Elena Fedorova1,2, Irina Ermakova1,3, Sergey Novikov1

1I.P.
Pavlov Institute of Physiology, Russian Academy of Sciences,
2Institute of Experimental Medicine, Russian Academy of Medical Sciences,
3Institute of Cytology, Russian Academy of Sciences

E-mail: ellena_fedorova@mail.ru

Aggressive behaviour represents universal type of social Fig 1. Testosterone influence on β-glucuronidase Table 4. Combinatorial genotype-dependent Our data on genotype- and sex-specific ratios of principal
communication system in Animal Kingdom. Aggression activity in voided urine, kidneys, salivary and MUP profiles during male mice ontogenesis MUP fractions (A, C, D, E) in CBA/LacY and C57BL/6JY
takes place between at least two animals after an intensive mice may provide new insights into mechanisms of genetic
exchange of information via several sensory modalities:
preputial glands from male mice of Genotype Age, Fractions Consequential Ratio of principal and epigenetic regulation of Mup gene cluster, which result
visual, acoustic, tactile, and chemosensory. Thus, genetic different genotypes weeks fractions’ fractions
in expression of specific bouquets of MUPs. We propose that
A B C D E F G H order
analysis of aggression is rather problematic due to complex various volatile pheromonally active ligands (e.g. SBT,
Kidneys CBA/LacY
Urine
and highly dynamic interactions between animals. In mice, 30000 C57BL/6JY 250
3 + − + + − + + + GCAHFD 0.45:0.40:0.15 HMH, DHB) represent «letters» of the chemical «language
like in many other mammals, the leading sensory modality is 25000 200 of communication» in Mus musculus L. and that,
20000
4 + + + + − + + + CADGBHF 0.60:0.25:0.15
olfaction, and much of the information is conveyed by highly 150
correspondingly, specific combinations of MUPs efficiently
F.U.
F.U.

15000
C57BL/6JY
organize these «letters» into readable «words» that encode
100
specific ligands, pheromones, which have the ability to 10000
50 8 + + + + − + + + CADBGFH 0.55:0.25:0.20
5000
initiate innate (genetically programmed) behavioural 0 0 sex, physiological state, age, and genotype differences
responses from receiver (Novikov, 1993). Sham-operated Castrated Castrated+TP Sham-operated Castrated Castrated+TP
12 + + + + − + + + CADBGFH 0.55:0.25:0.20 (Novikov et al., 2009).
The model of pheromonally mediated aggression in Mus Preputial glands Salivary glands Together with the data showing that vomeronasal neurons
musculus L. was introduced in social behaviour analysis 35
1500 10000
3 + − − + + − + + EADGH 0.40:0:30:0:30 can be directly activated by MUPs (Chamero et al., 2007),
1200 8000
years ago (Kessler et al., 1975). However, until recently 900 6000 and that expression of pheromone receptors V2Rs is sex
+ + + + + − + + DCEABGH 0.50:0.25:0.20:0.05
F.U.

F.U.

genetical basis of differences in physiological activity of 600
mouse androgen-dependent pheromones such as 2-sec-butyl- 300
4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone 0
(HMH), and 2,3-dehydro-exo-brevicomin (DHB) (Novotny
et al., 1999; Perez-Miller et al., 2010) was poorly
understood.
In 2007 it was reported that the main fraction of mouse
urinary proteins with M.m.18-20 kDa, named «major urinary Fig 2. β-Glucuronidase activity in kidneys and
voided urine from male mice of
proteins» (MUPs), by itself and without pheromonal ligands
(SBT, HMH, DHB) could efficiently promote aggressive
reactions from the recipient (Chamero et al., 2007). It means
that MUPs are a powerful tool for genetical dissection of
10000
social behaviour (Novikov, 2007, 2008). 8000
In 2003 we put forward an idea that genes Mup, Gus and 6000
F.U.
4
4000
CBA/LacY
specific (He et al., 2008, 2010) and combinatorial (Silvotti et
2000
8 + + + + + − + + DECABGH 0.45:0.35:0.15:0.05 al., 2007), our results provide valuable insights into fine
0
Sham-operated Castrated+TP Sham-operated Castrated Castrated+TP
molecular mechanisms of olfactory coding of intermale
12 + + + + + − + + DECABGH 0.60:0.20:0.10:0.10 aggression in Mus musculus L. based on specific subsets
(modules) of MUPs combinations and their interplay with
Quantitative evaluation of 8 protein fractions A-H with regard to complementary sets of V2Rs.
age and genotype revealed significant interstrain differences in We suggest that androgen- and genotype-dependent activity
MUPs expression patterns in male mice during postnatal
of kidney metallopeptidase meprin (EC 3.4.24.18), which
development. According to their relative values at various
probably participates in degradation of MUPs (Beynon et al.,
ontogenetic stages, we divided these MUP fractions into two
different genotypes subsets that probably reflect their different functional significance. 1996), is tightly linked with pheromonal activity of several
The first subset, which could be characterized as «ontogenetically oligopeptides (Tsujikawa, Kashiwayanagi, 1999; Kimoto et
Kidneys Urine stable», consists of fractions A, C, D, and E; the second subset al., 2005, 2007). This peptidase is encoded by Mep1a gene
500
400
consists of fractions B, F, G, and H, which are significantly mapped to chromosome 17 near the main histocompatibility
300 influenced by the age factor (Chemical Senses, in preparation). locus H-2. Taking into account that H-2 complex also

F.U.

Mep1a form an «aggressive behaviour gene network» 4000

2000
200

100
participates in olfactory-mediated social recognition in mice
(Novikov, 2003). Gus gene is located on chromosome 5 and 0 0 (Boehm, 2006, 2009; Brennan, Kendrick, 2006; Rodriguez,
encodes β-glucuronidase (GUS, EC 3.2.1.31), which
CBA/LacY CBAB6F1 B6CBAF1 C57BL/6JY CBA/LacY CBAB6F1 B6CBAF1 C57BL/6JY Table 5. Combinatorial genotype-dependent Boehm, 2009; Tirindelli et al., 2009; Kwak et al., 2010), we
Genotype Genotype
activates latent aggression-promoting cues in mouse urine MUP codes for gender and physiological propose for the first time that Gus, Mup, Mep1a, and H2
(Ingersoll et al., 1982). Mep1a is mapped to chromosome 17 state in mice genes represent an «aggressive behaviour gene net» in Mus
and encodes meprin (EC 3.4.24.18), a metallopeptidase that musculus L. (Figure 4).
Genotype Physiological Fraction Consequential Ratio of principal
probably participates in degradation of MUPs (Beynon et al., state fractions’ fractions
1996). Mup multigene locus is located on chromosome 4. In Table 1. Correlation between plasma A B C D E F G H order Fig 4. Proposed «aggressive behaviour gene net»
the current study, we examined the idea of the «gene testosterone level and β-glucuronidase activity in females + + + − − + + + CABFGH 0.80:0.20
in Mus musculus L.
network» in details. Quantitative analysis of plasma kidneys and voided urine from male mice of
testosterone level, MUPs content and GUS activity in voided different genotypes castrates + + + − − + + + CABFGH 0.50:0.50
urine, kidney, preputial and salivary glands in male mice of C57BL/6JY
different genotypes revealed a strong correlation between castrated+T + + + + − + + + CADBFGH 0.40:0.35:0.25
Kidneys Urine
plasma testosterone level, GUS activity, and differential Genotype males + + + + − + + + CADBFGH 0.40:0.30:0.30
pattern of Mup gene expression. F.U. F.U./mg tissue F.U.
females + − − + + − + + EADHG 0.50:0.35:0.15
CBA/LacY +0.670* +0.702* +0.851**

Key words: pheromones, major urinary proteins, β- castrates + − − + + − + + EDAHG 0.60:0.35:0.05

CBAB6F1 +0.348 +0.390 +0.327
glucuronidase, aggressive behaviour, MUP combinatorial CBA/LacY
pattern, laboratory mice, olfactory code castrated+T + + + + + − + + DEACBGH 0.45:0.30:0.15:0.10
B6CBAF1 -0.109 -0.074 +0.243

males + + + + + − + + DEACBGH 0.55:0.20:0.15:0.10

C57BL/6JY +0.158 +0.026 +0.382

*p<0.05 Quantitative evaluation of 8 protein fractions A-H with regard to

**p<0.01 genotype and sex revealed that the concentration-based consecutive
order of different MUP fractions varies with sex and androgenic
state. In other words, presence and concentration of various MUP  Our study revealed highly complex tissue- and genotype-
fractions form an individual combinatorial pattern, which might specific androgen influence on expression of Mup and Gus
reflect the genotype- and sex-specific olfactory signature of the genes.
Fig 3. Content of MUP fractions in male mice of animal. It is noticeable that the ratios of principal fractions in  We demonstrate that 8 analyzed MUP fractions form two
different genotypes testosterone-treated castrates and sham-operated males are almost distinct subsets (modules) according to stability of their ratio
the same, especially in the case of C57BL/6JY mice. We suggest during ontogenesis. The «adult proportion» profile of different
Subjects, housing conditions and urine collection that our data on genotype-specific ratios of principal MUP fractions MUPs is genotype-specific, appears very soon after weaning
3,5
CBA/LacY may reflect the number of genes, which code proteins of the given and resembles a «bar code».
Experiments were performed during winter and spring periods
3 fraction. Thus, stability of the «MUPs ratio» may be regarded as a  Concentration-based consecutive order of different MUP
using males and females of two highly inbred and genealogically C57BL/6JY
new feature of testosterone-dependent Mup genes expression fractions varies with sex, genotype and androgenic state.
unrelated strains of laboratory mice, CBA/LacY and C57BL/6JY, 2,5
(Chemical Senses, in preparation). Presence and concentration of various MUP fractions form an
and their F1 hybrids from reciprocal crosses (n=365). Animals 2
mg/ml

individual combinatorial pattern, which might reflect the

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, and stored
at –18º C.
Three sets of experiments were performed. In the first set, we
studied β-glucuronidase activity in male mice urine, kidney, and
two pheromone-producing organs, preputial and salivary glands. In
the second set of experiments, we studied the influence of genotype
on MUPs production in male mice during postnatal development.
In the third set, effects of male castration and testosterone therapy
on MUPs excretion profile were investigated.
Experimental procedures and testosterone measurement
For the first and third sets of experiments, six-week old male mice
were castrated using standard technique under ether anesthesia. At
the age of eight weeks some animals were implanted with silicone
capsules («Dow Corning», USA) containing crystalline
testosterone propionate (TP) («Sigma», USA). This procedure
provided the daily TP dose of 27.1 μg and these animals formed the
«castrated+TP» group. The plasma testosterone concentration was
estimated by radioimmunoassay using standard kits («Sorin»,
France).
MUPs characterization and β-glucuronidase activity
measurement
MUPs were analyzed by electrophoresis in polyacrylamide gel
(PAGE) (Churakov et al., 1992; Churakov, Novikov, 2000).
Separation of native proteins was performed using 0.1 M tris-
acetate buffer, pH 5.5. Samples were prepared by 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
MUP fractions were evaluated using Calibration Kit proteins
(«Sigma», USA). Quantitative analysis of MUP fractions was
performed using GelScan XL densitometer («Pharmacia»,
Sweden). The activity of native lysosomal β-glucuronidase (EC
3.2.1.31) was measured in voided urine and homogenated samples
of kidney, preputial and salivary glands by the rate of
phenolphthalein-β-D-glucuronide hydrolysis («Sigma», USA), and
expressed in Fishman’s units (F.U.) according to standard method.
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 «genotype» as experimental factors (GraphPad Software
Inc, San Diego, CA). Significance level was set at P0.05.
1,5 genotype- and gender-specific olfactory signature of the animal.
Table 6. Combinatorial genotype-dependent Our results show that this modular organization of MUPs
1
MUP codes in F1 progeny from pattern is common for two genealogically unrelated strains
0,5 CBA/LacY and C57BL/6JY and thus, probably, for mice in
reciprocal crosses general.
0
Gender Genotype Fraction Consequential Ratio of principal  We suggest that these specific subsets (modules) of MUPs
A B C D E F G H
fractions’ fractions combinations are subsequently screened and recognized by
MUP fractions order
A B C D E F G H complementary vomeronasal neurons of the recipients, and that
these processes constitute the basis of pheromonal coding in
CBAB6F1 + + + + + + + + DCEABGHF 0.40:0.25:0.20:0.15
Mus musculus L.
Male
 Taken together, our data demonstrate that MUPs may represent
B6CBAF1 + + + + + + + + DACEBGHF 0.45:0.25:0.15:0.15
a very promising tool for genetic dissection of olfactory-
mediated aggressive behaviour in Mus musculus L.
Table 2. Correlation between plasma CBAB6F1 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10
Female
testosterone level and concentrations of MUP B6CBAF1 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10
fractions in male mice of different genotypes
In urine of male and female F1 progeny from reciprocal crosses, all
8 MUP fractions are present. Concentration-based «MUP
Fraction
formulae» are with high significance similar within sex:
Spearman’s rank-order correlation coefficients are rs=0.91 in males The authors are grateful to Gennady Churakov (University of
Genotype A B C D E F G H
and rs=0.98 in females (P<0.01). Ratios of principal fractions are Münster, Germany), Anatoly Philimonenko (Institute of Molecular
CBA/LacY -0.604 +0.846* +0.698 +0.122 -0.467 - +0.682 +0.116 also similar within sex, which is especially obvious in females of Genetics, Prague, Czech Republic) and Valentina Sukonina (Umeå
both genotypes (Table 6). However, at the moment it is not clear University, Sweden) for collection of experimental data and helpful
CBAB6F1 +0.777 +0.746* +0.027 +0.501 +0.519 +0.672 +0.148 +0.631 how the presence and ratios of different MUP fractions are defined discussions, and to Sergey Mylnikov (St. Petersburg State
in F1 animals and to what extent parental patterns are inherited University, Russia) for his invaluable help in statistical data
B6CBAF1 -0.650 +0.054 +0.455 +0.687 +0.502 +0.693 +0.948* +0.942* analyses.
(Chemical Senses, in preparation).
C57BL/6JY +0.295 +0.703 +0.717 +0.597 - +0.707 +0.454 +0.024
The obtained data revealed distinct interstrain differences in β- The work was supported by Russian Foundation for Basic
Research (projects 02-04-49273, 04-04-63050, 07-04-01762) and
glucuronidase activity in kidney, urine and salivary glands (Figure
Russian State Science and Technology Program «Priority Frontiers
*p˂0.05 1). Importantly, urine from testosterone-treated castrates and
shame-operated C57BL/6JY males, which were previously in Genetics» (project 2.152 to SN).
characterized as «pheromone-deficient» mice (Novikov, 1988), has
a very low level of β-glucuronidase activity (Figures 1, 2). Elena Fedorova is deeply grateful to the Chromosome Research
journal for awarding her with the 2008 Scholarly Manuscript Prize,
However, it was previously shown that incubation of urine samples
Table 3. Correlation between plasma from C57BL/6JY with commercial GUS increases the activity of which allowed her to participate in this ECRO Congress.
testosterone level and proportions of MUP aggression-promoting pheromones (Novikov, 1993). Thus, GUS
fractions in male mice of different genotypes activity is an important factor in formation of the individual
combination of pheromones and, consequently, in the process of
communication.
Fraction Our results also demonstrate that olfactory coding by MUPs in Mus
musculus L. is probably based on a modular principle (Tables 4-6).
We suggest that ratios and concentrations of MUP fractions in the Chamero P, Marton TF, Logan DW, Flanagan K, Cruz JR, Saghatelian A, Cravatt
Genotype A B C D E F G H
BF, Stowers L. Identification of protein pheromones that promote aggressive
given combination define behavioural response of the recipient behaviour. Nature. 2007. 450:899-902. He J, Ma L, Kim SS, Nakai J, Yu CR.
CBA/LacY -0.603 +0.867* +0.697 -0.016 -0.557 - +0.736 -0.011 mouse: some MUPs combinations can promote stereotype Encoding gender and individual information in the mouse vomeronasal organ.
intermale aggression, others can initiate mating-like behaviour. The Science. 2008. 320:535-538. Kimoto H, Sato K, Nodari F, Haga S, Holy TE,
CBAB6F1 +0.644 +0.204 -0.557 -0.282 +0.101 +0.497 -0.349 +0.253 proposed GUS and MUP models can effectively trace the Touhara K. Sex- and strain-specific expression and vomeronasal activity of mouse
multicomponent chemosensory pathways from gene(s) to ESP family peptides. Curr. Biol. 2007. 17:1879-1884. Novikov SN, Churakov GA,
Philimonenko AA, Ermakova II, Fedorova EM, Burkot IA. The pattern of major
B6CBAF1 -0.667 -0.144 +0.407 +0.613 +0.463 +0.684 +0.945* +0.950* olfactory-mediated physiological and social behaviour responses in urinary proteins (MUPs) expression during postnatal ontogenesis of the laboratory
laboratory mice via pheromones. These models represent a mouse depends on genotype and sex. Ontogenez. 2009. 40:261-269. Novikov SN.
C57BL/6JY -0.339 +0.739 +0.328 +0.353 - +0.641 -0.198 -0.775* valuable methodological approach for dissection of aggressive Pheromones and Reproduction in Mammals: Physiological Aspects. 1988. Nauka
behaviour phenotypes using pheromones as a fine natural tool and Publishing House. 169 pp. Novikov SN. The genetics of pheromonally mediated
intermale aggression in mice: current status and prospects of the model. Behav.
*p˂0.05 lead to our better understanding of how brain translates chemical Genet. 1993. 23:505-508. Silvotti L, Moiani A, Gatti A, Tirindelli R. Combinatorial
information encoded by odorant mixtures (Novikov, 2007, 2008). co-expression of pheromone receptors, V2Rs. J. Neurochem. 2007. 103:1753-1763.