Olfactory sex discrimination persists, whereas the preference for urinary odorants from estrous females disappears in male mice after vomeronasal organ removal

Abstract

Based on observed changes in the social context for the display of ultrasonic vocalizations, scent marking, aggression, and mounting behavior by male mice with a null mutation of the transient receptor potential 2 ion channel, it was proposed recently that a primary function of the mouse vomeronasal organ (VNO)/accessory olfactory system is sex discrimination. We tested this hypothesis directly by studying the ability of male mice to discriminate between urinary odors of conspecifics of the two sexes and in different endocrine states using habituation-dishabituation tests. Male mice from which the VNO had been surgically removed (VNOx) resembled sham-operated controls (VNOi) in their ability to discriminate between volatile urinary odors from estrous females versus gonadally intact males, as well as between urinary odors from estrous versus ovariectomized females and from gonadally intact versus castrated males. When physical access to stimuli was permitted, VNOi control males strongly preferred to investigate volatile and nonvolatile urinary odorants from estrous females as opposed to intact males, whereas VNOx males showed no such preference. Mating performance in tests with estrous females was equivalent in VNOi and VNOx subjects. Both groups of males preferred to mount an estrous female instead of a castrated male. Our results suggest that the VNO is not required for sex discrimination but instead detects the nonvolatile components of opposite-sex urine that may be used to help prolong contact with individuals that produce these chemosignals.

Figure 1.

Representative photomicrographs show sagittal sections through the AOB of male mice that had received either a sham removal of the vomeronasal organ or a bilateral surgical removal of the vomeronasal organ. A, B, Brain sections were stained with SBA-HRP. C, D, Brain sections immunolabeled for Fos protein are shown from VNOi and VNOx males exposed for 90 min to soiled bedding from estrous females before being killed. E, Mean ± SEM counts of Fos-IR cells in AOB layers are compared from VNOi and VNOx males exposed to soiled bedding, as well as from VNOi males exposed to clean bedding. Induction of Fos in mitral and granule cell layers was significantly higher in VNOi males exposed to soiled bedding compared with the other groups. For each cell layer, means with different letters (a, b) are significantly different (Fisher's least significant difference tests, p < 0.01). The absence of SBA-HRP staining in the glomerular layer (B) and the background levels of Fos-IR cells in the AOB mitral and granule cell layers (D) were taken as evidence of successful VNO removal. Gl, Glomerular cell layer; Mi, mitral cell layer; Gr, granule cell layer. Scale bar, 100 μm.

Figure 2.

Effect of bilateral removal of the vomeronasal organ or sham operation on the ability of male mice to discriminate volatile urinary odors from gonadally intact male versus estrous female mice when urine was presented outside the home cage (A) or to discriminate volatile and nonvolatile components of these two kinds of urine when presented inside the home cage (B). In both situations, subjects received a habituation-dishabituation test during which investigation times were recorded in nine total trials that consisted of three presentations of water, followed by three presentations of one of the urinary odors, followed by three presentations of the second urinary odor. The test was repeated for each subject on a separate day but with the order of the odors reversed. Data from the two tests were combined for each subject, and the mean ± SEM seconds that VNOi and VNOx males spent investigating stimuli is shown for each trial, with Urine One representing the first odor presented and Urine Two representing the second odor presented. ^*,#p < 0.05, two-tailed Wilcoxon test comparisons with the third presentation of the previous stimulus for the respective groups. The time that subjects spent investigating the same two of these urinary odors when presented simultaneously outside (C, left) or inside (C, right) the home cage is also shown. ^†p < 0.05, two-tailed paired Student's t test. The number of subjects in each group that was tested under each condition is shown in parentheses.

Figure 3.

Effect of bilateral removal of the vomeronasal organ or sham operation in male mice on the percentage of mounts directed toward an estrous female versus a castrated male stimulus with intact male urine swabbed on the back when presented simultaneously for 20 min in the subjects' home cages (top). The dotted horizontal lines show the 50% preference for each type of stimulus. The total number of mounts per test displayed by VNOi and VNOx subjects toward both stimulus mice is shown at the bottom. Results for each test are based only on male subjects that displayed more than two mounts toward either stimulus animal (the number of subjects is given across the x-axis).

Figure 4.

Effect of bilateral removal of the vomeronasal organ or sham operation on the ability of male mice to discriminate volatile and nonvolatile urinary odors (presented inside the home cage) from estrous versus ovariectomized (Ovex.) females (A) or from gonadally intact versus castrated males (B). Subjects were given habituation-dishabituation tests as described in Figure 2, and the data are similarly presented as the mean ± SEM seconds that VNOi and VNOx males spent investigating stimuli on each trial. ^*,#p < 0.05, two-tailed Wilcoxon test comparisons with the third presentation of the previous stimulus for the respective groups. The time that subjects spent investigating gonadally intact male versus estrous female urine when presented simultaneously inside the home cage is also shown (C). ^†p < 0.05, two-tailed paired Student's t test. The number of subjects in each group that was tested under each condition is shown in parentheses.