Developmental Changes in Lectin-Binding Patterns of Three Nasal Sensory Epithelia in Xenopus laevis

Authors

  • Daisuke Endo,

    1. Faculty of Agriculture, Laboratory of Veterinary Anatomy, Iwate University, Morioka, Iwate, Japan
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  • Yoshio Yamamoto,

    1. Faculty of Agriculture, Laboratory of Veterinary Biochemistry and Cell Biology, Iwate University, Morioka, Iwate, Japan
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  • Nobuaki Nakamuta,

    1. Faculty of Agriculture, Laboratory of Veterinary Anatomy, Iwate University, Morioka, Iwate, Japan
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  • Kazuyuki Taniguchi

    Corresponding author
    1. Faculty of Agriculture, Laboratory of Veterinary Anatomy, Iwate University, Morioka, Iwate, Japan
    • Faculty of Agriculture, Laboratory of Veterinary Anatomy, Department of Veterinary Sciences, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan
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    • Fax: +81-19-621-6209


Abstract

The nasal cavity of adult Xenopus laevis (X. laevis) is composed of a series of three compartments: principal, middle, and inferior chambers. The principal chamber is lined with olfactory epithelium (OE), middle chamber with middle chamber epithelium (MCE), and inferior chamber with vomeronasal epithelium (VNE). In the present study, we examined developmental changes of lectin-binding patterns of the OE, MCE, and VNE by the use of four biotinylated lectins; DSL, DBA, PNA, and UEA-I. From Stage 59, just after the beginning of metamorphosis, the stainings of the free border for DBA and UEA-I were decreased in the OE and MCE, respectively, but the stainings of secretory granules (SGs) in the OE became intense. From Stage 63, sensory cells positive for DSL were increased in these three epithelia, and positive stainings for UEA-I and DBA increased in the SGs and Jacobson's glands (JGs), respectively. In addition, from 3 months after the end of metamorphosis, the stainings of sensory cells for PNA, DBA, and DSL changed in the OE, MCE, and VNE, respectively, and those of the SGs, Bowman's glands, and JGs also changed for several lectins. The present results showed that glycoconjugates expressed in three epithelia and their associated glands changed during and after the end of metamorphosis. These findings may indicate that the functional maturation of each epithelium depends not only on the maturation of sensory cells, but also on the maturation of the SGs in supporting cells of the OE and their associated glands after the end of metamorphosis. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

Most vertebrates have distinct two olfactory systems: the main and vomeronasal olfactory systems (Wenzel and Sieck,1966; Halpern and Martínez-Marcos,2003). These two systems show different specificities to odoriferous molecules. The main olfactory system perceives ordinary odorants. The vomeronasal olfactory system is widely recognized to perceive pheromones (Wysocki et al.,1980; Halpern,1987; Singer,1991), although this system has been recently reported to receive also prey and general odorants (Placyk and Graves,2002; Baxi et al., 2006).

Xenopus laevis (X. laevis) is an anuran amphibian and lives in water throughout life. The nasal cavity of anuran amphibians, such as Xenopus, Pipa, Bufo, and Alytes, consists of a series of three compartments, that is, principal, middle, and inferior chambers (Parsons,1967). The principal chamber (PC) is the largest and opens anteriorly at the external naris and posteriorly at the choana leading to the oral cavity. The middle chamber (MC) is situated ventrolaterally to the rostral half of the PC, and communicates dorsally with the PC through a narrow slit. The inferior chamber (IC) is dorsoventrally flattened, anteroposteriorly elongated, and situated below the PC (Oikawa et al.,1998). Although most frogs have only two types of nasal sensory epithelia as in the case of the majority of other tetrapods (Eisthen,1992), frogs in Pipidae including X. laevis have three types of sensory epithelium in the nasal cavity in the adult, that is, the PC is lined with the olfactory epithelium (OE), the IC with the vomeronasal epithelium (VNE), and the MC with a unique sensory epithelium called the middle chamber epithelium (MCE) (Föske,1934). These three sensory epithelia are separated from one another by a non-sensory epithelium. In the nasal cavity of larval X. laevis, the OE and VNE are observed from the early developmental stages, and the MCE appears from Stage 51 or Stage 52, that is, around 21 days after hatching (Hansen et al.,1998).

By electron microscopy, it has been reported that the OE of X. laevis contains ciliated and microvillous sensory cells in the larval stage and during metamorphosis but only ciliated sensory cells in the adult, the MCE ciliated and microvillous sensory cells, and the VNE microvillous sensory cells throughout life (Hansen et al.,1998; Oikawa et al.,1998). In addition, the lectin histochemical method has been applied to characterize olfactory systems in X. laevis by detection of glycoconjugates, because the glycoconjugates play very important roles in cell-to-cell communication of neurons (Key and Giorgi,1986; Barber,1989; Sharon and Lis,1989; Hofmann and Meyer,1991; Riddle et al.,1993). In the adult X. laevis, Hofmann and Meyer (1991) applied lectin histochemistry using Soybean agglutinin (SBA) and observed that SBA binds to sensory cells in the MCE and VNE. Suzuki et al. (1999) reported that sensory cells in the OE and MCE exhibited different staining patterns for several lectins in X. laevis. Saito and Taniguchi (2000) revealed in detail the expression patterns of glycoconjugates in the olfactory pathways from the OE, MCE, and VNE to the olfactory bulb by lectin histochemistry in X. laevis. However, it is still unknown how the characteristics of these sensory cells change during metamorphosis.

In the present study, therefore, we examined developmental changes in lectin-binding patterns of the nasal sensory epithelia in X. laevis by lectin histochemistry to reveal functional changes of the OE, MCE, and VNE during development.

MATERIALS AND METHODS

All procedures were approved by the local animal ethical committee of Iwate University.

Materials

X. laevis of both sexes were purchased from Watanabe Breedings (Hyogo, Japan). Embryos were obtained by the mating of adult X. laevis after chorionic gonadotropin stimulation. Embryos and larvae were reared in tap water maintained at 23°C. Embryos and larvae were staged according to the table of Nieuwkoop and Faber (1967). In the present study, we examined larvae at stages from 50 to 66, which ranged from just before the formation of the middle chamber to the end of metamorphosis. To examine whether lectin-binding patterns change after the end of metamorphosis, we also used young adults at 3 and 6 months of age, and mature adults over 1.5 years of age. Animals used in the present study are shown in Table 1. Larvae and young adults at 3 months of age were decapitated and fixed by immersion in Zamboni's fixative (4% paraformaldehyde, 0.5% picric acid in 0.1 M phosphate buffer; pH 7.4). Young adults at 6 months of age and mature adults were anesthetized by pentobarbital (60 mg/kg; intraperitoneal injection) and transcardially perfused with Ringer's solution followed by Zamboni's fixative. After decapitation, the upper jaws were decalcified in Plank-Rychlo's solution (Plank and Rychlo,1952). The rostral portion of the upper jaw of each animal was embedded in paraffin, sectioned serially at 5 μm and processed for lectin histochemistry. Some sections were stained with hematoxylin–eosin (HE) stain.

Table 1. Number of specimens used in this study and their respective stages of development (Nieuwkoop and Faber,1967)
StageAgeN
5015 days12
5324 days3
5532 days5
5741 days6
5945 days6
6148 days6
6351 days6
6658 days5
3 months4
6 months4
Adult>1.5 years4

Lectin Histochemistry

The sections were deparaffinized with xylene and rehydrated in a graded series of ethanol. They were incubated with 3% H2O2 in methanol at room temperature for 30 min to eliminate endogenous peroxidase and washed in 0.01 M phosphate buffered saline (PBS; pH 7.4) for 5 min at three times. Then, sections were incubated with 1% bovine serum albumin in PBS at room temperature for 30 min. The sections were washed in PBS, and labeled with the biotinylated lectins; DSL, DBA, PNA, and UEA-I (Vector, Burlingame, CA) at 4°C overnight. The lectins used in the present study were selected according to our previous results (Suzuki et al.,1999) to show their distinct binding patterns. Details of these lectins used in the present study are shown in Table 2. After washing in PBS, the sections were incubated with avidin-biotin peroxidase complex (Elite ABC kit; Vector) at room temperature for 30 min. After washing in PBS, peroxidase activities were detected by the incubation with 0.05 M Tris-HCl (pH 7.6) containing 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.006% H2O2 at room temperature for 15 min. After washing in distilled water, the sections were dehydrated, cleared, mounted with coverslips, and examined with a light microscope.

Table 2. Binding specificities of lectins
Biotinylated-lectinConcentration (mg/mL)Binding specificity
  1. Fuc, Fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine.

Datura stramonium lectin (DSL)4.0 × 10−3β-GlcNAc
Dolichos biflorus agglutinin (DBA)1.0 × 10−2α-GalNAc
Peanut agglutinin (PNA)4.0 × 10−3β-Gal(1,3), GalNAc
Ulex europeus agglutinin-I (UEA-I)2.0 × 10−2α-Fuc

RESULTS

The nasal cavity of X. laevis at Stage 50 consisted of two chambers: PC and IC. At Stage 53, the MC was formed at the anterior region of the PC (Fig. 1A,B). The OE in the PC was equipped with Bowman's glands (BGs) from Stage 59, and the VNE in the IC was also equipped with Jacobson's glands (JGs) from Stage 50 whereas the MCE in the MC was devoid of such associated glands.

Figure 1.

Photomicrographs of HE-stained sections containing three sensory epithelia in the left nasal cavity of X. laevis at Stage 53. At anterior part of external naris, middle chamber (MC), and inferior chamber (IC) are formed (A). At the posterior part of external naris, principle chamber (PC) is observed (B). Boxed areas correspond to Fig. 1C–E, respectively, at higher magnification. PC, MC, and IC are lined with olfactory epithelium (OE; C), middle chamber epithelium (MCE; D), and vomeronasal epithelium (VNE; E), respectively. Three epithelia consist of supporting, basal, and sensory cells. Nuclei of supporting cells are in superficial layer (Sp), and those of sensory cells are distributed in middle layer (Se), and basal cells are on surface of basal lamina. Arrows indicate sensory cells. Arrowheads indicate supporting cells. Scale bars: 100 μm for A and B, 20 μm for C–E.

The OE, VNE, and MCE consisted of three types of cells: sensory, supporting, and basal cells (Fig. 1C–E). These cells were observed at all stages examined, and could be distinguished by localizations and shapes of their nuclei in the epithelia. Nuclei of sensory cells were distributed in the middle layer of the epithelia and were round in shape. Nuclei of supporting cells were distributed in the superficial layer and were elliptical in shape. Small basal cells with irregular nuclei were scattered in the basal layer. In addition, the OE had secretory granules (SGs) in the apical part of supporting cells at all stages examined.

Lectin-Binding Patterns

For four lectins, positive stainings were observed in the sensory cells and on the free borders of the three epithelia, the SGs and BGs in the OE, and JGs in the VNE. We evaluated numbers or intensities of these stainings at each stage examined. Lectin-binding patterns observed in the present study are summarized in Tables 3 and 4.

Table 3. Developmental changes of lectin-binding patterns in the three nasal sensory epithelia of X. laevis
LectinEpithelium st.50st.53st.55st.57st.59st.61st.63st.663 m6 mAdult
  • OE, olfactory epithelium; MCE, middle chamber epithelium; VNE, vomeronasal epithelium; FB, free border; st, stage; m, months; Se, sensory cells.

  • –: There are no positive sensory cells/Free border is negative.

  • ±: A few sensory cells are positive/Free border is faintly positive.

  • +: Some sensory cells are positive/Free border is intensely positive.

  • ++: Many sensory cells are positive.

  • +++: Most sensory cells are positive.

  • *

    *: MCE is not yet formed.

DSLOESe±±±±±±+++++++++++
FB+++++++++++
MCESe*±±±±±+++++++++++
FB*++++++++++
VNESe++++++++++++++++
FB++++++++++±
DBAOESe±±±±±±±±±±±
FB++++±±±±±±+
MCESe*±±±±±±±++++++
FB*++++++++++
VNESe+++++++++++
FB+++++++++++
PNAOESe++
FB±±++
MCESe*
FB*
VNESe
FB
UEA-IOESe±±±±±±±±±±±
FB±±±±±±±±±±±
MCESe*
FB*±±±
VNESe
 FB
Table 4. Developmental changes of lectin-binding patterns of the secretory granules in supporting cells and the Bowman's glands in the olfactory epithelium or Jacobson's glands in the vomeronasal epithelium of X. laevis
LectinEpithelium st.50st.53st.55st.57st.59st.61st.63st.663 m6 mAdult
  • OE, olfactory epithelium; MCE, middle chamber epithelium; VNE, vomeronasal epithelium; SGs, Secretory granules; BGs, Bowman's glands; JGs, Jacobson's glands; st, stage; m, months.

  • –: There are no positive secretory granules/Glands are negative.

  • ±: A few secretory granules are positive/Glands are faintly positive.

  • +: Some secretory granules are positive/Glands are moderately positive.

  • ++: Many secretory granules are positive/Glands are intensely positive.

  • *

    *: Bowman's glands are not yet formed.

DSLOESGs
BGs****±±±±±+++
VNEJGs++++++++++++
DBAOESGs++++++++
BGs****±±
VNEJGs±±±±±±++++++++
PNAOESGs++++
BGs****
VNEJGs
UEA-IOESGs±±+++++
BGs****+++
VNEJGs±±±++

DSL: In the OE, faintly positive stainings were observed in the middle layer from Stage 50 to Stage 61 (Fig. 2A). At Stage 63, several sensory cells were intensely positive in the lower half of the OE. At Stage 66 and 3 months, many sensory cells were positive in the lower half of the OE (Fig. 2B). At 6 months and in the adult, most of the sensory cells were positive (Fig. 2C). The free border of the OE was intensely positive at all stages examined (Fig. 2A–C). Immediately beneath the apical surface of the OE, the stainings were arranged in a broken line from Stage 63 to the adult (Fig. 2B,C). There were no positive SGs in the apical part of supporting cells of the OE at all stages examined. BGs were faintly positive from Stage 59 to 3 months, intensely positive at 6 months, and moderately positive in the adult (Fig. 3A,B). In the MCE, faintly positive stainings were observed in the sensory cells from Stage 53 to Stage 61 (Fig. 2D). At Stage 63, several sensory cells were positive in the lower layer of the MCE. At Stage 66 and 3 months, many sensory cells were positive in the lower half of the MCE (Fig. 2E). At 6 months and in the adult, most of the sensory cells were positive (Fig. 2F). Punctate stainings were observed in the upper half of the MCE from Stage 53 to 3 months. The free border of the MCE was intensely positive at all stages examined (Fig. 2D–F). In the VNE, several sensory cells were positive from Stage 50 to Stage 61. The punctate stainings were also observed at these stages (Fig. 2G). Most of the sensory cells were positive from Stage 63 to 6 months (Fig. 2H), but only a few sensory cells were positive in the adult (Fig. 2I). The free border of the VNE was intensely positive from Stage 50 to 6 months, but faintly positive in the adult (Fig. 2G–I). In addition, JGs were intensely positive at 6 months (Fig. 3C), but moderately positive at the other stages (Fig. 3D).

Figure 2.

Stainings of DSL in the olfactory epithelium (OE; A–C), middle chamber epithelium (MCE; D–F), and vomeronasal epithelium (VNE; G–I). In the OE and MCE, faintly positive stainings are observed in the sensory cells at Stage 53 (A and D). Many sensory cells in the lower half of the OE are positive at 3 months (B and E). Most of the sensory cells are positive in the adult (C and F). The free borders are intensely positive in both OE and MCE at Stage 53, Stage 66, and in the adult (A–F). In the VNE, several sensory cells are positive at Stage 53 (G), and most of the sensory cells are positive at Stage 66 (H), but only several sensory cells are positive in the adult (I). The free border of the VNE is intensely positive at Stage 53 and Stage 66 whereas faintly positive in the adult (G–I). Arrows indicate positive sensory cells. Arrowheads indicate punctate stainings. Double arrows indicate stainings arranged in a broken line immediately beneath the apical surface of the OE. Scale bars: 20 μm for A–I.

Figure 3.

Stainings of DSL in the Bowman's glands (BGs) in the OE (A and B) and the Jacobson's glands (JGs) in the VNE (C and D). BGs are faintly positive at Stage 63 (A), but moderately positive in the adult (B). JGs are intensely positive at 6 months (C), but moderately positive in the adult (D). Scale bars: 20 μm for A and B and 40 μm for C and D.

DBA: In the OE, faintly positive stainings were observed in a few sensory cells from Stage 50 to the adult (Fig. 4A–C). The free border of the OE was intensely positive from Stage 50 to Stage 57, faintly positive from Stage 59 to 6 months, and intensely positive in the adult (Fig. 4A–C). Several SGs were positive in the apical part of supporting cells of the OE at Stage 55 and Stage 63, but many SGs were positive from Stage 57 to Stage 61 (Fig. 5A,B). No positive SGs were observed at the other stages (Fig. 5C). BGs were negative from Stage 59 to 3 months, but faintly positive at 6 months and in the adult. In the MCE, faintly positive stainings were observed in the sensory cells from Stage 53 to Stage 66. Punctate stainings were also observed in the upper half of the MCE from Stage 53 to Stage 63 (Fig. 4D). At 3 months and 6 months, several and many sensory cells were positive in the lower half of the MCE, respectively (Fig. 4E). In the adult, most of the sensory cells were positive (Fig. 4F). The free border of the MCE was intensely positive at all stages examined (Fig. 4D–F). In the VNE, several sensory cells were positive at all stages examined. Punctate stainings were observed from Stage 53 to Stage 61. The free border of the VNE was intensely positive at all stages examined (Fig. 4G–I). In addition, JGs were faintly positive from Stage 50 to Stage 61, moderately positive at Stage 63 and Stage 66, and intensely positive from 3 months to the adult (Fig. 5D–F).

Figure 4.

Stainings of DBA in the OE (AC), MCE (DF), and VNE (GI). There are faintly positive sensory cells in the OE at Stage 53 (A). At 3 months and in the adult, a few sensory cells are positive (B and C). The free border of the OE is intensely positive at Stage 53, faintly positive at 3 months, and intensely positive in the adult (A–C). In the MCE, the sensory cells are faintly positive at Stage 53 (D), and several sensory cells in lower region are positive at 3 months (E), and most of the sensory cells are positive in the adult (F). In the VNE, several sensory cells are positive at Stage 53, 3 months, and in the adult (G–I). The free borders of the MCE and VNE are intensely positive at Stage 53, 3 months, and in the adult (D–I). Arrows indicate positive sensory cells. Arrowheads indicate punctate stainings. Scale bars: 20 μm for A–I.

Figure 5.

Stainings of DBA in the secretory granules (SGs) in the apical part of supporting cells of the OE (AC) and JGs (DF). A few SGs are positive at Stage 55 (A), but many SGs are positive at Stage 59 (B). No positive SGs are observed in the adult (C). JGs are faintly positive at Stage 53 (D), moderately positive at Stage 66 (E), and intensely positive in the adult (F). Arrowheads indicate positive SGs. Scale bars: 20 μm for A–C, 30 μm for D, and 40 μm for E and F.

PNA: In the OE, no sensory cells were positive from Stage 50 to 3 months (Fig. 6A,B), but several sensory cells were positive at 6 months and in the adult (Fig. 6C). The free border of the OE was negative from Stage 50 to Stage 63, faintly positive at Stage 66 and 3 months, and intensely positive at 6 months and in the adult (Fig. 6A–C). Immediately beneath the apical surface of the OE, the stainings were arranged in a broken line from 6 months to the adult (Fig. 6C). No positive SGs were observed in supporting cells of the OE from Stage 50 to Stage 66. Although many SGs were positive at 3 months, only a few SGs were positive at 6 months and in the adult (Fig. 6A–C). There were no positive BGs at all stages examined. In the MCE and VNE of all animals examined, sensory cells and the free borders were negative. In addition, JGs were negative at all stages examined.

Figure 6.

Stainings of PNA in the OE (AC). No sensory cells are positive in the OE at Stage 53 and 3 months (A and B), but several sensory cells are positive in the adult (C). Many SGs in the apical part of supporting cells of the OE are positive at 3 months (B), and several SGs are positive in the adult (C). The free border of the OE is negative at Stage 53, but faintly positive at 3 months, and intensely positive in the adult (A–C). Arrows indicate positive sensory cells. Arrowheads indicate positive SGs. Double arrows indicate stainings arranged in a broken line immediately beneath the apical surface of the OE. Scale bars: 20 μm for A–C.

UEA-I: In the OE, a few sensory cells and the free border were faintly positive from Stage 50 to the adult. Positive SGs in supporting cells of the OE were none from Stage 50 to Stage 59, a few at Stage 61 and Stage 63, several at Stage 66 and 3 months, many at 6 months, but only a few in the adult (Fig. 7A–C). BGs were negative from Stage 59 to Stage 66, and moderately positive from 3 months to adult (Fig. 7D–F). In the MCE, there were no positive sensory cells at all stages examined. The free border of the MCE was faintly positive from Stage 53 to Stage 57, and negative from Stage 59 to the adult. In the VNE, sensory cells and the free border were negative at all stages examined. JGs were faintly positive at Stage 50, Stage 66, and 3 months and moderately positive at 6 months and in the adult. At the other stages, no positive stainings were observed in the JGs (Fig. 7G–I).

Figure 7.

Stainings of UEA-I in the SGs (AC) of the OE, BGs (DF), and JGs (GI). No SGs are positive at Stage 53 (A). Many SGs are positive at 6 months (B), and several SGs are positive in the adult (C). BGs are negative at Stage 66 (D), but moderately positive at 3 months and in the adult (E and F). JGs are negative at Stage 63 (G), but faintly positive at 3 months (H), and moderately positive in the adult (I). Arrowheads indicate positive SGs. Scale bars: 20 μm for A–F, 40 μm for G–I.

DISCUSSION

Most amphibians change their morphological and ecological characteristics during metamorphosis dramatically. Metamorphosis of X. laevis takes place approximately from Stage 58 to Stage 66 (Nieuwkoop and Faber,1967). Although positive stainings for several lectins were observed in the sensory cells and on the free borders of the OE, MCE, and VNE from Stage 50, changes in lectin-binding patterns were not observed from Stage 50 to Stage 57. However, punctate stainings for DSL and DBA appeared in the upper half of the MCE and VNE from Stage 50 or Stage 53. Because it has been reported that subcellular organelles such as lysosomes, Golgi apparatuses, and mitochondria contain various glycoproteins and glycosphingolipids (Chen et al.,1985; Gillard et al.,1993), these punctate reactions are thought to be caused by the glycoconjugates of these subcellular organelles. From their distributions within epithelia, it is most likely that the punctate stainings were attributed to the subcellular organelles of the supporting cells. Although it was not possible in the present study to determine which type of cells contained these subcellular organelles, glycoconjugate expressions may change only in subcellular organelles of the MCE and VNE, but not in the sensory cells and on the free borders of the OE, MCE, and VNE before metamorphosis.

From Stage 59, just after the beginning of metamorphosis, stainings of the free border for DBA and UEA-I decreased in the OE and MCE, respectively. The SGs in the apical part of supporting cells of the OE were positive for DBA from Stage 55 to Stage 63. It has been reported by electron microscopy that sensory cells in the OE show surface blebbing and degenerating organelles by Stage 57, and size and electron density of the SGs in supporting cells of the OE increase from Stage 55 to Stages 63–65 (Hansen et al.,1998). Therefore, characteristics of the sensory and supporting cells may change around the beginning of metamorphosis.

From Stage 63, positive stainings for DSL were observed in the sensory cells in the OE, MCE, and VNE. These stainings increased gradually in the sensory cells located from basal to middle layers in the OE and MCE. These findings agree to the reports that most sensory cells in the OE are replaced by new sensory cells during metamorphosis and that the neuronal replacement occurs from the basal layer to the middle layer (Mackay-Sim and Kittel,1991; Higgs and Burd,2001). Although there is no report on the neuronal replacement in the MCE, these binding patterns for DSL may suggest the replacement of immature sensory cells to mature sensory cells. In addition, positive stainings for UEA-I continuously increased in the SGs of the OE, and stainings for DBA became intense in JGs. These lectin-binding patterns may indicate that mature sensory cells increase in number and that characteristics of the SGs change during metamorphosis, although functions of each epithelium are immature even at the end of metamorphosis. In addition, stainings arranged in a broken line were observed immediately beneath the apical surface of the OE from Stage 63. These stainings coincide well with the terminal web reported previously (Hansen et al.,1998).

From 3 months after birth, binding patterns changed dramatically in several lectins. Although positive sensory cells for DSL increased continuously in the OE and MCE, they decreased in the VNE. Positive sensory cells for DBA and PNA first appeared at 3 months in the MCE and OE, respectively. Functionally, the OE and MCE perceive volatile and water-soluble odorants, respectively (Vogler and Schild,1999; Mezler et al.,2001). The VNE is generally thought to perceive pheromones (Brennan,2001; Halpern et al.,2003), though the VNE is also suggested to perceive some odorants (Placyk et al.,2002; Baxi et al.,2005). Conceivably, these changes in lectin-binding patterns in the sensory cells of the OE, MCE, and VNE may suggest that these three epithelia differentiate functionally after the end of metamorphosis.

In the OE, the SGs of supporting cells were positive for UEA-I and PNA from Stage 61 and at 3 months, respectively. On the other hand, the SGs were positive for DBA at Stage 55 but became negative at Stage 66 and remained negative after the end of metamorphosis to the adult. These changes in lectin-binding patterns for UEA-I, PNA, and DBA in the SGs may reflect the maturation process of the SGs after the end of metamorphosis. In addition, the stainings for DSL and UEA-I became intense in BGs after the end of metamorphosis. Because the SGs of supporting cells and BGs contribute to form the mucous layer on the surface of the OE to provide a stable environment for olfactory function in terrestrial life (Getchell and Getchell,1992), changes in lectin-binding patterns of the SGs of supporting cells and BGs after the end of metamorphosis may also reflect the functional maturation of the OE after the end of metamorphosis. On the other hand, JGs continuously changed the lectin-binding patterns for several lectins after the end of metamorphosis. JGs secrete mucus on the surface of the VNE to be involved in receipt of the pheromone. The pheromones provide various kinds of information about social and reproductive status of other individuals (Brennan,2001; Halpern and Martínez-Marcos,2003). Because sexual maturation of X. laevis completes about 8–12 months after birth (Hirsch,2002), continuous changes in lectin-binding patterns in JGs may parallel with the sexual maturation of the frog. In conclusion, functional maturation of the OE, MCE, and VNE may depend not only on the maturation of sensory cells of these epithelia but also on the maturation of the SGs in supporting cells of the OE, BGs, and JGs after the end of metamorphosis.