Histological definition of the vomeronasal organ in humans and chimpanzees with a comparison to other primates.код для вставкиСкачать
THE ANATOMICAL RECORD 267:166 –176 (2002) Histological Definition of the Vomeronasal Organ in Humans and Chimpanzees, With a Comparison to Other Primates TIMOTHY D. SMITH,1,2* KUNWAR P. BHATNAGAR,3 KRISTIN L. SHIMP,1 JONATHAN H. KINZINGER,1 CHRISTOPHER J. BONAR,4 ANNIE M. BURROWS,2,5 MARK P. MOONEY,2,6 AND MICHAEL I. SIEGEL2 1 School of Physical Therapy, Slippery Rock University, Slippery Rock, Pennsylvania 2 Department of Anthropology, University of Pittsburgh, Pittsburgh, Pennsylvania 3 Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky 4 Cleveland Metroparks Zoo, Cleveland, Ohio 5 Department of Physical Therapy, Duquesne University, Pittsburgh, Pennsylvania 6 Department of Oral Medicine and Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania ABSTRACT The vomeronasal organ (VNO) is a chemosensory structure that has morphological indications of functionality in strepsirhine and New World primates examined to date. In these species, it is thought to mediate certain socio-sexual behaviors. The functionality and even existence of the VNO in Old World primates has been debated. Most modern texts state that the VNO is absent in Old World monkeys, apes, and humans. A recent study on the VNO in the chimpanzee (Smith et al., 2001b) challenged this notion, demonstrating the need for further comparative studies of primates. In particular, there is a need to establish how the human/chimpanzee VNO differs from that of other primates and even nonhomologous mucosal ducts. Histochemical and microscopic morphological characteristics of the VNO and nasopalatine duct (NPD) were examined in 51 peri- and postnatal primates, including humans, chimpanzees, five species of New World monkeys, and seven strepsirhine species. The nasal septum was removed from each primate and histologically processed for coronal sectioning. Selected anteroposterior intervals of the VNO were variously stained with alcian blue (AB)-periodic acid-Schiff (PAS), PAS only, Gomori trichrome, or hematoxylin-eosin procedures. All strepsirhine species had well developed VNOs, with a prominent neuroepithelium and vomeronasal cartilages that nearly surrounded the VNO. New World monkeys had variable amounts of neuroepithelia, whereas Pan troglodytes and Homo sapiens had no recognizable neuroepithelium or vomeronasal nerves (VNNs). Certain unidentified cell types of the human/chimpanzee VNO require further examination (immunohistochemical and electron microscopic). The VNOs of P. troglodytes, H. sapiens, and New World monkeys exhibited different histochemistry of mucins compared to strepsirhine species. The nasopalatine region showed great variation among species. It is a blind-ended pit in P. troglodytes, a glandular recess in H. sapiens, a mucous-producing duct in Otolemur crassicaudatus, and a stratified squamous passageway in all other species. This study also revealed remarkable morphological/ histochemical variability in the VNO and nasopalatine regions among the primate species examined. The VNOs of humans and chimpanzees had some structural similarities to nonhomologous ciliated gland ducts seen in other primates. However, certain distinctions from the VNOs of other primates or nonhomologous epithelial structures characterize the human/ chimpanzee VNO: 1) bilateral epithelial tubes; 2) a superiorly displaced position in the same plane as the paraseptal cartilages; 3) a homogeneous, pseudostratified columnar morphology with ciliated regions; and 4) mucous-producing structures in the epithelium itself. Anat Rec 267:166 –176, 2002. © 2002 Wiley-Liss, Inc. Key words: vomeronasal complex; histochemistry; mucous glands; mucins; VNO Grant sponsor: College of Health and Human Services, Slippery Rock University; President’s Academic Initiatives, Slippery Rock University. *Correspondence to: Dr. Timothy D. Smith, School of Physical Therapy, Slippery Rock University, Slippery Rock, PA 16057. Fax: (724) 738-2113. E-mail: email@example.com © 2002 WILEY-LISS, INC. Received 6 June 2001; Accepted 5 March 2002 DOI 10.1002/ar.10095 Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). 167 PRIMATE VOMERONASAL ORGAN AND NASOPALATINE DUCT TABLE 1. Age group, preservation, and source of the primate species investigated species* Mirza coquereli (Coquerel’s dwarf lemur) Microcebus murinus (Grey lesser mouse lemur) Eulemur mongoz (Mongoose lemur) Galago moholi (Mohol’s galago) Galagoides demidoff (Dwarf galago) Otolemur crassicaudatus (Fat-tailed bushbaby) O. garnettii (Garnett’s bushbaby) Callithrix jacchus (White ear-tufted marmoset) Leontopithecus rosalia (Golden lion tamarin) L. chrysomelas (Golden-headed lion tamarin) Saguinus geoffroyi (Geoffroy’s tamarin) Alouatta caraya (Black howler monkey) Homo sapiens (Human) Pan troglodytes (Common chimpanzee) n Agea Preservationb Sourcec 1 2 1 1 1 4 4 2 3 1 6 1 21 3 1 3 1 1 1 3 3 3 1, 2 3 1, 2, 3 3 2, 3 2, 3 a a b a a b b b b b b a c, d a, b DUPC DUPC CMZ DUPC DUPC DUMC DUMC DUMC CMZ CMZ CMZ CMZ SRU UP, CMZ *Classification according to Nowak (1999). 1, neonate; 2, infant/juvenile; 3, adult. b a, frozen, later immersed in formalin; b, immersed in formalin after death; c, embalmed; d, immersed in Bouin’s solution. c CMZ, Cleveland Metroparks Zoo; DUMC, Duke University Medical Center; DUPC, Duke University Primate Center; SRU, Slippery Rock University; UP, University of Pittsburgh. a For more than a century, it has been thought that humans possess at least a postnatal remnant of the vomeronasal organ (VNO) (Kölliker, 1877; Potiquet, 1891), whereas other Old World primates lack any trace of the VNO postnatally (Loo, 1973; Zingeser, 1984; Ankel-Simons, 2000). This has led to confusion regarding the homology of the human VNO to that of other mammals (see Smith et al., 2001a). Recent work has shown that chimpanzees also possess a VNO during postnatal ontogeny (Smith et al., 2001b), which partly resolves this issue. At the same time, such findings hint that the degree of variability of the VNO among primates may be greater than previously thought. In a recent review, Smith et al. (2001a) sought to define character states to account for the variation. However, delineating the unique characteristics of the human or chimpanzee VNO has remained difficult. Humans and chimpanzees have VNOs that are more superiorly positioned compared to those of other primates (Smith et al., 2001b). This atypical location, combined with a markedly different (simplified) epithelial morphology creates a difficulty in that such a structure may be easily confused with other mucosal structures, such as gland ducts or remnants of the nasopalatine duct (NPD) (see Jacob et al., 2000; Bhatnagar and Smith, 2001; Smith et al., 2001c). It is therefore essential to morphologically define the “VNO character state” of chimpanzees and humans. Such a clarification will augment a reexamination of other catarrhine primates for VNO presence or absence, and will ultimately lead to a better understanding of the evolution of this chemosensory system among the Haplorhini (Old World monkeys, New World monkeys, tarsiers, apes, and humans). Among all haplorhine primates, the human VNO has received the most attention to date. The function of the human VNO is highly debated (Grosser et al., 2000; Wysocki and Preti, 2000; Bhatnagar and Smith, 2001; Kouros-Mehr et al., 2001), in part due to the scarcity of comparative studies to place the structure in proper context (Smith et al., 2001a). Certain histochemical and morphological aspects of the human VNO differ markedly from those of other mammals (Roslinski et al., 2000; Bhatnagar and Smith, 2001). Mucin produced in the human VNO appears histochemically similar to that of respira- tory epithelium (acidic and neutral mucin production), and the VNO appears to act both in secretion (via goblet cells and other intraepithelial glands) and transport (Roslinski et al., 2000; Bhatnagar and Smith, 2001). In contrast, the vomeronasal glands of most other mammals are more elaborate and produce mucins that differ histochemically (primarily neutral mucins) from other glands of the nasal cavity (Roslinski et al., 2000). Few studies have compared the histochemistry of the VNO among primates (Hunter et al., 1984), and the histochemical characteristics of the VNO in chimpanzees are still poorly understood. It is also unclear how the VNO and remnants of the NPD in humans and chimpanzees compare to those of other primate species or to other duct-like structures within the nasal mucosa. The present study examines the vomeronasal and NPD regions of the nasal septum among primates in order to provide a clearer morphological/histochemical definition of the VNO in humans and chimpanzees. MATERIALS AND METHODS Cadaveric nasal septal tissues from 51 primates (adults except as noted; see Table 1), including Pan troglodytes (two juveniles, one adult), Homo sapiens (one juvenile, 20 adults), Saguinus geoffroyi (three neonates, two juveniles, one adult), Leontopithecus rosalia (two neonates, one juvenile), Leontopithecus chrysomelas, Callithrix jacchus, Alouatta caraya, Mirza coquereli (one neonate), Microcebus murinus, Eulemur mongoz (one neonate), Galago moholi (one neonate), Galagoides demidoff (one neonate), Otolemur garnettii, and O. crassicaudatus were examined. Tissues were obtained from gross anatomy laboratories, research laboratories, Duke University Primate Center, and the Cleveland Metroparks Zoo. Initial methods of preservation involved freezing, embalming, or immersion in 10% buffered formalin (Table 1), but all specimens were stored for at least 2 days in 10% buffered formalin prior to decalcification and histological processing. Either the entire head or one-half of the head was processed in the perinatal primates. In all other cases, resected septa were used. Tissues were decalcified in a formic acid solution (formic acid-sodium citrate or Cal-Ex II; Fisher Scientific, Pittsburgh, PA), dehydrated in an ethanol series, cleared 168 SMITH ET AL. Figure 1. 169 PRIMATE VOMERONASAL ORGAN AND NASOPALATINE DUCT TABLE 2. Morphological and histochemical observations on the primate species investigated Epithelial morphology Species Mirza coquereli Microcebus murinus Eulemur mongoz Galago moholi Galagoides demidoff Otolemur crassicaudatus Otolemur garnettii Callithrix jacchus Leontopithecus rosalia L. chrysomelas Saguinus geoffroyi Alouatta caraya Homo sapiens Pan troglodytes NPD VNO NP VND VNO AB PAS AB PAS StSq StSq StSq StSq StSq StSq StSq StSq StSq NA StSq StSq PSt,C StSq,PSt,C StC StC StC StC StC StSq, K StSq, K StSq St-Sq/StC NA StC StC StC StC StC, NE SiC, NE StC, NE StC, NE SiC/StC, NE StC/PSt, NE StC/PSt, NE PSt, NE SiC/StC/PSt, NE SiC/StC/PSt, NE SiC/StC/PSt, NE StC/PSt, NE? PSt,C PSt,C – – – – – ⫹⫹ – – – NA – – ⫹⫹ ⫹⫹ – – – – – ⫹⫹ – – – NA – – ⫹⫹ ⫹⫹ – – – – – ⫹⫹a – ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ a AB⫹ seen only in region of the VNO duct. NP, nasopalatine duct region (duct or remnant); VND, vomeronasal organ duct; VNO, vomeronasal organ; AB, alcian blue; PAS, periodic acid-Schiff; C, ciliated; K, keratinized cells at apex; NE, neuroepithelium; PSt, pseudostratified, columnar; SiC, simple cuboidal/columnar; StC, stratified cuboidal/columnar; StSq, stratified squamous; ⫹, restricted patches of tissue with AB/PAS positivity; ⫹⫹, widespread AB/PAS positivity seen; –, no AB/PAS positivity seen; NA, relevant tissue not available. in xylene, embedded in paraffin, and serially sectioned in the coronal plane at 10 –25 m. Every 10th section was mounted on glass slides and alternately stained with hematoxylin-eosin and Gomori trichrome protocols. The tissues were examined for the presence of the VNO using a Leica photomicroscope. In each specimen, both the nasopalatine and more posterior regions were examined for structures resembling either the human/chimpanzee VNO (Johnson et al., 1985; Smith et al., 2001a) or the generalized mammalian VNO (Ciges et al., 1977; Breipohl et al., 1979). The beginning and end points of the VNO were identified, and the 1st, 25th, 50th, 75th, and 100th percentiles of the VNO length were noted. Unstained sections that approximated each anteroposterior (or rostrocaudal) percentile were mounted on glass slides and stained with a combined alcian blue (AB)-periodic acid-Schiff (PAS) protocol to ascertain the presence of acidic (AB⫹) or neutral (PAS⫹) mucins (Humason, 1979). Selected sections from each species were also mounted on glass slides and stained with PAS only, to verify whether AB may have masked the affinity of PAS⫹ structures. Each series was examined at ⫻25 to ⫻1,000 by light microscopy to describe the histological structure of the VNOs and other mucosal structures. Numerous aspects of the primate VNOs in the present study were compared to those of humans as previously described (Roslinski et al., 2000; Bhatnagar and Smith, 2001). In addition, the apex of the VNO epithelium was carefully examined using human and chimpanzee specimens, and cilia were clearly seen in all sections. Cilia were examined with regard to circumferential distribution (i.e., to what extent they lined the apex of the VNO epithelium in coronal cross-section) and with regard to anteroposterior distribution (i.e., whether they were seen at a particular anteroposterior level). Associated glandular tissues were described by examining sections stained with an AB/PAS protocol. Characteristics of the tissues were then compared among species at similar anatomical levels. To control for the possibility that PAS⫹ structures contained glycogen and not mucins, a series of sections containing the VNO were soaked in diastase of malt solution at 37° for 1 hr (Humason, 1979). The slides were then stained with another control series of adjacent sections in PAS. Fig. 1. A: Coronal section showing the vomeronasal duct (open arrow) and (slightly posteriorly on the other side) the VNO in a neonatal Galago moholi. The section is posterior to the connection of the vomeronasal duct and the nasopalatine duct (NPD). B: Coronal section of the VNO in Mirza coquereli (neonate) showing the precocious development of the organ. Note the thicker, medial sensory epithelium. C: The VNO of an adult Otolemur crassicaudatus, showing the thicker, medioventral neuroepithelium (VNNE) and thinner, lateral nonsensory epithelium (NSE) and adjacent VNNs. D: The NSE of an adult Otolemur crassicaudatus. Note the nonciliated, simple columnar epithelium. E: The VNNE of an adult Otolemur crassicaudatus. Note the dense population of receptor cells (white arrows) and microvillar apex (small open arrows). F: Coronal section showing the VNOs of a 1-month-old Saguinus geoffroyi. G: Note that in the magnified view sparse receptor cells (white arrows) can be seen in the VNO epithelium. H: The VNO of an adult Callithrix jacchus exhibited a uniform sensory epithelium (white arrows indicate receptor nuclear layer) at nearly all anteroposterior levels. I: Posteriorly, the VNO of L. rosalia (4-month-old shown) resembled that of strepsirhines (small arrows indicate medioventral neuroepithelium). J: The VNO of a juvenile chimpanzee shows the typical enlarged lumen (L ⫽ lumen) and abundant communicating glands (GD ⫽ gland duct). K: The region of the gland duct is enlarged to show certain nonciliated, unidentified cells with round nuclei (small arrows). NS ⫽ nasal septum; VNC ⫽ vomeronasal cartilage. Scale bars: A ⫽ 300 m; B, I, J ⫽ 150 m; C ⫽ 150 m; D, E, H, and K ⫽ 40 m; F ⫽ 600 m; G ⫽ 30 m. RESULTS VNOs were found in all primates, although with variable morphology (Fig. 1). In several specimens, epithelial damage (interpreted as freezing artifacts that occurred in two of the specimens that were frozen before fixation) prevented certain morphological observations, as noted below. VNOs of all strepsirhine species exhibited a well defined vomeronasal neuroepithelium (VNNE ) at all ages 170 SMITH ET AL. Figure 2. PRIMATE VOMERONASAL ORGAN AND NASOPALATINE DUCT 171 (Fig. 1A–D) and nonsensory epithelium (NSE) (Fig. 1E; Table 2). The VNO epithelia of New World species were more highly variable. A clear VNNE was found in small patches in neonatal S. geoffroyi and Leontopithecus spp., but was more widely distributed in juveniles and adults (Fig. 1F, G, and I). In both genera, VNNE could be found on all sides of the VNO (superior, inferior, medial, and lateral) and was sometimes interrupted by segments of NSE. Callithrix jacchus was characterized by a homogeneous sensory lining in the VNO (Fig. 1H), with only minute portions of NSE. In all callitrichids, receptor populations appeared to be more dense toward the posterior half of the VNO. Neonatal and juvenile L. rosalia showed a remarkable morphology in the posterior one-fourth of the VNO, in which the organ resembled that of strepsirhines, with a medial VNNE and a lateral NSE (Fig. 1I). The VNO epithelium of A. caraya was difficult to interpret due to apparent damage from freezing (disrupted epithelia), but some small segments of stratified or pseudostratified NSE could be seen. In addition, VNNs superior to the VNO (and ascending in an anteroposterior direction from the VNO) suggested the likely existence of a VNNE in disrupted regions. The NSE of the VNO in all strepsirhines and New World monkeys was generally nonciliated epithelium of variable morphology (Fig. 1D; Table 2), but isolated ciliated cells were sometimes seen—for example, in the more posterior portions of NSE in Otolemur spp. Vomeronasal ducts were lined with stratified cuboidal epithelia in all strepsirhine and New World monkeys, but had some apparent keratinization in some species (Table 2). In addition, the vomeronasal duct of O. crassicaudatus had intraepithelial mucous glands in the duct wall. The VNOs of P. troglodytes and H. sapiens were more simplified oval or round tubes (Figs. 1J, and 2A and F). Initially, these were lined with stratified cuboidal epithelia, and made a transition to pseudostratified, columnar epithelia within 50 –100 m. In most specimens, the pseudostratified columnar epithelia were nonciliated for the first several (anterior) sections, and later sections contained cilia projecting into the lumen from all sides, with small gaps of nonciliated cells (Fig. 2D and E). As reported previously (Smith et al., 1998), some human VNOs were not suitable for identifying cilia due to distortion or apparent tissue decay that obscured the epithelial apex of the VNO. Using more recently sectioned material (Smith et al., 2001c), three human septa were suitable for serial anteroposterior examination for distribution of cilia. All chimpanzees had at least some discernable cilia, but only one had sufficient preservation to reveal that cilia were present in all anteroposterior sectional levels (Fig. 2B–E). The anteroposterior distribution of cilia varied among humans. In the 2-year-old human, the anterior two-thirds of the VNO was nonciliated. In most of the posterior one-third, the apex of the VNO epithelium was almost completely ciliated, with small patches of nonciliated cells. A 77-year-old male human exhibited nearly continuous cilia (circumferentially) in the VNO for most of the length following the short, stratified cuboidal “duct” (Fig. 2G), although the posterior-most sections had no cilia (Fig. 2I). A 79-year-old male exhibited variation in the circumferential distribution of cilia, from nearly continuous to patchy. Both of these adults displayed markedly different distributions of cilia on the right and left VNOs, with one side having a more continuous circumferential presence of cilia. In both P. troglodytes and H. sapiens, the presence of dark lines suggestive of basal bodies were also seen at the apex of ciliated cells (Fig. 2D and G). Basal cells could also be seen, and gaps in the line of basal bodies (Fig. 2D) were seen where goblet cells or at least one other type of columnar cell intervened among ciliated cells. The latter cells were more rare than other types, were nonciliated, and had nuclei within the middle region of the VNO. In one juvenile P. troglodytes, certain of these cells had round, dark nuclei and were sparsely scattered throughout the epithelium (Fig. 1J–K). There were no apparent unmyelinated nerves in the adjacent lamina propria. There were some structures resembling the VNOs of humans and chimpanzees in the other primates—specifically unilateral, ciliated gland ducts. In general, most gland ducts were lined with simple cuboidal epithelia and were nonciliated, but in one strepsirhine (E. mongoz) a unilateral epithelial duct that bore some resemblance to the human/chimpanzee VNO was seen. This duct ended 70 m anterior to the sectional level of the NPD and thus had no direct connection to the VNO. It was found in the same sectional plane and was spatially separated from the lamina transversalis anterior. The duct was clearly ciliated and had associated AB–/PAS– glandular tissue. The left VNO of one L. rosalia neonate also ended in a ciliated gland duct; cilia were observed in gland ducts entering the superior pole of the VNO in C. jacchus. In all strepsirhines and New World primates, the NPD was lined with stratified (usually squamous) epithelium (Fig. 3; Table 2). In P. troglodytes, a partially patent NPD was seen, completely fused on the oral (anteroinferior) aspect (Figs. 3E and 4J). It was mainly lined with stratified squamous epithelium, although large patches of respiratory (pseudostratified columnar) epithelia were noted in one specimen, and seromucous glands were seen to empty to the surface through both types of epithelia. In H. sapiens, the only vestige of the NPD was a small fossa located within an elongated recess (Fig. 3F), the nasopal- Fig. 2. A: A coronal section showing the VNOs of a juvenile chimpanzee, which exhibit a spatial separation from the paraseptal cartilage (PC). B and C: The chimpanzee VNO had multiple glandular communications (open arrows ⫽ glands; GD ⫽ gland duct; GL ⫽ gland) throughout most of the anteroposterior extent of the VNO (B ⫽ 1st percentile; C ⫽ 50th percentile). In the left VNO of one specimen (a juvenile chimpanzee) it was possible to observe cilia (open arrows) and basal bodies (arrow heads) throughout the entire anteroposterior extent (D ⫽ 75th percentile; E ⫽ 100th percentile). Basal cells (BCs) were seen, and gaps in (D) basal bodies revealed goblet cells (GCs) and at least one other nonciliated cell type. F: A coronal section showing the location of the VNO in a 2-year-old human. G: VNO in a 77-year-old male. In some humans, a continuous row of cilia (open arrows) could be seen on all inner surfaces of the VNO (G ⫽ 100 m posterior to the VNO opening in the subject shown), and numerous glands (GLs) could be seen to empty into the VNO throughout most of the anteroposterior extent (H ⫽ 50th percentile of same adult human). I: In most humans, however, some nonciliated portions of the VNO were seen, as shown in the 100th percentile of the VNO in the same adult human. L ⫽ lumen of VNO; NS ⫽ nasal septum. Scale bars: A and F ⫽ 600 m; B, C, and H ⫽ 150 m; D and G ⫽ 30 m; E and I ⫽ 40 m. 172 SMITH ET AL. Fig. 3. A: The region of the NPD in a neonatal Eulemur mongoz, just posterior to its communication with the vomeronasal duct (*). A, B, and D: The NPD was lined with stratified squamous epithelium in all strepsirhine species. B and C: An adult Otolemur crassicaudatus was unique in possessing intraepithelial mucous glands (open arrows) in both the vomeronasal duct and NPD. The vomeronasal duct of most species was stratified cuboidal, as shown in (D) an infant Saguinus geoffroyi, although Otolemur was unique in possessing vomeronasal ducts (*) mostly lined with (C) a keratinized, stratified squamous epithelium. The NPD of the chimpanzee was blind-ended, penetrating only partially through the superior palate ((E) juvenile chimpanzee). The NPD of Pan troglodytes was mostly lined with a stratified squamous epithelium that had numerous seromucous glands (open arrows) emptying through it (GD ⫽ gland ducts). No NPD was found in humans ((F) 79-year-old male); there was only a remnant in the form of a shallow groove, the NPR. The NPR was lined with respiratory epithelium and had numerous seromucous glands (open arrows) in the lamina propria. Scale bars: A and F ⫽ 600 m; B and E ⫽ 300 m; C and D ⫽ 150 m. atine recess (NPR) (Bhatnagar and Smith, 2001). Both the recess and fossa were lined with a highly glandular respiratory mucosa (Figs. 3F and 4H). Glands associated with the VNO were PAS⫹ and AB– in all strepsirhines examined, in both neonates and adults (Fig. 4A and C), whereas glands not communicating with the VNO lumen, but emptying into the nasal cavity directly, were AB⫹ and PAS⫹ (Fig. 4C). However, glands of the vomeronasal duct were AB⫹/PAS⫹ in O. crassicaudatus (Fig. 4B). In all primates, unicellular glands lining the nasal cavity (i.e., goblet cells) were AB⫹ and PAS⫹. In all New World monkeys, H. sapiens, and P. troglodytes, glands that communicated with the VNO were AB⫹/ PAS⫹, and some AB⫹ secretions were found in gland ducts or the VNO itself (Fig. 4D–G, and K). In all species represented at different ages, subadults were relatively less PRIMATE VOMERONASAL ORGAN AND NASOPALATINE DUCT PAS⫹ compared to adults. Glands were most abundant superiorly and posteriorly in S. geoffroyi and L. rosalia, but were otherwise sparse; sparse goblet cells also were seen, but not in all sections examined. Glands were similarly distributed and more abundant in C. jacchus. Unique rostral glands were seen in A. caraya, which coursed posteriorly via large ducts that connected to the VNO at its intersection with the NPD. Glands that emptied into the VNO of H. sapiens and P. troglodytes also were AB⫹/ PAS⫹, including intraepithelial glands (Fig. 4G and K). Glands associated with the nasopalatine region in H. sapiens and P. troglodytes were AB⫹/PAS⫹ (Fig. 4H–I). In both H. sapiens and P. troglodytes, adult specimens had more goblet cells and intraepithelial glands in the VNO than juveniles. Numerous adult humans and the adult chimpanzee had AB⫹/PAS⫹ material completely occluding the VNO lumen at some anteroposterior levels. The sections that were soaked in diastase of malt prior to staining still showed PAS positivity in all glands, indicating that the PAS⫹ structures contained neutral mucins. DISCUSSION Bhatnagar and Smith (2001) described two functional categories of VNOs: chemosensory and nonchemosensory. Morphologically, there appear to be more categories, or character states, of VNOs that may have phylogenetic, rather than strictly functional, significance (Smith et al., 2001a). VNOs of humans and chimpanzees are similar to each other and histologically distinct from the chemosensory VNOs seen in strepsirhines. Unfortunately, the human/chimpanzee VNO is minute (approximately 2–12 mm in length) and is more easily confused with other mucosal structures compared to the chemosensory VNO of strepsirhines. Proportionately, adult VNO length ranged from 16.4% to 19.7% of head length in Microcebus murinus, but from only 0.5% to 5.6% of head length in H. sapiens (Smith et al., 2001d). The VNO of humans and chimpanzees may be likened to some aspects of other mucosal structures in the nasal septum. For example, the human/chimpanzee VNO is similar to an exocrine gland duct in overall shape and because of the multiple seromucous glands that empty into its lumen. However, most seromucous gland ducts are nonciliated and simple cuboidal, and are not bilateral in nature (Smith et al., 2001a, b). The ciliated gland ducts found in E. mongoz and other species are more similar (see Fig. 6H in Smith et al., 2001a), but are unilateral structures found in variable locations. There are at least four cell types in the human/chimpanzee VNO: basal cells, ciliated cells, goblet cells, and at least one unidentified type of nonciliated columnar cell. Indications of basal bodies suggest that motile cilia, rather than long microvilli (as seen in the VNO of other mammals) or olfactory cilia (as seen in the olfactory epithelium), exist in the human and chimpanzee VNO. Although the embryogenesis of the VNO in chimpanzees has not been described, in humans the ontogeny of the VNO from a sensory structure (at 43 days to 12 weeks of embryonic development) to a duct-like, ciliated structure (cilia first appearing at 10 weeks fertilization age) can be clearly traced (Boehm and Gasser, 1993; Smith and Bhatnagar, 2000). Variability in the extent of anteroposterior or right vs. left presence of cilia among the few specimens examined emphasizes the need for further ontogenetic study of cilia in the VNO in H. sapiens and P. troglodytes. The ontogeny of the ciliated duct in E. mongoz is unclear, but unless it originated from 173 the anterior part of the primordial VNO, it simply represents a functional analog of the human/chimpanzee VNO. Ciliated gland ducts leading to the VNO in L. rosalia and C. jacchus strengthen this view, and suggest a function in secretion transport. One similarity between the human/chimpanzee VNO and that seen in other primates is in the vomeronasal duct, which consists of stratified cuboidal epithelium in all cases. The human and chimpanzee VNO may therefore be regarded as having a short duct, followed by pseudostratified, columnar epithelium. A noteworthy aspect of this duct in humans and chimpanzees is its glandular nature, with both external and intraepithelial glands. Intraepithelial glands of similar histochemistry also were seen in the vomeronasal duct of O. crassicaudatus. This presents an interesting point of comparison since O. crassicaudatus possesses the morphological structures (surrounding cartilaginous capsules, large venous sinuses) associated with the “vasomotor pumping” mechanism (Meredith and O’Connell, 1979) that is thought to aid VNO function (Hedewig, 1980). Thus, these secretions could potentially be drawn into the VNO in O. crassicaudatus and play a role in VNO function. On the other hand, it is noteworthy that these intraepithelial glands were histochemically more similar to glands of the nasal cavity proper than to the vomeronasal glands. It is therefore possible that they produced accessory secretions released to the nasal or oral cavities. It has recently been suggested (Jacob et al., 2000; Bhatnagar and Smith, 2001; Smith et al., 2001c) that some investigators attempting to study the human VNO have inadvertently examined the NPD region (containing only a small pit) rather than the VNO itself, as there are histological similarities between the two regions (Bhatnagar and Smith, 2001). This confusion may have occurred in H. sapiens, but there are clear distinctions between the human/chimpanzee VNO and the NPD of primates in general. The NPD, where present, shows adaptations for transport of substances between the oral and nasal cavities, and also to or from the VNO duct. In contrast, the nasopalatine region of P. troglodytes (blind-ended duct) and H. sapiens (minute pit) appears to provide accessory glandular tissue, unlike the other species examined (with the exception of O. crassicaudatus). The literature does not support the suggestion that only glands secreting neutral mucins are compatible with functional VNOs (e.g., see Salazar et al., 1997). However, the histochemical partitioning of neutral mucin secretions to the VNO and acidic/ neutral secretions to the remainder of the nasal cavity is striking in strepsirhines (and other mammals (see Roslinski et al., 2000)). In contrast, the VNO of New World monkeys was histochemically more similar to H. sapiens and P. troglodytes than to the strepsirhines, which may reflect general similarities among haplorhine primates. With the above features in mind, the VNO of humans and chimpanzees is defined by a suite of characteristics, as is the strepsirhine VNO. There is little overlap between these characteristics, rendering them useful character states. In order to exclude some similarities to seromucous gland ducts or specialized ciliated ducts, VNOs of H. sapiens and P. troglodytes are regarded as paired structures that are 1) bilateral epithelial tubes; 2) in a superiorly displaced position in the same plane as the paraseptal cartilages; 3) lined with a homogeneous, pseudostratified columnar morphology with ciliated regions; and 4) have internal mucous-producing structures (i.e., contain goblet 174 SMITH ET AL. Figure 4. PRIMATE VOMERONASAL ORGAN AND NASOPALATINE DUCT 175 Fig. 4. Histochemical variation of the vomeronasal and nasopalatine regions using AB-PAS staining, revealing acidic (AB) and neutral (PAS) mucins. A: A slightly oblique coronal section from a neonatal primate (Galago moholi) showing the NPD just posterior to the VNO opening, with the vomeronasal duct on the left side of the image and the VNO on the right side of the image. Note the absence of any AB⫹ (blue) stain adjacent to the VNO or its duct. B: A magnified coronal section showing the communication of the NPD to the VNO duct (open arrow) in an adult Otolemur crassicaudatus. Note the AB⫹ glands lining the ducts, which were unique in the species among strepsirhines. C: The VNO is shown in an adult Otolemur crassicaudatus. Note the PAS⫹ glands (open arrows) around the VNO, whereas the AB⫹ glands (small arrows) are found elsewhere, e.g., goblet cells along the respiratory epithelium. D: A neonatal Leontopithecus rosalia showing primarily AB⫹ glands communicating to the VNO; no glands were associated with the NPD. E: A 4-month-old L. rosalia showed somewhat more numerous AB⫹ (small arrows) and PAS⫹ (open arrow) glands, along with secretions in the lumen. F: The VNO of Callithrix jacchus showed more extensive AB⫹ glands than other callitrichids, shown here superior to the VNOs (small arrows). The (G) VNO and (H) NPR (a vestige of the duct) from a 2-year-old human are shown. I: The inset shows both regions and the distance between them. Note that the VNO has predominantly AB⫹ glands (G), whereas the NPR has both AB⫹ (small arrow) and PAS⫹ (open arrow) glands. The blind-ended NPD remnant (J) and VNO (K) of a juvenile chimpanzee had AB⫹ glands. L ⫽ VNO lumen; NPD ⫽ nasopalatine duct; NPR ⫽ nasopalatine recess; NS ⫽ nasal septum; PC ⫽ paraseptal cartilage; VNC ⫽ vomeronasal cartilage. Scale bars: A–D ⫽ 300 m; E and F ⫽ 150 m; G and H ⫽ 600 m; I ⫽ 800 m; J ⫽ 300 m; K ⫽ 150 m. cells or multicellular intraepithelial glands). It is possible to speculate that this character state may properly be regarded as a synapomorphic condition of hominoids, especially since epithelial tubes that are positionally similar to the human VNO have been located in orangutan and gorilla fetuses (C.S. Evans, personal communication). Functionally, this structure remains poorly understood, but highly distinctive aspects of the VNOs of humans and chimpanzees compared to other primates appear to indicate accessory glandular activity at minimum. The sparse, unidentified nonciliated cells seen in humans and chimpanzees offer little basis for speculation on their chemosensory function, especially in the absence of adjacent unmyelinated axons. Some such cells were roughly “receptor-like” in shape (e.g., in a chimpanzee (Fig. 1K)), yet these also may represent populations of other cells (such as leukocytes) known to occur in the NSE of the mammalian VNO (Adams and Weikamp, 1984; Carmanchahi et al., 1999). There is clearly a need to examine larger samples of fresh tissue from hominoids to gather immunohistochemical or electron microscopic data to clarify the identity of such cells. The data of the present study are limited in regard to functional interpretation, but may be in keeping with known trends regarding the central connection of the VNO in primates, the accessory olfactory bulb. The accessory olfactory bulb is absolutely and proportionately largest in strepsirhines, smaller in New World primates (Stephan et al., 1982), and only present prenatally in Old World primates (see Bhatnagar and Meisami, 1998; Meisami and Bhatnagar, 1998); the degree of neuroepithelial presence among primates in the present study appeared to correspond to those data. Interestingly, there also appeared to be a difference in the amount of VNNE in the neonatal strepsirhines compared to that in the two neonatal callitrichids (see also Evans and Grigorieva, 1994), although glandular development was incomplete at birth in all neonates. Previous efforts to locate a VNO in postnatal Old World primates may have been hampered by the expectation that the VNOs of Old World species would be located in a position similar to those of strepsirhines and New World species (i.e., near the palate, surrounded by vomeronasal cartilages). It is now clear that the VNO of humans and chimpanzees is positionally and histologically different from that of other primates; developmental evidence indicates that they are homologous structures (Starck, 1960; Smith and Bhatnagar, 2000)1. Recent attempts to find similar structures in postnatal Colobus guereza (Smith et al., 2001b) and Macaca spp. (Smith et al., 2001a), suggest that Cercopithecoids may lack the VNO postnatally (Jordan, 1972). It is necessary to reexamine the VNO using a broad spectrum of haplorhine primates, keeping in mind the unique character states exhibited by hominoids and New World monkeys. ACKNOWLEDGMENTS We are grateful to A.B. Taylor for arranging access to the marmoset tissues used in this study. All procedures related to processing the human material used for this study were reviewed and approved by the Human Studies Committee, Department of Anatomical Sciences and Neurobiology, University of Louisville, and the Institutional Review Board for the Protection of Human Subjects, Slippery Rock University. This is Duke University Primate Center publication #747. LITERATURE CITED Adams DR, Wiekamp MD. 1984. The canine vomeronasal organ. J Anat 138:771–787. Ankel-Simons F. 2000. 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