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.     

Hormonal control of odor preferences and urine-marking in male and female rats

Gonadectomized male and female rats show no preferences for the odors of conspecifics of the opposite sex and no urine-marking. Castrated males given testosterone propionate (TP) injections showed preferences for female odors over no odor as did males given estradiol benzoate (EB). Males given EB plus progesterone (P), P only, or oil (controls) showed no preferences for female odors. No group of ovariectomized females (TP, EB, EB+P, or oil injected) showed a preference for male odors over no odor. Males given TP, EB, or EB+P injections showed an increase in urine-marking while males given P or oil showed no marking. Females given TP injections showed an increase in marking but those given EB, EB+P or oil showed no marking. These results are discussed in relation to studies on the hormonal control of scent-marking in gerbils and sexual behaviour in rats.     

Role of the vomeronasal system in intersexual attraction in female mice

Although it is generally accepted that rodents' sociosexual behavior relies mainly on chemosignals, the specific roles played by the vomeronasal and olfactory systems in detecting these signals are presently unclear. This work reports the results of three experiments aimed at clarifying the role of the vomeronasal system on gender recognition and intersexual attraction, by analyzing the effects of lesions of the accessory olfactory bulbs (AOB) in chemically naïve female mice. The first experiment demonstrates that lesions of the AOB abolish the preference that females show for male-soiled bedding in tests in which the females can contact the bedding, thus having access to both volatile and involatile male chemosignals. The second experiment shows that airborne male-derived chemosignals are not attractive to intact, chemically naïve females but tend to be preferentially explored by females whose AOB has been lesioned. However, repeated exposure to male-soiled bedding has opposite effects in sham-operated and AOB-lesioned female mice. Whereas after this experience sham-operated females show an (acquired) attraction toward male airborne chemosignals, in AOB-lesioned females the same experience makes male-derived volatiles aversive. Finally, in the third experiment we have confirmed that our AOB-lesioned females are able to detect urine-borne male odorants, as well as to discriminate them from the synthetic terpene geraniol. These findings strongly suggest that in mice, the involatile male sexual pheromone that is intrinsically attractive is detected by the vomeronasal system of the females. In addition, the repeated experience of females with male-soiled bedding would probably allow the association of this pheromone, acting as unconditioned stimulus, with olfactory stimuli (odorants) that therefore would become conditioned attractors to the females.     

Characterization of 50-kHz ultrasonic vocalizations in male and female rats

Male and female rats emit ultrasonic vocalizations in reproductive encounters. While estrous bedding has been used to elicit vocalizations of males, the number of responses is variable. We report a reliable method to assess vocalizations using exposure to a stimulus animal. The stimulus rat is placed behind a wire barrier for 5 min, then removed. Vocalizations are then recorded for 5 min. Experiment 1 validated this method and it was used for subsequent experiments. In Experiment 2, male rats were castrated and tested for the restoration of vocalizations. In one group, males were allowed to copulate freely; in the other, females had vaginal masks to prevent ejaculation, but not mounting. Vocalizations were restored only in males allowed to ejaculate. In Experiment 3, we measured vocalizations in sexually nai;ve and sexually experienced males following exposure to either castrated (CAS) males, testosterone (T)-treated males, ovariectomized (OVX) females, or OVX females receiving estrogen plus progesterone (E+P). Males vocalized most after exposure to E+P females, whether they were sexually experienced or naive. However, the rate of vocalizations was significantly higher after exposure to E+P females when the males were sexually experienced. In Experiment 4, we measured vocalizations in females following exposure to CAS males, T-treated males, OVX females, or E+P females. Females vocalized most after exposure to T-treated males. Our results show that (1) sexual experience facilitates vocalizations in male rats, (2) vocalizations are highest after exposure to hormonally receptive conspecifics, and (3) ultrasonic signaling is a sensitive index for assessing the hormonal disposition of conspecifics.     

Sexual and olfactory preference in noncopulating male rats

In some species including rats, mice, gerbils, and rams, apparently normal males fail to copulate when repeatedly tested with receptive females. These animals are called "noncopulators (NC)," and the cause of this behavioral deficit is unknown. It has been shown that NC rats do not have hormonal alterations or deficits in the mechanisms that control penile function. The present study was designed to examine (Experiment 1) whether NC male rats prefer receptive females to sexually active males. In addition, the olfactory preference for bedding soiled from estrous or for anestrous bedding was investigated. These tests were performed in NC and copulating (C) male rats when the subjects were intact, gonadectomized (GDX), or GDX and treated with high doses of testosterone propionate (TP). Our results demonstrate that NC rats do not display sexual behavior even after high TP treatment. While C male rats have a clear preference for receptive females as opposed to a sexually active male, NC rats do not. In all hormonal conditions, the preference shown by NC rats for estrous bedding was significantly reduced in comparison to that seen in C rats. TP treatment in NC rats did not modify either partner or odor preference. In Experiment 2, we evaluated if NC rats are feminized and if it could be easier to induce feminine-like behavior by hormone treatment with estradiol benzoate (EB) or with EB plus progesterone (P) (EB+P). Odor preference for estrous or male bedding under these hormonal conditions was also compared. No differences between NC and C rats were found in feminine sexual behavior. In the olfactory test, we found that NC rats prefer odors from receptive females as opposed to male odors, but this preference is reduced compared to that of C rats. Males treated with EB or EB+P show no preference for female odors. These results demonstrate that treatment with EB or EB+P does not increase feminine sexual behavior in NC rats.     

Sexual behaviors of aging female rats: influence of age and hormonal state of male partners

Proceptive and receptive behaviors were examined in aging and young ovariectomized female rats treated with estradiol benzoate and progesterone. Young females approached males confined in wire mesh cages more frequently and exhibited more proceptive behaviors toward these males than did middle-aged females. Both middle-aged and young females approached young males more often than they approached old males, but young females showed greater preferences for young males and for males with high levels of testosterone than did middle-aged females. Young females also exhibited more proceptive behaviors than did middle-aged and old females during tests of sexual behavior. The frequencies of lordosis responses, however, did not differ among these three groups of females.     

Olfactory, partner preference and Fos expression in the vomeronasal projection pathway of sexually sluggish male rats

Sexually sluggish (S) male rats take a long time to ejaculate or sometimes they don't achieve ejaculation when tested on repeated occasions with receptive females. In order to further understand what factors might contribute to the inconsistent display of sexual behavior in these animals and determine if S and non-copulating males have a different neurobiological profile, the present study was design to characterize sex-related behaviors of S male rats. We tested their preference to physically interact between a sexually receptive female and a sexually active male. We also tested whether S males have a preference to investigate soiled bedding from females in estrous, anestrous or clean bedding. We also evaluated if the serum hormonal levels of testosterone (T) and estradiol (E) are different between copulating (C) and S male rats. Finally, we compared the neuronal activity of the vomeronasal projection pathway of C and S male rats exposed to estrous bedding. The results indicate that S male rats increased the percentage of animals displaying mounts and intromissions after repeated testing with receptive females but no increased was observed in the percentage of animals displaying ejaculations (a total of nine tests were performed). Both C and S males had a clear preference to interact with a sexually receptive female. Both groups showed a clear preference for bedding from estrous females as opposed to anestrous or clean bedding. However, the time investigating the estrous bedding was significantly lower for the S in comparison to C rats. No differences in the serum levels of T and E were found. The structures of the vomeronasal projection pathway were equally activated by estrous bedding in C and S male rats. The results indicate that S male rats do not have hormonal alterations or deficient processing of sexually relevant olfactory cues. Although S males showed a reduced preference for estrous bedding, no changes in preference for a receptive female were observed.     

Nutritional and social cues influence the onset of puberty in California voles

Weanling male California voles were housed individually in cages containing either bedding taken from their own mothers, from unrelated mothers, or clean woodshavings. Half of the males in each housing condition received fresh lettuce daily in addition to their standard diets. At 45 days of age males housed in clean bedding had heavier seminal vesicles than males housed in bedding from their own or an unrelated mother. However, males which received lettuce supplementation displayed large-sized seminal vesicles regardless of their housing condition. In a second study 45-47 day old females were housed either with an adult male, alone in the bedding of an adult male, or in clean bedding for 4 days. Again, one half of the females in each housing condition received lettuce daily in addition to their standard diet. Ovarian and uterine weights were heaviest in females that had direct contact with an adult male. Lettuce supplementation increased uterine weights in females housed alone in either clean or male-soiled bedding. Lettuce did not accelerate uterine growth in females co-habiting with males, presumably male contact causes maximal development. These data show that the ingestion of green vegetation may facilitate reproductive development in male and female voles, despite inhibitory social conditions.     

Effects of prenatal morphine exposure on rat heterotypical sexual behavior.

Prenatal exposure to morphine inhibits ovarian steroid-dependent lordosis behavior in female rats, and enhances certain components of male sexual behavior in male rats. In the present study, the effects of mid to late gestational morphine exposure on male sexual behavior in females and on female sexual behavior in males were examined in adult offspring. Gonadectomized male rats were injected at weekly intervals with 30 or 60 microg estradiol benzoate and 1.0 mg progesterone and tested for female sexual behavior with stimulus males on 2 consecutive weekly tests. Ovariohysterectomized (OVX) females were injected with 500 microg testosterone propionate (TP) daily for 15 days and tested for male sexual behavior with stimulus females on the last day of TP injection and 1 week later, after TP withdrawal. Prenatal morphine exposure increased the expression of male sexual behaviors in female rats, but it did not increase lordosis behavior in male rats. Thus, exposure to morphine during gestation alters male and female sexual behavior in young adult animals. Because prenatal morphine exposure both defeminized and masculinized adult sexual behavior in female rats, it is possible that female brain development is more vulnerable to prenatal insult such as opiate exposure.     

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.     

Effects of oestradiol 17B, progesterone and testosterone upon proceptivity and receptivity in ovariectomized common marmosets (Callithrix jacchus)

The sexual behaviour of eight pairs of marmosets was observed during blocks of 30 minute tests after the ovariectomized female partners were treated with physiological doses of testosterone, oestradiol and progesterone. The effects of an intra-vaginal progesterone treatment were also investigated. Pre-ovulatory levels of oestradiol significantly increased the females' proceptive and receptive tongue-flicking displays and reduced the percentage of mounts refused. Mid-luteal levels of progesterone, by comparison, virtually abolished proceptive and receptive tongue-flicking and significantly increased mount refusals. Testosterone treatment was without effect. Vaginal application of progesterone unlike subcutaneous implantation of the hormone did not significantly alter the females' sexual behaviour. These results indicate that central actions of oestradiol and progesterone upon proceptivity and receptivity may occur in this primate, although further research using intra-cranial implants of these steroids is required to test this hypothesis.     

Ventromedial hypothalamic damage and sexual proceptivity in female rats

Sexual proceptivity is indicated by behaviors which reflect feminine initiation with respect to the occurrence of mating behavior. In this study we used ovariectomized female rats, and tested for sexual behavior in a relatively large arena with a sexually active male tethered in one corner. This testing situation gave the unrestrained female control over the occurrence of sexual interaction, and the frequency with which a female approaches a sexually active male provides a measure of sexual proceptivity. We find that administration of estrogen and progesterone to neurologically intact female rats induces sexual receptivity and increases the frequency with which they approach a sexually active male. Bilateral destruction of the ventromedial hypothalamus renders females non-receptive and virtually eliminates the tendency for females to approach males. Bilateral sagittal knife cuts which bracket the ventromedial region produce similar effects. Apparently, the ventromedial hypothalamus is importantly involved in the control of both receptive and proceptive components of feminine sexual behavior.     

An active part of female mice in sexual interaction]

Proceptive behavior, according to Beach (1976), maintains and accelerates sexual interactions toward the end goal. Such distinctive type of proceptive behavior as darting or hopping of female rats is not apparent in mice. Nevertheless, it seems more reasonable that females may take an active part as much as their male partners also in this species. Twenty sexually experienced females in estrus were paired with 20 naive males of the same strain (ICR/JCL) and the pairs were observed for 6 hours. Eleven males of the 20 pairs successfully ejaculated. Females exhibited lordosis more frequently when they actively approached the male partners than when they were approached and mounted by males. This indicated that the rate of mating success was higher in the case of female's approach. Female's approaching behavior thus possibly plays a role as a proceptive behavior in mice.     

Female sexual behaviour: roles of gonadal hormones in the Syrian hamster

The sexual behaviour of ovariectomized female hamsters was examined after treatment with oestrogen alone and after the addition of progesterone. Both the proceptive and receptive components of behaviour were recorded at four oestrogen dose levels; progesterone dose was kept constant. Oestrogen alone caused dose-dependent increases in proceptive behaviour and, at the higher dose levels, a small increase in receptivity. The addition of progesterone increased proceptive behaviour in females primed with the lower dose of oestrogen but not the higher. Receptivity was markedly increased in all animals but the intensity of lordosis was oestrogen-dose dependent.     

Sexual maturation of female house mice: social inhibition

Young female laboratory mice reared in the presence of an adult male mouse or male bedding containing a pheromone reach sexual maturity earlier than control females. A pheromone produced by grouped female mice leads to long, irregular estrous cycles or anestrus. The present study demonstrated that a female-produced pheromone delays sexual maturation when females are grouped and that free social interaction and tactile contact among the grouped females are necessary for the production of this inhibitory pheromone. Both young and adult females caged in groups produced the inhibitory pheromone. This study provided additional support for the hypothesis that in female mice morphological and sexual development are under separate control mechanisms. Two pheromones may be active in reproductive processes of female mice: one exhibiting inhibitory effects and the other acceleratory effects.     

Female-biased sex difference in vasotocin-immunoreactive neural structures in the developing quail brain

The bed nucleus of the stria terminalis pars medialis (BSTM), medial preoptic nucleus (POM), and lateral septal region (LS) exhibit more vasotocin-immunoreactive (VT-ir) neural structures in male than in female adult quail. VT-ir cells and fibers in these regions are sensitive to gonadal steroids only in males. The insensitivity of adult female VT-ir neural structures to sex steroids is attributed to estradiol exposure during a critical period in embryonic life.Although the VT-ir system has been intensively examined in adult quail, information is limited in embryos and juveniles. Therefore, we herein investigated the development of VT-immunoreactive neural structures from embryonic day (E) 9 to adulthood with a particular focus on the BSTM, POM and LS of both sexes.VT-ir neural structures were more evident in female than in male embryos from E9 (BSTM and POM) and E11 (LS). This sex difference disappeared between E15 and post-hatch day 1 in the BSTM and POM, and during the first week of life in the LS. Male-biased sex differences in VT-ir structures appeared at puberty. Female-biased sexual dimorphism in the density of the VT-ir structures of BSTM was reflected by the stronger expression of VT mRNA in females than in males. However, the density of VT mRNA somata was comparable in the two sexes.The exposure of male embryos to estradiol resulted in the feminization of VT-ir neural structures in the BSTM, but not in the POM or LS at E11.Collectively, these results suggest that sex differences in VT-ir neural structures changes drastically throughout quail life. In embryos, endogenous estradiol may stimulate the expression of VT in females, resulting in a robust sex difference in VT-ir cells and fibers in favor of this sex.     

Sex versus sweet: opposite effects of opioid drugs on the reward of sucrose and sexual pheromones

Endogenous opioids mediate some reward processes involving both natural (food, sweet taste) and artificial (morphine, heroin) rewards. In contrast, sexual behavior (which is also reinforcing) is generally inhibited by opioids. To establish the role of endogenous opioids for a newly described natural reinforcer, namely male sexual pheromones for female mice, we checked the effects of systemic injections of the general opioid antagonist naloxone (1-10 mg/kg) and the agonist fentanyl (0.1- 0.5 mg/kg) in a number of behavioral tests. Naloxone affected neither the innate preference for male-soiled bedding (vs. female-soiled bedding) in 2-choice tests nor the induction of place conditioning using male pheromones as rewarding stimuli, although it effectively blocked the preference for consuming a sucrose solution. In contrast, fentanyl inhibited the preference for male chemosignals without altering sucrose preference. These results suggest that, in macrosmatic animals such as rodents, opioidergic inhibition of sexual behavior might be due, at least partially, to an impaired processing of pheromonal cues and that the hedonic value of sweet-tasting solutions and sexual pheromones are under different opioid modulation.     

The effects of beta-endorphin infusions into the amygdala on visual and olfactory sensory processing during sexual behaviour in the male rat.

Sexually experienced male rats infused bilaterally into the amygdala with 60 pmol beta-endorphin show decreased rate of precopulatory investigation of the female and delayed intromission latency, but copulation is left unaltered. Such males are still able to discriminate between the odours of bedding from receptive and unreceptive females, demonstrating that beta-endorphin does not impair the ability to detect sexually relevant odours. Preventing visual cues emitted by females during proceptive behaviour (by treating them with haloperidol) delayed intromission latency but had no effect on preintromission investigation. Intra-amygdaloid beta-endorphin exacerbated the effects of this treatment on the intromission latency. Inducing anosmia in males (by applying zinc sulphate solution to the olfactory mucosa) decreased their anogenital investigation and delayed their intromission latency. These effects were not enhanced by intra-amygdaloid beta-endorphin. Allowing males to investigate and initiate the first intromission prior to intra-amygdaloid infusion had no effects on subsequent intromissions. However, if following an intromission with one female and an infusion of beta-endorphin, the male was presented with an unfamiliar female then the effects of intra-amygdaloid beta-endorphin on investigation and intromission returned. These results suggest that beta-endorphin in the amygdala interferes with the processing of female-specific olfactory information. Without this processed information, classification of the female as a sexual stimulus may be impeded and thus sexual arousal delayed.     

Female mouse maturation: effects of excreted and bladder urine from juvenile and adult males

A sequence of 4 experiments examined the effects of prepubertal and adult males on the sexual maturation of young female house mice. The results support 3 conclusions: (1) the presence of a prepubertal male or of urine from prepubertal males does not affect the timing of sexual maturation in young female house mice; (2) the maturation-accelerating pheromone produced by adult males is present in the bladder urine of intact adult males but is absent from both excreted and bladder urine of castrated males; and (3) young females caged with 7 prepubertal males or with a castrated adult male mature earlier than control females caged alone. Results indicating that the presence of a castrated male leads to earlier sexual maturation of young female mice differ from previous findings. A possible explanation for this contradictory result is based on the ability of young weanling female mice to acclimatize the thermoregulate when separated from the dam and litter-mates. A model for density-feedback population regulation in house mice involving pheromones produced by males and females is presented.