Melanocortin-5 receptor deficiency reduces a pheromonal signal for aggression in male mice

Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory effects of systemic melanocortin injections on aggressive behavior. Because the pheromone-producing preputial gland expresses the MC5R, we tested the hypothesis that decreases in preputial pheromones underlie the behavioral deficit. Here we show that MC5R deficiency decreases preputial and urine levels of the sex pheromones, alpha- and beta-farnesene, relative to wild-type mice. We also demonstrate that farnesenes potently stimulate aggression in mice. Moreover, farnesene-stimulated aggression is reduced in MC5R-deficient mice, relative to wild-type mice. Our results suggest that activation of the MC5R promotes aggression by increasing farnesene signaling.     

Sex- and gonad-affecting scent compounds and 3 male pheromones in the rat

This study was aimed at identifying sex pheromones of the rat (Rattus norvegicus). We characterized the volatiles and semivolatiles of rat preputial gland and voided urine by using gas chromatography-mass spectrometry (GC-MS) and quantified them by their GC areas (abundances) and percentage of GC areas (relative abundances). Although all the compounds other than 4-heptanone and phenol detected were shared by males and females, the quantities for some of these sex-common compounds exhibited sexual dimorphism and decreased with gonadectomy. Thus, these compounds might be sex pheromones. Among them, squalene from preputial glands and 2-heptanone and 4-ethyl phenol from urine were 3 major compounds. They were richer in males and could be suppressed by castration. Adding any of the 3 compounds (at a concentration higher than its physiological level in male urine) to castrated male urine (CMU) increased the attractiveness of CMU to sex-naive females. Adding the 3 together (at the levels in normal male urine) to CMU significantly increased the attractiveness of CMU to females. However, such combination did not fully restore females' preference for urine from intact males, suggesting that some other trace compounds such as 4-heptanone and phenol might also play some roles in sex attractiveness. Thus, squalene, 2-heptanone, and 4-ethyl phenol were indeed male pheromone molecules in rats. Our study also indicates that E,E-beta-farnesene and E-alpha-farnesene, both richer in females than males, might be putative female pheromones.     

Sex preference and species specificity of rodent (Mus musculus and Microtus arvalis pheromones.

The behavioural response to the sex pheromones in the externally voided urine of field voles (Microtus arvalis) and laboratory mice (CFLP, CBA strains) although specific for species showed no strain specificity. Bladder urine (free of accessory sex-gland secretions) and the preputial glands of CFLP and CBA mice contain sex attractants. Ether extracts made of blood of male CFLP mice attracted CFLP female mice.     

Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication

Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.     

Putative chemical signals about sex, individuality, and genetic background in the preputial gland and urine of the house mouse (Mus musculus)

To explore whether preputial gland secretions and/or urine from the house mouse (Mus musculus) can be used for coding information about sex, individuality, and/or the genetic background of strain [ICR/albino, Kunming (KM), and C57BL/6], we compared the volatile compositions of mouse preputial glands and urine using a combination of dichloromethane extraction and gas chromatography coupled with mass spectrometry (GC-MS). Of the 40 identified compounds in preputial gland secretions, 31 were esters, 2 sesquiterpens, and 7 alcohols. We failed to find any compound unique to a specific sex, individual, or strain. However, many low molecular weight compounds between the sexes, most compounds among individuals, and several compounds among the 3 strains varied significantly in relative ratios. These quantitative differences in preputial gland volatiles (analog coding) are likely to convey information about sex, individual, and the genetic background of mouse strain. We identified 2 new main and male-elevated compounds, 1-hexadecanol (Z=3.676, P=0.000, N=19 in ICR; Z=3.576, P=0.000, N=18) and 1-hexadecanol acetate (Z=3.429, P=0.000, N=19 in ICR; Z=3.225, P=0.001, N=18), which were eluted in GC chromatogram after the 2 sesquiterpens. They might also be potential male pheromones, in addition to the well-known E-beta-farnesene and E,E-alpha-farnesene. Additionally, a few compounds including 1-hexadecanol also varied with strains and might also code for genetic information. Of the 9 identified volatile compounds in male urine, (s)-2-sec-butyl-4,5-dihydrothiazole and R,R-3,4-dehydro-exo-brevicomin are known urine-originated male pheromones from previous studies. We also detected 6-hydroxy-6-methyl-3-heptanone, a male urinary pheromonal compound, which had not been directly detected by GC-MS previously. Chemical analysis shows that the genetically more closely related ICR and KM strains had a higher similarity in the volatile compositions of preputial glands and urine than that between ICR or KM and C57BL/6. R,R-3,4-dehydro-exo-brevicomin, in particular, was sensitive to genetic shifts and differed in relative abundance among the 3 strains, whereas (s)-2-sec-butyl-4,5-dihydrothiazole differed between ICR or Km and C57BL/6. Hence, these 2 compounds might code for information about their genetic background.     

Intrauterine position affects sex-accessory biochemistry in adult female mice

Female mouse fetuses that develop in utero implanted between two male fetuses (2M females) are shown as adults to have significantly elevated (P less than 0.01) levels of beta-glucuronidase activity in their preputial glands compared to that of OM females, females that had not been contiguous to males in utero, and 1M females, females contiguous in utero to only one male (mean +/- SEM, 2M females = 23,9 +/- 2.1, 1M females = 13.3 +/- 5.2, and OM females = 7.8 +/- 2.5 Modified Sigma Units/mg frozen weight). It is hypothesized that the increased enzymatic activity in the preputial glands of 2M females could be important in releasing steroid metabolites from voided urine. These metabolites could then act as pheromones and thus explain some of the described differences in sexual behavior correlated with intrauterine position.     

Attraction thresholds and sex discrimination of urinary odorants in male and female aromatase knockout (ArKO) mice

We previously found that both male and female aromatase knockout (ArKO) mice, which cannot synthesize estrogens due to a targeted mutation of the aromatase gene, showed less investigation of volatile body odors from anesthetized conspecifics of both sexes in Y-maze tests. We now ask whether ArKO mice are in fact capable of discriminating between and/or responding to volatile odors. Using habituation/dishabituation tests, we found that gonadectomized ArKO and wild-type (WT) mice of both sexes, which were tested without any sex hormone replacement, reliably distinguished between undiluted volatile urinary odors of either adult males or estrous females versus deionized water as well as between these two urinary odors themselves. However, ArKO mice of both sexes were less motivated than WT controls to investigate same-sex odors when they were presented last in the sequence of stimuli. In a second experiment, we compared the ability of ArKO and WT mice to respond to decreasing concentrations of either male or female urinary odors. We found a clear-cut sex difference in urinary odor attraction thresholds among WT mice: WT males failed to respond to urine dilutions higher than 1:20 by volume, whereas WT females continued to respond to urine dilutions up to 1:80. Male ArKO mice resembled WT females in their ability to respond to lower concentrations of urinary odors, raising the possibility that the observed sex difference among WT mice in urine attraction thresholds results from the perinatal actions of estrogen in the male nervous system. Female ArKO mice failed to show significant dishabituation responses to two (1:20 and 1:80) dilutions of female urine, perhaps, again, because of a reduced motivation to investigate less salient, same-sex urinary odors. Previously observed deficits in the preference of ArKO male and female mice to approach volatile body odors from conspecifics of either sex cannot be attributed to an inability of ArKO subjects to discriminate these odors according to sex but instead may reflect a deficient motivation to approach same-sex odors, especially when their concentration is low.     

四川短尾鼩包皮腺的挥发性化学成分分析

哺乳动物的包皮腺分泌物对个体间性引诱及繁殖行为的信息交流和种群调节有着重要的作用。本研究以四川短尾鼩（Anourosorex squamipes）为研究对象对劳亚食虫目动物包皮腺挥发性化学成分和化学通讯功能进行报道。采用顶空固相微萃取—气质联用（HS-SPME-GC/MS）的方法,分析四川短尾鼩雄体包皮腺中挥发性物质的化学组成。结果表明：（1）四川短尾鼩雄体包皮腺的挥发性化学成分主要含烷烃类、醇类、酮类、醛类、醚类、酯类、酸类、芳香烃类等45种化合物;（2）成年雄性四川短尾鼩的包皮腺中挥发性成分有39种,幼年雄性含有28种,说明四川短尾鼩成年雄体的包皮腺中挥发性化学成分多于幼年雄体;（3）成年雄体的包皮腺分泌物中含有4种特有的挥发性化学成分,幼年雄体中特有成分仅为1种,表明四川短尾鼩包皮腺中的挥发性化学成分存在年龄差异;（4）四川短尾鼩包皮腺分泌物中不同化学成分相对含量不同,同种化学成分在不同个体间的相对含量存在差异。四川短尾鼩雄性包皮腺挥发性化合物种类丰富,个体、年龄差异明显。推测挥发性化合物中丁酸（Butanoicacid）、乙酸乙酯（Ethyl acetate）、苯酚（Phenol）为四川短尾鼩的信息素。本研究为进一步验证该物种的信息素成分及其传递机制提供了基础数据。     

Melanocortin-5 receptor deficiency promotes defensive behavior in male mice

The endogenous melanocortin, alpha-melanocyte-stimulating hormone (alpha-MSH), is a neurohormone secreted by the neurointermediate lobe of the pituitary. Alpha-MSH promotes intermale aggression in mice by influencing pheromone secretion, but the role of specific melanocortin receptors has not been determined. We assessed mice made deficient in the gene for the melanocortin-5 receptor (MC5R) to determine its role in pheromone-regulated behavior. In heterotypic pairs assessed in the social interaction test (SIT), MC5R-deficient mice exhibited less aggressive behavior and more defensive behavior than their wild-type opponents. By contrast, when assessed in homotypic pairs and against stimulus animals in the SIT, MC5R-deficient and wild-type mice behaved similarly. Moreover, urine from MC5R deficient mice stimulated more aggression than did urine from wild-type mice. The results suggest that MC5R deficiency disinhibits an aggression-suppressing pheromonal signal.     

Social status, activity and preputial glands of wild and domestic house mice

Scent marking by deposition of urine, and the preputial glands, of adult, male, wild house mice, Mus mmmlus L., were studied and compared with those of an outbred domestic strain. The preputial glands of dominant wild mice were always heavier than those of subordinates. No dominance relationships could be established among the domestic mice. For study of scent marking each mouse was observed singly in a residential maze. Dominant wild mice marked more than the subordinates. The domestic mice scent-marked much less even than the subordinate wild mice. Subordinate wild mice spent less time than the dominant wild mice outside the nest; but the number of excursions outside the nest made by the subordinates resembled that of the dominants. Hence social status influenced the pattern of movements in a structured environment.     

Are MUPs a Toxic Waste Disposal System?

Male house mice produce large quantities of major urinary proteins (MUPs), which function to bind and transport volatile pheromones, though they may also function as scavengers that bind and excrete toxic compounds (‘toxic waste hypothesis’). In this study, we demonstrate the presence of an industrial chemical, 2,4-di-tert-butylphenol (DTBP), in the urine of wild-derived house mice (Mus musculus musculus). Addition of guanidine hydrochloride to male and female urine resulted in an increased release of DTBP. This increase was only observed in the high molecular weight fractions (HMWF; > 3 kDa) separated from male or female urine, suggesting that the increased release of DTBP was likely due to the denaturation of MUPs and the subsequent release of MUP-bound DTBP. Furthermore, when DTBP was added to a HMWF isolated from male urine, an increase in 2-sec-butyl-4,5-dihydrothiazole (SBT), the major ligand of MUPs and a male-specific pheromone, was observed, indicating that DTBP was bound to MUPs and displaced SBT. These results suggest that DTBP is a MUP ligand. Moreover, we found evidence for competitive ligand binding between DTBP and SBT, suggesting that males potentially face a tradeoff between eliminating toxic wastes versus transporting pheromones. Our findings support the hypothesis that MUPs bind and eliminate toxic wastes, which may provide the most important fitness benefits of excreting large quantities of these proteins.     

Differential expression of the melanocortin-4 receptor in male and female C57BL/6J mice

The melanocortin 4 receptor is a member of melanocortin receptors of G-protein-coupled receptors. By binding to melanocortin receptor agonists or antagonists, MC4R participates in the regulating of food intake, weight, energy homeostasis and sexual behavior. By activating the protein kinase A and leptin-melanocortin signalling pathways, MC4R mediates the amplification of signals from the hypothalamo–pituitary–adrenal and hypothalamo–pituitary–thyroid axes. This process permits peripheral information about the status of energy metabolism to be transmitted to the central nervous system. The hypothalamic nuclei then integrate these signals to evoke the appropriate reaction. We found that different sexes exhibited distinct metabolic regulation abilities, likely due to differences in these signalling pathways. MC4R plays a key role in coordinating the afferent messages from the peripheral and regulatory signals by controlling food intake and energy expenditure. To probe the disparities in metabolism and weight regulation between the sexes, we analyzed the expression of MC4R in different tissues from male and female mice by qRT-PCR and immunofluorescence. The results show that the expression of MC4R in brain and kidney is higher in female mice than in male mice, but in the livers, the result is opposition. Additionally, in both sexes, the expression of MC4R is higher in the brain than in the kidneys, and its expression in the liver is lowest, in males, the expression of MC4R in the testis is higher than that in the kidneys. These data show that the expression of MC4R exist different between sexes mice.     

Female mice deficient in alpha-fetoprotein show female-typical neural responses to conspecific-derived pheromones

The neural mechanisms controlling sexual behavior are sexually differentiated by the perinatal actions of sex steroid hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estradiol, that exposure to prenatal estradiol completely defeminized the potential to show lordosis behavior in adulthood. Furthermore, AFP-KO females failed to show any male-directed mate preferences following treatment with estradiol and progesterone, indicating a reduced sexual motivation to seek out the male. In the present study, we asked whether neural responses to male- and female-derived odors are also affected in AFP-KO female mice. Therefore, we compared patterns of Fos, the protein product of the immediate early gene, c-fos, commonly used as a marker of neuronal activation, between wild-type (WT) and AFP-KO female mice following exposure to male or estrous female urine. We also tested WT males to confirm the previously observed sex differences in neural responses to male urinary odors. Interestingly, AFP-KO females showed normal, female-like Fos responses, i.e. exposure to urinary odors from male but not estrous female mice induced equivalent levels of Fos protein in the accessory olfactory pathways (e.g. the medial part of the preoptic nucleus, the bed nucleus of the stria terminalis, the amygdala, and the lateral part of the ventromedial hypothalamic nucleus) as well as in the main olfactory pathways (e.g. the piriform cortex and the anterior cortical amygdaloid nucleus), as WT females. By contrast, WT males did not show any significant induction of Fos protein in these brain areas upon exposure to either male or estrous female urinary odors. These results thus suggest that prenatal estradiol is not involved in the sexual differentiation of neural Fos responses to male-derived odors.     

Adpatations for urinary marking in rodents: prepuce length and morphology.

Urine marking was examined in house mice, deermice, gerbils and hamsters. The frequency of urine deposition varied with the species, and a correlation is suggested between the propensity of males to mark in this fashion and prepuce length and morphology. We postulate an adaptational advantage for a long penis sheath and for a particular configuration of the prepuce; i.e. to act as a wick for the deposition of urinary pheromones.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

Chronic exposure of cat odor enhances aggression,urinary attractiveness and sex pheromones of mice

To test whether predator odor exposure negatively affects the behavior of prey, we exposed three groups of male house mice (Mus musculus) to the odors of cat (Felis catus) urine, rabbit (Oryctolagus cuniculus) urine and water (control), respectively, for consecutive 58 days and investigated how the treatments affected the response, aggressiveness, dominance, urinary attractiveness to females and pheromone composition of male mice. Compared to mice exposed to rabbit urine or water, those exposed to cat odor did not show any response habituation to the cat odor and became more aggressive, increased mark urine production and were more attractive to females when the latter were tested with their urine. Furthermore, gas chromatography coupled with mass spectrometry analysis revealed coincident elevations of the well-known male pheromones, E,E-α-farnesene, E-β-farnesene, R,R-dehydro-exo-brevicomin or S-2-sec-butyl-dihydrothiazole. In addition, rabbit urine exposure increased urinary attractiveness to females and pheromonal levels of the males in comparison with the mice exposed to water. This could be related to olfactory enrichment of heterospecific chemosignals, suggesting that predator odors were more beneficial. In light of these anti-intuitional findings in the chemical interaction between cats and mice, we conclude that predator odor affects prey more profoundly than previously believed and that its impact may not always be negative.     

Protein-bound male urinary pheromones: differential responses according to age and gender

The attractive properties of male urinary pheromones were tested on adult or prepubertal male and female mice. An androgen-dependent protein is present in adult male urine (major urinary protein, MUP) which has been suggested to be a pheromone-binding protein. We tested the pheromonal properties of the protein-bound volatiles in a test of attractiveness. These molecules, that co-purify with MUP, attract females and repel adult males. In prepubertal animals, females are repelled and males are attracted by the same stimuli. These results are similar to those obtained by others with adult male whole urine. Therefore MUP binds molecules with a pheromonal activity, and these molecules are sufficient to act as male signals.      

Behavioral and electrophysiological experiments suggest that the antennular outer flagellum is the site of pheromone reception in the male helmet crab Telmessus cheiragonus

Sexually competent females of Telmessus cheiragonus (helmet crab) release two pheromones that elicit grasping and copulation behaviors in males (Kamio et al., 2000, 2002, 2003). Our study aimed to use behavioral and electrophysiological techniques to identify the site of reception of these sex pheromones. In behavioral experiments, either the inner or the outer flagella of the antennules were ablated bilaterally from male crabs, and responses of male crabs to female odor were examined. When the inner flagella were surgically ablated, the sexual response (i.e., grasping and copulation behavior) of male crabs was not significantly changed relative to control animals that had their second antennae ablated. In contrast, the sexual response was significantly reduced when the outer flagella of the antennules were ablated, suggesting that the outer flagellum is the receptor organ that detects the sex pheromones. In electrophysiological experiments, urine, which in females contains the pheromone that elicits grasping behavior by males but does not contain the pheromone eliciting copulation, whose release site is not known, was tested. Female and male urine as well as shrimp extract evoked phasic responses of chemosensory afferents innervating aesthetasc sensilla on the outer flagellum of male crabs. The response of the afferents had significantly higher magnitude and lower threshold when female urine was applied. Thus, behavioral and electrophysiological observations suggest that in male helmet crabs, the outer flagellum of the antennule is the chemosensory organ that detects female sex pheromone.     

Genetic and endocrine studies of the pregnancy-blocking pheromone of mice.

Secretion of the pregnancy-blocking pheromone was stimulated by injection of depo-testosterone cypionate into females and males of inbred strains of mice which do not normally secrete the pheromone. Testosterone treatment of SJL males altered pheromone secretion so that pregnancies were blocked when the stud male was of the same inbred strain; an event that does not normally occur. Injection of epiandrosterone, androstenedione, androsterone or testosterone significantly increased pheromone secretion in SJL females, but progesterone and dehydroepiandrosterone were ineffective. Kidney weights were significantly increased by administration of androgen metabolites and the possibility of the kidney being the site of pheromone synthesis is discussed. The preputial gland can be excluded as the site of pheromone synthesis since males which are hemizygous for the Tabby-J gene and have no preputial glands blocked pregnancies as effectively as their normal littermates. Preliminary results are also presented concerning the isolation of the pregnancy-blocking pheromone from urine. Urine was analysed by gas chromatography and a peak was observed whose concentration could be correlated with secretion of the pheromone, although the compound(s) has not been identified or tested for biological activity.     

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.