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Ultrastructural observations on the keratohyalin granules of the rat oral epithelium
ULTRASTRUCTURAL OBSERVATIONS ON THE KERATOHYALIN GRANULES OF THE RAT ORAL EPITHELIUM
Electron Microscope
Unit. Department of Dental Surgq, P.O. Box 147, Liverpool.
Unlvcrsit) L60 3BX
of Liverpool.
Pcmhrokc
Place.
Summary---When examined, after various fixatives. the kcratohyalin granules (KHG) of the rat oral mucosa showed morphological variability and a heterogeneous nature. In buccal epithelium. the KHG were circular and oval with no close contact with tonohlaments and were coated with ribosomes. They consisted of two types of material exhibiting different reactions to the fixatives. The first was electron-translucent when fixed in glutaraldehyde alone but was electron-dense when osmium tetroxide was included in the lixation procedure. This material was found as circular or elongated surface blebs and as oval regions in the body of the granule and was thought to be lipoprotein. The second type of material was electron-dense with all the fixatives and was thought to be protein rich in amino acids containing -SH groups. Some KHG of the tongue epitheiium had the same structure as the KHG of the buccal epithelium. Others were more irregular. coated with ribosomes and were in contact vvith tonofilaments. A third type of material appeared in the tongue epithelium KHG that was of moderate electron-density, Occasional KHG demonstrated all three types of material. The palatal epithelium KHG were irregular. uniformly electron-dense and were in intimate contact with the tonofilaments. When fixed with osmium tetroxide to which sucrose was added to the buffer. the KHG were less electron-dcnsc. Intra-nuclear granules. often coated with ribosomes. exhibited the same reactions to the different fixations as the bleb material. Keratohyalin-like unclusions were seen in the mitochondria. From the observations. it is concluded that thcrc arc no well defined types of KHG based on morphology. IUTRODUCTION
The structure of the rat oral cpithelium exhibits a wide range of variation. The degree of keratinization varies according to the site and there are also morphological variations in the keratohyalin granules associated with keratinization of the epithelium. In ultrastructural studies. keratohyalin granules (KHG) are usually described as spherical or irregular masses of elcctrondense material with no limiting membrane, bordered by ribosomes and frequently associated with tonohlaments. There have been several attempts to classify keratohyalin granules on their morphological appearance. Chen and Meyer (1971) describe two types: (I) the epidermal type which are irregular in shape, usually surrounded by ribosomcs and associated with tonofilaments found m regions undergoing complete orthokeratinization: and (2) the cheek type occurring only in the oral epithelium. These granules were largcr. more regular and surrounded by ribosomes but without the prominent association with the tonofilaments shown by type (I) granules. Farbman (1966) reported four types of KHG: t I) globular granules made up of homogeneous elcctronopaque material. (3) irregular granules also of homogeneousmaterial. (3) heterogeneous granules consisting of electron-dense material plus 21 second slightly less
dense material. and (4) dense keratohyalin material arranged as a dense rim around an electron-translucent. amorphous centrc. Jessen (1970) classified granules into two types which he calls: (I) “single granules” containing osmophilic and oxidizable material; (2) “composite granules” consisting of one component seen to bc clcctron-dense after fixation with glutaraldchyde~~osmium fixation and digestible with pepsin. together with a second component thought to be identical to single granule material. lntranuclcar granules with a morphology resembling KHG have been described by Matoltsy and Parakkal (1967) in the epidermis, by Oehmkc and Pctry (1964) in oesophageal epithelium and in the oral epithelium by Farbman (1966). Squier (196X) and Jcssen (1970). The latter suggested that the intra-nuclear kcratohyalin-like material is identical to the cytoplasmic “single granules”. In previous studies. there has been variation in fixation techniques and in the sites from which the tissue has taken. The purpose of this study was to observe the morphology of KHG in the eipthelium from three regions of the rat oral cavity-the mucosd of the hard palate the tongue and the buccal mucosa following fixation by eight different techniques. This made it
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possible to assess the influence of the preparation techniques on the observed structure and morphology of the KHG. The fixatives were selected as ones widely used in ultrastructural studies and possessing different fixation properties. Methanol was used as a dehydrating agent because it has a relatively less solvent action than other commonly used dehydrating agents. The superiority of methanol in the dehydration of aldehyde fixed tissues has been reported (Feder, 1960).
MATERIALS
AND METHODS
Two sibling, adult, Wistar rats were killed and small pieces of tissue were immediately excised. Care was taken to ensure that the pieces of tissue were no more than I mm thick in one dimension. Tissue was taken from the dorsal surface of the tongue, the hard palate and the buccal mucosa and subjected to the following methods of fixation: I. Fixation for 1 hr in 2.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4 (Sabatini. Bensch and Barnett. 1963). 2. Fixation for 1 hr in 4.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4. 3. Fixation for 1 hr in 6.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4. 4. Fixation for 1 hr in 6.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4 followed by post-fixation with I per cent osmium tetroxide in veronal acetate buffer at pH 7.2 for 0.5 hr (Palade, 1952). 5. Fixation for I hr in 6.25 per cent glutaraldehyde post-fixed with I per cent osmium tetroxide in veronal acetate buffer, containing 4.5 per cent (w/v) of sucrose for 0.5 hr (Caulfield, 1957). 6. Fixation for 0.5 hr in 1 per cent osmium tetroxide in vcronal acetate buffer at pH 7.2 (Palade, 1952). 7. Fixation for 0.5 hr in 1 per cent osmium tetroxide in Verona1 acetate buffer containing 4.5 per cent (WI v) of sucrose per ml of fixative. 8. Fixation for I hr in 0.6 per cent potassium permanganate in veronal acetate buffer at pH 7.4 (Luft. 1956). Tissue from a IO-day-old-rat was also fixed in 6.25 per cent glutaraldehyde and post-fixed in osmium tetroxide as in 4 above. All tissues were dehydrated in methanol and embedded in Araldite. Sections 2 pm in thickness were examined under phase contrast and areas suitable for thin sectioning were selected. Sections stained in P.A.S. and toluidine blue were examined by light microscopy. Sections approximately 60 nm thick were cut on an LKB ultramicrotome with a glass or a diamond knife, collected on copper grids and stained with a 25 per cent uranyl acetate in methanol solution. Some sections were left unstained. The grids were examined in a Philips E.M. 300 electron microscope.
OBSERVATIONS
Hisrolo~q~. Using the classification of the degree of keratinization as dctcrmined with the Mallory stain (Alvdres and Meyer. 1971). the rat buccal epithelium was “incompletely” orthokeratinized in sections stained with P.A.S. and toluidinc blue. In the stratum corncum there was a very thin inner layer of keratin which corresponded to a layer which stained like hair with Mallory’s stain and a wider inner layct which stained like the cellular layers. The two keratin layers were evident in the electron microscope (Fig. 7). The stratum granulosum was well developed with many KHG granules.
Glutur~u/dch~dt~,fisurior~. In the electron microscope. the KHG showed a similar appearance when fixed in the 3 different concentrations of glutaraldchydc and these will be described together. The KHG were oval or occasionally circular in shape varying in size from 0.5 to 4.5 itrn and their outline was not always regular. The body of the granules consisted of two types of material (Fig. I. Plate I). one being electron-trnnslucent and the other being electron-dense. The clectron-translucent material occurred in various distributions and varying proportions in the granules (Fig. 2. Plate I). Sometimes the electron-translucent material was within the body of the KHG as a small number of ovalor round bodiesofvarying sizes and irregular distribution (Fig. 2). In others (Fig. 3. Plate I). these bodies wero more numerous and mote uniform in size and distributed more evenly. In Fig. I shovvs an almost complete band of electron-translucent material at the periphery of a granule. but in other granules (Fig. 3) this electron-translucent material appeared as an incomplete band giving the granule an irregular outline with elongated blchs of electron-translucent material on the surface which in some instances appeared circular (Fig. 4. Plate I ). Occasionally small granules consisting of a large proportion of ciectron-translucent material were seen (Fig. 5. Plate I). but it was thought that these could rcprcscnt oblique sections through ;I surface bleb. Ribosomes wcrc closely associated with the keratohyalin granules (Fig. I ) and formed a surface layer on the granules irrcspcctivc of which type of material lay immediately inside (Fig. 4). Tonofilamcnts were present close to the granules but little direct contact with the granules was observed. The tonofilaments were scattered randomly through the cells and not organized into discrctc bundles. Small intra-nuclear granules were observed consisting of electron-translucent material only and bearing a close resemblance to the electron-translucent material of the cy toplasmic granules (Fig. 6. Plate 1). Fi.wfiou with 6.25 /XV WU1 ~~/uttrrn/dch u/c ~111d o,snkm tc/r.o.rirk. The KHG in the buccal cpithclium fixed in 6.25 per cent plutaraldehyde and post-fixed in
Ultrastructural
observations on keratohyalin granules
osmium tetroxide were circular (Fig. 7, Plate 2) and. like the intra-nuclear granules, consisted of electrondense material alone (Fig. 8, Plate 2). The KHG consisted of uniformly electron-dense material and were surrounded by layers of ribosomes of variable thickness which seemed to be incorporated into the body of the KHG granules. At the surface, groups of ribosomes projected from the KHG into the cytoplasm. There was no close association of tonofilaments and KHG. The intra-nuclear granules (Fig. 8) had an identical appearance to the cytoplasmic granules and were also surrounded by ribosomes. Occasionally, the KHG were situated close to mitochondria (Fig. 9, Plate 2) and had an irregular outline similar to those seen following glutaraldehyde fixation above (Fig. 4) but with ;I uniformly dense structure. Fi.uation with glutaraldehyde and buffered osmium ~ctroride conruining sucrose. The KHG appeared electron-dense and circular or oval in outline (Fig. 10. Plate 2). The peripheral layer of some of the granules appeared to consist of ribosomes (Fig. 10) apparently embedded in the surface of the granule. The intra-nuclear keratohyalin-like material was electron-dense. Some KHG in this specimen (Fig. 11, Plate 2) were faintly mottled and possessed surface blebs. The circular areas within the granules and the blebs were slightly less-electron dense than the material surrounding the KHG. Firution with buffered tetroxide. The keratohyalin granules appeared irregularly shaped, electron-dense and covered by layers of ribosomes (Fig. 12, Plate 3). Occasionally small granules were seen surrounded by a relatively large area of high ribosome density. The intranuclear bodies were electron-dense. Fir~tion with ht@red osmium tetroxide containing .S~UYW.The granules were very irregularly shaped and electron-dense. The intra-nuclear bodies were electrondense. In some unstained sections (Figs. 13 and 14, Plate 3), the periphery of occasional KHG was more electron-dense than the interior (Fig. 13, Plate 3) whilst other KHG were made up of electron dense-bodies surrounded by a less electron-dense material (Fig. 14. Plate 3). Fiuution with potassium permanganate. The KHG were variable in outline (Fig. 15, Plate 3). Occasional Webs and bodies within the granules were more electron-dense than the matrix or body of the KHG. The intra-nuclear bodies appeared electron-dense.
Histoloyy. The tongue epithelium was mostly “incompletely” orthokeratinized on light microscopic examination although the epithelium was much thicker than that of the buccal epithelium. The stratum granulosum was thicker and contained many more KHG than the cheek epithelium. The stratum corneum was also much thicker than in the cheek. Regions of almost “complete” keratinization were present on the dorsal surfaces of the filiform papillae.
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Ultrustructural ohserwtions Fi.uarion wirh glutaruldehydc. Fixed in the varying concentrations of glutaraldehyde. the KHG displayed a similar structure to those of the buccal epithelium (Fig. 16, Plate 3). The intranuclear granules, consisting of electron-translucent material, were more numerous than in the buccal epithelium (Fig. 17, Plate 3). Both the electron-translucent and electron-dense regions were bordered by ribosomes at the surfaces (Fig. 16). KHG were present with the electron-translucent blehs embedded in a peripheral zone of moderate electron density (Fig. 18, Plate 4). The main bulk of the KHG material was of high electron density. Fixation with 6.25 per cent glutaraldv/~~rlc~ and osmium tetroride. The KHG displayed a very variable appearance. Some were circular, electron-dense and surrounded by ribosomes throughout the body ol some of the small KHG (Fig. 19, Plate 4). Some of the KHG showed no association with the tonolilaments (Fig. 19) while others showed a distinct link with tonofilaments (Fig. 20. Plate 4). Bundles of tonofilamentx ran from the desmosome attachment plaque into direct contact with one edge of some KHG which were less circular in shape (Fig. 21. Plate 4). The material in contact with the tonofilaments was of moderate electron density compared to the main body of the granule (Fig. 21). The intra-nuclear bodies were of high electron density (Fig. 22. Plate 4) as were the large circular hlebs of the KHG. The central zone of the granule was less electron-dense. Often the blebs were elongated to form a surface zone of extremely electron-dense material which in some cases was complete and in others apparently forming a cap over the incomplete. granule. Fiwtion with glutaruldehydr und hr#&d osmi~rm tetroxide with sucrose. The large blebs of the irregular11 shaped KHG were of less electron density than the central zone of the granule (Fig. 24. Plate 5). The granules were often elongated (Fig. 24). Tonofilaments were attached to the material of moderate electron density. The electron-translucent blebs were contcd with ribosomes on the exterior surface of the granule. There were no ribosomes on the surface of the blcb in contact with the main bulk of the granule which was of moderate electron density. Another KHG (Fig, 74 left) consisted of electron-dense material with two caps of less dense material. The intra-nuclear granules were electron-translucent (Fig. 23). Fix&on with osmium tetroxidr. The KHG wcrc‘ elongated in the upper layers of the stratum granulosum and were of uneven density (Fig. 25. Plate 5). The blebs, where present, and the intra-nuclear granules were electron-dense but the central areas of the circular granules are much less electron-dense. Fisution with hufired o.smium tetro uidc corutrirnml sucrose. These granules exhibited the three types 01 material of moderate electron density on the surface (Fig. 26, Plate 5). The intra-nuclear bodies were elcctron-translucent.
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S. A. Kempson
Fixution with potu.ssium prrmungunatr. The electrondense KHG had various shapes often with denser material on the surface of the KHG (Fig. 27, Plate 6). The intra-nuclear bodies were electron-dense and circular.
Palatal rpithelium Histology. The epithelium of the hard palate was moderately thick and showed “‘complete” orthokeratinization. The stratum corneum stained uniformly throughout its thickness when examined in the light microscope. Ultrustructurul ohservutions Fixation with gluturuldehyde with 6.25 prr cent gluturuldehvde und with osmium tetroxide post-jixation, with o&urn tetroxidejxution and potassium permanganutr. The different methods of fixation had much less effect on the morphology of the KHG in the hard palate than in the tongue and cheek epithelium. The KHG in the palate were electron-dense and irregularly shaped (Fig. 28, Plate 6). often juxtaposed to mitochondria (Fig. 32, Plate 7) and in close contact with thick bundles of tonofilaments. The surfaces (Fig. 29, Plate 6) not in contact with the tonofilaments showed a border of ribosomes. Fixation with gluturuldehydr osmium trtroxidr and buffered osmium tetroxide with sucrose. The KHG appeared less electron-dense in the specimens fixed in glutaraldehyde and osmium tetroxide and buffered osmium tetroxide containing sucrose (Fig. 30, Plate 6), and were bordered with a coat of ribosomes. This appearance was very similar to the specimens fixed in osmium tetroxide buffered with sucrose. The palate tissue fixed in the other conditions all showed a similar appearance that was more electron-dense than the sucrose buffered specimens. Intru-nucleur bodies. Intra-nuclear bodies were rarely seen in the palate but occasionally electrondense bodies were seen in a specimen of hard palate from a lo-day-old rat fixed in 6.25 per cent glutaraldehyde post-fixed in osmium tetroxide. Ten-day-old rut palate. The lo-day-old rat palate displayed features that were not typical of the keratinized epithelium of the mature rats; for example, the KHG were nearly circular and did not show much contact with the tonofilaments although they seemed to emerge from the centre of one KHG which appeared to be made up of ribosomes (Fig. 31, Plate 6). Some granules of the lo-day-old rat palate epithelium showed very dense circular bodies embedded in a less dense granule. The KHG became larger with a more irregular outline in the two most superficial cell layers of the stratum granulosum. Mitochondria and krratohyu?alin-like muterial. Keratohyalin-like material was seen within the mitochondria (Figs. 33 and 34, Plate 7) of tongue and cheek epithehum. Small circular bodies of electron-dense material were seen within the body of mitochondria in the cells of the stratum granulosum. The mitochondria appeared to be degenerating in this layer. This material
had exactly the same appearance as the cytoplasmic keratohyalin granules. These inclusions had no limiting membrane (Fig. 34, Plate 7). Although keratohyalin-like material was not seen within the mitochondria of the hard palate mucosa, the KHG themselves often formed a halo around the mitochondria. DISCUSSION
This study demonstrates the morphological variability and heterogeneous nature of the KHG of the rat oral epithelium when different fixatives are employed. Histochemical and biochemical studies have given conflicting results on the composition of KHG (Cohen 1967; Matoltsy and Matoltsy 1970; Ugel and Idler 1972). The KHG of the tongue and cheek epithelium showed surface blebs and round or oval bodies within the granule body that behaved in a different way from the main body of the KHG. These’blebs have previously been called “dense homogeneous deposits” (Fukuyama and Epstein. 1971) and “single granules” (Jessen, 1970. 1973). The term “dense homogeneous deposits” is not favoured as, under certain fixation conditions, the “deposits” are not dense. The term “single granules” implies discrete, independent bodies. There was no evidence that the granules in the cytoplasm with the same behaviour as surface blebs were discrete granules and they could be sections of surface blebs in a different plane. Therefore this report has utilized the term “bleb” to describe the circular or elongated bodies found at the surface of the KHG. The blebs showed variable degrees of incorporation into the body of the granule, some being almost completely immersed in the main body of keratohyalin material. The very variable appearance of the blebs was comparable to that of three other cell components: (a) the intra-nuclear granules, (b) the round or oval granules that give the KHG their mottled appearance,(c) the keratohyalin-like mitochondrial inclusions (Kimura, 1972). These components and the blebs shared the variations in electron density resulting from the differing preparation methods. The main body of the KHG material in which the round or oval bodies were embedded appeared electron dense following all fixation procedures. This material, with the “single granules”. forms the “composite granules” (Jessen, 1970). This component has been reported (Jessen, 1970) as digestible in pepsin and non-oxidizable with hydrogen peroxide. In the tongue epithelium KHG. the third type of material. that of moderate density, was seen in all the fixation conditions except from potassium permanganate. The bundles of tonofibrils in contact with the KHG in the tongue were usually in contact with this third type of material. The intra-nuclear granules increase in size in the more superficial cells (Jessen, 1970). At the same time nuclei in the more superficial stratum granulosum cells show a rarefaction of their nuclear material. It is sug-
Ultrastructural
observations
gested that breakdown products of the nuclei may accumulate to form the intra-nuclear granules in a similar way to the keratohyalin like inclusions of the mitochondria although no clear evidence for this has been observed. Glutaraldehyde preserves proteins, and in particular nucleoproteins, well as it is thought to cross-link polypeptides at reactive side groups (Hayat, 1970). Glutaraldehyde is incapable of rendering lipids insoluble in organic solvents and therefore failed to show cellular membranes. Osmium tetroxide is soluble in certain lipids and can be taken up in an unreduced form by fully saturated lipids (Hayat. 1970). Osmium reacts with some amino acids owing to the reducing potential of -SH groups (Fukuyama. Wier and Epstein. 1972). The reactivity of osmium tetroxide is modified hy osmolarity (Palade, 1952) which could explain the appearance of the bleb material when sucrose was added to the osmium tetroxide fixatives. This effect could also be explained by the incomplete osmication of the tissue due to the retarding effect which added sucrose has on the rate of osmium tetroxide penetration into tissues (HagstrBm and Bdhr, 1960). Potassium permanganate has a larger oxidative potential than osmium tetroxide but its useful oxidative properties are largely confined to preserving lipo -protein complexes (Pease, 1964). From the observations of the KHG under different fixation conditions. it is suggested that the bleb material found in the KHG. the nuclei and the mitochondria consist of lipoprotein material. The main body of the granules is thought to consist ofprotein material rich in amino acids containing -SH. It has been reported that part of protein component of KHG is synthesized dr uoco in the stratum granulosum (Fukuyama and Epstein, 1971). Keratohyalin-like inclusions have been reported in mitochondria of stratum granulosum cells of the epidermis (Matoltsy and Parakkal, 1967) although it was undecided whether they were inside or outside the outer mitochondrial membrane. Keratohyalin-like mitochondrial inclusions have been described recently (Kimura, 1972). Thin sections of neonatal rat tongue were oxidized with hvdrogen peroxide and it was found that mitochondrial keratohyalin-like inclusions behaved the same way as the KHG blebs. It is possible that as the mitochondria degenerate in the stratum granulosum the lipid component of the mitochondrial membrane form part of the KHG. Other cell membranes may also contribute material to the KHG. The significance oflipids in KHG and keratinization has not yet been fully realized. KHG are almost completely digested after incubation with lipase (Cohen. 1967) and it was suggested that they were composed of complex lipids not removed by alcohol or during routint embedding. In the epidermis, lipids have great zignificance in normal keratinization (Swanbeck. I959: Rothman. 1964). Nicolaides (1964) found histochemitally that intense esterase activity in the upper cells of the epidermis was concurrent with the disintegration of the cell organelles. suggesting that these estcrases
on keratohyalin
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granules
attack the lipids and cause the membrane to disintegrate. The fate of the degraded lipid is obscure. From these observations. it is concluded that there are no clear-cut types of KHG based on morpholog), as has been suggested (Chen and Meyer, 1971: Farbman. 1966; Jessen. 1970). Instead there appears to be a gradation in the morphology related to the position and presumably function of the epithelium. The biochemical composition of the KHG probably also shows a gradation related to the “incomplete” orthokeratinized cheek to the “complete” orthokeratinized hard palate. A~h,~ol~l~,~/~e,,~(,~tf,s~ This
work
v+as supported
hy research
grant numhcr 2OY f’rom the United Liverpool Hospitals. I am indebted to Dr. J. .Appleton and Dr. C\‘. R. T!ldcsIL’) for their wluahlc advice.
Alwres 0. F. and Meyer J. 1971. Variable feuturca and regional differences in oral epithelium. In: C‘urwr~t C‘otrc’t’/~lr c?/ thv Hisrolq~ (!f t/w 01~1 Mwow (Edited hq Squier C. A. and Meyer J.) Chap. 3. p. Y7. Charles (‘. Thomas. Illinois. U.S.A. Caulfield J. B. 19.57. EfTects of varying the vehicle ~OIosmium t&oxide fiwtion. J. hiop/~!~ hirw/l~,r,~ (‘lvol 3, 827 830. Chen S. Y. and Meyer J. 1971. Regional dlfferenccs in tonofilaments and kerntohyalin granules. lo: Clfw~,,,t Ciwwpl\ of t/w Ni.srolo~g~~of ORI/ Mu~o.su (Edited hy Squier. C‘. A. and Mcycr. Julia) Chap. 6. p. 114. Ch:trle\ C. Thomas. Illinois. U.S.A. Cohen L. lY67. Presence of lipids in kerntohyalin ~ranulcs of human gmgiw. J. dr,lt. RCA.46, 630. Farhmnn A. I. 1966. Morphological variahilit> of keratohqalin. jIr~trr. Rw. 154, 17.5 286. Fedcr N. 1960. Some modifications in conventional tcchniques of tissue preparation. J. Hi~toc~hw. (‘\~t~w/~cw. 8.
309 Fukuyamn K. and Epstein W. L. lY71. The inhibltion of RNA and protein synthesis in granular cells h! actinomytin-D and puromqcin. J. inwst. Derm. 56, 21 I 212. Fukuyama K.. Wier K. .A. and Epstein W. L. 1972. Dense homogeneous deposits of’ keratohyalin granules in new born rat epidcrmls. J. ultrrrstrwt. Rrs. 38, I6 26. Halat M. A. 1970. Fixation. In: Pri~rci/~/c\LIEU/‘/‘~hticp~~\ of ICicctrwn51 kroswp I‘: Biological applicatwns. vol. 1. Chap. I. 2nd edition. van Nostrund Reinhold C’ompanq. Neu York.
Jessen H.
1970. Two types of keratohyalln granules. ./. ~rlrrc~.~rrrc~r. Rrs. 33, 95. I 15. Jessen H. 1973. Flectron cytochemical dem\)nstratwn of aulphqdrql groups in keratohyalin granulea and in the pcripheral envelope of cornified cells. Hi.~oc’/wmi~ 33, IS 29. Kimurn M. 1972. Keratohyalin granule-llke mitochondrial inclusion m the neonatal rat tongue and oesophageal epithelia. J. Elcc.tr.m .Micmsc. (‘T’oli!~) 21. 191 194. Lufi J. H. 1956. Permanganate: a new fixative [or electron microscop!. J. hio/~h>~ hioc~lwvr. C‘~to/.2, 799 X02. Matoltsq A. G. and Matoltsy M. N. 1970. The chemical nature of keratohyalin granule5 of the epldcrmis. .I. (c/r /TiCA. 47. 5’) 7 hoi.
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Matoltsy A. G. and Parakkal P. F. 1967. Keratinization. In: Ultrastructure ofNormal and Abnormal Skin (Edited by Zelickson A. S.) pp. 76104, Lea and Febiger. Philadelphia, U.S.A. Nicolaides N. 1964. Lipids, membranes and the human epidermis In: Thr Epidermis. (Edited by Montdgna, W. and Lobitz W. C.) Chap. 16. pp. 51 I-538, Academic Press, New York. Oehmke H. J. and Petry G. 1964. Beobachtungen iiber das Auftreten von Keratohyalingranuia in Zellkern. Expcrimrntia 20, 422-423. Palade G. E. 1952. A study of fixation for electron microscopy. J. rxp. Med. 95, 285-298. Pease D. C. 1964. Fixation. In: Histological techniques for Electron Microscopy, Chap. 3, 2nd Edition. Academic Press. New York.
Rothman S. 1964. Keratinization in historical perspective. In: The Epidrrmis (Edited by Montagna W. and Lobitz W. C.) Chap. 1, pp. l-14, Academic Press, New York. Sabatini D. D.. Bensch K. and Barnett R. J. 1963. Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymic activity by aldehyde fixation. J. &I Biol. 17, 19-58. Squier C. A. 1968. Ultrastructural observations on the keratinization process in rat buccal epithelium. Arch orul Bio/. 13, 11451451. Swanbeck G. 1959. Macromolecular organisation of epidermal keratin: an x-ray diffraction study of the horny layer from normal. ichthydotic and psoriatic skin. Acta. drrm. Vewr. suppl. 43, I-37. Ugel A. R. and Idler W. 1972. Further characterization of bovine keratohyalin. J. cell biol. 52, 453-464.
R&mr%Quand examinees, apres diverses variations des fixatifs. les granules keratohyalines (GKH) de la muqueuse orale du rat ont montrees une variabilite morphologique et une nature heterogene. Dans l’epithelium buccal les GKH Ctaient circulaires ou ovales, sans contact intime avec les tonofilaments et Ctaient doublees de ribosomes. 11sconsistaient en deux types de materiel exhibant differentes reactions aux fixatifs. Le premier Ctait transparent aux electrons, quand fixt dans la glutaraldehyde seule, mais opaque aux electrons quand le tetroxyde d’osmium Ctait inclu dans le pro&de de fixation. Ce materiel se presentait comme des vesicules superficielles circulaires ou allongees et comme des regions ovales dans le corps des granules et on supposait qu’elles Ctaient des lipo-proteines. Le deuxieme type de materiel etait opaque aux electrons avec tous les fixatifs et on le considerait d’ttre riche en proteines dans les aminoacides contanant les groupes -SH. Quelques GKH de l’epithelium de la langue avaient la mime structure que les GKH de l’epithelium buccal. D’autres etaient plus irrtgulires, doubles avec des ribosomes et etaient en contact avec destonofilaments. Un troisieme type de materiel apparaissait dans les GKH de l’epithelium de la langue, que Ctait dune densite electronique mod&e. Occasionnellement les GKH montraient tous les trois types de materiel, Les GKH de l’epithelium palatal Ctaient irregulieres, uniformement opaques aux electrons et etaient en contact intime avec les tonifilaments..Quand fix&es avec du tetroxyde d’osmium uaquel on ajoutait de la sucrose au tampon, les GKH etaient moins opaques aux electrons. Les granules intra-nucleaires, souvent doublees avec des ribosomes, exhibaient les memes reactions aux differents fixatifs, par ex: le materiel vesiculeux. Des inclusions d’aspect kerato-hyalinique ont et6 remarquees dans les mitochondries. De ces observations on conclut qu’il n’y a pas de types bien d&finis de GKH basees sur la morphologie. Zusammenfassung-Die Keratohyalinkornchen (KHG) der Mundschleimhaut der Ratte zeigten bei Priifung nach Benutzung verschiedener Fixiermittel morphologische Variabilitlt und ein heterogenes Wesen. In bukkalem Epithel waren die KHG kreisfijrmig und oval, in keinem engen Zusammenhang mit Tonofasern, und sie waren mit Ribosomen bedeckt. Sie bestanden aus zwei Materialarten, welche verschiedene Reaktionen zu den Fixiermitteln zeigten. Die erste war elektronendurchscheinend, wenn in Glutaraldehyd, allein, fixiert, war aber elektronendicht, wenn Osmiumperoxyd in dem Fixierverfahren enthalten war. Es wurde festgestellt, darj dieses Material kreisfiirmige oder langgestreckte Oberfliichenbllschen und ovale Stellen in dem Kiirper des Kornchens waren und man meinte, daD sie Lipoprotein seien. Die zweite Materialart war mit allen Fixiermitteln elektronendicht und es wurde angenommen, da13 sie proteinreich in Aminsluren mit Gehalt von -SH Gruppen sei. Einige KHG’s des Zungenepithels hatten die gleiche Struktur wie das KHG des bukkalen Epithels. Andere waren irregularer, mit Ribosomen bedeckt und waren in Bertihrung mit Tonofasern. Eine dritte Materialart erschien in dem Zungenepithel KHG, welche mlDige Elektronendichte be&. Gelegentliches KHG zeigte alle drei Materialarten. Die Gaumenepithel KHG waren irregular, einheitlich elektronendicht und standen in enger Verbindung mit den Tonofasern. Die KHG waren weniger elektronendicht, wenn sie mit Osmiumtetroxyd fixiert waren, bei dem Saccharose dem Puffer zugeftigt war. Inter-Kern-Kornchen, die oft mit Ribosomen ilberzogen waren, zeigten die gleichen Reaktionen auf verschiedene Fixiermittel wie das Bllschenmaterial. Einschltisse von einer Keratohyalin iihnlichen Art wurden in der Mitochondrie festgestellt. Aus den Beobachtungen wird der Schlulj gezogen, da13 es keine gut definierten Arten von KHG gibt. die auf Morphologie gegrtindet werden.
Ultrastructural
observations
on keratohyalin
granules
lOI7
S. A. Kempson
PLATE 1
The electron
micrographs
show sections
Figs. l-6.
stained
with uranyl
Glutaraidehyde-fixed
acetate,
unless stated otherwise.
buccal epithelium
Fig. 1. An oval KHG consisting of a peripheral band of electron-translucent material (ET). The body of the granule is made up of small electron-translucent bodies embedded in electron-dense (ED) material. The surface of the granule is coated with ribosomes (R). x 50,000 Fig. 2. A KHG
in which the electron-translucent material consists of oval bodies within the KHG. is no peripheral zone of electron-translucent material. x 50,000
There
Fig. 3. The electron-translucent (ET) bodies within the granule are small and evenly distributed. The surface of the KHG is irregular with elongated blebs (B) of electron-translucent material. x 50,000 Fig. 4. The peripheral Fig. 5. A small KHG
blebs are circular. consisting
Ribosomes
coat both types of material
entirely of electron-translucent material of ribosomes (R). x 50.000
in the KHG.
x 50,000
(ET). with a surrounding
Fig. 6. The small electron-translucent intra-nuclear granule (I) bears a close resemblance translucent material of the cytoplasmic granules. x 25.000
layer
to the electron-
Ultrastructural
Observations
on Keratohyalm
Granules
PLATE
1
A.O.B. f.p. 1018
S. A. KEMPSON
PLATF 2
Ultrastructural
observations
on keratohyalin
PLA E
Figs. 7-9. Figs. 10 and
Buccal epithelium
II. Buccal epithelium
IOIU
granules
2
fixed with glutaraldehyde
fixed with glutaraldehyde sucrose.
and osmium
and buffered
tetroxide.
osmium
tetroxide
contammg
Fig. 7. A low power electron micrograph showing cells of the stratum granulosum (SG) and stratum corneum (SC). The KHG are electron-dense and circular. The innermost layer and the surface layers of the stratum corneum are more electron-dense than the intermediate layers. x 8000 Fig. 8. The intra-nuclear Fig. 9. The KHG
granule
(I), consists of electron-dense material cytoplasmic KHG (K). x 25,000
is juxtaposed to a mitochondrion the KHG is uniformly
with a strong
(M). The outline of the KHG electron-dense. x 50,000
resemblance is irregular
Fig. 10. Buccdl epithelium fixed with glutaraldehyde and buffered osmium tetroxide containing The KHG are electron-dense. Ribosomes (R) are embedded in the surface of the more granule. x 50,000 Fig. Il. A faintly mottled granule showing surface granule are composed of slightly less electron-dense
to the although sucrose. circular
blebs (B). The blebs and circular areas wrthin the material than the surrounding material. x 50.000.
1020
S. A. Kempson
PLATI
Fig. 12. Buccal epithelium
containing
electron-dense
3
KHG
fixed wtth osmium
tetroxide.
x 50.000
Fig. 13. Buccal epithelium fixed with buffered osmium tetroxide containing sucrose. The periphery KHG is more electron-dense than the intertor. Unstained x 50.000
of the
Fig. 14. Buccal epithelium fixed with buffered osmium tetroxide containing sucrose. The electron-dense oval bodies are embedded in less electron-dense material. Unstained x 50.000 Fig. 15. Buccal epithelium fixed with potassium permanganate. The large intra-nuclear granules the same degree of electron-density as the cytoplasmic KHG (K). x 25.000
(I) exhibit
Fig. 16. Tongue epithelium fixed with glutaraldehyde. The peripheral circular blebs of electron-translucent material are embedded in electron-dense material, which makes up the main bulk of the granule. The KHG is coated with a layer of ribosomes. x 50.000 Fig. 17. Tongue
epithelium fixed with glutaraldehydc. The intranuclear granules (I) are electron-translucent and similar in appearance to the cytoplasmic KHG (K). x 25.000.
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE 3 A.O.B. f.p. 1020
S. A. KEMPSON
PLATE
4
Ultrastructural
observations
on keratohyalin
granules
PLATER Figs. 19-22.
Tongue
epithelium
fixed with glutaraldehyde
and osmium
tetroxide.
Fig. 18. Tongue epithelium fixed with glutaraldehyde. The KHG displays electron-translucent blebs (B) embedded in a peripheral zone of material of moderate electron density (MD). The main body of the granule is electron dense. x 50,000 Fig. 19. Ribosomes
(R) are seen throughout the body of the small KHG. The KHG are electron-dense and show no close association with the tonofilaments (T). x 50,ooO
Fig. 20. A section showing the tonofilaments (T) in close contact with the KHG (K). In this case. the tonotilaments link the KHG with the desmosome (D) attachment plaque. x 50,000 Fig. 21. The region of the KHG into which the tonofilaments appear to be inserted is of moderate density in comparison with the body of the granule. x 50,000 Fig. 22. The intra-nuclear
granule
(I) is electron-dense (B) of the cytoplasmic
and has the same appearance KHG. x 25.000
electron
as the surface
blcbs
I022
S. A. Kempson
PLATL 5 Figs. 23 and 24. Tongue
epithelium
fixed with glutaraldehyde-buffered sucrose.
osmium
tetroxide
and containing
Fig. 23. The nucleus contains several intra-nuclear granules (I), consisting of electron-translucent which bears a close resemblance to the cytoplasmic granules (K). x 25,000
material
Fig. 24. The section displays the three types of material seen in the KHG. The circular surface blebs of the elongated granule are electron-translucent. The main body of the elongated KHG consists of moderately electron-dense (MD) material. Tonofilaments (T) are in contact with the main body of the elongated KHG. The second type of granule seen in this cell has a main body of electron-dense material (ED) with peripheral regions of moderate electron density. x 50.000 Fig. 25. Tongue
epithelium
fixed with osmium
tetroxide. x 50.000
The KHG
are elongated
and electron-dense.
Fig. 26. Tongue cpithelium fixed with sucrose buffered osmium tetroxide. A KHG exhibiting the three types of material. an electron-translucent bleh. a peripheral lone of moderate density and an electrondense central region. x 50.000
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE 5
A.O.B. f.p.1022
S. A. KEMPSON
PLATE 6
Ultrastructural
observations
PLATl.
Fig. 27. Tongue Fig. 2X. Palatal
on kcratohyalin
granules
6
epithelium fixed with potassium permanganatc. A large KHG with surface material of greater electron density than the central body. x 50.000 epithelium
fixed with osmium tetroxide. Irregularly shaped tionship to the tonofilaments (T). x 50.000
KHG
hlchs
01
(K) \vlth a clobc rck-
Fig. 29. Palatal epithelium fixed with glutaraldehyde and osmium tctroxide. The surlaces of the KHG (K) not in contact with the tonofilaments (T) have a surface layer of ribosomes (R). x 50.000 Fig. 30. Tongue epithelium fixed with glutaraldehyde-buffcrcd osmium KHG (K) consist of material of moderate electron-densit). The KHG (R). x 50.000
tetroxidc containing have dcnsc bordcl-s
Fig. 31. Epithelium of a IO-day old rat fixed Nith glutaraldehydc and osmium tctroxidc. (T) emerge from the centre of the KHG (K). Rihosomes (R) make up a large proportion dense KHG. x 50.000
sucro~. The of riboaomca Tonofilament\ of the electron
1024
S. A. Kempson
PLATk
Figs. 33 and 34. Tongue Frg. 32. Palatal
epithelium
cpithclium
7
fixed with glutaraldehyde
fixed with glutaraldehyde. The KHG (M) x 50.000
Fig. 33. A small clcctron-dense Fig. 34. The keratohyalin-like
body (t) with a similar appearance (M). x 50,000 mitochondrial
inclusron
and osmium
(K) is juxtaposed to KHG
tetroxide.
with the mitochondria
is within
(T) shows no limiting
a mitochondrion
membrane.
x 80,000
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE
A.O.B.
1
f.p. 1024
Electron Microscope
Unit. Department of Dental Surgq, P.O. Box 147, Liverpool.
Unlvcrsit) L60 3BX
of Liverpool.
Pcmhrokc
Place.
Summary---When examined, after various fixatives. the kcratohyalin granules (KHG) of the rat oral mucosa showed morphological variability and a heterogeneous nature. In buccal epithelium. the KHG were circular and oval with no close contact with tonohlaments and were coated with ribosomes. They consisted of two types of material exhibiting different reactions to the fixatives. The first was electron-translucent when fixed in glutaraldehyde alone but was electron-dense when osmium tetroxide was included in the lixation procedure. This material was found as circular or elongated surface blebs and as oval regions in the body of the granule and was thought to be lipoprotein. The second type of material was electron-dense with all the fixatives and was thought to be protein rich in amino acids containing -SH groups. Some KHG of the tongue epitheiium had the same structure as the KHG of the buccal epithelium. Others were more irregular. coated with ribosomes and were in contact vvith tonofilaments. A third type of material appeared in the tongue epithelium KHG that was of moderate electron-density, Occasional KHG demonstrated all three types of material. The palatal epithelium KHG were irregular. uniformly electron-dense and were in intimate contact with the tonofilaments. When fixed with osmium tetroxide to which sucrose was added to the buffer. the KHG were less electron-dcnsc. Intra-nuclear granules. often coated with ribosomes. exhibited the same reactions to the different fixations as the bleb material. Keratohyalin-like unclusions were seen in the mitochondria. From the observations. it is concluded that thcrc arc no well defined types of KHG based on morphology. IUTRODUCTION
The structure of the rat oral cpithelium exhibits a wide range of variation. The degree of keratinization varies according to the site and there are also morphological variations in the keratohyalin granules associated with keratinization of the epithelium. In ultrastructural studies. keratohyalin granules (KHG) are usually described as spherical or irregular masses of elcctrondense material with no limiting membrane, bordered by ribosomes and frequently associated with tonohlaments. There have been several attempts to classify keratohyalin granules on their morphological appearance. Chen and Meyer (1971) describe two types: (I) the epidermal type which are irregular in shape, usually surrounded by ribosomcs and associated with tonofilaments found m regions undergoing complete orthokeratinization: and (2) the cheek type occurring only in the oral epithelium. These granules were largcr. more regular and surrounded by ribosomes but without the prominent association with the tonofilaments shown by type (I) granules. Farbman (1966) reported four types of KHG: t I) globular granules made up of homogeneous elcctronopaque material. (3) irregular granules also of homogeneousmaterial. (3) heterogeneous granules consisting of electron-dense material plus 21 second slightly less
dense material. and (4) dense keratohyalin material arranged as a dense rim around an electron-translucent. amorphous centrc. Jessen (1970) classified granules into two types which he calls: (I) “single granules” containing osmophilic and oxidizable material; (2) “composite granules” consisting of one component seen to bc clcctron-dense after fixation with glutaraldchyde~~osmium fixation and digestible with pepsin. together with a second component thought to be identical to single granule material. lntranuclcar granules with a morphology resembling KHG have been described by Matoltsy and Parakkal (1967) in the epidermis, by Oehmkc and Pctry (1964) in oesophageal epithelium and in the oral epithelium by Farbman (1966). Squier (196X) and Jcssen (1970). The latter suggested that the intra-nuclear kcratohyalin-like material is identical to the cytoplasmic “single granules”. In previous studies. there has been variation in fixation techniques and in the sites from which the tissue has taken. The purpose of this study was to observe the morphology of KHG in the eipthelium from three regions of the rat oral cavity-the mucosd of the hard palate the tongue and the buccal mucosa following fixation by eight different techniques. This made it
IO1 I
S. A. Kempson
1012
possible to assess the influence of the preparation techniques on the observed structure and morphology of the KHG. The fixatives were selected as ones widely used in ultrastructural studies and possessing different fixation properties. Methanol was used as a dehydrating agent because it has a relatively less solvent action than other commonly used dehydrating agents. The superiority of methanol in the dehydration of aldehyde fixed tissues has been reported (Feder, 1960).
MATERIALS
AND METHODS
Two sibling, adult, Wistar rats were killed and small pieces of tissue were immediately excised. Care was taken to ensure that the pieces of tissue were no more than I mm thick in one dimension. Tissue was taken from the dorsal surface of the tongue, the hard palate and the buccal mucosa and subjected to the following methods of fixation: I. Fixation for 1 hr in 2.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4 (Sabatini. Bensch and Barnett. 1963). 2. Fixation for 1 hr in 4.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4. 3. Fixation for 1 hr in 6.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4. 4. Fixation for 1 hr in 6.25 per cent glutaraldehyde in cacodylate buffer at pH 7.4 followed by post-fixation with I per cent osmium tetroxide in veronal acetate buffer at pH 7.2 for 0.5 hr (Palade, 1952). 5. Fixation for I hr in 6.25 per cent glutaraldehyde post-fixed with I per cent osmium tetroxide in veronal acetate buffer, containing 4.5 per cent (w/v) of sucrose for 0.5 hr (Caulfield, 1957). 6. Fixation for 0.5 hr in 1 per cent osmium tetroxide in vcronal acetate buffer at pH 7.2 (Palade, 1952). 7. Fixation for 0.5 hr in 1 per cent osmium tetroxide in Verona1 acetate buffer containing 4.5 per cent (WI v) of sucrose per ml of fixative. 8. Fixation for I hr in 0.6 per cent potassium permanganate in veronal acetate buffer at pH 7.4 (Luft. 1956). Tissue from a IO-day-old-rat was also fixed in 6.25 per cent glutaraldehyde and post-fixed in osmium tetroxide as in 4 above. All tissues were dehydrated in methanol and embedded in Araldite. Sections 2 pm in thickness were examined under phase contrast and areas suitable for thin sectioning were selected. Sections stained in P.A.S. and toluidine blue were examined by light microscopy. Sections approximately 60 nm thick were cut on an LKB ultramicrotome with a glass or a diamond knife, collected on copper grids and stained with a 25 per cent uranyl acetate in methanol solution. Some sections were left unstained. The grids were examined in a Philips E.M. 300 electron microscope.
OBSERVATIONS
Hisrolo~q~. Using the classification of the degree of keratinization as dctcrmined with the Mallory stain (Alvdres and Meyer. 1971). the rat buccal epithelium was “incompletely” orthokeratinized in sections stained with P.A.S. and toluidinc blue. In the stratum corncum there was a very thin inner layer of keratin which corresponded to a layer which stained like hair with Mallory’s stain and a wider inner layct which stained like the cellular layers. The two keratin layers were evident in the electron microscope (Fig. 7). The stratum granulosum was well developed with many KHG granules.
Glutur~u/dch~dt~,fisurior~. In the electron microscope. the KHG showed a similar appearance when fixed in the 3 different concentrations of glutaraldchydc and these will be described together. The KHG were oval or occasionally circular in shape varying in size from 0.5 to 4.5 itrn and their outline was not always regular. The body of the granules consisted of two types of material (Fig. I. Plate I). one being electron-trnnslucent and the other being electron-dense. The clectron-translucent material occurred in various distributions and varying proportions in the granules (Fig. 2. Plate I). Sometimes the electron-translucent material was within the body of the KHG as a small number of ovalor round bodiesofvarying sizes and irregular distribution (Fig. 2). In others (Fig. 3. Plate I). these bodies wero more numerous and mote uniform in size and distributed more evenly. In Fig. I shovvs an almost complete band of electron-translucent material at the periphery of a granule. but in other granules (Fig. 3) this electron-translucent material appeared as an incomplete band giving the granule an irregular outline with elongated blchs of electron-translucent material on the surface which in some instances appeared circular (Fig. 4. Plate I ). Occasionally small granules consisting of a large proportion of ciectron-translucent material were seen (Fig. 5. Plate I). but it was thought that these could rcprcscnt oblique sections through ;I surface bleb. Ribosomes wcrc closely associated with the keratohyalin granules (Fig. I ) and formed a surface layer on the granules irrcspcctivc of which type of material lay immediately inside (Fig. 4). Tonofilamcnts were present close to the granules but little direct contact with the granules was observed. The tonofilaments were scattered randomly through the cells and not organized into discrctc bundles. Small intra-nuclear granules were observed consisting of electron-translucent material only and bearing a close resemblance to the electron-translucent material of the cy toplasmic granules (Fig. 6. Plate 1). Fi.wfiou with 6.25 /XV WU1 ~~/uttrrn/dch u/c ~111d o,snkm tc/r.o.rirk. The KHG in the buccal cpithclium fixed in 6.25 per cent plutaraldehyde and post-fixed in
Ultrastructural
observations on keratohyalin granules
osmium tetroxide were circular (Fig. 7, Plate 2) and. like the intra-nuclear granules, consisted of electrondense material alone (Fig. 8, Plate 2). The KHG consisted of uniformly electron-dense material and were surrounded by layers of ribosomes of variable thickness which seemed to be incorporated into the body of the KHG granules. At the surface, groups of ribosomes projected from the KHG into the cytoplasm. There was no close association of tonofilaments and KHG. The intra-nuclear granules (Fig. 8) had an identical appearance to the cytoplasmic granules and were also surrounded by ribosomes. Occasionally, the KHG were situated close to mitochondria (Fig. 9, Plate 2) and had an irregular outline similar to those seen following glutaraldehyde fixation above (Fig. 4) but with ;I uniformly dense structure. Fi.uation with glutaraldehyde and buffered osmium ~ctroride conruining sucrose. The KHG appeared electron-dense and circular or oval in outline (Fig. 10. Plate 2). The peripheral layer of some of the granules appeared to consist of ribosomes (Fig. 10) apparently embedded in the surface of the granule. The intra-nuclear keratohyalin-like material was electron-dense. Some KHG in this specimen (Fig. 11, Plate 2) were faintly mottled and possessed surface blebs. The circular areas within the granules and the blebs were slightly less-electron dense than the material surrounding the KHG. Firution with buffered tetroxide. The keratohyalin granules appeared irregularly shaped, electron-dense and covered by layers of ribosomes (Fig. 12, Plate 3). Occasionally small granules were seen surrounded by a relatively large area of high ribosome density. The intranuclear bodies were electron-dense. Fir~tion with ht@red osmium tetroxide containing .S~UYW.The granules were very irregularly shaped and electron-dense. The intra-nuclear bodies were electrondense. In some unstained sections (Figs. 13 and 14, Plate 3), the periphery of occasional KHG was more electron-dense than the interior (Fig. 13, Plate 3) whilst other KHG were made up of electron dense-bodies surrounded by a less electron-dense material (Fig. 14. Plate 3). Fiuution with potassium permanganate. The KHG were variable in outline (Fig. 15, Plate 3). Occasional Webs and bodies within the granules were more electron-dense than the matrix or body of the KHG. The intra-nuclear bodies appeared electron-dense.
Histoloyy. The tongue epithelium was mostly “incompletely” orthokeratinized on light microscopic examination although the epithelium was much thicker than that of the buccal epithelium. The stratum granulosum was thicker and contained many more KHG than the cheek epithelium. The stratum corneum was also much thicker than in the cheek. Regions of almost “complete” keratinization were present on the dorsal surfaces of the filiform papillae.
IOI?
Ultrustructural ohserwtions Fi.uarion wirh glutaruldehydc. Fixed in the varying concentrations of glutaraldehyde. the KHG displayed a similar structure to those of the buccal epithelium (Fig. 16, Plate 3). The intranuclear granules, consisting of electron-translucent material, were more numerous than in the buccal epithelium (Fig. 17, Plate 3). Both the electron-translucent and electron-dense regions were bordered by ribosomes at the surfaces (Fig. 16). KHG were present with the electron-translucent blehs embedded in a peripheral zone of moderate electron density (Fig. 18, Plate 4). The main bulk of the KHG material was of high electron density. Fixation with 6.25 per cent glutaraldv/~~rlc~ and osmium tetroride. The KHG displayed a very variable appearance. Some were circular, electron-dense and surrounded by ribosomes throughout the body ol some of the small KHG (Fig. 19, Plate 4). Some of the KHG showed no association with the tonolilaments (Fig. 19) while others showed a distinct link with tonofilaments (Fig. 20. Plate 4). Bundles of tonofilamentx ran from the desmosome attachment plaque into direct contact with one edge of some KHG which were less circular in shape (Fig. 21. Plate 4). The material in contact with the tonofilaments was of moderate electron density compared to the main body of the granule (Fig. 21). The intra-nuclear bodies were of high electron density (Fig. 22. Plate 4) as were the large circular hlebs of the KHG. The central zone of the granule was less electron-dense. Often the blebs were elongated to form a surface zone of extremely electron-dense material which in some cases was complete and in others apparently forming a cap over the incomplete. granule. Fiwtion with glutaruldehydr und hr#&d osmi~rm tetroxide with sucrose. The large blebs of the irregular11 shaped KHG were of less electron density than the central zone of the granule (Fig. 24. Plate 5). The granules were often elongated (Fig. 24). Tonofilaments were attached to the material of moderate electron density. The electron-translucent blebs were contcd with ribosomes on the exterior surface of the granule. There were no ribosomes on the surface of the blcb in contact with the main bulk of the granule which was of moderate electron density. Another KHG (Fig, 74 left) consisted of electron-dense material with two caps of less dense material. The intra-nuclear granules were electron-translucent (Fig. 23). Fix&on with osmium tetroxidr. The KHG wcrc‘ elongated in the upper layers of the stratum granulosum and were of uneven density (Fig. 25. Plate 5). The blebs, where present, and the intra-nuclear granules were electron-dense but the central areas of the circular granules are much less electron-dense. Fisution with hufired o.smium tetro uidc corutrirnml sucrose. These granules exhibited the three types 01 material of moderate electron density on the surface (Fig. 26, Plate 5). The intra-nuclear bodies were elcctron-translucent.
1014
S. A. Kempson
Fixution with potu.ssium prrmungunatr. The electrondense KHG had various shapes often with denser material on the surface of the KHG (Fig. 27, Plate 6). The intra-nuclear bodies were electron-dense and circular.
Palatal rpithelium Histology. The epithelium of the hard palate was moderately thick and showed “‘complete” orthokeratinization. The stratum corneum stained uniformly throughout its thickness when examined in the light microscope. Ultrustructurul ohservutions Fixation with gluturuldehyde with 6.25 prr cent gluturuldehvde und with osmium tetroxide post-jixation, with o&urn tetroxidejxution and potassium permanganutr. The different methods of fixation had much less effect on the morphology of the KHG in the hard palate than in the tongue and cheek epithelium. The KHG in the palate were electron-dense and irregularly shaped (Fig. 28, Plate 6). often juxtaposed to mitochondria (Fig. 32, Plate 7) and in close contact with thick bundles of tonofilaments. The surfaces (Fig. 29, Plate 6) not in contact with the tonofilaments showed a border of ribosomes. Fixation with gluturuldehydr osmium trtroxidr and buffered osmium tetroxide with sucrose. The KHG appeared less electron-dense in the specimens fixed in glutaraldehyde and osmium tetroxide and buffered osmium tetroxide containing sucrose (Fig. 30, Plate 6), and were bordered with a coat of ribosomes. This appearance was very similar to the specimens fixed in osmium tetroxide buffered with sucrose. The palate tissue fixed in the other conditions all showed a similar appearance that was more electron-dense than the sucrose buffered specimens. Intru-nucleur bodies. Intra-nuclear bodies were rarely seen in the palate but occasionally electrondense bodies were seen in a specimen of hard palate from a lo-day-old rat fixed in 6.25 per cent glutaraldehyde post-fixed in osmium tetroxide. Ten-day-old rut palate. The lo-day-old rat palate displayed features that were not typical of the keratinized epithelium of the mature rats; for example, the KHG were nearly circular and did not show much contact with the tonofilaments although they seemed to emerge from the centre of one KHG which appeared to be made up of ribosomes (Fig. 31, Plate 6). Some granules of the lo-day-old rat palate epithelium showed very dense circular bodies embedded in a less dense granule. The KHG became larger with a more irregular outline in the two most superficial cell layers of the stratum granulosum. Mitochondria and krratohyu?alin-like muterial. Keratohyalin-like material was seen within the mitochondria (Figs. 33 and 34, Plate 7) of tongue and cheek epithehum. Small circular bodies of electron-dense material were seen within the body of mitochondria in the cells of the stratum granulosum. The mitochondria appeared to be degenerating in this layer. This material
had exactly the same appearance as the cytoplasmic keratohyalin granules. These inclusions had no limiting membrane (Fig. 34, Plate 7). Although keratohyalin-like material was not seen within the mitochondria of the hard palate mucosa, the KHG themselves often formed a halo around the mitochondria. DISCUSSION
This study demonstrates the morphological variability and heterogeneous nature of the KHG of the rat oral epithelium when different fixatives are employed. Histochemical and biochemical studies have given conflicting results on the composition of KHG (Cohen 1967; Matoltsy and Matoltsy 1970; Ugel and Idler 1972). The KHG of the tongue and cheek epithelium showed surface blebs and round or oval bodies within the granule body that behaved in a different way from the main body of the KHG. These’blebs have previously been called “dense homogeneous deposits” (Fukuyama and Epstein. 1971) and “single granules” (Jessen, 1970. 1973). The term “dense homogeneous deposits” is not favoured as, under certain fixation conditions, the “deposits” are not dense. The term “single granules” implies discrete, independent bodies. There was no evidence that the granules in the cytoplasm with the same behaviour as surface blebs were discrete granules and they could be sections of surface blebs in a different plane. Therefore this report has utilized the term “bleb” to describe the circular or elongated bodies found at the surface of the KHG. The blebs showed variable degrees of incorporation into the body of the granule, some being almost completely immersed in the main body of keratohyalin material. The very variable appearance of the blebs was comparable to that of three other cell components: (a) the intra-nuclear granules, (b) the round or oval granules that give the KHG their mottled appearance,(c) the keratohyalin-like mitochondrial inclusions (Kimura, 1972). These components and the blebs shared the variations in electron density resulting from the differing preparation methods. The main body of the KHG material in which the round or oval bodies were embedded appeared electron dense following all fixation procedures. This material, with the “single granules”. forms the “composite granules” (Jessen, 1970). This component has been reported (Jessen, 1970) as digestible in pepsin and non-oxidizable with hydrogen peroxide. In the tongue epithelium KHG. the third type of material. that of moderate density, was seen in all the fixation conditions except from potassium permanganate. The bundles of tonofibrils in contact with the KHG in the tongue were usually in contact with this third type of material. The intra-nuclear granules increase in size in the more superficial cells (Jessen, 1970). At the same time nuclei in the more superficial stratum granulosum cells show a rarefaction of their nuclear material. It is sug-
Ultrastructural
observations
gested that breakdown products of the nuclei may accumulate to form the intra-nuclear granules in a similar way to the keratohyalin like inclusions of the mitochondria although no clear evidence for this has been observed. Glutaraldehyde preserves proteins, and in particular nucleoproteins, well as it is thought to cross-link polypeptides at reactive side groups (Hayat, 1970). Glutaraldehyde is incapable of rendering lipids insoluble in organic solvents and therefore failed to show cellular membranes. Osmium tetroxide is soluble in certain lipids and can be taken up in an unreduced form by fully saturated lipids (Hayat. 1970). Osmium reacts with some amino acids owing to the reducing potential of -SH groups (Fukuyama. Wier and Epstein. 1972). The reactivity of osmium tetroxide is modified hy osmolarity (Palade, 1952) which could explain the appearance of the bleb material when sucrose was added to the osmium tetroxide fixatives. This effect could also be explained by the incomplete osmication of the tissue due to the retarding effect which added sucrose has on the rate of osmium tetroxide penetration into tissues (HagstrBm and Bdhr, 1960). Potassium permanganate has a larger oxidative potential than osmium tetroxide but its useful oxidative properties are largely confined to preserving lipo -protein complexes (Pease, 1964). From the observations of the KHG under different fixation conditions. it is suggested that the bleb material found in the KHG. the nuclei and the mitochondria consist of lipoprotein material. The main body of the granules is thought to consist ofprotein material rich in amino acids containing -SH. It has been reported that part of protein component of KHG is synthesized dr uoco in the stratum granulosum (Fukuyama and Epstein, 1971). Keratohyalin-like inclusions have been reported in mitochondria of stratum granulosum cells of the epidermis (Matoltsy and Parakkal, 1967) although it was undecided whether they were inside or outside the outer mitochondrial membrane. Keratohyalin-like mitochondrial inclusions have been described recently (Kimura, 1972). Thin sections of neonatal rat tongue were oxidized with hvdrogen peroxide and it was found that mitochondrial keratohyalin-like inclusions behaved the same way as the KHG blebs. It is possible that as the mitochondria degenerate in the stratum granulosum the lipid component of the mitochondrial membrane form part of the KHG. Other cell membranes may also contribute material to the KHG. The significance oflipids in KHG and keratinization has not yet been fully realized. KHG are almost completely digested after incubation with lipase (Cohen. 1967) and it was suggested that they were composed of complex lipids not removed by alcohol or during routint embedding. In the epidermis, lipids have great zignificance in normal keratinization (Swanbeck. I959: Rothman. 1964). Nicolaides (1964) found histochemitally that intense esterase activity in the upper cells of the epidermis was concurrent with the disintegration of the cell organelles. suggesting that these estcrases
on keratohyalin
1015
granules
attack the lipids and cause the membrane to disintegrate. The fate of the degraded lipid is obscure. From these observations. it is concluded that there are no clear-cut types of KHG based on morpholog), as has been suggested (Chen and Meyer, 1971: Farbman. 1966; Jessen. 1970). Instead there appears to be a gradation in the morphology related to the position and presumably function of the epithelium. The biochemical composition of the KHG probably also shows a gradation related to the “incomplete” orthokeratinized cheek to the “complete” orthokeratinized hard palate. A~h,~ol~l~,~/~e,,~(,~tf,s~ This
work
v+as supported
hy research
grant numhcr 2OY f’rom the United Liverpool Hospitals. I am indebted to Dr. J. .Appleton and Dr. C\‘. R. T!ldcsIL’) for their wluahlc advice.
Alwres 0. F. and Meyer J. 1971. Variable feuturca and regional differences in oral epithelium. In: C‘urwr~t C‘otrc’t’/~lr c?/ thv Hisrolq~ (!f t/w 01~1 Mwow (Edited hq Squier C. A. and Meyer J.) Chap. 3. p. Y7. Charles (‘. Thomas. Illinois. U.S.A. Caulfield J. B. 19.57. EfTects of varying the vehicle ~OIosmium t&oxide fiwtion. J. hiop/~!~ hirw/l~,r,~ (‘lvol 3, 827 830. Chen S. Y. and Meyer J. 1971. Regional dlfferenccs in tonofilaments and kerntohyalin granules. lo: Clfw~,,,t Ciwwpl\ of t/w Ni.srolo~g~~of ORI/ Mu~o.su (Edited hy Squier. C‘. A. and Mcycr. Julia) Chap. 6. p. 114. Ch:trle\ C. Thomas. Illinois. U.S.A. Cohen L. lY67. Presence of lipids in kerntohyalin ~ranulcs of human gmgiw. J. dr,lt. RCA.46, 630. Farhmnn A. I. 1966. Morphological variahilit> of keratohqalin. jIr~trr. Rw. 154, 17.5 286. Fedcr N. 1960. Some modifications in conventional tcchniques of tissue preparation. J. Hi~toc~hw. (‘\~t~w/~cw. 8.
309 Fukuyamn K. and Epstein W. L. lY71. The inhibltion of RNA and protein synthesis in granular cells h! actinomytin-D and puromqcin. J. inwst. Derm. 56, 21 I 212. Fukuyama K.. Wier K. .A. and Epstein W. L. 1972. Dense homogeneous deposits of’ keratohyalin granules in new born rat epidcrmls. J. ultrrrstrwt. Rrs. 38, I6 26. Halat M. A. 1970. Fixation. In: Pri~rci/~/c\LIEU/‘/‘~hticp~~\ of ICicctrwn51 kroswp I‘: Biological applicatwns. vol. 1. Chap. I. 2nd edition. van Nostrund Reinhold C’ompanq. Neu York.
Jessen H.
1970. Two types of keratohyalln granules. ./. ~rlrrc~.~rrrc~r. Rrs. 33, 95. I 15. Jessen H. 1973. Flectron cytochemical dem\)nstratwn of aulphqdrql groups in keratohyalin granulea and in the pcripheral envelope of cornified cells. Hi.~oc’/wmi~ 33, IS 29. Kimurn M. 1972. Keratohyalin granule-llke mitochondrial inclusion m the neonatal rat tongue and oesophageal epithelia. J. Elcc.tr.m .Micmsc. (‘T’oli!~) 21. 191 194. Lufi J. H. 1956. Permanganate: a new fixative [or electron microscop!. J. hio/~h>~ hioc~lwvr. C‘~to/.2, 799 X02. Matoltsq A. G. and Matoltsy M. N. 1970. The chemical nature of keratohyalin granule5 of the epldcrmis. .I. (c/r /TiCA. 47. 5’) 7 hoi.
1016
S. A. Kempson
Matoltsy A. G. and Parakkal P. F. 1967. Keratinization. In: Ultrastructure ofNormal and Abnormal Skin (Edited by Zelickson A. S.) pp. 76104, Lea and Febiger. Philadelphia, U.S.A. Nicolaides N. 1964. Lipids, membranes and the human epidermis In: Thr Epidermis. (Edited by Montdgna, W. and Lobitz W. C.) Chap. 16. pp. 51 I-538, Academic Press, New York. Oehmke H. J. and Petry G. 1964. Beobachtungen iiber das Auftreten von Keratohyalingranuia in Zellkern. Expcrimrntia 20, 422-423. Palade G. E. 1952. A study of fixation for electron microscopy. J. rxp. Med. 95, 285-298. Pease D. C. 1964. Fixation. In: Histological techniques for Electron Microscopy, Chap. 3, 2nd Edition. Academic Press. New York.
Rothman S. 1964. Keratinization in historical perspective. In: The Epidrrmis (Edited by Montagna W. and Lobitz W. C.) Chap. 1, pp. l-14, Academic Press, New York. Sabatini D. D.. Bensch K. and Barnett R. J. 1963. Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymic activity by aldehyde fixation. J. &I Biol. 17, 19-58. Squier C. A. 1968. Ultrastructural observations on the keratinization process in rat buccal epithelium. Arch orul Bio/. 13, 11451451. Swanbeck G. 1959. Macromolecular organisation of epidermal keratin: an x-ray diffraction study of the horny layer from normal. ichthydotic and psoriatic skin. Acta. drrm. Vewr. suppl. 43, I-37. Ugel A. R. and Idler W. 1972. Further characterization of bovine keratohyalin. J. cell biol. 52, 453-464.
R&mr%Quand examinees, apres diverses variations des fixatifs. les granules keratohyalines (GKH) de la muqueuse orale du rat ont montrees une variabilite morphologique et une nature heterogene. Dans l’epithelium buccal les GKH Ctaient circulaires ou ovales, sans contact intime avec les tonofilaments et Ctaient doublees de ribosomes. 11sconsistaient en deux types de materiel exhibant differentes reactions aux fixatifs. Le premier Ctait transparent aux electrons, quand fixt dans la glutaraldehyde seule, mais opaque aux electrons quand le tetroxyde d’osmium Ctait inclu dans le pro&de de fixation. Ce materiel se presentait comme des vesicules superficielles circulaires ou allongees et comme des regions ovales dans le corps des granules et on supposait qu’elles Ctaient des lipo-proteines. Le deuxieme type de materiel etait opaque aux electrons avec tous les fixatifs et on le considerait d’ttre riche en proteines dans les aminoacides contanant les groupes -SH. Quelques GKH de l’epithelium de la langue avaient la mime structure que les GKH de l’epithelium buccal. D’autres etaient plus irrtgulires, doubles avec des ribosomes et etaient en contact avec destonofilaments. Un troisieme type de materiel apparaissait dans les GKH de l’epithelium de la langue, que Ctait dune densite electronique mod&e. Occasionnellement les GKH montraient tous les trois types de materiel, Les GKH de l’epithelium palatal Ctaient irregulieres, uniformement opaques aux electrons et etaient en contact intime avec les tonifilaments..Quand fix&es avec du tetroxyde d’osmium uaquel on ajoutait de la sucrose au tampon, les GKH etaient moins opaques aux electrons. Les granules intra-nucleaires, souvent doublees avec des ribosomes, exhibaient les memes reactions aux differents fixatifs, par ex: le materiel vesiculeux. Des inclusions d’aspect kerato-hyalinique ont et6 remarquees dans les mitochondries. De ces observations on conclut qu’il n’y a pas de types bien d&finis de GKH basees sur la morphologie. Zusammenfassung-Die Keratohyalinkornchen (KHG) der Mundschleimhaut der Ratte zeigten bei Priifung nach Benutzung verschiedener Fixiermittel morphologische Variabilitlt und ein heterogenes Wesen. In bukkalem Epithel waren die KHG kreisfijrmig und oval, in keinem engen Zusammenhang mit Tonofasern, und sie waren mit Ribosomen bedeckt. Sie bestanden aus zwei Materialarten, welche verschiedene Reaktionen zu den Fixiermitteln zeigten. Die erste war elektronendurchscheinend, wenn in Glutaraldehyd, allein, fixiert, war aber elektronendicht, wenn Osmiumperoxyd in dem Fixierverfahren enthalten war. Es wurde festgestellt, darj dieses Material kreisfiirmige oder langgestreckte Oberfliichenbllschen und ovale Stellen in dem Kiirper des Kornchens waren und man meinte, daD sie Lipoprotein seien. Die zweite Materialart war mit allen Fixiermitteln elektronendicht und es wurde angenommen, da13 sie proteinreich in Aminsluren mit Gehalt von -SH Gruppen sei. Einige KHG’s des Zungenepithels hatten die gleiche Struktur wie das KHG des bukkalen Epithels. Andere waren irregularer, mit Ribosomen bedeckt und waren in Bertihrung mit Tonofasern. Eine dritte Materialart erschien in dem Zungenepithel KHG, welche mlDige Elektronendichte be&. Gelegentliches KHG zeigte alle drei Materialarten. Die Gaumenepithel KHG waren irregular, einheitlich elektronendicht und standen in enger Verbindung mit den Tonofasern. Die KHG waren weniger elektronendicht, wenn sie mit Osmiumtetroxyd fixiert waren, bei dem Saccharose dem Puffer zugeftigt war. Inter-Kern-Kornchen, die oft mit Ribosomen ilberzogen waren, zeigten die gleichen Reaktionen auf verschiedene Fixiermittel wie das Bllschenmaterial. Einschltisse von einer Keratohyalin iihnlichen Art wurden in der Mitochondrie festgestellt. Aus den Beobachtungen wird der Schlulj gezogen, da13 es keine gut definierten Arten von KHG gibt. die auf Morphologie gegrtindet werden.
Ultrastructural
observations
on keratohyalin
granules
lOI7
S. A. Kempson
PLATE 1
The electron
micrographs
show sections
Figs. l-6.
stained
with uranyl
Glutaraidehyde-fixed
acetate,
unless stated otherwise.
buccal epithelium
Fig. 1. An oval KHG consisting of a peripheral band of electron-translucent material (ET). The body of the granule is made up of small electron-translucent bodies embedded in electron-dense (ED) material. The surface of the granule is coated with ribosomes (R). x 50,000 Fig. 2. A KHG
in which the electron-translucent material consists of oval bodies within the KHG. is no peripheral zone of electron-translucent material. x 50,000
There
Fig. 3. The electron-translucent (ET) bodies within the granule are small and evenly distributed. The surface of the KHG is irregular with elongated blebs (B) of electron-translucent material. x 50,000 Fig. 4. The peripheral Fig. 5. A small KHG
blebs are circular. consisting
Ribosomes
coat both types of material
entirely of electron-translucent material of ribosomes (R). x 50.000
in the KHG.
x 50,000
(ET). with a surrounding
Fig. 6. The small electron-translucent intra-nuclear granule (I) bears a close resemblance translucent material of the cytoplasmic granules. x 25.000
layer
to the electron-
Ultrastructural
Observations
on Keratohyalm
Granules
PLATE
1
A.O.B. f.p. 1018
S. A. KEMPSON
PLATF 2
Ultrastructural
observations
on keratohyalin
PLA E
Figs. 7-9. Figs. 10 and
Buccal epithelium
II. Buccal epithelium
IOIU
granules
2
fixed with glutaraldehyde
fixed with glutaraldehyde sucrose.
and osmium
and buffered
tetroxide.
osmium
tetroxide
contammg
Fig. 7. A low power electron micrograph showing cells of the stratum granulosum (SG) and stratum corneum (SC). The KHG are electron-dense and circular. The innermost layer and the surface layers of the stratum corneum are more electron-dense than the intermediate layers. x 8000 Fig. 8. The intra-nuclear Fig. 9. The KHG
granule
(I), consists of electron-dense material cytoplasmic KHG (K). x 25,000
is juxtaposed to a mitochondrion the KHG is uniformly
with a strong
(M). The outline of the KHG electron-dense. x 50,000
resemblance is irregular
Fig. 10. Buccdl epithelium fixed with glutaraldehyde and buffered osmium tetroxide containing The KHG are electron-dense. Ribosomes (R) are embedded in the surface of the more granule. x 50,000 Fig. Il. A faintly mottled granule showing surface granule are composed of slightly less electron-dense
to the although sucrose. circular
blebs (B). The blebs and circular areas wrthin the material than the surrounding material. x 50.000.
1020
S. A. Kempson
PLATI
Fig. 12. Buccal epithelium
containing
electron-dense
3
KHG
fixed wtth osmium
tetroxide.
x 50.000
Fig. 13. Buccal epithelium fixed with buffered osmium tetroxide containing sucrose. The periphery KHG is more electron-dense than the intertor. Unstained x 50.000
of the
Fig. 14. Buccal epithelium fixed with buffered osmium tetroxide containing sucrose. The electron-dense oval bodies are embedded in less electron-dense material. Unstained x 50.000 Fig. 15. Buccal epithelium fixed with potassium permanganate. The large intra-nuclear granules the same degree of electron-density as the cytoplasmic KHG (K). x 25.000
(I) exhibit
Fig. 16. Tongue epithelium fixed with glutaraldehyde. The peripheral circular blebs of electron-translucent material are embedded in electron-dense material, which makes up the main bulk of the granule. The KHG is coated with a layer of ribosomes. x 50.000 Fig. 17. Tongue
epithelium fixed with glutaraldehydc. The intranuclear granules (I) are electron-translucent and similar in appearance to the cytoplasmic KHG (K). x 25.000.
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE 3 A.O.B. f.p. 1020
S. A. KEMPSON
PLATE
4
Ultrastructural
observations
on keratohyalin
granules
PLATER Figs. 19-22.
Tongue
epithelium
fixed with glutaraldehyde
and osmium
tetroxide.
Fig. 18. Tongue epithelium fixed with glutaraldehyde. The KHG displays electron-translucent blebs (B) embedded in a peripheral zone of material of moderate electron density (MD). The main body of the granule is electron dense. x 50,000 Fig. 19. Ribosomes
(R) are seen throughout the body of the small KHG. The KHG are electron-dense and show no close association with the tonofilaments (T). x 50,ooO
Fig. 20. A section showing the tonofilaments (T) in close contact with the KHG (K). In this case. the tonotilaments link the KHG with the desmosome (D) attachment plaque. x 50,000 Fig. 21. The region of the KHG into which the tonofilaments appear to be inserted is of moderate density in comparison with the body of the granule. x 50,000 Fig. 22. The intra-nuclear
granule
(I) is electron-dense (B) of the cytoplasmic
and has the same appearance KHG. x 25.000
electron
as the surface
blcbs
I022
S. A. Kempson
PLATL 5 Figs. 23 and 24. Tongue
epithelium
fixed with glutaraldehyde-buffered sucrose.
osmium
tetroxide
and containing
Fig. 23. The nucleus contains several intra-nuclear granules (I), consisting of electron-translucent which bears a close resemblance to the cytoplasmic granules (K). x 25,000
material
Fig. 24. The section displays the three types of material seen in the KHG. The circular surface blebs of the elongated granule are electron-translucent. The main body of the elongated KHG consists of moderately electron-dense (MD) material. Tonofilaments (T) are in contact with the main body of the elongated KHG. The second type of granule seen in this cell has a main body of electron-dense material (ED) with peripheral regions of moderate electron density. x 50.000 Fig. 25. Tongue
epithelium
fixed with osmium
tetroxide. x 50.000
The KHG
are elongated
and electron-dense.
Fig. 26. Tongue cpithelium fixed with sucrose buffered osmium tetroxide. A KHG exhibiting the three types of material. an electron-translucent bleh. a peripheral lone of moderate density and an electrondense central region. x 50.000
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE 5
A.O.B. f.p.1022
S. A. KEMPSON
PLATE 6
Ultrastructural
observations
PLATl.
Fig. 27. Tongue Fig. 2X. Palatal
on kcratohyalin
granules
6
epithelium fixed with potassium permanganatc. A large KHG with surface material of greater electron density than the central body. x 50.000 epithelium
fixed with osmium tetroxide. Irregularly shaped tionship to the tonofilaments (T). x 50.000
KHG
hlchs
01
(K) \vlth a clobc rck-
Fig. 29. Palatal epithelium fixed with glutaraldehyde and osmium tctroxide. The surlaces of the KHG (K) not in contact with the tonofilaments (T) have a surface layer of ribosomes (R). x 50.000 Fig. 30. Tongue epithelium fixed with glutaraldehyde-buffcrcd osmium KHG (K) consist of material of moderate electron-densit). The KHG (R). x 50.000
tetroxidc containing have dcnsc bordcl-s
Fig. 31. Epithelium of a IO-day old rat fixed Nith glutaraldehydc and osmium tctroxidc. (T) emerge from the centre of the KHG (K). Rihosomes (R) make up a large proportion dense KHG. x 50.000
sucro~. The of riboaomca Tonofilament\ of the electron
1024
S. A. Kempson
PLATk
Figs. 33 and 34. Tongue Frg. 32. Palatal
epithelium
cpithclium
7
fixed with glutaraldehyde
fixed with glutaraldehyde. The KHG (M) x 50.000
Fig. 33. A small clcctron-dense Fig. 34. The keratohyalin-like
body (t) with a similar appearance (M). x 50,000 mitochondrial
inclusron
and osmium
(K) is juxtaposed to KHG
tetroxide.
with the mitochondria
is within
(T) shows no limiting
a mitochondrion
membrane.
x 80,000
Ultrastructural
Observations
on Keratohyalin
Granules
PLATE
A.O.B.
1
f.p. 1024