Histochemistry of Mucosubstances in the Mantle of the Clam, Mercenaria mercenaria. II. MucoSubstances in the Second Marginal Fold
1969; Wiley; Volume: 88; Issue: 3 Linguagem: Inglês
10.2307/3224710
ISSN2325-5145
Autores Tópico(s)Calcium Carbonate Crystallization and Inhibition
ResumoThe results of a variety of histochemical reactions on mucosubstances in neutral formalin-fixed tissues indicate that in the second fold of the mantle edge there are two mucous cell types, one secreting a sulfated acid mucopolysaccharide or glycosaminoglycan and the other forming a neutral mucosubstance. The neutral mucosubstance was PAS-positive prior to and following diastase digestion. It also exhibited a positive PAS reaction following staining with alcian blue at pH 2.5, both prior to and following hyaluronidase digestion. For the most part, the glycosaminoglycan stained intensely with alcian blue at pH 2.5 and somewhat less intensely at pH 1.0. Portions of the material retained alcianophilia in solutions of magnesium chloride up to 1.0 molar, and after testicular hyaluronidase digestion. The material also reacted strongly with iron diamine and aldehyde fuchsin when these reagents were followed by alcian blue. Methylation extinguished alcianophilia in the acid mucosubstance, but it was partially restored following saponification. The need for information on the role of mucus in invertebrates has been pointed out previously (Jakowska, 1965, 1966; Jeanloz, 1966; Hillman, 1968). It has been shown by a number of workers that invertebrates, particularly mollusks, possess a wide array of complex mucosubstances which presumably play an important role in the life functions of the animal (e.g., Gabe, 1962; Healy, 1963; Pedersen, 1965; Hillman, 1968). The mantle of the quahog clam, Mercenaria mercenaria, is particularly rich in a variety of mucosubstances (Hillman & Shuster, 1962; Hillman, 1964, 1968) and was thus chosen for an investigation of the formation, secretion, and functions of these products in the clam. The mantle margin of the quahog M. mercenaria terminates in four folds. The first or outermost fold is principally secretory in function, and is responsible for the formation of the shell. In the first paper in this series, a moderate to strongly acidic, sulfated mucosaccharide was described in this fold (Hillman, 1968). Because of its location beneath the portion of the mantle which secretes the shell, and its similarity to dermatan sulfate, it was postulated that this material may play a role in the calcification of the clam shell. The work described here presents the results of histochemical reactions for mucosubstances in the second marginal fold. MATERIALS AND METHODS Specimens of the clam M. mercenaria were collected from Duxbury Bay, Massachusetts periodically throughout the year so that any seasonal effects in the secretion of mucosubstances could be observed. The clams were fixed for 48 hr in 10% formalin-2% calcium acetate containing 1% cetylpyridinium chloride, following which the mantle was removed for routine paraffin embedding and transverse sectioning at 5 j. 1 The author gratefully acknowledges the skillful technical assistance of Mrs. Darlene Harrington and Mr. Eric Pape. TRANS. AMER. MICROSC. Soc. 88(3): 420-425. 1969. This content downloaded from 207.46.13.21 on Tue, 27 Sep 2016 05:13:10 UTC All use subject to http://about.jstor.org/terms HILLMAN-MUCOSUBSTANCES IN MERCENARIA MANTLE 421 For general histological visualization, sections were stained in iron hematoxylin and eosin. Most of the reactions used for mucosubstances were discussed in detail in the work on the first marginal fold (Hillman, 1968). To distinguish between the various glycosaminoglycans and neutral mucosubstances the reactions described below were employed. Sections were subjected to 0.1% aqueous Azure A (AzA) at pH 1.0 and 4.0 (after Spicer, 1960). Strongly acid sulfated mucosaccharides stain metachromatically below pH 1.5. Sialomucins stain blue to purple above pH 3.0 and weakly acid mucosaccharides stain blue to purple below pH 4.5. Alcian blue (AB) at pH 1.0 and 2.5 (Spicer, 1960) was used to distinguish between strongly acid mucosubstances, which stain well at pH 1.0 but weakly or not at all at pH 2.5, and weakly acid and non-sulfated sialomucins, which exhibit alcianophilia at pH 2.5. Tissue sections were also exposed to varied concentrations of magnesium chloride in alcian blue solutions (Scott, 1960; Scott et al., 1964). Sulfated mucopolysaccharides stain selectively at MgCl2 concentrations of 0.2 M or greater. Chondroitin sulfates lose their alcianophilia at 0.5 M while heparin and other strongly acidic sulfomucins retain alcianophilia up to 1.0 M MgCl2. The periodic acid-Schiff (PAS) reaction with diastase controls was used to visualize polysaccharides and mucosaccharides containing hexoses or deoxyhexoses with vic-glycol groups. These groups color magenta to red with this reaction. For reasons which are not yet fully understood, this reaction does not usually take place with many sulfated acid mucopolysaccharides even though these materials contain oxidizable vic-glycol groups. For this reason, sections were subjected to an alcian blue (pH 2.5)/PAS reaction (after Mowry & Winkler, 1956; Spicer, 1960). At pH 2.5 neutral polysaccharides and mucosaccharides color magenta. Alcianophilic, periodate-unreactive acid mucosaccharides stain blue, while alcianophilic periodate-reactive mucosaccharides stain purple-blue. The alcian blue/PAS reaction was used following testicular hyaluronidase digestion and buffer-incubated controls (Lepnoi & Stoward, 1965). Testicular hyaluronidase hydrolyzes hyaluronic acid and chondroitin 4and 6-sulfates. Loss of alcianophilia indicates material containing these substances. The high iron diamine/alcian blue (HID/AB) reaction of Spicer (1965) was used to stain sulfated mucosaccharides, which color black to purple following this procedure. Tissues were also treated with the meta diamine/alcian blue (MD/AB) and periodic acid-meta diamine/alcian blue (PMD/AB) reactions (Spicer, 1965). Those substances with vic-hydroxyls adjacent to acid groups lose alcianophilia following periodate oxidation and exposure to meta diamine. Without the periodate oxidation meta diamine should have no effect and the same results should be achieved as with alcian blue alone. The alcian blue/safranin (AB/S) reaction of Spicer (1960) and the aldehyde fuchsin/alcian blue (AF/AB) technique of Spicer & Meyer (1960) were also employed. With alcian blue/safranin, strongly acidic, sulfated mucosaccharides should color red because of the greater affinity for safranin by these substances. Weakly acidic sulfated mucosubstances, sialmoucins, and hyaluronic acid color blue. Aldehyde fuchsin should react much the same as safranin, coloring the strongly acidic glycosaminoglycans purple to blue-purple. Other acidic mucosubstances will color blue. The periodic acid-phenylhydrazine-Schiff (PAPS) reaction (Spicer, 1961) was used to color periodate reactive acidic mucosaccharides red. A technique not used on sections for the previous study (Hillman, 1968), This content downloaded from 207.46.13.21 on Tue, 27 Sep 2016 05:13:10 UTC All use subject to http://about.jstor.org/terms 422 TRANS. AMER. MICROSC. SOC., VOL. 88, NO. 3, JULY 1969 FIG. 1. Mucus cells of the second fold in the mantle of M. mercenaria. The dark cells (AMP) produce a sulfated glycosaminoglycan while the lighter, granular cells (NMS) secrete a neutral mucosubstance. Stained with alcian blue/PAS. X 600. was methylation blockage of basophilia and subsequent saponification (Spicer, 1960). For this reaction sections were exposed for 4 hr in a solution of 0.4 ml concentrated HCI in 50 ml absolute methanol at 60 C. The tissues were then rinsed and stained with the alcian blue/PAS reaction at pH 2.5. Control serial sections of the tissues were stained with the AB/PAS reaction without prior methylation. Some of the methylated sections were then saponified in a 1% KOH solution in 70% ethanol. Methylation should esterify the carboxyl groups on glycosaminoglycans and hydrolytically remove any sulfate esters if they are present. The alcian blue should then have no effect. Saponification should deesterify the carboxyl groups and restore alcianophilia. Alcianophilia blocked by methanolytic removal of sulfate esters is not restored by saponification. RESULTS AND DISCUSSION The results of the histochemical reactions for mucosubstances (Table I) indicated that there are probably two types of mucous cells in the second marginal fold of the quahog mantle (Fig. 1). The first cell type produces a glycosaminoglycan of varying acidity depending upon factors which have not yet been determined. It was thought at first that there were a number of different glycosaminoglycan-producing cells in the second fold but the morphology of the cells is quite constant, so the variations in results will have to be explained in another way. The results of the histochemical reactions as shown in Table I are admittedly subjective and express the opinion of the author as to the average intensity of the reaction. A low rating is not necessarily due to a low color intensity, but This content downloaded from 207.46.13.21 on Tue, 27 Sep 2016 05:13:10 UTC All use subject to http://about.jstor.org/terms HILLMAN-MUCOSUBSTANCES IN MERCENARIA MANTLE 423 423
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