Molecular biology of pheromone perception in mammals

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct sensory organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the vomeronasal organ. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the vomeronasal organ might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.     

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

Type-specific inositol 1,4,5-trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel,TRPC2

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Origin and Evolution of the Gene Family of Proteinaceous Pheromones,the Exocrine Gland-Secreting Peptides,in Rodents

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.     

Expression of candidate pheromone receptor genes in vomeronasal neurons

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VON). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the VNO. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the VNO might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.      

Combinatorial coexpression of neural and immune multigene families in mouse vomeronasal sensory neurons

The vomeronasal organ (VNO) is a chemosensory organ specialized in the detection of pheromones in higher vertebrates. In mouse and rat, two gene superfamilies, V1r and V2r vomeronasal receptor genes, are expressed in sensory neurons whose cell bodies are located in, respectively, the apical and basal layers of the VNO epithelium. Here, we report that neurons of the basal layer express another multigene family, termed H2-Mv, representing nonclassical class I genes of the major histocompatibility complex. The nine H2-Mv genes are expressed differentially in subsets of neurons. More than one H2-Mv gene can be expressed in an individual neuron. In situ hybridization with probes for H2-Mv and V2r genes reveals complex and nonrandom combinations of coexpression. While neural expression of Mhc class I molecules is increasingly being appreciated, the H2-Mv family is distinguished by variegated expression across seemingly similar neurons and coexpression with a distinct multigene family encoding neural receptors. Our findings suggest that basal vomeronasal sensory neurons may consist of multiple lineages or compartments, defined by particular combinations of V2r and H2-Mv gene expression.     

Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons

The mechanisms that underlie axonal pathfinding of vomeronasal neurons from the vomeronasal organ (VNO) in the periphery to select glomeruli in the accessory olfactory bulb (AOB) are not well understood. Neuropilin-2, a receptor for secreted semaphorins, is expressed in V1R- and V3R-expressing, but not V2R-expressing, postnatal vomeronasal neurons. Analysis of the vomeronasal nerve in neuropilin-2 (npn-2) mutant mice reveals pathfinding defects at multiple choice points. Vomeronasal sensory axons are severely defasciculated and a subset innervates the main olfactory bulb (MOB). While most axons of V1R-expressing neurons reach the AOB and converge into distinct glomeruli in stereotypic locations, they are no longer restricted to their normal anterior AOB target zone. Thus, Npn-2 and candidate pheromone receptors play distinct and complementary roles in promoting the wiring and patterning of sensory neurons in the accessory olfactory system.     

Timing of neuronal intermediate filament proteins expression in the mouse vomeronasal organ during pre- and postnatal development. An immunohistochemical study

Several types of intermediate filament proteins are expressed in developing and mature neurons; they cooperate with other cytoskeletal components to sustain neuronal function from early neurogenesis onward. In this work the timing of expression of nestin, peripherin, internexin, and the neuronal intermediate filament triplet [polypeptide subunits of low (NF-L), medium (NF-M), and high (NF-H) molecular weight] was investigated in the developing fetal and postnatal mouse vomeronasal organ (VNO) by means of immunohistochemistry. The results show that the sequence of expression of intermediate filament proteins is internexin, nestin, and NF-M in the developing vomeronasal sensory epithelium; internexin, peripherin, and NF-M in the developing vomeronasal nerve; and nestin, internexin and peripherin, NF-L, and NF-M in the nerve supply to accessory structures of the VNO. At sexual maturity (2 months) NF-M is only expressed in vomeronasal neurons and NF-M, NF-L and peripherin are expressed in extrinsic nerves supplying VNO structures. The differential distribution of intermediate filament proteins in the vomeronasal sensory epithelium and nerve is discussed in terms of the cell types present therein. It is concluded that several intermediate filament proteins are sequentially expressed during intrauterine development of the VNO neural structures in a different pattern according to the different components of the VNO.     

达乌尔黄鼠犁鼻器和副嗅球的组织结构及嗅球c-Fos表达的季节变化

啮齿动物的犁鼻器和副嗅球与社会通讯和生殖行为有关,主嗅球影响其觅食行为。达乌尔黄鼠（Spermophilus dauricus）是一种具有较低社会行为的储脂类冬眠动物。本研究用组织学和免疫组织化学方法探究了其犁鼻器和副嗅球的结构特点及嗅球神经元活动对季节变化的适应。结果发现,达乌尔黄鼠犁鼻器具有较大的血管,犁鼻器管腔外侧为非感觉性的呼吸上皮（Respiratory epithelium,RE）,内侧为感觉上皮（Sensory epithelium,SE）,RE较SE薄,靠近管腔处为假复层柱状上皮。选取犁鼻器中间部位比较,发现SE的厚度、长度及感觉细胞密度均无性别差异。副嗅球位于主嗅球后方背内侧,由6层细胞构成。侧嗅束穿过副嗅球,位于颗粒细胞层之上。雄性达乌尔黄鼠较雌性有更长的僧帽细胞层和颗粒细胞层。春季（3月）和冬季（1月）达乌尔黄鼠主嗅球的嗅小球层、僧帽细胞层和颗粒细胞层的c-Fos-ir神经元密度显著低于夏季（7月）和秋季（10月）,且冬季外网织层的c-Fos-ir神经元密度显著低于夏季和秋季,说明达乌尔黄鼠在冬季和春季的嗅觉神经活动较弱,呈现出对冬眠的生理性适应。这些结果丰富了动物犁鼻器和副嗅球的形态学资料,并有助于理解冬眠动物嗅觉系统对季节变化和冬眠的适应。     

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca²⁺. Two family members, ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose. We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.     

Processing by the main olfactory system of chemosignals that facilitate mammalian reproduction

This article is part of a Special Issue “Chemosignals and Reproduction”.Most mammalian species possess two parallel circuits that process olfactory information. One of these circuits, the accessory system, originates with sensory neurons in the vomeronasal organ (VNO). This system has long been known to detect non-volatile pheromonal odorants from conspecifics that influence numerous aspects of social communication, including sexual attraction and mating as well as the release of luteinizing hormone from the pituitary gland. A second circuit, the main olfactory system, originates with sensory neurons in the main olfactory epithelium (MOE). This system detects a wide range of non-pheromonal odors relevant to survival (e.g., food and predator odors). Over the past decade evidence has accrued showing that the main olfactory system also detects a range of volatile odorants that function as pheromones to facilitate mate recognition and activate the hypothalamic-pituitary-gonadal neuroendocrine axis. We review early studies as well as the new literature supporting the view that the main olfactory system processes a variety of different pheromonal cues that facilitate mammalian reproduction.     

The vomeronasal organ and adjacent glands express components of signaling cascades found in sensory neurons in the main olfactory system

The vomeronasal organ (VNO) is a sensory organ that influences social and/or reproductive behavior and, in many cases, the survival of an organism. The VNO is believed to mediate responses to pheromones; however, many mechanisms of signal transduction in the VNO remain elusive. Here, we examined the expression of proteins involved in signal transduction that are found in the main olfactory system in the VNO. The localization of many signaling molecules in the VNO is quite different from those in the main olfactory system, suggesting differences in signal transduction mechanisms between these two chemosensory organs. Various signaling molecules are expressed in distinct areas of VNO sensory epithelium. Interestingly, we found the expressions of groups of these signaling molecules in glandular tissues adjacent to VNO, supporting the physiological significance of these glandular tissues. Our finding of high expression of signaling proteins in glandular tissues suggests that neurohumoral factors influence glandular tissues to modulate signaling cascades that in turn alter the responses of the VNO to hormonal status.     

Projection pattern of vomeronasal neurons to the accessory olfactory bulb in goats

Goats have a well-developed vomeronasal (VN) system and exhibit pheromone-induced reproductive facilitation, but there are no reports on the projection pattern of VN neurons in this species. Rodent, guinea pig and opossum accessory olfactory bulbs (AOBs) have been shown to have a segregated pattern of projection of the VN neurons, which express the two alpha-subtypes of the G-protein, namely Gi2 and Go, to the rostral and caudal regions of the AOB, respectively. In this study we investigated the projection pattern of VN nerve terminals by immunocytochemical staining of the goat vomeronasal organ (VNO) and the AOB with antibodies to Gi2 and Go. Gi2-immunoreactivity was found on the luminal surface of the sensory epithelium of the VNO, and in the VN nerve and glomerular layer throughout the AOB. On the other hand, Go-immunoreactivity was not identified in either the VNO or the VN nerve layer of the AOB. These results indicate that the projection pattern of VN neurons from the VNO to the AOB in the goat is considerably different from that in rodents which show a distinct segregated pattern.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.     

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.     

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.