Interlobular excretory ducts of mammalian salivary glandsStructural and histochemical review.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 288A:498 –526 (2006) Interlobular Excretory Ducts of Mammalian Salivary Glands: Structural and Histochemical Review BERNARD TANDLER,1* CARLIN A. PINKSTAFF,2 AND CARLETON J. PHILLIPS3 1 Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, Ohio 2 Department of Anatomy, West Virginia University, Morgantown, West Virginia 3 Department of Biological Sciences, Texas Tech University, Lubbock, Texas ABSTRACT In the major salivary glands of mammals, excretory ducts (EDs) succeed striated ducts. They are for the most part interlobular in position, although their proximal portions sometimes are on the periphery of a lobule, where they occasionally retain some of the structural features of striated ducts. Based on a survey of a broad range of mammalian species and glands, the predominant tissue type that composes EDs is pseudostratiﬁed epithelium. In some species, there is a progression of epithelial types: the proximal EDs are composed of simple cuboidal or columnar epithelium that, in the excurrent direction, usually gives way to the pseudostratiﬁed variety. Secretory granules are visible in the apical cytoplasm of the principal cells of the EDs of only a few species, but histochemistry has shown the presence of a variety of glycoproteins in these cells in a spectrum of species. Moreover, the latter methodology has revealed the presence of a variety of oxidative, acid hydrolytic, and transport enzymes in the EDs, showing that, rather than simply acting as a conduit for saliva, these ducts play a metabolically active role in gland function. It is difﬁcult to describe a “typical” mammalian ED because it can vary along its length and interspeciﬁc variation does not follow a phylogenetic pattern. Moreover, in contrast to intercalated and striated ducts, ED cellular features do not exhibit a relationship to diet. Anat Rec Part A 288A:498 –526, 2006. © 2006 Wiley-Liss, Inc. Key words: submandibular gland; parotid gland; sublingual gland; excretory ducts; interlobular ducts; glycoproteins; histochemistry The present review of interlobular excretory ducts (EDs) in mammalian salivary glands is the third in a series that includes reviews of intercalated ducts (Tandler et al., 1998c) and secretion by striated ducts (Tandler et al., 2001). Our approach in each has been to summarize the literature (which sometimes is abundant) on the subject, present a substantial amount of previously unpublished comparative information from 250 or more species of mammals, and use evolutionary, systematic, and ecological information to interpret the comparative data set. In some instances, it has been possible to posit new, testable hypotheses about the function of salivary gland ducts (Phillips, 1996). Over the past decade, it has become increasingly clear that salivary glands have played a vital role in mamma© 2006 WILEY-LISS, INC. lian evolution and adaptive radiation (Phillips, 1996; Phillips and Tandler., 1996). To some extent, this role involves diet and the ability of particular species to access speciﬁc nutrient resources (Phillips et al., 1998; Tandler and Phil- *Correspondence to: Bernard Tandler, Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106. Fax: 216-368-3204. E-mail: email@example.com Received 8 June 2005; Accepted 29 December 2005 DOI 10.1002/ar.a.20319 Published online 12 April 2006 in Wiley InterScience (www.interscience.wiley.com). EXCRETORY DUCTS OF SALIVARY GLANDS Fig. 1. Sublingual: human being (Homo sapiens). Light micrograph of a parafﬁn-embedded ED. Note the thick covering of ﬁbrous connective tissue (CT), a part of an interlobular septum. Masson’s trichrome. Magniﬁcation ⫽ 200⫻. lips, 1998). In a broader context, however, salivary gland functions encompass a truly remarkable range of actions. Among these, salivary pheromones are signiﬁcant because they can include proteins that enable kin recognition, affect mate selection (premating reproductive isolation), and have a fundamental role in speciation (Wickliffe et al., 2002). With all these factors in mind, the importance of comparative morphological and ultrastructural data is obvious. Although some of the information reported here comes from animals prepared under laboratory conditions (B.T.), much more originates from specimens collected in the wild by one of the authors (C.J.P.), ﬁeld-ﬁxed according to previously described procedures (Phillips, 1985; Tandler et al., 1998c) and deposited as vouchers in one of three established research collections (Natural Science Research Laboratory of the Texas Tech Museum, Division of Mammals of the Carnegie Museum of Natural History, and the Nebraska State Museum of the University of Nebraska-Lincoln). These vouchers serve as a permanent foundation for species identiﬁcation and other relevant information about specimens from which our salivary gland data were obtained. Species data from the literature are reported here as provided by the original authors; in a number of instances, we have only common names (species unknown or unveriﬁable). Much of the published data is based on tissue samples for which there are no voucher specimens. Thus, those workers who use the data collected here (other than the bulk of those presented in Table 6) must keep in mind that species identiﬁcation cannot always be veriﬁed. The excurrent duct system of parotid and submandibular salivary glands consists of intercalated duct (ID), stri- 499 Fig. 2. Parotid: Hart’s little fruit bat (Enchisthenes hartii). Survey electron micrograph of a transitional duct. Although this duct actually is situated within an interlobular septum, it is positioned at the edge of a lobule and superﬁcially still possesses the appearance of a striated duct. In many species and glands, proximal EDs are structurally indistinguishable from striated ducts. Magniﬁcation ⫽ 1600⫻. ated duct (SD), excretory duct, and main excretory duct (MED) (Tandler, 1993a); in many species of rodents, a further duct segment, the granular convoluted tubule (granular duct), is interposed between intercalated and striated ducts (Gresik, 1994). In the sublingual gland of many mammals, the intercalated ducts shade into ducts that probably are EDs, but which may include patches of striated cells in their walls (Riva et al., 1988); in certain species such as the ferret, sublingual secretory endpieces drain directly into EDs without intervening intercalated or striated ducts (Jacob and Poddar, 1989). In the cat sublingual gland, endpieces often are in direct continuity with the MED (Tandler and Poulsen, 1977), so that this gland lacks intralobular and interlobular ducts altogether. Most published ultrastructural studies of entire parotid and submandibular glands include descriptions of IDs and SDs, but more often than not omit any description of the intraglandular EDs. In contrast, many detailed descriptions of the MED have appeared. This morphological neglect of the EDs is surprising because the physiology of these ducts may be of some importance in salivary homeostasis. In this review, we describe the structure of EDs in a variety of mammalian species. Despite their deceptively simple histology in human beings and in common laboratory animals, these ducts show a degree of variation across the spectrum of mammalian orders. Although in most mammals, EDs lack distinctive structural features (such as the basal striations of SDs), they are readily identiﬁed in both parafﬁn sections and in epoxy semithin sections by their extralobular (interlobular) 500 TANDLER ET AL. Fig. 3. Submandibular: African yellow bat (Scotophilus dingani). An ED that consists of pseudostratiﬁed epithelium, but the principal cells are low cuboidal. Magniﬁcation ⫽ 2,000⫻. Fig. 4. Parotid: gray “four-eyed” opossum (Philander opossum). A portion of an ED immediately distal to the striated duct at the left. The ED here consists of simple columnar epithelium. Magniﬁcation ⫽ 3,200⫻. position and their relatively thick mantle of ﬁbrous connective tissue (Fig. 1). It needs to be emphasized, however, that in many small-sized species of mammals, the major salivary glands are tightly compacted so that interlobular ducts are difﬁcult to locate. In certain species, especially among bats, we have noted that an entire major salivary gland (usually the parotid gland) consists of just a single lobule, so that such organs perforce lack interlobular ducts. If the most proximal portion of an MED in a monolobular gland is fortuitously included in a given section, we considered it to be functionally analogous to bona ﬁde EDs and included it in our study (Fig. 2). In recent textbooks of human histology, smaller EDs are described as consisting of pseudostratiﬁed epithelium that, as the ducts increase in caliber, become stratiﬁed columnar epithelium. Actually, at any given level of human EDs (and in many other species as well), they are composed of a melange of epithelial types, including simple cuboidal, simple columnar, pseudostratiﬁed, stratiﬁed cuboidal, and stratiﬁed columnar (Figs. 3–5), in no particular pattern (Tandler and Riva, 1986; Tandler, 1988), although a given ED can consist wholly of just one type or one epithelial type may predominate. In this review, we use the conventionally described histological organization of human EDs as the exemplar of these ducts and detail deviations from this arbitrary (nonphylogenetic) standard. The transition between striated ducts and EDs is not necessarily abrupt. Sometimes there are ducts of intermediate morphology, usually at the edge of a given lobule, and continuing between lobules. Such intermediates usually display a modicum of basal striations, although some proximal EDs sometimes exhibit all of the structural trappings of striated ducts (Fig. 6). These transitional ducts are not dealt with in detail in this review. However, we do take into account bona ﬁde EDs that resemble striated ducts, at least in their proximal portions. In many species, the morphogenesis of the major salivary glands is incomplete at the time of birth (Young and van Lennep, 1978). For example, in the neonatal mouse, the parotid gland consists of a canalized branching duct system and terminal buds. Sialographic studies have shown that in this species, the organization of the peripheral duct system is developed by 3 weeks postnatally, and that there are strain-speciﬁc differences in the ductular patterns (Domon and Kurabayashi, 1987). In the submandibular gland of the adult mouse, EDs account for ⬃ 1.7% of total gland volume (Pardini and Taga, 1996) and in the rat for ⬃ 1%. EDs constitute 2.8% of the volume of the rat parotid gland (Ježek et al., 1996). EXCRETORY DUCTS OF SALIVARY GLANDS 501 Fig. 5. Submandibular: Geoffroy’s long-nosed bat (Anoura geoffroyi). An ED that consists of stratiﬁed columnar epithelium. Magniﬁcation ⫽ 12,100⫻. In the submandibular gland of the South American sea lion (Otaria byronia), EDs represent 1.3% of the fractional volume, whereas in the parotid gland of the same animal they represent just 0.6% (Fava-de-Moraes et al., 1966); the same percentages apply respectively to the submandibular and parotid glands in the Southern fur seal, Arctocephalus australis (Fava-de-Moraes et al., 1966). The fractional volume of EDs in the human submandibular gland is not ﬁxed, but increases with age, ranging from ⬃ 0.5% during years 16 –25 to ⬃ 1.0% during years 86 –95 (calculated from Scott, 1977). In the rat submandibular gland (and probably in major salivary glands of other species as well), the ducts are at their largest at the gland hilus and, as they undergo several orders of branching, they become smaller in diameter (Lorber, 1991). These ducts and their covering of collagen are considered by Lorber (1991) to constitute a structural framework for the ﬂuctuant parenchyma that helps to maintain the patency of endpiece, ductular, and vascular structures in the face of cervical and mandibular movements that potentially could occlude them. The presence of abundant circumferential elastic ﬁbers in the connective tissue that surrounds the EDs (Phang and Rannie, 1982; Lorber, 1992) might be important in the mechanical interplay between ducts and their connective tissue milieu (Lorber, 1992). In an ultrastructural study of EDs in human parotid and submandibular glands, Lantini et al. (1990) describe the walls of these ducts as consisting of tall principal cells and widely scattered basal cells (and thus could properly be described as consisting of pseudostratiﬁed epithelium). The principal cells lack basal striations, but their lateral surfaces are highly plicated and interlocked. The irregularity of these lateral cell surfaces is amply conﬁrmed by scanning electron microscope (SEM) studies of EDs that have been divested of their basement membranes and that have been fractured to reveal their entire proﬁle (Riva et al., 1990, 1993; Riva, 1992) (Fig. 7). It is the abundant slender ridges on these external surfaces that are responsible for the lateral interfoliation of the principal cells. The 502 TANDLER ET AL. Fig. 6. Submandibular: minipig (Sus scrofa). The basal portion of a proximal ED that still retains the basal conﬁguration of a striated duct. Magniﬁcation ⫽ 15,800⫻. lateral folds house the transport enzyme, K⫹-pNPPase (Lantini et al., 1990). The luminal surfaces of the principal cells frequently protrude into the duct lumen—these protrusions are different from the apical blebs that are often observed in SDs and that in most cases represent ﬁxation artifacts (Tandler et al., 2001)—and that contain cytoplasm indistinguishable from that in lower reaches of the cells. SEM reveals that these ED protrusions are covered by stubby microvilli (Lantini et al., 1990). Although the principal cells contain many mitochondria, these organelles show no preferential location within the cytoplasm (Lantini et al., 1990). Despite their irregular distribution, the mitochondria are metabolically active, as shown by histochemical demonstration of dehydrogenase (Table 1) and oxidase (Table 2) activities. Typically, the principal cells include scattered cisternae of rough endoplasmic reticulum (RER) as well as some smooth endoplasmic reticulum (SER). In the principal cells of EDs in the accessory submandibular gland of the long-winged bat, Miniopterus magnater, there are many membranous crystalloids that appear to be localized specializations of the SER (Fig. 8) (Tandler et al., 1997a). We Fig. 7. Submandibular: human (Homo sapiens). A portion of the wall of an ED viewed in proﬁle by SEM. Note the vertically oriented lateral folds on the principal cells, as well as the numerous short microvilli on the luminal surface of these cells. Magniﬁcation ⫽ 5,000⫻. Micrograph courtesy of Alessandro Riva. have hypothesized that these organelles might be involved in steroid metabolism and conceivably could be involved in reproductive behavior in this species. Prominent lipofuscin deposits frequently are present in the supranuclear cytoplasm of ED cells; these undoubtedly represent lysosomal structures. Acid phosphatase, the marker enzyme for lysosomes, has been cytochemically identiﬁed in the ED cells of many mammals (Table 3). Other acid hydrolases also have been identiﬁed in ED cells (Table 4). The apical protrusions include numerous empty-appearing vesicles, but in some cells these vesicles have a moderate density. Cytokeratins, components of the cytoskeleton, are a prominent feature of the apical cytoplasm of EDs in many different species. These are intermediate ﬁlament proteins that have been classiﬁed into at least 20 subtypes according to their molecular weights and isoelectric points. Both monoclonal and polyclonal antibodies have been used to identify cytokeratins in salivary glands; in fact, more than 60 different antibodies have been used in salivary gland studies. Table 5 is a compendium of species 503 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 1. Enzyme Dehydrogenases of ED cells Common Name Gland References P,SL,SM P SL SM SM P P,SM SL P,SL,SM P,SL,SM P,SL,SM P SL Succinic dehydrogenase cat dog Syrian hamster SL,SM Harrison, 1974 Kawakatsu et al., 1959b, 1962 Kawakatsu et al., 1959b Kawakatsu et al., 1959b, Arvy, 1963 Triantafyllou et al., 1999 Kawakatsu et al., 1959b, 1962 Kawakatsu et al., 1959b, 1962 Kawakatsu and Mori, 1962 Kawakatsu et al., 1959b Smith, 1969 Kawakatsu et al., 1959b Ferguson, 1967 Kawakatsu et al., 1959b; Ferguson, 1967; Vetter, 1969; Shiba et al., 1972 Hill and Bourne, 1954; Kawakatsu et al., 1959b; Arvy, 1963; Shklar and Chauncey, 1963; Ferguson, 1967; Vetter, 1969; Mira et al, 1971; Shiba et al., 1972; Tomich and Eversole, 1972; Chomette et al., 1981 Shapiro, 1967 rat P SL SM Ferguson, 1967 Ferguson, 1967; Chomette et al., 1981 Ferguson, 1967; Mira et al., 1971; Chomette et al., 1981 rat P,SL,SM Ferguson, 1967 rat P,SL SM Ferguson, 1967 Ferguson, 1967; Chomette et al., 1981 rat mouse black-eared tufted marmoset white-eared tufted marmoset Philippine tarsier human being SM SM P Mira et al., 1971; Tomich and Eversole, 1972 Tajima et al., 1979 Miraglia et al., 1974 P Miraglia et al., 1974 P,SL,SM P,SM Smith, 1969 Sirigu et al., 1982b black-eared tufted marmoset white-eared tufted marmoset human being P Miraglia et al., 1974 P Miraglia et al., 1974 P,SM Sirigu et al., 1982b pig SM Booth and Polge, 1976 pig SM Flood, 1973 pig SM Flood, 1973 pig SM Flood, 1973 rat P SM Ferguson, 1967 ferret guinea pig human being mouse Philippine tarsier rabbit rat SM Isocitrate dehydrogenase ␣-Ketoglutarate dehydrogenase Malate dehydrogenase Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase 3␤-Hydroxy-5-enesteroid dehydrogenase 5␤-3␤-Hydroxysteroid dehydrogenase D5-3␤-Hydroxysteroid dehydrogenase 5␤-3␣-Hydroxysteroid dehydrogenase 11␤-Hydroxysteroid dehydrogenase 504 TANDLER ET AL. TABLE 1. Dehydrogenases of ED cells (continued) Enzyme Common Name Gland References 17␤- Hydroxysteroid dehydrogenase human being P,SM Sirigu et al., 1982b rat P,SL SM Ferguson, 1967 Ferguson, 1967; Høyer and Møller, 1977 black-eared tufted marmoset white-eared tufted marmoset rat P Miraglia et al., 1974 P Miraglia et al., 1974 SM Mira et al., 1971; Tomich and Eversole, 1972 ferret rat SM SM Triantafyllou et al., 1999 Mira et al., 1971; Tomich and Eversole, 1972; Chomette et al., 1981 cat ferret rat P,SL,SM SM P SM Syrian hamster SL,SM Harrison, 1974 Triantafyllou et al., 1999 Peagler and Redman, 1999 Mira et al., 1971; Tomich and Eversole, 1972; Høyer and Møller, 1977 Shapiro, 1967 black-eared tufted marmoset white-eared tufted marmoset cat dog ferret Syrian hamster rat P Miraglia et al., 1974 P Miraglia et al., 1974 P,SL,SM P SM SL,SM SM Harrison, 1974b Kawakatsu et al., 1962 Triantafyllou et al., 1999 Shapiro, 1967 Mira et al., 1971; Høyer and Møller, 1977; Chomette et al., 1981 Alcohol dehydrogenase Glycerol-3-phosphate dehydrogenase Lactate dehydrogenase NADH dehydrogenase NADPH dehydrogenase P, parotid; SL,sublingual; SM, submandibular. and glands in which cytokeratin ﬁlaments have been identiﬁed in the principal cells of EDs by cytochemical and immunocytochemical means. Some of the cited studies include the identiﬁcation of cytokeratins in a variety of salivary gland tumors. The ED basal cells are irregularly cuboidal as seen by transmission electron microscopy (Lantini et al., 1990), but as viewed by SEM they are cup-shaped, with their concave surface facing the overlying principal cells (Riva et al., 1992). This concave surface possesses slender folds that are interlocked with those on the principal cells, with occasional desmosomes binding the two disparate cell types together. Their basal surface often forms irregular projections into the duct surroundings that are quite evident with SEM (Brocco and Tamarin, 1979). Hemidesmosomes on these projections bind the basal cells, hence the ED in toto, to the basement membrane. Sato and Miyoshi (1999) have devised a method for isolating the intact duct system of the rat submandibular gland, a technique that in the future might facilitate the examination of EDs by SEM. Variations on this basic structure, some minor, some major, are common in various species. Pinkstaff (1980) has reviewed ED epithelial types in a variety of mammals, a compendium based for the most part on parafﬁn-embedded material. The histological structure of EDs, based largely on light microscopic observations of semithin sections stained with toluidine blue (indicated by an asterisk) in many species, is given in Table 6; where electron microscopic observations are available, these are included. EDs in the parotid gland of the olive baboon for the most part resemble those in human parotid gland, but their lateral walls show less interplication (Tandler and Erlandson, 1976). In the little brown bat, Myotis lucifugus, the basal portion of the principal cells closely resembles that of SD cells in that they have basal striations consisting of highly folded plasma membranes and vertically oriented mitochondria (Fig. 9) (Tandler and Cohan, 1984). The tall columnar light cells (terminology of Tamarin and Sreebny, 1965) of laboratory rat submandibular glands show interleaﬁng of their basal portions of a complexity greater than that seen in the SDs of the same gland; the basal cells are similar to those in human beings. Secretory granules are present in the apical cytoplasm of EDs in various salivary glands of certain species (Fig. 10, Table 6). In the little brown bat, for example, cells with 505 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 2. Oxidases in ED cells Enzyme Common Name Gland References Cytochrome oxidase cat dog ferret mouse guinea pig Philippine tarsier human being rabbit rat P,SM SL P SL SM SM P,SL,SM P,SL,SM P,SL,SM P SL SM P,SM P SL SM Harrison, 1974b; Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Harrison, 1974 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Mori and Mizushima, 1965 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Triantafyllou et al., 1999 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Smith, 1969 Kawakatsu et al., 1964b Mori and Mizushima, 1965 Kawakatsu et al., 1964a, 1964b; Mori and Mizushima, 1965 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Burstone, 1959; Kawakatsu et al., 1964b; Mori and Mizushima, 1965 Kawakatsu et al., 1964b; Mori and Mizushima, 1965; Tomich and Eversole, 1972 Monoamine oxidase dog rat guinea pig P,SL,SM SL,SM P,SM Fujiwara et al, 1966 Almgren et al., 1966 Kawakatsu et al., 1961 P, parotid; SL, sublingual; SM, submandibular. Fig. 8. Accessory submandibular: long-winged bat (Miniopterus magnater). A crystalloid in a principal cell of an ED. These structures appear to be local specializations of the SER. Magniﬁcation ⫽ 14,400⫻. granules alternate with cells that lack these secretory products (Fig. 11) (Tandler and Cohan, 1984). The controversial hormone, parotin, has been shown by immunocytochemistry to be localized in the supranuclear cytoplasm of ED cells in the bovine parotid gland (Takano and Suzuki, 1971); presumably this factor is sequestered in secretory granules. Because ED granules have never been isolated and biochemically analyzed, knowledge of their composition, especially of their glucoconjugates, is based almost entirely on histochemistry. Periodic acid-Schiff (PAS)-positive glycoconjugates that may be either neutral or sialoglycoconjugates have been shown in EDs of several species: the sublingual gland of the vampire bat, Desmodus rotundus (DiSanto, 1960); buccal and submandibular glands of the giant anteater, Myrmecophaga tridactyla (Meyer et al., 1993); the parotid and submandibular glands of the lesser anteater (genus Tamandua, species not cited); the parotid gland of the giant anteater (Quintarelli and Dellovo, 1969); the submandibular glands of the buffalo, Bubalus bubalis (Pal and Chandra, 1979); the parotid gland of the little brown bat, Myotis lucifugus (Pinkstaff et al., 1982); and the lingual serous glands and lingual mucous glands of the ferret (Poddar and Jacob, 1979). Acidic glycoconjugates have been localized in the ED cells of several mammalian species. For example, alcian blue (pH 2.5)-positive glycoconjugates occur in the ED cells of the giant anteater (Meyer et al., 1993) and in the lingual mucous glands of the ferret (Poddar and Jacob, 1979); these acidic glycoconjugates apparently are sialoglycoconjugates. Sulfated glycoconjugates are found in ED cells of the vampire bat sublingual gland (DiSanto, 1960), in the buccal glands of the giant anteater (Meyer et al., 1993), and in the zygomatic glands of the ferret (Jacob and Poddar, 1978). Acid glycoconjugates that stain metachromatically with toluidine blue have been reported as occurring in ED cells of the vampire bat sublingual gland (DiSanto, 1960). The use of monoclonal antibodies to study glycoconjugates, primarily glycoproteins, has facilitated the localization of speciﬁc glycoconjugates in ED cells. Both H type 1 and H type 2 antigens of ABO blood group glycoproteins have been localized in the ED cells of the human subman- 506 TANDLER ET AL. TABLE 3. Acid phosphatase in ED cells Common Name Gland References cat dog cow rabbit human being SM P,SM P,SM P,SM P SM P P SM P SM Harrison, 1974 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1959 Chauncey and Quintarelli, 1959; Kawakatsu and Mori, 1962 Miraglia et al., 1974 Miraglia et al., 1974 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961; Shklar and Chauncey, 1963; Arvy, 1963; Bogart, 1970; Tomich and Eversole, 1972; Chomette et al., 1981; Isaacson, 1986 black-eared tufted marmoset white-eared tufted marmoset sheep rat P, parotid; SL, sublingual; SM, submandibular. TABLE 4. Acid hydrolases in ED cells Enzyme ␤-Galactosidase ␤-Glucosidase ␤-Glucuronidase Common name Gland References dog sheep pig rabbit human being P,SM SM SM P,SM P rat guinea pig SM SM SM Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1961 Chauncey and Quintarelli, 1959; Kawakatsu and Mori, 1962 Rutenberg et al., 1958; Shklar and Chauncey, 1963 Kawakatsu et al., 1960 human being SM Kawakatsu and Mori, 1962 human being rat mouse SM SM SM Kawakatsu and Mori, 1962 Chomette et al., 1981; Kawakatsu et al., 1960 rat SM Tomich and Eversole, 1972 rabbit rat SL,SM SM Vitaioli et al., 1983 Vitaioli et al., 1981 N-acetyl-␤-glucosaminidase Arylsulphatase P, parotid; SL, sublingual; SM, submandibular. dibular gland (Liu et al., 1998). Class I and class II major histocompatibility complex (MHC) antigens have been localized in ED cells of rat parotid, sublingual, and submandibular glands (Matthews et al., 1992). Polymorphic epithelial mucin has been localized by monoclonal antibodies against MAM-3 and MAM-6 antigens in the columnar and basal cells of the EDs of the human parotid and submandibular glands; columnar cells are labeled by antibody against MAM-6, whereas basal cells are labeled by antibodies against MAM-3 (Yamada et al., 1989b, 1991; Mori et al., 1992b). E-cadherin, a transmembrane glycoprotein that is involved in cellular adhesion, has been identiﬁed in human parotid and submandibular glands in the membranes of ED columnar cells and is strongly expressed in the basal cells (Yamada et al., 1996, 1999). E-cadherin is linked to actin molecules by ␤-catenin, which is a cytoplasmic protein that has been localized in the basal cells of the human parotid and submandibular gland EDs (Yamada et al., 1999). Variant isoforms of another transmembrane glycoprotein, CD44, have been identiﬁed in basal cells of EDs of human parotid glands (Terpe et al., 1994); no function for CD44 in salivary glands has been advanced. Human epithelial-related antigen (hERA), another transmembrane glycoprotein, has been identiﬁed in the columnar cells of the EDs of human parotid and submandibular glands, whereas it is basal cells in the sublingual and minor salivary mucous glands that are strongly labeled for this factor (Yamada et al., 1997). Human lung and gastric cancer antigen that closely resembles sialylated Lewis antigen has been localized in the basal cells of EDs of human submandibular (Tsuzi et al., 1989) and parotid glands (Shinohara et al., 1996). Using a monoclonal antibody (JSE3), Durban et al. (1994) have localized a glycoprotein in the ED cells of mouse parotid, sublingual, and submandibular glands. Therkildsen et al. (1994) have used monoclonal antibodies to study the simple mucintype carbohydrate antigens Tn, sialosyl-Tn, T, sialosyl-T, T, H, and A in human salivary glands. They found inconsistent staining for Tn, T, and sialosyl-T, H, and A antigens in the ED cells of human parotid, sublingual, sub- 507 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 5. Microﬁlaments and Intermediate Filaments in EDs Cytokeratins Common Name Gland Human being P SG Golden hamster Syrian hamster Rabbit Dog Horse Pig Cow Goat Japanese macaque Rat Mouse Guinea pig Cat SM SG SM SM SM SG SG SG SG SG SG SG SG SM SG References Caselitz et al., 1981a, 1981b, 1986; Burns et al., 1988; Dardick et al., 1988 Mori et al., 1985b, 1989; Hosaka et al., 1985; Born et al., 1987; Geiger et al., 1987; Takai et al., 1988; Gustafsson et al., 1988; Lee et al., 1990; Mori, 1991; Hamakawa et al., 1999 Nakai et al., 1985; Marshak and Leitner, 1987 Takai et al., 1985a; Mori et al., 1985a; Mori, 1991 Hamakawa et al., 1999 Ogawa et al., 2001 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a; Mori, 1991 Takai et al., 1985a,b; Hamakawa et al., 1999 Takai et al., 1985a,b; Hamakawa et al., 1999 Takai et al., 1985a; Mori et al., 1985b; Mori, 1991 Marshak et al., 1987; Hamakawa et al., 1999 Mori 1991 P, parotid; SG, unidentiﬁed salivary gland; SM, submandibular. mandibular, and labial salivary glands, and strong staining of basal cells in all gland types for T and sialosyl-T antigens, but no staining of duct cells in any gland type with antibody against sialosyl-Tn antigen. In addition to glycoconjugates, several peptide growth factors have been histochemically identiﬁed in ED cells in salivary glands of a variety of mammals. For example, basic ﬁbroblast growth factor has been immunohistochemically localized in the ED cells of the rat parotid, sublingual, and submandibular glands (Amano et al., 1993). Nerve growth factor (NGF) and its mRNA have been found in scattered cells in the EDs of the mouse sublingual gland (Ayer-Le Lievre et al., 1989). Scattered epidermal growth factor (EGF)-immunoreactive cells have been reported in the EDs of rat submandibular glands (Sakabe et al., 1988). EGF has been demonstrated in ED cells of the human submandibular gland using ultrastructural staining methods (Cossu et al., 2000) and EGF receptors occur in the ED cells of human salivary glands (Yamada et al., 1989a). Transforming growth factor ␤1 (TGF-␤1) has been localized in the ED cells of the rat parotid, sublingual, and submandibular glands (Amano et al., 1991), but no TGF-␤1 mRNA was found in the ED cells, suggesting that the ducts take up TGF-␤1 rather than synthesize it. Activin A belongs to the TGF-␤ superfamily; weak immunoreactivity for this growth factor has been localized in the ED cells of the rat parotid, sublingual, and submandibular glands (Bläuer et al., 1996). Transforming growth factor ␣ (TGF-␣) immunoreactivity has been found in the ED cells of the submandibular glands of mice, hamsters, guinea pigs, rabbits, Yucatan pigs, dogs, and rhesus monkeys (Ikematsu et al., 1997) and in human parotid, sublingual, and submandibular glands (Ogbureke et al., 1995). TGF-␣ has been localized in the ED cells of the rat parotid, sublingual, and submandibular glands; this peptide is not present in duct cells 4 weeks postnatally, but is present by 9 weeks (Wu et al., 1993). Because both NGF and EGF are found in secretory granules in the granular convoluted tubules of mice and other rodents (Gresik, 1994), it is likely that all of the aforementioned ED growth factors similarly are housed in secretory granules. The protease, glandular kallikrein, has been immunocytochemically identiﬁed in secretory granules in the striated ducts of certain salivary glands (reviewed by Tandler et al., 2001). In salivary glands, this enzyme may be involved in control of electrolyte and water transport, salivary duct secretion, and regulation of blood ﬂow in these organs. Kallikrein has been localized in ED cells by histochemical means (Table 7); it always is located in the subluminal cytoplasm of the duct cells, suggesting that, as in striated ducts, it is sequestered in secretory granules. This appears to be the case in the submandibular EDs of the pig where the apical granules are reported to contain kallikrein (Dietl et al., 1978). Other esteroproteases have been localized in EDs (Table 8). EDs of both the parotid and submandibular glands of the slow loris, Nyctecebus coucang, are heavily granulated (Tandler et al., 1996). These granules show a spectrum of sizes ranging from 0.2 m to ⬎ 12 m in diameter. Giant granules also are present in the SDs of these glands. Bundles of cytoﬁlaments (presumably actin) are connected to the limiting membranes of the giant granules in whatever kind of duct and presumably are involved in hauling these outsized granules to the luminal membrane for exocytosis. Large cytoplasmic inclusions are present in the ED cells of the parotid gland of the African mole rat, Tackyoryctes splendens (Fig. 12) (Tandler et al., 1998b). The inclusions consist of low-density cytosol in which are embedded myriad short ﬁlaments; all membranous organelles are excluded from each inclusion (Fig. 13). It is not known at present if these inclusions are in some way correlated with either the ecology or behavior of this fossorial species. Crystalloids are present in the taller ED cells in the sublingual gland of the African multimammate rodent, Praomys natalensis (Toyoshima and Tandler, 1991), and in the 508 TANDLER ET AL. TABLE 6. Structural characteristics of EDs in mammalian salivary glands Classiﬁcation† METATHERIA (marsupials) Philander opossum EUTHERIA Order Insectivora Suncus murinus Order Scandentia Dasypus novemcinctus Order Chiroptera (Suborder Megachiroptera) Cynopterus sphinx Epomophorus wahlbergi Gland Type of Epithelium P Coa & Ps SM Psa Shrewb,c SM Co House shrew P SM Co Coa Unpublished Unpublished Nine-banded armadillo SM SCub Shackleford, 1963a Short-nosed fruit bat P SM Wahlberg’s epauletted fruit bat AP Ps Cu3Co & Psa Psa PP SM AP PP SM Ps Psa Ps Ps Psa PP Psa AP PP SM Psa Psa Psa PP Ps P Ps PP AP PP SM PP SM C Psa Co & Ps Ps & Coa Coa Co & Psa ASM P PSM ASM P PSM P Co3Cu Cu & Ps Co & Psa Cu Co Co Cu3Co & Psa Co & Psa Psa Cu3Co & Psa Psa Cua Coa Cu3Psa Cu3Psa Coa Coa Common Name§ Grey ’four-eyed’ opossum Megaerops ecaudatus Temminck’s tailless fruit bat Myonycteris relicta East African little collared fruit bat Rousettus amplexicaudatus Rousettus (Lissonycteris) angolensis Rousettus (Stenonycteris) lanosus Rousettus leschenaulti Eonycteris spelaea Angolan rousette Macroglossus sobrinus Hill’s long-tongued fruit bat (Suborder Microchiroptera) Cardioderma cor Megaderma lyra Heart-nosed bat Greater false vampire bat Megaderma spasma Malayan false vampire bat Leschenault’s rousette Long-tongued dawn fruit bat Rhinolophus acuminatus SM P SM Rhinolophus afﬁnis Rhinolophus coelophyllus Rhinolophus fumigatus Rhinolophus lepidus Rüppell’s horseshoe bat Rhinolophus pumilio Hipposideros armiger Horseshoe bat Himalayan roundleaf bat Hipposideros diadema Diadem roundleaf bat SM P P SM SM P SM P SM Cu & Co & Psa Co & Psa Unusual features Prox. basal striations References Unpublished Unpublished Some basal striations Goblet cells Unpublished Unpublished Unpublished Unpublished Goblet cells Goblet cells Some goblet cells Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Some goblet cells Basal striations Unpublished Unpublished Unpublished Unpublished Unpublished Basal striations Long microvilli Basal striations Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Some basal striations Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished 509 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 6. Structural characteristics of EDs in mammalian salivary glands (continued) Classiﬁcation† Common Name§ Hipposideros gentilis Saccopteryx bilineata Greater sac-winged bat Gland Type of Epithelium a P SM SM P SM Ps Psa Psa Co* Co* Noctilio leporinus Mormoops blainevilli Pteronotus parnellii Greater bulldog bat Blaineville’s leaf-chinned bat Parnell’s mustached bat SM PSM P Cu Psa Psa Macrotus waterhousi California leaf-nosed bat SM P SM Psa Cu3Coa Cu & Co & Psa SM SM P SM Co prox Psa Psa Psa SM P SM Psa Psa Psa prox. P Psa Phyllostomus aphylla Phyllostomus discolor Phyllostomus elongatus Phyllostomus hastatus Phyllostomus latifolius Tonatia bidens Pale spear-nosed bat Lesser spear-nosed bat Pallas’s spear-nosed bat Guianan spear-nosed bat Greater round-eared bat Unusual features a SM SM Ps Basal striations Alternating light & dark cells Basal striations Basal striations Sparse granules Basal striations; nerve terminals Occasional dense granules Tonatia silvicola D’Orbigny’s round-eared bat P Psa Trachops cirrhosus Fringe-lipped bat SM P SM Erophylla sezekorni Anoura geoffroyi Buffy ﬂower bat Geoffroy’s long-nosed bat SM P SL SM Glossophaga soricina Long-tongued nectar bat P Leptonycteris nivalis Saussure’s long-nosed bat SM SM Cu3Co3Psa Psa Ps Bundles of nerve terminals Co & Ps Ps* Ps* Psb* & Some basal SCo striations Cu prox & Psa Psa Psa Microvilli Monophyllus redmani Leach’s single leaf bat Carollia perspicillata Short-tailed leaf-nosed bat P SM P Coa Coa Ps Ametrida centurio Ariteus ﬂavescens White-shouldered bat Jamaican ﬁg-eating bat SM SM P SM Psa Psa Cu3Coa Cua Some basal striations; numerous hypolemmal nerve terminals Prominent microvilli References Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tandler et al., 1999 Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Phillips et al., 1977; Unpublished Unpublished Unpublished Tandler et al., 1988 Unpublished Unpublished Unpublished Unpublished 510 TANDLER ET AL. TABLE 6. Structural characteristics of EDs in mammalian salivary glands (continued) Classiﬁcation† Common Name§ Gland Type of Epithelium Unusual features Artibeus cinereus Gervais’s fruit bat P Ps Artibeus jamaicensis Jamaican fruit bat SM P Artibeus lituratus Chiroderma villosum Enchisthenes hartii Big fruit bat Sturnira lilium Yellow epauletted bat P SM Uroderma bilobatus Tent-building bat Desmodus rotundus Common vampire bat P SM ASM Cu3Co3Psa Cu & Psa Resembles SD; branching dark cells Cu* Co & Psa Cua Ps Prox: like SD Dist: numerous mitochondria a Co3Ps Cua prox & Psa Co3Cu Cua Microvilli Co & Ps PSM Psa Brush border Ps Cu Cu Coa Brush border Hart’s little fruit bat SM SM SM P Diaemus youngi Natalus strameus White-winged vampire bat Mexican funnel-eared bat Chalinolobus argentatus Silvered bat P SL SM P Brazilian brown bat SL SM SM P Coa Cua Cu & Psa Co P P SL Psa Psa Psa SM Co & Psa ASM P Coa Ps ASM P PSM P P SM Coa Psa Coa Psa Coa Coa & Psa P Coa Chalinolobus humeralis Eptesicus brasiliensis Eptesicus lynni Hesperoptenus blanfordi Myotis annectans Myotis lucifugus Lynn’s brown bat Little brown bat Myotis montivagus Myotis muricula Pipistrellus mimus Pipistrellus pulveratus Pipistrellus coromandra Indian pipistrelle Pipistrellus kuhli Scotophilus dingani Tylonycteris pachypus a European pipistrelle P SM P Ps Psa Ps African yellow bat Club-footed bat SM ASM Ps?Cu Cu P SM Co & Psa Psa Many mitochondria Numerous mitochondria Mitochondrion: desmosome complexes; basal striations Prox. basal striations Basal striations Alternating non-granular & granular cells; basal folds Basal striations Some basal striations Small dense granules References Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tandler et al., 1990a Tandler et al., 1990a Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tandler and Cohan, 1984 Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Azzali et al., 1986 Unpublished Tandler et al., 1995 Unpublished Unpublished 511 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 6. Structural characteristics of EDs in mammalian salivary glands (continued) Classiﬁcation† Tylonycteris pachypus Common Name§ Club-footed bat Harpiocephalus harpia Hairy-winged tube-nosed bat Murina cyclotis Tube-nosed bat Murina leucogaster Miniopterus inﬂatus Gland Type of Epithelium ASM Cu a Tube-nosed bat Greater long-ﬁngered bat P SM P SM SM ASM Co & Ps Psa Ps Co Co & Ps Ps Miniopterus magnater Western long-ﬁngered bat ASM Ps Miniopterus schreibersi Schreiber’s long-ﬁngered bat ASM Ps Molossus molossus Velvety free-tailed bat P PSM P Cu proxa Cu & Co Ps Tadarida thersites Free-tailed bat AP Ps SM SCoa P Ps SM Ps Order Primates Nycticebus coucang Slow loris Saguinas fuscicollis Ateles paniscus Tamarin Spider monkey P P Cu PS Macaca mulatta Rhesus monkey SM Psb Papio cynocephalus Baboon SM Psb Homo sapiens Order Carnivora Felis catus Mustela putorius Mustela siberica Human being Domestic cat Ferret Siberian weasel Unusual features Hypolemmal nerve terminals SER crystalloids; hypolemmal nerve terminals Hypolemmal nerve terminals A few granulated cells Single row of apical granules Some basal striations Many goblet cells P SM SCo PS SCo P C SM Psa P Cu?Cob SL SCub SM Cu?Cob P Cu SL C Tandler et al., 1995 Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tandler et al., 1998 Unpublished Many granules; some giant Many granules; some giant b References Many goblet cells Vesiculated tall cells Goblet cells Paracrystalloid granules Small secretory granules Tandler et al., 1996 Tandler et al., 1996 Unpublished Leeson, 1969 Pinkstaff, 1980; Boshell and WiIlborn, 1983 Boshell and Willborn, 1983 Scott, 1977 Paz Ossorio et al.,1975; Tandler and Riva, 1986; Riva et al., 1990 Tandler, 1978 Menghi et al., 1989 Tandler , 1993b, Tandler and Poulsen, 1976 Poddar and Jacob, 1977 Poddar and Jacob, 1977 Poddar and Jacob, 1977 Matsunaga, 1992 Matsunaga, 1992 512 TANDLER ET AL. TABLE 6. Structural characteristics of EDs in mammalian salivary glands (continued) Gland Type of Epithelium Pig P P C Ps Glycogen Minipig SM Co One-humped camel P NA Basal striations; numerous granules Goblet cellsb P Classiﬁcation† Order Artiodactyla Sus scrofa Camelus dromedarius Common Name§ Bos indicus Zebu P SCu & SCob SCu & SCob NA Bos taurus Cow SM Psb Ovis aries Order Lagomorpha Lepus europaeus Sheep P NA European hare P Ps Oryctolagus cuniculus Rabbit SM Co3Ps SM Z Order Rodentiac Puget Sound deer mouse Chipmunk Desert rat Sagebrush deer mouse Western ﬂying squirrel Beechey ground squirrel Grey squirrel P SL SM P Psb Psb Psb Psb SL Psb SM Psb P SL SM SL SM P Cub Cob Cob Psb Psb Cu & Co & Psb Cob Psb Cob Cob Cob SL SM P SL SM b P Ps Psb Co & Psb Co & Psb Coa Psa Acomys percivali Cricetomys gambianus Spiny mouse Giant pouched rat SM P SM SM P Tachyoryctes splendens African mole-rat SM P Psa Co SM Co Lined squirrel Unusual features Many goblet cellsb Many goblet cells Goblet cellsb Light cells & vacuolated cells Blebs on tall cells Granular cells Some basal striations Many dark cells Some basal striations References Imai et al., 1978 Boshell and Wilborn, 1978 Unpublished Nawar and ElKhaligi, 1975 Mansouri and Atri, 1994 Mansouri and Atri, 1994 Vignoli and Nogueira, 1981 Shackleford and Klapper, 1962 Quintarelli, 1963 Suzuki et al., 1985 Toyoshima and Tandler, 1986 Gargiulo et al., 1996 Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Some basal striations Cuboidal principal cells Basal striations Large, ﬁlamentous inclusions Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tandler et al., 1998b Unpublished 513 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 6. Structural characteristics of EDs in mammalian salivary glands (continued) Classiﬁcation† Common Name§ Gland Type of Epithelium Ps Ps Ps Ps Coa Tatera nigricauda Ondatra zibethicus Castor canadensis Naked soled gerbil Muskrat North American beaver Marmota monax Ground hog SM SM P SM SL Proechimys cuvieri Apodemus agrarius Cuvier’s spiny rat Striped ﬁeld mouse SM SM Coa Coa Lophuromys sikapusi Harsh-furred mouse P Ps Praomys natalensis African multi-mammate rodent Laboratory rat SL Co P Co SL variable SM Ps Rattus norvegicus Mus musculus Laboratory mouse SM Ps Zelotomys hildegardeae Tachoryctes splendens Broad-headed mouse African mole-rat SL P Ps Ps Zygodontomys reigi Cane mouse SM Co Myocastor coypus Nutria P NA Unusual features Basally branched dark cells Basal striations Some globular leukocytes Cytoplasmic crystalloids Tall dark vesiculated cells Catalasepositive rods Luminal membranes positive for carbonic anhydrase I &2 Filamentous inclusions Basal striations Many goblet cellsb References Unpublished Unpublished Unpublished Unpublished Pinkstaff, 1980 Unpublished Unpublished Unpublished Toyoshima and Tandler, 1991 Jeẑek et al., 1996; Unpublished Garrett and Anderson, 1991 Tamarin and Sreebny, 1965 Hanker et al., 1977 Ogawa et al., 1992 Gutierrez Marin et al. 1990 Unpublished Tandler et al., 1998 Unpublished Dix, 1969 † Generic and speciﬁc names are based on Wilson and Reeder (1993). In a few instances, we have corrected or updated generic or speciﬁc names from the original published nomenclature. In published work other than our own, we are unable to conﬁrm the identiﬁcations. § Where there is no generally accepted common name, this designation is omitted. AP, accessory parotid; ASM, accessory submandibular; P, parotid; PSM, principal submandibular; SL, sublingual; SM, submandibular; VB, ventral buccal; Z, zygomatic; Co, simple columnar epithelium; Cu, simple cuboidal epithelium; Ps, pseudostratiﬁed epithelium; SCo, stratiﬁed columnar epithelium; SCu, stratiﬁed cuboidal epithelium; NA, not available; SD, striated duct; ? , proceeding in a distal direction; dist, distal; prox, proximal; a Based on semithin epoxy sections; b Based on parafﬁn sections; cInsectivore and rodent data without genus and species identiﬁcation are based on slides from the collection of the late Arnold Tamarin (no voucher specimens were available for species identiﬁcation). EDs of the accessory submandibular glands of the longwinged bat, Miniopterus magnater (Tandler et al., 1997a). EDs in the parotid glands of seven species of fruit bats in the genus Artibeus resemble SDs in the same gland, but occasionally include one or several dark cells in a given sectioned duct (Fig. 14) (Tandler et al., 1997c). Dark cells constitute 4.7% of the cells in the wall of the proximal EDs in the rat submandibular gland and 7.2% of the cells in the more distal EDs (Sato and Miyoshi, 1998). The nature of dark cells is unclear. They conceivably may be ﬁxation artifacts; they also might represent a distinct phase in the cell life cycle or physiological activity. In some glands, dark cells might represent a unique cell type. This especially appears to be the case in the EDs of sublingual glands of the woodchuck, where dark cells with branched bases alternate with light cells of more typical morphology (Pinkstaff, 1980). The subject of dark cells is more fully dealt with elsewhere in the context of the striated duct (Tandler et al., 2001). In the sublingual glands of various mammals, endpieces either empty into IDs, which lead into EDs without the intervention of SDs, or empty directly into EDs. In the retrolingual (equivalent to the sublingual) gland of the European hedgehog, intercalated ducts gradually become EDs, changing from simple cuboidal to simple columnar epithelium in the process (Tandler, 1986). In this gland, most of the principal cells of the ED display a subluminal zone that contains an abundance of moderately dense 514 TANDLER ET AL. granules, which measure ⬃ 0.2 m in diameter. Intermingled with these granulated cells are a few rare cells that lack granules. At a point at which the ED becomes pseudostratiﬁed are occasional mitochondrial aggregates, wherein adjacent organelles are joined by periodic crossbridges. The ductular picture that emerges in minor salivary glands is not unlike that occurring in the sublingual glands. Endpieces can join intercalated ducts that progress into EDs without an obvious line of demarcation (Tandler et al., 1970; Takeda et al., 1978) or that may be directly connected with EDs (Nagato et al., 1998). EDs in human palatal glands run a twisted helical course (Maximuk and Shertyuk, 1990). In von Ebner’s glands of the rabbit (Toyoshima and Tandler, 1986), the most proximal duct cells contain dense serous-type granules that exhibit a lacuna of lower density. Granules in the more distal ducts of these glands differ from their predecessors, having a compound structure consisting of a dense portion and a portion of lesser density. Cells of the latter type occasionally include a dilated cistern of RER with a moderately dense content. Some ciliated cells are present in the most distal EDs of human von Ebner’s glands (Azzali et al., 1989). In certain minor glands, e.g., Weber’s glands of the rat tongue (Nagato et al., 1997) and the midlingual glands of the vampire bat (Tandler et al., 1997d), EDs may lead directly onto an oral surface, in a sense acting as the MED of such organs. In the latter glands, some of the duct cells are ﬁtted with cilia with typical 9⫹2 structure (Tandler et al., 1997d). Human anterior lingual glands (glands of Blandin and Nuhn) have EDs (Tandler et al., 1954) that resemble those Fig. 9. Submandibular: laboratory rat (Rattus norvegicus). An ED consisting of tall simple columnar epithelial cells. Note the highly folded cell base. These folds are too slender to accommodate mitochondria, hence the cells lack obvious basal striations. Magniﬁcation ⫽ 2,300⫻. Fig. 11. Parotid: little brown bat (Myotis lucifugus). A principal cell in an ED that is devoid of granules, but that contains an abundance of smooth-surfaced vesicles and tubules. This cell is ﬂanked by cells that have an abundance of small dense granules. Magniﬁcation ⫽ 24,500⫻. Fig. 10. Submandibular: minipig (Sus scrofa). The subluminal cytoplasm of several ED cells contains many small dense secretory granules. Magniﬁcation ⫽ 12,700⫻. 515 EXCRETORY DUCTS OF SALIVARY GLANDS TABLE 7. Kallikrein in ED cells Common name cat rat mouse guinea pig pig sheep crab-eating macaque Japanese macaque rhesus monkey grivet human being Gland References P SM P SL SM SM SM SM SM P,SL,SM P,SL,SM P,SL,SM P,SL,SM P SM Maranda et al., 1978 Maranda et al., 1978; Garrett et al., 1985 Arnold, 1985 Ørstavik et al., 1975; Arnold, 1985 Ørstavik et al., 1975; Gutkowska et al., 1983; Arnold, 1985 Penschow and Coghlan, 1993 Schachter et al., 1978 Dietl et al., 1978 Trahair and Ryan, 1989 Arnold, 1984, 1985 Yahiro and Miyoshi, 1996 Arnold, 1984 Arnold, 1984 Ørstavik et al., 1980; Kimura and Moriya, 1984, 1986 Ørstavik et al., 1980 P, parotid; SL, sublingual; SM, submandibular. TABLE 8. Esteroproteases in ED cells Common Name mouse rat Phillipine tarsier crab-eating macaque Gland References SL SM P,SL P,SM P,SL,SM Gresik and Barka, 1983 Lexow et al., 1979 Arnold, 1985 Smith, 1969 Arnold, 1985 P, parotid; SL, sublingual; SM, submandibular. in the human parotid and submandibular glands (Lantini et al., 1990), with a few structural deviations. The EDs in these lingual glands [and in human labial glands as well (Tandler et al., 1970)] often include patches of SDs that are indistinguishable from those in human major glands (Tandler, 1993b). The pyramidal basal cells often exhibit a primary (solitary) cilium, a structural feature more often associated with basal cells in the rodent MED (Higashi and Sasa, 1985; Higashi et al., 1994). Compared to principal cells, basal cells show relatively little variation among mammalian species. Their precise nature has aroused some controversy. Based on their stainability with antibodies to S100b protein and to various keratins (Palmer et al., 1985; Caselitz et al., 1986; Palmer, 1986; Born et al., 1987; Dardick et al., 1987, 1988; Geiger et al., 1987; Gustafsson et al., 1988; Shinohara et al., 1989) that stain endpiece myoepithelial cells (Dairkee et al., 1985), at least some of the basal cells have been labeled as myoepithelium or as “modiﬁed myoepithelium” by some authors (Mori et al., 1992a). Other authors have relied simply on the morphology of basal cells to justify their identiﬁcation as myoepithelial cells (Chaudhry et al., 1987). Because myoepithelial cells are involved in the histogenesis of a variety of salivary gland tumors (Batsakis et al., 1983; Dardick and van Nostrand, 1985; Nikai et al., 1986; Gustafsson et al., 1989), it is of some importance to determine whether the ductular basal cells are in fact of myoepithelial origin. In a more recent report, Dardick et al. (1991) state that S100 protein antibodies stain nerve ﬁbers that are closely applied to salivary parenchyma, rather than myoepithelium. Other workers have pointed out the unreliability of S100 protein as a marker of myoepithelium (Gillett et al., 1990; Möller and Hellmén, 1994). The use of antikeratin antibodies to identify myoepithelial cells probably is similarly ﬂawed, because various antibodies of this type label basal cells of stratiﬁed epithelia, irrespective of the source of this tissue (Pallesen et al., 1987; Maeda and Sueishi, 1989); the labeling of basal cells in tissues such as stratiﬁed squamous epithelium that clearly lack myoepithelial components makes evident the unreliability of keratin staining as a label for myoepithelium. When all is said and done, the best marker for myoepithelial cells remains actin (Archer and Kao, 1968; Drenckhahn et al., 1977; Yoshihara et al., 1988; Norberg et al., 1992). Using an antiactin antibody, Nilsen and Donath (1981) found a few positive cuboidal cells in the “larger” ducts of the human submandibular and parotid glands, but Gugliotta et al. (1988) failed to stain basal cells in EDs of several different human salivary glands with a similar antibody, although myoepithelial cells associated with endpieces and intercalated ducts were positive. Thus, it appears that basal cells of EDs probably are not related to myoepithelial cells. The absence from basal cells of the cytoplasmic machinery normally associated with secretion or electrolyte transport (although their histochemistry reveals the presence of various transport enzymes) suggests that a major function of these cells in the parotid and submandibular glands is to bind the ductular epithelium to the basement membrane through the agency of their hemidesmosomes. In some species, other cell types are intermingled with the principal cells of the EDs. These include the aforementioned dark cells, and vesiculated, brush, and goblet cells as well. Tall dark vesiculated cells characterized by a dense cytosol and numerous small empty-appearing vesicles are present in the EDs of the rat submandibular gland (Tamarin and Sreebny, 1965). These cells have basal striations that match those in their more conventional neighbors. Tuft cells have a unique structure that allows their ready identiﬁcation: they have many thick blunt microvilli with very long rootlets, among which are numerous electron-lucent vesicles as well as microtubules (Sato and Miyoshi, 1997). The function of these cells is unknown but they might act as chemoreceptors (Höfer et al., 1996). Scattered goblet cells are present in the EDs of both parotid and submandibular glands primarily of primates and of megachiropteran bats, but also occur in certain 516 TANDLER ET AL. Fig. 13. Parotid: African mole rat (Tachyoryctes splendens). A cytoplasmic inclusion at higher magniﬁcation. It consists of a zone of moderate- to low-density cytosol in which short ﬁlaments and occasional vesicles are embedded. All conventional cytoplasmic organelles are excluded, even though the inclusion lacks a circumscribing membrane. Magniﬁcation ⫽ 34,800⫻. Fig. 12. Parotid: African mole rat (Tachyoryctes splendens). Top: Photomicrograph of an ED in semithin section in which many constituent cells contain a large lucent cytoplasmic inclusion. Toluidine blue. Magniﬁcation ⫽ 300⫻. Bottom: Low-magniﬁcation electron micrograph of a portion of the wall of an ED showing the size and extent of the cytoplasmic inclusions. Magniﬁcation ⫽ 2,500⫻. ungulates (Shackleford, 1963b), as well as in a variety of other species. In some glands, the EDs occasionally include clusters of goblet cells within their walls (Fig. 15). All ductular goblet cells are of typical morphology and are reported to be PAS-positive. This is the case in oxen and pig parotid glands (Munhoz, 1971), in buffalo submandibular glands (Pal and Chandra, 1979), in tapir submandibular glands (Quintarelli and Dellovo, 1969), in rhesus monkey (Macaca mulatta) parotid and submandibular glands (Boshell and Wilborn, 1983; Stephens et al., 1986), in cow, guinea pig, and rhesus monkey parotid glands, in cow and pig sublingual glands (Shackleford and Klapper, 1962), and in the submandibular gland of the miniature pig (Pinkstaff, 1972). These PAS-positive glycoconjugates may be either neutral or sialoglycoconjugates, or both. Alcian blue (pH 2.5)-positive glycoconjugates in goblet cells of EDs are believed to be sialoglycoconjugates and have been reported to occur in the parotid glands of oxen and pigs (Munhoz, 1971), in buffalo submandibular glands (Pal and Chandra, 1979), in lingual serous glands and lingual mucous glands of the ferret (Poddar and Jacob, 1979), and in the parotid and submandibular glands of the rhesus monkey (Stephens et al., 1986). Sulfated glycoconjugates have been reported in the goblet cells of EDs in oxen and pig parotid glands (Munhoz, 1971), in buffalo submandibular glands (Pal and Chandra, 1979), and in lingual serous and lingual mucous glands of the ferret (Poddar and Jacob, 1979). It is obvious that goblet cells in EDs add a mucus component to the saliva, even in ostensibly pure serous glands. Oncocytes occasionally are present in the EDs of human salivary glands (Riva et al., 1988; Lantini et al., 1990). These cells characteristically contain a plethora of structurally modiﬁed mitochondria to the virtual exclusion of the other cytoplasmic organelles (Tandler, 1966; Riva and Tandler, 2000; Tandler and Hoppel, 2004). Little is known of the physiology of oncocytes arising in normal salivary glands. Like other parenchymal components of salivary glands, EDs are subject to invasion by mononuclear wandering cells, usually lymphocytes (Takeda and Fujimura, 1985), but sometimes neutrophils (Fig. 16); the duct walls in the parotid gland of the harsh-furred mouse, Lophuromys EXCRETORY DUCTS OF SALIVARY GLANDS 517 Fig. 14. Parotid: Jamaican fruit bat (Artibeus jamaicencis). An ED with many irregular dark cells. Magniﬁcation ⫽ 2,900⫻. Micrograph courtesy of Toshikazu Nagato. sikapusi, occasionally include globular leukocytes (Fig. 17), which have also been observed in the parotid gland of the cow (Gurusinghe and Birtles, 1985). Intruding wandering cells can be identiﬁed by their high nucleus-tocytoplasm ratio and by their total lack of desmosomal connections to surrounding epithelial cells. In the rhesus monkey, Macaca fascicularis, labial salivary glands are subject to invasion by bacteria (Nair and Schroeder, 1985). In this event, ducts are surrounded by lymphoid tissue and are heavily inﬁltrated by lymphocytes and blast-forming T-cells, in some cases to a point where the duct epithelium is partially or wholly supplanted by lymphoid cells. The nuclei in the ducts of human labial glands have immunohistochemically detectable human ␤-defensin1 (hBD-1), an antimicrobial peptide (Sahasrabudhe et al., 2000); this agent also coats the luminal surface of the duct cells. In contrast to the minor glands that evince inﬂammatory responses, the numerous bacteria that occupy the lumina (including those of EDs) of the entire parenchymal tree of the accessory submandibular gland of the clubfooted bat, Tylonycteris pachypus, fail to elicit an immunological response; mononuclear cells are absent from the duct walls and lumina and are present in the surrounding connective tissue in only modest numbers (Tandler et al., 1995). In this instance, the bacteria may be acting as symbionts. In a few rare cases, EDs in human parotid glands form cysts that contain large crystalloids (Kurashima, 1983; Takeda and Ishikawa, 1983). These crystalloids are rich in sulfur (Takeda and Ishikawa, 1983). Solid, homogeneously dense, usually eosinophilic deposits sometimes are present in EDs of both major and minor human salivary glands (Scott, 1978, 1987). Sebaceous differentiation frequently occurs in salivary glands; this metaplasia often involves the EDs (Gnepp, 1983; Van Esch et al., 1986; Martinez-Madrigal and Micheau, 1989). In such metaplastic ducts, a part of or the entire duct wall may be replaced by sebaceous cells. In human beings, such metaplastic foci are of importance because they can give rise (although very rarely) to sebaceous neoplasms (Gnepp, 1983). In vitamin A-deﬁcient rats, EDs in the submandibular gland undergo squamous metaplasia (Trowbridge, 1969). A similar process can occur in human salivary glands (Martinez-Madrigal and Micheau, 1989) during inﬂammation, especially if the latter condition is associated with calculi (Isacsson and Lundquist, 1982). In connection with neoplasia, the basal cells of EDs are considered by some authors (Regezi and Batsakis, 1977; Batsakis, 1980) to constitute a population of semipleuripotential reserve cells that are the histogenic precursors of such salivary gland tumors as mucoepidermoid and squamous carcinomas (reviewed by Dardick et al., 1990). Dardick (1998) presents evidence that militates against this concept. It appears that every cell type that makes up salivary parenchyma is capable of self-replication. Not only basal cells of EDs, but principal cells as well, are capable of division (Komiya and Fukushima, 1991; Redman, 1995) and thus are potential progenitors of neoplastic cells. It is extremely difﬁcult to arrive at a conclusion concerning innervation of EDs because most studies that deal with salivary gland innervation simply lump all extraacinar parenchymal elements together under the rubric “ducts.” Only rarely are EDs speciﬁed in such studies. According to Garrett (1976), cholinergic nerves form a loose association with extralobular ducts, i.e., EDs, but adrenergic nerves have not been observed in such a relationship. The cholinergic innervation of EDs can vary even in related species (Rossoni et al., 1981). In rat salivary glands, both calcitonin gene-related peptide (CGRP) (Soinila et al., 1989) and met5-enkephalin-arg6-gly7-leu8 (MEAGL) (Soinila et al., 1991) immunoreactive nerve ﬁbers are present in close relation to interlobular ducts. Gland segments proximal to the EDs are directly inner- 518 TANDLER ET AL. Fig. 16. Sublingual: broad-headed mouse (Zelotomys hildegardae). A neutrophil has invaded the wall of an ED. The duct lumen is ﬁlled with dense material, which may have attracted the neutrophil. Magniﬁcation ⫽ 7,500⫻. Fig. 15. Parotid: tailless fruit bat (Megaerops ecaudatus). Two goblet cells at the junction of a striated duct (left) with an ED (right). Magniﬁcation ⫽ 6,200⫻. vated by hypolemmal nerve terminals (discussed by Garrett and Kidd, 1993). Such terminals previously have not been reported in EDs of any salivary gland. In our comparative studies, we have encountered two species that exhibit profuse hypolemmal innervation of their EDs. In the short-tailed leaf-nosed bat (Carollia perspicillata), bundles of naked axons and terminals are ubiquitous throughout the ED wall (Fig. 18). As in several other bat species (Tandler and Phillips, 1995), these hypolemmal terminals are shrouded by mitochondria in the adjacent duct cells. In the fringe-lipped bat, Trachops cirrhosis, the accessory submandibular gland has a peculiar architecture (Tandler et al., 1997b). The secretory endpieces, which have a follicular structure, empty into ducts consisting of simple cuboidal epithelium that probably represent intercalated ducts; the epithelium gradually increases in height to form what we interpret to be EDs. Ensconced in the walls of these presumptive EDs are bundles of hypolemmal nerve terminals. Unlike those in Carollia, these show no special relationship to mitochondria in surrounding cells. The signiﬁcance of ED innerva- tion is unknown, but sodium and potassium transport in the MED is under sympathetic control (Schneyer, 1976); such a function probably can safely be extrapolated to EDs. Knowledge of the function of EDs is conﬁned wholly to experimental animals and is based almost entirely on micropuncture and microperfusion studies (Schneyer et al., 1972) and on histochemistry (Pinkstaff, 1980, 1993). The production of saliva in major salivary glands is a two-step process: the endpiece-intercalated duct complex elaborates saliva that has plasma-like concentrations of electrolytes, then electrolytes are resorbed from this primary saliva as it passes through the ductal tree to render the ﬁnal saliva hypotonic (Thaysen, 1960; reviewed by Young, 1979). Early micropuncture studies involved collection of primary saliva from the endpiece-intercalated duct complex and from distal parts, i.e., the MED, of the duct system (e.g., Martinez et al., 1966). By comparing the two samples, it is apparent that there is a net resorption of electrolytes as the saliva passes through the duct system. The presence of Na⫹,K⫹-ATPase at the base of ED cells in human, feline, and rodent salivary glands provides the enzymatic basis for such activity (Winston et al., 1988, 1990; Peagler and Redman, 1999). The physiological events involved in this process have been reviewed in detail by Schneyer et al. (1972), Martinez (1987), and Young et al. (1987). It needs to be pointed out that as- EXCRETORY DUCTS OF SALIVARY GLANDS Fig. 17. Parotid: harsh-furred mouse (Lophuromys sikapusi). A globular leukocyte is ensconced in the wall of an ED. Magniﬁcation ⫽ 5,900⫻. sumed transport functions of EDs are based on extrapolations from studies on the MED (Young et al., 1987). In minor salivary glands that lack ductular segments other than EDs, the ﬁnal saliva usually is isotonic, suggesting that in these small glands the EDs do not play a regulatory role in salivary composition. Pinkstaff (1980, 1993) has reviewed the histochemical and cytochemical literature on ED enzymes. Such studies belie the general impression that EDs merely are passive conduits for saliva. ED cells in various species evince substantial enzymatic activity, as determined histochemically (Tables 1, 2, 3, 4, 8, and 9). For example, ED cells exhibit carbonic anhydrase (CA) activity (Table 9); this enzyme is involved in several physiological processes, including bicarbonate ion production, providing H⫹ and bicarbonate for other ions in transmembrane transport and in the secretion of macromolecules. This enzyme may be demonstrated by metal capture methods or by immunohistochemical methods; using immunohistochemical methods, it has been possible to identify at least four isozymes of carbonic anhydrase: CA-i, CA-II, CA-III, and CA-VI (Table 9). Interestingly, there are signiﬁcant species differences in the localization of the various carbonic anhydrase isozymes. One species may be entirely devoid of staining for an isozyme while other species may exhibit staining for it. There are differences between staining reactions in the different glands of the same species, and 519 Fig. 18. Submandibular: short-tailed leaf-nosed bat (Carollia perspicillata). A bundle of hypolemmal nerve terminals within the wall of an ED. The varicosities contain both small clear synaptic vesicles and larger dense-cored ones. The axons contain obvious neurotubules. Note the relationship between the nerve bundle and the mitochondria in the abutting cells. Magniﬁcation ⫽ 11,900⫻. it is also of interest to note that in the EDs of some species the columnar cells of stratiﬁed columnar or pseudostratiﬁed epithelia may be unstained but the basal cells will be intensely stained, or both columnar cells and basal cells are stained. For example, in the submandibular gland of the guinea pig, the basal cells of the EDs are strongly stained, whereas the columnar cells are negative to only very faintly positive. In the horse parotid, sublingual, and submandibular glands, intense activity of carbonic anhydrase III is seen in basal cells of EDs, but basal cells are unstained for either carbonic anhydrase I or II. The possibility exists that basal cells function entirely differently from columnar cells in terms of ionic transport. Further evidence that EDs are involved in transport phenomena is provided by immunohistochemical and histochemical examination of these duct segments for phosphatases. The most intensely studied phosphatase in ED cells of salivary glands has been Na⫹,K⫹-adenosine triphosphatase (Na⫹,K⫹-ATPase). This is a component of the sodium pump and has been localized in sites of ﬂuid and ion transport. Na⫹,K⫹-ATPase activity has been localized in the basolateral plasma membranes of ED cells in the cat parotid and sublingual glands (Winston et al., 1990) and in the cat submandibular gland (Winston et al., 520 TANDLER ET AL. TABLE 9. Carbonic anhydrases (CA) in ED cells Enzyme Common name Gland References Syrian hamster guinea pig human beings horse mouse SM SM P,SM P,SL,SM SL SM P SL SM Noda et al., 1986b Noda et al., 1986b Noda et al., 1986a, 1986c Asari et al., 1991a Hennigar et al., 1983 Hennigar et al., 1983; Noda et al., 1986b Peagler et al., 1998; Asari et al., 1991b Hennigar et al., 1983; Redman et al., 2000 Hennigar et al., 1983; Noda et al., 1986b; Ogawa et al., 1992; Redman et al., 2000 P,SL,SM P,SL SM SL,SM SM SL SM P SM Noda et al., 1986a, 1986b Spicer et al., 1990 Noda et al., 1986b; Spicer et al., 1990 Asari et al., 1991a Asari et al., 1989 Hennigar et al., 1983; Spicer et al., 1990 Hennigar et al., 1983; Noda et al., 1986b Peagler et al., 1998 Noda et al., 1986b P SM SL,SM Asari et al., 1991b Nishita et al., 1989 Spicer et al., 1990 SM P,SL,SM Asari et al., 1989, 1993 Asari et al., 1991a P SL,SM Peagler et al., 1998 Redman et al., 2000 CA-I rat CA-II human beings guinea pig horse cow mouse rat CA-III rat mouse guinea pig cow horse CA-VI rat P, parotid; SL, sublingual; SM, submandibular. 1990; Garrett et al., 1992), in human parotid, sublingual, and submandibular glands (Winston et al., 1988), in mouse parotid and sublingual glands (Winston et al., 1988), in mouse submandibular glands (Sims-Sampson et al., 1984; Winston et al., 1988; Kurihara et al., 1996), and in the rat parotid, sublingual, and submandibular glands (Winston et al., 1988). Na⫹,K⫹-ATPase has been studied in the ED cells of the postnatally developing rat parotid gland (Peagler and Redman, 1999). Another ATPase has been localized primarily on the microvilli of ED cells of the rat parotid and submandibular glands; this is an ectoATPase and may be identical to a membrane glycoprotein called C-CAM 105 (Murphy et al., 1994). Two other phosphatases in ED cells are nucleoside diphosphatase, which is found in what appears to be the area of the Golgi complex in the cat parotid and submandibular glands, and thiamine pyrophosphatase that is also localized in the area of the Golgi complex in the cat submandibular gland ED cells (Harrison, 1974). Clearly, Pinkstaff’s (1980) suggestion that “EDs do contribute in some way to the ﬁnal product of salivary gland secretion” is well founded. In the broad view, the mammalian ED is variable in terms of histology, histochemistry, and ultrastructure. Indeed, for this reason, it is impossible to describe a “typical” ED. This circumstance is noteworthy because it raises an evolutionary issue: is the absence of a conserved pattern indicative of the adaptive importance of the ED, or is it due to its relatively low functional importance? We tested its potential adaptive importance by mapping ED data on a phylogenetic tree. In this exercise, we did not ﬁnd patterns associated with diet, ecology, or evolutionary lineage. Our interpretation is that species variation with regard to EDs does not reﬂect “adaptive” responses involving this duct. Therefore, it seems possible that the variation in EDs is a reﬂection of the relative lack of evolutionary constraints or of selection pressures. It is apparent that the ED differs from both SDs and IDs. The latter two salivary gland ducts reﬂect evolutionary history and diet (Tandler et al., 1998c, 2001). For instance, the ID in some bats varies according to hardbodied (chitinous) versus soft-bodied insects that constitute their diets (Tandler and Phillips, 1998). The ED data for the same species do not exhibit any discernible patterns or differences associated with diet. ACKNOWLEDGMENTS The authors appreciate the professional assistance of several colleagues who participated in ﬁeldwork, conﬁrmed species identiﬁcation of voucher specimens, or both. In particular, they thank Duane A. Schlitter, Stephen Williams, and Hugh H. Genoways. Toshikazu Nagato and Kuniaki Toyoshima participated in some of the studies on which this review is based. Carol Ayala and Thomas J. Slabe provided technical assistance for some aspects of this work, which was supported in part by a grant from the National Institute of Dental Research (to B.T. and C.J.P.) and Hofstra University HCLAS grants (to C.J.P.), as well as by ﬁnancial support from the Texas Tech University and Illinois State University Departments of Biological Sciences (to C.J.P.) and the Texas Tech EXCRETORY DUCTS OF SALIVARY GLANDS Institute of Environmental and Human Health (to B.T.). 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