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A morphological study of the vomeronasal organ and the accessory olfactory bulb in the Korean roe deer, Capreolus pygargus
Acta Histochemica 116 (2014) 258–264
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Acta Histochemica journal homepage: www.elsevier.de/acthis
A morphological study of the vomeronasal organ and the accessory olfactory bulb in the Korean roe deer, Capreolus pygargus Changnam Park a , Meejung Ahn b , Jae-Yuk Lee c , Sang Lee c , YoungMin Yun a,c , Yoon-Kyu Lim a , Kazumi Taniguchi d , Taekyun Shin a,∗ a Laboratory of Veterinary Anatomy, College of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju 690-756, Republic of Korea b Department of Anatomy, School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea c Jeju Wildlife Rescue Center, Jeju 690-121, Republic of Korea d Laboratory of Veterinary Anatomy, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
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Article history: Received 8 May 2013 Received in revised form 2 August 2013 Accepted 4 August 2013 Keywords: Accessory olfactory bulb Lectin Roe deer Vomeronasal organ
a b s t r a c t The vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the Korean roe deer (Capreolus pygargus) were studied histologically to evaluate their morphological characteristics. Grossly, the VNO, encased by cartilage, has a paired tubular structure with a caudal blind end and a rostral connection through incisive ducts on the hard palate. In the VNO, the vomeronasal sensory epithelium (VSE) consists of galectin-3-positive supporting cells, protein gene product (PGP) 9.5-positive receptor cells, and basal cells. The vomeronasal respiratory epithelium (VRE) consists of a pseudostratified epithelium. The AOB strata included a vomeronasal nerve layer (VNL), a glomerular layer (GL), a mitral/tufted cell layer, and a granular cell layer. All lectins used in this study, including Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA), labeled the VSE with varying intensity. In the AOB, both the VNL and the GL reacted with BSI-B4, SBA, and WGA with varying intensity, but not with UEA-I. This is the first morphological study of the VNO and AOB of the Korean roe deer, which are similar to those of goats. © 2013 Elsevier GmbH. All rights reserved.
Introduction The vomeronasal system (VNS), an olfactory system, consists of the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), the vomeronasal amygdala, and nerves connecting them all (Takami, 2002; Halpern and Martinez-Marcos, 2003; Yokosuka, 2012). The VNS is present in mammals, reptiles, amphibians (Taniguchi and Saito, 2011), but morphological and functional differences clearly exist even between closely related species such as mice and rats (Takigami et al., 2000; Tirindelli et al., 2009). Depending on the species, the VNS is related to a variety of functions, including reproduction through the perception of pheromones (Gelez and Fabre-Nys, 2004; Keller et al., 2009).
Abbreviations: AOB, accessory olfactory bulb; BSI-B4, Bandeiraea simplicifolia agglutinin isolectin B4; GL, glomerular layer; GrL, granular cell layer; M/TcL, mitral/tufted cell layer; PGP, protein gene product; SBA, soybean agglutinin; UEAI, Ulex europaeus agglutinin I; VNL, vomeronasal nerve layer; VNO, vomeronasal organ; VNS, vomeronasal system; VRE, vomeronasal respiratory epithelium; VSE, vomeronasal sensory epithelium; WGA, Triticum vulgaris wheat germ agglutinin. ∗ Corresponding author. E-mail address: [email protected] (T. Shin). 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.08.003
In the past several decades, the morphological characteristics of the VNOs of various animals have been studied, including those in mice (Salazar et al., 2001; Salazar and Sanchez Quinteiro, 2003), rats (Salazar and Sanchez Quinteiro, 1998; Lee et al., 2012), golden hamsters (Taniguchi et al., 1992; Taniguchi, 2008) and cats (Salazar and Sanchez-Quinteiro, 2011). Furthermore, comparative morphological studies have been conducted on the VNOs of domestic animals including horses and cattle (Lee et al., 2003; Taniguchi and Mikami, 1985), goats (Takigami et al., 2000), pigs (Park et al., 2012a) and sheep (Salazar et al., 2007). However, little is known about the VNOs of wild ruminants apart from the Scandinavian moose (Vedin et al., 2010). The behavioral characteristics of wild roe deer have been studied under natural conditions (McLoughlin et al., 2007; Richard et al., 2008). On Jeju Island, Korea, the Korean roe deer Capreolus pygargus inhabits fields and meadows, particularly on Halla mountain (Han et al., 2007). Few studies have addressed their morphological characteristics apart from a genetic identification study (Han et al., 2007). Hence, as a first step to understanding the morphological characteristics of this animal, we histologically analyzed two VNS organs, the VNO and AOB of the Korean roe deer.
C. Park et al. / Acta Histochemica 116 (2014) 258–264 Table 1 Description of Korean roe deer used in this study. Age* (years)
Weight (kg)
Sex
2 3 3 3 3 3
20 25 21 21 23 22
Male Male Male Male Male Male
*
The estimated age was calculated by the number of horn branches.
Materials and methods Tissue preparation VNO and AOB samples from seven (six male, one female) roe deer C. pygargus (Han et al., 2007) were obtained from the Jeju Wildlife Rescue Center. Their ages were determined to be two to three years old, respectively (Table 1), based on the number of horn branches (spikes). For light microscopy, the VNO and AOB were removed immediately after death and fixed in 10% buffered formalin for 48 h. All experimental procedures were conducted in accordance with Jeju National University Guidelines for the Care and Use of Laboratory Animals. Histological examination Formalin-fixed VNOs were trimmed and decalcified in sodium citrate–formic acid solution with several changes of the solution, until the bony pieces softened as shown in our previous study (Park et al., 2012b). Then, decalcified VNO and olfactory bulbs containing AOB were dehydrated in a graded ethanol series (70%, 80%, 90%, 95%, and 100%), cleared in xylene, embedded in paraffin, and sectioned at a thickness of 5 m. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E). To visualize the AOB nerve fiber tract and strata, Kluver–Barrera staining (0.1% Luxol Fast Blue and 0.1% Cresyl violet) was applied to paraffin sections (Kluver and Barrera, 1953; Geisler et al., 2002). The morphological study of VSE and AOB was accomplished in six male roe deer because only one sample of female VNO was obtained and both the VNO and AOB in the female were histoloigcally similar those of the males.
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endogenous peroxidase activity. Then non-specific binding was blocked with 10% normal goat serum (ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA), washed in phosphate-buffered saline (PBS, pH 7.4) for 1 h, and then allowed to react with the rat anti-galectin-3 antibody (1:1000) for 1 h at room temperature. After washing in PBS, the sections were reacted for 45 min with biotinylated rabbit anti-rat IgG (1:100; Vector Laboratories). Rabbit polyclonal antibody to PGP 9.5 (1:800) was reacted with a biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories). After another wash in PBS, the sections were incubated for 45 min with the avidin–biotin peroxidase complex (ABC Elite Kit; Vector Laboratories) prepared according to the manufacturer’s instructions. After washing in PBS, the peroxidase reaction was developed for 3 min using a diaminobenzidine substrate (DAB Kit; Vector Laboratories), prepared according to the manufacturer’s instructions. Sequentially, the sections were counterstained with hematoxylin for 30 s, washed in running tap water for 20 min, dehydrated through a graded ethanol series, cleared with xylene, and mounted with Canada balsam (Sigma–Aldrich, St. Louis, MO, USA). Lectin histochemistry The following lectins (all of which were purchased from Sigma–Aldrich, St. Louis, MO, USA) were used: Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA). Lectins are commonly used to identify receptor cells and/or their matching area in the AOB (Taniguchi et al., 1993; Salazar and Sanchez Quinteiro, 2003). Lectin histochemistry was performed as in our previous studies (Park et al., 2012b). In brief, the paraffin-embedded VNO and AOB were sectioned to a thickness of 5 m using a microtome. The sections were mounted on glass microscope slides, and the paraffin was removed. Then the sections were rehydrated. Endogenous peroxidase activity was blocked through 30 min incubation with 0.3% hydrogen peroxide in methanol. After three washes with PBS, the sections were incubated with either BSI-B4-peroxidase (diluted 1:50), SBA-peroxidase (diluted 1:100), UEA-I-peroxidase (1:200), or WGA-peroxidase (1:100) for 3 h at room temperature. Signals were developed using a DAB substrate kit (Vector). The sections were counterstained with hematoxylin before mounting. Negative controls were processed through omission of lectin during the staining procedure to clarify the reactivity of lectin histochemisty.
Antibodies Results To confirm the presence of receptor cells and supporting cells in the VSE, immunohistochemistry was performed using a rabbit polyclonal antibody to protein gene product 9.5 (PGP 9.5) or a rat anti-galectin-3 monoclonal antibody, respectively. The rat anti-galectin-3 monoclonal antibody was purified by affinity chromatography from the supernatant of hybridoma cells (clone TIB-166TM , M3/38.1.2.8. HL.2; American Type Culture Collection, Manassas, VA, USA) and used at a final concentration of 1–5 g/mL for immunohistochemistry. This antibody has been used to detect galectin-3 in the tissues of various animals including ungulates, cows (Kim et al., 2008), and pigs (Park et al., 2012b). Rabbit polyclonal antibody to PGP 9.5 (Biotrend, Cologne, Germany) has been used to detect receptor cells in the VNOs of humans and rats (Johnson et al., 1994; Witt et al., 2002). Immunohistochemistry Sections (5 m) of paraffin-embedded tissue were deparaffinized and heated in a microwave (800 W) in citrate buffer (0.01 M, pH 6.0) for 5 min. After cooling the slides, the sections were exposed to aqueous 0.3% hydrogen peroxide for 20 min to block
Gross anatomy The VNO had a prominent tubular-shaped structure about 80 mm long on the floor of the nasal cavity, adjacent to the vomer (Fig. 1, boxed area). The ducts originated from medially located openings in the rostral hard palate caudal to an incisive papilla (Fig. 1, inset, hollow arrows). From the openings, the VNO was arranged under the nasal septum bilaterally and stretched over two-thirds of the nasal cavity. The vomeronasal nerves (Fig. 1, arrows) were observed in sagittal sections through the nasal septum; they innervated the AOB (Fig. 1, circled area). Histological features of the VNO The morphological features of the VNO are shown at low magnification in Fig. 2A. A lamina of cartilage wrapped around the whole structure and connective tissue, glands, vessels, and nerves making up the soft tissue of the VNO were organized around the vomeronasal duct. The vomeronasal glands were most consistently found to communicate with the lumen of the VNO at two locations:
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In the lamina propria, there were enlarged blood vessels and numerous vomeronasal glands (Fig. 2A). The ductal epithelium and acini of the vomeronasal glands were mainly located under the VRE. On the medial side, the vomeronasal nerve bundles were positioned under the VSE (Fig. 2A).
Immunohistochemical analysis of the VNS epithelium and the lamina propria
Fig. 1. Gross anatomy of the rostral part of the head of the adult Korean roe deer (Capreolus pygargus) on the left lateral side. The maxilla and the nasal conchae were removed. The vomeronasal nerves (VNn, arrows) derived from the vomeronasal organ (VNO, boxed area) project in the dorsocaudal direction. The circled area marks the position of the accessory olfactory bulb. Inset shows the incisive papilla (hollow arrows) connected to the incisive duct in the palate. OB: olfactory bulb, Asterisk: nasopharynx. Scale bar = 1 cm.
through short ducts located at the dorsal junction of the VSE and the VRE, and through short ducts located at the ventral junction of the VSE and VRE (Fig. 2A, boxed area). The VSE was located in the medial portion of the VNO and consisted of supporting cells, receptor cells, and basal cells (Fig. 2B). The free border of the VSE was adjacent to the lumen of the vomeronasal duct (Fig. 2B, arrowheads). The VRE was located in the lateral portion of the VNO and consisted of pseudostratified epithelium (Fig. 2C).
In the VSE, galectin-3 immunoreactivity was observed in supporting cells (Fig. 3A, arrows), as well as in apices of the epithelium (Fig. 3A). In addition, thin processes of galectin-3-positive cells reached the basal layer (Fig. 3A, arrowheads). In contrast, no galectin-3 reactivity was observed in the receptor cells or in the basal cells. In the lamina propria, the ductal epithelium of the vomeronasal glands was positive for galectin-3 (Fig. 3A, hollow arrow). PGP 9.5 expression was detected in receptor cells in the middle layer of the VSE (Fig. 3B, arrows), but not in supporting cells or basal cells (Fig. 3B). PGP 9.5-positive cells extended some dendrites to the free border (Fig. 3B, arrowheads). The axonal bundles of PGP 9.5positive cells were spread across the lamina propria (Fig. 3B, hollow arrow).
Histological features of the AOB The olfactory bulb was obliquely located in the rostral portion of the forebrain (Fig. 1). The AOB was an oval-shaped structure located in the dorso-medial part of the main olfactory bulb (Fig. 4A). Microscopically, it was possible to divide the main olfactory bulb into several layers, including the olfactory nerve layer, glomerular layer
Fig. 2. Histology of the vomeronasal organ of the Korean roe deer. (A) Cross-section through the ventral part of the nasal septum. The vomeronasal cartilage (C) encapsulates the lamina propria. The vomeronasal nerve bundles (N) are mainly spread under the vomeronasal sensory epithelium (VSE). There are large blood vessels (bv) and the vomeronasal glands (gl) under the vomeronasal respiratory epithelium (VRE). At the dorsal (boxed area) and ventral parts of the lumen, the vomeronasal gland ducts open into the junction between the VSE and the VRE. RE: respiratory epithelium. (B) Higher magnification of the VSE. The VSE is composed of three layers that are discernible (SC, supporting cell; RC, receptor cell; BC, basal cell). Dashed line marks the basement membrane. Arrowheads indicate the free border. (C) Higher magnification of the VRE. The VRE consists of non-sensory, ciliated pseudostratified epithelium. (A–C), H–E staining. (A), scale bar = 250 m; (B) and (C), scale bars = 25 m.
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Fig. 3. Immunohistochemical localization of galectin-3 and PGP 9.5 in the vomeronasal sensory epithelium. (A) In the VSE, galectin-3 is expressed in the supporting cells (arrows). Galectin-3 positive cells extend thin processes to the basal cell layer (arrowheads) as well as to the luminal apices of the supporting cells. In the lamina propria, the ductal epithelium of the vomeronasal glands is weakly positive for galectin-3 (hollow arrow). (B) PGP 9.5-positive cells are arranged in the middle layer of the VSE (arrows). Some dendrites of PGP 9.5-positive cells reach the free border (arrowheads). PGP 9.5-positive axons are spread in the lamina propria (hollow arrow). Scale bars = 25 m.
(GL), external plexiform layer, mitral cell layer, and granular cell layer (GrL) (Fig. 4B). In the AOB (Fig. 4C), four layers including the vomeronasal nerve layer (VNL), GL, mitral/tufted cell layer (M/TcL), and granular cell layer (GrL) were found, although the demarcation between M/TcL and GrL was not obvious. The thickness of the AOB (Fig. 4C) was thinner than that of the main olfactory bulb (Fig. 4B). Both the VNL and GL were well organized (Fig. 4C). In the M/TcL,
the mitral/tufted cells with round or oval shape were not clearly organized in rows. The granular cells were dispersed in the GrL (Fig. 4C). Because of the population and distribution of the M/Tcells and the granular cells, it was difficult to distinguish the boundary between M/TcL and GrL. In Luxol Fast Blue–Cresyl violet staining (Fig. 4D), the lateral olfactory tract was clearly identified under the GrL, and the VNL and GL were also well visualized.
Fig. 4. Histological findings of the olfactory bulb and accessory olfactory bulb located in the rostral part of the telencephalon. (A) The bulb is divided into the main olfactory bulb (left rectangle) and the accessory olfactory bulb (right rectangle). (B) Higher magnification of the main olfactory bulb in (A). The olfactory nerve cell layer (ONL) is the first layer below which lies the glomerular layer (GL). Under the GL, several layers follow: the external plexiform layer (ePL), the mitral cell layer (ML), and the granular cell layer (GrL). (C) Higher magnification of the accessory olfactory bulb in (A). It is divided into the vomeronasal nerve layer (VNL), the glomerular layer (GL), the mitral/tufted cell layer (M/TcL), the granular cell layer (GrL) and the lateral olfactory tract (LOT). (D) Kluver–Barrera-stained parasagittal section of the accessory olfactory bulb. Each AOB strata is visualized. The lateral olfactory tract (LOT) is under the base of the GrL. (A–C), H&E staining. (D) Kluver–Barrera staining. (A) Scale bar = 1000 m; (B–D) scale bars = 100 m.
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Fig. 5. Lectin histochemistry of BSI-B4 (A), SBA (B), UEA-I (C), and WGA (D) in the VSE of Korean roe deer. (A–D), counterstained with hematoxylin. Scale bars = 25 m.
Lectin histochemistry of the VSE and AOB BSI-B4 staining weakly labeled the perikarya of receptor cells and basal cells, but not supporting cells (Fig. 5A). SBA labeled receptor cells but neither supporting cells nor basal cells (Fig. 5B). UEA-I stained supporting (but not receptor or basal) cells in the VSE (Fig. 5C). WGA stained receptor cells intensely (but not supporting or basal cells) (Fig. 5D). In summary, in the AOB, the VNL and GL positively reacted with three lectins (BSIB4, SBA, and WGA), with heterogeneous staining in the whole AOB; these layers did not react with UEA-I (Fig. 6A–D). There was no specific staining in the negative control (not illustrated).
The lectin binding patterns in this study are summarized in Table 2. Discussion This is the first descriptive morphological study of the VNOs and AOBs of Korean roe deer. The morphological characteristics of the VNO are similar to those of other ruminants, including Scandinavian moose (Vedin et al., 2010), sheep (Salazar et al., 2003), and goats (Takigami et al., 2000; Mogi et al., 2007). In mice (Salazar and Sanchez Quinteiro, 2003), there are apical and basal receptor cell zones in the VSE, which project to the anterior and posterior AOB,
Fig. 6. Sagittal section of the accessory olfactory bulb labeled by BSI-B4 (A), SBA (B), UEA-I (C), and WGA (D) in Korean roe deer. The vomeronasal nerve layer and glomerular layer are positive for BSI-B4, SBA, and WGA, but not UEA-I: Counterstained with hematoxylin. Scale bars = 200 m.
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Table 2 Lectin binding patterns in the vomeronasal system of Korean roe deer. Lectin (concentration, g/mL)
Structure
BSI-B4 (5.0 × 10−1 )
SBA (1.0 × 10−2 )
UEA-I (2.0 × 10−2 )
WGA (1.0 × 10−2 )
VSE
Supporting cells Receptor cells Basal cells
− + ++
− ++ −
++ − −
− +++ −
AOB
Vomeronasal nerve layer Glomerular layer
+ ++
+ +
− −
+++ +++
The intensity of lectin binding in each structure is expressed as follows: −, negative; +, weak; ++, moderate; +++, intense.
respectively. These projections form two distinct VNO–AOB pathways (Halpern et al., 1998). In contrast, there is only a single VNS pathway between the VNO and AOB of cats (Salazar and SanchezQuinteiro, 2011), sheep (Salazar et al., 2007), pigs (Salazar et al., 2000), and goats (Takigami et al., 2000). In the present study, we found one zone of receptor cells in the VSE of roe deer, with no division of AOB revealed by lectin histochemistry, indicating the formation of a single VNO–AOB pathway. Thus, we postulate that the VNS of roe deer contains a single VNO–AOB pathway, similar to those of the other animals mentioned above. As for the lectin binding patterns in the VNOs of ruminants including sheep, the VSE has been shown to be positive for UEA-I, BSI-B4, Dolichos biflorus agglutinin (DBA), and Lycopersicon esculentum agglutinin (LEA) (Salazar et al., 2000). However, the specific VSE cells that each lectin stains have not been described. In the present study, we found that each lectin bound differently to the various cells, namely, receptor cells, supporting cells, and basal cells. Briefly, receptor cells were positive for BSI-B4, SBA, and WGA, but not UEA-I, whereas supporting cells were only positive for UEAI. The functional role of each lectin in the VSE of the roe deer remains to be elucidated in further studies. There are four AOB strata layers in sheep (Salazar et al., 2003, 2007) and goats (Takigami et al., 2000; Mogi et al., 2007), including the VNL, GL, M/TcL, and GrL. The miral/tufted cells did not align to form M/TcL in both species, which is a similar pattern in the M/TcL of Korean roe deer. In the present study, we identified four layers in Korean roe deer, although we did not observe a clear demarcation between M/TcL and GrL. This finding suggests that the AOB of roe deer is structurally similar to that of most Artiodactyla such as goats and sheep. As for the functional implication of VSE in roe deer, we have tested the immunoreactivity of PGP9.5, which has been used as a mature neuron-specific marker in VSE receptor cells in rat (Johnson et al., 1994) and pig (Park et al., 2012b). In the present study, we have found that PGP9.5 was immunodetected in the receptor cell bodies and their dendrites in the free border, suggesting that receptor cells in the VSE were fully matured and functioning to sense odoriferous substances. In conclusion, this is the first morphological and descriptive study of the VNS of Korean roe deer. Our study confirms that there is only a single VNO–AOB pathway. The general features of the VNO and the four strata of the AOB are similar to those of ruminants. Acknowledgement This study was supported by a research grant of Jeju National University in 2013. References Geisler S, Heilmann H, Veh RW. An optimized method for simultaneous demonstration of neurons and myelinated fiber tracts for delineation of individual trunco- and palliothalamic
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A morphological study of the vomeronasal organ and the accessory olfactory bulb in the Korean roe deer, Capreolus pygargus Changnam Park a , Meejung Ahn b , Jae-Yuk Lee c , Sang Lee c , YoungMin Yun a,c , Yoon-Kyu Lim a , Kazumi Taniguchi d , Taekyun Shin a,∗ a Laboratory of Veterinary Anatomy, College of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju 690-756, Republic of Korea b Department of Anatomy, School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea c Jeju Wildlife Rescue Center, Jeju 690-121, Republic of Korea d Laboratory of Veterinary Anatomy, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
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Article history: Received 8 May 2013 Received in revised form 2 August 2013 Accepted 4 August 2013 Keywords: Accessory olfactory bulb Lectin Roe deer Vomeronasal organ
a b s t r a c t The vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the Korean roe deer (Capreolus pygargus) were studied histologically to evaluate their morphological characteristics. Grossly, the VNO, encased by cartilage, has a paired tubular structure with a caudal blind end and a rostral connection through incisive ducts on the hard palate. In the VNO, the vomeronasal sensory epithelium (VSE) consists of galectin-3-positive supporting cells, protein gene product (PGP) 9.5-positive receptor cells, and basal cells. The vomeronasal respiratory epithelium (VRE) consists of a pseudostratified epithelium. The AOB strata included a vomeronasal nerve layer (VNL), a glomerular layer (GL), a mitral/tufted cell layer, and a granular cell layer. All lectins used in this study, including Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA), labeled the VSE with varying intensity. In the AOB, both the VNL and the GL reacted with BSI-B4, SBA, and WGA with varying intensity, but not with UEA-I. This is the first morphological study of the VNO and AOB of the Korean roe deer, which are similar to those of goats. © 2013 Elsevier GmbH. All rights reserved.
Introduction The vomeronasal system (VNS), an olfactory system, consists of the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), the vomeronasal amygdala, and nerves connecting them all (Takami, 2002; Halpern and Martinez-Marcos, 2003; Yokosuka, 2012). The VNS is present in mammals, reptiles, amphibians (Taniguchi and Saito, 2011), but morphological and functional differences clearly exist even between closely related species such as mice and rats (Takigami et al., 2000; Tirindelli et al., 2009). Depending on the species, the VNS is related to a variety of functions, including reproduction through the perception of pheromones (Gelez and Fabre-Nys, 2004; Keller et al., 2009).
Abbreviations: AOB, accessory olfactory bulb; BSI-B4, Bandeiraea simplicifolia agglutinin isolectin B4; GL, glomerular layer; GrL, granular cell layer; M/TcL, mitral/tufted cell layer; PGP, protein gene product; SBA, soybean agglutinin; UEAI, Ulex europaeus agglutinin I; VNL, vomeronasal nerve layer; VNO, vomeronasal organ; VNS, vomeronasal system; VRE, vomeronasal respiratory epithelium; VSE, vomeronasal sensory epithelium; WGA, Triticum vulgaris wheat germ agglutinin. ∗ Corresponding author. E-mail address: [email protected] (T. Shin). 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.08.003
In the past several decades, the morphological characteristics of the VNOs of various animals have been studied, including those in mice (Salazar et al., 2001; Salazar and Sanchez Quinteiro, 2003), rats (Salazar and Sanchez Quinteiro, 1998; Lee et al., 2012), golden hamsters (Taniguchi et al., 1992; Taniguchi, 2008) and cats (Salazar and Sanchez-Quinteiro, 2011). Furthermore, comparative morphological studies have been conducted on the VNOs of domestic animals including horses and cattle (Lee et al., 2003; Taniguchi and Mikami, 1985), goats (Takigami et al., 2000), pigs (Park et al., 2012a) and sheep (Salazar et al., 2007). However, little is known about the VNOs of wild ruminants apart from the Scandinavian moose (Vedin et al., 2010). The behavioral characteristics of wild roe deer have been studied under natural conditions (McLoughlin et al., 2007; Richard et al., 2008). On Jeju Island, Korea, the Korean roe deer Capreolus pygargus inhabits fields and meadows, particularly on Halla mountain (Han et al., 2007). Few studies have addressed their morphological characteristics apart from a genetic identification study (Han et al., 2007). Hence, as a first step to understanding the morphological characteristics of this animal, we histologically analyzed two VNS organs, the VNO and AOB of the Korean roe deer.
C. Park et al. / Acta Histochemica 116 (2014) 258–264 Table 1 Description of Korean roe deer used in this study. Age* (years)
Weight (kg)
Sex
2 3 3 3 3 3
20 25 21 21 23 22
Male Male Male Male Male Male
*
The estimated age was calculated by the number of horn branches.
Materials and methods Tissue preparation VNO and AOB samples from seven (six male, one female) roe deer C. pygargus (Han et al., 2007) were obtained from the Jeju Wildlife Rescue Center. Their ages were determined to be two to three years old, respectively (Table 1), based on the number of horn branches (spikes). For light microscopy, the VNO and AOB were removed immediately after death and fixed in 10% buffered formalin for 48 h. All experimental procedures were conducted in accordance with Jeju National University Guidelines for the Care and Use of Laboratory Animals. Histological examination Formalin-fixed VNOs were trimmed and decalcified in sodium citrate–formic acid solution with several changes of the solution, until the bony pieces softened as shown in our previous study (Park et al., 2012b). Then, decalcified VNO and olfactory bulbs containing AOB were dehydrated in a graded ethanol series (70%, 80%, 90%, 95%, and 100%), cleared in xylene, embedded in paraffin, and sectioned at a thickness of 5 m. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E). To visualize the AOB nerve fiber tract and strata, Kluver–Barrera staining (0.1% Luxol Fast Blue and 0.1% Cresyl violet) was applied to paraffin sections (Kluver and Barrera, 1953; Geisler et al., 2002). The morphological study of VSE and AOB was accomplished in six male roe deer because only one sample of female VNO was obtained and both the VNO and AOB in the female were histoloigcally similar those of the males.
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endogenous peroxidase activity. Then non-specific binding was blocked with 10% normal goat serum (ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA), washed in phosphate-buffered saline (PBS, pH 7.4) for 1 h, and then allowed to react with the rat anti-galectin-3 antibody (1:1000) for 1 h at room temperature. After washing in PBS, the sections were reacted for 45 min with biotinylated rabbit anti-rat IgG (1:100; Vector Laboratories). Rabbit polyclonal antibody to PGP 9.5 (1:800) was reacted with a biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories). After another wash in PBS, the sections were incubated for 45 min with the avidin–biotin peroxidase complex (ABC Elite Kit; Vector Laboratories) prepared according to the manufacturer’s instructions. After washing in PBS, the peroxidase reaction was developed for 3 min using a diaminobenzidine substrate (DAB Kit; Vector Laboratories), prepared according to the manufacturer’s instructions. Sequentially, the sections were counterstained with hematoxylin for 30 s, washed in running tap water for 20 min, dehydrated through a graded ethanol series, cleared with xylene, and mounted with Canada balsam (Sigma–Aldrich, St. Louis, MO, USA). Lectin histochemistry The following lectins (all of which were purchased from Sigma–Aldrich, St. Louis, MO, USA) were used: Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA). Lectins are commonly used to identify receptor cells and/or their matching area in the AOB (Taniguchi et al., 1993; Salazar and Sanchez Quinteiro, 2003). Lectin histochemistry was performed as in our previous studies (Park et al., 2012b). In brief, the paraffin-embedded VNO and AOB were sectioned to a thickness of 5 m using a microtome. The sections were mounted on glass microscope slides, and the paraffin was removed. Then the sections were rehydrated. Endogenous peroxidase activity was blocked through 30 min incubation with 0.3% hydrogen peroxide in methanol. After three washes with PBS, the sections were incubated with either BSI-B4-peroxidase (diluted 1:50), SBA-peroxidase (diluted 1:100), UEA-I-peroxidase (1:200), or WGA-peroxidase (1:100) for 3 h at room temperature. Signals were developed using a DAB substrate kit (Vector). The sections were counterstained with hematoxylin before mounting. Negative controls were processed through omission of lectin during the staining procedure to clarify the reactivity of lectin histochemisty.
Antibodies Results To confirm the presence of receptor cells and supporting cells in the VSE, immunohistochemistry was performed using a rabbit polyclonal antibody to protein gene product 9.5 (PGP 9.5) or a rat anti-galectin-3 monoclonal antibody, respectively. The rat anti-galectin-3 monoclonal antibody was purified by affinity chromatography from the supernatant of hybridoma cells (clone TIB-166TM , M3/38.1.2.8. HL.2; American Type Culture Collection, Manassas, VA, USA) and used at a final concentration of 1–5 g/mL for immunohistochemistry. This antibody has been used to detect galectin-3 in the tissues of various animals including ungulates, cows (Kim et al., 2008), and pigs (Park et al., 2012b). Rabbit polyclonal antibody to PGP 9.5 (Biotrend, Cologne, Germany) has been used to detect receptor cells in the VNOs of humans and rats (Johnson et al., 1994; Witt et al., 2002). Immunohistochemistry Sections (5 m) of paraffin-embedded tissue were deparaffinized and heated in a microwave (800 W) in citrate buffer (0.01 M, pH 6.0) for 5 min. After cooling the slides, the sections were exposed to aqueous 0.3% hydrogen peroxide for 20 min to block
Gross anatomy The VNO had a prominent tubular-shaped structure about 80 mm long on the floor of the nasal cavity, adjacent to the vomer (Fig. 1, boxed area). The ducts originated from medially located openings in the rostral hard palate caudal to an incisive papilla (Fig. 1, inset, hollow arrows). From the openings, the VNO was arranged under the nasal septum bilaterally and stretched over two-thirds of the nasal cavity. The vomeronasal nerves (Fig. 1, arrows) were observed in sagittal sections through the nasal septum; they innervated the AOB (Fig. 1, circled area). Histological features of the VNO The morphological features of the VNO are shown at low magnification in Fig. 2A. A lamina of cartilage wrapped around the whole structure and connective tissue, glands, vessels, and nerves making up the soft tissue of the VNO were organized around the vomeronasal duct. The vomeronasal glands were most consistently found to communicate with the lumen of the VNO at two locations:
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In the lamina propria, there were enlarged blood vessels and numerous vomeronasal glands (Fig. 2A). The ductal epithelium and acini of the vomeronasal glands were mainly located under the VRE. On the medial side, the vomeronasal nerve bundles were positioned under the VSE (Fig. 2A).
Immunohistochemical analysis of the VNS epithelium and the lamina propria
Fig. 1. Gross anatomy of the rostral part of the head of the adult Korean roe deer (Capreolus pygargus) on the left lateral side. The maxilla and the nasal conchae were removed. The vomeronasal nerves (VNn, arrows) derived from the vomeronasal organ (VNO, boxed area) project in the dorsocaudal direction. The circled area marks the position of the accessory olfactory bulb. Inset shows the incisive papilla (hollow arrows) connected to the incisive duct in the palate. OB: olfactory bulb, Asterisk: nasopharynx. Scale bar = 1 cm.
through short ducts located at the dorsal junction of the VSE and the VRE, and through short ducts located at the ventral junction of the VSE and VRE (Fig. 2A, boxed area). The VSE was located in the medial portion of the VNO and consisted of supporting cells, receptor cells, and basal cells (Fig. 2B). The free border of the VSE was adjacent to the lumen of the vomeronasal duct (Fig. 2B, arrowheads). The VRE was located in the lateral portion of the VNO and consisted of pseudostratified epithelium (Fig. 2C).
In the VSE, galectin-3 immunoreactivity was observed in supporting cells (Fig. 3A, arrows), as well as in apices of the epithelium (Fig. 3A). In addition, thin processes of galectin-3-positive cells reached the basal layer (Fig. 3A, arrowheads). In contrast, no galectin-3 reactivity was observed in the receptor cells or in the basal cells. In the lamina propria, the ductal epithelium of the vomeronasal glands was positive for galectin-3 (Fig. 3A, hollow arrow). PGP 9.5 expression was detected in receptor cells in the middle layer of the VSE (Fig. 3B, arrows), but not in supporting cells or basal cells (Fig. 3B). PGP 9.5-positive cells extended some dendrites to the free border (Fig. 3B, arrowheads). The axonal bundles of PGP 9.5positive cells were spread across the lamina propria (Fig. 3B, hollow arrow).
Histological features of the AOB The olfactory bulb was obliquely located in the rostral portion of the forebrain (Fig. 1). The AOB was an oval-shaped structure located in the dorso-medial part of the main olfactory bulb (Fig. 4A). Microscopically, it was possible to divide the main olfactory bulb into several layers, including the olfactory nerve layer, glomerular layer
Fig. 2. Histology of the vomeronasal organ of the Korean roe deer. (A) Cross-section through the ventral part of the nasal septum. The vomeronasal cartilage (C) encapsulates the lamina propria. The vomeronasal nerve bundles (N) are mainly spread under the vomeronasal sensory epithelium (VSE). There are large blood vessels (bv) and the vomeronasal glands (gl) under the vomeronasal respiratory epithelium (VRE). At the dorsal (boxed area) and ventral parts of the lumen, the vomeronasal gland ducts open into the junction between the VSE and the VRE. RE: respiratory epithelium. (B) Higher magnification of the VSE. The VSE is composed of three layers that are discernible (SC, supporting cell; RC, receptor cell; BC, basal cell). Dashed line marks the basement membrane. Arrowheads indicate the free border. (C) Higher magnification of the VRE. The VRE consists of non-sensory, ciliated pseudostratified epithelium. (A–C), H–E staining. (A), scale bar = 250 m; (B) and (C), scale bars = 25 m.
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Fig. 3. Immunohistochemical localization of galectin-3 and PGP 9.5 in the vomeronasal sensory epithelium. (A) In the VSE, galectin-3 is expressed in the supporting cells (arrows). Galectin-3 positive cells extend thin processes to the basal cell layer (arrowheads) as well as to the luminal apices of the supporting cells. In the lamina propria, the ductal epithelium of the vomeronasal glands is weakly positive for galectin-3 (hollow arrow). (B) PGP 9.5-positive cells are arranged in the middle layer of the VSE (arrows). Some dendrites of PGP 9.5-positive cells reach the free border (arrowheads). PGP 9.5-positive axons are spread in the lamina propria (hollow arrow). Scale bars = 25 m.
(GL), external plexiform layer, mitral cell layer, and granular cell layer (GrL) (Fig. 4B). In the AOB (Fig. 4C), four layers including the vomeronasal nerve layer (VNL), GL, mitral/tufted cell layer (M/TcL), and granular cell layer (GrL) were found, although the demarcation between M/TcL and GrL was not obvious. The thickness of the AOB (Fig. 4C) was thinner than that of the main olfactory bulb (Fig. 4B). Both the VNL and GL were well organized (Fig. 4C). In the M/TcL,
the mitral/tufted cells with round or oval shape were not clearly organized in rows. The granular cells were dispersed in the GrL (Fig. 4C). Because of the population and distribution of the M/Tcells and the granular cells, it was difficult to distinguish the boundary between M/TcL and GrL. In Luxol Fast Blue–Cresyl violet staining (Fig. 4D), the lateral olfactory tract was clearly identified under the GrL, and the VNL and GL were also well visualized.
Fig. 4. Histological findings of the olfactory bulb and accessory olfactory bulb located in the rostral part of the telencephalon. (A) The bulb is divided into the main olfactory bulb (left rectangle) and the accessory olfactory bulb (right rectangle). (B) Higher magnification of the main olfactory bulb in (A). The olfactory nerve cell layer (ONL) is the first layer below which lies the glomerular layer (GL). Under the GL, several layers follow: the external plexiform layer (ePL), the mitral cell layer (ML), and the granular cell layer (GrL). (C) Higher magnification of the accessory olfactory bulb in (A). It is divided into the vomeronasal nerve layer (VNL), the glomerular layer (GL), the mitral/tufted cell layer (M/TcL), the granular cell layer (GrL) and the lateral olfactory tract (LOT). (D) Kluver–Barrera-stained parasagittal section of the accessory olfactory bulb. Each AOB strata is visualized. The lateral olfactory tract (LOT) is under the base of the GrL. (A–C), H&E staining. (D) Kluver–Barrera staining. (A) Scale bar = 1000 m; (B–D) scale bars = 100 m.
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Fig. 5. Lectin histochemistry of BSI-B4 (A), SBA (B), UEA-I (C), and WGA (D) in the VSE of Korean roe deer. (A–D), counterstained with hematoxylin. Scale bars = 25 m.
Lectin histochemistry of the VSE and AOB BSI-B4 staining weakly labeled the perikarya of receptor cells and basal cells, but not supporting cells (Fig. 5A). SBA labeled receptor cells but neither supporting cells nor basal cells (Fig. 5B). UEA-I stained supporting (but not receptor or basal) cells in the VSE (Fig. 5C). WGA stained receptor cells intensely (but not supporting or basal cells) (Fig. 5D). In summary, in the AOB, the VNL and GL positively reacted with three lectins (BSIB4, SBA, and WGA), with heterogeneous staining in the whole AOB; these layers did not react with UEA-I (Fig. 6A–D). There was no specific staining in the negative control (not illustrated).
The lectin binding patterns in this study are summarized in Table 2. Discussion This is the first descriptive morphological study of the VNOs and AOBs of Korean roe deer. The morphological characteristics of the VNO are similar to those of other ruminants, including Scandinavian moose (Vedin et al., 2010), sheep (Salazar et al., 2003), and goats (Takigami et al., 2000; Mogi et al., 2007). In mice (Salazar and Sanchez Quinteiro, 2003), there are apical and basal receptor cell zones in the VSE, which project to the anterior and posterior AOB,
Fig. 6. Sagittal section of the accessory olfactory bulb labeled by BSI-B4 (A), SBA (B), UEA-I (C), and WGA (D) in Korean roe deer. The vomeronasal nerve layer and glomerular layer are positive for BSI-B4, SBA, and WGA, but not UEA-I: Counterstained with hematoxylin. Scale bars = 200 m.
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Table 2 Lectin binding patterns in the vomeronasal system of Korean roe deer. Lectin (concentration, g/mL)
Structure
BSI-B4 (5.0 × 10−1 )
SBA (1.0 × 10−2 )
UEA-I (2.0 × 10−2 )
WGA (1.0 × 10−2 )
VSE
Supporting cells Receptor cells Basal cells
− + ++
− ++ −
++ − −
− +++ −
AOB
Vomeronasal nerve layer Glomerular layer
+ ++
+ +
− −
+++ +++
The intensity of lectin binding in each structure is expressed as follows: −, negative; +, weak; ++, moderate; +++, intense.
respectively. These projections form two distinct VNO–AOB pathways (Halpern et al., 1998). In contrast, there is only a single VNS pathway between the VNO and AOB of cats (Salazar and SanchezQuinteiro, 2011), sheep (Salazar et al., 2007), pigs (Salazar et al., 2000), and goats (Takigami et al., 2000). In the present study, we found one zone of receptor cells in the VSE of roe deer, with no division of AOB revealed by lectin histochemistry, indicating the formation of a single VNO–AOB pathway. Thus, we postulate that the VNS of roe deer contains a single VNO–AOB pathway, similar to those of the other animals mentioned above. As for the lectin binding patterns in the VNOs of ruminants including sheep, the VSE has been shown to be positive for UEA-I, BSI-B4, Dolichos biflorus agglutinin (DBA), and Lycopersicon esculentum agglutinin (LEA) (Salazar et al., 2000). However, the specific VSE cells that each lectin stains have not been described. In the present study, we found that each lectin bound differently to the various cells, namely, receptor cells, supporting cells, and basal cells. Briefly, receptor cells were positive for BSI-B4, SBA, and WGA, but not UEA-I, whereas supporting cells were only positive for UEAI. The functional role of each lectin in the VSE of the roe deer remains to be elucidated in further studies. There are four AOB strata layers in sheep (Salazar et al., 2003, 2007) and goats (Takigami et al., 2000; Mogi et al., 2007), including the VNL, GL, M/TcL, and GrL. The miral/tufted cells did not align to form M/TcL in both species, which is a similar pattern in the M/TcL of Korean roe deer. In the present study, we identified four layers in Korean roe deer, although we did not observe a clear demarcation between M/TcL and GrL. This finding suggests that the AOB of roe deer is structurally similar to that of most Artiodactyla such as goats and sheep. As for the functional implication of VSE in roe deer, we have tested the immunoreactivity of PGP9.5, which has been used as a mature neuron-specific marker in VSE receptor cells in rat (Johnson et al., 1994) and pig (Park et al., 2012b). In the present study, we have found that PGP9.5 was immunodetected in the receptor cell bodies and their dendrites in the free border, suggesting that receptor cells in the VSE were fully matured and functioning to sense odoriferous substances. In conclusion, this is the first morphological and descriptive study of the VNS of Korean roe deer. Our study confirms that there is only a single VNO–AOB pathway. The general features of the VNO and the four strata of the AOB are similar to those of ruminants. Acknowledgement This study was supported by a research grant of Jeju National University in 2013. References Geisler S, Heilmann H, Veh RW. An optimized method for simultaneous demonstration of neurons and myelinated fiber tracts for delineation of individual trunco- and palliothalamic
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