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mise à jour du
8 août 2010
Behav Brain Res
Behavioral characterization of non-copulating male rats with high spontaneous yawning frequency rate


Portillo W, Camacho F, Eguibar JR, Paredes RG.
Instituto de Neurobiología, Universidad Nacional Autónoma de México
Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren


An important number of Sprague-Dawley males selected by strict inbreeding process for their high spontaneous yawing frequency (HY) fail to copulate after repeated exposure to receptive females. These HY males that fail to mate are called non-copulators (HYNC). The causes of this behavioral deficit are still unknown.
The aim of the present study was to make a detailed behavioral characterization of these animals by evaluating: their partner preference between a sexually receptive female as opposed to a sexually active male; their ability to detect food related odors and their preference for sexually relevant chemosensory cues between bedding from estrous females, bedding from sexually active males and clean bedding. We also evaluated whether these males had alterations in motor function using a rotarod or in their general reward system mediated by opioids by injecting them with 1mg/kg of morphine to evaluate if they develop conditioned place preference (CPP). At the end of these behavioral tests, we measured their plasmatic levels of testosterone (T).
Together, these results will contribute to elucidate the causes of their deficient copulatory performance. Both HYNC and HY copulators (HYC) males showed a clear preference for receptive females as opposed to sexually active males. As well, both groups of animals had a similar ability to detect food related odors. HYC males had a clear preference for estrous female odors as opposed to male or clean bedding, but HYNC males spend the same amount of time sniffing estrous, anestrous, male and clean bedding. In both, HYC and HYNC, morphine induced CPP suggesting that in these males the reward system is functional. No differences were found in motor coordination or in T levels between HY and HYNC males.
The behavioral deficit in HYNC male rats cannot be explained by an alteration in: partner preference, food related odor recognition, motor coordination, general reward system, or differences in plasmatic levels of T. However, HYNC males present clear deficits in recognizing sexually relevant odors. These results could, at least in part, explain the deficient execution of copulatory pattern in HYNC males.
Yawning is a stereotypical behavioral pattern that is commonly associated with other behaviors such as grooming, penile erection, stretching, and arousal [47]. Several research groups have demonstrated an association between yawning and sexual excitement [29]. For example, systemic administration of dopamine agonists elicits yawning and penile erections in rats, mice, monkeys, and humans (review in 118]). In high-yawning (HY) rat subline, a strong correlation between spontaneous yawning and penile erection has been demonstrated [22]. Penile erections increase from 0.4 erections/h in the non-yawning rats to about 2erections/h in the HY males that exhibit 26-3Oyawns/h. The dopamine (DA) agonists; apomorphine and bromocriptine also increase yawning as well as penile erection in low-yawning (LY) and HY sublines [22]. The DA induced yawning and penile erection is blocked by D2 or D3 antagonists suggesting that this effect is mediated by D2/D3 receptors' family [10-12]. Opiate receptors appear also to be involved in the control of yawning, morphine injected systemically or in the paraventricular nucleus (PVN) of the hypothalamus blocks apomorphine, oxytocin [27] and N methyl-D-aspartic acid (NMDA) [28] induced penile erection and yawning.
Yawning is sexually dimorphic, adult males yawn more frequently than females [13,30], suggesting that hormones might play a role in the incidence of this behavior. Castration disrupts sexual behavior, penile erections, and yawning [13,20,35]. The administration of testosterone restores yawning and penile erection frequency to pre surgery levels [34,35]. Cessation of testosterone treatment or administration of testosterone simultaneously with the non-steroidal antiandrogen hydroxyfiutamide decreases yawning frequency [13]. In female monkeys, treatment with dihydrotestosterone (a nonaromatizable androgen) induces high levels of yawning from a baseline level of 0.3-4.7 yawns per 30 min after treatment. Female monkeys treated simultaneously with dihydrotestosterone and with the androgen receptor antagonist flutamide show a reduced yawn frequency of just 1.9 yawns per 30 min [19]. Taken together, these observations suggest that androgens are important modulators of yawning and penile erections.
Sprague-Dawley rats that differed in their spontaneous yawning frequency [46] were selectively bred. The High-Yawning (HY) subline has a mean of 20 spontaneous yawns per hour, and the Low-Yawning (LY) one has only an average of 2 yawns/h [17,46]. We have observed that a high proportion of HY males fail to mate after repeated exposure to sexually active females, and we refer to them as HY non-copulators (HYNC). The cause of this behavioral deficit is unknown. Studies in our research group previously demonstrated that Wistar non-copulating male rats (NC) show a reduced sexual motivation for a receptive female when given the opportunity to physically interact with them or when they can only see, smell, and hear her [37,38]. We also demonstrated that NC rats showed a reduced preference for chemosensory relevant olfactory cues from estrous females [37,38].
The aim of the present study was to make a detailed behavioral characterization of HYNC and evaluate if they do not mate due to alterations in: their partner or olfactory preference for sexually receptive females; in their motor coordination or in their plasmatic levels of testosterone. Several lines of evidence indicate that opioids are release during sexual behavior mediating the rewarding properties of mating in males and females rodents as evaluated by conditioned place preference (CPP). For example, systemic administration of the opioid antagonist naloxone blocks the reward state induced by ejaculations in males and females [32]. Moreover, it is well established that the opiate agonist morphine (1 mg/kg, ip) reliably induces CPP. Therefore, we evaluated if this treatment can induce CPP in HYNC to determine if the general reward system mediated by opioids is functional in these males.
5. Discussion
In rodents, the expression of sexual behavior depends on a careful selection of an appropriate conspecific with whom to interact. Sexually active males spend more time near estrous females than near males [2,14,37,38]. This preference is observed even if the females are anesthetized of if they are placed behind a screen, and the male can only smell them [8,43]. Data from the present study show that both HYC and HYNC males have a clear preference for a sexually receptive female as opposed to a sexually active male. Sexually active males and females emit many kinds of stimuli to attract the opposite sex. In rodents, olfactory clues are involved in the appropriate selection of sexual partner [4,8]. When tested in a T-maze, adult male rats prefer to investigate the arm containing odors from estrous versus anestrous females. Likewise, when given free access to soiled bedding, male rats prefer to investigate estrous female bedding versus that from sexually active males [4,14]. In the present study, we demonstrate that HYC males show a clear preference for the female odors as opposed to male or clean bedding. However, HYNC males spend the same amount of time sniffing estrous, anestrous, male or clean bedding. Thus, HYNC did not show a preference for the odors from a receptive female. The inability of the HYNC males to prefer the estrous female odors cannot be explained for their lack of heterosexual experience. Others and we demonstrated [2,38] that without sexual experience male rats show a clear preference for estrous female odors over odors from sexually active males.
The sexually relevant clues are detected by the main and accessory olfactory system. In mice, lesions of the main olfactory epithelium completely abolished sexual behavior in both sexual experience and sexually naive males [24]. In rats and hamster's with lesions in the main and accessory olfactory bulbs decrease their sexual performance [15,26,48]. Thus, the appropriate odor detection is a crucial requirement for sexual behavior performance in rodents. In our previous studies in Wistar male rats we demonstrated that NC in the same way, that HYNC males did not show a preference for the estrous odors. Wistar NC males had deficits in the neuronal processing of the sexual relevant clues. For example, sexually active males exposed to estrous bedding show a significant increase in the number of Fos-IR cells in the brain structures that integrate the vomeronasal projection system. Such as; the accessory olfactory bulbs, the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area as opposed to clean bedding. Wistar NC males failed to show a Fos response when exposed to estrous beeding indicating a possible deficit in the processing of chemosensory relevant olfactory cues by the vomeronasal projection system [38]. Further studies need to evaluate the functional integrity of this olfactory system in HYNC males.
In mammals sexual behavior is intrinsically rewarding, independent of any other consequence that the behavior might have [1]. The conditioned place preference (CPP) procedure can measure approach responses to an environment where a reinforcing (positive or negative) event has occurred and can be used to reveal incentive motivational responses to reward-related stimuli [32,45]. Different research groups including ours have shown that sexual behavior induces a change of preference in male and female rats [3,7,32]. The reward state induced by mating in both male and female rats is mediated by opioids (review in [32]). Previous studies done in our research group have demonstrated that one ejaculation has the same reward value that systemic morphine (1 mg/kg) in sexually active males [7]. Data from the present study show that, HYC and HYNC male rats treated with morphine develop a clear CPP indicating that the lack of sexual behavior is not due to alterations in their general reward system. It would be interesting to evaluate if sexually relevant odors can induce CPP in male rats in the same way as in mice [36].
As described in Section I yawning is an androgen dependent behavior in various mammalian species including rats [13,19,20,30,34,35]. Its frequency decreases when males are gonadectomized and systemic administration of testosterone (T) or dihydrotestosterone treatment restores spontaneous yawning [13,19,20,30,34,35]. The results of the present experiments show that plasma T levels are not significantly different between HYC and HYNC males and thus, differences in T are not the cause of the behavioral deficit in HYNC. However, it should be noted that T potentiates DA [6,21], and ACTH-induced yawning in rats and guinea pigs [42].
Estradiol derived from T aromatization modulates male sexual behavior. Estrogen treatment of gonadectomized male rats restores mounts and intromissions although not ejaculation [5]. Progesterone is another hormone involved in sexual behavior and some studies proposed that progesterone act synergistically with androgen to modulate male sexual response. Administration of progesterone agonist RU486 in male rats during the postnatal period significantly reduces sexual behavior when the males reach adulthood [25]. Studies in progesterone receptor knockout mice (PRKO) showed that males have a reduced mount frequency in comparison to the wild-type [33]. Future studies are needed to evaluate if HYNC males have alterations in their plasmatic levels of estrogen and progesterone.
The reason for the high incidence of non-copulating males in the HY strain is unknown. Yawning is strongly modulated by stress (30,31], foot shock and swimming increased the number of yawns in rats [44], and in primates yawing increases in response to social stress [9]. High levels of stress can lead to alterations in sexual performance and yawning [9,40,41,44]. Chronic stress induced by cold water immersion and electric foot shocks increased the number of mounts, as well as, intromission and ejaculation latencies. It also induced a significant decrease in the number of ejaculations with respect to the non-stressed males [40]. Interestingly, preliminary observations from our lab indicate that basal corticosterone levels are similar in HY and LY males. We analyze the effects on yawning after i.c.v. injection of ACTH124 administration (doses 1, 3, 6,9 and 12mg/kg). Yawning did not increase in HY with all doses tested, but significantly increased in LY ones [16]. Future studies are needed to evaluate corticosterone and adrenocorticotropic hormone levels in HYNC males.
Rhees et al. [41] demonstrated that 24% of the males born to non-stressed females did not mate, and the percentage increased to 60% in males born to stress mothers. Future studies are needed to evaluate if the high incidence of non-copulators in the HY colony is because these males are more susceptible to high levels of stress during maternal care. In fact, preliminary data from our laboratory demonstrates that HY mothers develop a deficient maternal behavior. For example, they spent less time inside the nest, retrieved the pups in an atypical way (from pup's mouth, tail, belly or legs) and the retrieving is faster than the one observed in Sprague-Dawley dams. Also, offspring's weight of HY mothers is lower during lactation and weaning in comparison to those of Sprague-Dawley mothers. These data together indicate that HY mothers have a disorganized maternal care (Ugarte et al., unpublished data).
In conclusion, HY rats are a subline with several behavioral and neuroendocrine characteristics that allow us to analyze in detail the environmental and genetic background that might contribute to alterations in sexual behavior. The results of the present experiment indicate that HYNC males showed no preference for sexually relevant cues from female rats.