Elena Fedorova1,2, Irina Ermakova1,3, Sergey Novikov1

Pavlov Institute of Physiology, Russian Academy of Sciences, 2Institute of Experimental Medicine, Russian Academy of Medical Sciences, 3Institute of Cytology, Russian Academy of Sciences
1I.P.

E-mail: ellena_fedorova@mail.ru

Aggressive behaviour represents universal type of social communication system in Animal Kingdom. Aggression takes place between at least two animals after an intensive exchange of information via several sensory modalities: visual, acoustic, tactile, and chemosensory. Thus, genetic analysis of aggression is rather problematic due to complex and highly dynamic interactions between animals. In mice, like in many other mammals, the leading sensory modality is olfaction, and much of the information is conveyed by highly specific ligands, pheromones, which have the ability to initiate innate (genetically programmed) behavioural responses from receiver (Novikov, 1993). The model of pheromonally mediated aggression in Mus musculus L. was introduced in social behaviour analysis 35 years ago (Kessler et al., 1975). However, until recently genetical basis of differences in physiological activity of mouse androgen-dependent pheromones such as 2-sec-butyl4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and 2,3-dehydro-exo-brevicomin (DHB) (Novotny et al., 1999; Perez-Miller et al., 2010) was poorly understood. In 2007 it was reported that the main fraction of mouse urinary proteins with M.m.18-20 kDa, named «major urinary proteins» (MUPs), by itself and without pheromonal ligands (SBT, HMH, DHB) could efficiently promote aggressive reactions from the recipient (Chamero et al., 2007). It means that MUPs are a powerful tool for genetical dissection of social behaviour (Novikov, 2007, 2008). In 2003 we put forward an idea that genes Mup, Gus and Mep1a form an «aggressive behaviour gene network» (Novikov, 2003). Gus gene is located on chromosome 5 and encodes β-glucuronidase (GUS, EC 3.2.1.31), which activates latent aggression-promoting cues in mouse urine (Ingersoll et al., 1982). Mep1a is mapped to chromosome 17 and encodes meprin (EC 3.4.24.18), a metallopeptidase that probably participates in degradation of MUPs (Beynon et al., 1996). Mup multigene locus is located on chromosome 4. In the current study, we examined the idea of the «gene network» in details. Quantitative analysis of plasma testosterone level, MUPs content and GUS activity in voided urine, kidney, preputial and salivary glands in male mice of different genotypes revealed a strong correlation between plasma testosterone level, GUS activity, and differential pattern of Mup gene expression. Key words: pheromones, major urinary proteins, βglucuronidase, aggressive behaviour, MUP combinatorial pattern, laboratory mice, olfactory code

Fig 1. Testosterone influence on β-glucuronidase activity in voided urine, kidneys, salivary and preputial glands from male mice of different genotypes
Kidneys
30000 25000 20000 CBA/LacY C57BL/6JY 250 200

Table 4. Combinatorial genotype-dependent MUP profiles during male mice ontogenesis
Genotype Age, weeks Consequential Ratio of principal fractions’ fractions order A B C D E F G H + − + + − + + + GCAHFD + + + + − + + + CADGBHF + + + + − + + + CADBGFH + + + + − + + + CADBGFH + − − + + − + + EADGH + + + + + − + + DCEABGH + + + + + − + + DECABGH + + + + + − + + DECABGH 0.45:0.40:0.15 0.60:0.25:0.15 0.55:0.25:0.20 0.55:0.25:0.20 0.40:0:30:0:30 0.50:0.25:0.20:0.05 0.45:0.35:0.15:0.05 0.60:0.20:0.10:0.10 Fractions

Urine
150

3 4 C57BL/6JY 8

15000
10000 5000 0

100 50
0

Sham-operated Castrated

Castrated+TP

Sham-operated Castrated

Castrated+TP

12 3 4 CBA/LacY 8

1500 1200

Preputial glands

Salivary glands
10000
8000

F.U.

F.U.

900 600 300 0

6000 4000 2000 0

Sham-operated

Castrated+TP

Sham-operated Castrated

Castrated+TP

12

Fig 2. β-Glucuronidase activity in kidneys and voided urine from male mice of different genotypes
Kidneys
10000 8000 500 400

Urine
F.U.
300 200 100 0

F.U.

6000 4000 2000 0
CBA/LacY CBAB6F1 B6CBAF1 C57BL/6JY

Quantitative evaluation of 8 protein fractions A-H with regard to age and genotype revealed significant interstrain differences in MUPs expression patterns in male mice during postnatal development. According to their relative values at various ontogenetic stages, we divided these MUP fractions into two subsets that probably reflect their different functional significance. The first subset, which could be characterized as «ontogenetically stable», consists of fractions A, C, D, and E; the second subset consists of fractions B, F, G, and H, which are significantly influenced by the age factor (Chemical Senses, in preparation).

CBA/LacY CBAB6F1 B6CBAF1 C57BL/6JY

Genotype

Genotype

Table 5. Combinatorial genotype-dependent MUP codes for gender and physiological state in mice
Genotype Physiological state Consequential Ratio of principal fractions’ fractions order A B C D E F G H + + + − − + + + CABFGH + + + − − + + + CABFGH 0.80:0.20 0.50:0.50 0.40:0.35:0.25 0.40:0.30:0.30 0.50:0.35:0.15 0.60:0.35:0.05 0.45:0.30:0.15:0.10 0.55:0.20:0.15:0.10 Fraction

Our data on genotype- and sex-specific ratios of principal MUP fractions (A, C, D, E) in CBA/LacY and C57BL/6JY mice may provide new insights into mechanisms of genetic and epigenetic regulation of Mup gene cluster, which result in expression of specific bouquets of MUPs. We propose that various volatile pheromonally active ligands (e.g. SBT, HMH, DHB) represent «letters» of the chemical «language of communication» in Mus musculus L. and that, correspondingly, specific combinations of MUPs efficiently organize these «letters» into readable «words» that encode sex, physiological state, age, and genotype differences (Novikov et al., 2009). Together with the data showing that vomeronasal neurons can be directly activated by MUPs (Chamero et al., 2007), and that expression of pheromone receptors V2Rs is sex specific (He et al., 2008, 2010) and combinatorial (Silvotti et al., 2007), our results provide valuable insights into fine molecular mechanisms of olfactory coding of intermale aggression in Mus musculus L. based on specific subsets (modules) of MUPs combinations and their interplay with complementary sets of V2Rs. We suggest that androgen- and genotype-dependent activity of kidney metallopeptidase meprin (EC 3.4.24.18), which probably participates in degradation of MUPs (Beynon et al., 1996), is tightly linked with pheromonal activity of several oligopeptides (Tsujikawa, Kashiwayanagi, 1999; Kimoto et al., 2005, 2007). This peptidase is encoded by Mep1a gene mapped to chromosome 17 near the main histocompatibility locus H-2. Taking into account that H-2 complex also participates in olfactory-mediated social recognition in mice (Boehm, 2006, 2009; Brennan, Kendrick, 2006; Rodriguez, Boehm, 2009; Tirindelli et al., 2009; Kwak et al., 2010), we propose for the first time that Gus, Mup, Mep1a, and H2 genes represent an «aggressive behaviour gene net» in Mus musculus L. (Figure 4).

F.U.

F.U.

Table 1. Correlation between plasma testosterone level and β-glucuronidase activity in kidneys and voided urine from male mice of different genotypes
Kidneys Genotype F.U. CBA/LacY CBAB6F1 B6CBAF1 C57BL/6JY +0.670* +0.348 -0.109 +0.158 F.U./mg tissue +0.702* +0.390 -0.074 +0.026 F.U. +0.851** +0.327 +0.243 +0.382 Urine

females castrates C57BL/6JY

Fig 4. Proposed «aggressive behaviour gene net» in Mus musculus L.

castrated+T + + + + − + + + CADBFGH males females castrates CBA/LacY castrated+T + + + + + − + + DEACBGH males + + + + + − + + DEACBGH + + + + − + + + CADBFGH + − − + + − + + EADHG + − − + + − + + EDAHG

*p<0.05 **p<0.01

Fig 3. Content of MUP fractions in male mice of different genotypes
Subjects, housing conditions and urine collection Experiments were performed during winter and spring periods using males and females of two highly inbred and genealogically unrelated strains of laboratory mice, CBA/LacY and C57BL/6JY, and their F1 hybrids from reciprocal crosses (n=365). Animals were kept in groups of four in standard polypropylene cages T-2 («Velaz», Czech Republic) under conditions of an inverted light cycle (day – 12 h, night – 12 h). Urine samples were taken from animals by gentle abdomen massage, individually collected in plastic 2 ml Eppendorf tubes at the same time of the day, and stored at –18º C. Three sets of experiments were performed. In the first set, we studied β-glucuronidase activity in male mice urine, kidney, and two pheromone-producing organs, preputial and salivary glands. In the second set of experiments, we studied the influence of genotype on MUPs production in male mice during postnatal development. In the third set, effects of male castration and testosterone therapy on MUPs excretion profile were investigated. Experimental procedures and testosterone measurement For the first and third sets of experiments, six-week old male mice were castrated using standard technique under ether anesthesia. At the age of eight weeks some animals were implanted with silicone capsules («Dow Corning», USA) containing crystalline testosterone propionate (TP) («Sigma», USA). This procedure provided the daily TP dose of 27.1 μg and these animals formed the «castrated+TP» group. The plasma testosterone concentration was estimated by radioimmunoassay using standard kits («Sorin», France). MUPs characterization measurement and β-glucuronidase activity
3,5 CBA/LacY

3
2,5

C57BL/6JY

Quantitative evaluation of 8 protein fractions A-H with regard to genotype and sex revealed that the concentration-based consecutive order of different MUP fractions varies with sex and androgenic state. In other words, presence and concentration of various MUP fractions form an individual combinatorial pattern, which might reflect the genotype- and sex-specific olfactory signature of the animal. It is noticeable that the ratios of principal fractions in testosterone-treated castrates and sham-operated males are almost the same, especially in the case of C57BL/6JY mice. We suggest that our data on genotype-specific ratios of principal MUP fractions may reflect the number of genes, which code proteins of the given fraction. Thus, stability of the «MUPs ratio» may be regarded as a new feature of testosterone-dependent Mup genes expression (Chemical Senses, in preparation).

mg/ml

2 1,5
1

0,5 0
A B C D E F G H

Table 6. Combinatorial genotype-dependent MUP codes in F1 progeny from reciprocal crosses
Gender Genotype

MUP fractions
CBAB6F1 Male B6CBAF1

Consequential Ratio of principal fractions’ fractions order A B C D E F G H + + + + + + + + DCEABGHF 0.40:0.25:0.20:0.15 + + + + + + + + DACEBGHF 0.45:0.25:0.15:0.15 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10 + + + + + + + + CAEDBGHF 0.35:0.30:0.25:0.10

Fraction

Table 2. Correlation between plasma testosterone level and concentrations of MUP fractions in male mice of different genotypes
Fraction Genotype CBA/LacY CBAB6F1 B6CBAF1 A B C D E F G H

CBAB6F1 Female B6CBAF1

 Our study revealed highly complex tissue- and genotypespecific androgen influence on expression of Mup and Gus genes.  We demonstrate that 8 analyzed MUP fractions form two distinct subsets (modules) according to stability of their ratio during ontogenesis. The «adult proportion» profile of different MUPs is genotype-specific, appears very soon after weaning and resembles a «bar code».  Concentration-based consecutive order of different MUP fractions varies with sex, genotype and androgenic state. Presence and concentration of various MUP fractions form an individual combinatorial pattern, which might reflect the genotype- and gender-specific olfactory signature of the animal. Our results show that this modular organization of MUPs pattern is common for two genealogically unrelated strains CBA/LacY and C57BL/6JY and thus, probably, for mice in general.  We suggest that these specific subsets (modules) of MUPs combinations are subsequently screened and recognized by complementary vomeronasal neurons of the recipients, and that these processes constitute the basis of pheromonal coding in Mus musculus L.  Taken together, our data demonstrate that MUPs may represent a very promising tool for genetic dissection of olfactorymediated aggressive behaviour in Mus musculus L.

-0.604 +0.846* +0.698 +0.122 -0.467

+0.682 +0.116

+0.777 +0.746* +0.027 +0.501 +0.519 +0.672 +0.148 +0.631 -0.650 +0.054 +0.455 +0.687 +0.502 +0.693 +0.948* +0.942* +0.707 +0.454 +0.024

In urine of male and female F1 progeny from reciprocal crosses, all 8 MUP fractions are present. Concentration-based «MUP formulae» are with high significance similar within sex: Spearman’s rank-order correlation coefficients are rs=0.91 in males and rs=0.98 in females (P<0.01). Ratios of principal fractions are also similar within sex, which is especially obvious in females of both genotypes (Table 6). However, at the moment it is not clear how the presence and ratios of different MUP fractions are defined in F1 animals and to what extent parental patterns are inherited (Chemical Senses, in preparation). The obtained data revealed distinct interstrain differences in βglucuronidase activity in kidney, urine and salivary glands (Figure 1). Importantly, urine from testosterone-treated castrates and shame-operated C57BL/6JY males, which were previously characterized as «pheromone-deficient» mice (Novikov, 1988), has a very low level of β-glucuronidase activity (Figures 1, 2). However, it was previously shown that incubation of urine samples from C57BL/6JY with commercial GUS increases the activity of aggression-promoting pheromones (Novikov, 1993). Thus, GUS activity is an important factor in formation of the individual combination of pheromones and, consequently, in the process of communication. Our results also demonstrate that olfactory coding by MUPs in Mus musculus L. is probably based on a modular principle (Tables 4-6). We suggest that ratios and concentrations of MUP fractions in the given combination define behavioural response of the recipient mouse: some MUPs combinations can promote stereotype intermale aggression, others can initiate mating-like behaviour. The proposed GUS and MUP models can effectively trace the multicomponent chemosensory pathways from gene(s) to olfactory-mediated physiological and social behaviour responses in laboratory mice via pheromones. These models represent a valuable methodological approach for dissection of aggressive behaviour phenotypes using pheromones as a fine natural tool and lead to our better understanding of how brain translates chemical information encoded by odorant mixtures (Novikov, 2007, 2008).

The authors are grateful to Gennady Churakov (University of Münster, Germany), Anatoly Philimonenko (Institute of Molecular Genetics, Prague, Czech Republic) and Valentina Sukonina (Umeå University, Sweden) for collection of experimental data and helpful discussions, and to Sergey Mylnikov (St. Petersburg State University, Russia) for his invaluable help in statistical data analyses. The work was supported by Russian Foundation for Basic Research (projects 02-04-49273, 04-04-63050, 07-04-01762) and Russian State Science and Technology Program «Priority Frontiers in Genetics» (project 2.152 to SN). Elena Fedorova is deeply grateful to the Chromosome Research journal for awarding her with the 2008 Scholarly Manuscript Prize, which allowed her to participate in this ECRO Congress.

MUPs were analyzed by electrophoresis in polyacrylamide gel (PAGE) (Churakov et al., 1992; Churakov, Novikov, 2000). Separation of native proteins was performed using 0.1 M trisacetate 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 MUP fractions were evaluated using Calibration Kit proteins («Sigma», USA). Quantitative analysis of MUP fractions was performed using GelScan XL densitometer («Pharmacia», Sweden). The activity of native lysosomal β-glucuronidase (EC 3.2.1.31) was measured in voided urine and homogenated samples of kidney, preputial and salivary glands by the rate of phenolphthalein-β-D-glucuronide hydrolysis («Sigma», USA), and expressed in Fishman’s units (F.U.) according to standard method.

C57BL/6JY +0.295 +0.703 +0.717 +0.597

*p˂0.05

Table 3. Correlation between plasma testosterone level and proportions of MUP fractions in male mice of different genotypes
Fraction Genotype CBA/LacY A B C D E -0.557 F G H

-0.603 +0.867* +0.697 -0.016 +0.644 +0.204 -0.557 -0.667

+0.736 -0.011

Statistical data analyses Biochemical data were expressed as means ± standard error. An ordinary parametric t-Student test and nonparametric Spearman correlation analyses were performed. The results were also analyzed using the two-way analysis of variance (ANOVA) with «age» and «genotype» as experimental factors (GraphPad Software Inc, San Diego, CA). Significance level was set at P0.05.
CBAB6F1 B6CBAF1 -0.282 +0.101 +0.497 -0.349 +0.253 -0.144 +0.407 +0.613 +0.463 +0.684 +0.945* +0.950* +0.641 -0.198 -0.775*

C57BL/6JY -0.339 +0.739 +0.328 +0.353

*p˂0.05

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