In contrast to Kimchi et al. (2007)
, disabling vomeronasal signaling either by surgical VNO removal (Experiments 1 and 3) or by lesioning the AOB (Experiment 2) did not significantly increase mounting or pelvic thrusting behaviors displayed by female mice toward either an estrous female or a castrated, urine swabbed male. In Experiment 1 these male-typical sexual behaviors were completely absent in ovary-intact female mice (as used in Kimchi et al., 2007
), and were only minimally expressed in ovariectomized E2
+P-treated subjects. Considerably more mounting and pelvic thrusting behaviors, more often directed towards the stimulus female than the male, were displayed when ovariectomized VNOi females were treated with TP. In Experiment 2, AOBi and AOBx subjects showed equivalent increases in male-typical sex behavior when ovariectomized and given TP, and a similar effect of TP was seen in both VNOi and VNOx females studied in Experiment 3. Previous studies (Herrada and Dulac, 1997
; Halem et al., 2001
; He et al., 2008
) showed that aspects of VNO sensory neuron function are sexually differentiated in rodents. Taken together, however, our results provide no indication that female-typical VNO signaling inhibits the expression of male-typical sex behavior in female mice.
In Experiments 1 and 2, the numbers of mounts and pelvic thrusts displayed by ovariectomized female subjects with male-typical circulating levels of testosterone were much lower than in testes-intact male mice. Thus while female mice possess the neural circuitry controlling the display of male-typical sexual behavior, a sex difference exists in the response of this circuitry to the activational effect of testosterone in adulthood. This sex difference is presumably the result of organizational effects of perinatal exposure to testosterone in the male (Phoenix et al., 1959
), and as shown in Experiments 1 and 2, has nothing to do with an inhibitory effect of tonic VNO signaling in the female.
Previous studies in rats (Ball, 1940
; Beach, 1942
; Pfaff, 1970
; Södersten, 1972
; Emery and Sachs, 1975
; Fang and Clemens, 1998) and mice (Edwards and Burge, 1971
) showed that adult administration of estradiol or testosterone activated male-typical mounting behavior in ovariectomized females that were tested with an estrous female. Likewise, in Experiment 3 maximal, equivalent levels of male-typical sex behavior were seen in VNOi and VNOx female mice when they were given TP following ovariectomy and tested separately with an estrous female. Neonatal administration of TP further augmented the ability of female mice to display male-typical mating behavior in later life (Manning and McGill, 1974
), a finding that is, again, consistent with the traditional view that male-typical brain and behavioral sexual differentiation results from the perinatal action of testosterone in the male nervous system. Several more recent studies suggest that a degree of male-typical, perinatal sex steroid signaling also contributes to the ability of normal female mice to display appreciable mounting and pelvic thrusting behavior in adulthood. Thus female mice in which estradiol signaling was reduced due to a null mutation of the estrogen receptor-alpha (Wersinger et al., 1997
) or the aromatase (Bakker et al., 2002
) genes showed significantly less male-typical mounting behavior than wild-type females following adult ovariectomy and testosterone or estradiol treatment. Conversely, female alpha-fetoprotein knockout mice, whose brains are exposed fetally to increased estradiol, showed increased mounting behavior toward estrous stimulus females following adult ovariectomy and estradiol treatment (Bakker et al., 2006
). Finally, ovariectomized female mice with a null mutation of the androgen receptor gene showed significantly less mounting behavior than wild-type control females following adult ovariectomy and treatment with estradiol (Sato et al., 2004
). Our results reaffirm the ability of adult testosterone to stimulate appreciable male-typical sexual behavior in female mice, and provide no support for the suggestion (Kimchi et al., 2007
) that VNO signaling inhibits activity of the circuitry controlling this behavior in females.
Habituation/dishabituation urinary odor discrimination tests in Experiments 1-3 yielded two important findings. First, females with or without a functional VNO or AOB were able to distinguish between volatile male and female urinary odors as well as urinary odors from males in different endocrine states. Similar results were previously obtained in VNOi and VNOx male mice (Pankevich et al., 2004
). These results argue against the previous suggestion (Stowers et al., 2002
; Kimchi et al., 2007
) that a functional VNO is necessary for sex discrimination in both male and female mice. Second, previous studies (Pankevich et al., 2004
; Keller et al., 2006
) were disregarded by Kimchi et al. (2007)
because, they argued, blood clots resulting from VNO removal surgery likely occluded the nasal sinuses so as to render subjects anosmic. Clearly, the fact that lesioned and sham-operated subjects in all three of the present experiments successfully discriminated between different volatile urinary odors indicates that they were not anosmic. Additionally, histological analysis of the snouts of VNOx and VNOi animals from Experiments 1 and 3 revealed that even when small blood clots were present (in 4 out of 36 females given VNOx or VNOi surgery), investigation times in the habituation/dishabituation tests were not attenuated compared to subjects that were completely free of sinus blood clots at the completion of the study.
When male and female stimulus animals were introduced individually in separate tests (Experiment 3) we observed more mounting and pelvic thrusting toward the female stimulus animals in both VNOi and VNOx subjects than was seen in Experiments 1 and 2 when social stimuli were presented simultaneously. Interestingly, Fang and Clemens (1999)
reported that ovariectomized E2
+P treated female rats showed robust female-oriented mounting behavior, which was dramatically reduced in the presence of a male rat. Little pelvic thrusting behavior was displayed by female subjects toward castrated male stimulus animals in any of our studies. This likely reflects the resistance to female mounts shown by outbred Swiss Webster male mice, even after castration. As shown in Figure S4
, while VNOi and VNOx subjects mounted both male and female stimulus animals a similar number of times, the total time spent mounting the female (~12.7% and ~12.5% of the total time, respectively) versus the male (~0.3% and ~0.6% respectively) stimulus animal was dramatically different, indicating that subjects typically were not able to sustain male mounts long enough to show pelvic thrusting behavior.
It is unclear why robust male-typical sexual behavior was observed in female mice lacking a functional VNO whereas little mounting or pelvic thrusting behavior was observed in control females studied by Kimchi et al. (2007)
. It is standard practice to monitor sexually dimorphic behaviors in gonadally intact subjects as well as gonadectomized subjects given adult sex hormones (Becker et al., 2005
); however, Kimchi et al. conducted their experiments only in ovary-intact females. It is noteworthy that the TRPC2−/−
females studied by Kimchi et al. had significantly elevated plasma levels of free testosterone compared to heterozygous controls. While these levels were much lower than those typically observed in testes-intact male mice, the possibility remains that TRPC2−/−
females (and perhaps VNOx females as well) were more responsive to testosterone than other groups of females. When we clamped circulating testosterone in our ovariectomized VNOx and VNOi subjects as well as in our ovariectomized AOBx and AOBi subjects, no enhancing effect of disrupted VNO signaling on the expression of male-typical sexual behavior was seen. Thus the presence of circulating testosterone as opposed to the absence of VNO signaling was the main factor controlling the expression of male-typical sex behavior in our mice.
Kimchi et al. (2007)
conducted their experiments using either TRPC2−/−
mice bred on a mixed C57Bl/6J × 129/Sv genetic background or VNOx mice from either this mixed background or a pure C57Bl/6J inbred strain, whereas our experiments were carried out using Swiss Webster outbred mice. Our results show that VNO-mediated inhibition of neural circuits controlling male-typical sex behavior is not a general characteristic of female mice, regardless of strain.
Although we observed no effect of disrupted VNO signaling on male-typical mating behavior, we did observe several other behavioral effects that corroborate previous reports of the effects of disrupting VNO function in mice of both sexes. In Experiment 1, we corroborated the report of Keller et al. (2006)
that VNOx female mice showed significantly reduced lordosis quotients compared to VNOi subjects following treatment with ovarian hormones. A similar reduction in lordosis behavior was also seen in AOBx female mice (Experiment 2), suggesting that VNO signaling plays an essential role in regulating the neural circuits that control female-typical mating behavior. In Experiment 3 both VNOi and VNOx subjects preferred to investigate volatile male versus estrous female urinary odors, presumably after their detection by the main olfactory system. However, when given direct nasal access to the stimuli, only VNOi subjects showed this preference. These observations support our previous conclusion (Pankevich et al., 2004
; Keller et al., 2006
) that VNO signaling motivates mice of both sexes to prolong their contact with opposite-sex non-volatile pheromones so as to facilitate reproductive success.