ISSN 1062-3604, Russian Journal of Developmental Biology, 2009, Vol. 40, No. 4, pp. 204–211. © Pleiades Publishing, Inc.

, 2009.
Original Russian Text © S.N. Novikov, G.A. Churakov, A.A. Philimonenko, I.I. Ermakova, E.M. Fedorova, I.A. Burkot, 2009, published in Ontogenez, 2009, Vol. 40, No. 4,
pp. 261–269.

DEVELOPMENTAL GENETICS

The Pattern of Major Urinary Proteins (MUPS) Expression

during Postnatal Ontogenesis of the Laboratory Mouse Depends
on Genotype and Sex1, 2
S. N. Novikov, G. A. Churakov, A. A. Philimonenko, I. I. Ermakova,
E. M. Fedorova, and I. A. Burkot
I.P. Pavlov Institute of Physiology, Russian Academy of Sciences, Saint Petersburg, nab. Makarova, 6, 199034 Russia
Received June 8, 2007; in final form, November 12, 2008

Abstract—We investigated the specific pattern of major urinary proteins (MUPs) expression in 3-, 4-, and
12-week old mice of CBA/LacY and C57BL/6JY inbred strains using polyacrylamide gel electrophoresis.
Quantitative evaluation of 8 protein fractions A-H with regard to sex, age, and genotype of the animals is pre-
sented for the first time. Actual problems of genetic control and neuroendocrine regulation of MUPs expression
during ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulation
information exchange via pheromones, we put forward the idea that the genetically determined structure of the
olfactory code of the definitive type is formed at an early ontogenetic stage on the basis of the MUPs combina-
torial pattern.
DOI: 10.1134/S1062360409040031

Key words: pre- and postpubertal periods, laboratory mice, major urinary proteins, sex differences, phero-
mones, olfactory image, MUPs combinatorial pattern, structure of the olfactory code.

12 One of the most mysterious sides in the life activity of
various rodents is associated with the phenomenon of
“physiological proteinuria” (Parfentjev, 1932). Despite the
fact that the molecular-genetic basis and physiological
mechanisms of the phenomenon itself were studied in detail
(Hastie et al., 1979; Berger, Szoka, 1981; Knopf et al., 1983;
Kuhn et al., 1984; McIntosh, Bishop, 1989; Al-Shavi et al.,
1992), the functional meaning of the sharply raised level of
protein excretion in urine remained until recently the most
difficult puzzle in the biology of this numerous systematic
group.
The first reports regarding a possible regulatory role of
proteins found in urine of the house mouse, and particu-
larly of MUP (major urinary protein) complex fractions, in
the processes of intrapopulation informational exchange
via pheromones appeared in the beginning of the 1990th
(Böcskei et al., 1992; Bacchini et al., 1992; Churakov et al.,
1992; Robertson et al., 1993). These data caused many
detailed experimental and population genetic investigations
of the structural and functional features of MUPs fractions
(Robertson et al., 1996, 1997; Utsumi et al., 1999; Chura-
kov, Novikov, 2000; Marchlewska-Koj et al., 2000; Baba-
lyan, Novikov, 2001; Hurst et al., 2001, 2005; Marie et al.,
2001; Timm et al., 2001; Daev, Sverdlova, 2002; Sharrow et
al., 2002, 2005; Novikov, 2003; Armstrong et al., 2005;
1 The work was supported by the Russian Foundation for Basic
Research (projects no. 02-04-49273, 04-04-63050).
2 The article was translated by the authors.
Cavaggioni et al., 2006; Macek et al., 2006; More, 2006;
Stopkova et al., 2007).
It is an accepted hypothesis that excretion of MUPs
with urine is typical for males and is directly linked to the
androgenic status of an organism (Szoka, Paigen, 1978,
1979; Berger, Szoka, 1981; Hayakawa et al., 1983).
Meanwhile, as we have demonstrated earlier, in laboratory
mice the qualitative composition of MUPs of castrated
males is identical to that of females, and the MUPs pattern
is determined genetically (Churakov, Novikov, 2000).
These data allow us to put forward a question about the
role of genetic and age-related factors in the establishment
of sex differences in MUPs composition during postnatal
ontogenesis of the laboratory mouse.
The aim of our research was to perform a comparative
quantitative analysis of MUPs expression between males
and females of laboratory mice of two genotypes during
the pre- and postpubertal periods of ontogenesis.
MATERIALS AND METHODS
The experiments were performed using males and
females of two highly inbred and genealogically unrelated
laboratory mice strains, CBA/LacY and C57BL/6JY (n =
102). Animals were kept in groups of 4–6 individuals in
standard polypropylene cages T-2 (“Velaz”, Czech Repub-
lic) in the inverted light cycle conditions (day—12 h,
night—12 h). Our experiments were performed in spring.

204
 THE PATTERN OF MAJOR URINARY PROTEINS (MUPS) EXPRESSION 205

Table 1. Age dynamics of MUPs fractions content (mg/ml) in urine of CBA/LacY male and female laboratory mice

MUPs Age of males, weeks Age of females, weeks

K K
fraction 3 4 12 3 4 12
A 0.015 ± 0.0042 0.02 ± 0.007 0.45 ± 0.075 30.0 0.020 ± 0.0033 0.060 ± 0.0187 0.27 ± 0.070 13.5
B 0 0.03 ± 0.013 0.23 ± 0.019 7.7 – – – –
C 0 0.07 ± 0.050 0.51 ± 0.082 7.3 – – – –
D 0.014 ± 0.0031 0.16 ± 0.101 2.97 ± 0.141 212.1 0.008 ± 0.0014 0.028 ± 0.0062 0.24 ± 0.047 30.0
E 0.018 ± 0.0046 0.06 ± 0.037 1.06 ± 0.045 58.9 0.021 ± 0.0056 0.075 ± 0.0212 0.56 ± 0.103 26.7
F – – – – – – – –
G 0.010 ± 0.0029 0.02 ± 0.007 0.15 ± 0.012 15.0 0.010 ± 0.0021 0.031 ± 0.0101 0.07 ± 0.006 7.0
H 0.005 ± 0.0009 0.01 ± 0.003 0.13 ± 0.009 26.0 0.008 ± 0.0019 0.014 ± 0.0044 0.04 ± 0.005 5.0
ΣA–H 0.064 ± 0.0116 0.37 ± 0.213 5.49 ± 0.127 85.8 0.068 ± 0.0103 0.210 ± 0.0561 1.19 ± 0.213 17.5
Note: K is the factor of the fraction content increase.

Urine was taken from 3-, 4, and 12-week-old mice by
gentle abdomen massage, individually collected in plastic
2 ml Eppendorf tubes always at the same time of the day,
and stored at –18°C.
MUPs were analyzed by electrophoresis in polyacryla-
mide gel. Separation of native proteins was performed
using 0.1 M tris-acetate buffer, pH 5.5. Samples were pre-
pared 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 Coo-
massie G-250 (“Serva”, Germany). Molecular weights of
MUPs were evaluated using Calibration Kit proteins
(“Sigma”, USA).
Total urinary protein concentration was determined by
the Bradford method (Bradford, 1976). Quantitative anal-
ysis of the fractionated MUPs was performed at the Bio-
chemistry Department of the N.I. Vavilov Research Insti-
tute of Plant Industry RAAS using a GelScan XL densito-
meter (“Pharmacia”, Sweden).
Statistical analyses of the experimental data were per-
formed with the GraphPad Prism 4 software (GraphPad
Software, USA).
RESULTS
Age dynamics of MUPs expression in male and female
laboratory mice of CBA/LacY strain. As shown in Table 1,
males of this genotype have a markedly heterogeneous
combination of MUP fractions already at the 3rd week of
ontogenesis; when they are 4-week-old, fractions B and C
appear in their urine. Partial value of these fractions
decreases from 25.7 to 13.4% during the analyzed period
(Fig. a), while their absolute values progressively increase
to the 12th week of ontogenesis (Table 1).
Total content of MUP fractions increases in males
85.8-fold to the 12th week; the minimal factor of incre-
ment belongs to fraction C (äë = 7.3), the maximal one to
fraction D (äD = 212.1). Percentage of this major fraction
in the general MUP pool increases from 22.6% in prepu-
bertal 3-week-old animals to 54.1% in 12-week-old males
(p < 0.01) (Fig. a).
In contrast to males, females of this genotype do not
have fractions B and C in their MUP complex (Table 1);
partial values of the other five fractions remain relatively
stable while their absolute values rise steadily (Fig. 1b).
Total content of MUPs fractions in females increases
17.5-fold to the 12th week; the minimal increment factor
belongs to fraction H (äH = 5.0), the maximal one to frac-
tions D and E (äD = 30.0, äE = 26.7). Percentage of the
major fraction E in the general MUP pool grows from
31.9% in prepubertal 3-week-old animals to 47.8% in
12-week-old females (p < 0.01).
Generally, the rate of increment of MUPs fractions
content in urine of CBA/LacY males is 4.9 times higher
than that of females (Table 1).
Age dynamics of MUPs expression in male and female
laboratory mice of C57BL/6JY strain. As demonstrated in
Table 2, already 3-week-old animals have a heterogeneous
combination of MUP fractions; fraction B appears in urine
of 4-week-old males of this genotype.
Total MUPs fractions content increases 123.6-fold in
males during the analyzed period; the minimal increment
factor belongs to fraction G (äG = 13.8) and the maximal
one to fraction D (äD = 265.0). The partial value of this
fraction differs considerably in pre- and postpubertal peri-
ods and comprises correspondingly 7.9, 7.1 and 15.6% in
3-, 4-, and 12-week-old animals. Percentage of fraction C
which prevails in adult animals (41.7%) comprises 21.1%
in prepubertal 3-week-old males (Fig. 1c).
Contrary to males, females of this strain do not have
fraction D; fraction B appears in their MUP complex
when they are 4 weeks old (Table 2). Total content of
MUPs increases 38.6-fold to the 12th week; the minimal
increment factor belongs to fraction H (äH = 5.0), the
maximal one to fraction A (äA = 84.0), and percentage of
the major fraction C virtually is stable (Fig. 1d).

RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY Vol. 40 No. 4 2009

206 NOVIKOV et al.

‡ b
100

80

60

40

20

0
c d
100

80

60

40

20

0
3 4 12 3 4 12

Fig. Percentages of MUPs fractions (Y axis, %) of male (a, c) and female (b, d) laboratory mice of CBA/acY (a, b) and
C57BL/6JY (c, d) strain at various ontogenetic points (X axis, weeks). A – ( ), B – ( ), C – ( ), D – ( ), E – ( ),
F−( ), G – ( ), H – ( ).

Generally, the rate of increment of MUPs fractions
content in urine of C57BL/6JY males is 3.2 times higher
than that of females (Table 2).
Results of two-factor dispersion analysis demonstrate
that age significantly influences the absolute values of
MUPs fractions but has practically no effect on their par-
tial values (Tables 3, 4).
DISCUSSION
Postnatal ontogenesis of the house mouse Mus muscu-
lus L. is traditionally divided into several periods. Of par-
ticular importance is the so-called “socialization” stage,
when young mice leave their nests and begin to feed
actively; this 5–7 days long stage is over by the 4–5th week
of ontogenesis. Immediately after that, hierarchical relation-
ships between males are established. This process is associ-
ated with a dramatic increase of the level of aggressiveness
(McKinney, Desjardins, 1973; Barkley, Goldman, 1977),
physiologically based upon a change of the neuroendocrine
state of the animal, and first of all upon the manifold
increase of testosterone production by testes (Selmanoff et
al., 1977a, b; Jean-Faucher, 1978). This relatively long
period is over by the 7–8th week, and it is accompanied by
maturation of a number of enzymatic systems that take part

Table 2. Age dynamics of MUPs fractions content (mg/ml) in urine of C57BL/6JY male and female laboratory mice
MUPs Age of males, weeks Age of females, weeks
K K
fraction 3 4 12 3 4 12
A 0.005 ± 0.0009 0.007 ± 0.0013 0.68 ± 0.157 136.0 0.005 ± 0.0014 0.007 ± 0.0016 0.42 ± 0.125 84.0
B 0 0.006 ± 0.0017 0.51 ± 0.111 85.0 0 0.007 ± 0.0037 0.13 ± 0.040 18.6
C 0.006 ± 0.0014 0.018 ± 0.0078 1.43 ± 0.182 238.3 0.022 ± 0.0134 0.041 ± 0.0295 0.99 ± 0.197 45.0
D 0.002 ± 0.0003 0.004 ± 0.0008 0.53 ± 0.152 265.0 – – – –
E – – – – – – – –
F 0.002 ± 0.0002 0.002 ± 0.0004 0.09 ± 0.031 45.0 0.003 ± 0.0014 0.004 ± 0.0016 0.06 ± 0.019 20.0
G 0.008 ± 0.0013 0.009 ± 0.0030 0.11 ± 0.022 13.8 0.006 ± 0.0030 0.007 ± 0.0034 0.04 ± 0.010 6.7
H 0.004 ± 0.0005 0.004 ± 0.0010 0.07 ± 0.010 17.5 0.006 ± 0.0025 0.007 ± 0.0035 0.03 ± 0.006 5.0
ΣA–H 0.028 ± 0.0030 0.050 ± 0.0140 3.46 ± 0.628 123.6 0.043 ± 0.0205 0.072 ± 0.0406 1.66 ± 0.370 38.6
Note: K is the factor of the fraction content increase.

RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY Vol. 40 No. 4 2009

 THE PATTERN OF MAJOR URINARY PROTEINS (MUPS) EXPRESSION 207

Table 3. Results of the two-factor dispersion analysis of dynamics of absolute values of MUPs fractions during ontogenesis
of male and female laboratory mice of CBA/LacY and C57BL/6JY strains

Source of variability Degrees of freedom, df Mean square, Ms F test, F Significance, p

Males CBA
Age 2 11.84 830.1 <0.0001
Fraction 6 3.396 238.2 <0.0001
Interaction 12 2.910 204.1 <0.0001
Random deviations 175 0.01426
Females CBA
Age 2 0.9527 50.51 <0.0001
Fraction 4 0.2428 12.87 <0.0001
Interaction 8 0.1655 8.775 <0.0001
Random deviations 180 0.01886
Males C57BL/6
Age 2 3.608 107.6 <0.0001
Fraction 6 0.5266 15.7 <0.0001
Interaction 12 0.5115 15.25 <0.0001
Random deviations 119 0.03355
Females C57BL/6
Age 2 0.6924 29.14 <0.0001
Fraction 5 0.2577 10.84 <0.0001
Interaction 10 0.2209 9.296 <0.0001
Random deviations 72 0.02376

in metabolism of testosterone and cell receptors to dihy-
drotestosterone (Minetti et al., 1986; Murono, Washburn,
1989).
Our results are in accordance with published data
about the age dynamics of MUPs mRNAs in hepatocytes
of the laboratory mouse (Derman, 1981; Barth et al.,
1982). At the same time, discrimination between pre- and
postpubertal periods allowed us to reveal significant inter-
strain differences in specifics of concentration changes of
similar protein fractions in males and females.
Progressive enlargement of MUPs fractions content
during ontogenesis is typical for both strains of mice
(Tables 1, 2). It is noteworthy that start concentration
values of most of the fractions in immature 3-week-old
CBA/LacY animals is much higher than that in
C57BL/6JY mice of the same age. We have demon-
strated earlier that these differences smooth over in males
by their 8th week, and the factor of rank correlation
between absolute values of the same fractions in juvenile
4-week-old and mature 8-week-old animals of CBA/LacY
strain has a highly significant positive value (rs = +0.96,
p < 0.01) (Churakov, Novikov, 2000). These data indicate
that the pattern of MUPs percentages in immature mice of
this genotype corresponds to that in mature animals. It is
known that MUPs are structurally and molecularly identi-
cal in juvenile and mature animals of CBA/LacY strain
and that MUPs synthesis is androgen-dependent. Taking
these data into account, we propose that genetic control of
the “adult way” of biosynthesis of these proteins in early
ontogenesis, and formation of the corresponding “adult”
MUPs pattern as a feature of males of this genotype, chro-
nologically precede the abrupt increase of testosterone
level in blood at the 30th day of postnatal ontogenesis
(Selmanoff et al., 1977b; Jean-Faucher et al., 1978).
Our results confirm the existing hypothesis that expres-
sion of fractions B and C in CBA/LacY strain and of frac-
tion D in C57BL/6JY strain is androgen-dependent
(Churakov, Novikov, 2000). Importantly, 3-week-old ani-
mals of both strains lack fraction B, and in CBA/Lac strain
fraction C is also absent. On the other hand, in the latter
strain fraction D is expressed already at the 3rd week,
while androgen-dependent fraction B is absent in the
MUP complex. This could either suggest an enhanced bio-
synthesis of proteins, which make up the electrophoretic

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208 NOVIKOV et al.

Table 4. Results of the two-factor dispersion analysis of dynamics of MUPs fractions percentages during ontogenesis of male
and female laboratory mice of CBA/LacY and C57BL/6JY strains

Source of variability Degrees of freedom, df Mean square, MS F test, F Significance, p

Males CBA
Age 2 0.0 0.0 ns
Fraction 6 0.4467 6.290 <0.0001
Interaction 12 0.08342 1.175 ns
Random deviations 175 0.01426
Females CBA
Age 2 0.0 0.0 ns
Fraction 4 0.6343 12.25 <0.0001
Interaction 8 0.04813 0.9292 ns
Random deviations 180 0.05180
Males C57BL/6
Age 2 0.0 0.0 ns
Fraction 6 0.1770 2.482 <0.05
Interaction 12 0.04651 0.6521 ns
Random deviations 119 0.07133
Females C57BL/6
Age 2 0.0 0.0 ns
Fraction 5 0.5643 5.903 <0.0001
Interaction 10 0.01820 0.1904 ns
Random deviations 72 0.09560
Note: ns indicates nonsignificant values.

fraction D, or indicate the existence of inter-strain varia-
tions in the expression of fraction B. In favor of the first
suggestion is the fact that fraction D is the one whose con-
tent increases sharply in 12-week-old animals (Table 1); in
favor of the second one are obvious inter-strain differences
in dynamics of the fraction B concentration during onto-
genesis (Tables 1, 2).
It should be noted that in contrast to laboratory mice of
other strains, males of C57 family have a relatively low
level of plasma testosterone (Sustarsic, Wolfe, 1976; Sel-
manoff et al., 1977b; Stalvey, Payne, 1983; Diuzhikova,
1994) and an increased tissue sensitivity to its physiologi-
cally active metabolite, dihydrotestosterone (Bartke,
1974). This probably explains the sharp increase of the
intensity of MUPs expression from the 4th to the
8th weeks of ontogenesis in C57BL/6JY mice, and prima-
rily of the androgen-dependent fraction D (Table 2). It is
also known that males of C57BL/6JY strain, in contrast to
those of CBA/Kw strain, have a lower level of functional
activity of thyroid gland (Marchlewska-Koj et al., 1974).
Taking into account the data regarding thyroxin-depen-
dent expression of Mup genes (Knopf et al., 1983;
Kuhn et al., 1984), we propose that our results demon-
strate the inter-strain differences of the level of this hor-
mone during postnatal ontogenesis of the laboratory
mouse (Minetti et al., 1986). Growth hormone and prolac-
tin could also play an essential role in generation of inter-
sex differences in dynamics of the same MUPs fractions
during ontogenesis (Michael et al., 1980; Norstedt, Palm-
iter, 1984; Johnson et al., 1995).
Mechanisms of hormonal control of expression of cer-
tain protein fractions during female ontogenesis, particu-
larly of fraction E in CBA/LacY strain and fraction H in
C57BL/6JY strain, could be of special interest with regard
to further studies of functional and structural features of
MUPs. Fraction E, which is the major biochemical marker
of the CBA/LacY strain, is expressed in both males and
females (Table 1). On the other hand, its content correlates

RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY Vol. 40 No. 4 2009

 THE PATTERN OF MAJOR URINARY PROTEINS (MUPS) EXPRESSION
negatively with the level of plasma testosterone (rs =
−0.98,  < 0.05). Percentage of this fraction in mature
males increases sharply after castration, and in mature
females it comprises more than 50% of the total MUPs
pool (Churakov, Novikov, 2000). Our data suggest that the
MUP genes, which encode fraction E proteins, are par-
tially repressed by testosterone, whose physiological role
at different stages of ontogenesis depends on genotype
(Akhmerova et al., 2002; Akhmerova, 2006).
With regard to the minor fraction H in C57BL/6JY ani-
mals it was shown earlier (Churakov, Novikov, 2000) that
in mature males its concentration negatively correlates
with the level of plasma testosterone (rs = –0.78, p < 0.05)
and that its percentage progressively decreases during
ontogenesis (Fig. 1c and 1d).
Thus, our analysis indicates a complex and hetero-
chronic nature of MUPs fractions expression during post-
natal ontogenesis in males and females of laboratory mice
of two genealogically unrelated strains CBA/LacY and
C57BL/6JY, which have different physiological activity of
androgen-dependent pheromones (Novikov, 1988).
Here we demonstrate for the first time the evidence of
the coordinate and age-unrelated pattern of expression of
the Mup cluster genes, which are responsible for the per-
centages of individual MUP isoforms. These data are in
accordance with the current idea of genetic regulatory
mechanisms of coordination and differential expression of
Mup genes (Chamero et al., 2007; Logan et al., 2008;
Mudge et al., 2008).
Further studies of MUPs, in our opinion, will be asso-
ciated with unraveling the functional basis of the olfactory
code and with investigations of patterns of its formation
during the house mouse ontogenesis (Novikov et al.,
2008). Our model is based upon the idea of competitive
binding of certain MUPs fractions with low-molecular
ligands of pheromonal nature (Novikov, 2003). Using an
analogy with a letter code (the alphabet), we propose that
pheromonal ligands correspond to chemical symbols (let-
ters), while given combination of MUP isoforms makes
from these letters a semantically meaningful message (an
olfactory image) about individual features of the donor
(genotype, sex, age, hierarchical status etc) (Novikov,
2007; Novikov et al., 2008).
Data regarding the inheritance of Mup genes expres-
sion pattern in F1 progeny from direct and reciprocal
crosses could be of interest in studies of the olfactory code
formation: it was shown that while CBAB6F1 and
B6CBAF1 animals of the same sex have an almost ideal
likeness of phenotypes, the phenotypic markers E and F
are expressed in both males and females of these geno-
types independently from the direction of the cross
(Churakov, Novikov, 2000). These data could indicate that
genes, which control MUPs expression in F1 progeny, are
not Y-linked, that the trait itself is not under the maternal
influence, and that it is inherited additively by F1 heterozy-
gotes.
The fundamental issue of the modifying role of the
environment, and specifically of pheromonal imprinting,
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
209
in formation of the sexual selectivity on the basis of MUPs
pattern in males and females of house mouse Mus muscu-
lus L. during ontogenesis requires further investigations.
ACKNOWLEDGMENTS
Authors are deeply grateful to Dr. S.V. Myl’nikov
for his invaluable help with the statistical data manipu-
lation and critical discussions during the preparation of
the manuscript.
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