Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium

Abstract

Terrestrial vertebrates have evolved two anatomically and mechanistically distinct chemosensory structures: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Although it has been generally thought that pheromones are detected through the VNO, whereas other chemicals are sensed by the MOE, recent evidence suggests that some pheromones may be detected through the MOE. Odorant receptors in the MOE are coupled to the type 3 adenylyl cyclase (AC3), an enzyme not expressed in the VNO. Consequently, odorants and pheromones do not elicit electrophysiological responses in the MOE of AC3-/- mice, although VNO function is intact. Here we report that AC3-/- mice cannot detect mouse milk, urine, or mouse pheromones. Inter-male aggressiveness and male sexual behaviors are absent in AC3-/- mice. Furthermore, adenylyl cyclase activity in membranes prepared from the MOE of wild-type mice, but not AC3-/- mice, is stimulated by 2-heptanone, a mouse pheromone. We conclude that signaling through AC3 in the MOE is obligatory for male sexual behavior, male-male aggressiveness, and the detection of some pheromones.

Figure 1.

Pheromone detection defects in the MOE of AC3^−/−mice. A, Pheromone-stimulated EOG responses from adult AC3^+/+and AC3^−/−mice. Male mouse urine was diluted 20-fold and mouse milk was diluted 50-fold in water; farnesene (500 μm) and heptanone (50 μm) were diluted in mineral oil. B, Summary of the mean EOG amplitudes in response to pheromones. AC3^+/+mice (n= 10) exhibited significantly greater EOG responses to all agents compared with AC3^−/−mice (n= 6): urine, p< 0.0001; milk, p< 0.0001; farnesene, p< 0.0001; heptanone, p< 0.0001. C, AC3^−/−mice are unable to detect male urine, female urine, milk, or farnesene. Detection of pheromones was monitored using the odorant-habituation assay described in Materials and Methods. The ratio of the number of times the mouse sniffed a pheromone-soaked cotton swab compared with the number of times it sniffed a water-soaked cotton swab on initial exposure is an indication of the ability of the animal to detect a specific substance. Cotton swabs were laced with 50 μl of farnesene (500 μm), male urine (20-fold diluted), female urine (20-fold diluted), or mouse milk (50-fold diluted). There were significant differences in the ability of AC3^−/−(n= 15) and AC3^+/+(n= 13) mice to detect farnesene (p< 0.001), male urine (p< 0.001), female urine (p< 0.001), and mouse milk (p< 0.001). Error bars represent ±SEM.

Figure 2.

Sexual behavioral defects in AC3^−/−mice. A, AC3^−/−mice display no male–male aggressive behavior. There were significant differences between AC3^−/−(n= 17) and AC3^+/+(n= 16) mice in anogenital area investigation (p< 0.0001), attack number (p< 0.0001), and attack duration (p< 0.0001). B, AC3^−/−male mice do not exhibit sexual behavior toward females. There were significant differences between AC3^+/+(n= 14) and AC3^−/−(n= 15) mice in anogenital area investigation (p< 0.0001), mounting number (p< 0.001), and mounting duration (p< 0.001) assayed during a 15 min observation period. C, Male–male aggressive behaviors were ablated in ZnSO₄-treated male mice. There were significant differences in anogenital area investigation (p< 0.001), attack number (p< 0.05), and attack duration (p< 0.01) between control (n= 5) and ZnSO₄-treated (n= 7) mice. D, Male–female sexual behaviors were ablated in ZnSO₄-treated male mice. There were significant differences in anogenital investigation duration (p< 0.05) and mounting frequency (p< 0.01) between control (n= 6) and ZnSO₄-treated (n= 7) mice. Error bars represent ±SEM.

Figure 3.

ZnSO₄treatment of the MOE destroys olfactory receptor neurons without affecting the function of the VNO. A, Pheromone-stimulated EVG responses from sham and ZnSO₄-treated mice. B, Pheromone-stimulated EOG response in the MOE from sham and ZnSO₄-treated mice. Farnesene (500 μm) and 2-heptanone (50 μm) were diluted in mineral oil. C, Cellular structure was visualized by Hoechst nuclear counterstaining (blue), and sensory neurons were immunostained using an antibody against β-tubulin III (red). Scale bar, 40 μm. The MOE was treated with ZnSO₄as described in Materials and Methods.

Figure 4.

Adenylyl cyclase activity is stimulated by 2-heptanone in membranes from the MOE of AC3^+/+but not AC3^−/−male mice. Stimulation of adenylyl cyclase activity by 2-heptanone in the MOE of AC3^+/+but not AC3^−/−male mice is shown. 2-Heptanone was diluted in DMSO. Data are presented as mean ± SEM of triplicates. The adenylyl cyclase activity is expressed as a percentage relative to controls in AC3^+/+and AC3^−/−male mice.