms of physiology/behavior
and pheromonal attributes, the evolutionarily related
mound-building mouse (Mus spicilegus) has received
attention only recently due to its divergent behavioral traits
related to olfaction. To date, no chemical studies on urinary
volatile compounds have been performed on M. spicilegus.
The rationale for our investigations was to determine if
there are differences in urinary volatiles of intact and
castrated M. spicilegus males and to explore further whether
this species could utilize the same or structurally similar
pheromones as the male house mouse, M. domesticus. The
use of capillary gas chromatography/mass spectrometry
(GC-MS) together with sorptive stir bar extraction sampling
enabled quantitative comparisons between the intact and
castrated M. spicilegus urinary profiles. Additionally,
through GC-MS and atomic emission (sulfur-selective)
detection, we identified qualitative molecular differences
between intact M. spicilegus and M. domesticus. A series of
volatile and odoriferous lactones and the presence of
coumarin were the unique features of M. spicilegus, as was
the notable absence of 2-sec-butyl-4,5-dihydrothiazole (a
prominent M. domesticus male pheromone) and other sulfur-
containing compounds. Castration of M.spicilegus males
eliminated several substances, including δ-hexalactone and
γ-octalactone, and substantially decreased additional com-
pounds, suggesting their possible role in chemical commu-
nication. Some other M. domesticus pheromone components
were also found in M. spicilegus urine. These comparative
chemical analyses support the notion of metabolic similar-
ities as well as the uniqueness of some volatiles for M.
spicilegus, which may have a distinct physiological function
in reproduction and behavior.
Keywords Mus spicilegus .Mus domesticus .Urinary
volatile profile .Gas chromatography/mass spectrometry .
Stir bar extraction .Pheromones
Introduction
There is an evolutionary connection between the common
house mouse (Mus domesticus) and the mound-building
mouse (Mus spicilegus), with two pairs of closely related
species and subspecies in the phylogenetic tree: Mus
musculus domesticus and Mus musculus musculus in one
pair and M. spicilegus and Mus macedonicus in the second
one (Bonhomme et al. 1984; Sage et al. 1993). The M.
domesticus living environment is interwoven with human
habitats, whereas M. spicilegus species is feral, and in some
cases, at least during the summer period, M. spicilegus may
live in a close contact with M. musculus musculus (Orsini
et al. 1983; Sokolov et al. 1998; Simeonovska-Nikolova
2007). Additionally, in its social structure and behavior, M.
spicilegus has diverged far from the M. domesticus. Unlike
M. domesticus,M. spicilegus individuals are monogamous
(Patris and Baudoin 1998; Dobson and Baudoin 2002;
Baudoin et al. 2005; Gouat and Féron 2005) and cooper-
atively build colonial mounds for overwintering (Orsini
et al. 1983; Garza et al. 1997; Poteaux et al. 2008).
J Chem Ecol
DOI 10.1007/s10886-009-9628-2
H. A. Soini :D. Wiesler :S. Koyama :M. V. Novotny (*)
Department of Chemistry, Institute for Pheromone Research,
Indiana University,
Bloomington, IN 47405, USA
e-mail: novotny@indiana.edu
C. Féron :C. Baudoin
Laboratoire d’Ethologie Expérimentale et Comparée,
Université Paris 13,
99 Avenue Jean-Baptiste Clément,
93430 Villetaneuse, France
Moreover, M. spicilegus males display intense paternal care
(Patris and Baudoin 2000; Féron and Gouat 2007).
M. spicilegus individuals appear to rely on olfaction in
their social behavior and mate selection (Patris and Baudoin
1998; Patris et al. 2002; Heth et al., 2001,2003). In this
species, social and kin recognition through olfactory cues
have been observed in several studies (Gouat et al. 1998;
Dobson and Baudoin 2002; Baudoin et al. 2005; Busquet
and Baudoin 2005; Todrank et al. 2005; Colombelli-Negrel
and Gouat 2006). The odors, which are used for social
communication in mice, have a number of glandular and
nonglandular sources: preputial glands, ear glands, plantar
glands, anal glands, coagulating glands, submaxillary
glands, urine, and feces (Brown 1985). The salivary
androgen-binding proteins (Laukaitis et al. 1997; Talley
et al. 2001) and tear fluid peptides (Kimoto et al. 2005) also
have been reported as sources of chemical messengers in
M. domesticus. In the house mouse, the urine-mediated
chemical signals have been relatively well-characterized over
the years (for reviews, see Novotny 2003; Hurst and Beynon
2004). Behavioral tests have revealed that the odor cues
(expected to originate mainly from urine) are used in
communication (Patris and Baudoin 1998; Féron and Gheusi
2003; Busquet and Baudoin 2005; Colombelli-Negrel and
Gouat 2006). Thus far, there have been no complementary
reports for M. spicilegus on the chemical nature of such
olfactory cues; to date, the chemical constituents used in
communication and scent sources for M. spicilegus remain
unknown.
The first purpose of this study was to structurally
characterize and quantify individual chemical constituents
of the urinary volatile profiles for the intact versus castrated
M. spicilegus males. The second goal was to qualitatively
compare the findings to the previously well-characterized
male M. domesticus urinary compounds.
A quantitative comparison of the intact and castrated M.
spicilegus male volatile profiles was performed to explore
the metabolic end products that could be produced under
testosterone control. Some of the endocrinologically con-
trolled urinary constituents may act as chemical messengers
regulating various reproductive and social functions. Addi-
tionally, the urinary volatile profiles of female and male M.
spicilegus individuals were qualitatively compared in order
to explore gender roles related to the urine-mediated
chemical communication, such as those used in mate
selection and individual recognition. This chemical charac-
terization is expected to provide clues to the observed
differences in social and behavioral characteristics between
the two species.
To facilitate this study, we used the stir bar aqueous
extraction method (Baltussen et al. 1999,2002), which was
followed by solventless sample introduction into a gas
chromatograph-mass spectrometer (GC-MS) instrument.
This methodology is compatible with screening for volatile
organic compounds at low concentrations in biological
samples and is well suited for compound identification and
quantitative comparisons (Soini et al. 2005). In addition, a
combination of gas chromatography with atomic emission
detection (GC-AED) was utilized for the highly sensitive
sulfur compound profiling. In this report, we also take our
previously determined characteristics of the male M.
domesticus (ICR, C57Black/B6 and C57Black/B10) urinary
volatile components and qualitatively compare them with
the new M. spicilegus chemical information obtained in this
study. Analytical approaches for the M. domesticus and M.
spicilegus samples were identical. All analyses were
performed in the same laboratory.
Methods and Materials
Experimental Animals M. spicilegus mice were fifth-
generation animals from a population collected in Gyön-
gyös, Hungary in October 1999.From the time of
collection, the genealogy of every individual was known,
and all breeding pairs had been formed in a way to avoid
inbreeding. They were bred at the University of Paris 13
(University of Paris-Nord at Villetaneuse) under laboratory
conditions (20±1°C) with a 14:10 h L/D cycle. Food
(mouse pellets type M20, Special Diet Services, Witham,
Essex, UK), water, and bedding material (sawdust and
cotton) were provided. Mice were weaned at 28 days of age
and housed in same-sex sibling groups from 35 days of age.
Males and females were 3–6 months old when they were
used as urine donors.
For the male–female mouse comparisons, six males and
six females were isolated in standard polycarbonate cages
(26×16 cm and 14 cm high) 1 week before urine collection
in order to eliminate the social dominance effect (Féron and
Baudoin 1993,1998). With the male presence being
required to induce sexual receptivity in M. spicilegus
females (Féron and Gheusi 2003), we assumed that all the
isolated females were in anoestrus.
For the comparisons between intact and castrated males,
isolation occurred 3 weeks prior to urine collection. Twenty
males were isolated in standard polycarbonate cages (26×
16 cm, 14 cm high). Six of them were then castrated under
anesthesia induced by an intraperitoneal injection of a mixture
of ketamine (Imalgène 500, Merial, France, 100 mg/kg) and
xylazine (Rompun 2%, Bayer, Puteaux, France, 5 mg/kg).
Male M. domesticus urinary volatile profiles from inbred
ICR albino mice, C57BL /B6 and C57BL /B10 black mice
(Jackson Laboratories, Bar Harbor, ME, USA) were used
for qualitative comparisons. The data obtained for M.
domesticus have been previously reported by Harvey et al.
(1989) and Novotny et al. (2007).
J Chem Ecol
Urine Collection Animals were introduced individually into
a clean polycarbonate cage and surveyed for urination at least
every 5 min. Fresh individual urine was quickly collected with
a syringe and frozen (−20°C). A preliminary study showed
that this was the best process to handle these sensitive mice.
Ethical Note The experiments complied with the current
French laws (authorization 93-0033 for C. Féron; laboratory
approval was secured from the Prefecture of Seine Saint Denis
(prefectorial decree 02-2651), complying with the Associa-
tion for the Study of Animal Behaviour/Animal Behaviour
Society Guidelines for the Use of Animals in Research.
Reagents and Analytical Methods All compound identifi-
cations were verified by comparisons to authentic stand-
ards. Standard compounds were purchased from Aldrich
Chemical Company (Milwaukee, WI, USA), except 3-
octen-2-one and β-farnesene were from TCI America
(Portland, OR, USA) or synthesized in our laboratory
according to the previously described synthetic methods for
dehydro-exo-brevicomin (Wiesler et al. 1984), sec-butyl-
4,5-dihydrothiazole (North and Pattenden 1990), and 6-
hydroxy-6-methyl-3-heptanone (Novotny et al. 1999).
Twister™stir bars (10 mm in length, 0.5 mm film
thickness, 24-μl polydimethylsiloxane volume) were used
as the sorptive extraction devices. They were purchased
from Gerstel GmbH (Mülheim an der Ruhr, Germany).
Volatile and semivolatile compounds were extracted from
0.2 ml of urine in 20-ml capped glass vials for 60 min with
a Twister™stir bar. The urine samples were first diluted
with 2.0 ml water (high-purity OmniSolv® water, EM
Science, Gibbstown, NJ, USA). As an internal standard,
8 ng of 7-tridecanone (Aldrich, Milwaukee, WI, USA) was
added in 10μl of ethanol to each vial. Stirring speed was
800+rpm on the Variomag Multipoint HP 15 stirplate (H+P
Labortechnic, Oberschleissheim, Germany). Prior to extrac-
tion, all glassware were washed with acetone and dried at
80°C. After extraction, stir bars were rinsed with a small
amount of distilled water, dried gently on a paper tissue, and
placed in the Thermal Desorption Autosampler (TDSA) tube
(Gerstel GmbH, Mülheim an der Ruhr, Germany) for the GC
or GC-MS analysis. GC equipment for the sulfur compound
analysis consisted of an Agilent GC Model 6890 instrument
with an Atomic Emission Detector (Model G2350A from
Agilent Technologies, Wilmington, DE, USA), and a
Thermal Desorption Autosampler (Gerstel) operated in a
splitless mode. The separation capillary column was HP-
5MS (30 m×0.25 mm, i.d., 0.25μm film thickness) from
Agilent. Samples were thermally desorbed in a TDSA
automated system, followed by injection into the column
with a cooled injection assembly. Temperature program for
desorption was 20°C (0.5 min), then 60°C/min to 280°C
(hold 10 min). Temperature of the transfer line was set at
280°C. The injector was cooled with liquid nitrogen to−60°C
and, after desorption and cryotrapping, was heated at 12°C/s
to 280°C with the hold time of 10 min. The injector inlet was
operated in the solvent-vent mode, with a vent pressure
of 14 psi, a vent flow of 30 ml/min, and a purge flow of
50 ml/min. The GC temperature program was 40°C (0.5 min)
then 2°C/min to 200°C (hold 10 min). The carrier gas head
pressure was 14 psi for a flow rate of 1.2 ml/min. The GC unit
was operated in the constant-flow mode. The emission lines
for carbon (193 nm), sulfur (181 nm), and nitrogen (174 nm)
were monitored during the atomic plasma emission detection.
The GC-MS instrument used for the compound identi-
fication was an Agilent 6890 N gas chromatograph
connected to the 5973i MSD mass spectrometer (Agilent
Technologies). The GC column was a narrow-bore capillary
with 180μm, i.d.×20 m DB-5MS (0.18μm film thickness,
Agilent Technologies, Wilmington, DE, USA). The inlet head
pressure was 12.5 psi for the helium flow of 0.7 ml/min. The
system operated in the constant-flow mode. The temperature
program was 50°C (2 min) at 4°C /min to 200°C (hold 1 min).
Positive electron ionization (70 eV) mode was used with the
scanning rate of 4.51 scans per second over the mass range of
35–350 amu. The MSD transfer line temperature was set at
280°C. The ion source and quadrupole temperatures were set
at 230°C and 150°C, respectively. The TDSA-injector sample
introduction setting was identical to that described above in
connection with the GC-AED system, except that the injector
trapping temperature was set at −80°C.
Quantitative Evaluations and Statistical Analyses As the
basis for quantitative comparisons of urinary chromatographic
profiles of the fresh urine collected on plates, the peak area
integration was performed, and peak areas were normalized by
dividing with the peak area of internal standard (7-tridecanone)
for each separated component. Either GC-MS total-ion
chromatograms (TIC) or selected-ion chromatograms obtained
after the postrun modification of TICs were used for
calculations. Normalized peak areaswere statistically evaluated
for the intact and castrated mouse groups. Student’sttest was
employed for pairwise comparisons. The sulfur compound
profiles from the GC-AED were compared in a qualitative
manner between the intact and castrated males. The quanti-
tative data obtained in this study for the male M. spicilegus
urine samples were compared semiquantitatively with the
female M. spicilegus urine and qualitatively with the male M.
domesticus urine data obtained previously in this laboratory.
Results
Male M. spicilegus urinary volatile compound profiles by
GC-MS featured more than 100 components. Approximately
60 compounds showed sufficient spectral intensity and
J Chem Ecol
Table 1 Comparison of male M. spicilegus urinary compounds in intact (I) and castrated (C) samples
Compound Retention
time (min)
Comparison of
levels (I) vs. (C)
Student’s
ttest P*
Intact mean ±SD
(normalized peak area)
N=14
Castrated mean ±SD
(normalized peak area)
N=6
5,5-Dimethyl-2-ethyl-4,5-dihydrofuran
(DHF 1)
3.52 I>C<0.001 464,084±224,499 51,261± 29,647
Z-5,5-Dimethyl-2-ethylidenetetrahydrofuran
(DHF 2)
4.97 I>C<0.001 177,204±88,099 34,820± 15,200
2-Heptanone 5.24 C=0 3,933,911± 4,598,416 0
E-5,5-Dimethyl-2-ethylidenetetrahydrofuran
(DHF 3)
5.72 I>C0.002 76,468± 43,006 12,070± 6,267
p-Cymene 9.13 I>C0.028 5,367,885±1,995,779 3,344,710± 699,330
3-Octen-2-one 9.60 >0.05 89,763± 80,251 146,340± 152,691
Dehydro-exo-brevicomin (DHB) 9.93 >0.05 340,327± 351,343 104,202± 123,621
γ-Hexalactone
b
9.99 I>C0.023 4,535,674±2,436,519 1,998,715± 592,374
Acetophenone 10.46 I>C0.013 525,485±435,655 30,341± 12,777
o-Toluidine 10.61 >0.05 1,441,690±1,919,224 37,787±37,458
δ-Hexalactone
b
11.37 C=0 426,477± 357,873 0
Undecane 11.76 I>C0.01 2,155,122± 756,483 1,123,143± 676,228
Nonanal 11.91 I>C0.018 3,688,456± 1,621,797 1,921,820± 247,488
Phenylacetone
a
12.55 >0.05 1,406,846± 1,805,201 136,475± 227,297
A lactone
b,a
13.38 I>C0.001 312,203±110,744 83,383± 34,966
Undecalactone
a,b
13.63 I>C0.032 218,191±177,650 45,713± 28,089
4-Ethylphenol
b
13.95 I>C0.003 298,778±91,715 135,636± 116,851
A methyl toluate
a,b
14.28 >0.05 653,578± 453,522 4,109,225± 6,503,658
Octanoic acid 14.39 >0.05 304,124± 281,096 122,047± 133,034
Decanal 15.42 >0.05 1,647,466± 1,005,640 860,987± 342,581
Unidentified m/z 121
b
15.63 I=0 0 373,429± 196,797
N-Phenylformanilide
a
15.65 C=0 4,635,124± 5,249,321 0
Unidentified m/z 140
b
15.83 C=0 431,407± 265,044 0
2-Coumaranone
b
16.24 C>I <0.001 3,979,595±1,610,512 44,592,400± 32,762,900
A lactone
a,b
16.96 I=0 0 679,252± 326,535
γ-Octalactone
b
17.16 C=0 341,615± 179,053 0
A lactone
a,b
17.29 >0.05 454,554± 185,246 901,687± 977,437
Nonanoic acid 17.64 >0.05 120,761± 90,627 1,099,975± 2,101,058
Decanol 17.68 >0.05 2,025,997±1,869,935 738,656±643,914
δ-Nonyl-δ-valerolactone
a,b
17.82 >0.05 922,899± 2,019,056 55,588±21,976
A ketone
a
17.9 I>C0.036 6,645,912±5,406,927 1,531,829± 749,722
Indole 18.16 >0.05 621,379± 681,142 320,436± 253,647
Coumarin
b
18.35 I=0 0 285,687± 253,621
γ-Nonenolactone
b
19.77 C>I0.048 419,497±193,279 928,870± 878,601
A lactone
a,b
20.45 I=0 0 1,314,727± 712,105
An acid
a,b
20.90 >0.05 254,023± 156,938 528,732± 722,600
Undecanol 21.05 >0.05 4,985,273± 3,841,959 1,594,441± 1,155,482
N-(Methylthio)methylaniline
a
21.92 I>C0.01 2,068,105± 1,468,134 288,766± 414,582
Dodecanal 22.15 >0.05 2,108,451 ± 1,246,395 1,308,000±463,597
Geranylacetone 23.31 >0.05 1,037,403± 651,760 898,834±1,411,928
β-Farnesene 23.47 C=0 313,220± 357,030 0
m/z 125 δ-Lactone
a,b
23.68 I>C0.003 366,068±177,288 97,930± 89,840
Unknown
b
23.78 I=0 0 157,963± 107,125
Unknown
b
23.97 I=0 0 562,598± 243,505
Dodecanol 24.19 I>C0.028 12,682,500±7,735,907 4,775,774± 2,980,101
J Chem Ecol
purity for the quantitative comparisons. Among these, 30
were identified, 15 were tentatively identified, while ten
remain unknown (Table 1).
The characteristic feature for the profiles from M. spicilegus
was the prominent presence of γ- and δ-lactones, ketones,
alcohols, and acids. Characteristic urinary components found
in M. domesticus are three dihydrofuran compounds (MW
126), which were shown previously to originate from a
puberty-accelerating pheromone, 6-hydroxy-6-methyl-3-
heptanone (Novotny et al. 1999) and its lactol form (see
Table 1for 5,5-dimethyl-2-ethyl-4,5-dihydrofuran, Z-5,5-
dimethyl-2-ethylidenetetrahydrofuran and E-5,5-dimethyl-2-
ethylidenetetrahydrofuran). These three furan derivatives were
present in male M. spicilegus at levels comparable to those for
M. domesticus reported earlier (Harvey et al. 1989; Novotny
et al. 2007). In castrated M. spicilegus, the levels of these
furan derivatives were significantly lower (P<0.002) than in
intact M. spicilegus males. Another M. domesticus pheromone
compound, dehydro-exo-brevicomin, also present in female
urine in small amounts (Harvey et al. 1989; Jemiolo et al.
1991), was found at lower levels in urine from the intact and
castrated male and also in female M. spicilegus urine (data not
shown). Castration did not change dehydro-exo-brevicomin
levels in M. spicilegus significantly in contrast to the
suppressed levels of dehydro-exo-brevicomin in the samples
from castrated M. domesticus (Novotny et al. 1980; Harvey
et al. 1989). Trace levels of the dominant male mouse
pheromone for M. domesticus,β-farnesene (Harvey et al.
1989), were detected in the intact male M. spicilegus urine but
were not seen in female or castrated animals. N-(Methylthio)
methylaniline was the only identified sulfur compound in the
GC-MS TIC urine profiles from M. spicilegus, being more
abundant in the intact male mouse urine when compared to
the mouse urine of castrates (P<0.01). The representative
structures of the main compounds distinguishing the two
species are shown in Fig. 1.
Table 1 (continued)
Compound Retention
time (min)
Comparison of
levels (I) vs. (C)
Student’s
ttest P*
Intact mean ±SD
(normalized peak area)
N=14
Castrated mean ±SD
(normalized peak area)
N=6
Unknown
b
24.99 >0.05 1,587,800± 1,972,417 53,771± 131,711
Unknown
b
25.51 I=0 0 3,411,073± 1,893,080
Dodecanoic acid 26.87 >0.05 988,836± 1,058,383 451,964±413,834
An acid
a,b
27.34 I=0 0 8,786,688± 3,876,541
Unknown
b
33.66 I=0 0 8,514,168± 3,430,211
N,N-Dimethyldodecanamide
a,b
34.30 >0.05 9,643,776± 4,582,043 6,694,015± 6,896,710
Unknown
b
34.69 I=0 0 3,201,660± 2,890,689
Palmitic acid 37.37 I>C0.042 2,142,529±1,710,270 585,610±167,857
Unknown
b
38.30 I=0 0 2,264,620± 793,599
Unknown
b
38.82 I=0 0 13,472,067± 3,126,728
*P<0.05 accepted significance; statistical significance for the compound level differences between intact (I) and castrated (C) male M. spicilegus
urine
a
Tentatively identified
b
Unique urinary compounds for M. spicilegus, not found in M. domesticus
S
N
S
N
H3C
S
S
CH3
SCH3
HSCH3
H
O
O
OO
O
O
OO
CH3S
SSCH3
OO
2-sec-butyl-4,5-dihydrothiazole (SBT)
2-isopropyl-4,5-dihydrothiazole (IPT)
dimethyl disulfide
bis(methylthio)methane
M. domesticus
M. spicilegus
coumarin
2-coumaranone
methyl (methylthio)methyl disulfide
γ octalactone
γ-hexalactone
δ-hexalactone
Fig. 1 Distinguishing chemical structures of volatile compounds in
M. spicilegus and M. domesticus male urine
J Chem Ecol
Castration of M. spicilegus males affected the volatile
profiles in three ways: certain compounds, apparently under
endocrine control, disappeared; while the levels of other
compounds increased; and a set of previously undetected
compounds became apparent (see Table 1). Figure 2
illustrates the representative urinary volatile profiles for
intact and castrated M. spicilegus males and intact male M.
domesticus. In addition to β-farnesene, castration removed
2-heptanone, δ-hexalactone, N-phenylformanilide, and γ-
octalactone from the set of urinary volatiles. Some
compound levels decreased after castration, including γ-
hexalactone (P<0.02) and N-(methylthio)methylaniline
(P<0.01), shown as normalized peak areas with standard
deviation (SD) in Table 1. In contrast, after castration,
levels of 2-coumaranone, a unique compound for M.
spicilegus, increased (P<0.001). The absolute amounts of
2-coumaranone in castrated male urine were about 300±
200 ng/ml (standard deviation, SD, N=6), while in the
intact mouse urine, the levels were just 2±2 ng/ml (SD,
N=14). In addition, other constituents, including several
lactones, were found at higher levels, and new compounds
such as coumarin and several late-eluting lactones appeared
in the urine of castrated males (see Table 1). Figure 3shows
a comparison of selected compound levels affected by
castration. Individual variation for some of the compounds
appeared relatively large (>70%, relative standard deviation
(RSD), N=6–14), while some compounds varied only
within the range of 12–30% (RSD, N=6–14) among the
sampled individuals. Typically, the variation due to the
sampling method was only 5–10% (RSD, N=4).
Qualitative comparisons in the GC-AED sulfur-selective
profiles did not reveal any clear differences between intact
5.00 10.00 15.00 20.00 25.00 30.00 35.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
5.00 10.00 15.00 20.00 25.00 30.00 35.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
Mus spicilegus
Intact (I)
Mus spicilegus
Castrated ( C )
5.00 10.00 15.00 20.00 25.00 30.00 35.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
Mus domesticus
Intact
Time (min)
2-sec-butyl-4,5-
dihydrothiazole
2-coumaranone
TIC
-hexalactone
Fig. 2 Representative urinary
(GC-MS total ion current)
profiles of aintact and
bcastrated male M. spicilegus.
cA comparative profile for male
intact M. domesticus
J Chem Ecol
and castrated animals. Relatively low-level (sub-picogram),
sulfur-containing compounds (data not shown) were
detected but could not be identified structurally.
Several of the previously reported M. domesticus urinary
compounds (Novotny et al. 1990a,b,2007) also were
present in M. spicilegus profiles, including three character-
istic dihydrofurans. It is notable that the M. domesticus
male dominance signaling pheromone compound, 2-sec-
butyl-4,5-dihydrothiazole, and its “structural relative,”2-
isopropyl-4,5-dihydrothiazole (Novotny et al. 1985), were
not detected in the M. spicilegus urine. Linear sulfur
compounds, such as dimethyl disulfide, bis(methylthio)
methane, and methyl(methylthio)methyl disulfide, typical
for M. domesticus (Novotny et al. 2007), were also absent in
M. spicilegus urinary volatile profiles.Furthermore, few M.
spicilegus urinary ketones were present compared to those
identified in M. domesticus urine (Novotny et al. 2007).
Discussion
Since castrated male M. spicilegus did not show β-farnesene,
2-heptanone, δ-hexalactone, N-phenylformanilide, or γ-
octalactone among the urinary volatiles, it is suggested that
the metabolic pathways involving these compounds may be
under endocrine control. Behavioral tests with these com-
pounds would be necessary to show what are the possible
chemo-signaling and physiological functions of these com-
pounds for male and female M. spicilegus. Castration also
significantly affected the production of lactones, thus
demonstrating an endocrine feedback for lactone biosynthesis.
There is a strong possibility that some of the lactones may be
mediators of chemical communication that involve reproduc-
tion. Furthermore, lactone levels were particularly varied
among the individual intact males, suggesting that the lactone
profile could be related to individual recognition. In their pure
form, many of the lactones exhibit fruit- or berry-like aromas
(Gatfield et al. 1993), which are relatively subtle odors in
human perception, as opposed to the pungent smell in the M.
domesticus male urine caused by the sulfur-containing
compounds. The same unique lactone compounds were
found in the female M. spicilegus urine in our qualitative
screening (unpublished experiments).
Lactone biosynthesis involves C-18 hydroxyl fatty acids
as precursors, which undergo β-oxidation steps followed by
lactonization (Albrecht et al. 1992). Additionally, 9,10-oleic
acid has been reported as a precursor for γ-dodecalactones
in the yeast cultures (Haffner and Tressl 1996). This may
imply that the metabolism of fatty acids leading to urinary
lactone end products could play a prominent role in M.
spicilegus metabolism. Contrarily, M. domesticus shows
little presence of urinary lactones. The occurrence of
urinary lactones previously has been reported in female
and male pine voles (Microtus pinetorum) (Boyer et al.
1989). In female pine voles, γ-octalactone exhibited the
greatest urinary level changes among the volatile com-
pounds after estrogen treatment or ovariectomy.
Other biological sources for lactones have been reported
for insects and microorganisms, such as the cephalic gland
γ-octalactone of giant honeybee workers (Apis laboriosa;
Blum et al. 2000) and δ- and γ-lactones emitted by marine
Alphaproteobacteria (Dickschat et al. 2005). In these two
studies, lactones were hypothesized as potential chemical
signaling compounds within the giant honeybee colony and
bacterial culture, respectively.
Urinary ketones in intact male M. spicilegus interestingly
were sparse (Table 1). M. domesticus urinary ketones (e.g.,
2-heptanone, 6-methyl-5-hepten-3-one, 5-hepten-2-one)
previously have been found to correlate with the major
histocompatibility complex mouse haplotypes (Novotny
et al. 2007).
M. spicilegus originate from Eastern Europe. Several
diagnostic genetic loci separate M. spicilegus and M.
domesticus species (Bonhomme et al. 1984; reviewed in
Sokolov et al. 1998). The genes in these loci code for
enzymatically active proteins. Alcohol and malate dehy-
0
100000
200000
300000
400000
500000
600000
700000
800000
DHF (1) DHF (2) DHF (3)
castrated
intact
0
2000000
4000000
6000000
8000000 castrated
intact
N-(methylthio)- palmitic acid
meth
y
laniline
γ-hexalactone
Fig. 3 Effect of castration on selected compound levels in urine (see
Table 1). Y-axis corresponds to normalized peak areas. Error bars
indicate standard deviation (SD)
J Chem Ecol
drogenases, esterases, carbonic anhydrase, and mannose
and glucose phosphate isomerases are among these pro-
teins. Consequently, some of the distinguishing coded
enzymes may impact the metabolic pathways that lead to
the excretion of urinary substances.
In addition to the lactones, urinary coumarin appears as
an interesting “metabolic marker compound”for the M.
spicilegus species. The presence of coumarin and abun-
dance of 2-coumaranone in the urine of castrated M.
spicilegus male also represent distinguishing metabolic
pathways not found in M. domesticus. Coumarin could
originate from different plants (reviewed in Bourgaud et al.
2006). In mammalian systems, coumarin generally exhibits
toxic effects and is oxidatively metabolized (detoxified) by
the cytochrome P450 mono-oxygenase enzyme system in
liver microsomes (Creaven et al. 1965; Lewis and Lake
2002). The specific mouse enzyme for coumarin elimina-
tion through 7-hydroxylation is CYP2A5 (Miles et al.
1990). The CYP2A5 enzyme is also known to be inhibited
by lactones and 2-coumaranone (Juvonen et al. 1991,
2000). In a study within the M. domesticus strains, P450
enzymes have been found genetically altered among these
strains (Wood 1979). Furthermore, a single autosomal gene
locus Gpi-1 (glucose phosphate isomerase-1) was found
responsible for the differential hydroxylase activity of P450
(Wood and Taylor 1979). Since Gpi-1 also was found as
one of the distinguishing genetic loci between M. spicilegus
and M. domesticus (Bonhomme et al. 1984), the vastly
different urinary volatile profiles of these species could be
due, in part, to differential P450 oxidase activity, among
other metabolic routes. For example, the distinguishing
Es-2 loci controlling esterases have been found especially
active in the kidney (Ruddle et al. 1969) and thus likely to
affect some of the urinary metabolite excretion. However,
separate genetic studies and metabolic mapping are necessary
to link the genetic sources for the observed metabolic profile
differences between M. spicilegus and M. domesticus, as
exemplified by the urinary coumarin levels observed in this
study. Furthermore, it seems desirable to investigate how
these genetically induced changes in the urinary volatile
constituents could facilitate chemical communication and
social behavior in the “scent world”of the M. spicilegus
species.
In summary, quantitative comparisons of the urinary
volatile profiles for male M. spicilegus mice reveal several
compounds that have previously shown biological activity
as components of male-produced pheromones in the M.
domesticus species. These similarities suggest that the two
mouse species carry a certain genetic linkage that may be
utilized in chemo-signaling. On the other hand, the total
absence of the prominent M. domesticus male aggression
pheromone, 2-sec-butyl-4,5-dihydrothiazole, in the M.
spicilegus urine and the presence of unique δ- and γ-
lactones and coumarin seem to indicate that these species
have developed some distinctly separate metabolic path-
ways involving urinary constituents. Castration of M.
spicilegus males removed δ-hexalactone and γ-octalactone
among the identified urinary constituents. Their testosterone
control suggests a possible involvement in chemical commu-
nication within the species. Behavioral tests are in progress to
define possible roles of several urinary volatile organic
compounds in M. spicilegus chemical communication.
Acknowledgments This work was sponsored jointly by the Lilly
Chemistry Alumni Chair and the Indiana University METACyt
Initiative, partially funded through a grant from the Lilly Endowment,
Inc., as well as the French National Center for Scientific Research and
the French Ministry for Higher Education and Research (University of
Paris 13). We wish to thank Mr. K.E. Bruce (Institute for Pheromone
Research, Indiana University) for assistance with the analyses and data
collection and Professor A. Hefetz (Tel Aviv University, Israel) for
comments on a previous version of the manuscript.
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