Membrane coating granules in nonkeratinizing oral epithelium

JOURNAL

OF U L T R A S T R U C T U R E

RESEARCH

60, 212-220 (1977)

Membrane Coating Granules in Nonkeratinizing Oral Epithelium C. A. SQUIER Department of Oral Pathology and Collegiate Research Laboratories, College of Dentistry, University of Iowa, Iowa City, Iowa 52242 Received November 3, 1976 Examination of a variety of nonkeratinized oral epithelia with the electron microscope reveals the presence of a population of small cytoplasmic granules, approximately 0.2 ~ m in diameter, t h a t appear in the Golgi region of the prickle cell layers, migrate to the superficial aspect of ceils in the midepithelial region and t h e n apparently fuse with the cell m e m b r a n e in the t h i r d quarter of the epithelium. Serial sections cut through the granules show t h a t they are autonomous structures and not infoldings of the cell membrane. These granules are similar in size, distribution, and behavior to the " m e m b r a n e coating granules" of keratinized oral epithelium and epidermis and may represent an homologous organelle in nonkeratinized epithelium.

Among the cytological features that characterize a keratinizing stratified squamous epithelium is the presence of a population of small, lamellated granules that first make their appearance in the prickle cell layer of the epithelium. Although these granules have been referred to by a variety of names (20, 37), the term most frequently, if not most appropriately, used is that of "membrane coating granule"

cervical epithelium (10), it has also been claimed that such granules merely represent fortuitous sections through infoldings of the convoluted plasma membrane of adjacent epithelial cells (3). In an attempt to resolve the conflict this paper examines the appearance of membrane coating granules from a variety of nonkeratinized oral epithelia and describes the results of cutting serial sections through them.

(21). The occurrence of membrane coating granules in a variety of mammalian keratinized tissues such as epidermis (23, 29), oral epithelium (4, 8) and esophageal epithelium (26) is well documented but their representation in nonkeratinizing epithelia is more debatable. In keratinized tissues the granules are first seen in the upper spinous cells, after which they migrate to the superficial borders of the cells and, in the upper granular cells, fuse with the cell plasma membrane. As a consequence the contents of the granules, a series of lamellate structures, appear in the intercellular spaces of the upper granular and lower keratin layers. While granules of a similar size and distribution to those of keratinized tissues have been described in nonkeratinized buccal epithelium (12, 14, 30, 31, 41), lingual epithelium (27) and

MATERIALS AND METHODS Biopsies from buccal, labial and alveolar mucosa and from the floor of the m o u t h were t a k e n from h u m a n volunteers; other specimens were t a k e n from monkey buccal and labial mucosa. Tissues were fixed either in 3% phosphate-buffered glutaraldehyde followed by postfixation with 1% osmium tetroxide or in 1% collidine-buffered osmium tetroxide. All specimens were dehydrated in ascending concentrations of ethanol and embedded in Epon. Sections were cut on a Cambridge-Huxley ultramicrotome using a diamond knife; series of up to 15 sections were cut from blocks of h u m a n buccal and monkey alveolar mucosa and picked up on filmed single hole grids. All sections were stained with lead citrate and uranyl acetate and examined in an AEI EM6B or Siemens Elmiskop 101 electron microscope. RESULTS

The thickness of the epithelium varied considerably, from less than 100 t~m in the floor of the mouth to more than 500 t~m in 212

Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0022-5320

MEMBRANE COATING GRANULES IN ORAL EPITHELIUM the buccal epithelium. Classification of the epithelium into recognizable cell layers is difficult because well-defined strata, as seen in epidermis or keratinized oral epithelium, are absent (Fig. 1). There is a well-defined layer of cuboidal basal cells succeeded by several rows of larger, slightly flattened cells representing the prickle cell layer. The remainder of the epithelium, amounting to about half of its thickness, consists of flattened, nucleated cells with an essentially similar morphology. In all the epithelia examined small granules, first seen in the prickle cell layer, became predominantly arranged along the superficial plasma membrane of the cells at the midlevel of the epithelium (Fig. 2) and had disappeared from the cells making up the outer quarter of the epithelium. The majority of the granules consisted of a central dense amorphous core, surrounded by an electron-lucent zone which was bounded by a trilaminar membrane (Fig. 3a). Irregular dense strands sometimes extended radially from the core to the membrane, giving the granules a ~spoked" appearance; in rare instances a granule contained several parallel lamellae (Fig. 3c). Most granules had a circular profile with an average diameter of 170.6 t~m (SD +_ 70.3) although some granules tended to be more elliptical, with dimensions approximating 340 by 130 t~m (Fig. 3b). At the level where the granules were first evident (usually the second row of cells in the prickle cell layer) they were invariably seen in the vicinity of Golgi systems that lie adjacent to the cell nuclei of this layer (Fig. 4a). These Golgi systems comprise flattened cisternae surrounded by small circular vesicles. Serial sections cut through such Golgi systems did not reveal any connection between the flattened cisternae and adjacent granules. In more superficial cells granules were only

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occasionally seen adjacent to a Golgi system (Fig. 4b). The arrangement of membrane coating granules along the superficial plasma membrane of the cells was evident at the midlevel of the epithelium; fusion of the granules with the plasma membrane occurred in approximately the third quarter of the epithelium. Figure 5 shows a series of profiles that are suggestive of the sequence of events during fusion. When granules that were adjacent to, but not actually fusing with, the plasma membrane were examined in serial section (Fig. 6) there was no evidence of any continuity; because of their small size it was usually possible to encompass a granule within three serial ultrathin sections. Cells making up the outer one quarter of the epithelium, and superficial to the region of granule fusion, showed a well-defined thickening on the cytoplasmic aspect of their plasma membranes. The transition from cells with a normal plasma membrane to those with thickening was abrupt, membranes of adjacent cells often showing a marked difference in appearance. The intercellular spaces in the outer one quarter of the epithelium contained varying amounts of amorphous or fibrillar material and, on rare occasions, membrane-bounded vesicles. These vesicles did not have the thickened membrane characteristic of the immediately adjacent epithelial cells (Fig. 7), suggesting that they were not sections of cell processes but structures that had been extruded before membrane thickening took place. Once membrane thickening had occurred, there was no further change in the morphology of either the plasma membrane or the intercellular space of the remainder of the epithelium. DISCUSSION Serial sections left no doubt that the small cored granules described here do not represent an artefact of sectioning as El-

Fla. 1. A light micrograph of a 0.5 t~m thick, resin embedded section through nonkeratinized oral epithelium from h u m a n alveolar mucosa. Note the absence of a keratinized layer or other well-defined strata. × 150. FIG. 2. An electron micrograph of cells from the midlevel of nonkeratinized epithelium. Numbers of small granules (arrows) are situated close to the superficial cell membrane [(N) cell nucleus]. × 11 000. 214

MEMBRANE COATING GRANULES IN ORAL EPITHELIUM Labban and Kramer (3) have proposed, but rather a distinct cell organelle. Further evidence to support this assertion comes from experiments in which electrondense intercellular tracer substances may be seen surrounding the cells containing these granules but not within the granules themselves (34, 36) and from the demonstration of a reaction product for acid phosphatase within membrane coating granules in the upper prickle cells of nonkeratinized epithelium but not in the intercellular space at this level (31). Finally, there is the difficulty of explaining the consistent location of granules at only the superficial aspect of the epithelial cells if they represent cross sections of interdigitating cell processes. Accounts of morphologically similar granules in the cells of nonkeratinized cervical epithelium (10), oral sulcular epithelium (15) and in the "lesser keratinized" regions between the filiform papillae of human tongue (27) suggest that such granules may be a characteristic component of nonkeratinized stratified squamous epithelia and, by virtue of similarities in size and distribution, homologous with membrane coating granules of keratinized epithelium. Thus granules in both tissues are of a similar size, have a similar distribution within the epithelium and have certain chemical components in common. Cytochemical studies have identified hydrolytic enzymes in the granules of keratinized (9, 36, 40) and nonkeratinized epithelia (15, 31, 32). Similarly mucosubstances, possibly glycoproteins, have been demonstrated in both types of granules (13, 14). The major difference is the absence of internal lamellae in the nonkeratinized granule. The question that now arises is as to the origin, fate and function of the granules in nonkeratinized epithelium. There is a considerable amount of indirect evidence that the membrane coating granules of keratinized epithelia originate from the Golgi

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system of the cells (1, 6, 18, 21, 38, 39) and the frequent association of the granules with the Golgi complex seen in the present study would argue for such an origin in nonkeratinized epithelium. This conclusion is not invalidated by the failure to demonstrate continuity between these two different organelles in serial sections for, by the time the granules become identifiable by virtue of their central dense core they are likely to have budded off from the cisternae of the Golgi system. Supporting evidence comes from the cytochemical studies already mentioned in which both hydrolytic enzymes, that are packaged in the Golgi complex (22) and mucosubstances, that are elaborated in the Golgi complex in many tissues (25) have been identified in these granules. The fate of the granules is also comparable to that of membrane coating granules of keratinized epithelium, which have frequently been observed to fuse with the plasma membrane of cells in the upper granular layer and to extrude their lamellate contents into the intercellular space (8, 11, 19, 20). However, as the granules from nonkeratinized epithelium contain only amorphous material it is impossible to distinguish this from the existing unorganized material making up the intercellular substance and so assess the extent of their contribution to this region. The thickening h e r e observed on the cytoplasmic aspect of the membranes of the superficial cells has been reported in various nonkeratinized epithelia (7, 10, 12) and seems, as is the case in keratinized epithelium (5, 24) to be unconnected with the activities of the membrane coating granules. Nevertheless, it is of interest that this thickening has been attributed to the deposition of sulfur-rich protein from the regular, "single" keratohyalin granules which are often observed in nonkeratinized oral epithelium (31). Finally, there is the question of the role of the membrane coating granules in non-

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C. A. SQUIER

Fro. 3a. Typical form of a granule from nonkeratinized oral epithelium. There is a central dense core from which fine filaments radiate out towards the enclosing trilaminar membrane. (b) An elongate form of granule sometimes encountered in nonkeratinized oral epithelium, (c) A granule from nonkeratinized oral epithelium containing a lamellate structure. All micrographs × 185 000. FIG. 5. Micrographs of the superficial membrane of cells in the third quarter of the epithelium showing profiles suggestive of granules fusing with the cell membrane. All micrographs, × 100 000.

MEMBRANE COATING GRANULES IN ORAL EPITHELIUM

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Fro. ~ An extensive Golgi system adjacent to the nucleus (N) of a cell from the lower prickle cell layer of nonkeratinized oral epithelium. Several granules with dense cores (arrows) lie closeto the Golgivesicles. × 22 000. (b) A small Golgi system associated with granules (arrows) in a cell from the midlevel of nonkeratinized oral epithelium. × 24 000.

keratinized epithelium. The functions that have been attributed to the keratinized granules are as varied as their nomenclature but there is now good evidence that they contribute to the formation of an intercellular permeability barrier located in the superficial portion of epidermis and keratinized oral epithelium (2, 28, 34). The level in nonkeratinized epithelium at which the membrane coating granules fuse with the plasma membrane corresponds closely with the limit of penetration through this tissue of intercellular tracers, such as horseradish peroxidase and lanthanum, that have been introduced subepithelially (34, 35). It is therefore reasonable to suggest t h a t the membrane coating granules of nonkeratinized epithelium, like their counterparts in ker-

atinized epithelium, are involved in the formation of an intercellular permeability barrier in the superficial portion of the epithelium. However, the membrane coating granules of keratinized epithelium extrude organized lamellae into the intercellular space while the nonkeratinized granules secrete 0nly amorphous material apparently containing hydrolytic enzymes and glycoprotein. It is thus possible that the intercellular permeability barrier in nonkeratinized epithelium arises either from the contribution of new intercellular material or by enzymatic modification of the existing intercellular substance by the membrane coating granules. The author wishes to thank Lynn Rooneyfor her expert technical assistance and Julia Meyer, who

FIG. 6. Electron micrographs of three serial sections showing g r a n u l e s adjacent to the m e m b r a n e s of cells at the midlevel of the epithelium. Three g r a n u l e s which appear to be diametrically sectioned in b (arrows) appear only in grazing section in the i m m e d i a t e l y adjacent sections a and c (circles with arrows). Other granules, which appear in cross section in sections a and c are barely detectable in section b and are not present in the third section. There is no continuity between the g r a n u l e s and the cell m e m b r a n e at t h i s level in the epithelium. All micrographs, x 30 000.

MEMBRANE COATING GRANULES IN ORAL EPITHELIUM

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FIG. 7. A portion of intercellular space from the outer quarter of the epithelium. Several circular vesicles are present which lack the thickening evident on the internal aspect of the cell membrane; compare this with the unthickened membrane of deeper cells, illustrated in Fig. 5. × 90 000.

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