Succinic dehydrogenase activity in the epidermis ofNatrix piscator during its sloughing cycle

Succinic dehydrogenase activity, in the epidermis of Nairix piscutor in different stages of sloughing cycle, has been localized using a nitro-BT technique with appropriate controls. The staining properties of different layers in scale epidermis are similar to the corresponding layers in hinge epidermis.
In the stratum germinativum, the layers of undifferentiated epidermal cells in all stages of the sloughing cycle, and in the lacunar tissue of Stages 3,4 and 5, a positive though weak reaction for SDH activity reflects the active metabolic state of the cells in these layers. Loss of SDH activity in Stage 6 indicates an inactive metabolic state of the lacunar tissue cells, corresponding with their disintegration owing to the cessation of nutrients as a result of keratinization of cells in the underlying layers.
The Oberhautchen, mesos and alpha layers in all Stages, and the clear layer cells in Stages 5 and 6 (outer epidermal generation), the presumptive Oberhautchen, presumptive mesos layer and presumptive alpha layer in all stages of their differentiation, and the presumptive beta layer in Stages 3 and 4 (inner epidermal generation) all stain purple with nitro-BT technique even in sections incubated in the medium without the substrate-succinate. The reaction is inhibited by prior treatment with 0.1 M N-ethyl maleimide blocking protein-bound -SH groups. This suggests that the reaction is due to the presence of protein-bound -SH groups in these sites. The reduced intensity of reaction in the mature beta layer of the outer epidermal generation, and in the presumptive beta layer in Stages 5 and 6 of the inner epidermal generation, is due to simultaneous loss of their content of -SH groups with maturation and keratinization.     

Enzymes in the epidermis of Natrix piscator during its sloughing cycle

Acid phosphatase, non-specific esterase, alkaline phosphatase, monoamine oxidase and true lipase activities, in the epidermis of Natrix piscator in different stages of the sloughing cycle, have been localized using various histochemical techniques.
Different layers in scale epidermis have staining properties similar to corresponding layers in hinge epidermis.
Acid phosphatase and non-specific esterase activity in cell layers undergoing keratinization, and the lacunar tissue undergoing disintegration are associated with hydrolytic and catabolic wasting processes involving cell death. The activity of these enzymes in the clear layer is associated with the breaking down of the cementing substance resulting in the separation of clear layer from underlying tissue and facilitating the shedding of old slough.
Alkaline phosphatase activity in the stratum germinativum and undifferentiated epidermal cells has been associated with cell proliferation and differentiation. The presence of alkaline phosphatase in the lacunar tissue and clear layer has been correlated with the synthesis of mucopolysaccharides in these layers.
Monoamine oxidase and true lipase activity could not be located in the epidermis at any stage of the sloughing cycle.     

Lipid histochemistry of the epidermis of Natrix piscator during its sloughing cycle

The lipid histochemistry of the scale and hinge epidermis of the chequered water snake, Natrix piscator , throughout the sloughing cycle, has been described. The presence of comparatively high concentrations of phospholipids in the mesos layer and a-layer, in comparison to neutral lipids, has been associated with a permeability barrier to transcutaneous water flux. Free fatty acids, present in almost all epidermal layers and in eosinophilic granular cells, may protect the epidermis from bacterial and fungal attacks. Cholesterol, in addition to phospholipids, in various keratinized layers, is assumed to be derived from membranous structures of epidermal cells and is regarded as a stabilizer of the phospholipids in membranes.     

Morphogenesis of the digital pad lamellae in the embryo of the lizard Anolis lineatopus

The present study in the embryo of the lizard Anolis lineatopus describes the modality of cell proliferation responsible for the morphogenesis of the digital pad lamellae and of the epidermal stratification. After tritiated thymidine and 5-bromodeoxy-uridine administration, autoradiographic and immunocytochemical methods have been used. The lamellae originate as long, slightly slanted, undulations of the epidermis of fingers and toes. At an early stage, the epidermis consists of an outer periderm and a basal layer. Cell hypertrophy, and the prevalent cell proliferation in the longer side of the undulation with respect to the shorter side, generate the surface of the outer lamella. Under the peridermis, a shedding complex, composed by clear and oberhautchen layers, is formed and later determines the first intraepidermal shed. The first subperidermal layer derived from the basal layer is a clear layer and the first shed epidermis in the embryo is represented by periderm and clear layer. The heavily granulated clear layer in Anolis lineatopus represents the first epidermal layer produced in the embryonic epidermis, and is connected with the process of shedding. The spinulae of the underlying oberhautchen in the outer scale surface become long setae which grow toward the upper clear layer. Under the shedding complex a β-layer is produced. Autoradiographical study shows that the radioactivity stays in the basal layer for about 4 days before cells move to upper layers. At 6–8 days post-injection labelled cells are visible in the differentiated clear, oberhautchen and β-layers. Under the β-layer differentiating mesos cells are visible before the embryo hatches.     

Ultrastructure of the epidermis of the lizard (Lacerta vivipara) at the resting stage of the sloughing cycle

The ultrastructure of the epidermis of the lizard ( Lacerta vivipara ) one day after sloughing is described. The non-keratinized layers of the epidermis are essentially similar in structure to those of amphibians and mammals. The cells of the basal layer are not however separated from each other by the large spaces described in the amphibian (Farquhar & Palade, 1965). The middle layers of the epidermis at this stage of the sloughing cycle produce neither the characteristic mucous granules found in amphibians nor the keratohyalin granules of mammals. A small number of granules corresponding in size and location to the "Odland bodies" of both mammalian and amphibian epidermis are, however, present. The intermediate layer cells also contain a number of bodies similar in appearance to those described by Farquhar & Palade as lysosomes in amphibian skin. These structures are both osmium iodide and acid phosphatase positive. Unlike the condition in amphibians and mammals, the cytoplasm of cells in the layer immediately beneath the keratinized strata is honeycombed with small vesicles, and contains large irregular vacuoles of uncertain content. Certain nonkeratinizing elements within the epidermis are tentatively interpreted as nerve terminations. Two morphologically distinct keratinized strata can be distinguished, the inner stratum consisting of flattened cells similar to those of the stratum corneum of mammalian epidermis; individual cell outlines cannot be distinguished in the outer stratum, which has a structure similar to that of avian feather keratin. A shallow surface zone of the outer keratinized stratum has been identified as the Oberhautchen. This consists of longitudinally disposed leaflets or laminae which are responsible for the sculptured pattern of the epidermal surface. The observations reported here provide a basis for analysis of changes occurring at other stages of the sloughing cycle.     

Structure and histochemistry of the dermis of a chequered water snake, Natrix piscator

The histology and the protein, carbohydrate, lipid and enzyme histochemistry of the dermis of the chequered water snake, Natrix piscator , have been examined throughout the sloughing cycle.
The structural organization and staining properties of the dermis do not show any changes in different stages of the cycle.
The dermis is divided into the stratum laxum and the stratum compactum. Acid mucopolysaccharide deposits in the stratum laxum, which possibly bind water, may be involved in prevention of desiccation.
The numerous elastin fibres in the hinge regions are related to the greater elasticity of the skin in these sites.     

Observations on the epidermis of desert-living iguanids

The epidermis of 146 specimens of Dipsosaurus dorsalis and 182 Uma notata collected throughout the active period of the animals' year has been examined. The morphology of the epidermis is essentially similar to previously described lacertilians but differs in the relatively great degree of development of the mesos layer and the complete keratinization of the lacunar tissue prior to sloughing. Analysis of sloughing frequency throughout the year suggests that species specific patterns may exist, but these do not correlate with any particular known ecologic datum. The patterns do not reflect the reproductive activity of the two species supporting previous experimental conclusions on the lack of effect of gonadial hormones on epidermal activity. There appears to be no evidence of association of femoral gland activity with epidermal activity in D. dorsalis, but the situation is not clearcut in U. notata. These data are discussed in the light of recent studies of the evolutionary origin of epidermal glands in lizards.     

Glycogen Distribution in Relation to Epidermal Cell Differentiation During Embryonic Scale Morphogenesis in the Lizard Anolis lineatopus

The differentiation of the epidermis during scale morphogenesis in the lizard Anolis lineatopus has been studied by autoradiographic and immunocytochemical techniques and by electron microscopy, in relation to mitotic activity and to the distribution of glycogen. The flat embryonic epidermis of the early embryo is transformed into symmetric epidermal papillae which progressively become asymmetric and eventually form scales with stratified epidermal and peridermal layers. Papilla asymmetrization and epidermal stratification derive from cell hypertrophy and multiplication in the “basal hypertrophic layer of the forming outer side of scales” (BLOS). Glycogen is scarce or absent during early stages of epidermis development. In the dermis no glycogen is found at any stage of scale morphogenesis. Glycogen particles 25–40 nm in size accumulate in hypertrophic basal cells and peridermal cells during scale development. Conversely cells in the forming inner side of scales do not accumulate glycogen, divide less frequently than in the outer side and do not form a β–keratinized layer. It is suggested that an osmotic effect related to glycogen deposition causes increased hydration of the BLOS, whose cells become swollen and contribute to the asymmetrization of the epidermal papillae. Glycogen decreases in suprabasal differentiating cells and disappears from the BLOS at the stage of complete keratinization of the scale, around the period of hatching. Terminal differentiation in the peridermis and suprabasal epidermal layers takes place by cell flattening and condensation of the nucleus and cytoplasm as typical for apoptotic cells.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Comparison of α and β keratin in reptiles

Summary The different patterns of keratin formation that have evolved in the class Reptilia are all variations of a common process. In Squamata (snakes and lizards), a sequence of layers composed of or keratin is formed periodically, after which the old epidermal generation is shed. In Chelonia (turtles and tortoises), the epidermis of the shell is composed of only keratin, whereas the skin of the neck and leg is composed exclusively of keratin. Molting in toto does not occur and shedding is a continuous process comparable to that in avian and mammalian epidermis. In Crocodilia (crocodiles, caimans, alligators) there is only a single layer of cornified cells, but the composition of the layer varies in different parts of the scale. The hinge regions have many of the morphological characteristics of and keratin whereas the center resembles keratin. The living cells beneath contain accumulations of keratohyalin.There are four ultrastructural characteristics of a keratinized layer: 1) cellular outlines remain distinct, 2) a thickened plasma membrane forms during keratinization, 3) 80 Å filaments embedded in an amorphous matrix can be seen, and 4) PAS-positive material accumulates in extracellular spaces between the desmosomes.The layer exhibits none of these features. Instead the cells more or less (depending on species) coalesce into a compact layer which becomes attenuated in the hinge regions. A 30 Å filament pattern can be seen.The mesos layer of squamates resembles the hinge region of crocodilians, exhibiting a combination of the characteristics of both and keratin.This study constitutes publication No. 464 from the Oregon Regional Primate Research Center, supported in part by NIH Grant No. FR-00163.     

Ultrastructural contributions to the identification of cell types in the lizard epidermal generation

The ultrastructure of the epidermis at different stages of the shedding cycle has been studied in Anolis carolinensis. Cells of the germinal layer are morphologically similar at all stages in the cycle. Immediately after leaving the germinal layer all daughter cells resemble one another closely. However, they later acquire specific ultrastructural features that enable them to be classified into six distinct fully differentiated types corresponding to the grouping previously set forth by light microscopy. A comparison of cytoplasmic filament size with the known X-ray diffraction data suggests that the Oberhautchen and β-layer contain a protein similar to that of avian feather; the protein in the α-layer and lacunar tissue is similar to that in mammalian hair, and the mesos layer cells probably contain a mixture of feather and hair-like proteins. The nature of the amorphous cytoplasmic material in the mature clear layer is as yet unknown.     

Ultrastructural contributions to an understanding of the cellular mechanisms involved in lizard skin shedding with comments on the function and evolution of a unique Lepidosaurian phenomenon

Previous reports on the fine structure of lizard epidermis are confirmed and extended by SEM and TEM observations of cell differentiation and the form of shed material from the American anole Anolis carolinensis. Attention is drawn to two issues: 1) the tips of the spinules arising from the mature oberhautchen are markedly curved; this morphology can be seen during differentiation; 2) the median keels of scales from all parts of the body show “naked” oberhautchen cells that lack characteristic spinules, but have a membrane morphology comprising a complex system of serpentine microridges. Maderson's ([1966] J. Morphol. 119:39–50) “zip-fastener” model for the role of the shedding complex formed by the clear layer and oberhautchen is reviewed and extended in the light of recent SEM data. Apparently periodic lepidosaurian sloughing permits somatic growth; understanding how the phenomenon is brought about requires integration of data from the organismic to the molecular level. The diverse forms of integumentary microornamentation (MO) reported in the literature can be understood by considering how the cellular events occurring during the renewal phase prior to shedding relate to the emergence of the form-function complex of the β-layer, which provides physical protection. Issues concerning the evolutionary origin of lepidosaurian skin-shedding are discussed. J. Morphol. 236:1–24, 1998. © 1998 Wiley-Liss, Inc.     

Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.     

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.     

Ultrastructure of the embryonic snake skin and putative role of histidine in the differentiation of the shedding complex.

The morphogenesis and ultrastructure of the epidermis of snake embryos were studied at progressive stages of development through hatching to determine the time and modality of differentiation of the shedding complex. Scales form as symmetric epidermal bumps that become slanted and eventually very overlapped. During the asymmetrization of the bumps, the basal cells of the forming outer surface of the scale become columnar, as in an epidermal placode, and accumulate glycogen. Small dermal condensations are sometimes seen and probably represent primordia of the axial dense dermis of the growing tip of scales. Deep, dense, and superficial loose dermal regions are formed when the epidermis is bilayered (periderm and basal epidermis) and undifferentiated. Glycogen and lipids decrease from basal cells to differentiating suprabasal cells. On the outer scale surface, beneath the peridermis, a layer containing dense granules and sparse 25-30-nm thick coarse filaments is formed. The underlying clear layer does not contain keratohyalin-like granules but has a rich cytoskeleton of intermediate filaments. Small denticles are formed and they interdigitate with the oberhautchen spinulae formed underneath. On the inner scale surface the clear layer contains dense granules, coarse filaments, and does not form denticles with the aspinulated oberhautchen. On the inner side surface the oberhautchen only forms occasional spinulae. The sloughing of the periderm and embryonic epidermis takes place in ovo 5-6 days before hatching. There follow beta-, mesos-, and alpha-layers, not yet mature before hatching. No resting period is present but a new generation is immediately produced so that at 6-10 h posthatching an inner generation and a new shedding complex are forming beneath the outer generation. The first shedding complex differentiates 10-11 days before hatching. In hatchlings 6-10 h old, tritiated histidine is taken up in the epidermis 4 h after injection and is found mainly in the shedding complex, especially in the apposed membranes of the clear layer and oberhautchen cells. This indicates that a histidine-rich protein is produced in preparation for shedding, as previously seen in lizard epidermis. The second shedding (first posthatching) takes place at 7-9 days posthatching. It is suggested that the shedding complex in lepidosaurian reptiles has evolved after the production of a histidine-rich protein and of a beta-keratin layer beneath the former alpha-layer.     

Physiological adaptation in relation to hyperosmotic stress in the epidermis of a fresh-water teleost Barbus sophor (Cypriniformes, Cyprinidae): a histochemical study.

The changes in distribution of mucopolysaccharides, glycogen and protein bound sulphydryl groups of cysteine in the various cellular components of the epidermis of Barbus sophor along with its structural alterations as a result of hyperosmotic stress, have been described using histochemical techniques. The hyperosmotic saline induces a cyclic secretion of acid mucopolysaccharides by the mucous cells. Simultaneously the polygonal cells also show a marked disturbance in the processes of mucogenesis and keratinization, indicating an inverse relationship between the degree of keratinization and the amount of mucus secreted by epidermis. The role of glycogen in the polygonal cells has been discussed in relation to disturbed mucogenesis. The appearance of intercellular spaces in basal layer and middle layer has been correlated with the passage for movement of increased amounts of nutrients through the skin.     

Ultrastructural studies of epidermis keratinization in grass snake embryos Natrix natrix L. (Lepidosauria, Serpentes) during late embryogenesis

The changes and biochemical features of the epidermis that accompany the differentiation and embryonic shedding complex formation in grass snake Natrix natrix L. embryos were studied ultrastructurally and immunocytochemically with two panels of antibodies (AE1, AE3, AE1/AE3; anti-cytokeratin, pan mixture, Lu-5 and PCK-26). All observed changes in the ultrastructure of the cells forming the epidermal layers were associated with the physiological changes that occurred in the embryonic epidermis, such as changing of the manner of nutrition and keratinization leading to the embryonic shedding complex formation. The layers that originated first (basal, outer and inner periderm and clear layer) differentiated very early and rapidly. Rapid differentiation was also observed in the layers that are very important for the functioning of the epidermis in Natrix embryos (oberhäutchen and beta-layers). They started to differentiate at developmental stage IX, and then fused and formed the embryonic shedding complex at developmental stage XI. During the embryonic development of the grass snake the smallest changes appeared in the ultrastructure of the cells in the mesos and alpha-layers because they perform supplementary functions in the process of embryonic molting. They were undifferentiated until the end of embryonic development and started to differentiate just before the first adult molting. AE1/AE3, anti-cytokeratin, pan mixture, Lu-5 and PCK-26 antibodies immunolabeled clear layer, oberhäutchen and beta-layers at the latest phase of developmental stage XI. It should be noted that these antibodies did not immunolabel the alpha-layer until hatching. The presence of alpha-keratin immunolabeling in layers that were keratinized, particularly in the oberhäutchen and beta-layers in embryos, indicated that they were not as hard as in fully mature individuals.     

Formation of α- and β-type keratin in lizard epidermis during the molting cycle

Summary The epidermis of Anolis carolinensis is renewed periodically by molting. Prior to the molt the distinct layers of the epidermis, namely, the Oberhäutchen, , mesos, and layers, are formed in sequence from a morphologically homogenous population of basal cells. The Oberhäutchen, the first cell layer to form, has spinules on the surface which interdigitate with the overlying cells of the clear layer. The cells of the Oberhäutchen develop 80 Å filaments similar to those in the cells of the layer. Beneath the Oberhäutchen is the layer, the cells of which develop membrane-bounded packets containing a homogenous material during the early stages of differentiation. Later 100–500 Å thick fibrils are formed in the membranebounded packets. The fully keratinized cells, however, are packed with filaments 30 Å in diameter separated by an electron dense amorphous matrix, very similar to -type keratin found in the feather rachis. The cells of the layer, which is immediately below the layer, contain 80 Å filaments very similar to the -type keratin found in hair cortex and keratinizing stratified epithelia of mammals. Large quantities of glycogen are found in the cells of each layer during their genesis. Even though a stratum granulosum is not found underneath the layer, the cells of the clear layer develop bodies which have histochemical and ultrastructural characteristics of keratohyalin granules. The old epidermis is then shed in toto at the junction of the clear layer (above) and the Oberhäutchen (below).This study constitutes publication No. 406 from the Oregon Regional Primate Research Center, supported in part by postdoctoral training fellowship 1-TIAM-5521-02 and NIH Grant No. FR-00163.     

Heat shock protein 25 plays multiple roles during mouse skin development

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.