Exocrine Gland-secreting Peptide Research Articles

Prelimbic cortex responds to male ultrasonic vocalizations in the presence of a male pheromone in female mice.

Sensory signals are critical to perform adaptive social behavior. During copulation, male mice emit ultrasonic vocalizations (USVs). Our previous studies have shown that female mice exhibit approach behavior toward sound sources of male USVs and that, after being exposed to a male pheromone, exocrine gland-secreting peptide 1 (ESP1), female mice exhibited a preference toward a particular type of male USVs. These findings suggest that male USVs modulate female courtship behavior. However, it remains unclear which brain regions and what cell types of neurons are involved in neuronal processing of male USVs. To clarify this issue, immediate early gene analysis, behavioral analysis, and neurochemical analysis were performed. The in situ hybridization analysis of c-fos mRNA in multiple brain regions showed that neurons in the prelimbic cortex were responsive to presentation of male USVs in the presence of ESP1. Furthermore, this study found that activity of prelimbic cortex was correlated with the duration of female exploration behavior toward a sound source of the USVs. Finally, by using double immunohistochemistry, the present study showed that the prelimbic neurons responding to the presentation of male USVs were presumably excitatory glutamatergic neurons. These results suggest that the prelimbic cortex may facilitate female courtship behavior in response to male USVs.

Origin and Evolution of the Gene Family of Proteinaceous Pheromones, the Exocrine Gland-Secreting Peptides, in Rodents.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Sexual rejection via a vomeronasal receptor-triggered limbic circuit

Mating drive is balanced by a need to safeguard resources for offspring, yet the neural basis for negative regulation of mating remains poorly understood. In rodents, pheromones critically regulate sexual behavior. Here, we observe suppression of adult female sexual behavior in mice by exocrine gland-secreting peptide 22 (ESP22), a lacrimal protein from juvenile mice. ESP22 activates a dedicated vomeronasal receptor, V2Rp4, and V2Rp4 knockout eliminates ESP22 effects on sexual behavior. Genetic tracing of ESP22-responsive neural circuits reveals a critical limbic system connection that inhibits reproductive behavior. Furthermore, V2Rp4 counteracts a highly related vomeronasal receptor, V2Rp5, that detects the male sex pheromone ESP1. Interestingly, V2Rp4 and V2Rp5 are encoded by adjacent genes, yet couple to distinct circuits and mediate opposing effects on female sexual behavior. Collectively, our study reveals molecular and neural mechanisms underlying pheromone-mediated sexual rejection, and more generally, how inputs are routed through olfactory circuits to evoke specific behaviors.

Exocrine Gland-Secreting Peptide 1 Is a Key Chemosensory Signal Responsible for the Bruce Effect in Mice

The Bruce effect refers to pregnancy termination in recently pregnant female rodents upon exposure to unfamiliar males [1]. This event occurs in specific combinations of laboratory mouse strains via the vomeronasal system [2, 3]; however, the responsible chemosensory signals have not been fully identified. Here we demonstrate that the male pheromone exocrine gland-secreting peptide 1 (ESP1) is one of the key factors that causes pregnancy block. Female mice exhibited high pregnancy failure rates upon encountering males that secreted different levels of ESP1 compared to the mated male. The effect was not observed in mice that lacked the ESP1 receptor, V2Rp5, which is expressed in vomeronasal sensory neurons. Prolactin surges in the blood after mating, which are essential for maintaining luteal function, were suppressed by ESP1 exposure, suggesting that a neuroendocrine mechanism underlies ESP1-mediated pregnancy failure. The single peptide pheromone ESP1 conveys not only maleness to promote female receptivity but also the males' characteristics to facilitate memorization of the mating partner.

A Labeled-Line Neural Circuit for Pheromone-Mediated Sexual Behaviors in Mice

In mice, various instinctive behaviors can be triggered by olfactory input. Despite growing knowledge of the brain regions involved in such behaviors, the organization of the neural circuits that convert olfactory input into stereotyped behavioral output remains poorly understood. Here, we mapped the neural circuit responsible for enhancing sexual receptivity of female mice by a male pheromone, exocrine gland-secreting peptide 1 (ESP1). We revealed specific neural types and pathways by which ESP1 information is conveyed from the peripheral receptive organ to the motor-regulating midbrain via the amygdala-hypothalamus axis. In the medial amygdala, a specific type of projection neurons gated ESP1 signals to the ventromedial hypothalamus (VMH) in a sex-dependent manner. In the dorsal VMH, which has been associated with defensive behaviors, a selective neural subpopulation discriminately mediated ESP1 information from a predator cue. Together, our data illuminate a labeled-line organization for controlling pheromone-mediated sexual behavioral output in female mice.

Self-Exposure to the Male Pheromone ESP1 Enhances Male Aggressiveness in Mice

Exocrine gland-secreting peptide 1 (ESP1) released into male tear fluids is a male pheromone that stimulates sexually receptive behavior in female mice via the vomeronasal sensory system. ESP1 also induces c-Fos expression in male brain regions distinct fromthose in females. However, behavior in males following ESP1 exposure has not been examined. In the present study, we show that ESP1, in conjunction with unfamiliar male urine, enhances male aggression via the specific vomeronasal receptor V2Rp5. In addition, male mice that secrete ESP1 but lack V2Rp5 exhibit a lower level of aggressiveness than do mice that express V2Rp5. These results suggest that ESP1 not only acts as a male pheromone in both sexes but also serves as an auto-stimulatory factor that enhances male aggressiveness by self-exposure. Finally, re-activation of ESP1-induced c-Fos-positive neurons by using the designer receptor exclusively activated by designer drug (DREADD) approach resulted in enhancement of sexual and aggressive behaviors in female and male mice, respectively, indicating that sexually dimorphic activation in the brain is a neural basis for the sex-specific behavioral responses to ESP1.

Proteins and peptides as pheromone signals and chemical signatures

Proteins and peptides are used as pheromones by many invertebrates and vertebrates, on land as well as underwater. These molecules are used as pheromones by aquatic animals such as squid, sea hares, and newts. Males in some terrestrial salamander species use high molecular weight glycopeptide pheromones transferred directly to the nostrils of the female. In Drosophila fruit flies, sex peptides in the male's seminal fluid change the female's behaviour so that after mating she rejects other males and starts to lay eggs. The rapid evolution of Drosophila sex peptides and salamander peptide pheromones suggests that sexual conflict may be involved. In mice, exocrine gland-secreting peptide 1 (ESP1) secreted from the male's tear glands is transferred to the female's nose through physical contact during investigation of the facial areas during courtship. ESP1 activates a narrowly specific vomeronasal receptor, leading to receptive behaviour in the female. The urine marks of male territorial house mice contain high concentrations of highly variable major urinary proteins (MUPs). The MUPs bind small molecule pheromones and other odorants, slowly releasing them and thus greatly prolonging the attractive volatile lifetime of the signal. One of the MUPs, darcin, is the same in all male house mice: it is a pheromone in itself. When the female comes into contact with darcin in a scent mark it prompts a learned attraction to both the male's individual chemical signature mixture and the location of the scent mark. Proteins and peptides may also contribute to the highly variable chemical profiles that differ between individual mammals, for example, and that are used as cues to allow individuals to be distinguished.

Expression and purification of mouse peptide ESP4 in Escherichia coli

Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members.

A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system

Animals display a repertoire of different social behaviours. Appropriate behavioural responses depend on sensory input received during social interactions. In mice, social behaviour is driven by pheromones, chemical signals that encode information related to age, sex and physiological state. However, although mice show different social behaviours towards adults, juveniles and neonates, sensory cues that enable specific recognition of juvenile mice are unknown. Here we describe a juvenile pheromone produced by young mice before puberty, termed exocrine-gland secreting peptide 22 (ESP22). ESP22 is secreted from the lacrimal gland and released into tears of 2- to 3-week-old mice. Upon detection, ESP22 activates high-affinity sensory neurons in the vomeronasal organ, and downstream limbic neurons in the medial amygdala. Recombinant ESP22, painted on mice, exerts a powerful inhibitory effect on adult male mating behaviour, which is abolished in knockout mice lacking TRPC2, a key signalling component of the vomeronasal organ. Furthermore, knockout of TRPC2 or loss of ESP22 production results in increased sexual behaviour of adult males towards juveniles, and sexual responses towards ESP22-deficient juveniles are suppressed by ESP22 painting. Thus, we describe a pheromone of sexually immature mice that controls an innate social behaviour, a response pathway through the accessory olfactory system and a new role for vomeronasal organ signalling in inhibiting sexual behaviour towards young. These findings provide a molecular framework for understanding how a sensory system can regulate behaviour.

Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

Backbone and side-chain 1H, 15N and 13C assignments of mouse peptide ESP4

A peptide or a small protein released from an exocrine gland or in urine is utilized as a chemosignal that elicits social or reproductive behavior in mice. Recently, we identified the male-specific peptide, exocrine gland-secreting peptide 1 (ESP1), in mouse tear fluids that enhanced female sexual receptive behavior, and determined the three dimensional structure. ESP1 appears to be a member of multigene family that consists of 38 genes in mice, which we call the ESP family. ESP4, a member of the ESP family, is expressed in various exocrine glands, and shows the highest sequence similarity with ESP1. Here, we report the NMR assignments of ESP4 which provides a basis for NMR analyses of this protein. Our results will give insight into structural relationships within the ESP family.

G protein Gαo is essential for vomeronasal function and aggressive behavior in mice

The rodent vomeronasal organ (VNO) mediates the regulation of species-specific and interspecies social behaviors. We have used gene targeting to examine the role of the G protein Gαo, encoded by the gene Gnao1, in vomeronasal function. We used the Cre-loxP system to delete Gαo in those cells that express olfactory marker protein, which includes all vomeronasal sensory neurons of the basal layer of the VNO sensory epithelium. Using electrophysiology and calcium imaging, we show that the conditional null mice exhibit strikingly reduced sensory responses in V2R receptor-expressing vomeronasal sensory neurons to specific molecular cues, including MHC1 antigens, major urinary proteins, and exocrine gland-secreting peptide. Gαo is also vital for vomeronasal sensing of two N-formylated mitochondrially encoded peptides derived from NADH dehydrogenase 1. Furthermore, we show that Gαo is an essential requirement for the display of male-male territorial aggression as well as maternal aggression in mice. Finally, we show that Gαo-dependent maternal aggression can be induced by major urinary proteins. These cellular and behavioral phenotypes identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein pheromones by sensory neurons of the VNO.

The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor

Various social behaviours in mice are regulated by chemical signals called pheromones that act through the vomeronasal system. Exocrine gland-secreting peptide 1 (ESP1) is a 7-kDa peptide that is released into male tear fluids and stimulates vomeronasal sensory neurons in female mice. Here, we describe the molecular and neural mechanisms that are involved in the decoding of ESP1 signals in the vomeronasal system, which leads to behavioural output in female mice. ESP1 is recognized by a specific vomeronasal receptor, V2Rp5, and the ligand-receptor interaction results in sex-specific signal transmission to the amygdaloid and hypothalamic nuclei via the accessory olfactory bulb. Consequently, ESP1 enhances female sexual receptive behaviour upon male mounting (lordosis), allowing successful copulation. In V2Rp5-deficient mice, ESP1 induces neither neural activation nor sexual behaviour. These findings show that ESP1 is a crucial male pheromone that regulates female reproductive behaviour through a specific receptor in the mouse vomeronasal system.

Molecular characterization of vomeronasal sensory neurons responding to a male-specific peptide in tear fluid: Sexual communication in mice

Abstract Pheromonal signals received by the vomeronasal organ (VNO) have been shown to elicit various behavioral and physiological responses that are typically stereotyped and preprogrammed. Recently, we found a novel male-specific peptide, named exocrine gland-secreting peptide 1 (ESP1), that is secreted in tear fluid and stimulates the VNO in mice. Excreted ESP1 appears to be transferred to the female VNO, where it induces c-Fos expression and elicits an electrical response in a small subset of vomeronasal sensory neurons (VSNs). We report here the identification of molecular components expressed in ESP1-stimulated VSNs by double-staining with c-Fos. We found that the c-Fos-induced cells were localized amongst the Gαo-expressing VSNs. Furthermore, the ESP1 signal was received by VSNs expressing a single type of vomeronasal receptor type 2 (V2Rp5). Finally, double in situ hybridization of the V2Rp5 and various members of the M1 and M10 families of major histocompatibility complex (MHC) class Ib molecules revealed that V2Rp5-expressing VSNs can express multiple MHC molecules. These results suggest that a V2R rather than MHC molecule is mainly responsible for the recognition of ESP1. The identification of the putative sex-pheromone ESP1 and its cognate receptor therefore will help clarify the molecular mechanisms underlying pheromone recognition in the mouse vomeronasal system.

Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons

In mammals, social and reproductive behaviours are modulated by pheromones, which are chemical signals that convey information about sex and strain. The vomeronasal organ, located at the base of the nasal septum, is responsible for mediating pheromone information in mice. Two classes of putative pheromone receptor gene families, V1R and V2R, are expressed by vomeronasal sensory neurons in mutually segregated epithelial zones of the vomeronasal organ. Although numerous studies have suggested that pheromones originate from urine, direct recordings of behaving mice have shown that neuronal firing in the vomeronasal system is modulated by physical contact with the facial area. Here we identify a male-specific 7-kDa peptide secreted from the extraorbital lacrimal gland. This peptide, which we named exocrine gland-secreting peptide 1 (ESP1), is encoded by a gene from a previously unrecognized large family clustered in proximity to the class I major histocompatibility complex (MHC) region. ESP1 is secreted from the eyes and is transferred to the female vomeronasal organ, where it stimulates V2R-expressing vomeronasal sensory neurons and elicits an electrical response. Our results indicate that mice respond to sex-specific peptides released from exocrine glands through the vomeronasal system during direct contact.