0300-9629/93 $6.00+ 0.00 0 1993 Pergamon Press Ltd zyxwvut C’omp. Biochem. Physiol. Vol. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 104A, No. 3, pp. 443448, 1993 Printed in Great Britain zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA MINI REVIEW THE CALCIFIED DERMAL LAYER IN ANURANS R. C. TOLEDO*~ and C. JARED*$ *Laboratory of Cellular Biology, Instituto Butantan Av. Vital Brazil, 15OO-CEP05504, Sao Paulo; TDepartment of Biological Sciences, Universidade Cidade de Slo Paulo (UNICID), SHo Paulo, Brazil (Received 28 April 1992; accepted 5 June 1992) INTRODUCTION “he vertebrate skin is basically composed of epidermis and dermis. The epidermis lies closer to the surface. It is ectodetmal in origin, and is a : tratified epithelium, in which a number of structures of importance to the animal are formed: receptor organs, glands, scales, hairs, feathers, horns and nails. ‘Yhedermis, lying immediately beneath the epidermis, is mesodermal in origin. It is basically a connective tissue, in which collagen and elastic fibers are pre- dominant. The dermis also contains blood vessels, irerve and muscle fibers, glands and portions of cpidet-mal derivatives such as hairs and feathers. Its structural characteristics depend on the mode of life of the animal in question, and on the phylo- genetic position which it occupies (Moss, 1972). In the amphibians, and in vertebrates in general, the dermis (Fig. 1) is made up of two layers or strata, the stratum spongiosum and the stratum compactum- or the papillar and reticular dermis respectively. The stratum spongiosum-that closer to the outer surface --is mostly made up of loose connective tissue. In amphibians it contains not only collagen and elastic fibers but also nerves, blood vessels, chromatophores and multicellular glands. The stratum compacturn lies tleeper in the dermis and is made up of dense c.onnective tissue. The collagen fibers in this stratum lie, in general, in bundles parallel to the body surface of the animal. The dermis reaches its basal limit at the t ela subcutanea (or hypodermis) characterized by loose connective tissue which, in the anurans, lines the lymphatic spaces. The calcified layer-also known as the GS (from the initials of “Ground Substance”) layer (Elkan, 1976) was first described by Eberth (1869). While using roots of madder (Rubia tinctorum L.) to stain bones of frogs, Eberth, and a few years later Kastschenko (1882), noted that the staining also showed up a particular layer in the dermis of the animals. The layer usually lies between the stratum spongiosum and the stratum compacturn in the anurans, especially in those of a more terrestrial habitat. :Author to whom all correspondence should be addressed. CHEMICAL COMPOSlTlON The calcified layer has no cellular structure. It is composed of an amorphous ground substance, together with calcium salts. This ground substance is fairly highly concentrated in the stratum spongiosum of the amphibian skin (Moss, 1972) and may be defined as the extravascular, extracellular and extra- fibrillar phase of the dermis (Pearce and Grimmer, 1970). It is made up of a well-hydrated gel, poly- saccharide in nature, in which the collagen and elastic fibers are dispersed. The polysaccharide component is made up of glycosaminoglycans, in general covalently linked with proteins. The presence of sulfate and/or carboxyl radicals makes the glycosaminoglycan molecules intensely negative. The dermis usually contains hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate and heparin. These glycosamino- glycans join up with protein compounds to make more complex substances known as proteoglycans (Alberts et al., 1986). In the calcified layer of toads of the species Bufo bufo, hyaluronic acid has been identified in combination with sodium, and dermatan sulfate and chondroitin-6-sulfate are also present (Elkan, 1968). Quantitative analysis of the calcium in the calcified layer in Bufo marinus and Xenopus laevis has revealed respective percentages of 28.3 and 2.7% (Elkan, 1968). Techniques used to detect sodium show it to accumulate in the calcified layer in frogs of species Rana nigromaculuta. It is suggested that the calcified layer in this species must also contain sialic acid and cations Co’+ and Cu’+, in addition to Ca2+ (Imamura et al., 1965). In the toad B. marinus, the layer contains neutral lipids of unknown function (Sampson et al., 1987). The electron microscope shows the existence of electron-dense crystals in the region of the calcified layer in the skin of B. marinus. These crystals contain a high concentration of calcium and phosphorus (Sampson et al., 1987). Ultrastructural examination of the layer in B. bufo has revealed the presence of vesicles of up to 1 pm in diameter, with acicular crystals, possibly made of hydroxy- apatite (Verhaagh and Greven, 1981). These vesicles, known as matrix vesicles, observed in certain tissues of other animals, have proved to be wrapped in a 443