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.
Introduction
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.
