In order to identify the molecule(s) that elicit inherent sexual attraction to male mouse urine, female house mice (Mus musculus domesticus) were presented with a choice between a male versus a female urine stimulus. These were streaked onto filter papers in the shape of scent marks, placed 28 cm apart on the Perspex ceiling of a clean test chamber, such that females could make nasal contact with each stimulus by stretching upright on their hind legs (full details are given in Materials and Methods); wild mice normally spend very little time in this central area of the arena, preferring to stay close to the outer walls. We used a large number of wild-derived female house mice in oestrus or proestrus as subjects throughout this study (bred in captivity to control experience). This ensures that any attraction to male scent is a general response across normal genetically-variable female mice rather than a response that is specific to a laboratory strain, and has not been influenced by many generations of artificial selection for successful breeding in the laboratory. It also avoids the problem of unnatural genetic similarity between subjects and stimulus donors that would be implicit with inbred laboratory mice [22]. All male scent donors were unrelated to the subject females, singly housed (not subordinates), in good health and their scents were not competitively countermarked [8], so should be attractive to females. In order to standardize the stimuli used in different fractionation experiments, we used pooled urine from C57BL/6 (B6) inbred mice after first confirming that female attraction to male urine from this inbred strain was as strong as that towards urine from many different wild-derived house mice (Figure 1A).
Figure 1 Female sexual attraction to male urine. (A) Total time spent under urine stimuli from males (blue bars) and from matched females (pink bars), together with the difference in time spent under male minus female stimulus (circles), plotted as means ± standard error of mean. Significant P values indicate greater attraction to the male stimulus (matched pair t-tests of log transformed data (t) or Wilcoxon matched pair tests (z) when transformed data did not approximate normality). Wild: urine from a random selection of wild males and females (n = 15); B6: normal intact B6 urine (n = 12); low molecular weight (LMW) urine fraction (< 3 kDa, n = 12): high molecular weight (HMW) urine fraction (≥ 3 kDa, n = 12); equal protein: male urine diluted to the same protein concentration as female urine (n = 12); water (open bars): two water stimuli (n = 10). The breakdown of time sniffing each stimulus and time under each stimulus not sniffing is shown in Figure S1. (B) Intact urine, HMW and LMW fractions from C57BL/6 (B6) male and female mice resolved by 15% one dimensional SDS-PAGE. Major urinary proteins (MUPs) were the only major protein bands observed, including a male-specific 18893Da MUP (darcin) which shows unusually high mobility for its size on reducing SDS-PAGE and appears as a band equivalent to 16 kDa [32]. Full size image
Inherent sexual attraction through scent contact
Candidate molecules in male mouse urine that are detected on scent contact include many low molecular weight (LMW) androgen-dependent volatiles detected through V1R receptors in vomeronasal sensory neurons [23–25]. In addition, V2R vomeronasal receptors detect high molecular weight (HMW) involatile proteins and peptides including major urinary proteins (MUPs) [26, 27], exocrine-gland secreting peptides (ESPs) [28] and synthetic peptides that emulate major histocompatibility complex (MHC) peptide ligands [29]. All of these molecules have been implicated in sexual signalling in the mouse, although ESPs are secreted in tears and saliva rather than in urine [30]. Separation of urine into HMW and LMW fractions by centrifugation through a 3 kDa molecular filter revealed that the LMW fraction stimulated little interest but the HMW fraction stimulated similar attraction to intact urine (Figure 1A). This was due largely to the time spent under the stimulus without sniffing (Additional File 1: Figure S1 shows the time under the stimulus broken down into sniffing and not sniffing), a more robust measure of attraction than the time spent sniffing in order to gather information from the scent [17, 31]. The main components of the HMW fraction are urinary proteins (essentially all MUPs, Figure 1B) and their bound ligands (Table 1). The male stimulus contained approximately five times more urinary protein than the female stimulus (the HMW fractions of both sexes contained approximately double the concentration of urinary protein in intact urine). However, higher protein concentration per se was not responsible for the greater attraction to male scent: dilution of intact male urine to the same protein concentration as female urine did not eliminate the attraction response (Figure 1A).
Table 1 Relative concentration (counts adjusted relative to standard) of two urinary volatiles in intact B6 urine and in high molecular weight (HMW ≥ 3 kDa) and low molecular weight (LMW < 3 kDa) fractions. Full size table
As the main components of the HMW fraction consisted of a mixture of different MUP isoforms and their bound ligands [32, 33], these were further separated by anion exchange chromatography into four specific charge group fractions (I - IV) that contained different MUPs. The same four fractions were collected from B6 female urine, although female fractions I and IV contained very low levels of protein (Figure 2, Table 2). MUPs predominated in these fractions. Intact mass analysis by mass spectrometry confirmed that male fractions I and IV contained distinct MUPs expressed predominantly or exclusively by males (masses 18645Da and 18893Da, respectively, corresponding to known Mup genes that show sex-specific expression in the B6 mouse strain [33]). The MUPs in fractions II and III were expressed by both sexes (Figure 2) albeit at different concentrations (Table 2).
Table 2 Protein concentration of each charge fraction from B6 urine and combined stimulus. Full size table
Figure 2 Separation of urinary proteins in B6 mouse urine by anion exchange chromatography. Urinary proteins from (A) adult male and (B) adult female B6 mice were separated by strong anion-exchange chromatography, monitored by ultraviolet (UV) absorbance (central trace, blue or pink line). Four specific fractions corresponding to peak UV absorbance were collected from male urine, together with the corresponding fractions from female urine (I - IV). The protein masses in each of these four fractions, along with unfractionated MUPs, were analysed by electrospray ionisation mass spectrometry (outer traces), confirming that each mass corresponded to a known MUP mass in B6 mice [28]. Male fractions I and IV each contained a single male-specific MUP with a mass that was not detectable in matching female fractions. Resolution of each fraction on SDS-PAGE confirmed the absence of MUPs in female fractions I and IV, while male fraction IV contained the male-specific high mobility 18893Da MUP, darcin. Full size image
A mixture of these four MUP-containing fractions (excluding all other protein-free fractions) presented at concentrations similar to that in intact urine was highly effective in eliciting much greater time near the male stimulus (Figure 3A). The difference was due to the time under the stimulus but not sniffing rather than to active sniffing behaviour (Additional File 1: Figure S2 shows the breakdown of the time sniffing and not sniffing) and was thus similar to the response to the complete HMW fraction. When each charge fraction was tested separately, fractions II and III failed to stimulate significant preference for the male fraction (Figure 3A), although the MUP concentration was much greater in the male than in the female fraction in both cases (2.4 and 5.6 times greater, respectively; Table 2). Male fractions I and IV both stimulated greater attraction than the equivalent female fractions. As this might have been due to the absence of detectable MUP in female fractions I and IV (which would not be the case for intact urine signals), we tested the attraction to each of these male fractions when paired with female fraction III (the most attractive female fraction when tested against the equivalent male fraction). Although male fraction I contained five times more protein than female fraction III, the male fraction was not more attractive (Figure 3B). By contrast, male fraction IV elicited strong attraction compared to female fraction III (Figure 3B); these male and female fractions contained approximately equal concentrations of urinary protein but very different MUP isoforms.
Figure 3 Female sexual attraction to urine fractions separated by anion exchange chromatography containing different major urinary proteins (MUPs). Total time spent under urine stimuli from B6 males (blue bars) and from matched females (pink bars), together with the difference in time spent under male minus female fraction (circles), plotted as means ± standard error of mean. Significant P values indicate greater attraction to the male stimulus (matched pair t-tests of log transformed data (t) or Wilcoxon matched pair tests (z) when log transformed data did not approximate normality); n.s. = no significant attraction to male. (A) Response to urine fractions containing different MUPs (separation shown in Figure 2). I: 18645Da MUP (in male fraction only); II: 18709Da MUP (both sexes); III: 18694Da and 18713Da MUPs (both sexes); IV: 18893Da MUP (darcin, male only). The protein concentration in each male and female fraction is given in Table 2. (B) Male fractions I and IV were subsequently tested against female fraction III. For each test n = 12 except fraction I which bordered on significance and sample size was increased to n = 17. Full size image
In house mice, most urinary MUP isoforms are encoded by Mup genes in the central region of the Mup cluster on chromosome 4 where there has been recent rapid expansion of gene-pseudogene pairs [33]. Variation in expression of these MUP isoforms, which differ in sequence from each other by only a few amino acids, underlies the strong individual variation in MUP profiles between wild mice. These central Mup genes encode the urinary MUPs expressed by both sexes as well as the 18645Da isoform in male fraction I. While expression of the 18645Da MUP is male-specific among laboratory strains [33], it is expressed only by some wild mice and male-specificity varies between wild MUP genotypes (Figure 4). By contrast, the 18893Da MUP isoform in male fraction IV is encoded by a gene in the peripheral region of the Mup cluster where there is considerably greater divergence between Mup genes [33]. This protein is variously known as the '18893Da MUP', 'Peripheral region 2 MUP 17', 'Class A MUP 24' or 'MUP20' but may be best referred to according to the Mouse Genome Informatics Database (MGI) nomeclature: (MGI: Mup20, major urinary protein 20, MGI:3651981; Ensembl reference Gene: Mup20, ENSMUSG00000078672). This MUP is consistently expressed in the urine of male but not female wild mice across a large number of genotypes ([32] and unpublished observations). It shows unusually high mobility on reducing gel electrophoresis (Figure 1B), and is responsible for binding most of the male-specific volatile 2-sec-butyl 4,5 dihydrothiazole (thiazole) in mouse urine [32]. In order to highlight its unusual characteristics compared to all other known MUPs, and its role in female sexual attraction (see below), we named this 18893Da MUP as darcin (after Jane Austen's hero in Pride and Prejudice).
Figure 4 Variation in expression of the 18645Da major urinary protein (MUP) among laboratory and wild mice with different MUP genotypes. Electrospray ionisation mass spectra of urine samples with MUP Intensity expressed relative to the highest peak in each spectrum. Blue: male profiles; pink: female profiles; dashed lines highlight 18645Da and 18893Da. All laboratory mice examined to date exhibit strong male-biased expression of an 18645Da MUP (examples A and B). Some wild mouse MUP genotypes result in similar male-biased expression of an 18645Da MUP (C), some have no detectable expression of this mass (D and E) while some express the protein in both sexes (F). Note that the atypical 18893Da MUP (darcin) does not give a signal on electrospray ionisation mass spectrometry that reflects its abundance. Full size image
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The tight binding of thiazole to darcin considerably extends the release of this volatile pheromone from scent marks over many hours; when artificially displaced from MUPs, thiazole evaporates from scent marks within minutes [8]. Thus, greater attraction to male fraction IV may be due to the binding of thiazole (and possibly other volatile ligands) to darcin. In order to explore this possibility, we compared attraction to male urine that was freshly deposited with attraction to urine deposited 24 h or 7 days prior to testing. Virtually all (> 98%) thiazole is lost 24 h after deposition under similar conditions [32]. If females are attracted by volatile components specifically bound to darcin in male fraction IV, attraction should reduce as scent marks age and volatiles are lost. However, scent age had no significant effects on the strength of attraction to the male sample whether intact urine or HMW stimuli were used in tests (Figure 5) and females continued to show significant attraction even when urine deposits were aged by 7 days.
Figure 5 Female sexual attraction to intact urine or to the high molecular weight fraction of urine (≥ 3 kDa, HMW) according to stimulus age. Urine stimuli (10 μL) from male (blue bars) and female (pink bars) B6 mice were streaked onto 55 mm diameter glass microfibre filters cut in half and left to dry at room temperature for 5 min, 24 h or 7 days prior to testing. The effect of stimulus age and donor sex on response (log s + 1) to intact urine or to HMW was compared by repeated measures ANOVA with stimulus sex as a within-subjects factor and urine age as a between-subjects factor. P values on the graph show post-hoc t-tests which confirmed greater response to the male sample within each test. Log transformed data are plotted to illustrate the similarity of response between tests. Full size image
Attraction to recombinant darcin
Continued attraction to aged male urine does not rule out the possibility that females are sensitive to extremely low levels of thiazole that may still be bound to darcin or, perhaps, to other unidentified components in male MUP fraction IV. In order to test attraction to darcin itself, we constructed an artificial gene, codon optimized to drive high level heterologous expression of the recombinant protein in Escherichia coli (Figure 6 and Additional File 1: Figure S4 showing codon optimized DNA sequence and translation). The recombinant darcin (r-darcin) had atypical mobility on SDS-PAGE (Figure 7C), suggesting similar folding to the native protein. When presented alone, at a physiological level similar to that in normal wild male or B6 strain urine, r-darcin stimulated significant attraction relative to both female urine and to a buffer control [Figure 7A(b, c); Additional File 1: Figure S3 shows the breakdown of time sniffing and not sniffing]. Indeed, response levels were very similar to a B6 intact urine control test [Figure 7A(a)]. Tests of other recombinant MUPs (expressed using the same system - see Additional File 1: Figure S5 - showing codon optimized DNA sequence and translation) confirmed that females showed no attraction either towards recombinant 18694Da MUP (which does not have sex-specific expression) or towards recombinant 18645Da MUP [expressed by male but not female laboratory mice; Figure 7A(d,e)]. Note that response to the 18694Da r-MUP had abnormally high variability [Figure 7A(d)] because one female spent an unusual 85s resting under this stimulus. Excluding this trial, the mean time under the recombinant 18694Da MUP was 6.4 ± 1.3s, very similar to the time normally spent under water, buffer or female urine. This complete absence of response to other recombinant MUPs confirms that attraction to r-darcin was not due to simple unfamiliarity or to a contaminant from the expression system.
Figure 6 Expression and purification of recombinant darcin (r-darcin). E.coli BL21(λ)DE3 cells wer e transformed with a pET28b plasmid and at hourly intervals after induction cells were removed and lysed in water. The equivalent of 0.1 A600 of lysate was loaded into each lane of a 15%(w/v) polyacrylamide gel (panel A). The cell lysate was passed through a 1.2 mm filter and applied directly to a NiNTA metal affinity column column equilibrated with 50 mM sodium phosphate, 10 mM imidazole, 0.3 M NaCl pH8.0. The column was washed in the same buffer containing 20 mM imidazole and bound r-darcin was eluted by increasing the imidazole concentration to 250 mM (panel B). The purified r-darcin was subjected to in gel digestion with endopeptidase LysC and the peptides were analysed by MALDI-ToF mass spectrometry. All LysC peptides of mass greater than 800Da were readily detected in the MALDI-ToF spectrum, confirming the expression of the correct protein (panel C). Full size image
Figure 7 Female sexual attraction to male urine is elicited by darcin. Total time spent under test male stimulus (blue bars: male urine; cyan bars: recombinant major urinary protein (MUP) alone; hatched bars: male urine plus recombinant MUP) and matched control stimulus (pink bars: BALB/c female urine; open bar: buffer), together with the difference in time spent under test minus control (circles), plotted as means ± standard error of mean. Significant P values indicate greater attraction to the test male stimulus [matched pair t-tests of log transformed data (t)]. Control tests using intact urine from C57BL/6 strain [B6, A(a)] or a random selection of n = 14 wild males [Wild, B(f)] confirmed greater attraction to male urine. No attraction was shown towards BALB/c male urine [Bc, B(g)] which contained an extremely low level of darcin. Recombinant darcin (r-darcin, 11 μg) stimulated significant attraction when presented alone [A(b,c)] and when added to male BALB/c urine [B(h)]. There was no attraction to other recombinant MUPs (r-18694Da; r-18645Da). SDS-PAGE of urine stimuli (C), equivalent to one-thirtieth of the amount used in behavioural tests. The different mobility of r-darcin relative to native darcin is a consequence of the C-terminal His-tag used for purification (see Additional File 1: Figure S4). Full size image
Under normal conditions, darcin would not be encountered alone but as a constituent of urine, which also contains a large number of volatile molecules. Airborne volatiles are likely to be important for alerting attention to the presence of a scent and stimulating the close contact investigation that is necessary to detect involatile proteins [8]. As we were testing the inherent attraction that females show in response to contact with a male's scent, trials sometimes had to be excluded because females did not make contact with one or both stimulus locations during the 10 min test. When r-darcin alone was tested against female urine, there was a strong response and most females contacted the stimuli during their normal exploration of the test arena (only 2/15 trials were excluded). However, when r-darcin was tested against just a buffer control, 9/20 trials were excluded due to lack of contact. While this may have been a chance effect, the absence of any urinary volatiles in the arena may also have contributed by failing to stimulate investigation during these trials. In order to confirm that darcin elicits female attraction in the normal context of intact male urine, we also tested sexual attraction to urine from an inbred laboratory strain that expresses this MUP at only trace levels. Several of the laboratory strains derived through the Castle lineage express extremely low and sometimes undetectable levels of darcin [22], with BALB/c males expressing darcin at < 0.5% of their total MUP output (Figures 8, 9A). These classic laboratory strains have been bred in captivity over a considerable number of generations [34], divorced from the normal pressures of sexual selection and the need to attract females through scent. By contrast, adult male wild mice consistently express a high level of darcin (typically 10-20% of MUP expressed similar to B6 males, Figure 9B). As predicted, females were strongly attracted to urine from a random selection of males recently derived from the wild, but failed to spend significantly more time near male BALB/c urine than near female urine [Figure 7B(f,g)]. However, addition of r-darcin to male BALB/c urine at a normal male level stimulated a normal level of attraction [Figure 7B(h)]. They showed no such attraction when other recombinant MUPs were added to male BALB/c urine [Figure 7B(i,j)], confirming that darcin alone stimulates greater attraction to male than to female urine.
Figure 8 Separation of urinary proteins in BALB/c mouse urine by anion exchange chromatography. Urinary proteins from (A) adult male and (B) adult female BALB/c mice were separated by strong anion-exchange chromatography, monitored by ultraviolet (UV) absorbance (central trace, blue line). Five specific fractions corresponding to peak UV absorbance were collected from male urine, together with the corresponding fractions from female urine (I - V). The protein masses in each of these five fractions, along with the starting material (SM), were analysed by electrospray ionisation mass spectrometry (outer traces). Male fractions I and V contained male specific major urinary proteins (MUPs) that were not detectable in the female fractions. Resolution of each fraction on SDS-PAGE confirmed that male fraction V contained a very low abundance of the male-specific high mobility 18893Da MUP, darcin, which was not detectable in the female fraction. See Figure 1 for explanation of the molecular weight labelling on SDS-PAGE. Full size image
Figure 9 Expression of darcin in C57BL/6, BALB/c and wild male mice. Panel A: Urine from male adult mice of C57BL/6 (B6) or BALB/c (Bc) strains was resolved on SDS-PAGE at different loadings, from normal (2 μg of protein) to heavily overloaded (20 μg of protein) lanes. The high mobility band corresponding to darcin was evident at all loadings in C57BL/6 mice, but was barely evident at 10 μg and 20 μg loadings from BALB/c mice. Densitometric analysis of this gel indicated that darcin constituted approximately 15% of the total major urinary protein (MUP) in C57BL/6 mice, but less than 0.5% of total MUP in BALB/c mice. Panel B: Protein (5 μg) from a number of adult, male, wild-derived mice was analysed by SDS-PAGE. The darcin band, confirmed by mass spectrometry to be darcin, was evident in every animal, at levels similar to that seen in C57BL/6 mice. We do not have an explanation for the doublet in animal ID5343, but it is possible that this mouse is heterozygous for two darcin genes - the upper band has tryptic peptides that match to the darcin sequence [32]. Full size image
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Thus, darcin acts as a sex pheromone that reliably elicits female sexual attraction to a male's urine scent, doubling the time spent near a male compared to a female urine mark. Unlike most mammalian scents that have been termed 'pheromones', we have shown that darcin meets the criteria widely accepted for the definition of the term pheromone [1, 5]: it is a species-specific molecule, produced deliberately for scent signalling, and elicits a clear behavioural response that is inherent and highly consistent across the large number of genetically variable wild-derived animals used in this study. It is also equally as effective in eliciting female attraction whether encountered in urine or presented alone. We expressed darcin and other MUPs as recombinant proteins to ascertain the role of these proteins in the absence of natural ligands. We cannot exclude the possibility that r-darcin carries exactly the same thiazole in the same stereochemical configuration as the mouse, but this is extremely unlikely. First, the ligand 2-sec-butyl 4,5 dihydrothiazole is not a central metabolite (the biosynthesis is not known) but is unlikely to be a metabolic pathway that is essential for life. Second, a comprehensive search of the metabolic pathways of E. coli reveals that the only thiazole known in E. coli metabolism is that related to biotin synthesis, an essential metabolic function that has been lost in mammals (hence it is a vitamin). Third, as one of the other recombinant MUPs tested (18694Da MUP) also binds thiazole in mouse urine [32] it provided a good control for putative contaminant ligands from E. coli, and clearly stimulated no response. Thus the response appears to be to darcin alone rather than to a darcin-ligand complex.
Although the response to darcin is inherent and highly consistent, this does not mean that female sexual attraction to male mice is inflexible. Nor does this mean that other components of male urine scent have no role to play in sexual attraction. Importantly, female attraction is to the individual owner of the scent encountered, not to males in general [17, 31]. As darcin is a single protein that is not polymorphic between males, it has no capacity itself for providing the individual-specific scent signatures that females need to recognize particular males, which is an essential component of selective mate choice. Instead, females learn an attraction to individual-specific airborne urinary volatiles on contact with male urine [17].
Learned attraction to individual-specific airborne volatiles
To test whether it is contact with darcin in male urine that stimulates this learned attraction to individual-specific airborne volatiles, we provided females with the opportunity to learn odours by pre-exposing them to a male and female urine stimulus prior to testing. We then tested female attraction to airborne volatiles from a male versus female urine stimulus when they were unable to contact the stimuli during the test. Using urine from males that expressed normal adult levels of darcin, females learned an attraction to airborne urinary volatiles if they had prior physical contact before the test either with urine from the same individual male [using stimuli from genetically diverse wild males, Figure 10A(a)], or with urine from a genetically identical male [Figure 10A(c)]. They did not learn any attraction if they were exposed only to the airborne urinary volatiles of the male before the test without any contact with involatile cues [Figure 10B(d,e)]. We also confirmed that females learned the specific airborne odours of the individual male on contact with male urine, as females pre-exposed to contact with urine from one male showed no subsequent attraction to airborne odours that came from a different genetically distinct individual male in the test [Figure 10A(b)].
Figure 10 Contact with darcin in male urine stimulates learning and subsequent attraction to airborne urinary odours specific to that individual male. Females were pre-exposed to full contact with urine test stimuli (A, C) or to airborne odours only (B) before being tested with airborne odours from a male versus female urine stimulus that could not be contacted during the test. Displayed is the total time spent under airborne urine stimuli from males (blue bars: urine; hatched bars: urine plus recombinant major urinary protein MUP) and females (pink bars), together with the difference in time spent under male minus female stimulus (circles), plotted as means ± standard error of mean. Significant P values indicate greater attraction to the male airborne stimulus (matched pair t-tests on log transformed data (t), or Wilcoxon matched pair tests (z) when transformed data did not approximate normality). Male urine stimuli: Wild1, Wild2 = random selection of wild-derived males; B6 = C57BL/6 inbred strain; Bc = BALB/c inbred strain; Bc + rd = r-darcin added to male BALB/c urine (11 μg in 10 μL urine); Bc + r94 = recombinant 18694Da MUP added to BALB/c urine (11 μg in 10 μL urine); Bc + r45 = recombinant 18645Da MUP added to male BALB/c urine (11 μg in 10 μL urine). A standard BALB/c female stimulus was used in all tests. Full size image
By contrast, prior contact with urine from BALB/c laboratory males, which express only trace levels of darcin (see above), failed to elicit any learned attraction to the males' airborne volatiles [Figure 10C(f)]. However, when r-darcin was added to male BALB/c urine to a level expressed by normal males, contact elicited a strong learned attraction to airborne volatiles from the stimulus [Figure 10C(g)]. This was not because r-darcin altered the airborne volatile profile such that the volatiles themselves became more attractive to females: a control test confirmed that airborne volatiles from this stimulus were not attractive if females had prior contact only with water [Figure 10C(h)]. Instead, contact with darcin stimulates females to learn the individual-specific airborne odour associated with that male's scent and subsequently show attraction only to that airborne odour; contact with r-darcin in BALB/c male urine led to no attraction to airborne odours from males with different individual airborne odour signatures [Figure 10C(i)]. The individual urinary volatile profiles of mice are complex, and are influenced by many genes including MHC [35, 36], as well as by non-genetic differences such as bacterial microflora and diet [37, 38]. Darcin itself may also have some influence on the pattern of airborne volatiles learned on contact: while contact with BALB/c male urine plus r-darcin resulted in strong attraction to volatiles from this same stimulus, it resulted in only weak attraction to airborne volatiles from BALB/c male urine without r-darcin [Figure 10C(g,j)]. As darcin binds and slowly releases the volatile thiazole (and possibly other volatiles) from male scent marks, it is likely to have induced a slight mismatch between volatiles in the manipulated and unmanipulated BALB/c urine. Finally, to confirm that learned attraction to airborne urinary volatiles is a specific response to darcin, other recombinant MUPs added to male BALB/c urine failed to stimulate any learned attraction to airborne volatiles from the stimulus [Figure 10C(k,l)].
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