Epidermal keratinization in the salamander and a comparison with other amphibia

In a variety of amphibians examined the stratum corneum was one cell in depth, although in Xenopus it was up to three cells deep. The flattened horny cells were closely fused together along their lateral membranes to form a continuous sheet. Disulphide bonds of keratin were most concentrated in the peripheral cytoplasm, but the interiors of the cornified cells were sufficiently well keratinized to prevent more than slight enzymatic cytolysis of the normal cell components. Characteristically large, weakly stainable, non-shrunken nuclear remnants were found in the salamander and frog horny layers, but the clawed toad had small pyknotic (parakeratotic) nuclei. The mature amphibian keratinocytes contained free fats, bound phospholipids, calcium and sulphydryl groups, together with acid phosphatase and non-specific esterase. Cornification appears to begin by a process of separate individual cell keratinization and lateral membranes of neighbouring cells only later become fused together. This differs from the process in higher vertebrates in which the cells undergoing keratinization form a uniform transitional layer in the epidermis. In the amphibian epidermis neighbouring cells occur in different stages of keratinization.     

A comparison of the epidermis and pigment cells of the crocodile with those in two lizard species

Keratinization and pigmentation in Crocodilus niloticus skin were compared with the conditions in the lizards Lacerta viridis and Anolis carolinensis. The epidermis, both in the crocodile and in lizards, is arranged to form a surface pattern of scales and narrower intervening hinge regions. Similar keratin-bound substances were found in the crocodile and lizard stratum corneum. Nevertheless, the greater uniformity in histological structure and in distribution of chemical substances throughout the depth of the crocodile stratum corneum was in marked contrast to the lizards, which showed morphological differences, and differences in intensities of chemical reactions in the horny cells laid down early and late in each keratinization cycle. In the crocodile, keratin-bound S-S and SH are uniformly distributed in the horny scales, but in the lizards the superficial cells have most S-S and the lowermost keratinized cells most SH. The loosely arranged horny cells in the crocodile are shed in small flakes as in mammals, in contrast to lizards which undergo periodic sloughs of a compact stratum corneum. In the lizards, the intermediate layer between two horny layer generations contains no detectable S-S and is probably unkeratinized, so that when these cells die a fission zone is formed. The crocodile scales each contain a raised pigmented papule in which melanin is introduced into the epidermal cells, and keratinization is also different from the neighbouring area. Guanophores and lipophores are absent in the crocodile, although present in the lizards. All contain prominent dermal melanophores.     

An electron microscopic study of the human epidermal keratinocyte

Summary 1. The epidermis of the flexor surface of the upper arm of human subjects was studied with the electron microscope. 2. The cytoplasm of the keratinocytes in the basal layer contained many tonofilaments, ribosomes and other cell organelles. The tonofilaments were arranged singly or in loose bundles and many were attached to the inner membrane of the desmosomes. Along the basal border of the cells pinocytotic vesicles could be seen at different stages of development. 3. The keratinocytes in the stratum spinosum differed from those in the basal layer in two main ways: (a) The tonofilaments were grouped together into large compact bundles known as tonofibrils and it was possible to determine a definite beading or cross banding along the length of some of the filaments. (b) The cells were assuming a flattened shape. 4. The keratinocytes in the stratum granulosum possessed large numbers of irregularly shaped keratohyaline granules. The granules were strongly osmiophilic and were always situated on a meshwork of tonofibrils. The keratohyaline granules had no internal structure. The nuclei and mitochondria showed evidence of degeneration. 5. The keratinocytes in the stratum corneum were long and flattened. The cell walls showed increased electron density and were considerably thickened. The cytoplasm was filled with closely packed fibres separated by a small amount of lucent matrix. The fibres were grouped together in bundles running in different directions within the flattened squames. The fibres had along their entire length alternating areas of high and low electron density. The keratohyalin granules had disappeared and nothing remained of the nuclei or the organelles. In the deepest cells of this region the fibres were sometimes loosely packed leaving large irregular open spaces. This area corresponded to the stratum lucidum. In the most superficial layers of the stratum corneum the fibres appeared to be breaking down so that little remained within the keratinocyte except large lucent spaces. The desmosomes showed distinct structural changes. 6. An attempt was made to correlate the structural changes in the different epidermal layers with the process of keratinization. The possible part that keratohyalin may play in the process of thickening of the cell walls was discussed. The relationship between the desmosome and its dynamic environment was considered.I wish to express my sincere thanks to Dr. David Hilding of the Department of Otolaryngology for the use of an R.C.A. electron microscope and other facilities in his laboratory. This research was supported by the United States Public Health Service and American Cancer Society grants. USPHS CA 04679-07, NB 03995.     

Calcium-modulated desmosome formation and sodium-regulated keratinization in frog skin cultures

The processes of desmosome formation and keratinization were studied in isolated frog skins cultured in a two-compartment (mucosal and serosal) chamber. Before culture, the skin fragments were trypsinized (stratum corneum together with some parts of stratum granulosum and spinosum were scraped off with forceps) allowing the stratum germinativum to remain on the dermis. When both the mucosal and the serosal culture media contained 1.5 mM calcium and 86 mM sodium concentrations, fully developed desmosomes were differentiated and no keratinization occurred. When the mucosal medium was lowered in two steps to a final calcium concentration of 0.5 mM by dilution with tridistilled sterile water, poorly developed desmosomes were formed, keratinocytes interdigitated and the keratinization was strongly enhanced. The calcium-dependent desmosome formation was affected by the salt gradient established across the skin. These two effects, modulated desmosome formation (calcium) and increased keratinization (sodium), were concomitant with but did not complement one another.     

Histochemistry of the tongue epithelium in four mammals with respect to keratinization

The tongue epithelium was examined in the laboratory rat, guinea pig, rabbit and Domestic cat, using light microscopical, histological fluorescent and histochemical methods. The distributions of the enzymes, acid and alkaline phosphatase were examined. Protein-bound phospholipid and calcium were investigated, together with thiol sulphydryl groups and cysteine disulphide bonds of proteins. A variety of different types of keratinization were shown in the various species, as well as in the same species in different regions of the tongue. The most strongly keratinized structures were the filiform and conical papillae which varied widely from species to species. Those of the rat dorsum were similar to papillae described previously in the House mouse and have strongly keratinized spines. The guinea pig showed some differences but also had keratinized spines. In contrast the rabbit papillae did not have spines but the horny layer over the posterior sides was hardened instead to form pointed edges. Human filiform papillae are similar to the rabbit without spines but the horny layer is less strongly keratinized. In the Domestic cat the conical papillae were also without spines but the horny layer on the anterior and posterior surface was hardened to form claw-like structures.     

Keratinization of rat vaginal epithelium. III. Effect of estradiol on keratinization

Effect of estradiol-17 beta on rat vaginal epithelial cells (VEC) was studied by transmission electron microscopy. During the normal estrous cycle in adult rats keratinization and exfoliation of VEC was observed only in the estrus phase, though tonofilament bundles were seen distributed in the cytoplasm of the VEC during the proestrus and, to a lesser extent, in the diestrus phase. Electron microscope data on adult ovariectomised, immature and neonatally estradiol-primed rats demonstrated keratinization of the VEC within 24 h of injection. Proliferative activity was observed in the basal cells in primed animals. The basal cells remained cuboidal whereas cells of intermediate and luminal layers became flattened. These cells showed irregular profiles and, therefore, enhanced numbers of cellular junctions (desmosomes) were observed compared to control groups. Many tonofilament bundles were seen in the cytoplasm of cells belonging to intermediate and luminal layers in primed animals.     

The structures of the tentacles of Rhabdopleura compacta (Hemichordata) with special reference to neurociliary control

Summary The tentacle of Rhabdopleura compacta (Hemichordata) consists of two layers of cells surrounding a central coelomic cavity. The two layers of cells are separated by a cell free basement lamella.The tentacles on the arms of Rhabdopleura bear three longitudinal rows of cilia. The ciliated cells are closely associated with bundles of nerve fibres, and between some of the cells and nerve fibres there are synapses. The peripheral regions of the ciliated cells are joined to one another by desmosomes. Tonofibrils join some of these desmosomes to the kinetosomes of the cilia.The nerve fibres are confined to the ectodermal layer and the muscle cells to the layer of cells within the basement lamella. In the ectodermal layer besides ciliated cells there are mucus cells, densely pigmented cells, and green bodies. The function of these last two types of cells is secretory. Most of the epithelial cells have microvilli upon their free borders.I wish to thank Professor J. Z. Young F. R. S. for enthusiastic advice and encouragement. Dr. R. Bellairs generously provided the facilities for electron microscopy. Mr. R. Moss gave excellent technical and photographic assistance. Dr. A. Stebbing of the Plymouth Marine Biological Laboratory helped me to obtain and to identify the specimens. Professor D. W. James kindly allowed me to use his facilities for interference microscopy.     

Plantar epidermis of the guinea pig and characteristics of the stratum corneum.

The guinea pig plantar epidermis was examined by light-microscopical histochemical methods and by transmission electron microscopy. Autolysis of cell structure was much less complete in guinea pig plantar horny layer than in the back, and stainable cytoplasm was retained in keratinized cells but organelles were lost except for some degraded ultrastructural remnants. By light microscopy the whole thickness of the horny layer showed bound phospholipid and bound cysteine, and there was a weak cystine reaction at the peripheries of the keratinized cells. In ultrastructure the keratohyalin contained slightly larger subparticles than in the back skin. The horny layer was not divisible into basal, intermediate and superficial regions as in hairy skin. The stratum lucidum of light microscopy was not defined in electron micrographs. Osmium-stained cytoplasmic material was retained in horny cells about to be desquamated, in contrast to the empty appearance of these cells in hairy skin. Epidermal cells in plantar skin have ultrastructural cytoplasmic processes which are longer than they are broad. In the horny layer these interdigitate with those of neighbouring cells and are held together by lateral demonsomal junctions. Probably this gives mechanical strength against shearing forces experienced by the plantar horny layer.     

A histochemical study of keratinization in the domestic fowl (Gallus gallus)

The microanatomy of the epidermis of the domestic fowl is described and related to the distribution of various histochemical constituents involved in keratinization.
The avian horny layer over the back is composed of a loose network of structurally solid horny cells. This is in contrast to most mammalian epidermal horny cells in which structural keratin is found only in the peripheral cytoplasm, and the interior of the keratinocyte contains soluble products of cytolysis with possibly some free keratin filaments dispersed in the fluid material.
The avian tarsal epidermal horny scales show similarities to both the scales of lizards and snakes and to mammalian tail scales which appear to be homologous structures.
It is suggested that a thin layer of cells containing no detectable disulphide bonds, found in the tarsal scale region of the young chick, is probably mechanically weak and may function as a fission plane for sloughing of the horny layer. A specialized epidermis and thickened horny layer is developed in the fowl on the plantar underside of the toes, but this is quite different in structure from the mammalian plantar epidermis.
The overlapping of zones rich in ribonucleic acid (RNA) and bound cysteine (SH) in the growing feather suggests that protein synthesis and the preparatory stages to keratin disulphide bonding normally occur concurrently in feather formation. This is in contrast to the growing hair which has a region rich in RNA followed immediately before it becomes keratinized by a discrete keratogenous zone weak in RNA but rich in bound cysteine.     

A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization

Summary A quantitative histochemical study was carried out on the distribution of protein thiol and disulphide groups in normal human plantar epidermal tissue. Histochemical demonstration of reactive groups was achieved by addition ofN-(4-aminophenyl) maleimide, subsequent diazotization and final coupling with a Nitro Red or chromotropic acid label as first described by Sippel. The quantitative reliability of the method was tested by absorption cytophotometry, and evaluated on the basis of the internal consistency of the results reported.Our histological observations and histophotometric data support accepted views on epidermal keratinization. A limited, though reproducible, amount of disulphide bonds was observed near the basement membrane. The free thiol concentration in basal and prickle cells was low and almost constant, but was higher in the granular cells, where deposition of sulphur-containing proteins on cell membranes is initiated. In Malpighian layers, disulphide cross-links only occurred just beneath the transition zone in thickened cell membranes. The staining pattern of the inner stratum corneum resembled a mosaic and was characterized by a sharp rise of the disulphide content, which exceeded the decrease in free thiol groups. The free thiol concentration decreased further throughout the cornified layers whilst the disulphide content remained fairly constant. Staining of thiol and disulphide groups together corresponded, within the limits of the standard error, to the sum of the thiol and disulphide concentrations when they were assayed separately in living and horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent site of disulphide cross-links.     

The electron microscopy of normal human oesophageal epithelium.

Oesophageal biopsies were studied with the electron microscope. Three layers were identified, as in the light microscopy of the oesophageal epithelium: basal, prickle and funtional cell layers. A continuous basement membrane separated the lamina propria from the basal cells. The basal cell membrane carried hemidesmosomes, desmosomes and microvillous processes. Their cytoplasm contained the usual organelles plus free ribosomes and tonofirbrils. Prickle cells contained glycogen rosettes and many tonofilaments, and their cell membrane many microvillous and demosomal processes, in places elaborated into desmosome fields. In both these layers there was a wide intercellular space containing some particulate and membranous debris. The flattened cells of the functional layer had fewer desmosomes and microvilli but abundant glycogen and tonofilaments, and a narrow intercellular space. Membrane coating granules first reaching a maximum in the functional cell layer appeared in the upper prickle cell layer and few persisted into the surface cells. The apical cell membrane of the most superficial cells was thickened and had few small microvillous processes, which were covered with a filamentous "fuzzy" coat. No keratohyaline granules were present. Papillae of lamina propria contained capillaries, some with a fenestrated endothelium.     

Ultrastructure of the human enamel organ

Summary The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated.Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions.A parallel differentiation of stratum intermedium — external enamel epithelium, and the ameloblast layer is demonstrated.The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.     

Distribution and nature of desmosomes in the bovine developing ruminal epithelium.

Desmosomes entirely similar to those of the deeper layers of ruminal epithelium are seen in the luminal layers of the early fetal ruminal epithelium. In the older fetuses, these desmosomes have morphological features that reveal to some extent the occurrence of processes of keratinization. This could indicate that the basis for cell keratinization is already present in the fetus and that postnatal keratinization corresponds mainly to the full development of the prenatally existing pattern of differentiation.     

Morphology of the oral disc of Bufo bufo (Salientia: Bufonidae) tadpoles

The fully developed oral disc of the tadpole of Bufo bufo consists of dorsal and ventral labia bearing, respectively, two and three ridges bearing numerous horny denticles, a horny beak provided with jaw sheath serrations, and large lateral papillae that are borne by two cutaneous plicae. As development progresses toward metamorphosis, these structures gradually regress until they disappear. Each cusped clavate labial denticle adheres, by means of a thin peduncle, to a similar labial denticle fixed in the lip and formed by a group of three or four cells that keratinize gradually and thus present remarkable differences in their morphology. Once all the cells of a group have been converted into horny tissue, the denticle sheds and is replaced by the underlying one. The beak serrations also are horny structures; each consists of a columnar band of cells which undergoes a gradual keratinization. The horny cells that detach themselves at intervals, being replaced by those of the underlying anlagen. The labial denticles and the beak serrations keratinize in two distinct ways. In the former, the desmosomal filaments appear to play an important role whereas, in the latter, the keratin seems to be synthesized “ex novo” by the ribosomes.     

Axillary granular parakeratosis

Axillary granular parakeratosis is an alteration of keratin characterized by a thick parakeratotic horny layer with abundant intracellular keratohyalin granules. It was first described in 1991 and since then 32 cases have been reported from USA, Europe and Australia. Lesions may affect intertriginous areas other than the axilla. The disease has apparently not been previously described in Latin America. Three overweight Colombian women were diagnosed with axillary granular parakeratosis. They presented encrusted, hyperkeratotic, hyperpigmented and pruriginous papules and plaques which affected both axillae in two women and only one in the other. Lesions had persisted for two and four months in two patients and for one year in the third. Clinical diagnoses were benign familiar pemphigus and tinea nigra. Skin biopsies showed a thick parakeratotic basophilic horny layer. Electron microscopy demonstrated a high content of keratohyalin granules. No Langerhans cells were demonstrated in the lesions using IHC for S-100 protein. No fungi were seen with the PAS stain. Infundibula showed thick horny plugs with changes similar to those seen in the epidermis. Dermal tissue showed few perivascular lymphocytes. These findings suggest that the disease has an irritative pathogenesis. Clinical histories indicated that the three women were overweight and used several types of antiperspirants. These factors plus local irritation and humidity apparently triggered the keratinization response.     

Age-related changes in the ultrastructural organization of the human gingival epithelium

By means of transmissive and scanning electron microscopy 103 gingival bioptates in practically healthy persons at the age of 18-80 years have been studied. At ageing essential changes take place in all structural elements of the epithelium. The basal membrane is intermittent and loose. In cytoplasm of the cells of the basal layer epithelium the amount of microfilaments increases essentially, and as a result it becomes electron opaque. Tonofibrillar fasciculi of the spinous layer cells are fragmented, their contours are indistinct. In cytoplasm of the granular layer cells amount of keratohyalin granules increases, their size becomes large and their typical form is lost. In cytoplasm of the basal, spinous and granular layer cells the amount of organells decreases. Mitochondria acquire the appearance of electron translucent cavities with discomplexic, and sometimes, destroyed cristae. Rather great changes occur in intercellular interrelations. In all the layers some intercellular spaces are widen, in the spaces formed isolated desmosomes and other debries of cellular structures are formed. Sharp changes of microrelief of the granular layer epitheliocytes are observed. The ultrastructural rearrangements of epitheliocytes, revealed in the human gingiva, demonstrate certain disturbances in keratinization processes, in mechanical firmness, as well as in barrier function of the epithelial layer.     

A sequential study of incomplete Freund's adjuvant-induced peritonitis in Atlantic cod

Development of diagnostic and prophylactic methodologies is dependent on knowledge of the host’s defence system and reaction to different vaccine adjuvants. Here we present a sequential morphological study of peritonitis and inflammatory cell processing of incomplete Freund’s adjuvant (IFA) in intraperitoneally injected Atlantic cod. The peritoneal tissue responses were characterised using necropsy, histology and electron microscopy. An extensive inflammatory response as characterised by leukocyte morphology and contents of enzymes, presence of apoptotic cells and IFN-γ-expressing cells was observed. Three days post injection, IFA droplets were surrounded by different types of inflammatory cells and two different patterns could be discerned. The first was characterised by flattened and concentrically arranged interdigitating cells connected by desmosomes and with macrophage-like cells (MLCs) predominant in the periphery. The second type possessed four stratified layers with an inner layer containing many apoptotic MLCs; a second layer containing flattened and shrunken cells and outer layers comprising moderately flattened cells and an outermost layer of mononuclear cells expressing IFN-γ. Oil was detected both inside and outside MLCs. The two types of processes, of which the second was clearly stratified, were similar to those observed in other teleosts, indicating a variety of reaction modes or alternatively sequential process development. The numerous dead MLCs contributed to inflammation.     

Ultrastructure of the silkworm testis. II. Ductus deferens]

Ultrastructure of the silkworm's ducts defferens is described after spermatozoa went out of lobulli testis and after the end of secretion. D. defferens is composed of an external layer of the ring and longitudinal muscles and of an internal layer of glandular cells. Both layers are separated by structurless lamina--tunica propria. Z-discs of irregular form are placed at the borders of sarcomeres. M-lines are absent. Sarcolemma intrudes into the muscle fibers at the level of Z-discs and gives origin to T-system the tubes of which together with sarcoplasmic reticulum form dyades. Structures like intercalate discs are observed in muscle tissue at the level of Z-discs. Glandular cells have unusually developed nucleoli, many ribosomes, lysosome-like and residual bodies. There are comb desmosomes between the glandular cells.     

Cytokeratin filaments and desmonsomes in the epithelioid cells of the perineurial and arachnoidal sheaths of some vertebrate species

Using electron microscopy and immunohistochemistry with a large panel of antibodies to various cytoskeletal proteins we have noted that the single- or multi-layered sheaths of epithelioid cells ("neurothelia") surrounding peripheral nerves (perineurial cells) or structures of the central nervous system, including the optic nerve (arachnoid cells), show remarkable interspecies differences in their cytoskeletal complements. In two anuran amphibia examined (Xenopus laevis, Rana ridibunda), the cells of both forms of neurothelia, i.e., perineurial and arachnoid, are interconnected by true desmosomes and are rich intermediate-sized filaments (IFs) of the cytokeratin type. Among higher vertebrates, a similar situation is found in the bovine and chicken nervous systems, in which the arachnoid cells of the meninges contain desmosomes and IFs of both the cytokeratin (apparently with restricted epitope accessibilities in the chicken) and the vimentin type, whereas the perineurial cells of many nerves contain cytokeratin IFs, often together with vimentin, but no desmosomes. In contrast, in rat arachnoidal and perineurial cells significant reactions have been observed neither for cytokeratins nor for desmosomes. In the human nervous system, cytokeratins and desmosomes have also not been seen in the various perineuria studied whereas desmosomes are frequent in arachnoidal cell layers which are dominated by vimentin IFs and only in certain small regions of the brain contain some additional cytokeratins. The occurrence of cytokeratins in the tissues found positive by immunohistochemistry has been confirmed by gel electrophoresis of cytoskeletal proteins, followed by immunoblotting. Our results emphasize both similarities and differences between the neurothelia on the one hand and epithelia or endothelia on the other, justifying classification as a separate kind of tissue, i.e., neurothelium. The observations of interspecies differences lead to the challenging conclusion that neither desmosomes nor cytokeratins are essential for the basic functions of neurothelial sheaths nor does the specific type of IF protein expressed in these cells appear to matter in this respect. The results are also discussed in relation to the cytoskeletal characteristics of other epithelioid tissues and of human neurothelium-derived tumors.     

The formation of a neo-intima in textile prostheses implanted in the aorta of rats and dogs

Summary The formation of a neo-intima in textile prostheses implanted in the rat and dog aorta was studied by means of light- and scanning electron microscopy. Two independent cellular layers (the superficial and deep ingrowth layers) developed on the free surface and under the fibrin layer initially deposited on the inner surface of the prostheses. The superficial ingrowth layer invades the prosthesis from both the proximal and distal aortic stumps and extends over the primary fibrin layer, or replaces it. This layer consists mainly of smooth muscle cells of the triangular aortic type covered by endothelial-like cells. The deep ingrowth layer originates from cellular elements of the prosthetic bed. Fibroblasts, myofibroblasts and spindle-shaped smooth muscle cells invade the fibrin layer through the interstices of the fabric structure of the prosthesis. Precursors of endothelial cells, however, are absent from this population. The superficial and the deep ingrowth layers may become joined by progressive replacement of the fibrin layer, but remain distinguishable because of their different cellular components.When a continuous cellular layer is established on the inner surface of the prosthesis, and this is then covered by endothelial-like cells, the neo-intima formed remains stable during long-term studies.