Sergey Novikov1, Irina Ermakova1, Elena Fedorova1, Anatoly Philimonenko2, Gennady Churakov3

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

E-mail: novikov47@yandex.ru

Major urinary proteins (MUPs) of the house mouse Mus musculus L. form Fig 1. Electrophoresis pattern of the major urinary
a large group of highly polymorphic acidic isoforms with molecular masses
of 18-20 kDa [Hurst, 2009]. MUPs are encoded by the Mup gene cluster,
proteins (MUPs) depends on mouse physiological
which is mapped to chromosome 4 and consists of about 35 genes and state, sex and genotype
pseudogenes [Logan et al., 2008; Mudge et al., 2008]. MUPs are mainly
synthesized in the liver and in several secretory tissues, such as nasal and
lachrymal glands. 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 [e.g. Knopf et al.,
1983].
MUPs are strongly associated with volatile pheromonally active ligands.
Moreover, MUPs by themselves can also convey essential olfactory
information about sex, social rank and individuality of donors [Chamero et
al., 2007, 2011, 2012]. Thus, MUPs are widely assumed to be an important
component in formation of the olfactory signature in Mus musculus L.
[Churakov et al., 1992; Churakov & Novikov, 2000; Hurst et al., 2001;
Hurst, 2009; Roberts et al., 2010; Janotova & Stopka, 2011; Kwak et al., Table 1. Influence of factors «age» and «genotype» on
2011]. Other physiological functions of MUPs until recently remained absolute and relative values of different MUP
poorly understood. However, during last decade there is rapidly growing
fractions (two-way ANOVA test)
experimental evidence that MUPs are involved in regulation of energy
expenditure and glucose metabolism [Miller et al., 2002; Lan et al., 2003; Absolute value
Dhabli et al., 2004; Feng et al., 2005; Baur et al., 2006; Van Schothorst et
Fraction Age Genotype Interaction
al., 2006; Elchuri et al., 2007]. For example, circulating concentrations of
A 0.001 0.05 ns
MUP-1 are markedly decreased in obese mice compared with the lean
B 0.001 ns ns
controls [Hui et al., 2009; Zhou et al., 2009]. It was suggested that the
mechanism of Mup gene repression in diet-induced hypercholesterolemic C 0.001 0.001 0.001
mice is based on differential action of testosterone on Mup gene cluster D 0.001 0.001 0.001
[Feng et al., 2005]. Moreover, presence of recombinant MUP-1 (rMUP-1) E 0.001 0.001 0.001
in db/db mice alleviates insulin resistance and glucose intolerance by F 0.001 0.001 0.001
improving mitochondrial function in skeletal muscles, thereby increasing G 0.001 ns Ns
energy expenditure and enhancing insulin signaling [Hui et al., 2009; Zhou H 0.001 0.001 0.001
et al., 2009]. These findings highlight the functional importance of Notes: ns – non significant
circulating lipid-binding proteins like MUPs as an emerging class of
humoral factors, which are critically involved in energy metabolism and
insulin sensitivity [Zhou et al., 2009].
In 2009 we reported for the first time that combinatorial expression of Mup Table 2. Combinatorial genotype-dependent MUP
genes during ontogenesis in mice is reflected in genotype-specific profiles during male mice ontogenesis
proportion of different androgen-dependent MUP fractions and thus can
create an individual olfactory signature [Novikov et al., 2009]. These data
Genotype Age, weeks Fractions
also mean that testosterone as a main steroid hormone acts differentially on
expression of Mup gene cluster in male mice. Now we examine these data A B C D E F
Fig 2. Electrophoresis pattern of MUPs resembles MUPs belong to the lipocalin superfamily, whose members are
characterized by a tertiary structure with a cup-shaped hydrophobic ligand-
«bar code» binding pocket surrounded by an eight-stranded beta-barrel [Flower et al.,
2000]. This structure gives lipocalins the ability to bind and transport a
wide variety of small lipophilic substances, including pheromones, fatty
acids, cholesterols, and prostaglandins. Noticeably, several members of the
lipocalin family, including retinol-binding protein 4 (RBP4), lipocalin-2,
and adipocyte fatty acid-binding protein (A-FABP), have recently been
shown to be important mediators of obesity-related insulin resistance and
glucose intolerance [Zhou et al., 2009].
Our results demonstrate that testosterone has a differential action on Mup
gene cluster that, moreover, takes place very early after weaning. In the
light of recent findings on the involvement of testosterone-dependent
MUP-1 in genetic and diet-induced hypercholesterolemic mice [Feng et al.,
2005; Zhou et al., 2009], our data provide a new methodological approach
for creation and development of selective immunochips: microarray
matrixes based on monoclonal antibodies to various rMUPs.
Our results demonstrate that olfactory coding by MUPs in Mus musculus
Coupled with recent trends of using MUPs as perspective biomarkers in
L. is probably based on a modular principle (Tables 1-3). We suggest that
diagnosis of experimental carcinogenesis [Duan et al., 2004; Chatterji &
ratios and concentrations of MUP fractions in the given combination
Borlak, 2007; Elchuri et al., 2007; Ritorto & Borlak, 2011], nephritis
define behavioral response of the recipient mouse: some MUPs
Relative value [Wenderfer et al., 2009], and parasitic diseases [Manivannan et al., 2010],
combinations can promote stereotype intermale aggression, others can
the presented material suggests high innovative potential of the mouse
Age Genotype Interaction initiate mating-like behavior. The proposed MUP model can effectively
MUP model in studies of socially significant human diseases.
ns 0.05 ns trace the multicomponent chemosensory pathways from gene(s) to
0.001 0.01 ns olfactory-mediated physiological and social behavior responses in
laboratory mice via pheromones. This model presents valuable
ns 0.001 ns
Fig 5. Localization of genes encoding mouse resistance (Hpcr1) and
methodological approach for functional dissection of social behavior
ns 0.01 ns
sensitivity (Hcs5) to hepatocarcinogenesis, -glucuronidase (Gsub)
phenotypes using pheromones as a fine molecular tool and leads to our
ns 0.001 ns
activity, -fetoprotein (Zhx2), and MUP s(Mup1, Mupm1).
better understanding how brain translates the chemical information
0.001 0.001 0.001
encoded by odorant mixtures [Fedorova et al., 2010; Novikov et al.,
0.001 0.05 ns
2010, 2011].
0.001 0.01 0.05
Fig 3. Proposed scanning of the MUP signature by
V2Rs
Concentration formula
Consequential Ratio of principal fractions
fractions’ order
G H

in details using different physiological models of C57BL/6JY and 3 + − + + − + + + GCAHFD 0.45:0.40:0.15

CBA/LacY mouse strains. The main aim of this work was to study the
4 + + + + − + + + CADGBHF 0.60:0.25:0.15
effects of ontogenetic stage and testosterone influences on individual C57BL/6JY
expression of different MUP fractions in male mice of these common 8 + + + + − + + + CADBGFH 0.55:0.25:0.20  We demonstrate that 8 analyzed MUP fractions form two distinct subsets
strains. (modules) according to stability of their ratio during ontogenesis. The
12 + + + + − + + + CADBGFH 0.55:0.25:0.20 Specific proportion «adult proportion» profile of different MUPs is genotype-specific,
Key words: major urinary proteins (MUPs), sexual dimorphism, 3 + − − + + − + + EADGH 0.40:0:30:0:30 appears very soon after weaning and resembles a «bar code».
testosterone, modular combinatorial pattern, postnatal ontogenesis,  Concentration-based consecutive order of different MUP fractions varies
+ + + + + − + + DCEABGH 0.50:0.25:0.20:0.05
laboratory mice, individual olfactory bar-code, biomarkers, immunochips, 4 with sex, genotype and androgenic state. Presence and concentration of
CBA/LacY
microarray on MUP matrixes 8 + + + + + − + + DECABGH 0.45:0.35:0.15:0.05 various MUP fractions form an individual combinatorial pattern, which
might reflect the genotype- and gender-specific olfactory signature of the
12 + + + + + − + + DECABGH 0.60:0.20:0.10:0.10
animal. Our results show that this modular organization of MUPs pattern
is common for two genealogically unrelated strains CBA/LacY and
Quantitative evaluation of 8 protein fractions A-H with regard to age and C57BL/6JY and thus, probably, for mice in general.
genotype revealed significant interstrain differences in MUPs expression Fig 4. The combinatorial pattern of MUPs can reflect  We predict that these specific subsets (modules) of MUPs combinations
patterns in male mice during postnatal development. According to their sex and physiological state of donors and create are screened and recognized by complementary vomeronasal neurons of
relative values at various ontogenetic stages, we divided these MUP the recipients, and that these processes constitute the basis of
Subjects, housing conditions and urine collection unique odor image pheromonal coding and subsequent olfactory decoding in Mus musculus
fractions into two subsets that probably reflect their different functional
significance. The first subset, which could be characterized as L.
Experiments were performed during winter and spring periods using males and  Our data confirm the notion that testosterone plays a key role in
«ontogenetically stable», consists of fractions A, C, D, and E; the second
females of two highly inbred and genealogically unrelated strains of laboratory regulation of the Mup gene cluster and imply that not only MUP-1 but
mice, CBA/LacY and C57BL/6JY (n=262). Animals were kept in groups of subset consists of fractions B, F, G, and H, which are significantly
influenced by the age factor [Novikov et al., 2009]. also other MUPs may contribute to physiological regulation of energy
four in standard polypropylene cages T-2 («Velaz», Czech Republic) under
expenditure.
conditions of an inverted light cycle (day – 12 h, night – 12 h). Urine samples
 Together with results of other studies, our data of Mup gene expression
were taken from animals by gentle abdomen massage, individually collected in
patterns during ontogenesis suggest high innovative potential of these
plastic 2 ml Eppendorf tubes at the same time of the day, and stored at –18º C.
proteins as sensitive biomarkers in different studies of socially
Table 3. Combinatorial genotype-dependent MUP significant human diseases.
Experimental procedures and testosterone determination
codes for gender and physiological state in mice
For the several sets of experiments, six-week old male mice were castrated
Genotype Physiological state Fraction Consequential Ratio of principal fractions
using standard technique under ether anesthesia. At the age of eight weeks fractions’ order
some animals were implanted with silicone capsules («Dow Corning», USA) A B C D E F G H

containing crystalline testosterone propionate (TP) («Sigma», USA). This females + + + − − + + + CABFGH 0.80:0.20
procedure provided the daily TP dose of 27.1 μg and these animals formed the
− −
“castrated+T” group. The plasma testosterone concentration was estimated by C57BL/6JY
castrates + + + + + + CABFGH 0.50:0.50

radioimmunoassay using standard kits («Sorin», France). castrated+T + + + + − + + + CADBFGH 0.40:0.35:0.25

The authors are grateful to Sergey Mylnikov (St. Petersburg State University,
Russia) for his help in statistical data analyses and to Valentina Sukonina
Protein determination and polyacrylamide gel electrophoresis males + + + + − + + + CADBFGH 0.40:0.30:0.30 (University of Gothenburg, Sweden) for collection of experimental data and
helpful discussions.
females + − − + + − + + EADHG 0.50:0.35:0.15
The work was supported by Russian Foundation for Basic Research (projects 02-
Total urinary protein concentration was determined by the standard method Table 4. MUPs as biomarkers in various
castrates + − − + + − + + EDAHG 0.60:0.35:0.05 04-49273, 04-04-63050, 07-04-01762) and Russian State Science and Technology
[Bradford, 1976]. MUPs were analyzed by electrophoresis in polyacrylamide
CBA/LacY physiological and genetic models Program «Priority Frontiers in Genetics» (project 2.152 to SN).
gel using both tris-acetate system (pH 5.5) according to Hainey & Bishop castrated+T + + + + + − + + DEACBGH 0.45:0.30:0.15:0.10
[1982] and tris-glycine system (pH 8.2) according to Davis [1964] in our Genotype/model Sex Age Sample MUPs’ level Ref.
modification. Samples were prepared by mixing aliquots of urine (2-10 μl) males + + + + + − + + DEACBGH 0.55:0.20:0.15:0.10
BALB/c, C57BL/6, C3H,
129/Ola M, F 8-9 months liver ↓Mup 1 mRNA Miller et al., 2002
with 0.1 M tris-HCl buffer, pH 7.4, containing 20% glycerin and 0.01 % caloriс restriction
bromphenol blue. The gels were stained with Coomassie G-250 («Serva», ↓Mup 1,3,4,5 mRNA
Lan et al.,
Quantitative evaluation of 8 protein fractions A-H with regard to sex and BTBR-ob/ob M 14 weeks liver
2003
Germany). Molecular weights of MUPs were evaluated using Calibration Kit Modular encoding of the odor signatures by major urinary proteins (MUPs) in mice may create an individual bar-code: from fundamental
proteins («Sigma», USA). Quantitative analysis of fractioned MUPs was
genotype revealed that the concentration-based consecutive order of B6C3F1
M 34 months liver ↓Mup 1 mRNA Dhabli et al., 2004 aspects of olfaction to innovative prospects in biomedicine
caloriс restriction
performed using GelScan XL densitometer («Pharmacia», Sweden). different MUP fractions varies with sex and androgenic state. In other C57BL/6 *Novikov S.N., *Ermakova I.I., *Fedorova E.M., **Philimonenko A.A.,***Churakov G.A.
Feng et al.,
words, presence and concentration of various MUP fractions form an diet-induced M 12 weeks liver ↓MUPs
2005
hypercholesterolemics
Statistical data analyses individual combinatorial pattern, which might reflect the genotype- and C57BL/6NIA
*Department of Sensory Physiology, I.P. Pavlov Institute of Physiology, Russian Academy of Sciences, Saint Petersburg, Russia, **Institute of Molecular Genetics,
v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic, ***Institute of Experimental Pathology/Molecular Neurobiology (ZMBE), University of
18-24 ↑ Mup 1 mRNA Baur et al., Münster, Münster, Germany
sex-specific olfactory signature of the animal. It is noticeable that the high-calorie diet M
months
liver
↑ Mup 3 mRNA 2006
+ resveratrol
Biochemical data were expressed as means standard error. An ordinary ratios of principal fractions in testosterone-treated castrates and sham- C57BL/6J up to 14 adipose Major urinary proteins (MUPs) of the mouse represent a large group of the structurally related odorant-binding proteins with molecular masses about 18-21 kDa.
F ↓Mup 1 mRNA Schothorst et al., 2006 MUPs are synthesized mainly in liver and excreted through the kidneys with urine. Nowadays MUPs are considered as a key component of the mouse olfactory
parametric t-Student test and nonparametric Spearman correlation analyses operated males are almost the same, especially in the case of C57BL/6JY dietary obesity weeks tissue signature and can provide essential information about the individuality of donors (Novikov et al., 2009; Roberts et al., 2010; Janotova, Stopka, 2011; Kwak et al.,
2011). Other physiological functions of the MUPs remain poorly understood. However, there is rapidly growing evidence that MUPs are involved in the regulation of
mice. We suggest that our data on genotype-specific ratios of principal ↓Mup 1 mRNA
were performed. The results were also analyzed using the two-way analysis of C57BL/6 Sod1 -/- M 18 months liver
↓MUP 1
Elchuri et al., 2007 glucose metabolism (Zhou et al., 2009) and can be used as sensitive biomarkers in early diagnosis of carcinogenesis (Chatterji, Borlak, 2007; Ritorto, Borlak, 2011),
nephritis (Wenderfer et al., 2009), and parasitic diseases (Manivannan et al., 2010). Using different physiological models, we found that individual MUPs form two
variance (ANOVA) with «age» and «genotype» as experimental factors MUP fractions may reflect the number of genes, which code proteins of C57BKS db/db liver ↓Mup 1 mRNA
distinct combinatorial protein subsets (modules) which appear in both sexes very soon after weaning and resemble genotype-specific «bar-code». The presented data
suggest modular principle of the MUPs recognition of chemicals in complex mixtures by selective binding of individual components. Taking into account the recent
(GraphPad Software Inc, San Diego, CA). Significance level was set at the given fraction. Thus, stability of the «MUPs ratio» may be regarded C57BL/6J M, F 10 weeks serum
Hui et al.,
2009
trends of MUPs application as perspective biomarkers in socially significant diseases, we announce here for the first time the new methodological strategy for
creation and development of sensitive biochips based on MUPs 3D matrix.
dietary obesity ↓MUP 1
P 0.05. as a new feature of testosterone-dependent Mup genes expression urine Novikov et al (2009) Russian J. Develop. Biol., 40, 204-211; Roberts et al (2010) BMC Biol., 8, 75; Janotova, Stopka (2011) J. Chem. Ecol., 37, 647-656; Kwak et al
C57BL/6 db/db up to 16 ↓Mup 1 mRNA Zhou et al., (2011) Chem. Senses, 36, 443-452; Zhou et al (2009) J. Biol. Chem., 284, 11152-11159; Chatterji, Borlak (2007) Proteomics, 7, 3980-3991; Ritorto, Borlak (2011) J.
[Chemical Senses, in preparation]. and dietary obesity
M
weeks
liver
↓MUP 1 2009 Proteome Res., 10, 3012-3030; Wenderfer et al (2009) Amer. J. Nephrol., 30, 450-458; Manivannan et al (2010) Infection & Immunity, 78, 618-628.