Darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male's odour
© Roberts et al; licensee BioMed Central Ltd. 2010
Received: 2 March 2010
Accepted: 3 June 2010
Published: 3 June 2010
Among invertebrates, specific pheromones elicit inherent (fixed) behavioural responses to coordinate social behaviours such as sexual recognition and attraction. By contrast, the much more complex social odours of mammals provide a broad range of information about the individual owner and stimulate individual-specific responses that are modulated by learning. How do mammals use such odours to coordinate important social interactions such as sexual attraction while allowing for individual-specific choice? We hypothesized that male mouse urine contains a specific pheromonal component that invokes inherent sexual attraction to the scent and which also stimulates female memory and conditions sexual attraction to the airborne odours of an individual scent owner associated with this pheromone.
Using wild-stock house mice to ensure natural responses that generalize across individual genomes, we identify a single atypical male-specific major urinary protein (MUP) of mass 18893Da that invokes a female's inherent sexual attraction to male compared to female urinary scent. Attraction to this protein pheromone, which we named darcin, was as strong as the attraction to intact male urine. Importantly, contact with darcin also stimulated a strong learned attraction to the associated airborne urinary odour of an individual male, such that, subsequently, females were attracted to the airborne scent of that specific individual but not to that of other males.
This involatile protein is a mammalian male sex pheromone that stimulates a flexible response to individual-specific odours through associative learning and memory, allowing female sexual attraction to be inherent but selective towards particular males. This 'darcin effect' offers a new system to investigate the neural basis of individual-specific memories in the brain and give new insights into the regulation of behaviour in complex social mammals.
See associated Commentary http://www.biomedcentral.com/1741-7007/8/71
Pheromones are specific chemical signals, produced for communication between individuals of the same species, that trigger a specific natural behaviour or physiological process . Signals can be single compounds or combinations of compounds in a precise ratio, detected either by smell or taste . Ubiquitous among invertebrates, pheromones are used to coordinate many aspects of social behaviour, including sexual recognition and attraction to bring opposite sex conspecifics together for mating . Vertebrates also make widespread use of chemical scent signals for within-species communication. However, the more complex individual-specific odours of mammals, combined with variable responses to scents that often depend on context and learning, have led many to suggest that mammalian scent signals generally do not trigger the specific inherent responses required to fulfil the classical definition of pheromones [2, 4, 5]. How then do mammals use scent signals to stimulate the individual-specific responses that underpin the typical flexibility of mammalian social behaviour?
Scents play an integral role in mediating reproductive interactions in many mammals. This includes not only the recognition and location of opposite sex conspecifics but also assessment of the suitability and, thus, the attractiveness of different individuals as potential mates [6, 7]. In common with many species, male house mice advertise their location, successful territory ownership and dominance through scent marks continually deposited around their defended territory . These urinary scents provide a broad range of individual-specific information on a male's genetic compatibility and quality that is used by females in mate selection, influencing the relative attractiveness of individual males and their scents [7, 9]. Females range over several male territories under natural conditions, strongly preferring to mate with dominant territory owners, and approach selected males when ready to mate [10–12].
Sexual attraction which causes an animal to spend more time in the vicinity of chemical signals from the opposite sex, and much greater interest in opposite sex scents, is common to many animals, from C. elegans to elephants [13–15]. Although the specific chemical signals involved have not been identified, previous studies have revealed that adult female mice are inherently attracted to spend more time near scents from adult males than those from other females or from castrated males, regardless of any prior familiarity with males or their odours, as long as females can contact the scent [16–18]. Contact with male scents appears to be rewarding to females  as this induces a conditioned place preference such that females will continue to spend more time in a location where they had previously encountered male soiled substrate, even after the scent has been removed . However, in addition to this inherent contact-mediated attraction, females also learn attraction to airborne volatiles emanating from male urine . Although female mice are readily able to discriminate between male and female airborne odours [18, 21], attraction to airborne odours is learned only on contact with the scent and is specific to the individual airborne urinary odour of the male . This suggests that: (a) there may be an unidentified sexual attraction pheromone that elicits inherent female attraction to spend time near adult male mouse urine; and (b) contact with this sex pheromone may stimulate associative learning of a male's airborne urinary odour as a conditioned stimulus, so that, subsequently, females are attracted to that specific male. Here we show that a single sex-specific protein expressed in adult male mouse urine elicits the highly repeatable inherent attraction of females to spend more time near male than near female urinary scent marks. This involatile protein also stimulates female learning and subsequent selective attraction to the airborne urinary volatiles of individual males. We show that a simple sex attraction pheromone can elicit a flexible response to individual-specific odours through stimulating learning and memory in a complex social mammal, allowing female sexual attraction to be both inherent, yet selective towards particular males.
Inherent sexual attraction through scent contact
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.
2-sec-butyl 4,5 dihydrothiazole
Protein concentration of each charge fraction from B6 urine and combined stimulus.
Combined fractions I-IV
Attraction to recombinant darcin
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  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 .
Learned attraction to individual-specific airborne volatiles
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)].
We have shown that female sexual attraction to spend time near male urine scent is an inherent response to a single male-specific protein pheromone, darcin, detected through physical contact. This involatile pheromone also stimulates females to remember the associated airborne volatile profile and, subsequently, also show attraction to this individual-specific airborne stimulus. Contact with darcin, whether presented alone or combined with urinary volatiles, consistently doubled the time spent near a male compared to a female scent mark. Response to the airborne volatiles learned in association with darcin was even stronger, tripling the time spent near to the source of the learned individual male's scent mark relative to female airborne scent. The consistency of response is all the more remarkable in view of our deliberate use of a large random selection of genetically heterogeneous wild-stock mice to ensure that responses reflect normal functional behaviour in this species, mirroring the attraction to male urine shown by females from seminatural populations . Adult males consistently express darcin at a high level in their urine and, under natural conditions, cover their territories with many hundreds of these small urine scent marks . Attraction to spend time near the source of a male's scent will thus be the primary mechanism that allows females to locate a selected mate, stimulated by contact with this protein pheromone. Indeed, a wild female ready to mate will return repeatedly to a site and wait for short periods for a male to return if he is not currently present  (and JL Hurst's other unpublished observations).
Inherent attraction to darcin in male urine ensures that females naturally discriminate between conspecific male and female scents, without any need for social learning. However, females fail to show the same inherent attraction to male airborne urinary volatiles, despite the prevalence of many androgen-dependent volatiles in male mouse urine [39–41] that allow females to discriminate readily between male and female airborne odours [18, 42]. Importantly, sexual attraction involves more than the simple recognition of opposite sex conspecifics, which could be achieved through any male-specific scent components. Females must assess an individual male's suitability as a mate, including both quality and genetic compatibility with the female's own genome  and then choose between those males available. The requirement for initial contact with the male-specific MUP darcin to stimulate attraction ensures that females gain full information from other involatile and volatile components of the male's scent during contact investigation, prior to showing any attraction to spend time near the male's scent. While some information may be encoded in airborne urinary odours, such as male infection status , we have shown that female mice depend on information encoded by other involatile MUPs in a male's urine to assess the individual attractiveness of a male according to several separate criteria: females use contact with individual-specific MUP patterns to recognize and assess the competitive ability of males from their territorial scent marks ; sharing of the same MUP type to avoid inbreeding with close kin ; and MUP homozygosity to avoid sharing nest sites with males that are likely to be inbred . All of this information provides important modulation of attraction to a particular male. Other involatile protein signals detected on contact  may further influence a male's attractiveness, although specific behavioural responses to other involatile scent components remain to be established.
Most importantly, contact with the male-specific pheromone darcin also stimulates females to learn and become attracted to the individual-specific airborne volatiles associated with this pheromone. Thus, this pheromone signal stimulates both inherent attraction on contact and the learned individual-specific attraction that is essential for choosiness in females. Intuitively, it has been assumed that attractant pheromones in terrestrial animals will be small airborne volatiles that can be detected at a distance from the scent source , with involatile peptides acting as sexual attractants only in aquatic animals such as newts and other amphibia, where they can transmit through water from signaller to receiver [47, 48]. However, we have uncovered a new mechanism used by terrestrial mammals such as mice, in which an involatile protein pheromone elicits the learning of associated airborne volatiles, which can then attract females to spend time near that male's scent even when they are not in direct nasal contact with the scent. The inherently attractive involatile component of male scent may itself be rewarding , as repeated contact with male soiled bedding in a particular location induces a conditioned preference for that location ; thus, females are attracted to male scent marks that they have previously contacted through the memory of their location. However, by also learning the airborne volatiles associated with the pheromone, they can then detect that male's scent wherever they encounter the same volatile profile. As many genes contribute to complex mammalian scents, the airborne volatiles learned are specific to that individual, unlike the attraction pheromone itself; this mechanism therefore allows inherent sexual attraction to be directed to particular males. We have shown previously that contact with just a single scent mark from a male induces a subsequent preference both to spend more time near to that individual scent owner and to gnaw at a barrier to get to that male . At present, we do not know whether females learn all the volatiles that contribute to an individual's airborne signature or focus only on a pattern or subset of male-specific volatiles expressed by each male. Nevertheless, the high level of many androgen-dependent volatiles in male mouse urine, together with female sensitivity to these odorants, will increase the chances that a learned male's scent will be detectable, particularly from a distance. Airborne volatiles thus play an important role in selective attraction to a particular male, but this learned attraction is driven by initial contact with darcin, which elicits an inherent attraction response. The rabbit mammary pheromone (2-methylbut-2-enal), which stimulates inherent searching and grasping for a nipple in newborn rabbits, is also a potent enforcer of odorant learning . A single brief pairing of any odorant with the mammary pheromone also leads to a rapid transfer of the mammary pheromone response to the odorant. Under natural conditions, pups are likely to learn the volatile odours of their mother associated with this pheromone, which then become additional direction cues for subsequent nursing.
The learning of individual odours during prior contact with scents from the opposite sex appears to be common across many mammals, resulting in memory and selective attraction to that scent owner [50–54]. However, previous studies have not suspected that this learning of individual scents may be driven by a specific pheromone, as we have shown here in mice. Notably, when inbred mice contact darcin, the airborne odours that are learned generalize to all males of that strain because they share the same genetically determined odortype. The importance of learning a particular individual male's scent, and selective attraction to this, is thus not apparent when using an inbred laboratory strain to assess the role of scents in the control and coordination of sexual behaviour. Nevertheless, individual selection is the key to female sexual behaviour outside the laboratory. However, the use of males from different laboratory strains has confirmed the importance of learning an individual male's scent in the context of pregnancy block. Exposure of recently mated laboratory females to LMW in urine constituents (< 12-14 kDa) from an unfamiliar strain male blocks pregnancy [29, 55]; importantly, though, the individual scent of the familiar stud male is learned during a brief sensitive period after mating and has no such inhibitory effect . Further research will be needed in order to establish whether darcin plays a role in stimulating this learning of a stud male's scent during mating or whether vagino-cervical stimulation alone provides sufficient reward to stimulate associative learning of a male's volatile urinary scent  regardless of the presence of darcin.
The involatile protein darcin is most likely to be detected through the accessory olfactory subsystem which detects scents that are actively pumped to the vomeronasal organ on nasal contact with the scent source [58, 59]. While unique vomeronasal receptors for darcin have yet to be established, a mixture of recombinant MUPs, that included darcin, activated vomeronasal sensory neurons expressing V2R receptors . Lesion of the accessory olfactory bulb (which receives input from vomeronasal neurons) is also known to abolish both the female innate preference for adult male soiled bedding and learned attraction towards airborne volatiles . By contrast, the airborne individual-specific volatiles detected at a distance from the scent source are most probably detected through the main olfactory subsystem. This is activated when females explore airborne chemicals emanating from male soiled bedding or urine [41, 61], with different patterns of glomerular activation in the main olfactory bulbs stimulated by airborne urinary odours from genetically distinct individuals . A major future challenge will be to understand the brain pathways and neural mechanisms by which the involatile pheromone darcin stimulates the learning and memory of individual-specific airborne odours and results in attraction to this conditioned airborne stimulus.
There has been much debate about whether the original concept of a 'pheromone'  has any useful meaning when applied to complex vertebrates such as mammals, because learning modulates most responses to odours, and the pattern of many scent components is specific to each individual [2, 4, 5]. Indeed, very few pheromones that trigger a defined instinctive and consistent response across genetically diverse individuals have ever been identified in mammals. Using careful molecular dissection closely coupled to assessment of a highly repeatable functional response among recently wild-derived animals, we provide evidence for the first identification of a male-specific signalling protein (darcin) that drives the inherent sexual attraction of female mice to spend time near a male's scent. Moreover, we show that contact with this protein pheromone stimulates associative learning of airborne urinary volatiles, resulting in targeted attraction to a specific individual male by choosy females. The ability of this invariant pheromone to stimulate a learned response towards individual-specific scents suggests that such pheromones may play a much more important role than previously recognized in driving flexible individual-specific social responses that are typical of mammals. Indeed, such pheromones could even underlie some complex, individual-specific responses of humans. Darcin is species-specific, like other MUPs expressed by mice, as would be expected for any pheromone that plays an important role in sexual attraction. However, MUP-like lipocalins show sex-specific expression in other rodents , while other scent components may be candidates for this role in more diverse species. Identifying pheromones with similar effects in other species could have important applications for manipulating mate selection in mammalian breeding programmes (among livestock, experimental animals or captive breeding for conservation). Further, the reliable response to darcin in mice could be used to investigate the neural basis of individual-specific memories in the brain that are fundamental to regulating social behaviour in mammals and other vertebrates.
Subjects and urine donors
The subjects were 469 captive bred adult female Mus musculus domesticus (F0-F2) aged 3-12 months, from a colony derived from wild ancestors captured from five different populations in the northwest of England, UK. Most females (77%) were used in only a single test over a total of 614 trials, but a small number were used in two (17%), three (4%) or four (2%) tests, each involving different scent stimuli, with 2-16 weeks between successive tests. Females were housed in 45 × 28 × 13 cm cages (MB1, North Kent Plastics, Rochester, UK) in single-sex small family groups (2-4 sisters per cage during the test period) in a different room from the urine donors. As we have previously shown that females show very similar attraction responses to urine stimuli from unfamiliar males whether completely naïve to adult male scents or after natural social and sexual experience , subjects were not isolated from contact with other adult male scents in the animal unit and soiled bedding from captive bred wild males was regularly added to female cages during the testing period to ensure normal oestrus cycling.
The urine donors were 17 male and 14 female C57BL/6 laboratory mice (Harlan, Loughborough, UK) aged 60 days - 9 months, 12 male and 17 female BALB/c mice (Harlan, UK) aged 4-9 months, and 33 captive bred adult male house mice derived from the same colony as the female subjects but unrelated. Males were housed singly in 43 × 11.5 × 12 cm cages (M3, North Kent Plastics, UK) to ensure that there were no subordinate males whose scent was unattractive to females. Females were housed in single sex small groups (2-4) in 45 × 28 × 13 cm cages. Urine was collected by holding the donor mouse by the scruff of the neck over a clean 1.5 ml Eppendorf tube. Urine from 5-8 individual donors of the same strain and sex was pooled for testing, using different combinations of donors to create each stimulus pool. Pooling also provided a standard female control stimulus that combined urine from donors in different stages of the oestrus cycle rather than reflecting a particular stage. Urine samples from individual captive-bred wild donor males were not pooled as each has a different individual genetic signature. Urine was collected up to 1-2 weeks prior to testing and stored at -20°C until use. A single large pool was used in each fractionation experiment to ensure that fractions were identical between behavioural replicates and biochemical analyses.
Throughout, all animals were housed on a reversed 12:12 h light cycle with lights off at 0800 h, and were maintained on Corn Cob Absorb 10/14 substrate with paper wool nest material and ad libitum access to water and food (Lab Diet 5002 Certified Rodent Diet, Purina Mills, MO, USA). Cardboard tubes and red plastic mouse houses (Techniplast, NJ, USA) were provided for home cage enrichment.
Assay of attraction to male scent stimuli
Three days prior to each trial, soiled nest material and substrate from wild-derived males was introduced into the subject female's home cage to induce oestrus during the preference test . Previous studies using this procedure on mice in our colony has shown that over 95% of females were likely to be in oestrus or proestrus during a test  and show a robust attraction to a male urine stimulus compared to an equivalent female stimulus when allowed to contact the urine stimuli . All testing was carried out during the dark phase of the light cycle under dim red lighting.
Tests were conducted in a clean 45 × 28 × 13 cm arena (MB1 cage base fitted with a perforated Perspex lid ) to which females were familiarized for 30 min immediately before each test. In most tests, females were presented with a choice between an unfamiliar male urine stimulus and an equivalent female control stimulus that they could contact, placed within two 55 mm diameter circles that were 25 cm apart on the underside of the Perspex arena lid. Test stimuli were deliberately placed in the central zone of the test arena, where wild mice normally spend little time, so that increased time in these sites reflected attraction to the scent. In order to mimic two typical mouse urine scent marks, 10 μL of each test stimulus was streaked on to a 55 mm diameter glass microfibre filter (GMF) cut in half. These were then stuck onto the lid with Sellotape (GMF were used because the release kinetics of urinary volatiles has been characterized previously from this substrate [32, 65]). The position of the male and female control stimulus was randomized but balanced to ensure that an equal number of each stimulus type was presented on each side. Trials lasted 10 min and female behaviour towards the two stimuli was recorded remotely on DVD in a neighbouring laboratory. Transcription of the DVD recordings was carried out blind to the relative position of the two stimuli during each trial which were marked as A and B.
In order to test sexual attraction to airborne urinary volatiles alone, and the influence of prior contact with male-specific urine components, females were first pre-exposed for 30 min in their home cage to a 55 mm GMF cut in half and streaked with 10 μL of each test stimulus (or water). Filter papers were held outside a mesh sphere that allowed full nasal contact with the test stimuli, or inside the mesh sphere for pre-exposure to airborne volatiles only, following the procedure in . Females were then tested in the arena as described above except that fresh test stimuli were suspended 2 cm above the perforated Perspex lid using a 5 cm diameter Perspex cylinder to ensure that mice could not physically contact the test stimuli during the test.
In order to assess sexual attraction, we tested whether females spent more total time under the male rather than the female or control stimulus (nose within the 55 mm diameter circle in which a test stimulus was presented) during the 10 min trials. Time under a stimulus consisted of time spent sniffing or not sniffing the stimulus, each of which were recorded separately to provide further insight into the female's response. Time spent sniffing up at the cage lid within each 55 mm circle indicates the female's interest in gaining further information from a scent source and is part of the information gathering process, thus most sniffing occurs in the first few minutes of a test. Time under the stimulus when not directly investigating the stimulus (defined as the subject's nose being within the 55 mm diameter circle but not sniffing up at the lid) reflects non-investigatory attraction to spend time close to the scent source, and is thus likely to be a more robust measure of attraction [17, 31]. In most cases, log transformation (s+1) of behaviour durations approximated normality (Kolmogorov-Smirnov and Shapiro-Wilks tests, P > 0.05) and allowed parametric analyses (matched pair t-tests to assess sexual attraction, parametric ANOVA to compare the log difference in response to the paired stimuli between tests). Non-parametric tests were used when log transformation did not approximate normality (Wilcoxon matched pair signed-ranks test). As it is widely established that females are more attracted to male than to female urine stimuli, and we needed to be conservative in concluding that manipulation of a urine signal eliminates this typical attraction to male urine, one-tailed statistical tests of greater attraction to male than to female or control stimuli were used throughout. In tests with scent contact, we also confirmed that the response observed after the first close sniff at a stimulus (when females may have gained involatile information from the scent) was the same as the response over the whole trial, and that time under a stimulus did not differ between the two stimuli prior to this first close contact sniff (data not presented).
Fractionation of urine stimuli
Samples were separated into HMW and LMW components using Vivaspin 500 centrifugal concentrators (Vivascience, Sartorious Group, Aubagne, France) with a 3000Da molecular weight cut-off. Vivaspins were washed twice using ddH2O (500 μL added and centrifuged at 13800 rpm for 15 min). Vivaspins were emptied of water and urine (500 μL) was added and centrifuged at 13800 rpm for 15 min. The LMW component was then pipetted into a clean capped 1.5 mL Eppendorf tube, labelled and stored at -20°C until use. Urine was added to the Vivaspin to make up to 500 μL and the process repeated. The HMW and LMW components were pipette into separate capped Eppendorf tubes, labelled and stored at -20°C until use. Protein concentrations of each fraction were determined with the Coomassie plus protein assay reagent kit (Pierce, UK).
Anion exchange chromatography
Purification of individual MUP peaks from C57BL/6 urine was achieved by high-resolution strong anion exchange chromatography on either the Duo-Flow (Biorad Laboratories, CA, USA) or the Äkta (GE Lifesciences, Bucks, UK) chromatography platforms. Both systems were fitted with a Resource Q column (GE Lifesciences, Vt = 6 mL), equilibrated with several column volumes of 50 mM MES buffer, pH 5.0. Pooled urine was desalted on 5 mL Sephadex G-25 columns previously equilibrated with MES buffer, pH 5.0, then passed through a 0.45 μm filter. Typically 1 mL (~10 mg) of desalted protein was applied to the column. Bound protein was eluted from the column using a linear salt gradient of 0 - 1 M NaCl. Fractions (3 mL) were collected and fractions corresponding to individual MUP peaks were pooled then concentrated and desalted to deionised water (~0.2-0.3 mL final volume) using Vivaspin centrifugal concentrators (3 kD molecular weight cut off, Vivascience, Hannover, Germany). Final protein concentrations were obtained by Coomassie dye binding assay. Purity and mass was confirmed by native and SDS-PAGE as well as electrospray ionisation mass spectroscopy.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SDS-PAGE was performed as described by Laemmli . Concentrated anion exchange fractions were mixed 1:1 with reducing sample buffer (100 mM DTT) and then heated for 5 min at 95°C. All samples were run at a constant voltage of 200 V on 15% gels. Protein bands were visualized with Coomassie brilliant blue stain.
Electrospray ionization mass spectrometry (ESI/MS)
Concentrated anion exchange separated MUP peaks were centrifuged at 11,000 g for 10 min. All analyses were performed on an Easy-nLC nano high performance liquid chromatography system (HPLC; Proxeon Biosystems, Odense, Denmark) coupled to a QToF micro mass spectrometer (Waters, Manchester, UK), fitted with an ESI source. Samples (10 μL) were desalted and concentrated on a C4 reverse phase trap (LC packings). MUPs were eluted at a flow rate of 0.8 μL/min using repeated 0 - 100% acetonitrile gradients. Data were gathered between 800 and 1600 Th and were processed and transformed to a true mass scale using MaxENT 1 maximum entropy software (Waters Micromass, Massachusetts, USA). All data sets were processed at 0.25 Da/channel over a mass range of 18400 -19000 Da, and a peak width of 0.75 Da was used to construct the damage model. The instrument was calibrated initially using a 500 fmol/μL solution of Glu fibrinopeptide (Sigma, Munich, Germany) in 50% acetonitrile and 0.2% formic acid and calibrated during runs with a 1 pmol/μL solution of horse heart myoglobin (Sigma), 2 mM DTT in 0.2% formic acid. All water used was HPLC grade (VWR, Pennsylvania, USA)
Hexane extraction of urine and analysis of volatiles was performed according to the methodology in , using a Thermo Fisher Scientific PolarisQ GC/MS.
Expression and purification of recombinant MUPs
The primary sequence of darcin was obtained from the sequence located under accession numbers NP_001012323/XP_355497 and was used to direct de novo gene synthesis for maximal expression in E. coli. The gene was codon optimised for expression in E. coli and cloned into pET28b via NcoI and XhoI restriction sites (Entelechon GmBH, Regensburg, Germany). The plasmid was used to transform BL21(λ)DE3 cells and darcin expressed in Luria Broth containing kanamycin (30 μg/mL). At OD600 nm of between 0.6-0.8, the expression of recombinant darcin (r-darcin) was induced by the addition of isopropyl β-D-1 thiogalactopyranoside to a final concentration of 1 mM. Five hours post-induction, cells were harvested by centrifugation at 2000 × g and the cell pellets stored at -20°C prior to further purification. Harvested cells were lysed using Bugbuster protein extraction reagent (Novagen, Nottingham, UK) containing Complete™ EDTA-free protease inhibitor cocktail (Roche, Burgess Hill, UK). Darcin, present in the soluble fraction of the bacterial cell lysate was purified by virtue of the hexahistidine tag on nickel affinity columns according to manufacturers protocols (Novagen). Column fractions containing r-darcin were pooled and dialysed against 50 mM phosphate, 20 mM NaCl pH7.4. This preparation was used without further processing. The purity of r-darcin was assessed by SDS-PAGE analysis and protein concentration was determined by protein assay. Similar workflows were applied to express the 18645Da MUP (accession GB/AAH91744.1) and 18694Da MUP (accession NP_001157998.1).
Recombinant darcin and other recombinant MUPs were tested alone (11 μg in 10 μL buffer) or added to stimulus urine samples (11 μg to 10 μL BALB/c male urine) in order to mimic the natural concentration of darcin observed in B6 males (approximately 10-14% of total MUP) and in wild male mice [22, 32]. To ensure that both the manipulated urine and control stimulus were treated equally, an equivalent volume of 50 mM phosphate, 20 mM NaCl buffer, pH7.4 was added to the corresponding control stimulus.
List of abbreviations
exocrine-gland secreting proteins
glass microfibre filter
high molecular weight
high performance liquid chromatography
low molecular weight
Mouse Genome Informatics Database
major urinary protein
major histocompatibility complex
C57BL/6 strain mice.
We thank Dr Richard Humphries, John Waters, Felicity Fair, Linda Burgess, Rachel Spencer and Sue Jopson for their technical help; Dr Kathryn Lilley for inspiring discussion of protein naming conventions; and Dr Paula Stockley for helpful discussions and comments on the draft manuscript. The study was funded by a research grant to JLH and RJB from the Biotechnology and Biological Science Research Council (BBC503897).
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