Attractive properties of sexual pheromones in mice: innate or learned?

It is generally assumed that chemical signals (sexual pheromones) constitute the primary stimulus for sexual attraction in many mammals. However, it is unclear whether these pheromones are volatile or nonvolatile and which sensory systems are involved in their detection (vomeronasal and/or olfactory). Moreover, it has been demonstrated that experience influences the behavioral response to sexual pheromones and the sensory systems implicated. In order to clarify this issue, the attractive properties of volatile and nonvolatile components of the male-soiled bedding have been analyzed in female mice that had no previous experience with adult male-derived chemical signals (chemically na?ve females) using two-choice preference tests. The results indicate that some nonvolatile male-derived substances exert an innate attraction to females, but volatiles derived from male-soiled bedding do not attract chemically nai;ve females. Therefore, the primary attractive sexual pheromone includes a nonvolatile compound (e.g. major urinary proteins, MUPs). On the other hand, male-derived volatiles become attractive to females because of repeated exposure to male-soiled bedding. This represents a Pavlovian-like associative learning in which previously neutral volatiles (very likely odorants) acquire attractive properties by association with the nonvolatile, innately attractive pheromone(s). These findings indicate that not only the sexual but also the 'chemical' experience (previous experience with sexual pheromones) has to be taken into account to interpret the role of chemicals as releaser or primer pheromones. The sensory systems involved in the detection of these stimuli and the neural basis of the odor-pheromone association are discussed.     

Attraction to male pheromones and sexual behaviour show different regulatory mechanisms in female mice

In rodents, female sexual behaviour is under hormonal control. The attraction females show for male-derived nonvolatile chemicals (pheromones) can be regarded as the first step of this behaviour, but it is unknown whether this attraction is also modulated by sexual steroids. To test this possibility, ovariectomized adult female mice with no experience of chemical signals from adult males were randomly assigned to four groups that received oil (control), progesterone, estradiol (E) or estradiol+progesterone (E+P) injections, respectively. Females were then tested for their attraction to male-soiled bedding and, subsequently, for their proceptive behaviour when confronted to adult males. Females showed attraction to male-soiled bedding irrespective of the hormonal treatment, whereas only those females treated with E or E+P showed proceptive behaviour. Therefore, in contrast to proceptive and copulatory behaviour, the female attraction to male pheromones is independent of sexual steroids, thus indicating that those parts of the vomeronasal system involved in this attraction do not respond to steroids. In summary, sexual behaviour in female mice can be seen as a two-step process. First, females are attracted by male pheromones, a process which is independent of their hormonal status. After encountering the males, females show proceptive behaviour only in estrous, when fertilization is more likely. The attraction exerted by male sexual pheromones promotes female autostimulation that might ensure anticipatory endocrine changes leading to ovulation by the time of sexual intercourse.     

Effect of bilateral accessory olfactory bulb lesions on volatile urinary odor discrimination and investigation as well as mating behavior in male mice

Previous research raises the possibility that urinary volatiles from estrous female mice activate mitral cells in the accessory olfactory bulb (AOB) of male mice following detection via the main olfactory epithelium as opposed to the vomeronasal organ. We asked whether bilateral lesions of the AOB would disrupt the ability of male mice to discriminate between urinary volatiles from mice of different sexes or endocrine states, or affect their interest in investigating these odors when they were presented sequentially in home-cage habituation/dishabituation tests. Males with either partial or complete bilateral lesions of the AOB resembled sham-operated control males in their ability to discriminate between ovariectomized and estrous female urinary volatiles as well as between male and estrous female urinary volatiles. However, males with either complete or partial AOB lesions spent significantly less time than sham-operated control males investigating urinary volatiles from estrous females, especially during tests when the alternative stimulus presented was male urine. Placement of AOB lesions failed to disrupt males' mating performance. Our results suggest that the incentive value of opposite-sex (female) volatile urinary odors which are initially detected by the main olfactory system is enhanced when they are further processed by the male's AOB.     

The vomeronasal organ: Primary role in mouse chemosensory gender recognition

Four experiments were conducted to determine the chemosensory modality that supports ultrasonic courtship vocalizations by male mice to females and to chemosignals from females. Both removal of the olfactory bulbs (Experiment 1) and removal of the vomeronasal organ (Experiments 2–4) produced similar deficits in the pattern of ultrasonic vocalizations elicited by conspecifics or their odors. Removal of the vomeronasal organ did not impair the ability to locate food buried under cage shavings. These results are consistent with the notion that the analysis of food related odors is subserved by olfaction and that vocalizations to sex chemosignals are elicited primarily by stimulation of the vomeronasal organ/accessory olfactory bulb. Removal of the vomeronasal organ did not induce seminal vesicle regression or lower plasma immunoreactive testosterone levels (Experiment 2) nor was an attempt to restore vocalizations with exogenous testosterone successful (Experiment 4). Thus the altered vocalization pattern following removal of the vomeronasal organ does not appear to arise as a motivational deficit mediated by androgens. Experiments 2 and 3 demonstrated that, in the absence of the vomeronasal organ, stimulation of other sensory systems can, to some extent, maintain the male's tendency to vocalize more to females or their odors than to males or their odors. However, this responsiveness to females may rely upon additional behavioral cues. Previous experience also plays a considerable role in the response to chemosensory gender cues by males who lack their vomeronasal organs. Removal of the vomeronasal organ prior to adult heterosexual encounters (Experiment 3) virtually eliminated the male's responsiveness to either anesthetized females or their chemosignals. Hence males require adult heterosexual experience with a functioning vomeronasal organ before other chemosensory systems acquire the ability to mediate gender recognition as measured by ultrasonic vocalizations.     

Sex and gonadal steroid modulation of pheromone receptor gene expression in the mouse vomeronasal organ

Non-volatile chemosignals in rodents are detected by unique receptors in the vomeronasal organ of the accessory olfactory system. Although the vomeronasal organ has been implicated in the regulation of sexually dimorphic behavioral and neuroendocrine functions, the underlying cellular mechanisms are undetermined. In previous studies we showed that exposure to soiled male bedding augmented immediate early gene immunoreactivity in neurons of the basal zone of the vomeronasal organ, an effect that depended on gender and sex steroid expression. To determine whether this effect could be due to differences in vomeronasal organ receptor expression, we examined two representatives (VR1 and VR4) from different subfamilies of the V2R family of receptors that are expressed in the basal zone of the vomeronasal organ. Adult Swiss-Webster male and female mice were gonadectomized and implanted with capsules containing 17β-estradiol, testosterone or neither steroid (control). Two weeks later vomeronasal organs were processed for in situ hybridization using probes from the N-terminal extracellular domains of VR1 and VR4. Expression of both VR1 and VR4 was significantly higher in males than in females. Estradiol, but not testosterone-treated, males had significantly lower levels of VR1 expression in the caudal vomeronasal organ compared with untreated gonadectomized males. In contrast, testosterone enhanced VR4 expression in males relative to similarly treated females. Despite these effects, we found no evidence that vomeronasal organ neurons express either androgen or estrogen receptors. These data show that expression of vomeronasal organ receptors in mice is sexually dimorphic and regulated by sex steroids. Thus, gonadal hormones may affect the response of vomeronasal organ neurons to chemosignals by altering levels of the receptors to which they bind.     

Olfactory cues and accessory olfactory bulb lesion: effects on sexual behavior in the cyclic female rat

The influence of olfactory cues and accessory olfactory bulb (AOB) lesion on female sexual behavior was studied in virgin female Wistar rats. In Experiment 1, it appeared that distance or contact exposure to male urine soiled bedding for 8 hours before testing increased sexual receptivity, i.e., the number of receptive females at 18:00-19:00 on proestrus. In Experiment 2, we observed that sexual receptivity at 18:00-19:00 on proestrus was not affected by AOB lesion as compared to sham-operated females. In Experiment 3 the effects of both AOB lesion and olfactory cues were analyzed. Sexual receptivity at 18:00-19:00 on proestrus did not significantly differ in sham-operated and accessory olfactory bulbectomized females both exposed to the odor of male urine. Regarding lordosis quotient in the three experiments, no significant difference was observed. Mechanisms whereby olfactory cues and/or AOB lesion modified female sexual behavior on proestrus in virgin female rats were discussed in the light of previous and present observations.     

Modification of odor investigation and discrimination in female opossums (Monodelphis domestica) following the ablation of the accessory olfactory bulbs

To determine whether the vomeronasal system of the Brazilian short-tailed opossum (Monodelphis domestica) is important to the response to conspecific chemical signals, the authors tested female opossums with conspecific odors, before and after ablation of their accessory olfactory bulbs (AOBs). Anesthesia and sham treatments did not modify females' discrimination of conspecific odors when tested against water, between male and female odors, or between different odors from the same male donors. Odor investigation was partially diminished following partial ablation of the AOB, and complete ablation of the AOBs further impaired the ability of females to discriminate between certain odors. These findings provide the first evidence for the importance of the vomeronasal system in the detection of chemosignals of known origin in opossums.     

Sex difference in Fos induced by male urine in medial amygdala-projecting accessory olfactory bulb mitral cells of mice

We previously reported that exposure to soiled male bedding induced Fos protein immunoreactivity (Fos-IR) in significantly more neurons of the vomeronasal organ (VNO) and medial amygdala of gonadectomized, estradiol-treated female than male mice whereas no such sex difference was seen in the intervening mitral cells of the accessory olfactory bulb (AOB). We asked whether a sexually dimorphic functional response to male urinary pheromones might be revealed in AOB mitral cells that project specifically to the medial amygdala. Gonadectomized mice of both sexes were treated with estradiol and 3 days later received bilateral injections of the retrograde tracer, Cholera toxin-B (CTB) into the medial amygdala. Five days later male urine or saline was applied nasally to each subject 90 min prior to sacrifice, and sections of the AOB were processed for double-label fluorescent immunocytochemistry for Fos protein and CTB. In both the rostral and caudal AOB, there were significantly more double-labeled mitral cells in female than in male subjects following exposure to male urine. A sex difference in the responsiveness of VNO sensory neurons seen previously to male soiled bedding is reflected in a parallel sex difference in the responsiveness of AOB mitral cells when only AOB cells that project to the amygdala are examined and when male urine as opposed to soiled male bedding is used as the activating stimulus.     

Volatile female odors activate the accessory olfactory system of male mice without physical contact

We previously reported that male mice are more attracted to volatile odors from intact female mice than from ovariectomized female mice. In the present study, we investigated male attraction to volatile odors from soiled bedding collected from the cages of estrous or ovariectomized female mice. There was no difference in the total time spent sniffing volatile odors from estrous and ovariectomized female mice, suggesting that female mice emit volatile odors which are not excreted into bedding. To test this possibility, we investigated c-Fos expression in the mitral cell layer and granule cell layer of the accessory olfactory bulb 60 min after exposure of male mice to volatile odors without physical contact. Volatile odors from an estrous female mouse significantly increased the total number of c-Fos positive cells in each of the rostral and caudal granule cell layer, but not in the mitral cell layer. After exposure to volatile odors from estrous bedding, the total number of c-Fos positive cells did not increase. Volatile odors from a male mouse did not increase the total number of c-Fos positive cells. Volatile odors from an ovariectomized female mouse increased c-Fos expression only in the caudal granule cell layer. These results suggest that female mice emit specific volatile odors which are not excreted into bedding, and that the volatile odors activate the accessory olfactory system of male mice without physical contact. To characterize the female-specific volatile odors, we conducted habituation-dishabituation tests. Whereas sham-operated male mice discriminated between volatile odors of estrous and ovariectomized female mice, vomeronasal organ-removed male mice did not. These results suggest that male mice discriminated whether or not female mice were ovariectomized, by volatile odors via the accessory olfactory system, and that the female-specific volatile odors are involved in reproduction.     

Activation of an anatomically distinct subpopulation of accessory olfactory bulb neurons by chemosensory stimulation.

Chemosensory cues known as pheromones play a key role in rodent reproductive physiology and social interactions. Pheromone molecules are detected by receptor cells located in the vomeronasal organ and conveyed exclusively to the accessory olfactory bulb, and then to limbic and hypothalamic sites for integration with other factors modulating reproductive physiology. We report here that chemosensory cues from the female mouse selectively activate a subpopulation of cells located in the anterior part of the accessory olfactory bulb of the male mouse. Exposure of male mice to female-soiled bedding resulted in a massive induction of c-fos expression, which was primarily confined to neurons located in the anterior part of the accessory olfactory bulb and was eliminated by removal of the vomeronasal organ. Exposure of the male to soiled bedding from a different stain of male mice also elevated c-fos expression, but immunoreactive cells were more evenly distributed along the anterior-posterior axis of the accessory olfactory bulb. No treatment effects were observed in the main olfactory bulb. Previous studies have indicated that vomeronasal receptor neurons are divided into two populations based on location within the organ, site of termination in the accessory olfactory bulb, second messenger content and putative pheromone receptor expression. The present study suggests that the two populations of vomeronasal receptor neurons detect different chemosensory stimuli. Since male mouse- and female mouse-specific urinary substances modulate different aspects of male mouse behavior, the present results suggest that anatomically segregated populations of vomeronasal organ receptor cells modulate distinct behavioral patterns.     

Acceleration and delay of puberty in female housemice: Methods of delivery of the urinary stimulus

A series of 7 experiments tested various methods for delivery of male and female urinary chemosignals which affect age of 1st estrus in young female mice. An initial experiment demonstrated that in and of themselves none of the delivery techniques used alters puberty without the accompanying urine stimulus. A series of 4 experiments with male urine revealed that acceleration of puberty occurred only when the females could achieve direct contact with the urine. Possible reception via the vomeronasal organ is suggested as an explanation for these data. In contrast, the urinary chemosignal from grouped females which delays 1st estrus produces its effect regardless of the mode of delivery. Reception of this chemosignal apparently may occur at the olfactory epithelium.     

Mouse vomeronasal organ: effects on chemosignal production and maternal behavior

Adult male mice excrete a urinary chemosignal that accelerates puberty in females, whereas group-housed female mice excrete a urinary chemosignal that delays puberty in young females. We found that: (1) the excretion of the puberty-acceleration chemosignal by males persisted in the absence of the vomeronasal organs and (2) the puberty-delay chemosignal was not present in the urine of group-housed females whose vomeronasal organs had been surgically removed (VNX), but was present in the urine of group-housed females subjected to sham surgery (SHAM). These results suggest that in males, vomeronasal chemoreception does not affect the excretion of the puberty-acceleration chemosignal, but that in females, the vomeronasal organ receives chemosignals that influence the excretion of the puberty-delay chemosignal. Additionally, we found no difference between SHAM and VNX females in rates of conception, litter size, pup growth, pup recognition, or maternal behavior, indicating that normal maternal processes are expressed in the absence of an intact accessory olfactory system.     

Male hamster copulatory responses to a high molecular weight fraction of vaginal discharge: effects of vomeronasal organ removal

The importance of the vomeronasal (accessory olfactory) system for the copulatory responses of male hamsters to a high molecular weight fraction (HMF) of vaginal discharge was assessed in animals that had their vomeronasal organs (VNO) removed. These organs were extirpated bilaterally using an oral approach through the palate so as to eliminate the peripheral afferents to the accessory olfactory bulb (AOB) with minimal or no damage to the main olfactory system. The selective peripheral deafferentation procedure was verified by applying horseradish peroxidase intranasally following intraperitoneal injections of epinephrine to facilitate the vomeronasal pumping mechanism that draws fluids into the VNO. Heavy, bilateral anterograde labeling was evident in the olfactory nerve afferents within the main olfactory bulb of males that had their VNO removed and of animals that received sham surgery. Sham-operated males also had heavy, bilateral labeling in the vomeronasal nerve afferents within the AOB, whereas no such labeling occurred among animals with bilateral removal of the VNO. In sham-operated animals, both the HMF and the unfractionated discharge significantly increased the incidence of intromission attempts toward anesthetized males (surrogate females) whose hindquarters were scented with these stimuli. The unfractionated discharge also produced a significant elevation of overt copulatory behavior in males with selective peripheral deafferentation of the vomeronasal system, whereas the HMF did not facilitate copulatory behavior in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of agonistic behavior by the dual olfactory system in male mice

This study analyzed the effects of deafferentation of the main, vomeronasal or both olfactory systems on the agonistic behavior of male mice in response to unfamiliar male intruder mice. In every animal, regardless of the technique used, disruption of the vomeronasal system led to consistent reductions in agonistic behavior. All animals receiving bulbectomies, vomeronasal tract transections or a combined treatment of vomeronasal cuts and ZnSO₄ nasal flush reliably suppressed agonistic responding toward the intruder. Animals treated with intranasal ZnSO₄ alone or those receiving control procedures continued to respond to the intruders at rates similar to pretreatment. These results indicate that the vomeronasal system is directly involved in the perception of the male chemosignals modulating agonistic behavior and, therefore, is capable of modulating the male's response to these signals.     

Differential plasticity of microglial cells in the rostrocaudal neuraxis of the accessory olfactory bulb of female mice following mating and stud male exposure

The formation of an olfactory recognition memory by female mice for the stud male pheromones requires two fundamental conditions: incidence of mating and retention of the stud male with the female for a critical 6h interval following mating. This biologically critical recognition memory results from plasticity of reciprocal dendrodendritic synapses in the accessory olfactory bulb (AOB). In this study, a microglia marker antibody (ionized calcium-binding adaptor protein, Iba1) was used to determine how mating and stud pheromones affect microglia in the AOB rostrocaudal axis in female mice. The results showed that compared with estrus and mating only, mating and pheromone exposure significantly increased Iba1 immunoreactivity in the AOB evidenced by increased complexity of ramified microglial processes characteristic of resting microglial morphological phenotype, particularly in the rostral AOB. The density of Iba1 staining after mating and stud pheromone exposure was higher in the rostral - compared to caudal - AOB and was most prevalent in the external plexiform layer, the site of reciprocal mitral-granule dendrodendritic synapses. While cells with activated phenotype were observed in caudal AOB during estrus, mating/pheromone exposure appeared to induce a morphological transformation to the resting microglia phenotype. Since previous evidence implicate the rostral AOB in processing pheromonal signals and microglial cells monitor active synapses, these observations have important functional implications for a potential role for microglia in processing pheromonal signals in the AOB during the formation of olfactory memory.     

Effects of dopaminergic drugs on innate pheromone-mediated reward in female mice: a new case of dopamine-independent "liking."

Male sexual pheromones are innately rewarding to adult female mice, but the role of dopamine in this natural reward is unknown. The authors have tackled this issue by assessing the effects of intraperitoneal injections of dopamine D1 (SCH 23390, 0.02- 0.05 mg/kg) and D2 (sulpiride, 20.00 mg/kg) antagonists, a dopamine releasing agent (amphetamine, 0.50 -2.00 mg/kg), and D1 (SKF 38393, 10.00 -20.00 mg/kg) and D2 (quinpirole, 0.20 -1.00 mg/kg) agonists on the chemoinvestigation displayed by female mice in male- versus female-soiled bedding 2-choice tests. Dopamine antagonists and quinpirole failed to affect the unconditioned preference displayed by females towards male chemosignals, whereas both amphetamine and SKF 38393 abolished it. Finally, D1 and D2 antagonists did not block the induction of operant place conditioning by male chemosignals. As the female mice were tested in their first encounter with male sexual pheromones, their behavior can only be influenced by the "liking" component of reward. Therefore, the results suggest that dopamine mediates neither the hedonic properties of male sexual pheromones nor the acquisition of conditioned place preference. However, dopamine acting on D1 receptors might inhibit female mice attraction towards male chemosignals.     

Olfactory and vomeronasal mediation of ultrasonic vocalizations in male mice

The effects of deafferentation of the vomeronasal, olfactory or both systems of male mice on ultrasonic vocalizations in response to a male or female stimulus were analyzed. Regardless of the technique used, disruption of the vomeronasal system led to consistent reductions in ultrasonic vocalizations. Each animal receiving vomeronasal tract cuts, bulbectomies or a combined treatment of vomeronasal tract transection and intranasal flush with ZnSO₄ markedly suppressed responding to the stimulus animals. Animals treated with intranasal ZnSO₄ alone or those receiving control procedures continued to respond to the female stimulus animals at rates similar to pretreatment. These results indicate that the vomeronasal system is directly involved in the perception of female mouse chemosignals and, hence, mediates the male's responses to these signals.     

Olfactory and vomeronasal system participation in male hamsters' attraction to female vaginal secretions.

The effects of olfactory (OLF) vomeronasal (VN), or combined deafferentations of male hamsters on their attraction to female hamster vaginal secretions (FHVS) were determined using 2 different attraction tests. In the first, FHVS was placed on one wall of a plastic test chamber, while in the second test, FHVS was rubbed onto 1 of 2 anesthetized castrate hamsters. OLF deafferentation abolished the males' attraction to FHVS in the first test but had no effect in the second. The persistence of FHVS attraction in the anesthetized castrate test depended on the VN system in that its subsequent deafferentation greatly attenuated the attraction. When the VN system was deafferented alone, FHVS attraction was significantly reduced only in males exhibiting severe mating behavior deficits. These results are interpreted to support the hypothesis that the OLF and VN systems may be preferentially responsive to volatile and non-volatile odorants, respectively. Our findings emphasize that both the OLF and VN systems participate in subserving males' attraction to FHVS and their mating with receptive females. Thus each chemosensory system may influence both sexual arousal and copulatory mechanisms.     

Role of nitric oxide in pheromone-mediated intraspecific communication in mice

Nitric oxide is known to take part in the control of sexual and agonistic behaviours. This is usually attributed to its role in neural transmission in the hypothalamus and other structures of the limbic system. However, socio-sexual behaviours in rodents are mainly directed by chemical signals detected by the vomeronasal system, and nitric oxide is abundant in key structures along the vomeronasal pathway. Thus, here we check whether pharmacological treatments interfering with nitrergic transmission could affect socio-sexual behaviour by impairing the processing of chemical signals. Treatment with an inhibitor of nitric oxide synthesis (Nω-Nitro-l-arginine methyl ester hydrochloride, L-NAME, 100 mg/kg) blocks the innate preference displayed by female mice for sexual pheromones contained in male-soiled bedding, with a lower dose of the drug (50 mg/kg) having no effect. Animals treated with the high dose of L-NAME show no reduction of olfactory discrimination of male urine in a habituation-dishabituation test, thus suggesting that the effect of the drug on the preference for male pheromones is not due to an inability to detect male urine. Alternatively, it may result from an alteration in processing the reinforcing value of pheromones as sexual signals. These results add a new piece of evidence to our understanding of the neurochemistry of intraspecific chemical communication in rodents, and suggest that the role of nitric oxide in socio-sexual behaviours should be re-evaluated taking into account the involvement of this neuromodulator in the processing of chemical signals.     

Vomeronasal organ removal and female mouse aggression: the role of experience

Removal of the vomeronasal organ (VNX) from female mice had little effect upon fecundity, nest building and pup retrieval. Surprisingly, VNX significantly affected aggression by lactating females. Normally, lactating females will attack and fight intruders placed into the home cage, especially if the intruder is an unfamiliar male. In Experiment 1, we determined that VNX prior to sexual experience totally eliminated this type of aggression. Intact females were highly aggressive, but much more so to unfamiliar than to familiar males. Copulation prior to surgery did not ameliorate the effects of VNX; again, none of the VNX females were aggressive (Experiment 2). In Experiment 3, attacks and fights by previously aggressive primiparous females also were eliminated or significantly suppressed following VNX. Even when the testing occurred later during the postpartum period and for extended periods of time, this same suppression of aggression held following VNX. We conclude that aggression by lactating female mice is under strict mediation by chemosignals detected by the vomeronasal organ.