THE ANATOMICAL RECORD 252:301–310 (1998) Fine Structure of Three Types of Olfactory Organs in Xenopus laevis TOSHIHIRO OIKAWA,1* KEIKO SUZUKI,2 TORU R. SAITO,1 KAZUAKI W. TAKAHASHI,1 AND KAZUYUKI TANIGUCHI2 1Department of Laboratory Animal Science, Nippon Veterinary and Animal Science University, Tokyo 180, Japan 2Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Iwate 020, Japan ABSTRACT There is no report on the fine structure of three types of olfactory organs in Xenopus laevis. Their functional assignments in olfaction are not yet established. The fine structure of three types of olfactory organs, olfactory epithelium (OE), vomeronasal organ (VNO), and middle chamber epithelium (MCE), was examined in Xenopus laevis by light and electron microscopy. The olfactory cells of the OE and the sensory cells of the VNO were equipped with cilia and microvilli, respectively, similar to terrestrial animals that possess both the OE and the VNO. On the other hand, the sensory cells of the MCE were classified into two types, the sensory cells with cilia and the sensory cells with microvilli, like those of the OE in fish. These findings suggest that the OE and the VNO in Xenopus laevis detect different kinds of odoriferous molecules in air, whereas the MCE is involved in the perception of odorants in water. Anat. Rec. 252:301–310, 1998. r 1998 Wiley-Liss, Inc. Key words: olfaction; Xenopus laevis; light and electron microscopy; olfactory organ types In vertebrates, at least two types of olfactory organs can occur, i.e., olfactory epithelium (OE) and vomeronasal organ (VNO). They perform functional assignments by perception of general odorants and pheromonal molecules, respectively (Abbott, 1984; Fleming et al., 1979; Halpern, 1987; Powers and Winans, 1975; Wysocki, 1979). Phylogenically, the OE exists in all vertebrates from fish to mammals (Andres, 1966; Cushieri and Bannister, 1975a,b; Graziadei, 1973,1977; Parsons, 1967). On the other hand, the VNO is absent in fish and first appears in amphibians as a diverticulum of the nasal cavity (Bertmar, 1981; Bruner, 1984; Burton et al., 1990; Kolnberger, 1971a). In reptiles, the VNO is well developed in terrestrial species, such as lizards and snakes (Kratzing, 1975; Parsons, 1970; Wang and Halpern, 1980a,b), but it is absent in aquatic crocodiles (Ciges et al., 1977). In mammals, this organ varies in its development among species. For example, it is well developed in rodents (Breipohl et al., 1979; Naguro and Breipohl, 1982; Oikawa et al., 1994; Taniguchi and Mochizuki, 1982,1983; Vaccarezza et al., 1981) but is absent in cetaceans adapted to aquatic life style (MackaySim et al., 1982). Thus, olfactory organs are assumed to undergo morphological modification to take part in different kinds of olfaction according to the life style of a species. r 1998 WILEY-LISS, INC. Amphibians are a good choice to examine the functional significance of these olfactory systems because of their adaptation to both aquatic and terrestrial life and because of the first occurrence of the VNO in them. Among amphibians, South African clawed frogs, Xenopus laevis, are particularly interesting, because they spend their entire lives in water and seem to be less adapted to terrestrial life than other anurans. Recent lectin-histochemical examinations revealed the presence of three different types of olfactory organs, i.e., OE, VNO, and middle chamber epithelium (MCE) in Xenopus laevis (Hofmann and Meyer, 1991). These olfactory organs are assumed to perform functional assignments in olfaction to perceive different kinds of odoriferous molecules. Their assumed functional assignments may be represented as differences in their fine structure, although there have been no reports on the fine structure of olfactory organs in Xenopus laevis. In the present study, therefore, the fine structure of three types of olfactory organs was examined in Xenopus *Correspondence to: Toshihiro Oikawa, Research Laboratories, Torii Pharmaceutical Co. Ltd., 1–2-1 Ohnodai, Midori-ku, Chibashi, Chiba 267, Japan. Received 25 October 1997; Accepted 19 May 1998 302 OIKAWA ET AL. Fig. 1. Light photomicrograph of the olfactory epithelium (OE; a), the vomeronasal organ (VNO; b), and the middle chamber epithelium (MCE; c). Each epithelium consists of sensory (olfactory) cells (O), supporting cells (Sp), and basal cells (B). Se, sensory epithelium. Magnification 3600. laevis by using light and electron microscopy from the viewpoint of phylogeny to discuss the significance of the functional assignments of these olfactory organs. MATERIALS AND METHODS South African clawed frogs, Xenopus laevis, were obtained from Hmamatsu Teaching Materials, Co. Ltd. (Shizuoka, Japan) and were kept in a glass aquarium maintained at average temperature of 22°C. A total of 11 adult frogs of both sexes were used for light and electron microscopic examinations. For light microscopy, five frogs were anesthetized by cooling on ice and sacrificed by cardiac perfusion with Ringer’s solution (0.0125% NaHCo3, 0.0125% CaCl2, 0.0075% KCl, 0.75% NaCl) followed by Bouin’s solution. The rostral portion of the upper jaw, including the whole nasal cavity, was removed, immersed in the same fixative for additional 24 hr, and decalcified in a mixture of 10% formic acid and 10% formalin. After decalcification, the materials were embedded in paraffin by using routine procedures, sectioned serially at 5 µm in the frontal plane, and stained with hematoxylin and eosin. For electron microscopy, six frogs were anesthetized as described above for light microscopy, and they perfused with Ringer’s solution followed by a modified Karnovsky’s solution (2% glutaraldehyde and 2.5% paraformaldehyde in 0.03 M cacodylate buffer, pH 7.4). The materials were immersed in the same fixative for 2 hr at 4°C, postfixed in 1% osmium tetroxide in 0.06 M cacodylate buffer for 1 hr at 4°C, and then embedded in Quetol-812 (Nisshin-EM, Tokyo, Japan) by using routine procedures. Ultrathin sections were cut on a Reichert-Nissei ULTRACUT-S ultramicrotome (Reichert-Jung, Nussloch, Germany), double stained with uranyl acetate and lead citrate, and examined with a HITACHI H-7100 (Tokyo, Japan) electron microscope. Semithin sections were cut at 2µm and were stained with 0.5% toluidine blue for light microscopic observations. RESULTS Subdivisions of the Nasal Cavity and Histological Structure of the Sensory Epithelia The entire nasal cavity in Xenopus laevis consisted of three chambers communicating with each other, i.e., the principal, middle, and inferior chambers. The principal chamber was the largest and opened anteriorly at the external naris and posteriorly at the choana leading into the oral cavity. The middle chamber was situated ventrolateral to the rostral half of the principal chamber and was subdivided into several recesses. The middle chamber communicated dorsally with the principal chamber through Fig. 2. Electron photomicrograph of the OE. Supporting cells (Sp) possess a large number of secretory granules (SG) in the apical cytoplasm. Magnification 33,000. Fig. 3. Electron photomicrograph of the apical part of the OE. Olfactory cells (O) are covered with cilia (Ci), and supporting cells (Sp) are covered with microvilli (Mv). Arrows indicate neurotubules. SG, secretory granule. Magnification 312,000. 304 OIKAWA ET AL. Fig. 4. Electron photomicrograph of the middle part of the OE. The olfactory cell extends its dendrite toward the apical surface. Supporting cells possess well-developed smooth endoplasmic reticulum (sER) in their cytoplasm. Magnification 37,000. Fig. 5. Electron photomicrograph of the basal part of the OE. The axons of olfactory cells penetrate the basement membrane. A, axon of olfactory cell; B, basal cell. Magnification 37,000. Fig. 6. Electron photomicrograph of the basal part of the OE. The basal cell (B) contacts with the basement membrane and surrounds the axons of olfactory cells (A). Magnification 37,000. a narrow slit. The inferior chamber was dorsoventrally flattened, anteroposteriorly elongated, and situated below the principal chamber. The inferior chamber ended blindly at its rostral portion and communicated caudally with the principal chamber. These three chambers possessed different types of sensory epithelia that were separated from one another by nonsensory respiratory epithelium. The principal chamber was lined with the OE on its dorsal-tomedial wall, and the inferior chamber was lined with the VNO on its medial wall. The middle chamber was lined medially with the MCE and was different from the OE and the VNO in the thickness of its epithelium. Histologically, the OE consisted of olfactory, supporting, and basal cells (Fig. 1a). Olfactory and supporting cells were elongated and were arrayed alternatively in the OE, whereas basal cells were small and were scattered sparsely on the basement membrane. Oval nuclei of supporting cells were arranged as a single layer in the upper one-third of the OE, and round nuclei of olfactory cells accumulated densely in the lower two-thirds. The VNO and MCE also consisted of three kinds of cells, i.e., sensory, supporting, and basal cells. They were conspicuously similar to the OE in the arrangement of their cells, but they were rather rich in the number of cells and were thicker than the OE (Fig. 1b,c). The OE, VNO, and MCE were 100 µm, 180 µm, and 140 µm in thickness, respectively. The OE contained numerous secretory granules in its apical portion and was equipped with its own associated glands (Bowman’s glands). The VNO was also equipped with its associated glands (Jacobson’s glands), whereas the MCE was devoid of such an associated gland. Nerve fibers issued from the OE and the VNO to form the olfactory nerve bundle and the vomeronasal nerve bundle, respectively. Nerve fibers from the MCE traveled along the olfactory nerve and took part in the formation of the olfactory nerve bundle. Fine structure of the OE, VNO and MCE The olfactory cells of the OE were bipolar neurons, the same as the sensory cells of the VNO and the MCE. Dendrites of the olfactory cells protruded their distal ends beyond the apical surface of adjacent supporting cells to form olfactory vesicles with long cilia on their apical surface (Fig. 2). These cilia were embedded in the thick mucous sheet on the surface of the OE. The olfactory cells contained numerous neurotubules, ciliary basal bodies, STRUCTURE OF OLFACTORY ORGANS IN XENOPUS 305 Fig. 7. Electron photomicrograph of the vomeronasal organ (VNO). The apical cytoplasm of the supporting cells (Sp) was occupied by a large number of mitochondria. Se, sensory cell. Magnification 33,000. and mitochondria in dendrites (Fig. 3) and contained rough endoplasmic reticulum (rER), Golgi apparatus, and lysosomes in the perikarya (Fig. 4). The cytoplasm of the olfactory cell tapered proximally to the nucleus and penetrated the basement membrane like the axon (Fig. 5). Columnar supporting cells of the OE were equipped with short microvilli on their apical surface (Fig. 3). These cells were characterized by a large number of vesicles with moderate electron density in the apical cytoplasm. These vesicles corresponded to the secretory granules that were observed under light microscopy in the OE. The perinuclear cytoplasm was occupied mostly by excessively developed smooth endoplasmic reticulum (sER) and also contained mitochondria, Golgi apparatus, rER, and bundles of microfilaments (Fig. 4). The basal cells of the OE were small with scanty cytoplasm and were almost the same as those of the VNO and the MCE in their ultrastructural features. They contained a few mitochondria and other, poorly developed cytoplasmic organelles, and they often extended their cytoplasm to surround the axons of olfactory cells (Fig. 6). On the other hand, the dendrites of the sensory cells of the VNO protruded slightly into the lumen and were equipped with a large number of long microvilli on their apical surface (Fig. 7). These microvilli were in close contact with mucous substance, which sparsely covered the VNO. These sensory cells contained centrioles in dendrites (Fig. 8) and sometimes displayed spirally arranged neurofilaments surrounded by well-developed sER in perikarya (Fig. 9). The other ultrastructural features were similar to those of olfactory cells of the OE. The apical surface of supporting cells of the VNO was covered with short microvilli in addition to long cilia with basal bodies and striated rootlets (Fig. 8). The apical cytoplasm of the supporting cells was occupied by a large number of mitochondria (Fig. 7), whereas the perinuclear cytoplasm contained the poorly developed rER and free ribosomes small in numbers (Fig. 9). The sensory cells of the MCE were divided into two types according to their apical morphology (Fig. 10). The first type of sensory cell (nonciliated sensory cell) was dominant and possessed microvilli on its apical surface and centrioles in its dendrites, similar to the sensory cells of the VNO (Figs. 11, 12). The second type of sensory cell (ciliated sensory cell) was covered with several cilia, similar to the olfactory cells of the OE, but formed no structure similar to the olfactory vesicles of the OE (Figs. 11, 12). Mucous substance was encountered scarcely on the surface of the MCE. The other ultrastructural features of both types of sensory cells were in similar to those of olfactory cells of the OE (Fig. 13). The supporting cells of the MCE were also classified into two types, i.e., ciliated and nonciliated supporting cells (Figs. 10, 11). Ciliated supporting cells resembled closely the supporting cells of the VNO in their apical morphology, long cilia, and short microvilli on their apical surface. Nonciliated supporting cells protruded into the lumen and bore several short microvilli on their apical surface. This 306 OIKAWA ET AL. Fig. 8. Electron photomicrograph of the apical part of the VNO. Sensory cells (Se) are covered with cilia (Ci), and supporting cells (Sp) are covered with microvilli (Mv). Arrows indicate neurotubules, and arrowheads indicate centrioles. Magnification 312,000. Fig. 9. Electron photomicrograph of the somata of the sensory cell of the VNO. Spirally arranged neurofilaments (Nf) are developed well in the perinuclear cytoplasm of the sensory cells. G, Golgi apparatus. Magnification 37,000. type of cell contained concentrically arranged sER in the supranuclear cytoplasm (Fig. 14). The other cytoplasmic organelles, such as mitochondria, rER, and Golgi apparatus, were developed moderately in both ciliated and nonciliated supporting cells. nostril that closed the principal chamber under water, allowing water exchange between the middle and inferior chambers, whereas this flap closed the middle and inferior chambers when outside of the water to enable the principal chamber to make contact with air. More recently, Hofman and Meyer (1991) distinguished three sets of olfactory systems, the MCE-ventral part of the MOB system, the VNO-AOB system, and the OE-dorsal part of the MOB system, according to their different in binding affinities to soybean agglutinin (SBA). They speculated that SBA-positive systems, the MCE-ventral part of the MOB system and the VNO-AOB system, were involved in the detection of nonvolatile water-born odors, whereas the SBA-negative, OE-dorsal part of the MOB system was involved in the detection of volatile air-born odors. Franceschini et al. (1992) also identified the same three olfactory systems by using SBA, but they did not agree with the hypothesis of Hofman and Meyer. Although the functional significance of three olfactory systems has not been established fully to date, at least it can be said that, in Xenopus laevis, the MCE plays a primary role in olfaction as well as the OE and the VNO. In the present study, details of the fine structure of the OE and the VNO in Xenopus laevis agreed well with those reported previously in anurans (Bloom, 1954; Burton, 1985; Franceschini et al., 1991; Kolnberger, 1971a,b; Mair et al., 1982; Menco, 1980; Reese, 1965; Scalia, 1976; DISCUSSION In general, vertebrates possess two different types of olfactory organs, the OE and the VNO. They project centrally through olfactory and vomeronasal nerves to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, and they constitute two distinct olfactory systems, the OE-MOB system and the VNO-AOB system (Barber and Raisman, 1974; Salazar et al., 1992; Scalia and Winans, 1975). The OE-MOB system performs the usual olfactory perception functions to respond to general odorants. On the other hand, the VNO-AOB system is thought to be specialized for the perception of pheromonal substances and to be involved in reproductive and/or social behaviors (Abbott, 1984; Fleming et al., 1979; Halpern, 1987; Powers and Winans, 1975; Saito et al., 1995; Wysocki, 1979). Thus, these two olfactory systems seem to play different roles in olfaction. In Xenopus laevis, the presence of three distinct region of olfactory mucosa was acknowledged by early authors (Altner, 1962; Föske, 1934). With regard to the significance of three nasal chambers, Altner described a skin flap in the Fig. 10. Electron photomicrograph of the middle chamber epithelium (MCE). Some sensory cells are covered with cilia and others are covered with microvilli. nSe, nonciliated sensory cell. Magnification 33,000. Fig. 11. Electron photomicrograph of the apical part of the MCE. The ciliated supporting cell (cSp) is covered with long cilia with basal bodies and striated rootlets. The nonciliated supporting cell (nSp) protrudes into the lumen and bears several short microvilli. Magnification 38,000. Fig. 12. Electron photomicrograph of the apical part of the MCE. Centrioles are encountered in the cytoplasm of the nonciliated sensory cell (nSe). Arrows indicate neurotubules, and arrowheads indicate centrioles. Ci, cilia; Mv, microvilli. Magnification 312,000. 308 OIKAWA ET AL. Fig. 13. Electron photomicrograph of the somata of the sensory cells of the VNO. These sensory cells contain mitochondria, rough endoplasmic reticulum, and Golgi apparatus in the cytoplasm as the olfactory cells of the olfactory epithelium. Magnification 37,000. Fig. 14. Electron photomicrograph of the supporting cells and dendrites of the sensory cells of the MCE. The smooth endoplasmic reticulum (sER) is arranged concentrically in the perinuclear cytoplasm of the supporting cell. Magnification 37,000. Taniguchi et al., 1996). The olfactory cells of the OE were covered with cilia, and the sensory cells of the VNO were covered with microvilli in Xenopus laevis, similar to other species that possess both the OE and the VNO. Because cilia and microvilli are recognized as receptor sites of olfaction (Graziadei, 1977; Halpern, 1987), the present findings seem to suggest that, ultrastructurally, the OE and the VNO are active in function and perceive general and pheromonal molecules, respectively, as in other species. The supporting cells of the OE were covered with microvilli and were characterized by a large number of secretory granules in their apical cytoplasm. These granules were also observed in the supporting cells of the OE of other amphibians, fish, lizards, and birds but not in mammals (Getchell and Getchell, 1992). In contrast, the supporting cells of the VNO possessed cilia but no secretory granules in the present study, although they are generally reported to possess microvilli but to lack cilia in mammals (Adams, 1986; Adams and Wiekamp, 1984; Bhatnagar et al., 1982; Kratzing, 1970; Loo and Kanagasuntheram, 1972; Luckhaus, 1969; Oikawa et al., 1994; Seiffert, 1971; Taniguchi and Mikami, 1985; Taniguchi and Mochizuki, 1982,1983; Taniguchi et al., 1992; Vaccarezza et al., 1981). Franceschini et. al. (1991) found ciliated supporting cells in the VNO of another frog, Rana esculenta, and hypothesized that these cilia play a role in the transmission of odorants to the VNO in place of the vomeronasal pump that is observed in some mammals. In the present study, cilia of the supporting cells of the VNO were equipped with basal bodies and long rootlets. These findings may lead to a reconfirmation of their hypothesis. On the other hand, there is no report on the fine structure of the MCE in Xenopus laevis. In the present study, the sensory cells of the MCE were classified into two types according to their apical morphology. One type of cell was covered with cilia, similar to those of the OE, and the other type was covered with microvilli, similar to the VNO. In fish that lack the VNO, some sensory cells of the OE are reported to be covered with cilia, whereas others are covered with microvilli (Breucker et al., 1979; Ichikawa and Ueda, 1977; Moran et al., 1992; Thommesen, 1983; Yamamoto and Ueda, 1977; Zeiske et al., 1976). Therefore, there seems to be a possibility that the primitive OE of fish remained in the middle chamber of Xenopus laevis as the MCE to detect odoriferous molecules in water and that it generated two kinds of olfactory organs, the OE and the VNO, during its phylogenetical development. Furthermore, the MCE lacked its own associated gland, like the OE of fish (Getchell and Getchell, 1992; Graziadei, 1973), and was barely covered by mucous substances on its apical surface. The secretory substances on the free surface of olfactory organs were essential for the olfactory perception of olfactory organs in terrestrial life, because odorants STRUCTURE OF OLFACTORY ORGANS IN XENOPUS must be dissolved in the mucous layer covering receptor sites of olfactory organs to be perceived as olfactory stimuli (Graziadei, 1977; Halpern, 1987). In fish, because odorants are dissolved in the surrounding water, the OE does not have to be covered with secretory substances. In terrestrial animals, Bowman’s glands and Jacobson’s glands seem to appear in the OE and the VNO, respectively, to cover the olfactory organs and to dissolve the odorants in air in secretory substances. The lack of associated glands in the MCE seems to suggest that the MCE is involved in olfactory perception in water. 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