Lectin histochemical studies on the olfactory epithelium and vomeronasal organ in the Japanese striped snake,Elaphe quadrivirgata

The olfactory epithelium and the vomeronasal organ of the Japanese striped snake were examined by lectin histochemistry. Of the 21 lectins used in the study, all lectins except succinylated‐wheat germ agglutinin (s‐WGA) showed similar binding patterns in the vomeronasal receptor cells and the olfactory receptor cells with varying intensities. The binding patterns of s‐WGA varied among individuals in the vomeronasal and olfactory receptor cells, respectively. Four lectins, Bandeiraea simplicifolia lectin‐II (BSL‐II), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), and Erythrina cristagalli lectin (ECL) stained secretory granules and the organelles in the olfactory supporting cells and did not stain them in the vomeronasal supporting cells. These results suggest that the glycoconjugate moieties are similar in the vomeronasal and olfactory receptor cells of the Japanese striped snake. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

The cell coat of the olfactory epithelium proper and vomeronasal neuroepithelium of the rat as revealed by means of the Ruthenium-red reaction

Summary The apical cell coat of the olfactory epithelium proper and the vomeronasal neuroepithelium of the rat was investigated electronmicroscopically by means of the Ruthenium-red reaction. In the olfactory epithelium proper, the cilia of receptor cells and microvilli of supporting cells possess a cell coat measuring approximately 10 nm in thickness. In the vomeronasal neuroepithelium, the apical cell coat is thicker than in the olfactory epithelium proper. On microvilli of vomeronasal receptor cells the cell coat varies in thickness from 15 to 20 nm, and on microvilli of supporting cells it measures approximately 75 nm. The functional implications of these findings are discussed.A portion of this study was presented at the 6^th European Anatomical Congress in Hamburg. This publication is dedicated to Prof. E. KlikaSupported by the Deutsche Forschungsgemeinschaft (Br 358/5-1).     

The prenatal maturity of the accessory olfactory bulb in pigs

The morphological development of the accessory olfactory bulb of the fetal pig was studied by classical and histo-chemical methods, and the vomeronasal organ and nasal septum were studied histochemically. Specimens were obtained from an abattoir and their ages estimated from their crown-to-rump length. The accessory olfactory bulb was structurally mature in fetuses of crown-to-rump length 21-23 cm, by which time the lectin Lycopersicum esculentum agglutinin stained the same structures as in adults (in particular, the entire sensory epithelium of the vomeronasal organ, the vomeronasal nerves, and the nervous and glomerular layers of the accessory olfactory bulb). These results suggest that the vomeronasal system of the pig may, like that of vertebrates such as snakes, be functional at birth.     

Lectin-binding pattern of neuroepithelial and respiratory epithelial cells in the mouse nasal cavity

Summary Sections from the nasal cavity of 12-day-old Swiss albino mice (NMRI strain) were subjected to lectin histochemistry. A panel of biotinylated lectins (Con A, WGA, s-WGA, PNA, SBA, DBA and UEA I) and a horseradish peroxidase-conjugated lectin (GSA II) showed marked differences in binding to the respiratory and the neuroepithelial cells. SBA (affinity for galactose andN-acetylgalactosamine), PNA (galactose) and WGA (sialic acids andN-acetylglucosamine) labelled the receptor neurons in the olfactory and vomeronasal epithelium. DBA (N-acetylgalactosamine) labelled a subgroup of about 5% of the olfactory receptor neurons, but most neurons in the vomeronasal organ. UEA I (fucose) and s-WGA (N-acetylglucosamine) intensely labelled the entire nerve cell population in the vomeronasal organ, but in the olfactory epithelium the labelling with these lectins was stratified. In the respiratory epithelium the ciliated cells were labelled with WGA and s-WGA, while the secretory cells bound most of the lectins. Thus different sugars are exposed on the surface of the different types of epithelia in the nasal cavity, providing a basis for selectivity in microbial attacks on these areas.     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

Summary The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested, Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.     

Cartilaginous torus epithelium of the vomeronasal organ of swine and sheep]

The vomeronasal organ consists of receptor cells of microvillous type, supporting and basal cells. According to their ultrastructural organization the microvillar cells are analogous to those in the main olfactory organ in the pig and have all signs of the receptor cell: microvilli at the top and centrioles in cytoplasm, as well as the central process getting off the cell body. Both in the pig and in the sheep the supporting cells contain in their apical region a number of basal bodies with cilia, getting them off. In the receptor zones of epithelium albuminous glands predominate, in the respiratory zones--mucous ones. A great amount of liquid mucus, excreted on the surface of the epithelium by numerous glands and supporting cells, apparently, facilitates adsorption and desorption of odorous molecules from the receptor cells after their stimulation. The cilia of the supporting cells probably from the stream of the vomeronasal mucus. The cartilagenous torus epithelium of the vomeronasal organ of the pig and sheep has in general a similar structural organization. This demonstrates general for Vertebrata receptor mechanisms of odorous substances, evidently connected with perception of feramones or contact olfaction.     

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: expression of AQP4 in neuronal sensory cells

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Organization of the vomeronasal organ in a plethodontid salamander

Salamanders in the family Plethodontidae show a unique behavior (nose-tapping) and have unique structures (nasolabial grooves) that may be used specifically to convey chemicals to the vomeronasal organ. The nasal structure of Plethodon cinereus was studied to determine if there is enhanced development of the vomeronasal organ compared with other salamander families that would correlate with use of these unique features. The vomeronasal organ in salamanders is found in a ventrolateral diverticulum of each main olfactory organ. P. cinereus has a more anteriorly placed vomeronasal organ within the diverticulum, and the posterior limit of each nasolabial groove is adjacent to the anterior limit of the vomeronasal organs. This suggests that the grooves deliver chemicals preferentially to the vomeronasal organs instead of to the main olfactory organs. In addition, the vomeronasal sensory epithelium is thickest anteriorly and is at its thinnest at about the level corresponding to the location of the vomeronasal organ in other salamander families. These adaptations suggest a specific mechanism of odorant delivery to the vomeronasal organ in plethodontid salamanders not found in other salamander families.     

Removal of the vomeronasal organ blocks the stress-induced hyperthermia response to alarm pheromone in male rats

Previously, we reported that male Wistar rats release alarm pheromone from their perianal region, which aggravates stress-induced hyperthermia (SIH) in pheromone-recipient rats. The subsequent discovery that this pheromone could be trapped in water enabled us to expose recipients to the pheromone in their home cages. Despite its apparent influence on autonomic and behavioral functions, we still had no clear evidence as to whether the alarm pheromone was perceived by the main olfactory system (MOS) or by the vomeronasal system. In this study, we investigated this question by exposing 3 types of recipients to alarm pheromone in their home cages: intact males (Intact), vomeronasal organ-excised males (VNX), and sham-operated males (Sham). The Intact and Sham recipients showed aggravated SIH in response to alarm pheromone, whereas the VNX recipients did not. In addition, the results of the habituation/dishabituation test and soybean agglutinin binding to the accessory olfactory bulb verified the complete ablation of the vomeronasal organ (VNO) with a functional MOS in the pheromone recipients. These results strongly suggest that male rats perceive alarm pheromone with the VNO.     

Distribution of beta 2-microglobulin in olfactory epithelium: a proliferating neuroepithelium not protected by a blood-tissue barrier

The olfactory neuroepithelium is unique in adult vertebrates in that bipolar sensory neurons are constantly dying and being replaced. The sensory neurons are also unusual because they are directly exposed to the external environment via their dendritic processes in the nasal cavity. Surveillance of this tissue by major histocompatibility complex (MHC) class I-restricted cytotoxic T cells would presumably serve as an important means of defense against foreign pathogens. Although adult brain shows a lack of class I molecules, it has not been reported if either proliferating neurons or sensory neurons in olfactory neuroepithelium also lack class I. To examine olfactory neuroepithelium, an antiserum against beta 2-microglobulin (beta 2-m), the invariant light chain associated with all class I molecules, was employed as a general probe in an immunocytochemical assay. beta 2-m was detected in columnar respiratory epithelium, blood vessel walls, and a small population of interstitial cells in the lamina propria, but no cell in the olfactory neuroepithelium stained for beta 2-m. Parallel patterns were obtained in the vomeronasal organ. These results suggest that lack of beta 2-m, and presumably class I, may be a general phenotype of neuronal cells regardless of their mitotic state or exposure to environmental antigens.     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

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.     

A classification schema for the vomeronasal organ in humans

The vomeronasal organ is a chemoreceptive structure located at the base of the nasal septum with direct axonal connections to the accessory olfactory bulb in many terrestrial vertebrates. Pheromones presumably bind to the vomeronasal organ and exert behavioral or physiologic responses, thereby allowing chemical communication between animals of the same species. The presence and function of the vomeronasal organ in humans is debated. A phenotypic classification schema for the human vomeronasal organ is described and applied to 253 human subjects who underwent nasal examination. Of these subjects, only 6 percent possessed a vomeronasal organ with 64 percent unilateral and 36 percent bilateral in appearance. No difference existed in gender, age, or race between those subjects with or without a vomeronasal organ. There is no evidence supporting involutional senescence of this structure. Future investigations should use this phenotypic schema for the vomeronasal organ to allow accurate comparisons of study populations.     

Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice

The septal organ represents one of the three chemosensory subsystems found in most vertebrate species. Analyzing the projection pattern of septal organ neurons using the OMP-GFP transgenic mouse line revealed that axons navigate in highly variable fiber tracks across the main olfactory epithelium toward the main olfactory bulb. All septal organ axons cross through the cribriform plate at a spatially defined site and terminate exclusively in the posterior, ventromedial aspect of the bulb. Here, one portion of axons forms a dense network on the medial side where they apparently enter glomeruli which are mainly innervated by axons of olfactory sensory neurons from the main olfactory epithelium. Another significant portion of the axons targets a few glomeruli which appear to receive input exclusively from the septal organ neurons.     

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae,Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

The vomeronasal organ and associated structures of the fetal African elephant, Loxodonta africana (Proboscidea, Elephantidae)

The vomeronasal organ (VNO) is a chemosensory structure of the nasal septum found in most tetrapods. Although potential behavioural correlates of VNO function have been shown in two of the three elephant species, its morphology in Loxodonta africana has not been studied. The development of the VNO and its associated structures in the African elephant are described in detail using serially sectioned material from fetal stages. The results show that many components of the VNO complex (e.g. neuroepithelium, receptor‐free epithelium, vomeronasal nerve, paravomeronasal ganglia, blood vessels, vomeronasal cartilage) are well developed even in a 154‐day‐old fetus, in which the VNO opens directly into the oral cavity with only a minute duct present. However, the vomeronasal glands and their ducts associated with the VNO were developed only in the 210‐day‐old fetus. Notably, in this fetus, the vomeronasal–nasopalatine duct system had acquired a pathway similar to that described in the adult Asian elephant; the VNOs open into the oral cavity via the large palatal parts of the nasopalatine ducts, which are lined by a stratified squamous epithelium. The paired palatal ducts initially coursed anteriorly at an angle of 45° from the oral recess and/or the oral cavity mucosa, and merged into the vomeronasal duct. This study confirms the unique characteristics of the elephant VNO, such as its large size, the folded epithelium of the VNO tube, and the dorsomedial position of the neuroepithelium. The palatal position and exclusive communication of the VNO with the oral cavity, as well as the partial reduction of the nasopalatine duct, might be related to the unique elephantid craniofacial morphogenesis, especially the enormous growth of the tusk region, and can be seen as autapomorphies.     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.