Doklady Biological Sciences, Vol. 378, 2001, pp. 208–209. Translated from Doklady Akademii Nauk, Vol.
378, No. 3, 2001, pp. 407–409.
Original Russian Text Copyright © 2001 by Babalyan, Novikov.
PHYSIOLOGY
The Pheromone Inhibiting Spermatogenesis in Laboratory Mice
Is Associated with the Urinary Protein Fraction
V. V. Babalyan and S. N. Novikov
Presented by Academician A.D. Nozdrachev May 10, 2000
Received October 4, 2000
Physiological proteinuria reaching sometimes 20 mg
of protein per 1 ml of urine a day in mature male house
mice is one of mysteries of rodent living activity [1].
The phenomenology and molecular and genetic basis
of proteinuria have been studied in detail; nevertheless,
the biological implication of an increased protein
excretion in this large systematic group is still an
enigma.
In the early 1990s, first reports appeared in which
close functional association was demonstrated between
urinary protein and physiological activity of phero-
mones controlling the rate of sexual maturation in
female laboratory mice [2].
In this study, we analyzed physiological effects of
the volatile compounds contained in both protein and
nonprotein urine fractions of six- to eight-month-old
male CBA/LacSto mice. To evaluate the physiological
activity of the fractions, a biotest was used, namely, the
count of morphologically abnormal spermia [3].
Low-molecular-weight compounds were separated
from the urinary proteins by salting-out with a satu-
rated solution of ammonium sulfate followed by cen-
trifugation. To obtain the lipophilic fraction, the super-
natant was four times extracted with diethyl ether, and
the solvent was removed with a nitrogen flow under
vacuum. The solid residual was dissolved in distilled
water to restore the initial volume.
Physiological activity of the fractions was evaluated
from their effect on spermatogenesis in 30-day-old
male CBAB6F1 mice after a 2-h exposure to a flow of
air containing the odorant in an olfactometer operating
in a definite mode [3].
The results obtained (table) indicate the following.
(1) In native urine of mature CBA/LacSto mice,
low-molecular-weight (volatile) compounds were
found. Their effect on the 30-day-old recipients led to
an increased frequency of abnormal spermia in the epi-
didymis.
Pavlov Institute of Physiology, Russian Academy of Sciences,
nab. Makarova 6, St. Petersburg, 199034 Russia
0012-4966/01/0506-0208$25.00 © 2001 MAIK “Nauka /Interperiodica”
(2) Physiologically active compounds are repre-
sented by substances of different polarity, which are
present in both lypophilic and hydrophilic urinary frac-
tions.
(3) After separation of the low-molecular-weight
and protein urinary fractions, the latter retains a high
pheromone activity.
Thus, we have demonstrated that, after multiple
extraction, the protein urinary fraction of mature male
CBA mice retains pheromone physiological activity
interfering with spermatogenesis in young CBAB6F1
mice. As early as in the mid-1970s, it was suggested
that the structure and function of urinary proteins (pep-
tides) of mature male mice were related to the physio-
logical activity of the androgen-dependent pheromones
that influence sexual maturation in females. [4]. This
suggestion was experimentally confirmed later: two
compounds accelerating sexual maturation were chemi-
cally identified as 2-(sec-buthyl)thiazoline and 2,3-dehy-
dro-exo-brevicomin and afterwards synthesized. They
were also found to be associated with the so-called
major urinary protein (MUP) with a molecular weight
of 18–19 kDa, which forms the basis of the protein
fraction [2].
Proteins of the MUP complex belong to a family of
phylogenetically ancient proteins that serve several
important functions in an organism, such as binding
and transport of hydrophobic physiologically active
compounds (hormones, vitamins, pheromones), inter-
action with membrane receptors of the target cells, for-
mation of functional complexes with various sub-
stances, etc. [5]. To date, at least 20 members of this
family have been identified; the best studied proteins
are fetoprotein, lactoglobulin, retinol-binding protein,
and apolipoprotein D. Despite a high variation in the
primary structure, lipocalins are characterized by a
conserved conformation of the protein molecule, which
is barrel-shaped with an open cuplike pocket and a
ligand-binding site situated within the “barrel” [2].
In the MUP complex, as many as 15 protein frac-
tions are distinguished. They differ in molecular
weight, amino-acid composition [6], and, probably, in
the affinity for the volatile pheromone-like physiologi-
cally active compounds. Both qualitative and quantita-
 THE PHEROMONE INHIBITING SPERMATOGENESIS IN LABORATORY MICE
Frequency of abnormal spermia in the caudal segment of the
epididymis (AC, %) in male CBAB6F1 mice exposed to vol-
atile components of the protein and nonprotein fractions of
urine of male CBA/LacSto mice
Variant of treatment N AC, %
VUC 3338 10.1 ± 0.52
P 3225 7.0 ± 0.45**
Lnf 3242 7.5 ± 0.46**
Hyd.Lnf 2170 7.8 ± 0.58**
Lip.Lnf 2623 4.7 ± 0.41*
CI 2572 2.8 ± 0.33
CO 4104 2.5 ± 0.24
Notes: VUC, volatile urinary components; P, urinary protein fraction;
Lnf, low-molecular-weight nonprotein urinary fraction; Lip.Lnf,
and Hyd.Lnf, lipophilic and hydrophilic low-molecular-weight
nonprotein urinary fractions, respectively; CO, intact males; CI,
exposure of male CBAB6F1 mice to water vapor.
* Significant differences from the variant CI at p < 0.05.
** Significant differences from the variant CI at p < 0.01.
tive compositions of the MUPs is highly polymorphic
[6]. We suggest, therefore, that the partial concentration
of physiologically active compounds in a solution are
determined by genetically determined differences in
the MUP expression and variations in ligand (phero-
mone) binding with one or another fraction, which, in
turn, depend on the structural or conformation proper-
ties of the protein molecule. In general, these properties
of the MUP complex determine the functional activity
of the pheromone “bouquet” serving as a vector
directed to the recipient organism.
The MUP structure is surprisingly similar to that of
the odorant-binding protein (OBP) playing a key role in
mechanisms of the olfactory reception, because OBP
transports the odorant molecules to the receptor cells of
the olfactory layer [7, 8]. Therefore, the reports in
which the special membrane receptors for OBP are
described [9], as well as the effect of pyrazine com-
pounds characterized by high affinity for OBP on the
functioning of slow sodium channels of the sensory
neurons [10] are of particular interest.
Note that a MUP homologue, α2u-globulin, isolated
from rat urine and injected i.c. has a pronounced anti-
estrogen effect [11] and causes changes in the levels of
gonadotropin, prolactin, and testosteron [12]. A more
surprising fact is that, in rats, a relatively high concen-
tration of α2u-globulin of extrahypophysial origin was
determined in cells of the anterior lobe of hypophysis
[13]. There is evidence indicating that one of the genes
controlling spermatogenesis and formation of the sper-
matozoon head in laboratory mice is linked to chromo-
some 4 [14]; the Mup genes responsible for synthesis of
DOKLADY BIOLOGICAL SCIENCES Vol. 378 2001
209
the MUP-complex proteins have been localized to the
same chromosome [15].
Comparison of these data with the results obtained
in our study leads to a proposal that the MUP and OBP
proteins are involved in a phylogenetically ancient
transport chain ensuring chemical communication and
reproduction regulation by means of pheromones in the
animal world. If so, the question arises as to whether
the artificial structural analogues of the MUP exhibit-
ing high affinity for the physiologically active com-
pounds can be used for directed (vector) regulation of
reproduction by means of olfactory stimulation.
ACKNOWLEDGMENTS
This work was partly supported by the Russian State
Program “Frontiers in Genetics” (project no. 2.152)
and the program “ASGL-Olfaction Pharmacology.”
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