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  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.
 204
 
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 thefact that the molecular-genetic basis and physiologicalmechanisms 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 mostdifficult puzzle in the biology of this numerous systematicgroup.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, inthe processes of intrapopulation informational exchangevia 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 manydetailed experimental and population genetic investigationsof 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 etal., 2002, 2005; Novikov, 2003; Armstrong et al., 2005;
 
1
 The work was supported by the Russian Foundation for BasicResearch (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 MUPswith urine is typical for males and is directly linked to theandrogenic status of an organism (Szoka, Paigen, 1978,1979; Berger, Szoka, 1981; Hayakawa et al., 1983).Meanwhile, as we have demonstrated earlier, in laboratorymice the qualitative composition of MUPs of castratedmales is identical to that of females, and the MUPs patternis determined genetically (Churakov, Novikov, 2000).These data allow us to put forward a question about therole of genetic and age-related factors in the establishmentof sex differences in MUPs composition during postnatalontogenesis of the laboratory mouse.The aim of our research was to perform a comparativequantitative analysis of MUPs expression between malesand females of laboratory mice of two genotypes duringthe pre- and postpubertal periods of ontogenesis.MATERIALS AND METHODSThe experiments were performed using males andfemales of two highly inbred and genealogically unrelatedlaboratory mice strains, CBA/LacY and C57BL/6JY (
 n
 =102). Animals were kept in groups of 4–6 individuals instandard 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.
 DEVELOPMENTAL GENETICS
 The Pattern of Major Urinary Proteins (MUPS) Expressionduring Postnatal Ontogenesis of the Laboratory Mouse Dependson Genotype and Sex
 
1, 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-, and12-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 expressionduring ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulationinformation exchange via pheromones, we put forward the idea that the genetically determined structure of theolfactory 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.
 
 RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
 
Vol. 40
 
No. 4
 
2009
 THE PATTERN OF MAJOR URINARY PROTEINS (MUPS) EXPRESSION205
 Urine was taken from 3-, 4, and 12-week-old mice bygentle abdomen massage, individually collected in plastic2 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 performedusing 0.1 M
tris
 -acetate buffer, pH 5.5. Samples were pre-pared by mixing aliquots of urine (2–10
µ
 l) with 0.1 M
 tris
 -HCl buffer, pH 7.4, containing 20% glycerin and0.01% 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 bythe 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 (GraphPadSoftware, USA).RESULTS
  Age dynamics of MUPs expression in male and femalelaboratory mice of CBA/LacY strain.
 As shown in Table 1,males of this genotype have a markedly heterogeneouscombination of MUP fractions already at the 3rd week of ontogenesis; when they are 4-week-old, fractions B and Cappear in their urine. Partial value of these fractionsdecreases from 25.7 to 13.4% during the analyzed period(Fig.
a
 ), while their absolute values progressively increaseto the 12th week of ontogenesis (Table 1).Total content of MUP fractions increases in males85.8-fold to the 12th week; the minimal factor of incre-ment belongs to fraction C (
 ä
 
ë
 = 7.3), the maximal one tofraction D (
 ä
 
D
 = 212.1). Percentage of this major fractionin 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 nothave fractions B and C in their MUP complex (Table 1);partial values of the other five fractions remain relativelystable while their absolute values rise steadily (Fig. 1
 b
 ).Total content of MUPs fractions in females increases17.5-fold to the 12th week; the minimal increment factorbelongs to fraction H (
 ä
 
H
 = 5.0), the maximal one to frac-tions D and E (
 ä
 
D
 = 30.0, ä
 
E
 = 26.7). Percentage of themajor fraction E in the general MUP pool grows from31.9% in prepubertal 3-week-old animals to 47.8% in12-week-old females (
  p
 < 0.01).Generally, the rate of increment of MUPs fractionscontent in urine of CBA/LacY males is 4.9 times higherthan that of females (Table 1).
  Age dynamics of MUPs expression in male and femalelaboratory mice of C57BL/6JY strain.
 As demonstrated inTable 2, already 3-week-old animals have a heterogeneouscombination of MUP fractions; fraction B appears in urineof 4-week-old males of this genotype.Total MUPs fractions content increases 123.6-fold inmales during the analyzed period; the minimal incrementfactor belongs to fraction G (
 ä
 
G
 = 13.8) and the maximalone to fraction D (
 ä
 
D
 = 265.0). The partial value of thisfraction differs considerably in pre- and postpubertal peri-ods and comprises correspondingly 7.9, 7.1 and 15.6% in3-, 4-, and 12-week-old animals. Percentage of fraction Cwhich prevails in adult animals (41.7%) comprises 21.1%in prepubertal 3-week-old males (Fig. 1
 c
 ).Contrary to males, females of this strain do not havefraction D; fraction B appears in their MUP complexwhen they are 4 weeks old (Table 2). Total content of MUPs increases 38.6-fold to the 12th week; the minimalincrement factor belongs to fraction H (
 ä
 
H
 = 5.0), themaximal one to fraction A (
 ä
 
A
 = 84.0), and percentage of the major fraction C virtually is stable (Fig. 1
 
 ).
 Table 1.
 Age dynamics of MUPs fractions content (mg/ml) in urine of CBA/LacY male and female laboratory miceMUPsfractionAge of males, weeksKAge of females, weeksK3 4 12 3 4 12A0.015
±
 0.00420.02
±
 0.0070.45
±
 0.07530.00.020
±
 0.00330.060
±
 0.01870.27
±
 0.07013.5B00.03
±
 0.0130.23
±
 0.0197.7C00.07
±
 0.0500.51
±
 0.0827.3D0.014
±
 0.00310.16
±
 0.1012.97
±
 0.141212.10.008
±
 0.00140.028
±
 0.00620.24
±
 0.04730.0E0.018
±
 0.00460.06
±
 0.0371.06
±
 0.04558.90.021
±
 0.00560.075
±
 0.02120.56
±
 0.10326.7F––G0.010
±
 0.00290.02
±
 0.0070.15
±
 0.01215.00.010
±
 0.00210.031
±
 0.01010.07
±
 0.0067.0H0.005
±
 0.00090.01
±
 0.0030.13
±
 0.00926.00.008
±
 0.00190.014
±
 0.00440.04
±
 0.0055.0
 Σ
 
A–H
 0.064
±
 0.01160.37
±
 0.2135.49
±
 0.12785.80.068
±
 0.01030.210
±
 0.05611.19
±
 0.21317.5
 Note:K is the factor of the fraction content increase.
 
 206
 RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
 
Vol. 40
 
No. 4
 
2009
 NOVIKOV et al.
 Generally, the rate of increment of MUPs fractionscontent in urine of C57BL/6JY males is 3.2 times higherthan that of females (Table 2).Results of two-factor dispersion analysis demonstratethat age significantly influences the absolute values of MUPs fractions but has practically no effect on their par-tial values (Tables 3, 4).DISCUSSIONPostnatal 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 feedactively; 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 neuroendocrinestate of the animal, and first of all upon the manifoldincrease of testosterone production by testes (Selmanoff etal., 1977a, b; Jean-Faucher, 1978). This relatively longperiod is over by the 7–8th week, and it is accompanied bymaturation of a number of enzymatic systems that take part
 1008060402001008060402003 4 12 3 4 12
 bc
 Fig.
Percentages of MUPs fractions (Y axis, %) of male (
 a
 ,
c
 ) and female (
 b
 ,
 ) laboratory mice of CBA/acY (
 a
 ,
b
 ) andC57BL/6JY (
 c
 ,
 ) strain at various ontogenetic points (X axis, weeks). A
  – (),
B
  – (),
C
  – (),
D
  – (),
E
  – (),
 F
 
 (),
G
  – (),
H
  – ().
 
Table 2.
Age dynamics of MUPs fractions content (mg/ml) in urine of C57BL/6JY male and female laboratory miceMUPsfractionAge of males, weeksKAge of females, weeksK3 4 12 3 4 12A0.005
±
 0.00090.007
±
 0.00130.68
±
 0.157136.00.005
±
 0.00140.007
±
 0.00160.42
±
 0.12584.0B00.006
±
 0.00170.51
±
0.11185.000.007
±
0.00370.13
±
0.04018.6C0.006
±
0.00140.018
±
0.00781.43
±
0.182238.30.022
±
0.01340.041
±
0.02950.99
±
0.19745.0D0.002
±
0.00030.004
±
0.00080.53
±
0.152265.0EF0.002
±
0.00020.002
±
0.00040.09
±
0.03145.00.003
±
0.00140.004
±
0.00160.06
±
0.01920.0G0.008
±
0.00130.009
±
0.00300.11
±
0.02213.80.006
±
0.00300.007
±
0.00340.04
±
0.0106.7H0.004
±
0.00050.004
±
0.00100.07
±
0.01017.50.006
±
0.00250.007
±
0.00350.03
±
0.0065.0
Σ
A–H
0.028
±
0.00300.050
±
0.01403.46
±
0.628123.60.043
±
0.02050.072
±
0.04061.66
±
0.37038.6
Note:K is the factor of the fraction content increase.

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