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Chem. Senses 26: 517-522, 2001
© Oxford University Press 2001

Vomeronasal Epithelial Cells of Human Fetuses Contain Immunoreactivity for G Proteins, Go{alpha} and Gi{alpha}2

Shigeru Takami, Maiko Yukimatsu, George Matsumura1 and Fumiaki Nishiyama

Department of Anatomy, School of Health Sciences, Kyorin University, Hachioji, Tokyo 192-8508 and 1 Department of Anatomy, School of Medicine, Kyorin University, Mitaka, Tokyo 181-8611, Japan

Correspondence to be sent to: S. Takami, Department of Anatomy, School of Health Sciences, Kyorin University, 476 Miyashita, Hachioji, Tokyo 192-8508, Japan. e-mail: takamis{at}kyorin-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two G protein subfamilies, Go{alpha} and Gi{alpha}2, were identified and localized immunohistochemically in the vomeronasal organ (VNO) of 5-month-old human fetuses. Immunoreactivity for Go{alpha} and Gi{alpha}2 was present in a subset of vomeronasal epithelial cells. Prominent immunoreactivity was observed in apical processes and their apical terminals facing onto the vomeronasal lumen. Nerve fibers associated with the VNO exhibited intense immunoreactivity for Go{alpha} and weak immunoreactivity for Gi{alpha}2. Since Go{alpha} and Gi{alpha}2 are characteristically expressed and coupled with putative pheromone receptors in rodent vomeronasal receptor neurons, the present results suggest the possibility that vomeronasal epithelial cells containing Go{alpha} and Gi{alpha}2 in human fetuses are chemosensory neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Most terrestrial vertebrates possess a vomeronasal system (VNS) that is anatomically separated from the main olfactory system. The VNS is known to be a pheromone-detecting system in many species. The sensory organ of the VNS, the vomeronasal organ (VNO), is a blind sac, situated bilaterally at the base of the nasal septum, and a liquid-filled lumen of the sac communicates with the nasal or oral cavity (Wysocki, 1979Go; Halpern, 1987Go; Meredith, 1991Go). Sensory cells of the VNS, vomeronasal receptor neurons (VRNs) (Takami et al., 1994Go, 1995aGo), are located within the sensory epithelium (SE) of the VNO. The VRNs are the first-order neurons of the VNS and bipolar in shape; every functional VRN has a single dendrite and an axon originating from its soma. The dendrite reaches the surface of the lumen of the VNO to form a dendritic terminal that bears microvilli (Graziadei, 1977Go; Halpern, 1987Go; Meredith, 1991Go). The axon leaves the SE, forming a group with neighboring axons, an axon bundle. All axons of VRNs make axon bundles, which are called the vomeronasal nerve (VNN), traveling toward the brain, and terminate on dendrites of second-order neurons of the VNS, in the accessory olfactory bulb (AOB) (Halpern, 1987Go).

Several chemical characteristics of VRNs have been reported using immunohistochemical (Mori, 1987Go; Schwarting and Crandall, 1991Go; Taniguchi et al., 1993Go; Johnson et al., 1994bGo; Takami et al., 1994Go, 1995bGo; Jia and Halpern, 1996Go) and lectin histochemical (Takami et al., 1992Go, 1994Go, 1995aGo; Ichikawa et al., 1994Go; Nakajima et al., 1998Go) techniques. Among these characteristics, the presence of immunoreactivity for two G protein subfamilies, Go{alpha} and Gi{alpha}2 (Jia and Halpern, 1996Go), is most directly related to the function of VRNs because these G proteins are expressed by VRNs and coupled with putative pheromone receptor molecules (Herrada and Dulac, 1997Go; Matsunami and Buck, 1997Go). In fact, VRNs are physiologically excited by pheromones/pheromone candidates (Inamura et al., 1999Go; Leinders-Zufall et al., 2000Go).

In the fetal life of humans, the VNO is recognizable as early as gestational week 8 (Kreutzer and Jafek, 1980Go). After that, the tubular-shaped VNO, which opens into the nasal cavity, the VNN and the AOB become well developed for many weeks (Pearson, 1942Go; Bossy, 1980Go; Kreutzer and Jafek, 1980Go). The VNO and VNN have been documented in 6-month-old fetuses (McCotter, 1915Go) and even at 28 weeks old (Nakashima et al., 1985Go). Several reports have demonstrated that neuron-specific molecules are immunolocalized in VRN-like cells that are present in the epithelium of fetal VNO (Boehm and Gasser, 1993Go; Johnson et al., 1994aGo; Kjaer and Hansen, 1996Go), called the vomeronasal epithelium (VE) (Takami et al., 1993Go). The above anatomical and immunohistochemical findings suggest that human fetuses contain a functional VNS, although no direct evidence exists to confirm this. To clarify that the VE contains VRNs, the present study aimed to examine whether vomeronasal epithelial cells (VECs) in 5-month-old human fetuses display immunoreactivity for Go{alpha} and Gi{alpha}2. Results of our study demonstrate that VRN-like cells in the VE of 5-month-old human fetuses contain Go{alpha}- and Gi{alpha}2-immunoreactive cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
We utilized tissues of nine Japanese 5-month-old fetuses (mean crown–rump length: 13.4 cm). These tissues were taken from human tissue specimens kept and authorized in the Department of Anatomy I, Kyorin University School of Medicine. They had been initially fixed in Formalin-based fixatives and stored in 70% ethanol for many years, so that their fixation/preservation condition seemed not to be perfect. The tissue blocks including the VNOs were dissected out from these specimens, and re-fixed in ice-cold Zamboni’s fixative overnight, cryoprotected in sucrose (15, 30%) in phosphate-buffered saline, and embedded in Tissue-Tek®, O.C.T. Compound (Sakura Finetechnical Co., Tokyo, Japan) as previously described (Takami et al., 1994Go, 1995aGo,bGo; Yamagishi et al., 1996Go). Fourteen-micrometer-thick cryostat sections were made by an electrical cryostat (Microm HM 500 OM, Carl Zeiss Co., Jena, Germany) in the frontal and sagittal planes.

The protein gene product 9.5 (PGP), which was originally isolated from human brains (Thompson et al., 1993Go), is a soluble protein of 27 kDa that is widely distributed in neurons and neuroendocrine cells (Wilson et al., 1988Go). In the olfactory system, PGP is contained in olfactory receptor neurons and VRNs (Takami et al., 1993Go; Taniguchi et al., 1993Go; Johnson et al., 1995Go; Takami et al., 1995bGo). Furthermore, PGP is contained in VECs of the human VNO (Takami et al., 1993Go; Kjaer and Hansen, 1996Go). Since the specimens used in this study were stored in Formalin/ ethanol solutions for many decades, we first examined whether these VNOs were appropriate for further immunohistochemical studies by use of an antiserum to PGP.

As a method for immunohistochemical labeling, we utilized a peroxidase-labeled streptavidin–biotin (PLSAB) method that is one of the most sensitive detection systems. Its details were previously described (Takami et al., 1995bGo). Briefly, primary antibodies used in this study were polyclonal antibodies against PGP raised in rabbits (Ultra Clone, Ltd, Wellow, UK) at a dilution of 1:2000, polyclonal rabbit IgG against Go{alpha} (Santa Cruz Biotech., Inc., Santa Cruz, CA) at a dilution of 1:1000, and two polyclonal rabbit IgGs against Gi{alpha}2 (Santa Cruz Biotech., Inc., and Wako Pure Chem., Osaka, Japan) at a dilution of 1:500. As a chromogen, aminoethyl carbazol was used throughout this study. Omission of the primary antibodies from the experimental protocols and preadsorption controls for two antibodies from Santa Cruz were carried out as negative controls. As positive-tissue controls, cryostat sections of VNOs of rats (Sprague–Dawley strain) were utilized.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The PLSAB method was successful to visualize PGP immunoreactivity in all specimens examined. In the VNOs, plenty of VRN-like VECs were PGP-immunoreactive (Figure 1a). PGP immunoreactivity was distributed throughout their somata and apical processes, some of which reached the luminal surface to form terminals (Figure 1b). Nerve fibers associated with the VNO were PGP-immunoreactive as well. Omission of the PGP antiserum resulted in no reaction products. These observations thus indicate that the specimens used here are liable for further immunohistochemical analyses, although the condition of fixation/ preservation of these tissues seemed not be perfect. Neighboring sections to those labeled with the PGP antiserum were immunolabeled with Go{alpha} and Gi{alpha}2 antisera.



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  		Figure 1 PGP immunoreactivity in the VNO of a 5-month-old fetus. (a) Low magnification of a sagittal section. Immunoreactivity for PGP is present in somata (small arrows) in the vomeronasal epithelium (VE), nerve fiber bundles (large arrow) and cells in the lamina propria (double arrows). Enlargement of the area enclosed by brackets are shown in (b). L, vomeronasal lumen. Bar = 100 mm. (b) The enlarged area in (a) showing PGP-immunoreactive cells in the VE. Immunoreactivity is distributed in somata (s), processes (small arrows) and their terminals (arrowheads) at the luminal surface. Bar = 20 mm.

Figure 2Go{alpha} immunoreactivity in the VNO of a 5-month-old fetus. (a) Medium magnification of a sagittal section showing Go{alpha}-immunoreactive somata (s) and process (small arrow) in the vomeronasal epithelium (VE). A small (medium arrow) and large (large arrows) nerve fiber bundles are present in the lamina propria (LP). Bar = 50 mm. Inset shows a preadsorption control for Go{alpha} immunoreactivity, frontal section. No specific reaction is observed in the VE. L, vomeronasal lumen. Bar = 50 mm. (b) Higher magnification of the VE, cut in a sagittal plane. Go{alpha}-immunoreactive somata (s) and processes (small arrows), some of which are continuous from the somata, are seen. A small nerve fiber bundle (medium arrow) just below the basement membrane is present in the lamina propria. Bar = 20 mm. (c) Vomeronasal epithelial cell showing a Go{alpha}-immunoreactive soma (s), an apical process (medium arrow) and its terminal (arrowhead). Neighboring processes belonging to other cells are also immunoreactive (small arrows). Bar = 20 mm.

Figure 3 Gi{alpha}2 immunoreactivity in the VNO of 5-month-old fetuses. (a) Medium magnification of a frontal section showing Gi{alpha}2-immunoreactive somata (s) and processes (small arrows) in the vomeronasal epithelium (VE). Some immunoreactive processes can be traced to the epithelial surface facing the vomeronasal lumen (L). Gi{alpha}2 immunoreactivity is also detected in nerve fibers (medium arrows) in the lamina propria (LP). Bar = 50 µm. The inset shows a negative control where the primary antiserum is omitted from the staining protocol, frontal section. No specific staining is observed in the VE. L, vomeronasal lumen. Bar = 50 µm. (b) Gi{alpha}2-immunoreactive VEC whose soma (s) is located in the basal layer of the VE. Immunoreactive processes (small arrows), one of which is derived form the soma apically, are also seen. Dotted lines indicate the relative location of the basement membrane of the VE. Bar = 20 µm. (c) Sagittal section containing cells with a spindle-shaped soma. Immunoreactivity for Gi{alpha}2 is present in apically located somata (s), processes (small arrows), their terminals (arrowheads) and basal processes (small arrows). Bar = 20 µm.

 
Go{alpha} immunoreactivity was present in a subset of VECs (Figure 2a). In general, the morphology of Go{alpha}-immunoreactive cells was similar to that of PGP-immunoreactive VECs. Go{alpha}-immunoreactive ovoid somata were distributed from the basal to the apical regions of the VE. Apical processes derived from the somata were also immunoreactive for Go{alpha} (Figure 2a,b). Terminals of apical processes of VECs were recognizable at the luminal surface of the VE (Figure 2c). Although we could not determine whether immunoreactive basal processes originated from the Go{alpha}-immunoreactive somata, Go{alpha}-immunoreactive fibers were present in the vicinity of the basement membrane (Figure 2a,b). Preadsorption control experiments resulted in no specific labeling in the above VECs, which were neuron-like cells, as well as nerve fibers in the lamina propria (Figure 2a, inset).

Gi{alpha}2 antisera labeled a small subset of VECs (Figure 3a–c) and nerve fiber bundles in the lamina propria (Figure 3a). There far fewer Gi{alpha}-immunoreactive VECs was than Go{alpha}-immunoreactive ones. Omission of the antisera from the experimental protocols and preadsorption control experiments (Figure 3a, inset) resulted in no specific reaction in the VE and lamina propria. Although some Gi{alpha}2-immunoreactive VECs were morphologically similar to Go{alpha}-immunoreactive VECs (Figure 3a,b), others had a spindle-shaped soma (Figure 3c). In addition to a single apical process, each of them tended to have a relatively thick basal process that could be traced down to the basement membrane (Figure 3c). The most intense immunoreactivity for Gi{alpha}2 was observed in apical processes and their terminals at the lumen, whereas moderate immunoreactivity was present in somata and basal processes.

As positive tissue controls, VNOs of adult rats were immunolabeled using Go{alpha} and Gi{alpha}2 antisera. The most intense immunoreactivity for Go{alpha} was present in the ‘mucomicrovillar complex’ (Takami et al., 1994Go, 1995aGo) of the vomeronasal sensory epithelium; intense immunoreactivity was found in the vomeronasal nerve bundles, and moderate immunoreactivity in VRNs (Figure 4a). Gi{alpha}2 antisera labeled the mucomicrovillar complex, VRNs and vomeronasal nerve bundles (Figure 4b). By contrast, nonsensory epithelium of the VNO did not exhibit any specific reaction (Figure 4b).



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  		Figure 4 Frontal sections of a rat VNO as positive tissue controls. (a) Go{alpha} immunoreactivity. The mucomicrovillar complex (curved arrow) of the vomeronasal sensory epithelium (VSE) contains the most intense immunoreactivity, and the somata and dendrites of VRNs exhibit weak immunoreactivity (arrows). Vomeronasal nerve bundles (VNB) in the lamina propria contain moderate immunoreactivity. Bar = 100 µm. (b) Gi{alpha}2 immunoreactivity. The mucomicrovillar complex (curved arrow) and isolated single dendritic terminals of vomeronasal receptor neurons (arrowheads) contain the most intense immunoreactivity in the VSE, whereas specific labeling is not detected in the apical surface of the vomeronasal nonsensory epithelium (VNE). Somata and dendrites of vomeronasal receptor neurons exhibit weak immunoreactivity (arrows). Bar = 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The results of the present study have demonstrated for the first time that VRN-like VECs in the human VNO contain immunoreactivity for Go{alpha} and Gi{alpha}2. Although there were relatively high background readings in the VE sections labeled with antisera to Go{alpha} and Gi{alpha}2, it is still convincing that immunoreactivity for them is present within the apical processes and their terminals of the VECs. Genes for these G proteins are contained in VRNs of rodents (Herrada and Dulac, 1997Go; Matsunami and Buck, 1997Go), and intense immunoreactivity for these G proteins are localized in their dendritic terminals (Jia and Halpern, 1996Go; Matsuoka et al., 2001Go). Our positive control experiments using rat VNOs have confirmed that the intense immunoreactivity for Go{alpha} and Gi{alpha}2 is localized in dendritic terminals of VRNs (see Figure 4). Thus, it is likely that genes for Go{alpha} and Gi{alpha}2 are also expressed in VECs of human fetuses and these G proteins are utilized within cellular components of these cells, including terminals of apical processes.

Adult rodent VRNs express putative pheromone receptors that are coupled with these Go{alpha} and Gi{alpha}2 (Herrada and Dulac, 1997Go; Matsunami and Buck, 1997Go). Physiological experiments using rats and mice have indicated that Go{alpha}- and/or Gi{alpha}2-containing VRNs in fact respond to putative pheromones. In adult rats, Go{alpha}- and Gi{alpha}2-containing VRNs responded differentially to pheromone-containing urine samples (Inamura et al., 1999Go). Six putative pheromones excited a subset of VRNs of mice which contain Gi{alpha}2 (Leinders-Zufall et al., 2000Go). However, there has been no direct evidence indicating that the VNS plays a role as a chemosensory system and detects pheromones in utero. In rodents, it has been proposed that fetal VNS is functional (Pedersen et al., 1983Go; Coppola and Millar, 1994Go). In humans, the presence of chemoreception in utero has been suggested (Schaal and Orgeur, 1992Go; Schaal et al., 1998Go), although neither the chemosensory system in human fetuses nor odorants/pheromones in the amniotic fluid have been identified. Functional VRNs of animals bear microvilli from dendritic terminals (Graziadei, 1977Go; Halpern, 1987Go) and recent immunoelectron microscopic studies have demonstrated that putative pheromones (Takigami et al., 1999Go), and Go{alpha} and Gi{alpha}2 (Matsuoka et al., 2001Go), are localized in dendritic terminals and microvilli of VRNs. Therefore, if one would claim that VECs of 5-month-old fetuses are functional chemosensory neurons, it is necessary to demonstrate that these VECs bear microvilli projecting from their terminals, and that pheromone receptors are identified and localized in these microvilli.

We have described that some Gi{alpha}2-immunoreactive VECs had a spindle-shaped soma, indicating that they are different from animal VRNs in that the basal process (axon) is very thin (Graziadei, 1977Go; Halpern, 1987Go) and hardly visualized by conventional light microscopy. However, VECs that are similar in shape to the above Gi{alpha}2-immunoreactive VECs have been reported in the VE of a 4-month-old baby (Johnson et al., 1994aGo) and of adult humans (Takami et al., 1993Go; Johnson et al., 1994aGo). Thus, it is likely that the spindle-shaped bipolar cells are generally present in human VE throughout life.

This paper has also shown that some nerve fibers associated with the VE are Go{alpha}/Gi{alpha}2-immunoreactive. Since vomeronasal nerve fibers of rodents exhibit Go{alpha}/Gi{alpha}2 immunoreactivity (Jia and Halpern, 1996Go), it is probable that the Go{alpha}/Gi{alpha}2-immunoreactive fibers demonstrated here are axons of VECs and a part of the VNN that is connected with the AOB (Humphrey, 1940Go; Bossy, 1980Go). In rodents, segregated projections of Go{alpha}/Gi{alpha}2-immunoreactive VNN to the AOB (Jia and Halpern, 1996Go) were present. To clarify the function of the VNS in humans, it is important to examine whether any topographical pattern of projection of Go{alpha}/Gi{alpha}2-immunoreactive VNN fibers is present human fetuses. Also, their AOB needs to be examined to determine whether synaptic terminals of VECs are identifiable.

In conclusion, the present study has revealed that VECs of 5-month-old human fetuses contained immunoreactivity for Go{alpha} and Gi{alpha}2. Moreover, it has shown the possibility that fetal VECs of humans are functional chemosensory neurons.


   Acknowledgments
 
This study was partially supported by a Frontier Research Grant from Ministry of Education of Japan (No. 088773), and a research grant for Science and Engineering 1997 from Shiseido Co. Ltd, Japan.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
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Accepted February 1, 2001


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