Gennady Churakov1, Anatoly Philimonenko2, Elena Fedorova3, Irina Ermakova3, Sergey Novikov3

1Institute
of Experimental Pathology/Molecular Neurobiology (ZMBE), University of Muenster, Muenster, Germany
2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
3Department of Sensory Physiology, I.P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg, Russia

E-mail: novikov47@yandex.ru

Major urinary proteins (MUPs) of the house mouse Mus
musculus L. form a large group of highly polymorphic
acidic isoforms with molecular masses of 18-20 kDa.
MUPs are encoded by the Mup genes cluster, which is
mapped to chromosome 4 and consists of about 35 genes
and pseudogenes. Numerous studies have established that
the synthesis of MUPs is regulated by a variety of steroid
and peptide hormones, including testosterone, estrogens,
growth hormone, and thyroxin. In their internal binding
pocket, MUPs can bind and transport diverse sets of
volatile pheromonally active ligands, such as 2-sec-butyl-
4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-
heptanone (HMH), and 2,3-dehydro-exo-brevicomin
(DHB) (Bacchini et al., 1992; Robertson et al., 1993;
Novotny et al., 1999; Perez-Miller et al., 2010). According
to several studies (Hurst et al., 1998; Cavaggioni et al.,
2008) these ligands bound to MUPs are released gradually
from the scent mark resulting in dynamic individual
chemical bouquet which creates specific olfactory signature
of the donor. Moreover, MUPs by themselves can also
convey essential olfactory information about sex, social
rank and individuality of donors (Chamero et al., 2007,
2011). Thus, MUPs are widely assumed to be a key
component in formation of the individual olfactory
signature in Mus musculus L. (Hurst et al., 2001;
Armstrong et al., 2005; Novikov et al., 2009; Roberts et al.,
2010; Kwak et al., 2011). However, fine molecular
mechanisms of olfactory encoding by MUPs and their
decoding by olfactory system of recipients remain to be
elucidated.
Fig 1. Electrophoresis pattern of the major
urinary proteins (MUPs) depends on mouse
physiological state, sex and genotype
Fig 2. Content of individual MUP fractions
in male mice of different genotypes
Table 3. Combinatorial genotype-
dependent MUP codes in F1 progeny from
reciprocal crosses
Gender Genotype Fraction Consequential Ratio of principal
fractions’ fractions
A B C D E F G H order
CBAB6F1 + + + + + + + + DCEABGHF 0.40:0.25:0.20:0.15
Male
B6CBAF1 + + + + + + + + DACEBGHF 0.45:0.25:0.15:0.15
CBAB6F1 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10
Female
B6CBAF1 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10
In urine of male and female F1 progeny from reciprocal
crosses, all 8 MUP fractions are present. Concentration-
based «MUP formulae» are with high significance similar
within sex: Spearman’s rank-order correlation coefficients
are rs=0.91 in males and rs=0.98 in females (P<0.01).
Ratios of principal fractions are also similar within sex,
which is especially obvious in females of both genotypes
(Table 3). However, at the moment it is not clear how the
presence and ratios of different MUP fractions are defined
in F1 animals and to what extent parental patterns are
inherited (Chemical Senses, in preparation).
Our data on genotype- and sex-specific ratios of principal
MUP fractions (A, C, D, E) in CBA/LacY and C57BL/6JY
mice may provide new insights into mechanisms of genetic
and epigenetic regulation of Mup genes cluster, which
result in expression of specific bouquets of MUPs. We
propose that various volatile pheromonally active ligands
(e.g., SBT, HMH, and DHB) represent «letters» of the
chemical «language of communication» in Mus musculus
L. and that, correspondingly, specific combinations of
MUPs efficiently organize these «letters» into readable
«words» that encode sex, physiological state, age, and
genotype differences (Novikov, 2003; Novikov et al.,
2009).
We suggest that MUPs are involved in formation of
individual olfactory signatures in mice by a decoding
algorithm within the vomeronasal organ in a way
analogous to an electronic bar scanning machine. Taking
into account that MUPs by themselves can efficiently
activate vomeronasal neurons (Chamero et al., 2007, 2011),
and that expression of pheromone receptors V2Rs is sex
specific (He et al., 2008, 2010) and combinatorial (Martini
et al., 2001; Silvotti et al., 2007), we propose that
molecular mechanisms of olfactory decoding of the unique
MUPs combination should be based on chemical
«dialogue» between individual subsets (modules) of MUPs
and complementary modules of V2Rs.

Fig 3. Electrophoresis pattern of MUPs

Key words: odorant-binding proteins, major urinary
proteins, pheromones, olfactory code, modular
combinatorial pattern, laboratory mice
C57BL/6JY
Recently we suggested that olfactory encoding by MUPs is
based on combinatorial and modular principles (Novikov et
al., 2011). In order to test this hypothesis in details, we
performed current study using laboratory mice of two
genealogically unrelated strains, CBA/LacY and
C57BL/6JY, under different physiological conditions and at
various ontogenetic stages.
Subjects, housing conditions and urine collection
Experiments were performed during winter and spring
periods using males and females of two highly inbred and
genealogically unrelated strains of laboratory mice,
CBA/LacY and C57BL/6JY, and their F1 hybrids from
resembles «bar code»
 We demonstrate that 8 analyzed MUP fractions form two
distinct subsets (modules) according to stability of their
ratio during ontogenesis. The «adult proportion» profile
of different MUPs is genotype-specific, appears very
soon after weaning and resembles a «bar code».
Table 1. Combinatorial genotype-dependent
 Concentration-based consecutive order of different MUP
MUP profiles during male mice ontogenesis fractions varies with sex, genotype and androgenic state.
Presence and concentration of various MUP fractions
Genotype Age, Fractions Consequential Ratio of principal form an individual combinatorial pattern, which might
weeks fractions’ fractions
A B C D E F G H order
reflect the genotype- and gender-specific olfactory
signature of the animal. Our results show that this
3 + − + + − + + + GCAHFD 0.45:0.40:0.15 modular organization of MUPs pattern is common for
two genealogically unrelated strains CBA/LacY and
4 + + + + − + + + CADGBHF 0.60:0.25:0.15
C57BL/6JY and thus, probably, for mice in general.
8 + + + + − + + + CADBGFH 0.55:0.25:0.20  We predict that these specific subsets (modules) of
Our results demonstrate that olfactory coding by MUPs in MUPs combinations are screened and recognized by
12 + + + + − + + + CADBGFH 0.55:0.25:0.20 Mus musculus L. is probably based on a modular principle complementary vomeronasal (V2R) neurons of the
(Tables 1-3). We suggest that ratios and concentrations of recipients, and that these processes constitute the basis of
3 + − − + + − + + EADGH 0.40:0:30:0:30
MUP fractions in the given combination define behavioural pheromonal coding and subsequent olfactory decoding in
response of the recipient mouse: some MUPs combinations Mus musculus L.
4 + + + + + − + + DCEABGH 0.50:0.25:0.20:0.05
CBA/LacY
can promote stereotype intermale aggression, others can
8 + + + + + − + + DECABGH 0.45:0.35:0.15:0.05 initiate mating-like behaviour. The proposed MUP model
can effectively trace the multicomponent chemosensory
12 + + + + + − + + DECABGH 0.60:0.20:0.10:0.10 pathways from gene(s) to olfactory-mediated physiological
and social behaviour responses in laboratory mice via
pheromones. This model presents valuable methodological
Quantitative evaluation of 8 protein fractions A-H with approach for functional dissection of social behaviour
regard to age and genotype revealed significant interstrain phenotypes using pheromones as a fine molecular tool and
differences in MUPs expression patterns in male mice leads to our better understanding how brain translates the The authors are grateful to Sergey Mylnikov (St.
during postnatal development. According to their relative chemical information encoded by odorant mixtures Petersburg State University, Russia) for his help in
values at various ontogenetic stages, we divided these MUP (Novikov et al., 2010; Fedorova et al., 2011). statistical data analyses and to Valentina Sukonina
fractions into two subsets that probably reflect their (University of Gothenburg, Sweden) for collection of
different functional significance. The first subset, which experimental data and helpful discussions.
could be characterized as «ontogenetically stable», consists The work was supported by Russian Foundation for Basic
of fractions A, C, D, and E; the second subset consists of Research (projects 02-04-49273, 04-04-63050, 07-04-
fractions B, F, G, and H, which are significantly influenced 01762) and Russian State Science and Technology Program
by the age factor (Novikov et al., 2009). Fig 4. Proposed scanning of the MUP «Priority Frontiers in Genetics» (project 2.152 to SN).
signature by V2Rs
Concentration formula

reciprocal crosses (n=289). Animals were kept in groups of

four in standard polypropylene cages T-2 («Velaz», Czech
Republic) under conditions of an inverted light cycle (day –
12 h, night – 12 h). Urine samples were taken from animals
by gentle abdomen massage, individually collected in
plastic 2 ml Eppendorf tubes at the same time of the day,
Table 2. Combinatorial genotype-dependent
MUP codes for gender and physiological
state in mice Chamero P, Marton TF, Logan DW, Flanagan K, Cruz JR, Saghatelian A, Cravatt BF, Stowers L. 2007.
Identification of protein pheromones that promote aggressive behaviour. Nature. 450:899-902. Chamero P,
Katsoulidou V, Hendrix P, Bufe B, Roberts R, Matsunami H, Abramowitz J, Birnbaumer L, Zufall F, Leinders-

and stored at –18º C. Genotype Physiological Fraction Consequential Ratio of principal

Zufall T. 2011. G protein Gαo is essential for vomeronasal function and aggressive behavior in mice. Proc.
Natl. Acad. Sci. USA. doi: 10.1073/pnas.1107770108. Churakov GA, Novikov SN. 2000. Heterogeneity and
differential expression of MUP proteins as a genetic basis of the physiological activity of androgen-
state fractions’ fractions
dependent pheromones. Dokl. Biol Sci. 375:549-552. Churakov GA, Philimonenko AA, Novikov SN. 1992.
A B C D E F G H order Genetic control and expression of the major urinary proteins (MUPs) in the laboratory mice: MUPs as a
Specific proportion potential pheromone messangers? Proc. 14th Meeting of the Association for Chemoreception Sciences
(AchemS). Sarasota, USA, p. 18. Fedorova E, Ermakova I, Novikov S. 2011. Major urinary proteins (MUPs)
females + + + − − + + + CABFGH 0.80:0.20 as a promising tool for analysis of the intermale aggression in mice. Chemical Senses 36:E47. He J, Ma L,
Protein determination and polyacrylamide gel Kim SS, Nakai J, Yu CR. 2008. Encoding gender and individual information in the mouse vomeronasal organ.
Science. 320:535-538. Kwak J, Josue J, Faranda A, Opiekun MC, Preti G, Osada K, Yamazaki K,
electrophoresis castrates + + + − − + + + CABFGH 0.50:0.50 Beauchamp GK. 2011. Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice.
C57BL/6JY Chem. Senses, 36:443-452. Novikov S, Fedorova E, Ermakova I. 2011. Towards unraveling the olfactory
code in Mus musculus: combinatorial encoding of age-, sex-, and genotype-specific olfactory signature by
castrated+T + + + + − + + + CADBFGH 0.40:0.35:0.25 major urinary proteins (MUPs). Chemical Senses. 36:E31.). Novikov SN, Churakov GA, Philimonenko AA,
Total urinary protein concentration was determined by the Ermakova II, Fedorova EM, Burkot IA. 2009. The pattern of major urinary proteins (MUPs) expression
standard method (Bradford, 1976). MUPs were analyzed during postnatal ontogenesis of the laboratory mouse depends on genotype and sex. Ontogenez. 40:261-269.
males + + + + − + + + CADBFGH 0.40:0.30:0.30 Novikov SN. 1988. Pheromones and Reproduction in Mammals: Physiological Aspects. Nauka Publishing
by electrophoresis in polyacrylamide gel using both tris- House. 169 pp. Roberts SA, Simpson DM, Armstrong SD, Davidson AJ, Robertson DH, Mclean L, Beynon
RJ, Hurst JL. 2010. Darcin: a male pheromone that stimulates female memory and sexual attraction to an
acetate system (pH 5.5) according to Hainey, Bishop females + − − + + − + + EADHG 0.50:0.35:0.15 individual male’s odour. BMC Biol. 8:75. Silvotti L, Moiani A, Gatti A, Tirindelli R. 2007. Combinatorial co-
expression of pheromone receptors, V2Rs. J. Neurochem. 103:1753-1763.
(1982) and tris-glycine system (pH 8.2) according to Davis
(1964) in our modification. Samples were prepared by castrates + − − + + − + + EDAHG 0.60:0.35:0.05

mixing aliquots of urine (2-10 μl) with 0.1 M tris-HCl
buffer, pH 7.4, containing 20% glycerin and 0.01 %
bromphenol blue. The gels were stained with Coomassie G-
250 («Serva», Germany). Molecular weights of MUPs
were evaluated using Calibration Kit proteins («Sigma»,
USA). Quantitative analysis of fractioned MUPs was
performed using GelScan XL densitometer («Pharmacia»,
CBA/LacY Fig 5. The combinatorial pattern of MUPs
castrated+T + + + + + − + + DEACBGH 0.45:0.30:0.15:0.10
can create unique odour image
males + + + + + − + + DEACBGH 0.55:0.20:0.15:0.10
Quantitative evaluation of 8 protein fractions A-H with Combinatorial encoding of mouse odour signatures by major urinary proteins (MUPs):
regard to sex and genotype revealed that the concentration- evidence for modular principle of their decoding?

Sweden). based consecutive order of different MUP fractions varies

CHURAKOV Gennady1, PHILIMONENKO Anatoly2, FEDOROVA Elena3,4,
ERMAKOVA Irina3,5, NOVIKOV Sergey3

with sex and androgenic state. In other words, presence and 1Institute of Experimental Pathology/Molecular Neurobiology (ZMBE), University of Muenster, Muenster, Germany, 2Institute of
Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3Department of Sensory Physiology,
concentration of various MUP fractions form an individual I.P. Pavlov Institute of Physiology, Russian Academy of Sciences, Saint Petersburg, Russia, 4Department of Molecular Genetics,
Institute of Experimental Medicine, Russian Academy of Medical Sciences, Saint Petersburg, Russia, 5Institute of Cytology,

combinatorial pattern, which might reflect the genotype- Russian Academy of Sciences, Saint Petersburg, Russia

Statistical data analyses
Biochemical data were expressed as means ± standard
error. An ordinary parametric t-Student test and
nonparametric Spearman correlation analyses were
performed. The results were also analyzed using the two-
way analysis of variance (ANOVA) with «age» and
and sex-specific olfactory signature of the animal. It is Major urinary proteins (MUPs) of the house mouse belong to the highly diverse lipocalin superfamily (Flower, 1996). As a typical
odorant-binding protein, MUP can bind a wide range of volatile pheromonally active ligands and thus can trigger several
behavioural and physiological responses in recipients. Nowadays MUPs are considered as a key component in olfactory signature
noticeable that the ratios of principal fractions in in Mus musculus: they can provide essential information about individuality and physiological state of donors (e.g., Roberts et al,
testosterone-treated castrates and sham-operated males are
2010; Wyatt, 2010; Janotova, Stopka, 2011; Kwak et al, 2011). However, fine mechanisms of olfactory coding by MUPs and
decoding MUP-odorant complexes remain to be elucidated (Novikov et al., 2011). Using electrophoresis in polyacrylamide gel
(PAGE), we examined differential protein profiles in urine of genealogically unrelated CBA/LacY and C57BL/6JY laboratory
almost the same, especially in the case of C57BL/6JY mice according to genotype, sex, age, and physiological status. Quantitative evaluation of eight MUPs’ fractions with different
electrophoretic mobility (A-H) reveals that specific combination and proportion of individual fractions form two distinct protein
mice. We suggest that our data on genotype-specific ratios subsets (modules). These subsets appear in both sexes very soon after weaning and resemble genotype-specific «bar codes»
(Novikov et al, 2009). Taking into account that MUPs by themselves can efficiently activate vomeronasal neurons (Chamero et al,
of principal MUP fractions may reflect the number of 2007, 2011) and that the expression patterns of vomeronasal receptors (V2Rs) are also combinatorial (Tirindelli et al, 2009) and
sex-specific (He et al, 2008), we propose that molecular mechanisms of olfactory decoding the unique MUPs combination should
genes, which code proteins of the given fraction. Thus,
be based on complementary chemical «dialogue» between individual subsets of MUPs and specific modules of V2Rs.

«genotype» as experimental factors (GraphPad Software
Inc, San Diego, CA). Significance level was set at P0.05.
stability of the «MUPs ratio» may be regarded as a new
Flower (1996) Biochem. J., 318, 1-14; Roberts et al (2010) BMC Biol., 8, 75; Wyatt (2010) J. Comp. Physiol. A., 196, 685-700;
Janotova, Stopka (2011) J. Chem. Ecol., 37, 647-656; Kwak et al (2011) Chem. Senses, 36, 443-452; Novikov et al (2011) Chem.
Senses, 36, E31; Novikov et al (2009) Russian J. Develop. Biol., 40, 204-211; Chamero et al (2007) Nature, 450, 899-902;
feature of testosterone-dependent Mup genes expression Chamero et al (2011) Proc. Natl. Acad. Sci. USA, 108, 12898-12903; Tirindelli et al (2009) Physiol. Rev. 89, 921-956; He et al
(2008) Science, 320, 535-656.

(Chemical Senses, in preparation). Supported by grants from Russian Foundation for Basic Research (projects 02-04-49273, 04-04-63050, 07-04-01762) and State
Science and Technical Program «Priority Directions of Genetics».