Sex-specific responses to urinary chemicals by the mouse vomeronasal organ

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.     

A novel mechanism regulating a sexual signal: The testosterone-based inhibition of female sex pheromone expression in garter snakes

Vertebrates communicate their sex to conspecifics through the use of sexually dimorphic signals, such as ornaments, behaviors and scents. Furthermore, the physiological connection between hormones and secondary sexual signal expression is key to understanding their dimorphism, seasonality and evolution. The red-sided garter snake (Thamnophis sirtalis parietalis) is the only reptile for which a described pheromone currently exists, and because garter snakes rely completely on the sexual attractiveness pheromone for species identification and mate choice, they constitute a unique model species for exploring the relationship between pheromones and the endocrine system. We recently demonstrated that estrogen can activate female pheromone production in male garter snakes. The purpose of this study was to determine the mechanism(s) acting to prevent female pheromone production in males. We found that castrated males (GX) are courted by wild males in the field and produce appreciable amounts of female sex pheromone. Furthermore, pheromone production is inhibited in castrates given testosterone implants (GX + T), suggesting that pheromone production is actively inhibited by the presence of testosterone. Lastly, testosterone supplementation alone (T) increased the production of several saturated methyl ketones in the pheromone but not the unsaturated ketones; this may indicate that saturated ketones are testosterone-activated components of the garter snake's skin lipid milieu. Collectively, our research has shown that pheromone expression in snakes results from two processes: activation by the feminizing steroid estradiol and inhibition by testosterone. We suggest that basal birds and garter snakes share common pathways of activation that modulate crucial intraspecific signals that originate from skin.     

The effects of sex on chemosensory communication in a terrestrial salamander (Plethodon shermani)

Although much evidence reveals sexually dimorphic processing of chemosensory cues by the brain, potential sex differences at more peripheral levels of chemoreception are understudied. In plethodontid salamanders, the volume of the vomeronasal organ (VNO) is almost twice as large in males as compared to females, both in absolute and relative size. To determine whether the structural sexual dimorphism in VNO volume is associated with sex differences in other peripheral aspects of chemosensation, we measured sex differences in chemo-investigation and in responsiveness of the VNO to chemosensory cues. Males and females differed in traits influencing stimulus access to VNO chemosensory neurons. Males chemo-investigated (“nose tapped”) neutral substrates and substrates moistened with female body rinses more than did females. Compared to females, males had larger narial structures (cirri) associated with the transfer of substrate-borne chemical cues to the lumen of the VNO. These sex differences in chemo-investigation and narial morphology likely represent important mechanisms for regulating sex differences in chemical communication. In contrast, males and females did not differ in responsiveness of VNO chemosensory neurons to male mental gland extract or female skin secretions. This important result indicates that although males have a substantially larger VNO compared to females, the male VNO was not more responsive to every chemosensory cue that is detected by the VNO. Future studies will determine whether the male VNO is specialized to detect a subset of chemosensory cues, such as female body rinses or female scent marks.     

Vomeronasal neuroepithelium and forebrain Fos responses to male pheromones in male and female mice

Male urinary pheromones modulate behavioral and neuroendocrine function in mice after being detected by sensory neurons in the vomeronasal organ (VNO) neuroepithelium. We used nuclear Fos protein immunoreactivity (Fos-IR) as a marker of changes in neuronal activity to examine the processing of male pheromones throughout the VNO projection pathway to the hypothalamus. Sexually naive male and female Balb/c mice were gonadectomized and treated daily with estradiol benzoate (EB) or oil vehicle for 3 weeks. Subjects were then exposed to soiled bedding from gonadally intact Balb/c males or to clean bedding for 90 min prior to sacrifice and processing of their VNOs and forebrains for Fos-IR. Male pheromones induced similar numbers of Fos-IR cells in the VNO neuroepithelium of oil-treated male and female subjects; however, EB-treated females had significantly more Fos-IR neurons in the VNO than any other group. There was an equivalent neuronal Fos response to male odors in the mitral and granule cells of the anterior and posterior accessory olfactory bulb of males and females, regardless of hormone treatment. In central portions of the VNO projection pathway (i.e., bed nucleus of the stria terminalis, medial preoptic area) neuronal Fos responses to male pheromones were present in female but absent in male subjects, regardless of hormone treatment. In a separate experiment, mating induced neuronal Fos-IR in these brain regions at levels in gonadally intact male subjects which were equal to or greater than those seen in ovariectomized females primed with estrogen and progesterone. This suggests that neurons in the central portions of the male's VNO pathway are capable of expressing Fos. Our results suggest that sexually dimorphic central responses to pheromones exist in mice that may begin in the VNO neuroepithelium.     

Pheromonal inhibition of male courtship behaviour in the brown tree snake, Boiga irregularis: a mechanism for the rejection of potential mates

Pheromones play a central role in coordinating the events leading up to copulation in snakes. We report here a novel pheromone system in the brown tree snake in which females release a pheromone that inhibits male courtship behaviour. In a previous study, we made observations of female brown tree snakes releasing cloacal secretions (CS) during courtship that appeared to cause courting males to cease courtship. All snakes have glands that release CS through ducts located along the cloacal orifice. Although CS have been studied for many years, their function in the mediation of snake behaviour has not been experimentally well determined. We examined the role of CS in the reproductive behaviour of male and female brown tree snakes. We conducted four experiments to test the effect of both male and female CS on brown tree snake behaviour under two behavioural contexts, courtship and male-male ritualized combat. Within each experiment, we compared the effects of CS to a control. Female CS caused a decrease in the time that males spent courting females and a decrease in the intensity of courtship compared with the control treatment. Male CS did not, however, affect the time that males spent displaying courtship or the intensity of that courtship. Neither male nor female CS had significant effects on male ritualized combat behaviour, including time that males spent in combat or the intensity of combat behaviours displayed. Furthermore, neither female nor male CS had an effect on female courtship versus controls. The inhibition of brown tree snake reproductive behaviours is specific to female CS inhibiting male courtship behaviour. This pheromone acts in concert with the female sex pheromone to regulate the events leading to copulation.Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

ZW,XY, and yet ZW: Sex chromosome evolution in snakes even more complicated

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in “basal” snakes such as pythons and boas, while heteromorphic and well differentiated in “advanced” (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non‐caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

Male-specific,sex pheromone-selective projection neurons in the antennal lobes of the mothManduca sexta

A subset of olfactory projection neurons in the brain of male Manduca sexta is described, and their role in sex pheromone information processing is examined. These neurons have extensive arborizations in the macroglomerular complex (MGC), a distinctive and sexually dimorphic area of neuropil in the antennal lobe (AL), to which the axons of two known classes of antennal pheromone receptors project. Each projection neuron sends an axon from the AL into the protocerebrum. Forty-one projection neurons were characterized according to their responses to electrical stimulation of the antennal nerve as well as olfactory stimulation of antennal receptors. All neurons exhibited strong selectivity for female sex pheromones. Other behaviorally relevant odors, such as plant volatiles, had no obvious effect on the activity of these neurons. Two broad physiological categories were found: cells that were excited by stimulation of the ipsilateral antenna with pheromones (29 out of 41), and cells that received a mixed input (inhibition and excitation) from pheromone pathways (12 out of 41). Of the cells in the first category, 13 out of 29 were equally excited in response to stimulation of the antenna with either the principal natural pheromone (bombykal) or a mimic of a second unidentified pheromone ('C-15') and were similarly excited by the natural pheromone blend. The remaining 16 out of 29 cells responded selectively, and in some cases, in a dose-dependent manner, to stimulation of the antenna with bombykal or C-15, but not both. Some of these neurons had dendritic arborizations restricted to only a portion of the MGC neuropil, whereas most had arborizations throughout the MGC. Of the cells in the second category, 9 out of 12 were excited by bombykal, inhibited by C-15, and showed a mixed response to the natural pheromone blend. For the other 3 out of 12 cells, the response polarity was reversed for the two chemically-identified odors. Two additional neurons, which were not tested with olfactory stimuli, were tonically inhibited in response to electrical stimulation of the ipsilateral antennal nerve. These observations suggest that some of the male-specific projection neurons may signal general pheromone-triggered arousal, whereas a smaller number can actively integrate inputs from the two know receptor classes (Bal- and C-15-selective) and may operate as 'mixture detectors' at this level of the olfactory subsystem that processes information about sex pheromones.     

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Innate gender-based proclivity in response to cytotoxicity and programmed cell death pathway

Many central nervous system (CNS) diseases display sexual dimorphism. Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon; however, CNS diseases of childhood and the elderly also demonstrate gender predominance and/or a sexually dimorphic response to therapies. Here we show that XY and XX neurons cultured separately are differentially susceptible to various cytotoxic agents and treatments. XY neurons were more sensitive to nitrosative stress and excitotoxicity versus XX neurons. In contrast, XX neurons were more sensitive to etoposide- and staurosporine-induced apoptosis versus XY neurons. The responses to specific therapies were also sexually dimorphic. Moreover, gender proclivity in programmed cell death pathway was observed. After cytotoxic challenge, programmed cell death proceeded predominately via an apoptosis-inducing factor-dependent pathway in XY neurons versus a cytochrome c-dependent pathway in XX neurons. This gender-dependent susceptibility is related to the incapacity of XY neurons to maintain intracellular levels of reduced glutathione. In vivo studies further demonstrated an incapacity for male, but not female, 17-day-old rats to maintain reduced glutathione levels within cerebral cortex acutely after an 8-min asphyxial cardiac arrest. This gender difference in sensitivity to cytotoxic agents may be generalized to nonneuronal cells, as splenocytes from male and female 16-18-day-old rats show similar gender-dependent responses to nitrosative stress and staurosporine-induced apoptosis. These data support gender stratification in the evaluation of mechanisms and treatment of CNS disease, particularly those where glutathione may play a role in detoxification, such as Parkinson's disease, traumatic brain injury, and conditions producing cerebral ischemia, and may apply to non-CNS diseases as well.     

Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action

Receptor cells of the vomeronasal organ (VNO) are thought to detect pheromone-like molecules important for reproductive physiology. Several compounds derived from male mouse urine have been demonstrated to affect endocrine events in female mice. In the present study, the ability of these compounds to affect VNO activity was tested. In dissociated VNO cells held under voltage clamp conditions, application of dehydro-exo-brevicomin (DHB) evoked an outward current at negative holding potentials and an inward current at positive holding potentials. Under current clamp, DHB reduced action potential firing. Since DHB application caused a decrease in membrane conductance, this compound appeared to act by reducing inward current through closing an ion channel. Biochemical experiments tested the effects of DHB and 2- (sec-butyl)-4,5-dihydrothiazole (SBT) on cAMP levels in the VNO. A mixture of DHB and SBT decreased cAMP levels in VNO sensory tissue and had no effect on VNO non-sensory tissue. The results suggest that pheromones have an inhibitory influence on action potential generation and on cAMP levels in receptor cells of the VNO.      

Seasonal morphometry of the vomeronasal organ in the marsupial mouse,Antechinus subtropicus

The vomeronasal system consists of a peripheral organ and the connected central neuronal networks. The central connections are sexually dimorphic in rodents, and in some species, parameters of the vomeronasal organ (VNO) vary with sex, hormonal exposure, body size and seasonality. The VNO of the dasyurid marsupial mouse, Antechinus subtropicus is presumed to be functional. The unusual life history (male semelparity) is marked by distinct seasonality with differences in hormonal environments both between males and females, and in males at different time points. Body size parameters (e.g., length, weight) display sexual dimorphism and, in males, a pronounced weight gain before breeding is followed by a rapid decline during the single, short reproductive season. VNO morphometry was investigated in male and female A. subtropicus to identify possible life cycle associated activity. The overall length of the VNO is positively correlated with the size of the animal. The amount of sensory epithelium exhibits a negative correlation, decreasing with increasing size of the animal. The effects of sex and breeding condition are not obvious, although they do suggest that sensory vomeronasal epithelium mass declines in the breeding period. The VNO may be more important in A. subtropicus before breeding when it may participate in synchronising reproduction and in the development of the male stress response. J. Morphol. 277:1517–1530, 2016. © 2016 Wiley Periodicals, Inc.     

Sexual differences in morphology and niche utilization in an aquatic snake,Acrochordus arafurae

Filesnakes (Acrochordus arafurae) are large (to 2 m), heavy-bodied snakes of tropical Australia. Sexual dimorphism is evident in adult body sizes, weight/length ratios, and body proportions (relative head and tail lengths). Dimorphism is present even in neonates. Two hypotheses for the evolution of such dimorphism are (1) sexual selection or (2) adaptation of the sexes to different ecological niches. The hypothesis of sexual selection is consistent with general trends of sexually dimorphic body sizes in snakes, and accurately predicts, for A. arafurae, that the larger sex (female) is the one in which reproductive success increases most strongly with increasing body size. However, the sexual dimorphism in relative head sizes is not explicable by sexual selection.The hypothesis of adaptation to sex-specific niches predicts differences in habitats and/or prey. I observed major differences between male and female A. arafurae in prey types, prey sizes and habitat utilization (shallow versus deep water). Hence, the sexual dimorphism in relative head sizes is attributed to ecological causes rather than sexual selection. Nonetheless, competition between the sexes need not be invoked as the selective advantage of this character divergence. It is more parsimonious to interpret these differences as independent adaptations of each sex to increase foraging success, given pre-existing sexually-selected differences in size, habitat or behavior. Data for three other aquatic snake species, from phylogenetically distant taxa, suggest that sexual dimorphism in food habits, foraging sites and feeding morphology, is widespread in snakes.     

Sex- and strain-specific expression and vomeronasal activity of mouse ESP family peptides

Male mice secrete exocrine-gland-secreting peptide 1 (ESP1) from the extraorbital lacrimal gland into tear fluid [1]. Other mice detect ESP1 through sensory neurons in the vomeronasal organ (VNO), a secondary olfactory system that senses pheromonal information, including sex, strain, and species. ESP1 is now known to be a member of a multigene family that encodes peptides of various lengths. We herein performed genomic and expression analyses of the ESP family. The ESP family consists of 38 members in mice and 10 members in rat but is absent from the human genome, suggesting rapid molecular evolution. In addition to the male-specific ESP1, we discovered one, which we designated ESP36, that, in adult BALB/c mice, is expressed only in the female extraorbital lacrimal gland. The sexually dimorphic expression is ensured by the release of testosterone after puberty. However, we observed dramatic differences in the expression levels of ESPs between strains. Finally, all ESPs elicited an electrical response in the vomeronasal epithelium but not in the main olfactory epithelium. Multielectrode recording of VNO activity demonstrated that ESP1 induces action potentials in vomeronasal neurons, leading to an increase in the spike firing rate, and that ESP1 is recognized by narrowly tuned vomeronasal sensory neurons. Sexual dimorphism and strain differences of ESPs and their reception in the VNO suggest that the ESP family can convey information about sex and individual identity via the vomeronasal system. The chemosensation of this nonvolatile peptide family by direct contact appears to be one of strategies for sociosexual communication in rodent species.     

Prenatal sex ratios and expression of sexually dimorphic traits in three snake species

Variation in intrauterine exposure to hormones associated with variation in the sex of litter mates has well-established and far-reaching effects on sexual development in some mammals. Research on this phenomenon in reptiles is scant, but suggests that lizards may follow the mammalian model whereas snakes may be affected differently. We examined sex-specific expression of four sexually dimorphic traits (tail length, head length, ventral scale count, swimming speed) in three species of snakes (Nerodia sipedon, Thamnophis sirtalis, T. sauritus) relative to litter sex ratios. We found little evidence that traits in either sex were masculinized or feminized in response to variation in litter sex ratio. The one significant result appeared best explained as a statistical artifact attributable to a single litter. Our results indicate that snakes are different from the one lizard studied to date. Unlike previous suggestions that prenatal hormonal mechanisms operate differently in snakes and lizards, however, the difference appears to be that development of sexually dimorphic traits in lizards is affected by litter sex ratios whereas in snakes it is not.     

Conditioned odor aversion induces social anxiety towards females in wild‐type and TrpC2 knockout male mice

Female‐emitted pheromonal inputs possess an intrinsic rewarding value for conspecific males, promoting approach and investigation of the potential mating partner. In mice these inputs are detected mainly by the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). We investigated the role of VNO‐mediated inputs in experience‐dependent plasticity of reproductive responses. We applied a sex‐specific conditioned odor aversion (COA) paradigm on adult, wild‐type (WT) male mice and on male mice impaired in VNO‐mediated signal transduction (TrpC2^?/?). We found that WT males, which underwent COA to female‐soiled bedding, lost their innate preference to female odors and presented lower motivation to approach a sexually receptive female. COA also abolished the testosterone surge normally seen following exposure to female odors. Moreover, the conditioned males displayed impairments in copulatory behaviors, which lasted for several weeks. Surprisingly, these males also exhibited phobic behaviors towards receptive females, including freezing and fleeing responses. In contrast, WT males which underwent COA specifically to male pheromones showed no change in olfactory preference and only a marginally significant elevation in intermale aggression. Finally, we show that TrpC2^?/? males were able to acquire aversion to female‐soiled bedding and presented similar behavioral alterations following COA in their responses to female cues. Our results demonstrate that the intrinsic rewarding value of female pheromones can be overridden through associative olfactory learning, which occurs independently of VNO inputs, probably through MOE signaling.     

Gall midge olfaction: Pheromone sensitive olfactory neurons in Contarinia nasturtii and Mayetiola destructor

This study describes the morphology and function of the antennal sensilla in two gall midge species, Contarinia nasturtii and Mayetiola destructor, where multi-component sex pheromones have been identified. Both species possess sensilla trichodea, s. coeloconica, s. chaetica and s. circumfila. Sensilla circumfila, which consist of several sensilla that bifurcate and fuse into one structure, are unique for the gall midges. In C. nasturtii s. circumfila are sexually dimorphic. In males, they form elongated loops suspended on cuticular spines, whereas in females they run like worm-like structures directly on the antennal surface. Single sensillum recordings demonstrated that olfactory sensory neurons housed in male s. circumfila in C. nasturtii responded to the female sex pheromone. In M. destructor, s. circumfila were attached to the antennal surface in both sexes, and displayed no response to sex pheromone components.A sexual dimorphism was also found in the number of s. trichodea per antennal segment in both C. nasturtii (male 1 vs. female 7) and M. destructor (male 13 vs. female 10). OSNs located in male M. destructor s. trichodea responded to the sex pheromone. This is the first gall midge single sensillum study, and the first demonstration of the functional significance of s. circumfila.     

Experiential and endocrine dependence of gonadotropin responses in male mice to conspecific urine

Previous research has shown that a urinary pheromone of female mice acts via the vomeronasal organ of the accessory olfactory system to elicit rapid release of luteinizing hormone (LH) in conspecific males. Several experiments were conducted to examine the importance of sexual experience for gonadotropin responses in male mice to female urine, male urine, saline, or mixtures of these stimuli. Both sexually naive and sexually experienced male mice had significantly higher plasma LH levels after presentations of female urine than after presentations of male urine. However, sexual experience appeared to increase the reliability of the short-latency gonadotropin response to female urine relative to a sexually neutral component of urine such as sodium chloride, and male urine appeared to suppress spontaneous LH secretion episodes in both naive and sexually experienced males. Subsequent experiments with sexually experienced subjects demonstrated that male mouse urine is a powerful suppressant of LH release in other males. Specifically, female mouse urine mixed with male urine failed to elicit LH responses in male subjects, whereas female urine mixed with saline was highly effective. Urine obtained from castrated male donors was as potent as urine from intact males in suppressing the gonadotropin response to female urine. The suppressive activity in male mouse urine thus does not appear to be critically dependent on gonadal hormones. The existence of a potent stimulatory pheromone in female urine and a potent suppressive pheromone in male urine makes male mice an excellent model system for studying the neural regulation of LH secretion.     

The genomic basis of vomeronasal-mediated behaviour

The vomeronasal organ (VNO) is a chemosensory subsystem found in the nose of most mammals. It is principally tasked with detecting pheromones and other chemical signals that initiate innate behavioural responses. The VNO expresses subfamilies of vomeronasal receptors (VRs) in a cell-specific manner: each sensory neuron expresses just one or two receptors and silences all the other receptor genes. VR genes vary greatly in number within mammalian genomes, from no functional genes in some primates to many hundreds in rodents. They bind semiochemicals, some of which are also encoded in gene families that are coexpanded in species with correspondingly large VR repertoires. Protein and peptide cues that activate the VNO tend to be expressed in exocrine tissues in sexually dimorphic, and sometimes individually variable, patterns. Few chemical ligand–VR–behaviour relationships have been fully elucidated to date, largely due to technical difficulties in working with large, homologous gene families with high sequence identity. However, analysis of mouse lines with mutations in genes involved in ligand–VR signal transduction has revealed that the VNO mediates a range of social behaviours, including male–male and maternal aggression, sexual attraction, lordosis, and selective pregnancy termination, as well as interspecific responses such as avoidance and defensive behaviours. The unusual logic of VR expression now offers an opportunity to map the specific neural circuits that drive these behaviours.