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 0012-4966/00/1112-$25.00 © 2000 MAIK “Nauka/Interperiodica0549
  Doklady Biological Sciences, Vol. 375, 2000, pp. 549–552. Translated from Doklady Akademii Nauk, Vol. 375, No. 1, 2000, pp. 130–133.Original Russian Text Copyright © 2000 by Churakov, Novikov.
 Although mammalian pheromones have been stud-ied for almost five decades, the genetic control of theirphysiological activity is still poorly understood. Thisproblem remains one of the most intricate in this newinterdisciplinary field of biology.Considerable polymorphism of the activities of androgen-dependent pheromones has been found indifferent mouse strains [1–3]. This has created a meth-odological basis for genetic studies aimed at revealingthe key molecular and biochemical mechanismsresponsible for the biogenesis, metabolism, and releaseof pheromones. Genetic models are promising tools forexperimental studies on the biochemical basis of theregulation of the pheromone physiological activity [4].According to one of the most promising moleculargenetic models [5], the physiological activities of androgen-dependent pheromones of the house mouse(
  Mus musculus
 L.), including
2-
 sec
 -butyldihydrothia-zol and 2, 3-dehydro-
 exo
 -brevicomin, are related to thelevel of a protein belonging to the major urinary protein(MUP) complex, which is excreted with urine. Individ-ual
 Mup
 genes mapped to chromosome 4 [6] have beenfound to control the biosynthesis and differentialexpression of certain androgen-dependent MUP frac-tions. The available data allow researchers to addressthe problem of the role of these genes in the regulationof the physiological activity of pheromones.The purpose of this study was to detect and analyzethe testosterone-induced expression of the
 Mup
 genecomplex in laboratory strains of mice with hereditarydifferences in the activities of androgen-dependentpheromones.We used male and female CBA/LacY andC57BL/6JY mice and their reciprocal F1 hybrids (
  N 
 =162). In experiments with changed endogenous test-osterone concentration, six-week-old males were cas-trated using the standard technique with ether anesthe-sia [3]. When castrated animals were eight weeks old,capsules (Dow Corning, United States) containingcrystalline testosterone propionate (TP) (Sigma, UnitedStates) were implanted into some of them. The capsuleswere implanted near the nape of animals anesthetizedwith ether; control animals were implanted capsuleswithout TP. The working part of the capsule was 10 mmin length, which corresponded to a daily TP dose of 27.1
µ
 g [6].The total protein concentration in urine was deter-mined according to Bradford. The endogenous
β
 -glu-curonidase (EC 3.2.1.31) was estimated by the standardFischmann’s phenolphthalein method. The plasma con-centration of testosterone was estimated by radioimmu-noassay using standard kits (Sorin, France). MUP pro-teins were fractionated by means of nondenaturing ver-tical gel electrophoresis in a tris-acetate buffer system(pH 5.5) [7]. Gel plates were scanned using a GelScanXL laser densitometer (Pharmacia, Sweden).The data obtained are shown in Figs. 1, 2 andTables 1, 2. The main results are summarized below.(1) Eight electrophoretic fractions (A–H) may bedistinguished in the MUP complexes of CBA andC57BL/6 mice. Fractions A, B, C, D, G, and H werecharacteristic of both strains. Fractions E and F wereunique; they may serve as phenotypic markers of theCBA (fraction E) and C57BL/6 (fraction F) strains.Castrated CBA males lacked fractions B and C, andcastrated C57BL/6 males, fraction D (Table 1). Castra-tion caused a decrease in the concentrations of individ-ual fractions of the MUP pool, except for the majorfraction E in CBA mice. Replacement testosteronetherapy recovered the MUP polymorphism to the levelobserved in animals subjected to false castration. Theadministration of TP caused a proportional increase inthe concentrations of all MUP fractions: in each strain,the coefficient of rank correlation between concentra-tions of individual protein fractions in variants I and IIIwas 1.(2) The content of MUP fractions constantlyincreased in the course of ontogeny in males of both
 PHYSIOLOGY
 Heterogeneity and Differential Expressionof MUP Proteins as a Genetic Basis of the PhysiologicalActivity of Androgen-Dependent Pheromones
 G. A. Churakov* and S. N. Novikov**
 Presented by Academician Yu.V. Natochin October 28, 1999Received December 29, 1999
 * Institute of Experimental Medicine, Russian Academyof Medical Sciences, St. Petersburg, Russia** Pavlov Institute of Physiology, Russian Academyof Sciences, nab. Makarova 6, St. Petersburg, 199064 Russia
 
 550
 DOKLADY BIOLOGICAL SCIENCES
 
Vol. 375
 
2000
 CHURAKOV, NOVIKOV
 strains (Table 2). Note that the initial concentrations of individual fractions in immature (four-week-old) CBAmice were an order of magnitude higher than inC57BL/6 mice of the same age. These differences dis-appeared by the age of eight weeks (in mature animals),but appeared again afterwards (
 P
 < 0.05). The rank cor-relation coefficients (
 
 
s
 ) between absolute concentra-tions of the same fractions in four- and eight-week-oldanimals were +
 0.96 (
 P
 < 0.01)
and +
 0.57 (
 P
 > 0.05)
inCBA and C57BL/6 mice, respectively. In other words,the specific MUP heterogeneity characteristic of adultanimals was expressed in CBA mice considerably ear-lier than in C57BL/6 mice.(3) We analyzed the inheritance of the protein spec-tra of 
 Mup
 products in direct and reciprocal F1 hybridsbetween the two strains. CBAB6F1 and B6CBAF1hybrids display an almost absolute phenotypic similar-ity within the same sex, with the unique fractions E andF being expressed in both males and females with anyof the two genotypes irrespective of the type of crossing(Fig. 2). This suggests that the genes that control MUPexpression are not linked to the Y chromosome; the traitis not influenced by the mother’s organism and exhibitsan additive inheritance in F1.Thus, comparative densitometric analysis of theexpression of MUP proteins (products of the
 Mup
 genecomplex) in mice differing with respect to genotype,sex, age, and physiological state allowed us to reducethe spectrum of proteins under study to five fractions:B, D, E, F, and H.
 Fraction B
 was absent in MUP electrophoreto-grams of CBA females (Fig. 2). In CBA males, its con-centration was positively correlated with the plasmatestosterone level (
 
 
s
 = +0.85,
P
 < 0.05), specific renal
 β
 -glucuronidase activity (
 
 
s
 = +0.91,
P
 < 0.05), and theactivity of this enzyme in urine (
 
 
s
 = +0.92,
P
 < 0.05).
 Fraction D
 was major in males from both strains(Table 1) and exhibited a high reactivity to testosterone.The concentration of this fraction drastically increasedin the course of ontogeny of C57BL/6 males (Table 2)and was almost absent in C57BL/6 females (Fig. 2).
 Fraction E
 was expressed in male and female CBAmice and F1 hybrids (Fig. 2). The concentration of thismarker fraction was negatively correlated with theplasma testosterone level (
 
 
s
 = –0.98,
P
 <
 0.05) and thespecific
β
 -glucuronidase activity in homogenates of thekidney of CBA males (
 
 
s
 = –0.83,
P
 
< 0.05).
 Table 1.
Concentrations (mg/ml) of MUP fractions in the urine of male CBA/LacY and C57BL/6JY miceFractionStrain CBAStrain C57BL/6males subjectedto false castration(the control group)castrated malescastrated malestreated with TPmales subjectedto false castration(the control group)castrated malescastrated malestreated with TPA0.72
±
 0.1080.18
±
0.0571.10
±
 0.1371.45
 ±
 0.1810.60
±
 0.2511.66
±
 0.256B0.27
±
 0.01900.37
±
 0.0550.91
±
0.0850.06
±
 0.0180.81
 ±
 0.132C0.64
±
 0.102 00.73
 ±
 0.0651.70
±
 0.1830.64
±
 0.2662.02
±
 0.140D3.32
±
 0.1951.53
±
0.3843.28
±
 0.1861.43
±
 0.15901.15
±
 0.275E1.20
±
 0.1632.41
±
0.4482.02
±
 0.234F0.25
±
 0.0340.04
±
0.0040.23
±
 0.061G0.11
±
 0.0200.03
±
 0.0110.13
 ±
0.0140.11
±
 0.0250.04
±
 0.0030.14
±
 0.040H0.08
±
 0.0160.04
 ±
0.0090.11
 ±
 0.0140.05
±
 0.0090.03
±
 0.0050.08
 ±
 0.0266.34
±
 0.5424.19
±
 0.8687.74
±
0.4085.90
±
 0.5401.40
±
 0.2806.09
±
 0.733
Σ
A–H
 CBA/LacYTPTPTPC57BL/6JYTP
 Fig. 1.
 Heterogeneity of mouse MUP proteins depending on the genotype an physiological state.
 
 DOKLADY BIOLOGICAL SCIENCES
 
Vol. 375
 
2000
 HETEROGENEITY AND DIFFERENTIAL EXPRESSION OF MUP PROTEINS551
 Fraction F
 was a unique minor fraction characteris-tic of the C57BL/6 strain and F1 hybrids.
 Fraction H
 was also minor in males and females witheach of the four genotypes studied (Fig. 2). The plasma tes-tosterone concentration was positively correlated with theconcentration of this fraction in B6CBAF1 males (
 
 
s
 =+0.95,
 P
 
< 0.05) and negatively correlated with it inC57BL/6 mice (
 
 
s
 = –0.78,
 P
 < 0.05).In summary, the results of the detailed analysis indi-cate that testosterone has a complex and multidirec-tional effect on the expression of 
 Mup
 genes in geneti-cally unrelated laboratory strains of mice, CBA andC57BL/6, which differ in the physiological activity of androgen-dependent pheromones [4]. Based on ourdata and the published data on pheromone ligand bind-ing to urine proteins in mice [5, 8], we may assume ahypothetical scheme of the modulation of the physio-logical activity of androgen-dependent pheromones.The
 Mup
 cluster contains more than 35 genes [7].Their activities are controlled by steroid hormones, inparticular, androgens, the level and balance of whichare determined by hereditary characteristics of ste-roidogenesis and testosterone metabolism [9, 10]. This,in turn, determines the qualitative and quantitativecomposition of MUPs, which are synthesized in theliver [11], bind to low-molecular-weight lipophiliccompounds, such as pheromones, and are excreted withurine. Differences between MUP fractions with respect
 Table 2.
 Age-related changes in the concentrations (mg/ml) of MUP fractions in the urine of male CBA/LacY and C57BL/6JY miceFractionStrain CBAStrain C57BL/64 weeks8 weeks12 weeks
 
 
CBA
 4 weeks8 weeks12 weeks
 
 
B6
 A0.02
±
 0.0070.08
±
 0.0150.45
±
 0.07523.60.007
±
 0.00130.24
±
 0.0750.68
±
 0.157103.5B0.03
±
0.0130.12
±
0.0200.23
±
0.0199.10.006
±
0.00170.17
±
0.0480.51
±
0.11186.3C0.07
±
0.0500.18
±
0.0240.51
±
0.0827.50.018
±
0.00780.50
±
0.1281.43
±
0.18278.4D0.16
±
0.1010.64
±
0.0912.97
±
0.14118.80.004
±
0.00080.21
±
0.0690.53
±
0.152152.6E0.06
±
0.0370.50
±
0.0471.06
±
0.04516.6F0.002
±
0.00040.05
±
0.0150.09
±
0.03143.0G0.02
±
0.0070.06
±
0.0070.15
±
0.0128.60.009
±
0.00300.08
±
0.0150.11
±
0.02212.3H0.01
±
0.0030.06
±
0.0070.13
±
0.00911.90.004
±
0.00100.05
±
0.0120.07
±
0.01016.00.37
±
0.2111.66
±
0.1695.49
±
0.12715.10.050
±
0.01401.31
±
0.3443.46
±
0.62868.7
Σ
A–H
HGEABD
×
DBAHGCFEFGHABCD
Male
CBAB6F1
CBAHGFED
Female
CBAB6F1+
×
Male
B6CBAF1
Female
B6CBAF1+
BACDFGHEGHABCDDCBAHGFECBAHGFED
Female
CBA
Male
C57BL/6
Female
C57BL/6
Male
CBA
Fig. 2.
Proportions (%) of MUP fractions in the total pool of MUPs in urine of male and female mice with different genotypes.

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