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 presented 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 combinatorial pattern. DOI: 10.1134/S1062360409040031 Key words: pre- and postpubertal periods, laboratory mice, major urinary proteins, sex differences, pheromones, olfactory image, MUPs combinatorial pattern, structure of the olfactory code.

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 particularly 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; Churakov, Novikov, 2000; Marchlewska-Koj et al., 2000; Babalyan, 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;
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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 Republic) in the inverted light cycle conditions (day—12 h, night—12 h). Our experiments were performed in spring.

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.

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THE PATTERN OF MAJOR URINARY PROTEINS (MUPS) EXPRESSION Table 1. Age dynamics of MUPs fractions content (mg/ml) in urine of CBA/LacY male and female laboratory mice MUPs fraction A B C D E F G H ΣA–H Age of males, weeks 3 0.015 ± 0.0042 0 0 0.014 ± 0.0031 0.018 ± 0.0046 – 0.010 ± 0.0029 0.005 ± 0.0009 0.064 ± 0.0116 4 0.02 ± 0.007 0.03 ± 0.013 0.07 ± 0.050 0.16 ± 0.101 0.06 ± 0.037 – 0.02 ± 0.007 0.01 ± 0.003 0.37 ± 0.213 12 K Age of females, weeks 3 4 0.060 ± 0.0187 – – 0.028 ± 0.0062 0.075 ± 0.0212 – 0.031 ± 0.0101 0.014 ± 0.0044 0.210 ± 0.0561 12 0.27 ± 0.070 – – 0.24 ± 0.047 0.56 ± 0.103 – 0.07 ± 0.006 0.04 ± 0.005 1.19 ± 0.213

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K 13.5 – – 30.0 26.7 – 7.0 5.0 17.5

0.45 ± 0.075 30.0 0.020 ± 0.0033 0.23 ± 0.019 7.7 – 0.51 ± 0.082 7.3 – 2.97 ± 0.141 212.1 0.008 ± 0.0014 1.06 ± 0.045 58.9 0.021 ± 0.0056 – – – 0.15 ± 0.012 15.0 0.010 ± 0.0021 0.13 ± 0.009 26.0 0.008 ± 0.0019 5.49 ± 0.127 85.8 0.068 ± 0.0103

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 polyacrylamide gel. Separation of native proteins was performed using 0.1 M tris-acetate buffer, pH 5.5. Samples were prepared 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 Coomassie 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 analysis of the fractionated MUPs was performed at the Biochemistry Department of the N.I. Vavilov Research Institute of Plant Industry RAAS using a GelScan XL densitometer (“Pharmacia”, Sweden). Statistical analyses of the experimental data were performed 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 increment 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 prepuRUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY

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 fractions 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 periods 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).
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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 partial values (Tables 3, 4). DISCUSSION Postnatal ontogenesis of the house mouse Mus musculus 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 relationships between males are established. This process is associated 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 fraction A B C D E F G H ΣA–H Age of males, weeks 3 0.005 ± 0.0009 0 0.006 ± 0.0014 0.002 ± 0.0003 – 0.002 ± 0.0002 0.008 ± 0.0013 0.004 ± 0.0005 0.028 ± 0.0030 4 0.007 ± 0.0013 0.006 ± 0.0017 0.018 ± 0.0078 0.004 ± 0.0008 – 0.002 ± 0.0004 0.009 ± 0.0030 0.004 ± 0.0010 0.050 ± 0.0140 12 0.68 ± 0.157 0.51 ± 0.111 1.43 ± 0.182 0.53 ± 0.152 – 0.09 ± 0.031 0.11 ± 0.022 0.07 ± 0.010 3.46 ± 0.628 K 136.0 85.0 238.3 265.0 – 45.0 13.8 17.5 123.6 Age of females, weeks 3 0.005 ± 0.0014 0 0.022 ± 0.0134 – – 0.003 ± 0.0014 0.006 ± 0.0030 0.006 ± 0.0025 0.043 ± 0.0205 4 0.007 ± 0.0016 0.007 ± 0.0037 0.041 ± 0.0295 – – 0.004 ± 0.0016 0.007 ± 0.0034 0.007 ± 0.0035 0.072 ± 0.0406 12 0.42 ± 0.125 0.13 ± 0.040 0.99 ± 0.197 – – 0.06 ± 0.019 0.04 ± 0.010 0.03 ± 0.006 1.66 ± 0.370 K 84.0 18.6 45.0 – – 20.0 6.7 5.0 38.6

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

in metabolism of testosterone and cell receptors to dihydrotestosterone (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 interstrain 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 demonstrated 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
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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 identical 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, chronologically 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 expression of fractions B and C in CBA/LacY strain and of fraction D in C57BL/6JY strain is androgen-dependent (Churakov, Novikov, 2000). Importantly, 3-week-old animals 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 biosynthesis of proteins, which make up the electrophoretic
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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 Males CBA Age Fraction Interaction Random deviations Females CBA Age Fraction Interaction Random deviations Males C57BL/6 Age Fraction Interaction Random deviations Females C57BL/6 Age Fraction Interaction Random deviations 2 5 10 72 0.0 0.5643 0.01820 0.09560 0.0 5.903 0.1904 ns <0.0001 ns 2 6 12 119 0.0 0.1770 0.04651 0.07133 0.0 2.482 0.6521 ns <0.05 ns 2 4 8 180 0.0 0.6343 0.04813 0.05180 0.0 12.25 0.9292 ns <0.0001 ns 2 6 12 175 0.0 0.4467 0.08342 0.01426 0.0 6.290 1.175 ns <0.0001 ns Degrees of freedom, df Mean square, MS F test, F Significance, p

Note: ns indicates nonsignificant values.

fraction D, or indicate the existence of inter-strain variations in the expression of fraction B. In favor of the first suggestion is the fact that fraction D is the one whose content 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 ontogenesis (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; Selmanoff et al., 1977b; Stalvey, Payne, 1983; Diuzhikova, 1994) and an increased tissue sensitivity to its physiologically 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 primarily 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-dependent expression of Mup genes (Knopf et al., 1983; Kuhn et al., 1984), we propose that our results demonstrate the inter-strain differences of the level of this hormone during postnatal ontogenesis of the laboratory mouse (Minetti et al., 1986). Growth hormone and prolactin could also play an essential role in generation of intersex differences in dynamics of the same MUPs fractions during ontogenesis (Michael et al., 1980; Norstedt, Palmiter, 1984; Johnson et al., 1995). Mechanisms of hormonal control of expression of certain protein fractions during female ontogenesis, particularly 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
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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 partially 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 animals 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 heterochronic nature of MUPs fractions expression during postnatal 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 percentages 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 associated 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 (letters), 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 expression 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 genotypes 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 heterozygotes. The fundamental issue of the modifying role of the environment, and specifically of pheromonal imprinting,
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in formation of the sexual selectivity on the basis of MUPs pattern in males and females of house mouse Mus musculus 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 manipulation and critical discussions during the preparation of the manuscript. REFERENCES
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