Co-expression of putative pheromone receptors in the sensory neurons of the vomeronasal organ

Abstract

Two large and divergent families of G-protein-coupled receptors (V1Rs and V2Rs) are expressed in subsets of neurons in the vomeronasal organ. These receptors are likely to mediate pheromone responses, but it appears that many V2R genes may encode expressed pseudogenes rather than functional proteins. Therefore we have raised antibodies to representative V2Rs and show labeling of vomeronasal neurons demonstrating that V2R genes encode expressed receptors. V2R immunoreactivity was detected at the sensory surface of the vomeronasal organ in dendritic terminals, indicating that these receptors are capable of directly interacting with pheromones and mediating physiological responses. Immunohistochemistry confirmed that three V2R receptors are expressed in small subsets of sensory neurons. However, surprisingly we found that a subfamily of V2R genes is broadly expressed in the Goalpha-layer of the vomeronasal organ and are coexpressed in the same cells as other V2Rs. This is in direct contrast to the main olfactory epithelium where sensory neurons express only a single receptor. Thus, our results suggest that different modes of the information processing may occur in the main and accessory olfactory systems.

Fig. 1.

Cellular distribution of the V2Rs. Representative rat V2Rs: Go-VN2 (a), Go-VN3 (b), and Go-VN4 (c) were studied by immunohistochemistry in coronal sections of rat VNO; strong staining of a small subset of VNO neurons was observed; dotted lines indicate the basal and luminal edge of the sensory epithelium. The number of antibody staining cells was quantitated and compared with in situ hybridization of corresponding V2R probes by counting positive cells in double-labeled sections. Similar numbers of cells were detected with antibodies and riboprobes for Go-VN2 and Go-VN3, but antibodies to Go-VN4 detected 4172 cells in 40 sections (4 rats), whereas only 1560 cells were positive by in situ hybridization. High magnification of the luminal region of the VNO stained with the anti-VN4 antibody (d) highlights labeling of sensory dendrites and knobs;arrows indicate immunopositive knobs and dendrites on the surface of the VNO epithelium. Scale bars: a–c, 100 μm; d, 25 μm.

Fig. 2.

V2R2 is expressed in the cell bodies and sensory dendrites of a large subset of VNO neurons. Anti-mouse V2R2 stained all neurons in the basal half of the VNO epithelium in mouse (a) and rat (b);c, higher magnification of the luminal region ofb showing staining of dendrites and knobs. Scale bars:a, 100 μm; b, 30 μm;c, 4 μm. d, Western blot of rat membrane protein extracts stained for anti-V2R2 immunoreactivity shows that this antibody recognizes an ∼100 kDa protein that is expressed in the VNO, but not MOE, olfactory bulb (OB), spleen (SP), or testis (TE); the position of the 94 kDa marker is indicated.

Fig. 3.

Specificity of antibodies to V2Rs.a, Western analysis of the four V2R-family antigens using the four antibodies; each antibody only recognizes the polypeptide against which it was raised. b, c, To demonstrate specificity of immunohistochemistry, the anti-V2R2 antibody was incubated with the polypeptide against which it was raised (b) or a mix of three polypeptides encoding the same region of Go-VN2, Go-VN3, and Go-VN4 (c). Preincubation with the V2R2 antigen abolished immunostaining (b), although preincubation with the other polypeptides had no effect on the pattern of immunohistochemistry (c). Scale bar, 100 μm.

Fig. 4.

Several V2R2 subfamily members are co-expressed in the same VNO neurons. In situ hybridizations with digoxigenin-labeled RNA probes to rat V2R2 subfamily members label most neurons in the basal half of the sensory epithelium of rat VNO. Similar patterns of labeling were observed for a coding sequence probe to rat V2R2a (a) and 3′ untranslated regions of rat V2R2a (b), rat V2R2 (c), and rat V2R2b (d). The development time of staining was 12 hr (a) and 60 hr (b–d).

Fig. 5.

The V2R2 subfamily and its homology.a, Southern blot analysis of rat genomic DNA cut withEcoRI, BglII, PstI,BamHI, SacI, PvuII, andXbaI and screened at high stringency using a single extracellular exon probe to mouse V2R2 indicates the existence of a family of closely related genes. b, Southern blot analysis of rat genomic DNA cut with EcoRI orPstI and screened at high stringency with PCR products corresponding to the 3′-noncoding region of rat receptors V2R2, V2R2a, and V2R2b indicates that these probes recognize different genes of the V2R2 subfamily. c, The extracellular regions of the full-length rat and mouse V2Rs (V2R, Go-VN, and VR) and related fish-receptors (Ca, GFB, and R) were aligned with ClustalW, and the results were analyzed with Prot-dist and are shown as a neighbor-joining tree. The length of the joining roots indicates the divergence between receptors. Pairwise comparisons between the extracellular domains of V2R2 and other V2Rs yielded values of ∼25% identity; for other V2Rs identity ranged from ∼30% identity e.g., for V2R1 and Go-VN2 to ∼50% identity e.g., for Go-VN2 and Go-VN4.

Fig. 6.

Co-expression of V2Rs in neurons of the VNO epithelium. Double-label immunohistochemistry and in situ hybridization directly demonstrates co-expression of V2Rs in rat VNO neurons. a–c, Images show double labeling with anti-V2R2 (green) and anti-Go-VN2 (a), anti-Go-VN3 (b), and anti-Go-VN4 (c) (red). Scale bar, 100 μm. Higher magnification image (Scale bar, 50 μm) of double label in situ hybridization (d) of V2R2 (red) and rat Go-VN3 (green) also shows co-expression.