An Histochemical and Ultrastructural Analvsis

Integument from blue and green areas of the variant blue frog were analyzed biochemically for pteridines and carotenoids. Solvent extraction and absorption spectrophotometry indicated that p carotene was greatly reduced in the blue skin, and present in high quantities in the green skin of the blue frog. Thin layer and paper chromatography indicated that the pteridines were almost totally lacking in the blue skin, and present in normal quantities in the green skin of the blue frog. Light and electron microscopy indicated that the xanthophore pigment cells were either greatly altered or absent from the blue integument and present in the green integument, The fine structure of the xanthphores of the green integument contained the normal ultrastructural components of xanthopores found in regular green integument. The blue integument contained a n abnormal cell type that occupied the position in the dermal chromatophore unit normally held by the xanthophores. The possibility of these cells being abnormal xanthophores or some other cell type is discussed. Blue pigmented variants in several ranid species have been recorded by several investigators (Berns and Uhler, '66; Martof, '62; Liu, '37). An adequate biochemical and morphological analysis of the integument of this variant form is lacking. Recently Bagnara et al. ('68) defined the dermal chromatophore unit as the functional integration of several morphologically distinct cells which provides the organism with its total overall pigmentary effect. This unit consists of a layer of melanin containing melanophores, overlain by a layer of crystal containing iridiophores, which in turn, is overlain by a layer of carotenoid-pteridine containing xanthophores. Normal green coloration is probably caused by the crystalline iridiophore layer scattering white light up through the yellow xanthophore layer which filters out the blue wavelengths and permits the green to pass. A hypothesis of this nature was first suggested by Fox and Vevers ('60) and more fully defined by Bagnara et al. ('68). Furthermore, it was pointed out (Bagnara et al., '68) that blue pigmentation could be produced by leaching out the xanthophore pigments with alcohol. The blue pigmentation in Rana could be explained by a lack of pigment from the xanthophores, or by a lack of the xanthophore cell type (Berns, '66). It is the purpose of this manuscript to examine the integument of the variant blue frog histochemically for the xanthophore pigments (carotenoids and pteridines), and morphologically for the xanthophores. The findings will be evaluated in light of the dermal chromatophore unit concept. MATERIALS AND METHODS Two blue specimens of Rana clamitans were used in these studies. Both specimens had blue and green areas in their integument. One specimen had a green mask (Berns and Uhler, '66) with blue pigmentation extending on the dorsal surface posteriorly from between the eyes to the anus. The other specimen was blue on the entire dorsal surface except for the dorsal surface of the legs, which was green. Samples of blue and green skin were removed from both specimens for the histochemical analysis. In the second specimen the 1 cm2 pieces of skin were cut in half, one half being used for histochemical analysis and the other half for morphological analysis. 169 J. MORPH., 132: 169-180. 170 M. W. BERNS AND K. S . NARAYAN Analysis for carotenoids and pteridines was performed in a two-step process: the carotenoids were first extracted from the skin, and then the pteridines were extracted. The skin to be utilized for the chemical analysis was placed in a 37" C incubator immediately upon removal from the animal, and desiccated for one hour after which it was weighed to the nearest tenth of a milligram. The skin was next placed in 5 ml of acetone/5 mg of dry skin weight and vigorously agitated with a glass stirring rod for 15 minutes. The acetone solution (which was yellow if carotenoids were present) was then placed in a spectrophotometer cuvette and the absorption curve run in a recording spectrophotometer. The absorption curve was then compared to a curve obtained from a pure sample of ,fi carotene. After extraction of the carotenoids the same piece of skin was placed in a solution of 20% NH,OH in a concentration of 2 ml NH40H/2 mg dry skin weight. All steps after carotenoid extraction were conducted in a dark room under safe light. The skin was left in the NH,OH for 24 hours to permit full extraction of the pteridines. After extraction the pteridine solution was subjected to thin-layer and paper chromatography. A 0.03 ml volume of the pteridine solutions (from blue and green skin) was placed along the base of the chromatogram sheet approximately 2 cm from the bottom of the sheet, and 2 cm apart. The sheet was next placed in a solvent solution of n-butyl alcohol, acetic acid, and distilled water ( 4 : 1 : 5), and run ascending for four to six hours. The chromatographic sheets were either no. 4 Whatman filter paper, or Eastman cellulose no. 6064 without fluorescence indicator. After removal from the solvent solution the chromatogram was air dried for ten minutes and then observed under ultraviolet light using a Wood's filter. The various fluorescent spots were noted with respect to RF values, color, and relative intensity in comparison to standardized samples obtained from normal green skin. In addition, a pure sample of isoxanthopterin was run as a control. The pieces of skin not used for the biochemical analysis were immediately fixed in 2% glutaraldehyde in phosphate buffer, washed in phosphate saline, post fixed in 2% OsOl in veronal acetate buffer, dehydrated in graded alcohol series containing uranyl acetate to absolute ethanol and propylene oxide. The tissue was infiltrated in Epon-Araldite and propylene oxide ( 1 : 1, pure epon-araldite (overnight) and embedded in fresh plastic. Sections were cut on a Porter Blum ultramicrotome with a glass knife as thick sections for light microscopy (1-2 p ) and as thin sections (GO120 mp thick) for electron microscopy. Thick sections were stained with toluidine blue and mounted in permount. Thin sections were stained in uranyl acetate and lead citrate, and examined and photographed with an RCA EMU 3G electron microscope.