Arch. histol. jap., Vol. 35, No. 3 (1973)p. 249-254

Department of Morphology, School of Dentistry of Bauru, Bauru, S.P. and Department

of Histology, Institute of Biomedicine, University of Sao Paulo,Sao Paulo, S. P., Brazil

An Amphoteric Character in Mast Cell Granules of Amphisbaenia

fuliginosa, a Snakelike Lizard

D. SOTTOVIA-FILHO, M. C. LEITE-RIBEIRO and L. C. U. JUNQUEIRA

Received November 22, 1972

Summary. By the use of haematoxylin-eosin and Masson's trichrome staining tech-

niques, and of histochemical tests with Alcian blue and metachromasia with toluidine blue,

the mast cell granules of the snakelike lizard Amphisbaenia fuliginosa are shown to

present an amphoteric character: they are stained by the acid as well by the basic dyes.This property is here discussed in function of the literature, with the main concern

directed toward the taxonomic position of this animal. The Amphisbaenidae seem to re-

present an intermediate stage between lizards and ophidians.The mast cell granules of lizards are basophilic and metachromatic; in the ophidian,

typical mast cells have not been evidenced yet, but a granular acidophilic cell has been

demonstrated in the connective tissue. The basophilic and acidophilic characters of these

two cells seem to be intermingled in the mast cell of the Amphisbaenidae.

In a general way, the identification of mast cells is made by the use of histo-chemical techniques that can detect acid mucopolysaccharides, mainly the sulfated

(heparin), in the cytoplasmic granules.Mast cells have been demonstrated by several authors in higher and lower verte-

brates. They have thus been evidenced in fishes (MICHELS, 1922b; BOLTON, 1933;STOLK, 1960a; ARVY, 1962); in amphibians (MAXIMOW, 1927; ARVY, 1962; SACERDOTEand PENNIZI, 1965); in lizards (STOLK, 1958; 1960b); in alligators (ARVY, 1962); in turtles

(JORDAN and FLIPPIN, 1913; MICHELS, 1922a); in birds (DANTSCHAKOFF, 1916; ARVY, 1955)and in mammals (NAGAYO, 1928; MICHELS, 1938; ARVY, 1955; 1962; MOTA et al., 1956;RILEY and WEST, 1956; GRUMBERG, 1965).

In our recent work using ophidians we noticed that mast cells could not be iden-tified in this animal group (SOTTOVIA-FILHO and JUNQUEIRA, 1972a; SOTTOVIA-FILHOet al., 1972b).

In continuing our phylogenetic studies of the mast cells in reptiles, we havedecided to investigate the presence as well as the histochemical behavior of thesecells in the Amphisbaenidae. The main interest in the choice of this animal was its

particular systematic position, between lizards and ophidians, which might give aclue to explain the absence of mast cells in ophidians, since they have been demon-strated in all the vertebrates which have been studied.

Material and Methods

Three samples of the species Amphisbaenia fuliginosa were used, two males andone female. The animals were sacrificed by decapitation and pieces of all of the mainorgans were obtained. The pieces were fixed in alcohol-formol and 4% basic leadacetate, both prepared according to SELYE (1965); Gendre's fluid and buffered neutral

249

250 D. SOTTOVIA-FILHO, M. C. LEITE-RIBEIRO and L. C. U. JUNQUEIRA:

formalin, according to CONN et al. (1965). The pieces were then embedded in paraffinand serial sections 7μ thick were cut.

The staining techniques used for the morphological analysis were: haematoxylin-eosin and Masson's trichrome, according to CONN et al. (1965). For the histochemicalstudy, the following reactions were examined: periodic acid-Schiff (PAS), accordingto MCMANUS and as described by LISON (1960); metachromasia with 0.01%, toluidineblue and acetic-Alcian blue staining, according to CONN et al. (1965); PAS plus Alcianblue, according to VIALLI and as described by LISON (1960).

Results

Mast cells have been evidenced by metachromasia with toluidine blue as wellas by Alcian blue staining in all the organs examined, mostly near blood vessels andnerve bundles (Fig. 1, 3). The cytoplasmic granules were PAS positive and, by

Fig. 1. Mast cells of Amphisbaenia near a nerve bundle (NB). Tolu-idine blue. ×250

Fig. 2. The same field of Fig. 1 (serial sections). Note the cyto-plasmic granules of mast cells stained by eosin (arrows). Hematoxylin

and eosin, ×250

Amphoteric Mast Cells of Amphisbaenia fuliginosa 251

Vialli's combined technique, PAS and Alcian blue positive granules were intermingledwithin the same cell.

Curiously, a certain acidophilia was recognized by haematoxylin-eosin and byMasson's trichrome techniques, as some granules were stained by eosin and by

ponceau-acid-fuchsin (Fig. 1, 2).

Discussion

The basophilia of mast cell granules is well demonstrated, being responsible forthe metachromasia. It is known that these staining properties are related to the

presence of a sulfated acid mucopolysaccharide, heparin (SYLVEN, 1954; HALE, 1957;LISON, 1960). The metachromatic reaction as well as the Alcian blue positiveness ofthe mast cell granules of the Amphisbaenia fuliginosa may then be related to the

presence of heparin.

Fis. 3. Section of Amphisbaenia tongue. Observe mast cells stainedby Alcian blue. ×250

Fig. 4. Oesophagus of snake. Groups of granular acidophilic cells

(arrows) in the lamina propria. The cytoplasmic granules stainedby ponceau-acid-fuchsin. Masson's trichrome. ×250

252 D. SOTTOVIA-FILHO, M. C. LEITE-RIBEIRO and L. C. U. JUNQUEIRA:

As to the results obtained by combined technique, PAS positive and Alcian blue

positive granules were found to coexist within the same cell. These different reac-tions have been attributed by several authors (HALE, 1957; ARVY and RANCUREL, 1958;TAKEDA, 1958; COMBS et al., 1965; GRUMBERG, 1965) to a variation in the compositionof the cell secretion. According to them the immature granules containing heparin

precursors (monosulfated heparin acid) would be PAS positive and after the esteri-fication of these precursors by sulfate radicals this positiveness would be lost. Themature granules (polysulfatic esters of heparin) would then be metachromatic andAlcian blue positive. Our results, obtained by Vialli's combined technique, could berelated to this same process.

In earlier works (SOTTOVIA-FILHO and JUNQUEIRA, 1972a; SOTTOVIA-FILHO et al.,1972b,) we could not evidence mast cells in the studied ophidians, by the use ofhistochemical and radioautographic methods nor by biological assays. On the otherhand, granular acidophilic cells were found in the connective tissue of these animals

(SOTTOVIA-FILHO et al., 1972c, d). The granular acidophilic cells present cytoplasmicgranules which are acidophilic with haematoxylin-eosin and Masson's trichrometechniques; they do not incorporate sulfur radioactive isotope (S35) nor are degran-ulated by compound 48/80.

The mast cells of Amphisbaenidae have an amphoteric staining character, i.e.,they can be stained by acidic dyes (eosin and ponceau-acid-fuchsin), basic dyes

(toluidine blue) as well as by Alcian blue which is considered an amphoteric dye(GURR, 1962).

The acidophilia might be related to the PAS positive material or other compo-nents which do not present acid mucopolysaccharidic nature. This is obviously ahypothesis that recquires deeper analysis for an eventual elucidation.

The position of the Amphisbaenidae in the order Squamata is subject of contro-versy. Some studious place them in the suborder Lacertilia while others locate themin a special suborder, the Amphisbenia (GRASSE, 1970).

According to GRASSE (1970), most of the taxonomists which classify the Amphi-sbaenidae in a special suborder consider them an evolutional stage between lizardsand ophidians. One of their basic arguments are the modifications in the scapularand pelvic archs, leading to the atrophy of limbs. From the evolutional standpoint,this atrophy begins in some representatives of the suborder Lacertilia (the Angui-morpha), being emphasized in the suborder Amphisbaenidae and reaching its maxi-mum in the suborder Ophidia.

Analysing the literature cited, it can be observed that typical mast cells havebeen evidenced in several animals of the class Reptilia, except for the suborderOphidia, where granular acidophilic cells were described.

If the Amphisbaenidae really represent an intermediate stage between lizardsand ophidians one might admit the hypothesis that in their mast cell granules thebasophilic and metachromatic properties of typical mast cells are intermingled withthe acidophilic character of the granular acidophilic cells. Comparing Figures 2 and4, a great structural similarity can be noticed between the granular acidophilic cellsof snakes and the mast cells of Amphisbaenia. fuliginosa.

Amphoteric Mast Cells of Amphisbaenia fuliginosa 253

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Dr. Dagoberto SOTTOVIA-FILHODepartamento de MorfologiaFaculdade de Odontologiade Bauru. Caixa Postal 73Bauru 17100-SP-Brasil