Doklady Biological Sciences, Vol. 391, 2003, pp. 318–321. Translated from Doklady Akademii Nauk, Vol.
391, No. 5, 2003, pp. 707–711.
Original Russian Text Copyright © 2003 by Novikov.
PHYSIOLOGY
Coordinated Expression of the Genes Gus and Mup as a Potential
Basis of the Functional Activity of the Androgen-Dependent
Pheromones of the House Mouse (Mus musculus L.)
S. N. Novikov
Presented by Academician A.D. Nozdrachev March 31, 2003
Received April 2, 2003
In one of the most interesting and promising models
of molecular physiology, the functional activity of the
androgen-dependent pheromones of house mouse Mus
musculus L. (in particular, 2-sec-butyl-4,5-dihydrothia-
zole, 3,4-dehydro-exo-brevicomin, and E,E-α- and
E-β-farnesenes) is related to the urinary proteins of the
major urinary protein (MUP) complex [1]. This com-
plex includes a group of low-molecular-weight acidic
proteins (m. w., 18–19 kDa; pI, 4.2–4.7). MUPs are
encoded by a cluster of Mup genes, located in chromo-
some 4 (linkage group VIII). To date, the Mup genes
have been cloned, and the multihormonal control of the
Mup expression in hepatocytes has been studied in
detail. Now the Mup system is a well-developed model
used in molecular genetics of mammals [2].
The existence of genotypic differences in the func-
tional activity of pheromones is commonly attributed to
the hereditarily determined deviations in the function of
some enzyme systems, in particular, the kidney acidic
hydrolase β-glucuronidase (GUS, EC 3.2.1.31). The
latter is encoded by the Gus gene (located in chromo-
some 5) with participation of sex steroids and involved
in the transformation of the immobilized chemosignal
(propheromone) in the form of glucuronides to its phys-
iologically active form [3].
This was the first study to present the entire picture
of the functioning of these two genetic systems as a
potential basis for the regulation of the physiological
activity of the androgen-dependent pheromones of
house mouse, based on the analysis of the characteristic
features of the expression of the protein products of
genes Mup and Gus as dependent on the androgen sta-
tus of the organism.
The study was performed in spring using mature
males of two highly inbred, genealogically unrelated
strains of mice with hereditarily determined differences
in the physiological activity of androgen-dependent
pheromones, namely, strains CBA/LacY and
Pavlov Institute of Physiology, Russian Academy of Sciences,
nab. Makarova 6, St. Petersburg, 199034 Russia
0012-4966/03/0708-0318$25.00 © 2003 MAIK “Nauka /Interperiodica”
C57BL/6JY (n = 48) [4]. The proteins of the MUP com-
plex were separated by SDS-PAGE with subsequent
densitometric analysis of fractions [2]. The activity of
lysosomal GUS in kidney homogenate and urine was
monitored by the rate of hydrolysis of phenolphthalein-
β-D-glucuronide (Sigma, United States) and expressed
in Fishman’s units [3]. The testosterone content of
blood plasma was determined radioimmunologically,
using standard kits (Sorin, France). The results were
statistically treated using standard procedures.
As seen from Fig. 1, the genotype significantly
affects the qualitative and quantitative composition of
MUPs: fractions A, B, C, D, G, and H were found in the
mice of both strains, whereas fractions E and F may
serve as genetic markers of the strains CBA (fraction E)
and C57BL/6 (fraction F). In general, the concentra-
tions of the MUP fractions were 5.49 + 0.127 and
3.46 ± 0.628 mg/ml in CBA and C57BL/6 males,
respectively.
Correlation analysis of the level of testosterone in
blood plasma, absolute content of different MUP frac-
tions, and the activity of GUS in kidney homogenates
and urine showed a significant positive correlation
Protein, mg/ml
4.0
3.5
Strain CBA
3.0
Strain C57BL/6
2.5
2.0
1.5
1.0
0.5
0
A B C D E F G H
Protein fractions
The concentrations of the protein fractions of the MUP
complex in the urine of male laboratory mice with different
genotypes.
 COORDINATED EXPRESSION OF THE GENES Gus AND Mup
Table 1. Correlation between the testosterone content in blood plasma and the content of individual MUP fractions in urine
of laboratory male mice of different genotypes
Genotype
A B C D
CBA –0.604 +0.846* +0.698 +0.122
C57BL/6 +0.295 +0.703 +0.717 +0.597
* P < 0.05.
between the testosterone level and the content of the
fraction B in CBA males (Table 1) and a positive cor-
relation between the testosterone content and the
GUS activity, also characteristic solely of CBA
males (Table 2).
It was shown earlier that a change in the endogenous
testosterone level in mature male mice, induced by cas-
tration, resulted in a drop of the absolute content of all
seven fractions in C57BL/6 mice and six fractions in
CBA mice [2]. Therapy with testosterone led to the res-
toration of the relative (partial) content of the fractions
to the level characteristic of sham-operated animals. In
this aspect, the expression of the proteins of three frac-
tions (B and C in CBA mice and D in C57BL/6 mice)
is of particular interest. These three fractions are com-
pletely absent in MUPs of females, disappear after cas-
tration of mature males, and are restored to the preop-
erational level after testosterone therapy [2]. Therefore,
the biosynthesis of proteins comprising these fractions
completely depends on the level of endogenous test-
osterone in the organism. It can be assumed the frac-
tions B, C, and D are the most interesting in terms of
studying the characteristics of affinity binding of MUPs
with the androgen-dependent pheromones [1].
The data presented in this study indicate that the
expression of two different proteins (the androgen-
dependent fraction B of the MUP complex and β-glu-
curonidase), coordinated with the testosterone level, is
characteristic of adult male CBA mice (with highly
active sex pheromones [4]), but not C57BL/6 mice.
Thus, we found a significant correlation between the
level of the hormone regulating the biosynthesis of sex
pheromones in house mouse [4] and the activity of
GUS involved in the transformation of immobilized
(latent) propheromone in the form of glucuronides in
the physiologically active form [3]. These data may
considerably broaden our notion on the molecular
genetic mechanisms regulating the physiological activ-
ity of natural compounds of this group, under the action
of steroids on genetic material (genes Gus and Mup).
A low activity of the androgen-dependent phero-
mones affecting the reproduction of C57BL/6 mice is
apparently related to the low testosterone level and the
genetically determined characteristics of steroidogene-
sis and hormonal reception [4]. Earlier, using the mod-
els of the pheromonal regulation of aggressive behavior
and spermatogenesis, we showed that excreted urine of
DOKLADY BIOLOGICAL SCIENCES Vol. 391 2003
319
Fraction
E F G H
–0.467 – +0.682 +0.116
– +0.707 +0.454 +0.024
mature males of this strain contained a large amount of
glucuronides: the incubation of the specimens with
commercial preparations of β-glucuronidase in vitro
resulted in a significant increase in the physiological
activity of the pheromone [4]. On the other hand, as was
shown in [3], the activity of native enzyme in C57BL/6
males was decreased by five and ten times in the kid-
neys and urine, respectively, compared to CBA males.
However, a possible universal role of GUS in the
regulation of the physiological activity of androgen-
dependent pheromones is questionable. A marked cor-
relation between the enzyme activity in the kidney and
urine of CBA males and a low correlation in C57BL/6
males (Table 2) may be indicative of not only hetero-
genic composition of the isozyme pool of GUS in
excreted urine, but also a high substrate specificity of
the enzyme in vivo, as well as the effect of the genotype
on its physical and chemical characteristics (in particu-
lar, the resistance to the specific inhibitor, glucaro-1,4-
lactone, a product of glucuronic acid metabolism [3]).
In this aspect, a comparative biochemical study of the
isozyme forms of this hydrolase in the kidney and
preputial gland (the main producers of a series of chem-
ically identified androgen-dependent pheromones of
house mouse [1]) would be especially interesting.
Based on preliminary data on the separation of the
proteins of the MUP complex using ion-exchange chro-
matography on DEAE cellulose, we assumed that each
of the electrophoretic fractions A–H is a mixture of iso-
morphic proteins, differing in the charge of the mole-
cule. This assumption is corroborated by the results of
a long-term work performed by scientists from the
United Kingdom, who studied the genetic polymor-
Table 2. Correlation between the testosterone content in
blood plasma and the activity of β-glucuronidase in kidneys
and urine of male laboratory mice of different genotypes
Kidneys
Genotype Urine
TA SA
CBA +0.670* +0.702* +0.851**
C57BL/6 +0.158 +0.026 +0.382
* P < 0.05, ** P < 0.01. TA, total enzyme activity, expressed in
Fishman’s units; SA, specific enzyme activity, expressed in Fish-
man’s units per mg tissue
320 NOVIKOV
phisms of MUPs in the populations of the wild form of
house mouse [5]. Using HPLC and mass-spectrometry,
it was shown that the proteins of the MUP complex,
which do not differ in molecular weight, may be present
in different chromatographic fractions. This may be
accounted for by their amino acid composition (in par-
ticular, the ratio between glycine, glutamine, and
lysine). In this case, insignificant amino acid substitu-
tions may result in significant conformational changes,
thereby affecting the functional properties of the pro-
tein molecule, the basis of the ligand-binding pocket of
which is formed by Val58, Ala107, Leu44, and Ala122
[6]. The authors of [6] assumed that the substitution of
Val58 and Ala107 with phenylalanine and Leu44 and
Ala122 with valine may reflect the characteristics of the
protein binding with one or another ligand. A good
experimental confirmation of this assumption was the
determination, in the MUP molecule, of the high-affin-
ity site for the known androgen-dependent pheromones
(2,3-dehydro-exo-brevicomin and 2-sec-butyl-4,5-
dihydrothiazole) and the creation, on the basis of these
data, of an artificial physiologically active hexapeptide
N-Glu–Glu–Ala–Arg–Ser–Met, which accelerates sex
maturation of female laboratory mice [7]. Therefore, it
can be assumed that the proteins contained in the elec-
trophoretic fractions A–H (Fig. 1) will differ, on the one
hand, in charge; on the other hand, in the composition
of the aforementioned amino acids.
Taken together, the above data and our results sug-
gest that the mechanisms of regulation of the functional
activity of the androgen-dependent pheromones in lab-
oratory mice may be the following.
The expression of the genes coding for the MUP
cluster is controlled by sex steroids (primarily, test-
osterone). The level, balance, and effectiveness of ste-
roid hormones depend on the genetically determined
characteristics of steroidogenesis (first of all, the activ-
ity of 3β-hydroxysteroid dehydrogenase/isomerse (EC
1.1.1.145) and the hormonal reception), as well as envi-
ronmental factors (in particular, the nature of the intrap-
opulation relations). The aforementioned factors affect
the qualitative and quantitative composition of MUPs,
which are synthesized in the liver and excreted from the
organism with urine. The differences in the characteris-
tics of the protein binding with physiologically active
ligands (determined, on one hand, by its structural and
conformational characteristics and, on the other hand,
by the activity of β-glucuronidase) will affect the abso-
lute and partial concentrations of these compounds in
the solution and the pheromone composition and deter-
mine the functional and informational properties of the
pheromonal “bouquet” on the whole (as a vector with
targeted effect on the recipient organism). The central
place in this scheme belongs to the idea on key roles of
(a) the ratio between different MUP fractions; (b) dif-
ferential expression of these proteins in hepatocytes [2];
and (c) significant differences in the nature of affinity
binding of the protein molecule with one or another
ligand [1]. Thus, according to our scheme, a limited
number of individual pheromone-like compounds
exists in the nature, and the mechanisms forming the
olfactory image are based on the combination of the
ratios between these physiologically active compounds
in the solution.
The genetic model based on a coordinated expres-
sion of the genes Gus and Mup under the action of tes-
tosterone probably represents the first and yet the only
example of a specific gene net whose nucleus consists
of these genes interacting at the level of their products
and thus affecting the physiological activity of the
androgen-dependent pheromone in rodents.
Within the framework of the genetic physiological
model put forward, particularly interesting are the data
on the presence, in excreted urine of house mouse, of
meprine, an androgen- and genotype-dependent metal-
lopeptidase (EC 3.4.24.18), the main function of which
is degradation of the proteins of the MUP complex [8].
The process of MUP degradation to individual oli-
gopeptides is the necessary and, possibly, the key stage
in the mechanism of effective action of the ligand–pro-
tein complex on the recipient organism. A similar con-
clusion on a pivotal role of the oligopeptides from urine
of male rats, weighing less than 5 kDa, in the regulation
of the activity of the c-Fos genes in the cells of addi-
tional olfactory bulb in females was made in [9].
In the context of the hypothesis on the possible role
of the protein product of the Gus gene, β-glucu-
ronidase, in the regulation of the functional activity of
house mouse pheromones, the study on uridine
5'-diphosphoglucuronyltransferase (UDP-glucurono-
syltransferase, EC 2.4.1.17) from olfactory lining
(UGTolf) is especially interesting [10]. The authors of
[10] reported on the key role of this enzyme in the
mechanisms of detoxification of xenobiotics and a
broad range of physiologically active volatile com-
pounds. Thus, UDP-glucuronosyltransferase fulfils a
protective function, on one hand, and is involved in
biotransformation of chemical compounds, on the other
hand.
One of the characteristic features of the uroproteins
of the MUP complex is related to their striking struc-
tural similarity with odorant-binding protein (OBP),
which carries the odorant molecules to the receptor cell
of the olfactory lining and plays a key role in the mech-
anisms of olfactory reception in terrestrial vertebrates
[11]. Interestingly, in addition to its protective and
transport functions, OBP is also involved in the primary
“recognition” (decoding) of the odorant. The data that
mRNA of the Mup genes is expressed in the cells of
mouse olfactory lining in the prepubertal period are
also interesting [12]. When comparing the data
reported in [1, 5, 6, 8, 11, 12] with our results, taking
into account the logic of this line of reasoning, a ques-
tion arises as to whether the MUP and OBP proteins
may be units of the same phylogenetically ancient cas-
cade, transducing informative chemosignals (phero-
mones) from one organism to another, and whether the
DOKLADY BIOLOGICAL SCIENCES Vol. 391 2003
 COORDINATED EXPRESSION OF THE GENES Gus AND Mup
process of information exchange between OBPs and
MUPs may be a sort of a dialogue in the language of
chemosignals. It should be added that the discovery, in
the mid-1990s, of high-affinity membrane receptors for
OBP in the cells of mammalian olfactory lining and
pulmonary tissue is suggestive of the existence of spe-
cific mechanisms of targeted axonal transport of physi-
ologically active ligands in the form of complex with
OBP or its polypeptide fragments as a result of endocy-
tosis.
The effect of sex pheromones on the reproduction
and various behavioral patterns in the majority of mam-
malian species is directly related to the function of the
vomeronasal (Jacobson’s) organ, a phylogenetically
ancient pair structure, located in the base of nasal sep-
tum [1, 4, 7]. This neuroanatomic structure is directly
projected via an additional olfactory bulb on three most
important parts of hypothalamus (the preoptical region,
as well as the ventromedial and premammilary nuclei)
[4]. Taking into account the existence of tight neuronal
connections between the vomeronasal organ and the
hypothalamic–pituitary complex, on the one hand, and
its key role in the regulation of the effect of sex phero-
mones on the organism, on the other hand, we can sug-
gest that further development of the experimental
model suggested in this study will lead to the develop-
ment of a highly effective system of vector control of
the reproductive function and sex behavior of mam-
mals, based on the principles of transnasal targeted
transport of physiologically and pharmacologically
active compounds to the brain in the form of artificial
protein complexes.
The development of the methods of intranasal intro-
duction of oligopeptides and drugs, associated with a
protein matrix [13–15], is a good empirical argument in
favor of this conclusion.
ACKNOWLEDGMENTS
I am grateful to G.A. Churakov, A.A. Filimonenko,
and V.E. Sukonina, who kindly provided the experi-
mental data necessary for the analysis, and I.A. Burkot,
who prepared the manuscript for publishing.
DOKLADY BIOLOGICAL SCIENCES Vol. 391 2003
321
The study was supported by the Russian State Sci-
ence and Technology Program “Frontiers in Genetics”
(project no. 2.152) and the Russian Foundation for
Basic Research (project no. 02-04-49273).
REFERENCES
1. Sharrow, S.D., Vaughn, J.L., Ž idek., L., et al., Protein
Sci., 2002, vol. 11, pp. 2247–2256.
2. Churakov, G.A. and Novikov, S.N., Dokl. Akad. Nauk,
2000, vol. 375, no. 1, pp. 130–133.
3. Novikov, S.N., Churakov, G.A., Filimonenko, A.A., and
Sukonina, V.E., Dokl. Akad. Nauk, 2000, vol. 374, no. 3,
pp. 408–410.
4. Novikov, S.N., Feromony i Razmnozhenie Mlekopitay-
ushchikh (Pheromones and Reproduction of Mammals),
Leningrad: Nauka, 1988.
5. Robertson, D.H.L., Hurst, J.L., Bolgar, M.S., et al.,
Rapid Commun. Mass Spectrom., 1997, vol. 11,
pp. 786−790.
6. Böcskei, Z., Groom, C.R., Flower, D.R., et al., Nature,
1992, vol. 360, pp. 186–188.
7. Mucignat-Caretta, C., Caretta, A., and Cavaggioni, A.,
J. Physiol., 1995, vol. 486, pp. 517–522.
8. Wu, C., Robertson, D.H.L., Hubbard, S.J., et al., J. Biol.
Chem., 1999, vol. 274, pp. 1108–1115.
9. Tsujikawa, K. and Kashiwayanagi, M., Biochem. Bio-
phys. Res. Commun., 1999, vol. 260, pp. 222–224.
10. Heydel, J.-M., Leclerc, S., Bernard, P., et al., Mol. Brain
Res., 2001, vol. 90, pp. 83–92.
11. Tegoni, M., Pelosi, P., Vincent, F., et al., Biochim. Bio-
phys. Acta, 2000, vol. 1482, pp. 229–240.
12. Utsumi, M., Ohno, K., Kawasaki, Y., et al., J. Neurobiol.,
1999, vol. 39, pp. 227–236.
13. Kovalenko, R.I., Zimina, O.A., Chernyshova, M.P., and
Nozdrachev, A.D., Dokl. Akad. Nauk, 2000, vol. 370,
no. 4, pp. 554–558.
14. Plate, N.A., Valuev, L.I., and Knyazhev, V.A., Vestn.
Ross. Akad. Nauk, 2001, vol. 71, pp. 899–914.
15. Shalyapina, V.G., Rakitskaya, V.V., Petrova, E.I., and
Mironova, V.I., Ross. Fiziol. Zh. im. I.M. Sechenova,
2002, vol. 88, pp. 1212–1218.