Journal of Chemical Ecology, Vol. 19, No. 3, 1993
CHEMISTRY OF ODORTYPES IN MICE: FRACTIONATION AND BIOASSAY
A L A N G. S I N G E R , I'* H I R O N O R I T S U C H I Y A , 2 J U D I T H L. W E L L I N G T O N , s G A R Y K. B E A U C H A M P , and KUNIO
l
YAMAZAKI ~
~Monell Chemical Senses Center 3500 Market Street Philadelphia, PA 19104 2Department of Dental Pharmacology Asahi University School of Dentistry 1851 Hozumi, Motosu, Gifu 501-02, Japan 3New Jersey State Aquarium 1 Riverside Drive Camden, NJ 08103 (Received July 15, 1992; accepted November 12, 1992) Abstract--Mice can discriminate samples of urine obtained from two groups of inbred mice that are genetically identical except in their major histocompatibility complex (MHC) haplotype (congenic mice), whereas they cannot distinguish urine samples from two genetically identical groups of mice. Chemical fractions of urine samples obtained from MHC congenic mice were tested in a Y-maze olfactometer using a method modified to accommodate the bioassay to chemical fractions that might differ in sensory properties from the unfractionated urine. Fractions depleted in protein by several methods were consistently discriminable by mice in the Y maze, providing a direct demonstration that the airborne MHC genotype information can be conveyed by volatile compounds alone. Key Words--Mouse, urine, social odor, individual identity, mating preference, major histocompatibility complex, H-2, class I proteins, odortype.
INTRODUCTION A m o u s e c a n d e t e c t the d i f f e r e n c e in t w o m i c e t h a t are g e n e t i c a l l y identical e x c e p t in a p o r t i o n o f c h r o m o s o m e 17, k n o w n as the m a j o r h i s t o c o m p a t i b i l i t y *To whom correspondence should be addressed. 569 0098 0331/93/0300-0569507.00/0 9 1993 Plenum Publishing Corporation
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complex (MHC) (Yamazaki et al., 1979). The role of the MHC in specifying individuality is well known in its effect on the immune response to transplanted tissues and pathogens. In particular, well-characterized translation products of the MHC, the so-called class I and class II membrane proteins, specify the context in which the immune system can identify proteins as self or nonself in origin. Several class I and class II molecules are encoded by an extraordinary number of alleles in some mammalian species, among which mice and humans have been most extensively investigated (Klein, 1986). In mice it appears that the genetic diversity of these alleles is maintained by mate selection substantially based on MHC genotype (Yamazaki et al., 1976; Potts et al., 1991), but how this identity is specified and why it is regulated by genes of the immune system are unknown. It is likely that the information that allows mice to distinguish the MHC type of their mate as different from their own is conveyed through chemosensory channels (Yamazaki et al., 1979). Such information can be encoded in airborne chemical signals emitted by a mouse and detected by olfaction or possibly other nasal chemical senses. Chemosensory identity is influenced by environmental factors, such as diet or rearing, so that even two genetically identical mice may be distinguishable (Bowers and Alexander, 1967). However, the existence of genetically determined chemosensory identity, or odortypes, has been rigorously demonstrated in a Y-maze olfactometer experiment using samples of urine from congenic mice that differed only at the MHC (Yamaguchi et al., 1981). Differences at other genetic loci, for example, in the sex chromosomes, are capable of specifying discriminable urinary odors in t h e Y maze, but training mice to make these distinctions takes significantly more trials, suggesting that odor differences related to MHC differences are, for whatever reason, more salient (Yamazaki et al., 1990). The chemistry of the MHC odortypes of mouse urine is the subject of the investigations described in this report. Several distinct mechanisms by which the MHC might specify odortype have been recognized: (1) The composition of volatile metabolites in urine may be determined by MHC selected commensal flora (Howard, 1977). (2) Metabolic processes producing a characteristic mixture of volatile compounds may be influenced by MHC genes, directly (Ivanyi, 1978) or indirectly (Schwende et al., 1984). (3) Characteristic volatile compounds may result from catabolism of MHC translation products (Boyse et al., 1991b). (4) Release of odorous compounds from the urine may be regulated by binding to MHC proteins in the urine (Boyse et al., 1987; Singh et al., 1987). (5) The MHC proteins in the urine may become airborne and be detected directly (Singh et al., 1987). In order to answer the question of how the MHC influences the odortype, we need to determine the nature of the chemical signals. We report here on experiments that had the aim of investigating the general chemical properties of these signals;
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the results provide direct evidence that odortype in the Y-maze olfactometer is transmitted by volatile molecules and that it does not require proteins. METHODS AND MATERIALS
Congenic pairs of urine donor panels consisted of 40-60 age-matched, inbred male mice selected so that the members of one panel differed significantly from the members of the second panel only in MHC haplotype. The modified bioassay procedure deemed necessary for testing chemical fractions, which is described in detail below, required that two such congenic pairs of panels be maintained. All panel mice were maintained under uniform conditions in the same animal room. New mice were acclimated for three weeks before using them as donors. Urine was obtained by gentle abdominal pressure and pooled with urine from other members of the panel; it was stored in a - 2 0 ~ freezer. Chemical fractionation procedures were performed separately on pooled samples of urine not more than eight weeks old from each of the four panels. The resulting fractions were stored at - 2 0 ~ and generally were tested within one week of preparation. Samples of urine were lyophilized for one to three days at a pressure of 0.05 torr. The sublimate was tested directly, and the residue was reconstituted to the original volume in water for bioassay. Gel permeation chromatography was performed on 5-ml samples of urine on a column of Sephadex G-15 eluted with deionized water. The fractions were analyzed for protein by SDS polyacrylamide gel electrophoresis (Laemmli, 1970). Proteins in samples of the urine were degraded by treatment with a high concentration (2 mg/ml) of Pronase for 1 hr at ambient temperature. The degradation of protein was confirmed by polyacrylamide gel electrophoresis (Singer et ah, 1988). The treated urine samples were tested without further work-up in the bioassay. Urinary proteins were precipitated and removed by addition of HC104 to the urine samples to a concentration of 0.24 M, heating at 90~ for 5 min, cooling, and centrifugation at 15,000g for 15 min. The supernatants obtained were tested directly. Dialysis was carried out on 5-ml samples of urine using cellulose tubing with a molecular weight cutoff of 3500 in 500 ml of deionized water for 72 hr at 4~ with two changes of water. The retentate and dialysate were lyophilized and the dried residues were redissolved in 5 ml of water for testing. Samples of urine from each of the panels were filtered at 5~ in a centrifuge through a membrane with a molecular weight cutoff of 3000. The ultrafiltrate was tested directly.
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Extractions were carried out on lyophilized urine by mixing with solvent (ether or ethanol) and filtering. The extracts were prepared from the filtrate for testing by evaporating the solvent to dryness in a rotary evaporator and reconstituting in distilled and deionized water for bioassay. The residues from the extractions were dried in the rotary evaporator to remove residual solvent and reconstituted in water as were the extracts. The Y-maze olfactometer, the training of mice, and the use of generalization to demonstrate that two samples of urine are of identical odortype have all been described in detail (Yamazaki et al., 1979; Yamaguchi et al., 1981). Air from outside the test room is blown into left and right odor boxes at the end of the two branches of the Y maze. Each odor box has a 3.6-cm Petri dish containing the sample. Left or fight placement of a sample is assigned randomly for each trial. An air current conducts the odor from each odor box through a tube into the connected Y-maze branch and then to the foot of the Y, where the subject mouse is initially located. At the beginning of the test, a gate confining the mouse and gates to each of the two branches of the Y are raised simultaneously. Each mouse is trained to choose one of the two odortypes of urine to be used in the experiment. Frequently a trained mouse chooses without pause or after sniffing at the entrance to the branches, or occasionally with brief retracing from one branch to the other. When the mouse has clearly entered one branch, generally 2-3 sec after the start of the test, the gates are lowered. If the mouse makes a choice that is to be rewarded, it receives a drop of water; if the mouse enters the wrong branch, or the trial is designated as unrewarded in the experimental design, the mouse is not rewarded. In any event, it is then returned to the start and within 30 sec, when the samples have been replaced and the drop of water renewed, another trial can begin. Bioassay of chemical fractions in the Y-maze olfactometer was conducted in two variations (see Discussion). Chemical fractions produced by the dialysis and lyophilization procedures were tested on mice trained to select one of the MHC types of the congenic pair of urine samples used to prepare the fractions. The mice were not rewarded for their choice of chemical fraction even if the choice was concordant with their training. The other chemical fractions were tested by a modified procedure that employed the sequence of sample presentation shown in Table 1. A fresh 0.3- to 0.5-ml sample was used for each testing session on a single mouse, which usually consisted of 48 trials. The chemical fractions were obtained from fractionation procedures performed identically on four samples of pooled urine: one from each of two pairs of panels of congenic mice. In the trials of fractions of urine from congenic mice following the scheme in Table 1, the mice were rewarded for the choice of haplotype source concordant with their training on the unfractionated urine. The trials of the fractions obtained from the second pair of congenic panels were never rewarded. Each of these sets of chemical fractions were usually tested on three or four different
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TABLE 1. BIOASSAYSAMPLE SEQUENCE
Trial
Sample type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
urine urine urine urine chemical fraction chemical fraction chemical fraction chemical fraction urine urine urine urine chemical fraction chemical fraction chemical fraction chemical fraction
Choice~ b vs. b vs. b vs. b vs. b~ vs. b~ v s . b~ vs. b2 vs. b vs. b vs. b vs. b vs. b~ vs. b~ vs. bl vs. b2 vs.
k k k k k~ k t
kI kz k k k k k~ k~ k~ k2
Result of correct response reward reward reward no reward reward~ rewardb rewardb no reward c reward reward reward no reward reward~' reward~ rewardb no reward''
"To exemplify the modified scheme used in testing chemical fractions from congenic mice, the following symbols are used in this table: b = pooled urine from two panels of B6 mice, which have H-2b; k = pooled urine from two panels of B6 H-2 k mice; bt and b2 = chemical fractions prepared from urine of first and second panels respectively of B6 mice; k~ and k 2 = chemical fractions prepared from urine of first and second panels respectively of B6 1t-2 ~ (congenic to B6) mice. hThese are the training trials scored in Table 2. CThese are the generalization trials scored in Table 2.
subject m i c e so that the u n r e w a r d e d , g e n e r a l i z a t i o n s a m p l e s f r o m the s e c o n d pair o f c o n g e n i c p a n e l s had b e e n t e s t e d in a b o u t 20 trials. T h e u n r e i n f o r c e d c h e m i c a l fraction s a m p l e s w e r e c o d e d so that the o p e r a t o r o f the b i o a s s a y w a s u n a w a r e o f the M H C h a p l o t y p e o f the urine d o n o r s and thus c o u l d not s y s t e m atically influence the o u t c o m e o f t h e s e trials. I f b o t h the r e w a r d e d trials and the u n r e w a r d e d trials e m p l o y i n g c h e m i c a l fractions g a v e a significant n u m b e r o f c h o i c e s c o n c o r d a n t with training ( P < 0.05 as d e t e r m i n e d f r o m a n o r m a l d i s t r i b u t i o n , t w o - t a i l e d ) , t h e n t h e y w e r e j u d g e d to c o n t a i n o d o r s c a p a b l e o f i n d i c a t i n g M H C h a p l o t y p e .
RESULTS T h e results are c o n t a i n e d in T a b l e 2. A p p l y i n g h i g h v a c u u m to the urine for three d a y s , u n d e r the c o n d i t i o n s o f l y o p h i l i z a t i o n , did not lead to a p p r e c i a b l e
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TABLE 2. DISCRIMINATIONOF CHEMICAL FRACTIONS OF CONGENIC MOUSE URINE SAMPLES
Concordance (%)a Fraction Lyophilization Sublimate Residue Gel permeation Fraction A Fraction A1 Fraction A2 Fraction A3 Fraction B Fraction C Pronase treated urine Supernatant from HCIO4 precipitation Dialysis Retentate Dialysate Ultrafiltrate of urine Extractions Ether extract residue Ethanol extract residue
Training trials
Generalization trials
nah na
52 80***
88*** 66*** 64*** 64*** 53 60* 84*** 76***
66* 74* 54 73* 38 62 83** 89**
na na 80***
55 77'** 82**
46 73** 87*** 76***
33 86** 79** 91 **
"Concordance values are mean percent of choices concordant with training on unfractionated urine (see Methods and Materials). The following symbols are used in this table: na = not applicable (training was conducted solely on the unfractionated urine); *** P < 0.001; ** P < 0.01; * P < 0.05.
loss o f activity w h e n the urine was reconstituted with water; no activity was r e c o v e r e d in the sublimate. Gel p e r m e a t i o n c h r o m a t o g r a p h y on a c o l u m n o f S e p h a d e x G-15 with the collection o f three fractions (A, B, and C in T a b l e 2) resulted in one active fraction. O n l y the earliest-eluting fraction, A, was active. This fraction contained the urinary protein as e x p e c t e d from the m e a s u r e d retention o f proteins o f k n o w n m o l e c u l a r w e i g h t and as c o n f i r m e d by S D S p o l y a c r y l a m i d e gel electrophoresis. Fractions e x p e c t e d to contain only l o w - m o l e c u l a r - w e i g h t c o m pounds ( < 1000 Da), B and C, w e r e not active. In a second fractionation s c h e m e in which the A portion o f the f o r m e r fractionation s c h e m e was collected in three smaller fractions, A 1 - A 3 , again the fraction containing the m a j o r urinary pro-
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teins was active. Activity was also obtained in fraction A3, corresponding in retention volume to the retention of compounds with molecular weight around 10,000. Urine treated with a high concentration of the protein-degrading enzymes, Pronase, was undiminished in activity in the Y-maze bioassay. The degradation of protein was confirmed by SDS polyacrylamide gel electrophoresis, which showed that less than 0.1% of the urinary protein remained in the enzymedigested urine. Perchloric acid precipitation apparently removed urinary protein, judging from the quantity of precipitate obtained. The remaining soluble material (supernatant after centrifugation) was discriminable in the Y-maze bioassay. On dialysis and ultrafiltration, the activity was likewise found in the fraction containing smaller molecules. Extraction of the residue from lyophilization recovered some active compounds when the extraction solvent was ethanol but none when the solvent was ether. In both cases the extracted residue was active.
DISCUSSION
The data from a large number of Y-maze experiments with a variety of inbred mouse MHC haplotypes indicate that, with the exception of some mutant inbred strains (Yamazaki et al., 1991), there is an odortype corresponding to each MHC genotype in mice (Boyse et al., 1991a). Assuming this is true, there must indeed be a very large number of odortypes corresponding to the exceptionally large number of MHC genotypes (Klein, 1986). How might a large number of odortypes be specified chemically? In the absence of any definite experimental results bearing on this question, we are considering two possibilities. First, we consider that there may be a specialized class of compounds, the biosynthesis of which is under relatively direct genetic regulation by the MHC. Odortype could be specified by a small number of closely related compounds with numerous possible variations in structure (Voznessenskaya et al., 1992). The possible specification of odortype by soluble class I-derived molecules in the urine (Singh et al., 1987), for example, is a mechanism in which odortype is differentiated by variations in a restricted class of compounds, that is, proteins. The other possibility to consider is that odortype is encoded by a less restricted mixture of chemically diverse secondary metabolites that varies in composition incidentally to variations in MHC genotype (Yamazaki et al., 1984). Most of the speculations on the mechanism of odortype production listed in the Introduction are based on the idea that odortype is encoded by quantitative variations in the composition of a diverse collection of metabolic by-products.
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This possibility is supported chemically by the fact that there are numerous and chemically diverse volatile compounds in mouse urine (Liebich et al., 1977; Schwende et al., 1986). The possibility that odortype is constituted by a mixture of compounds of diverse chemical functionality prompted us to elaborate on the design of the basic Y-maze experiment with generalization (Yamaguchi et al., 1981). With relatively crude fractionation techniques, such as dialysis or lyophilization, in which the urinary compounds are separated into two fractions of compounds with distinct differences in physical properties, the probability is high that the entire mixture of compounds making up the odortype will be found in one or the other fraction. Mice that had been trained to discriminate urine from congenic mice were indeed able to discriminate samples of one of the two fractions of these urines produced by either dialysis or lyophilization, whereas they were not able to discriminate the other fractions produced by these techniques (Table 2). This result indicates that all or most of the compounds that enable the trained mice to discriminate urine samples from congenic mice were contained in one of the two fractions resulting from each of these techniques that separate compounds on the basis of gross differences in properties. In testing for the biological activity of fractions produced by techniques such as chromatography that separate on the basis of relatively slight differences in chemical properties, we need to consider the probability that the compounds constituting the odortype, having different functional groups, would very likely be separated into two or more distinct fractions, and thus that the fractions would probably smell different from the unfractionated urine. In this case the mouse trained on whole urine could fail to recognize the fractions and might not respond to fractions actually containing important constituents of the odor. To circumvent this difficulty, the Y maze procedure was modified for testing chemical fractions by the inclusion of training on the fractions, after the mouse had learned to distinguish the odortypes of the unfractionated samples of urine. The unrewarded pairs of chemical fractions for discrimination, which could be coded so that the operator of the Y maze was not aware of their MHC haplotypes, were prepared from a second pair of congenic urine donor panels. This procedure has the added benefit of reducing the possibility of the mice learning an incidental distinction between the two haplotypes introduced accidentally during the fractionation process, because the second pair of chemical fractions is prepared independently. Use of the modified method for bioassay was validated by the results in Table 2. Subjects in the Y maze bioassay were able to discriminate the odors of a number of chemical fractions produced by diverse methods. The question of whether odortype is conveyed by a restricted class of specialized compounds or diverse metabolic by-products cannot be answered by these results, but the modified bioassay method should make possible the discrimination of odortypes
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of fractions containing only a few active components of a complex odorous mixture, if necessary. A question that was addressed by the data presented here is the question of the involvement of proteins in odortype. Since the signals of individuality detected in the Y maze are necessarily airborne, it might be assumed that only volatile molecules could transmit the odortype information in this apparatus, but it is possible that airborne proteins could be involved. A role for proteins in chemical communication has been established (Singer and Macrides, 1992), and recognizable soluble derivatives of MHC class I membrane proteins have been partially characterized in rat urine (Singh et al., 1988). As evidence that these proteins could become airborne, it is notable that airborne proteins from mouse urine have been demonstrated to cause allergic reactions in a room housing a mouse colony (Schumacher, 1980). The main objection to this argument is that these airborne proteins, which are probably released from dried samples as dust, would be present in extremely low concentration in the Y-maze bioassay, during which they could become airborne from solution in the urine in the Petri dish only by bubbles breaking at the surface forming droplets small enough to remain airborne. The direct involvement of proteins in conveying odortype is inconsistent with the results in Table 2. Whether proteins were eliminated by enzyme degradation, perchloric acid precipitation, dialysis, ultrafiltration, or ethanol extraction, the congenic pairs of fractions containing no protein were consistently discriminable in the Y-maze olfactometer. Proteins, however, might be less directly involved in odortype specification. One of the mechanisms of genetic regulation of individual odor listed in the Introduction holds that odortype is specified by a mixture of volatile metabolic by-products in which the composition is determined by the selective binding of some of the urinary metabolites to soluble MHC proteins. The plausibility of this mechanism is supported by the recent demonstrations of grooves in human class I membrane proteins (HLA-A and HLA-B proteins) that bind peptide antigens with some selectivity (Garrett et al., 1989; Hunt et al., 1992). The fact that the early-eluting gel permeation chromatographic fractions, which contain proteins, were discriminated in the Y maze indicates that proteins in the urine may normally bind the compounds constituting odortype, but from the other results it is clear that proteins are not necessary for the discrimination of urine samples from MHC congenic mice in the Y maze and that volatile compounds are capable of conveying the odortype. If urinary proteins are involved in specifying odortype, it is likely that their contribution is to select a distinct mixture of secondary metabolites from the blood serum for secretion by the kidneys (Singh et al., 1987). Another possible function attributed to putative binding proteins in the urine, that they specify the odortype by selectively binding compounds already present in the urine to make the odortype information more persistent (Boyse et al., 1987), is not consistent with the result that destruc-
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tion of the protein does not destroy the possibility of discriminating congenic samples of urine. REFERENCES BOWERS,J.M., and ALEXANDER,B.K. 1967. Mice: individual recognition by olfactory cues. Science 158:1208-1210. BOYSE, E.A., BEAUCHAMP,G.K., and YAMAZAKI,K. 1987. The genetics of body scent. Trends Genet. 3:97-102. BOYSE, E.A., BEAUCHAMP,G.K., BARD, J., and YAMAZAKI,K. 1991a. Behavior and the major histocompatibility complex of the mouse, pp. 831-846, in R. Ader, D.L. Felten, and N. Cohen (eds.). Psychoneuroimmunology, 2nd ed. Academic Press, San Diego. BOYSE, E.A., BEAUCHAMP,G.K., YAMAZAKI,K., and BARD, J., 1991b. Genetic components of kin recognition in mammals, pp. 148-161, in P.G. Hepper (ed.). Kin Recognition. Cambridge University Press, Cambridge, U.K. GARRETT, T.P.J., SAPER, M.A., BJORKMAN,P.J., STROMINGER,J.L., and WILEY, D.C. 1989. Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342:692696. HOWARD, J.C. 1977. H-2 and mating preferences. Nature 266:406-408. HUNT, D.F., HENDERSON, R.A., SHABANOWITE, J., SAKAGUCHI,K., MICHEL, l-I., SEVILIR, N., COX, A.L., APPELLA,E., and ENGELHARD,V.H. 1992. Characterization of peptides bound to the class I MHC molecule HLA-A2. l by mass spectrometry. Science 255:1261-1263. IVANY1,P. 1978. Some aspects of the H-2 system, the major histocompatibility system in the mouse. Proc. R. Soc. 202B: 117-158. KLEIN J. 1986. Natural History of the Major Histocompatibility Complex. Wiley, New York. LAEMMLI, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. LIEBICH, H.M., ZLATKIS, A., BERTSCH, W., VANDAHM,R., and WHITTEN, W.K. 1977. Identification of dihydrothiazoles in urine of male mice. Biomed. Mass Spectr. 4:69. POTTS, W.K., MANNING,C.J., and WAKELAND,E.K. 1991. Mating patterns in seminatural populations of mice influenced by MHC genotype. Nature 352:619-621. SCHUMACHER,M.J. 1980. Characterization of allergens from urine and pelts of laboratory mice. Mol. Immunol. 17:1087-1095. SCHWENDE, F.J., JORGENSON,J.W., and NOVOTNY, M. 1984. A possible chemical basis for histocompatibility-related mating preference in mice. J. Chem. Ecol. 10:1603-1615. SCHWENDE,F.J., WIESLER,D., JORGENSON,J.W., CARMACK,M., and NOVOTNY,M. 1986. Urinary volatle constituents of the house mouse, Mus musculus, and their endocrine dependency. J. Chem. Ecol. 12:277-296. SINGER, A.G., and MACRIDES,F. 1992. Lipocalycins associated with mammalian pheromones, in R.L. Doty and D. Muller-Schwarze (eds.). Chemical Signals in Vertebrates 6. Plenum, New York. In press. SINGER, A.G., CLANCY,A.N., MACRIDES,F., AGOSTA,W.C., and BRONSON,F.H. 1988. Chemical properties of a female mouse pheromone that stimulates gonadotropin secretion in males. Biol. Reprod. 38:193-199. SINGH, P.B., BROWN, R.E., and ROSER, B. 1987. MHC antigens in urine as olfactory recognition cues. Nature 327:161-164. SINGH, P.B., BROWN, R.E., and ROSER, B. 1988. Class I transplantation antigens in solution in body fluids and in the urine. J. Exp. Med. 168:195-211.
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VOZNESSENSKAYA,V.V., PARFYONOVA,V.M., and ZINKEVICH,E.P. 1992. individual odortypes, in R.L. Doty and D. Muller-Schwarze (eds.). Chemical Signals in Vertebrates 6. Plenum, New York. In press. YAMAGUCm, M., YAMAZAKI,K., BEAUCHAMP, G.K., BARD, J., THOMAS, L., and BOYSE, E.A. 1981. Distinctive urinary odors governed by the major histocompatibility locus of the mouse. Proc. Natl. Acad. Sci. U.S.A. 78:5817-5820. YAMAZAKI,K., BOYSE, E.A., MIKE, V., THALER, H.T., MATHIESON, B.J., ABBOTT,J., BOYSE, J., ZAYAS, Z.A., and THOMAS, L. 1976. Control of mating preferences in mice by genes in the major histocompatibility complex. J. Exp. Med. 144:1324-1335. YAMAZAKI,K., YAMAGUCHI,M., BARANOSKI,L., BARD, J., BOYSE, E.A., and THOMAS, L. 1979. Recognition among mice: evidence from the use of a Y-maze differentially scented by congenic mice of different major histocompatibility types. J. Exp. Med. 150:755-760. YAMAZAKI,K., BEAUCHAMP,G.K. THOMAS, L., and BOYSE, E.A. 1984. Chemosensory identity of H-2 heterozygotes. J. Mol. Cell. lmmunol. 1:79-82. YAMAZAKI, K., BEAUCHAMP,G.K., BARD, J., and BOYSE, E.A. 1990. Chemosensory identity and the Y chromosome. Behav. Genet. 20:157-165. YAMAZAKI,K., BEAUCHAMP,G.K., BARD, J., BOYSE, E.A., and THOMAS, L. 1991. Chemosensory identity and immune function in mice, pp. 211-225, in C.J. Wysocki and M.R. Kare (eds.). Chemical Senses: Genetics of Perception and Communication. Marcel Dekker, New York.
CHEMISTRY OF ODORTYPES IN MICE: FRACTIONATION AND BIOASSAY
A L A N G. S I N G E R , I'* H I R O N O R I T S U C H I Y A , 2 J U D I T H L. W E L L I N G T O N , s G A R Y K. B E A U C H A M P , and KUNIO
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YAMAZAKI ~
~Monell Chemical Senses Center 3500 Market Street Philadelphia, PA 19104 2Department of Dental Pharmacology Asahi University School of Dentistry 1851 Hozumi, Motosu, Gifu 501-02, Japan 3New Jersey State Aquarium 1 Riverside Drive Camden, NJ 08103 (Received July 15, 1992; accepted November 12, 1992) Abstract--Mice can discriminate samples of urine obtained from two groups of inbred mice that are genetically identical except in their major histocompatibility complex (MHC) haplotype (congenic mice), whereas they cannot distinguish urine samples from two genetically identical groups of mice. Chemical fractions of urine samples obtained from MHC congenic mice were tested in a Y-maze olfactometer using a method modified to accommodate the bioassay to chemical fractions that might differ in sensory properties from the unfractionated urine. Fractions depleted in protein by several methods were consistently discriminable by mice in the Y maze, providing a direct demonstration that the airborne MHC genotype information can be conveyed by volatile compounds alone. Key Words--Mouse, urine, social odor, individual identity, mating preference, major histocompatibility complex, H-2, class I proteins, odortype.
INTRODUCTION A m o u s e c a n d e t e c t the d i f f e r e n c e in t w o m i c e t h a t are g e n e t i c a l l y identical e x c e p t in a p o r t i o n o f c h r o m o s o m e 17, k n o w n as the m a j o r h i s t o c o m p a t i b i l i t y *To whom correspondence should be addressed. 569 0098 0331/93/0300-0569507.00/0 9 1993 Plenum Publishing Corporation
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complex (MHC) (Yamazaki et al., 1979). The role of the MHC in specifying individuality is well known in its effect on the immune response to transplanted tissues and pathogens. In particular, well-characterized translation products of the MHC, the so-called class I and class II membrane proteins, specify the context in which the immune system can identify proteins as self or nonself in origin. Several class I and class II molecules are encoded by an extraordinary number of alleles in some mammalian species, among which mice and humans have been most extensively investigated (Klein, 1986). In mice it appears that the genetic diversity of these alleles is maintained by mate selection substantially based on MHC genotype (Yamazaki et al., 1976; Potts et al., 1991), but how this identity is specified and why it is regulated by genes of the immune system are unknown. It is likely that the information that allows mice to distinguish the MHC type of their mate as different from their own is conveyed through chemosensory channels (Yamazaki et al., 1979). Such information can be encoded in airborne chemical signals emitted by a mouse and detected by olfaction or possibly other nasal chemical senses. Chemosensory identity is influenced by environmental factors, such as diet or rearing, so that even two genetically identical mice may be distinguishable (Bowers and Alexander, 1967). However, the existence of genetically determined chemosensory identity, or odortypes, has been rigorously demonstrated in a Y-maze olfactometer experiment using samples of urine from congenic mice that differed only at the MHC (Yamaguchi et al., 1981). Differences at other genetic loci, for example, in the sex chromosomes, are capable of specifying discriminable urinary odors in t h e Y maze, but training mice to make these distinctions takes significantly more trials, suggesting that odor differences related to MHC differences are, for whatever reason, more salient (Yamazaki et al., 1990). The chemistry of the MHC odortypes of mouse urine is the subject of the investigations described in this report. Several distinct mechanisms by which the MHC might specify odortype have been recognized: (1) The composition of volatile metabolites in urine may be determined by MHC selected commensal flora (Howard, 1977). (2) Metabolic processes producing a characteristic mixture of volatile compounds may be influenced by MHC genes, directly (Ivanyi, 1978) or indirectly (Schwende et al., 1984). (3) Characteristic volatile compounds may result from catabolism of MHC translation products (Boyse et al., 1991b). (4) Release of odorous compounds from the urine may be regulated by binding to MHC proteins in the urine (Boyse et al., 1987; Singh et al., 1987). (5) The MHC proteins in the urine may become airborne and be detected directly (Singh et al., 1987). In order to answer the question of how the MHC influences the odortype, we need to determine the nature of the chemical signals. We report here on experiments that had the aim of investigating the general chemical properties of these signals;
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the results provide direct evidence that odortype in the Y-maze olfactometer is transmitted by volatile molecules and that it does not require proteins. METHODS AND MATERIALS
Congenic pairs of urine donor panels consisted of 40-60 age-matched, inbred male mice selected so that the members of one panel differed significantly from the members of the second panel only in MHC haplotype. The modified bioassay procedure deemed necessary for testing chemical fractions, which is described in detail below, required that two such congenic pairs of panels be maintained. All panel mice were maintained under uniform conditions in the same animal room. New mice were acclimated for three weeks before using them as donors. Urine was obtained by gentle abdominal pressure and pooled with urine from other members of the panel; it was stored in a - 2 0 ~ freezer. Chemical fractionation procedures were performed separately on pooled samples of urine not more than eight weeks old from each of the four panels. The resulting fractions were stored at - 2 0 ~ and generally were tested within one week of preparation. Samples of urine were lyophilized for one to three days at a pressure of 0.05 torr. The sublimate was tested directly, and the residue was reconstituted to the original volume in water for bioassay. Gel permeation chromatography was performed on 5-ml samples of urine on a column of Sephadex G-15 eluted with deionized water. The fractions were analyzed for protein by SDS polyacrylamide gel electrophoresis (Laemmli, 1970). Proteins in samples of the urine were degraded by treatment with a high concentration (2 mg/ml) of Pronase for 1 hr at ambient temperature. The degradation of protein was confirmed by polyacrylamide gel electrophoresis (Singer et ah, 1988). The treated urine samples were tested without further work-up in the bioassay. Urinary proteins were precipitated and removed by addition of HC104 to the urine samples to a concentration of 0.24 M, heating at 90~ for 5 min, cooling, and centrifugation at 15,000g for 15 min. The supernatants obtained were tested directly. Dialysis was carried out on 5-ml samples of urine using cellulose tubing with a molecular weight cutoff of 3500 in 500 ml of deionized water for 72 hr at 4~ with two changes of water. The retentate and dialysate were lyophilized and the dried residues were redissolved in 5 ml of water for testing. Samples of urine from each of the panels were filtered at 5~ in a centrifuge through a membrane with a molecular weight cutoff of 3000. The ultrafiltrate was tested directly.
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Extractions were carried out on lyophilized urine by mixing with solvent (ether or ethanol) and filtering. The extracts were prepared from the filtrate for testing by evaporating the solvent to dryness in a rotary evaporator and reconstituting in distilled and deionized water for bioassay. The residues from the extractions were dried in the rotary evaporator to remove residual solvent and reconstituted in water as were the extracts. The Y-maze olfactometer, the training of mice, and the use of generalization to demonstrate that two samples of urine are of identical odortype have all been described in detail (Yamazaki et al., 1979; Yamaguchi et al., 1981). Air from outside the test room is blown into left and right odor boxes at the end of the two branches of the Y maze. Each odor box has a 3.6-cm Petri dish containing the sample. Left or fight placement of a sample is assigned randomly for each trial. An air current conducts the odor from each odor box through a tube into the connected Y-maze branch and then to the foot of the Y, where the subject mouse is initially located. At the beginning of the test, a gate confining the mouse and gates to each of the two branches of the Y are raised simultaneously. Each mouse is trained to choose one of the two odortypes of urine to be used in the experiment. Frequently a trained mouse chooses without pause or after sniffing at the entrance to the branches, or occasionally with brief retracing from one branch to the other. When the mouse has clearly entered one branch, generally 2-3 sec after the start of the test, the gates are lowered. If the mouse makes a choice that is to be rewarded, it receives a drop of water; if the mouse enters the wrong branch, or the trial is designated as unrewarded in the experimental design, the mouse is not rewarded. In any event, it is then returned to the start and within 30 sec, when the samples have been replaced and the drop of water renewed, another trial can begin. Bioassay of chemical fractions in the Y-maze olfactometer was conducted in two variations (see Discussion). Chemical fractions produced by the dialysis and lyophilization procedures were tested on mice trained to select one of the MHC types of the congenic pair of urine samples used to prepare the fractions. The mice were not rewarded for their choice of chemical fraction even if the choice was concordant with their training. The other chemical fractions were tested by a modified procedure that employed the sequence of sample presentation shown in Table 1. A fresh 0.3- to 0.5-ml sample was used for each testing session on a single mouse, which usually consisted of 48 trials. The chemical fractions were obtained from fractionation procedures performed identically on four samples of pooled urine: one from each of two pairs of panels of congenic mice. In the trials of fractions of urine from congenic mice following the scheme in Table 1, the mice were rewarded for the choice of haplotype source concordant with their training on the unfractionated urine. The trials of the fractions obtained from the second pair of congenic panels were never rewarded. Each of these sets of chemical fractions were usually tested on three or four different
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TABLE 1. BIOASSAYSAMPLE SEQUENCE
Trial
Sample type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
urine urine urine urine chemical fraction chemical fraction chemical fraction chemical fraction urine urine urine urine chemical fraction chemical fraction chemical fraction chemical fraction
Choice~ b vs. b vs. b vs. b vs. b~ vs. b~ v s . b~ vs. b2 vs. b vs. b vs. b vs. b vs. b~ vs. b~ vs. bl vs. b2 vs.
k k k k k~ k t
kI kz k k k k k~ k~ k~ k2
Result of correct response reward reward reward no reward reward~ rewardb rewardb no reward c reward reward reward no reward reward~' reward~ rewardb no reward''
"To exemplify the modified scheme used in testing chemical fractions from congenic mice, the following symbols are used in this table: b = pooled urine from two panels of B6 mice, which have H-2b; k = pooled urine from two panels of B6 H-2 k mice; bt and b2 = chemical fractions prepared from urine of first and second panels respectively of B6 mice; k~ and k 2 = chemical fractions prepared from urine of first and second panels respectively of B6 1t-2 ~ (congenic to B6) mice. hThese are the training trials scored in Table 2. CThese are the generalization trials scored in Table 2.
subject m i c e so that the u n r e w a r d e d , g e n e r a l i z a t i o n s a m p l e s f r o m the s e c o n d pair o f c o n g e n i c p a n e l s had b e e n t e s t e d in a b o u t 20 trials. T h e u n r e i n f o r c e d c h e m i c a l fraction s a m p l e s w e r e c o d e d so that the o p e r a t o r o f the b i o a s s a y w a s u n a w a r e o f the M H C h a p l o t y p e o f the urine d o n o r s and thus c o u l d not s y s t e m atically influence the o u t c o m e o f t h e s e trials. I f b o t h the r e w a r d e d trials and the u n r e w a r d e d trials e m p l o y i n g c h e m i c a l fractions g a v e a significant n u m b e r o f c h o i c e s c o n c o r d a n t with training ( P < 0.05 as d e t e r m i n e d f r o m a n o r m a l d i s t r i b u t i o n , t w o - t a i l e d ) , t h e n t h e y w e r e j u d g e d to c o n t a i n o d o r s c a p a b l e o f i n d i c a t i n g M H C h a p l o t y p e .
RESULTS T h e results are c o n t a i n e d in T a b l e 2. A p p l y i n g h i g h v a c u u m to the urine for three d a y s , u n d e r the c o n d i t i o n s o f l y o p h i l i z a t i o n , did not lead to a p p r e c i a b l e
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TABLE 2. DISCRIMINATIONOF CHEMICAL FRACTIONS OF CONGENIC MOUSE URINE SAMPLES
Concordance (%)a Fraction Lyophilization Sublimate Residue Gel permeation Fraction A Fraction A1 Fraction A2 Fraction A3 Fraction B Fraction C Pronase treated urine Supernatant from HCIO4 precipitation Dialysis Retentate Dialysate Ultrafiltrate of urine Extractions Ether extract residue Ethanol extract residue
Training trials
Generalization trials
nah na
52 80***
88*** 66*** 64*** 64*** 53 60* 84*** 76***
66* 74* 54 73* 38 62 83** 89**
na na 80***
55 77'** 82**
46 73** 87*** 76***
33 86** 79** 91 **
"Concordance values are mean percent of choices concordant with training on unfractionated urine (see Methods and Materials). The following symbols are used in this table: na = not applicable (training was conducted solely on the unfractionated urine); *** P < 0.001; ** P < 0.01; * P < 0.05.
loss o f activity w h e n the urine was reconstituted with water; no activity was r e c o v e r e d in the sublimate. Gel p e r m e a t i o n c h r o m a t o g r a p h y on a c o l u m n o f S e p h a d e x G-15 with the collection o f three fractions (A, B, and C in T a b l e 2) resulted in one active fraction. O n l y the earliest-eluting fraction, A, was active. This fraction contained the urinary protein as e x p e c t e d from the m e a s u r e d retention o f proteins o f k n o w n m o l e c u l a r w e i g h t and as c o n f i r m e d by S D S p o l y a c r y l a m i d e gel electrophoresis. Fractions e x p e c t e d to contain only l o w - m o l e c u l a r - w e i g h t c o m pounds ( < 1000 Da), B and C, w e r e not active. In a second fractionation s c h e m e in which the A portion o f the f o r m e r fractionation s c h e m e was collected in three smaller fractions, A 1 - A 3 , again the fraction containing the m a j o r urinary pro-
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teins was active. Activity was also obtained in fraction A3, corresponding in retention volume to the retention of compounds with molecular weight around 10,000. Urine treated with a high concentration of the protein-degrading enzymes, Pronase, was undiminished in activity in the Y-maze bioassay. The degradation of protein was confirmed by SDS polyacrylamide gel electrophoresis, which showed that less than 0.1% of the urinary protein remained in the enzymedigested urine. Perchloric acid precipitation apparently removed urinary protein, judging from the quantity of precipitate obtained. The remaining soluble material (supernatant after centrifugation) was discriminable in the Y-maze bioassay. On dialysis and ultrafiltration, the activity was likewise found in the fraction containing smaller molecules. Extraction of the residue from lyophilization recovered some active compounds when the extraction solvent was ethanol but none when the solvent was ether. In both cases the extracted residue was active.
DISCUSSION
The data from a large number of Y-maze experiments with a variety of inbred mouse MHC haplotypes indicate that, with the exception of some mutant inbred strains (Yamazaki et al., 1991), there is an odortype corresponding to each MHC genotype in mice (Boyse et al., 1991a). Assuming this is true, there must indeed be a very large number of odortypes corresponding to the exceptionally large number of MHC genotypes (Klein, 1986). How might a large number of odortypes be specified chemically? In the absence of any definite experimental results bearing on this question, we are considering two possibilities. First, we consider that there may be a specialized class of compounds, the biosynthesis of which is under relatively direct genetic regulation by the MHC. Odortype could be specified by a small number of closely related compounds with numerous possible variations in structure (Voznessenskaya et al., 1992). The possible specification of odortype by soluble class I-derived molecules in the urine (Singh et al., 1987), for example, is a mechanism in which odortype is differentiated by variations in a restricted class of compounds, that is, proteins. The other possibility to consider is that odortype is encoded by a less restricted mixture of chemically diverse secondary metabolites that varies in composition incidentally to variations in MHC genotype (Yamazaki et al., 1984). Most of the speculations on the mechanism of odortype production listed in the Introduction are based on the idea that odortype is encoded by quantitative variations in the composition of a diverse collection of metabolic by-products.
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This possibility is supported chemically by the fact that there are numerous and chemically diverse volatile compounds in mouse urine (Liebich et al., 1977; Schwende et al., 1986). The possibility that odortype is constituted by a mixture of compounds of diverse chemical functionality prompted us to elaborate on the design of the basic Y-maze experiment with generalization (Yamaguchi et al., 1981). With relatively crude fractionation techniques, such as dialysis or lyophilization, in which the urinary compounds are separated into two fractions of compounds with distinct differences in physical properties, the probability is high that the entire mixture of compounds making up the odortype will be found in one or the other fraction. Mice that had been trained to discriminate urine from congenic mice were indeed able to discriminate samples of one of the two fractions of these urines produced by either dialysis or lyophilization, whereas they were not able to discriminate the other fractions produced by these techniques (Table 2). This result indicates that all or most of the compounds that enable the trained mice to discriminate urine samples from congenic mice were contained in one of the two fractions resulting from each of these techniques that separate compounds on the basis of gross differences in properties. In testing for the biological activity of fractions produced by techniques such as chromatography that separate on the basis of relatively slight differences in chemical properties, we need to consider the probability that the compounds constituting the odortype, having different functional groups, would very likely be separated into two or more distinct fractions, and thus that the fractions would probably smell different from the unfractionated urine. In this case the mouse trained on whole urine could fail to recognize the fractions and might not respond to fractions actually containing important constituents of the odor. To circumvent this difficulty, the Y maze procedure was modified for testing chemical fractions by the inclusion of training on the fractions, after the mouse had learned to distinguish the odortypes of the unfractionated samples of urine. The unrewarded pairs of chemical fractions for discrimination, which could be coded so that the operator of the Y maze was not aware of their MHC haplotypes, were prepared from a second pair of congenic urine donor panels. This procedure has the added benefit of reducing the possibility of the mice learning an incidental distinction between the two haplotypes introduced accidentally during the fractionation process, because the second pair of chemical fractions is prepared independently. Use of the modified method for bioassay was validated by the results in Table 2. Subjects in the Y maze bioassay were able to discriminate the odors of a number of chemical fractions produced by diverse methods. The question of whether odortype is conveyed by a restricted class of specialized compounds or diverse metabolic by-products cannot be answered by these results, but the modified bioassay method should make possible the discrimination of odortypes
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577
of fractions containing only a few active components of a complex odorous mixture, if necessary. A question that was addressed by the data presented here is the question of the involvement of proteins in odortype. Since the signals of individuality detected in the Y maze are necessarily airborne, it might be assumed that only volatile molecules could transmit the odortype information in this apparatus, but it is possible that airborne proteins could be involved. A role for proteins in chemical communication has been established (Singer and Macrides, 1992), and recognizable soluble derivatives of MHC class I membrane proteins have been partially characterized in rat urine (Singh et al., 1988). As evidence that these proteins could become airborne, it is notable that airborne proteins from mouse urine have been demonstrated to cause allergic reactions in a room housing a mouse colony (Schumacher, 1980). The main objection to this argument is that these airborne proteins, which are probably released from dried samples as dust, would be present in extremely low concentration in the Y-maze bioassay, during which they could become airborne from solution in the urine in the Petri dish only by bubbles breaking at the surface forming droplets small enough to remain airborne. The direct involvement of proteins in conveying odortype is inconsistent with the results in Table 2. Whether proteins were eliminated by enzyme degradation, perchloric acid precipitation, dialysis, ultrafiltration, or ethanol extraction, the congenic pairs of fractions containing no protein were consistently discriminable in the Y-maze olfactometer. Proteins, however, might be less directly involved in odortype specification. One of the mechanisms of genetic regulation of individual odor listed in the Introduction holds that odortype is specified by a mixture of volatile metabolic by-products in which the composition is determined by the selective binding of some of the urinary metabolites to soluble MHC proteins. The plausibility of this mechanism is supported by the recent demonstrations of grooves in human class I membrane proteins (HLA-A and HLA-B proteins) that bind peptide antigens with some selectivity (Garrett et al., 1989; Hunt et al., 1992). The fact that the early-eluting gel permeation chromatographic fractions, which contain proteins, were discriminated in the Y maze indicates that proteins in the urine may normally bind the compounds constituting odortype, but from the other results it is clear that proteins are not necessary for the discrimination of urine samples from MHC congenic mice in the Y maze and that volatile compounds are capable of conveying the odortype. If urinary proteins are involved in specifying odortype, it is likely that their contribution is to select a distinct mixture of secondary metabolites from the blood serum for secretion by the kidneys (Singh et al., 1987). Another possible function attributed to putative binding proteins in the urine, that they specify the odortype by selectively binding compounds already present in the urine to make the odortype information more persistent (Boyse et al., 1987), is not consistent with the result that destruc-
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