Kyozo Watanabe

Ultrastructural changes in the tail muscles of anuran tadpoles during metamorphosis

SummaryThis report describes histochemical and ultrastructural studies of tail muscles in tadpoles of Rana japonica and Rana catesbeiana during metamorphosis, this process being accompanied by degeneration of the tail. Degeneration of individual tail muscles does not occur at the same time; this is true for both the red and white muscle fibres.The initial phase of degeneration showed mesenchymal macrophages first invading the muscle fibres and then sending out many long cytoplasmic processes which split the fibres apart.The disappearance of myofibrils during degeneration proceeds along at least two different mechanisms even within a single muscle fibre. In one type, the Z-band becomes diffuse and then disappears, resulting in fragmentation of the myofibrils at the sites previously occupied by the Z-bands. The second pattern of degeneration is characterized by disappearance of the Z-band followed by a fanning out of the myofilaments not associated with fragmentation of myofibrils. As atrophy of muscle fibres proceeds, acid phosphatase activity is localized in the perinuclear sarcoplasm. Macrophages show more intense acid phosphatase activity than do the muscle fibres. The formation of autophagic vacuoles is described and discussed.

Autolysis and heterolysis of the epidermal cells in anuran tadpole tail regression

Autolysis and heterolysis of the degenerating epidermis of the tail fin of Rana japonica tadpoles during spontaneous metamorphosis were observed by transmission and scanning electron microscopy. In the early climactic stages of metamorphosis (st. 19–20), the outermost epidermal cells developed vacuoles that were acid phosphatase positive and showed apparent breakdown of the cell membrane. The cells shrunk, perhaps due to the rupture of the cell membrane, and sloughed off without typical cornification. As tail resorption proceeded, autolysis of the epidermal cells spread towards the inner layers, in which some epidermal cells lost desmosomal junctions. They also displayed atrophic figures with condensed cytoplasm, breakdown of the cell membrane, and pycnotic nuclei. Lymphocytes, neutrophils and macrophages were already present in the basal layers of the premetamorphic epidermis (st. 10). Based on ultrastructural observation, blood cells could be distinguished from autolysing epidermal cells. Only a few blood cells were found in the early climactic stages of metamorphosis (st. 19–20), but the number of the blood cells, especially macrophages, greatly increased during the final stages of metamorphosis (st. 23–24). During the final stages, many macrophages were observed to phagocytose the autolysing epidermal cells by projecting slender pseudopodia into the inner epidermis. Macrophages also were observed to pass through the degraded basal lamella. These results suggest that not only autophagy but also heterophagy of the epidermal cells by the macrophages is a major process in the regression of the tail fin epidermis.

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

SummarySkins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.

Cytochemical studies of hydrogen peroxide production in the tadpole tail ofRana japonica during metamorphic climax

SummaryThe degeneration of tadpole tail tissue was investigated cytochemically by localizing the sites of hydrogen peroxide production. A cerium perhydroxide precipitation method was used. No reaction product was found in resting macrophages and intact muscle fibres during premetamorphosis. In the metamorphosis phase, extensive cerium precipitates were visualized on the outer surface of the plasma membrane of phagocytotic macrophages, fibroblasts, neutrophils, epidermal cells, muscle fibres, notochordal cells, nerve cells and capillary endothelial cells. The reaction products were localized on those parts of the plasma membranes of the macrophages that were in contact with those of adjoining cells. When catalase were added, the amount of deposits decreased. α-Tocopherol and indomethacin, but not dexamethasone, significantly inhibited the formation of the reaction products. These findings are taken to indicate that active oxygen is produced on the plasma membrane of activated macrophages and may play a role in the degeneration of the tail tissue.

Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature

SummaryThe histochemical activities of succinic dehydrogenase (SDH) and Ca++-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. ‘Red’ and ‘white’ muscle fibres, as distinguished by SDH, showed high and low Ca++-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K2-EDTA solution. The ultrastructural investigation of CA++-ATPase reaction at pH 7.4 by the Ca++-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and ‘Z’ bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb++ precipitation technique demonstrated Mg++-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail ‘red’ muscle fibres are possible ‘slow,’ and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, ‘red’ muscle fibres of the anuran tail musculature are not equivalent to ‘Type I’ fibres of higher chordates.

Electron-microscopical study of the operculum in anuran tadpole after extirpation of the right forelimb during metamorphosis.

SummaryThe process of histolysis and fenestration of the skin of the prospective opercular perforation region of Rana japonica after extirpation of the right forelimb was observed during metamorphosis by transmission and scanning electron microscopy.Epidermal cells of the belly of the tadpole, including the operculum, are extremely similar in their ultrastructure. Epidermal cells of the prospective opercular perforation region during metamorphosis become thin and vacuolated especially around the nucleus perhaps by autolysis, associated with lysosomal activity. The histolysis and formation of the perforation of the operculum occurs in the complete absence of forelimb. Macrophages containing phagosomes and lymphocytes or other blood cells are almost always found in the intercellular epidermis. Necrotic epidermal cells progressively separate by cleft formation and slough off without cornification.

Scanning electron microscopy of macrophages in the tail musculature of the metamorphosing anuran tadpole, Rana japonica

SummaryScanning electron microscopy was used to investigate the morphological changes of the tail musculature of the metamorphosing anuran tadpole, attention being focused on phagocytosis by macrophages. Muscle fibers were stained en bloc with silver and freeze-fractured during dehydration, or torn after drying. Samples were sputter-coated with gold-palladium and observed in both secondary electron- and back-scattered electron modes with a scanning electron microscope.Various cells were identified by the methods of secondary electron- and back-scattered electron images. Some macrophages lying between muscle fibers at prometamorphic stages possessed numerous finger-like projections and well-developed ruffles. During degeneration of muscle fibers macrophages collected in the degenerating region and invaded the space between the disordering myofibrils. In advanced stages the numbers of macrophages clearly increased on or around the degenerating muscle fibers. At the climactic stage fragmented muscles were entrapped and then engulfed by the macrophages. With the completion of phagocytosis, the macrophages became globular with reduction of the ridge-like ruffles. Macrophages may play a role not only in scavenging the fragmented muscle fibers, but also using their long processes in active formation of the fragments.

Satellite cells in the tail muscles of the urodelan larvae during development.

SummaryThe incidence and ultrastructure of satellite cells in the tail muscles of urodelan larvae were examined during development during which the number of satellite cells is gradually reduced. They are found more frequently in red than in the white fibres in all four stages examined (stage 53, 64, 66+ and juvenile). As development proceeds, intercellular space between satellite cell and muscle fibre is in general gradually extended and is mostly filled with basal lamina. Small muscle cells, satellite fibres, which are situated under the basal lamina of the parent fibre, are morphologically similar to satellite cells but contain a small amount of myofibrils. Three types of satellite fibres are distinguishable on the basis of differences in K2-EDTA-treated ATPase activity, width of Z line, and parent fibre type. Neuromuscular junctions are visible in satellite fibres.

Light and electron microscopic investigation of ATPase activity in musculature during anuran tail resorption

SummaryThe histochemical activity of adenosine triphosphatase (ATPase) was studied at light and electron microscopic levels in larval tail musculature of Rana catesbeiana and Rana ornativentris during late metamorphic stages. The presence of low, moderate or dark reaction of K2-EDTA-preincubated Ca++-ATPase was correlated with the variable degree of degeneration of white fibres even at the late stage of tail resorption. The reasons for an increase in this ATPase activity in degenerating white muscle fibres are discussed. Irrespective of the degree of degeneration, all red fibres showed high ATPase reaction. During myocytolysis, it is shown that the SR vesicles accumulate electron dense amorphous material. The degree of myofibrillar disintegration correlated with decrease in ultrastructural reaction product for Mg++-ATPase. Although grouped atrophy of muscle fibres (as seen in Xenopus laevis, den Hartog Jager et al., 1973, 1975) was absent in musculature of resorptive tails, ultrastructural characteristics including proliferation of SR and dilation of its vesicles represent alteration of the normal neural influence on the skeletal muscle fibres.

Transmission and Scanning Electron Microscopic Study of Adepidermal Granules of Teleosts and Amphibia

SummaryGranules in the adepidermal space of larvae of Salmo irideus, Hynobius tokyoensis and Rhacophorus buergeri, were observed by transmission and scanning electron microscopy. Adepidermal granules of S. irideus were smooth and spherical structures, those of H. tokyoensis were smooth and spherical, or oval, while in R. buergeri these granules appeared as single or grouped tangled strand-like or starfish-like structures under the scanning electron microscope. These adepidermal granules were spread all over the basal lamina in every animal investigated. The different sizes of adepidermal granules of S. irideus and H. tokyoensis seen under the transmission electron microscope are not the result of differently sectioned faces of granules, but the granules themselves exhibit different sizes. The probable functions of these granules are discussed.