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 0012-4966/03/0708-$25.00 © 2003 MAIK “Nauka/Interperiodica”0318
  Doklady Biological Sciences, Vol. 391, 2003, pp. 318–321. Translated from Doklady Akademii Nauk, Vol. 391, No. 5, 2003, pp. 707–711.Original Russian Text Copyright © 2003 by Novikov.
 In one of the most interesting and promising modelsof molecular physiology, the functional activity of theandrogen-dependent pheromones of house mouse
 Musmusculus
 L. (in particular, 2-
 sec
 -butyl-4,5-dihydrothia-zole, 3,4-dehydro-
 exo
 -brevicomin, and
 E 
 ,
  E 
 -
 α
 -
 and
  E 
 -
 β
 -farnesenes) is related to the urinary proteins of themajor urinary protein (MUP) complex [1]. This com-plex includes a group of low-molecular-weight acidicproteins (m. w., 18–19 kDa; pI, 4.2–4.7). MUPs areencoded by a cluster of 
 Mup
 genes, located in chromo-some 4 (linkage group VIII). To date, the
 Mup
 geneshave been cloned, and the multihormonal control of the
  Mup
 expression in hepatocytes has been studied indetail. Now the
 Mup
 system is a well-developed modelused in molecular genetics of mammals [2].The existence of genotypic differences in the func-tional activity of pheromones is commonly attributed tothe hereditarily determined deviations in the function of some enzyme systems, in particular, the kidney acidichydrolase
β
 -glucuronidase (GUS, EC 3.2.1.31). Thelatter is encoded by the
Gus
 gene (located in chromo-some 5) with participation of sex steroids and involvedin the transformation of the immobilized chemosignal(propheromone) in the form of glucuronides to its phys-iologically active form [3].This was the first study to present the entire pictureof the functioning of these two genetic systems as apotential basis for the regulation of the physiologicalactivity of the androgen-dependent pheromones of house mouse, based on the analysis of the characteristicfeatures of the expression of the protein products of genes
 Mup
 and
Gus
 as dependent on the androgen sta-tus of the organism.The study was performed in spring using maturemales of two highly inbred, genealogically unrelatedstrains of mice with hereditarily determined differencesin the physiological activity of androgen-dependentpheromones, namely, strains CBA/LacY andC57BL/6JY (
 n
 = 48) [4]. The proteins of the MUP com-plex were separated by SDS-PAGE with subsequentdensitometric analysis of fractions [2]. The activity of lysosomal GUS in kidney homogenate and urine wasmonitored by the rate of hydrolysis of phenolphthalein-
 β
 -
  D
 -glucuronide (Sigma, United States) and expressedin Fishman’s units [3]. The testosterone content of blood plasma was determined radioimmunologically,using standard kits (Sorin, France). The results werestatistically treated using standard procedures.As seen from Fig. 1, the genotype significantlyaffects the qualitative and quantitative composition of MUPs: fractions A, B, C, D, G, and H were found in themice of both strains, whereas fractions E and F mayserve as genetic markers of the strains CBA (fraction E)and C57BL/6 (fraction F). In general, the concentra-tions of the MUP fractions were 5.49
+
 0.127 and
 3.46
±
 0.628
 mg/ml in CBA and C57BL/6 males,respectively.Correlation analysis of the level of testosterone inblood plasma, absolute content of different MUP frac-tions, and the activity of GUS in kidney homogenatesand urine showed a significant positive correlation
 PHYSIOLOGY
 Coordinated Expression of the Genes
Gus
 and
 Mup
 as a PotentialBasis of the Functional Activity of the Androgen-DependentPheromones of the House Mouse (
  Mus musculus
 L.)
 S. N. Novikov
 Presented by Academician A.D. Nozdrachev March 31, 2003Received April 2, 2003
 Pavlov Institute of Physiology, Russian Academy of Sciences,nab. Makarova 6, St. Petersburg, 199034 Russia
 00.51.01.52.02.53.03.54.0ADCBGHEF
 Strain
 CBA
 Strain
 C57BL/6
 Protein fractionsProtein, mg/ml
 The concentrations of the protein fractions of the MUPcomplex in the urine of male laboratory mice with differentgenotypes.
 
 DOKLADY BIOLOGICAL SCIENCES
 
Vol. 391
 
2003
 COORDINATED EXPRESSION OF THE GENES
 Gus
 AND
 Mup
 319
 between the testosterone level and the content of thefraction B in CBA males (Table 1) and a positive cor-relation between the testosterone content and theGUS activity, also characteristic solely of CBAmales (Table 2).It was shown earlier that a change in the endogenoustestosterone level in mature male mice, induced by cas-tration, resulted in a drop of the absolute content of allseven fractions in C57BL/6 mice and six fractions inCBA mice [2]. Therapy with testosterone led to the res-toration of the relative (partial) content of the fractionsto the level characteristic of sham-operated animals. Inthis aspect, the expression of the proteins of three frac-tions (B and C in CBA mice and D in C57BL/6 mice)is of particular interest. These three fractions are com-pletely absent in MUPs of females, disappear after cas-tration of mature males, and are restored to the preop-erational level after testosterone therapy [2]. Therefore,the biosynthesis of proteins comprising these fractionscompletely depends on the level of endogenous test-osterone in the organism. It can be assumed the frac-tions B, C, and D are the most interesting in terms of studying the characteristics of affinity binding of MUPswith the androgen-dependent pheromones [1].The data presented in this study indicate that theexpression of two different proteins (the androgen-dependent fraction B of the MUP complex and
β
 -glu-curonidase), coordinated with the testosterone level, ischaracteristic of adult male CBA mice (with highlyactive sex pheromones [4]), but not C57BL/6 mice.Thus, we found a significant correlation between thelevel of the hormone regulating the biosynthesis of sexpheromones in house mouse [4] and the activity of GUS involved in the transformation of immobilized(latent) propheromone in the form of glucuronides inthe physiologically active form [3]. These data mayconsiderably broaden our notion on the moleculargenetic mechanisms regulating the physiological activ-ity of natural compounds of this group, under the actionof steroids on genetic material (genes
Gus
 and
 Mup
 ).A low activity of the androgen-dependent phero-mones affecting the reproduction of C57BL/6 mice isapparently related to the low testosterone level and thegenetically determined characteristics of steroidogene-sis and hormonal reception [4]. Earlier, using the mod-els of the pheromonal regulation of aggressive behaviorand spermatogenesis, we showed that excreted urine of mature males of this strain contained a large amount of glucuronides: the incubation of the specimens withcommercial preparations of 
β
 -glucuronidase
in vitro
 resulted in a significant increase in the physiologicalactivity of the pheromone [4]. On the other hand, as wasshown in [3], the activity of native enzyme in C57BL/6males was decreased by five and ten times in the kid-neys and urine, respectively, compared to CBA males.However, a possible universal role of GUS in theregulation of the physiological activity of androgen-dependent pheromones is questionable. A marked cor-relation between the enzyme activity in the kidney andurine of CBA males and a low correlation in C57BL/6males (Table 2) may be indicative of not only hetero-genic composition of the isozyme pool of GUS inexcreted urine, but also a high substrate specificity of the enzyme
in vivo
 , as well as the effect of the genotypeon its physical and chemical characteristics (in particu-lar, the resistance to the specific inhibitor, glucaro-1,4-lactone, a product of glucuronic acid metabolism [3]).In this aspect, a comparative biochemical study of theisozyme forms of this hydrolase in the kidney andpreputial gland (the main producers of a series of chem-ically identified androgen-dependent pheromones of house mouse [1]) would be especially interesting.Based on preliminary data on the separation of theproteins of the MUP complex using ion-exchange chro-matography on DEAE cellulose, we assumed that eachof the electrophoretic fractions A–H is a mixture of iso-morphic proteins, differing in the charge of the mole-cule. This assumption is corroborated by the results of a long-term work performed by scientists from theUnited Kingdom, who studied the genetic polymor-
 Table 1.
Correlation between the testosterone content in blood plasma and the content of individual MUP fractions in urineof laboratory male mice of different genotypesGenotypeFractionABCDEFGHCBA 0.604+0.846*+0.698+0.1220.467+0.682+0.116C57BL/6 +0.295+0.703+0.717+0.597+0.707+0.454+0.024
 *
P
 < 0.05.
 Table 2.
Correlation between the testosterone content inblood plasma and the activity of 
β
 -glucuronidase in kidneysand urine of male laboratory mice of different genotypesGenotypeKidneysUrineTASACBA+0.670*+0.702*+0.851**C57BL/6+0.158+0.026+0.382
 *
 P
< 0.05, **
P
 < 0.01. TA, total enzyme activity, expressed inFishman’s units; SA, specific enzyme activity, expressed in Fish-man’s units per mg tissue
 
 320
 DOKLADY BIOLOGICAL SCIENCES
 
Vol. 391
 
2003
 NOVIKOV
 phisms of MUPs in the populations of the wild form of house mouse [5]. Using HPLC and mass-spectrometry,it was shown that the proteins of the MUP complex,which do not differ in molecular weight, may be presentin different chromatographic fractions. This may beaccounted for by their amino acid composition (in par-ticular, the ratio between glycine, glutamine, andlysine). In this case, insignificant amino acid substitu-tions may result in significant conformational changes,thereby affecting the functional properties of the pro-tein molecule, the basis of the ligand-binding pocket of which is formed by Val58, Ala107, Leu44, and Ala122[6]. The authors of [6] assumed that the substitution of Val58 and Ala107 with phenylalanine and Leu44 andAla122 with valine may reflect the characteristics of theprotein binding with one or another ligand. A goodexperimental confirmation of this assumption was thedetermination, in the MUP molecule, of the high-affin-ity site for the known androgen-dependent pheromones(2,3-dehydro
 -exo
 -brevicomin and 2-
 sec
 -butyl-4,5-dihydrothiazole) and the creation, on the basis of thesedata, of an artificial physiologically active hexapeptideN-Glu–Glu–Ala–Arg–Ser–Met, which accelerates sexmaturation of female laboratory mice [7]. Therefore, itcan be assumed that the proteins contained in the elec-trophoretic fractions A–H (Fig. 1) will differ, on the onehand, in charge; on the other hand, in the compositionof the aforementioned amino acids.Taken together, the above data and our results sug-gest that the mechanisms of regulation of the functionalactivity of the androgen-dependent pheromones in lab-oratory mice may be the following.The expression of the genes coding for the MUPcluster is controlled by sex steroids (primarily, test-osterone). The level, balance, and effectiveness of ste-roid hormones depend on the genetically determinedcharacteristics of steroidogenesis (first of all, the activ-ity of 
3
 β
 -hydroxysteroid dehydrogenase/isomerse (EC1.1.1.145) and the hormonal reception), as well as envi-ronmental factors (in particular, the nature of the intrap-opulation relations). The aforementioned factors affectthe qualitative and quantitative composition of MUPs,which are synthesized in the liver and excreted from theorganism with urine. The differences in the characteris-tics of the protein binding with physiologically activeligands (determined, on one hand, by its structural andconformational characteristics and, on the other hand,by the activity of 
β
 -glucuronidase) will affect the abso-lute and partial concentrations of these compounds inthe solution and the pheromone composition and deter-mine the functional and informational properties of thepheromonal “bouquet” on the whole (as a vector withtargeted effect on the recipient organism). The centralplace in this scheme belongs to the idea on key roles of (a) the ratio between different MUP fractions; (b) dif-ferential expression of these proteins in hepatocytes [2];and (c) significant differences in the nature of affinitybinding of the protein molecule with one or anotherligand [1]. Thus, according to our scheme, a limitednumber of individual pheromone-like compoundsexists in the nature, and the mechanisms forming theolfactory image are based on the combination of theratios between these physiologically active compoundsin the solution.The genetic model based on a coordinated expres-sion of the genes
Gus
 and
 Mup
 under the action of tes-tosterone probably represents the first and yet the onlyexample of a specific gene net whose nucleus consistsof these genes interacting at the level of their productsand thus affecting the physiological activity of theandrogen-dependent pheromone in rodents.Within the framework of the genetic physiologicalmodel put forward, particularly interesting are the dataon the presence, in excreted urine of house mouse, of meprine, an androgen- and genotype-dependent metal-lopeptidase (EC 3.4.24.18), the main function of whichis degradation of the proteins of the MUP complex [8].The process of MUP degradation to individual oli-gopeptides is the necessary and, possibly, the key stagein the mechanism of effective action of the ligand–pro-tein complex on the recipient organism. A similar con-clusion on a pivotal role of the oligopeptides from urineof male rats, weighing less than 5 kDa, in the regulationof the activity of the
c-Fos
 genes in the cells of addi-tional olfactory bulb in females was made in [9].In the context of the hypothesis on the possible roleof the protein product of the
Gus
 gene,
β
 -glucu-ronidase, in the regulation of the functional activity of house mouse pheromones, the study on uridine5'-diphosphoglucuronyltransferase (UDP-glucurono-syltransferase, EC 2.4.1.17) from olfactory lining(UGT
 
olf 
 ) is especially interesting [10]. The authors of [10] reported on the key role of this enzyme in themechanisms of detoxification of xenobiotics and abroad range of physiologically active volatile com-pounds. Thus, UDP-glucuronosyltransferase fulfils aprotective function, on one hand, and is involved inbiotransformation of chemical compounds, on the otherhand.One of the characteristic features of the uroproteinsof the MUP complex is related to their striking struc-tural similarity with odorant-binding protein (OBP),which carries the odorant molecules to the receptor cellof the olfactory lining and plays a key role in the mech-anisms of olfactory reception in terrestrial vertebrates[11]. Interestingly, in addition to its protective andtransport functions, OBP is also involved in the primary“recognition” (decoding) of the odorant. The data thatmRNA of the
 Mup
 genes is expressed in the cells of mouse olfactory lining in the prepubertal period arealso interesting [12]. When comparing the datareported in [1, 5, 6, 8, 11, 12] with our results, takinginto account the logic of this line of reasoning, a ques-tion arises as to whether the MUP and OBP proteinsmay be units of the same phylogenetically ancient cas-cade, transducing informative chemosignals (phero-mones) from one organism to another, and whether the

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