Ultrastructural methods for localizing complex carbohydrates

ULTRASTRUCTURAL METHODS FOR L O C A L I Z I N G COMPLEX CARBOHYDRATES S. S. Spicer, MD, and B. A. Schulte, PhD Carbohydrate-rich macromolecules in mammalian tissue fall into two main groups, glycoproteins and glycosaminoglycans. Although glycoproteins occur mainly in epithelial cells and glycosaminoglycans in connective tissue interstices, exceptions are known. Chemical constituents that are characteristic of glycoproteins and provide a basis for their selective demonstration cytochemicallyinclude the sulfate ester, presumably bound to an internal sugar, the carboxyi group of terminal sialic acid, and the vicinal glycol of hexose (galactose or mannose) and deoxyhexose (fucose). Characteristic chemical components of glycosaminoglycans that are detectable cytochemically include the sulfate ester, usually linked to an aminosugar, and the carboxyl group of glucuronic or iduronie acid. Individual sugars of glycoconjugates, particularly those in a terminal position, are also identifiable cytochemically. Cytochemical methods are available to detect specifically one or more of the moieties characteristic of glycoproteins or glycosaminoglycans at the electron microscopic level. The sulfate esters can be localized specifically by pre-embedment staining with tile high-iron diamine (HID) method. Dialy~,ed iron (DI) and cationized ferritin stain both carboxyl groups and sulfate esters when similarly applied before embedment. In addition, these cationic reagents can be utilized to stain at least some sulfated glycoconjugates in post-embedment procedures carried out on ultrathin sections. Ruthenium red in the fixative solution localizes glycoconjugate and possibly other constituents on the cell surface or in the extracellular space. The periodic a c i d thiocarbohydrazide-silver proteinate (PA-T-SP) method and the tannic acid-uranyl acetate sequence carried out after embedment on epoxy thin sections demonstrate sugar residues of glycoconjugates, extending light microscopic periodic acid-Schiff (PAS) staining to the ultrastructural level. Further specificity can be achieved by pre- or post-embedment staining with conjugates of horseradish peroxldase or ferritin to a lectin with affinity for a specific sugar in a complex carbohydrate and by enzymatic digestion with specific glycosidase to decrease or increase staining with the above mentioned methods. DI-positive, HID-negative sites can be interpreted as containing nonsulfated, carboxylated complex carbohydrate, and IIID-positive sites as containing sulfated mucosubstance. DI- or IIID-positive sites lacking PA-T-SP reactivity, as a rule, consist of glycosaminoglycans, but infrequently contain periodate-negative glycoprotein. On the other hand, PA-T-SP staining and DI or HID non-reactivity evidence the presence of neutral glycoprotein, whereas PA-T-SP positivity plus basophilia indicates acidic glycoprotein. Examples of applications o f these methods are presented. The applications to normal tissue provide a basis for comparison with diseased specimens and point to the potential o f carbohydrate histochemistry for investigating and diagnosing pathologic change. H u m Pathol 13:343-354, 1982.

C a r b o h y d r a t e - r i c h m a c r o m o l e c u l e s - - r e f e r r e d to h e r e as c o m p l e x c a r b o h y d r a t e s , i n u c o s u b s t a n c e s o r g l y c o c o n j n g a t e s - - a r e f o u n d in b o t h i n t r a - a n d e x Received from the Departnlent of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29-125. Supported in part by NIl! grants AM-10956, AM-11028 and IIL19160. Address correspondence and reprint requests to Dr. Spiccr.

t r a c e l l u l a r sites in a w i d e w l r i e t y o f m a m m a l i a n tissues. T h e s e s u b s t a n c e s h a v e b e e n i m p l i c a t e d in t h e pathogenesis of respiratory diseases, including c h r o n i c b r o n c h i t i s , a s t h m a , a n d cystic fibrosis; in dig e s t i v e t r a c t d i s e a s e s s u c h as p e p t i c u l c e r ; a n d in m u c u s - p r o d t ~ c i n g c a r c i n o m a s a n d o t h e r n e o p l a s m s ~,z. T h e i n t e r e s t o f t h e p a t h o l o g i s t in u l t r a s t r u c t u r a l c a r b o h y d r a t e c y t o c h e m i s t r y p r e s u m a b l y c e n t e r s o n tire p o t e n t i a l o f this a p p r o a c h to p r o v i d e i n f o r m a t i o n o f v a l u e f o r c l a s s i f y i n g p a t h o l o g i c lesions a n d e x p l a i n i n g their genesis. The precise nature of the qualitative or q u a n t i t a t i v e c h a n g e s in c a r b o h y d r a t e - r i c h s u b s t a n c e s in t h e p a t h o l o g i c r e s p o n s e s has b e e n d i f f i c u l t to asc e r t a i n , p r i m a r i l y b e c a u s e o f lack o f a d e q u a t e c y t o chemical methodology. The methods for demonstrati n g g l y c o c o n j u g a t e s in e l e c t r o n m i c r o g r a p h s h a v e only recently been developed and are undergoing f u r t h e r m o d i l i c a t i o n ; t h e y h a v e n o t as y e t b e e n e x t e n s i v e l y a p p l i e d to d i s e a s e d tissues, q ' h i s r e v i e w c o n cerns mainly the currently available procedures for demonstration and differentiation of glycoconjugates in situ. T h e use o f t h e s e m e t h o d s to classify c a r b o h y d r a t e - r i c h s u b s t a n c e s h i s t o c h e m i c a l l y a n d to investig a t e t h e i r d i s t r i b u t i o n s in a n u m b e r o f n o r m a l tissue sites is d e s c r i b e d . A p p l i c a t i o n o f t h e m e t h o d s to tiss u e a l t e r e d b y d i s e a s e is i n c l u d e d to a l i m i t e d e x t e n t f o r t h e p u r p o s e o f s t r e s s i n g t h e p o t e n t i a l o f this a p p r o a c h f o r p a t h o l o g i c d i a g n o s i s . It can be r e a s o n a b l y a n t i c i p a t e d t h a t f l t r t h e r r e f i n e m e n t in t h e s p e c i f i c i t y of the cytochemical techniques for differentiating complex carbohydrates and elucidating their struct u r e s in situ will r e a c h a p o i n t w h e r e s u c h m e t h o d s can dentonstrate specific differences between normal a n d p a t h o l o g i c s p e c i m e n s as, f o r e x a m p l e , a d i f f e r e n c e in t h e c o m p o s i t i o n s o f t h e g l y c o c a l y x o f n e o plastic cells c o m p a r e d w i t h t h e i r cells o f o r i g i n .

CLASSIFICATION OF COMPLEX CARBOHYDRATES Carbohydrate-containing macromolecules show c o n s i d e r a b l e s t r u c t u r a l d i v e r s i t y . M a n y c o m p l e x systems of biochemical and histochemical nomenclature h a v e b e e n p r o p o s e d fox" t h e i r c l a s s i f i c a t i o n ? ,~ F o r t h e p u r p o s e o f a c c o m m o d a t i n g b i o c h e m i c a l l y a n t i hist o c h e m i c a l l y d e f i n e d c a t e g o r i e s , a s i m p l e classification s c h e m e is o u t l i n e d : COMPLEX CARBOIIYDRATES

I. l'olysaccharides A. ltomopolysaccharides (glycoge,0 B. Heteropolysaccharides (glycosaminoglycans)

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HUMAN PATHOLOGY--VOLUME 13, NUMBER 4 April 1982 II. Gl}'coconjugates A. Glycoproteins 1. Neutral 2. Acidic a. Sialylated b. Sulfated c. Sialylated and stdfated B. Proteoglycans 1. Containing uronic acid 2. Containing uronic acid anti sulfate C. Glycolipids 1. Ceramide monosaccharide (cerebroside) a. Neutral (galactocerebroside) b. Acidic, with carboxyls or carboxyls plus sulfates (phrenosin) 2. Ceramkle oligosaccharides a. Neutral (globoside) b. Acidic (ganglioside) The term complex carbohydrate is considered to encompass all carbohydrate-rich substances, and these m a c r 0 m o l e c u l e s are s u b d i v i d e d into two categories: the polysaccharides, which consist of only carbohydrate, and the glycoconjugates, which contain sugars linked to a non-carbohydrate moiety. In the latter category, the glycoproteins consist o f oligosaccharide side chains covalently bound to a protein backbone. Glycoconjugates o f a second class, the proteoglycans, contain glycosaminoglycans composed of repeating disaccharide units of a uronic acid and an acetylated amino sugar covalently linked to a protein backbone. Glycolipids consisting of a ceramide and one Or several sugars comprise a third category of glycoconjugate, which is theoretically demonstrable in appropriately processed tissue sections, but, presumably, is extracted during tissue processing and not detectable by post-embedment methods. HISTOCHEMICAL

TECHNIQUE

Biochemical and histochemical mefllods have provided insight into the structures o f carbohydratecontaining substances, but both approaches suffer limitations. Biochemical studies have been hampered by the inability to isolate these tissue moieties uncontaminated by o t h e r c o m p o n e n t s ? Additional problems are encountered in the subsequent purification of these substances because of their high" molecular weights and considerable polydispersity with respect to size and component sugars, s Histochemical methods, especially at the electron microscope level, have an advantage over biochemical techniques, in that they allow a precise localization of individual glycoconjugates to specific intra- and extracellular sites. 6 A major limitation of histochemical methods, however, Ires been a lack of sufficient specificity to allow the requisite detailed structural characterization o f the carbohydrate constituents. Until recently, histochemical methods served mainly to detect either the acid groups or the vicinal glycols o f sugars, both o f which occur selectively in c a r b o h y d r a t e moieties in sections of mammalian tissues. Procedures currently available, however, offer a prospect of

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achieving more precise information abont glycoconjugates in situ. T h e processing and staining of tissues tbr characterizing complex carbohydrates cytochemically are described in the following sections. FIXATION AND EMBEDMENT

Results obtainable with the available methods depend importantly on preparatory steps preceding staining. As the initial step, fixation markedly influences the reactivity o f complex carbohydrates. At the electron microscopic level glutaraldehyde provides optimal morphologic preservation but may impair cytochemical reactivity through impeding penetration of reagent or effecting masking o f reactive groups. A formaldehyde fixation of variable duration offers a possible compromise between preservation of structures and preservation of cytochemical reactivity. Aside from fixation, staining results vary according to whether the reaction is carried out before or after embedding. Pre-embedment staining procedures, the most widely used to date, involve applying to small peices o f finely minced tissue or cryostat sections the histochemical methods to be described. Tile tissues may be fixed or unfixed but, in general, are variably fixed in aldehyde solution prior to staining, postosmication, dehydration, and embedment in an epoxy resin. Pre-embedment histochemical staining procedures have proven useful for the ultrastructural localization of complex carbohydrates found in extracellular sites or on cell surfaces. A problem with the staining o f i n t r a c e l l u l a r locations by preembedment tecbniques arises through uncertainty wbether the staining reagents penetrate into the cell and to the reactive site. The inability to compare the staining results obtained with several histochemical methods directly on the same cell or intracelhflar organelle is a further disadvantage of pre-embedment procedures. In p o s t - e m b e d m e n t staining procedures, the various lfistochemical reagents are applied directly on thin sections cut from blocks o f tissue tlmt have been fixed in aldehyde solution and embedded in epoxy resins or a variety o f less cross-linked embedding media without postfixation by osmium tetroxide. Poste m b e d m e n t staining techniques c i r c u m v e n t the problems of diffusion o f staining reagents into the cell. They offer the distinct advantage that several staining reactions can be compared directly on adjacent thin sections, providing different information about a single structure. Knowledge could be acquired from such staining of adjacent thin sections about the nature and heterogeneity of secretory products within a single cytoplasmic storage granule, the site of biosynthetic steps involved in elaboration o f a secretory product, the relation of secretory granule material to glycocalyx components, and the biosynthesis and transport of glycoconjugate to the cell's glycocalyx. Cytochemical staining of uhratbin sections suf-

LOCALIZATION OF COMPI.EX CARBOIIYDRATES--SPtcER ANn SCHUL'I'E T A B L E 1.

ULTRASTRUCTURAL HISTOCHEMICAL METIIODS FOR LOCALIZING A N D CLASSIFYING COMPLEX C A R B O H Y D R A T E S * Reactive

Method

Dialyzed ironH's''s5 Cationized ferritin TM ttigh-iron diamine with or without thiocarbohydrazide-silver proteinate nat Periodic a c i d - t h i o c a r b o h y d r a z i d e - s i l v e r proteinate (PA-T-SPf "=~ Radioautography with ~SO'~zt'm

Component Carboxyls Sulfates Carboxyls Sulfates Sulfates Vic glycols in hexoses

Radioautography with aH suga& a-~

Tissue Motet)' Localized Intra- and extracelhdar glycosaminoglycans and acidic glycoconjugates Intra- and extracellular glycosaminnglycans and acidic glycoconjt, gates lntra- and extracellular sulfated glycosaminoglycans and glycoconjugates Intra- and extracellular glycoproteinst Sulfated complex carhohydrates with sufficient turnover Complex carLvohydrates with sufficient turnover

* Tissue macromoles reactive with the PA-"I'-SP method and lacking affinity for the cationic reagents (at both light and electron microscopic levels) and radioautographic evidence of radiosulfate incorporation can be classified as neutral gl)'coproteins. Those reacting with both the PA-T-SP method and cationic reagents are interpreted as acidic glycoproteins. Those lacking PA-T-SP reactivity but showing basophilia are regarded as glycosanfinoglycans or periodate-unreactive glycoproteins. t Glycolipids containing vicinal glycols could also be localized by this method if present.

fers, however, from the different problem o f penetration of the reagent into the embedding medium. Epoxy resins are penetrated poorly by most staining reagents, such as the dialyzed iron and iron diamine, stressed here, but are permeable to some reagents, for example, those of the periodic a c i d - t h i o c a r bohydrazide-silver proteinate sequence. Nevertheless,.colloidal iron, 7-~~ colloidal thorium, st~ and cationized ferritin ~~ have all been used not only as pre-embedment stains but also by application directly on thin sections cut from specimens embedded in Epon and/or methacrylate resin. The results with post-embedment staining have been variable but, in general, appear better on methacrylate than on epoxy sections. 7"st~ In 'addition to the penetration problems, there are also indicatitms that these resins or agents promoting their polymerization react with the stainable entities in tissue complex carbohydrates, thus reducing or blocking their demonstration cytochemically (unpublished observations). These problems call for investigation of the possibility of increasing the penetrability of epoxy resins tostaining reagents or of obtaining other less impenetrable embedding media. PROCEDURES FOR LOCALIZING AND DIFFERENTIATING COMPLEX CARBOHYDRATES

Methods for demonstrating tissue glycoconjugates in situ can be divided into five main categories. These methods were developed in large part from extension of light microscopic techniques to the electron microscopic level. T h e first group identifies axtionic groups characteristic o f acidic glycoconjugates by their binding o f electron-opaque cationic reagents. 7-ts Available methods serve to differentiate the entities containing only carboxyls, these posseSsing only sulfates, and those with both carboxyls and sulfates (Table 1). ~'-~3 It should be pointed out that, although the methods shown in Table 1 have been found from the experience gained in this laboratory to be applicable in a variety of sites, a number of alternative methods have been employed, including colloidal iron, 7 colloidal thorium, s ruthenium red, ~4

p h o s p h o t u n g s t i c acid, Is attd metal salts, t7 Like dialyzed iron, colloidal iron, colloidal thorium, and cationized ferritin TM are thought to react with both cartx)xyl and sulfate groups. The staining specificities of ruthenium red, phosphotungstic acid, and the cetylpyridinium chloride-ferric thiocyanate method have not been well established. A second type of procedure depends on visualizing the vicinal glycols that occur selectively in hexoses of glycoproteins (Table 1). In this approach a silver preparation reduced by aldehydes replaces tile Schiff reagent o f the light microscopic periodic a c i d - S c h i f f (PAS). method to visualize periodatee n g e n d e r e d aldehydes. T h e m e t h e n a m i n e - s i l v e r solution initially employed by Rambourg et al.19 has been modified, by Thi6ry, 2~ who substituted thiocarbohydrazide-silver proteinate to demonstrate the engendered aldehydes. High-resohttion r a d i o a u t o g r a p h y e m p l o y i n g either zsSO4= or 3H sugars prgvides a third means of localizing complex carbohydrates and obtaining information a b o u t . t h e i r compositions (Table 1). Ultrastrnctural radioautography has demonstrated the incorporation of 3sSO4= into both glycoproteins and glycosaminoglycans. 21'2z The incorporation o f aH sugars as revealed by uitrastructural radioautograph.y has served to localize glycoproteins and glycosaminoglycans3 i'm T h e incorporation o f 31-I sugars as revealed by ultrastructural radioautography has served to localize glycoproteins and glycosaminoglycans in both intra- and extracellular sites, m-25 In addition, 3H N-acetylmannosamine, a precursor o f sialic acid residues, has been used to label both intracellular and cell membrane-associated sialoglycoconjugates. 2s Ultrastructural radioautographic techniques have also provided information concerning the intracellular sites involved in the synthesis and secretion of the carbohydrate-containing substances. A fourth a p p r o a c h utilizes lectins that have binding affinities for specific sugars to detect these residues in tissue glycoconjugates. Many naturally

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IIUMAN PATIIOI.OGY--VOI.UME 13, NUMBER4 April 1982 TABLE 2.

INVESTIGATIONS THAT IIAVE UTILIZED LECTINS FOR ULTRASTRUCTURAL LOCALIZATION OF SPECIFIC SUGAR RESIDUES IN COMPLEX CARBOIIYDRATES Lectin Stain

Concouavalin A - h o r s e r a d i s h peroxidase seq u e n c e o r c o n c o n a v a l i n A - f e r r i t i n conjugate-~6-37 Limulus polyphemus lectln conjugated !o ferritin ~

Lotus tetragonolobus lectin conjugated to ferritin 3"~ Wheat germ lectin conjugated to horseradish pcroxidase or ferritiu z'J'z;'s3 Ricinus comrnunis lectin conjugated to horseradish peroxidase of ferritin *9's*m's* Peanut lectin conjugated to horseradish peroxidase or ferritin zSm

Reactive Sugar(s) M'annose

Glucose N-acetylglucosamine (?) N-acetylneuranfinic acidf Ftlcose N-acetylglncosamine T e r m i n a l galactose or Nacetylgalactosamine Terminal galactose

Tissue Moiety Densified by Stain* Intra- and extracclhdar glycoprotein containing mannnse, gh,cose or (?) N-acetylglucosamine Cell surface glycoproteins containing N-acetylneuraminic acid Cell surface glycSoproteins containing fucose Cell surface glycoproteins containing N-acetylglucosamine Cell surface glycoproteins containing terminal galactose or N-acetylgalactosamine Intra- and extracellular glycoproteins with ternfinal galactose residues

* Staining could conceivably be attributed also to glycolipid when present, as would be the case for pre-embedment staining. "t"Sialic acid invariably occupies a terminal position in oligosaccharide clmins.

occurring compounds tlmt lmve specific binding affinities for one or several sugars have been isolated and purified from both plant and animal sources. These lectins or agglutinins, when coupled to an enzyme or electron-opaque marker, provide a means of localizing specific sugar residues in complex carbohydrates at the uhrastructural level. As illustrated in Table 2, lecdns with various sugar-binding specificities have been employed in ultrastructural carbohydrate histochemistry. Concanavalin A was the first agglutinin utilized for uhrastructural histochemical studies3 G Concanavalin A has since been used frequently, either in a sequence of concanavalin A followed by horseradish peroxidase, which takes advantage o f the lectin's affinity for mannose or Nacetylglucosamine in Itorseradish peroxidase, 27-3~ or as a conjugate of concanavalin A to ferritin? '-3~ In addition, concanavalin A binds directly to g l u c o s e

residues in iron dextran and has been employed in conjunction with this electron-opaque m a r k e r ? 6"3~ The other lectins employed histochemically to date do not bind to carbohydrate moieties in horseradish peroxidase and cannot be used like concanavalin A in sequence with horseradish peroxidase, but rather must be utilized as a conjugate to either this enzyme29'~s or ferritin? ~'3~ T h e ultrastructural localization of specific sugar residues has been largely restricted to complex carb o h y d r a t e s associated with the external face o f plasma membranes (Table 2). This can be explained on tire basis that most of the lectin-staining methods are performed prior to embedment, and that the lectin does not penetrate the cell. Concanavalin A has been used, however, for post-embedment staining of tttin sections of glycol methacr)'late ~~ and E p o n araldite-embedded tissuesY Concanavalin A and peanut lectin conjugated to horseradish peroxidase Imve been utilized in this laboratory in both pre3r.38 and post-embedment (unpublished observations) staining at the ultra-structural level. Preliminary and somewhat v,~riable results to date have shown that the binding of these lectins can be demonstrated in intracellular sites in thin sections of Epon 812embedded tissues, but only after prior treatment of

346

sections with hydrogen peroxide or alcoholic sodium hydroxide. An a d d i t i o n a l c a t e g o r y o f p r o c e d u r e s for characterizing glycoconjugates cytochemically depends on digestion with glycosidases o f known specificity to impair or e n h a n c e staining by the aforementioned methods.(Yable 3). Loss or reduction of staining for cationic reagents after treatment with enzymes provides evidence as to the locations o f specific complex carbohydrates in tissues. Testicular llyaluronidase eliminates basophilia attributable to h y a l u r o n i c acid or ~:hondroitin-4- or 6-sulfate, whereas hyaluronidases o f bacterial or marine origin ablate basoplfilia of hyaluronic acid selectively.H'2-~m'4~ Enzymes that lmve broad specificities, such as chondroitinase ABC, serve in localizing the chondroitin sulfates and dermatan sulthte? 9,4~ I ieparitinase has been used to eliminate basoplfilia of heparan sulfate.4~ Loss of affinity for dialyzed iron or cationized ferritin after treatment of the tissue with neuraminidase, which cleaves the invariably terminal sialic acid residues, serves to localize sialoglycoconjugates.~s,ag-42 In addition to elimination o f affinity for cationic reagents, sialidase digestion has been shown to expose new binding sites for both Ricinus communis lectin and peanut lectin on erythrocyte plasmalemma, 43 thus demonstrating penuhimate galactose residues exposed to the lectin by the sialidase treatment. HISTOCHEMICAL IDENTIFICATION CLASSES O F C O M P L E X CARBOHYDRATES

OF

Identifying lfistochemically the classes o f complex carbohydrates outlined above depends on staining methods that react selectively with the different constituents characteristic o f the various substances. The glycosaminoglycans always contain the acidic carboxyl g r o u p o f u r o n i c acid and, except for hyaluronic acid, also possess sulfate esters and show basophilia accordingly. T h e glycosaminoglycans in cartilage occur covalently linked to protein, but hecause of failure to detect the protein portion his-

LOCALIZATION OF COMPLEX CARBOItYDRATES--SPICER AND SCllULTE TABLE 3. STUDIES EMPLOYING DIGESTION W I T H SPECIFIC ENZYMES TO I D E N T I F Y COMPLEX C A R B O H Y D R A T E S U L T R A S T R U C T U R A L L Y Enzyme-staining Sequence

Streptomycin hyaluronidase-cationic reagent3s.4~ Chondroitinase ABC--cationicreagent~'~~ }leparitinase-cationic reagent3'J Neuraminidase-cationic reagent~~ Neuraminidase--peanut lectin or Ricinus communis agglutinin conjugated to fer-

Substrate Specificity

Tissue Moiety Identified by Altered Staining Reaction*

Hyaluronicacid

Extracellular hyaluronicacid

Chondroitin 4- and 6-sulfate Dermatan sulfate ttyaluronic acid Most glycosaminoglycansincluding heparan sulfate N-acetylneuraminicacid N-acetylneuraminicacid

Extracelhdar proteoglycansor glycosaminoglycans Extracellular heparan sulfate Intra- and extracellular sialoglycoconjugate Penuhimate galactoseresidues in cell surfaceassociated glycoconjugates

ritin 43

* Stainingwithcationicreagents is abolishedafter enzymedigestionsbut stainingwith peanut lectin- peroxidaseconjugateis imparted after neuraminidasedigestionwhen penultimategalactose residues become terminal. tochemically, uncertainty exists as to w h e t h e r all glycosaminoglycans preserved in tissue sections comprise part of a proteoglycan. Some glycoproteins are neutral, but the majority possess acidic groups. The acidic glycoproteins appear to include some with sialic acid alone, others with sulfate alone, and a number with both types of acidic groups. Glycoproteins also contain a number of hexoses that are not present in glycosaminoglycans and occur in internal or terminal position in the oligosaccharide side chains. A constituent of glycoproteins of value for distinguishing them from proteoglycans is the periodate-reactive vicinal glycol present exclusively in hexoses of the glycoprotein's oligosacclmride side chains. Applying a battery of the uhrastructural procedures to tile analysis of the above-outlined complex carbohydrates in the various histologic sites enables an assessment of the class o f carbohydrate-rich substances present. Substances that have dialyzed iron H or high iron diamine t2"~s affinity but lack periodate reactivity as demonstrated by the periodic a c i d thiocarbohydrazide-silver proteinate method can g e n e r a l l y be c o n s i d e r e d g l y c o s a m i n o g l y c a n s or proteoglycans, although infi'equent sialoproteins lack periodic acid-thiocarbohydrazide-silver proteinate staining. On the other hand, glycoconjugates showing dialyzed iron or high-iron diamine reactivity as well as periodic acid-thiocarbohydrazide-silver proteinate reactivity can be interpreted as acidic glycoproteins or possibly glycolipids. The distinction between acidic complex carbohydrates with carboxyl groups and those containing sulfate esters can be made on the basis of the affinity of sulfates for both dialyzed iron and high-iron diamine as compared with the affinity o f carboxyls for dialyzed iron but not high-iron diamine. Asialoglycoproteins containing sulfate esters evidence high-iron diamine affinity and binding of a lectin with specificity for a terminal neutral sugar. Sulfated, sialylated glycoproteins are detected by their high-iron diamine affinity and lectin binding gained after sialidase digestion. Substances lacking tile capacity to bind cationic reagents but possessing periodic acid-thiocarbohydrazide-silver proteinate reactivity can be regarded as neutral glycoproteins. Components reacting strongly with lectins that bind

specifically to terminal hexoses or N-acetylated aminosugars fall into the glycoprotein class. EXAMPLES OF APPLICATION OF ULTRASTRUCTURAL METItODS FOR DEMONSTRATING COMPLEX CARBOHYDRATES

Biochemical events involved in synthesis of complex c a r b o h y d r a t e s , p a r t i c u l a r l y the activity o f glycosyl transferases, occur mainly at tile membranes of the rough endoplasmic reticuhtm and the Golgi complex. This location o f complex carbohydrate biosynthesis carries the implication t h a t macromolecules rich in carbohydrates are essentially components produced for export to the cell surface or extracellular matrix. T h e expected location o f c a r b o h y d r a t e - c o n t a i n i n g macromolecules would therefore include membranes or cisternae of the granular reticulum and the Golgi complex, tile matrix of secretory granules, tile plasmalemma, and the extracellular space. This generalization holds true in our experience except for the acidic complex carbohydrate evidenced by dialyzed iron staining in the outer intermembrane space and the cristae o f mitochondria. 4~ For lack of knowledge concerning the nature or biosynthetic source of tile mitochondrial glycoconjugate, it is not considered further in tiffs review. "File cytosol of all cells examined thus far fails to disclose a content o f glycoconjugate with any ultrastructural cytochemical method, in agreement with tile general consensus that free polysomes possess no carbohydrate biosynthetic activity. The following sections undertake to illustrate applications o f various cytochemical methods for in situ localization and characterization of the complex carbohydrate-rich macromolecules in various sites in normal or pathologic specimens, including the extracellular matrix, cell surface, secretory granules, and Golgi complex. GLYCOCONJ UGATES OF EXTRA CELL ULAR TISSUE SPACE

Stairting aldehyde-fixed specimens with dialyzed iron prior to embedntertt has disclosed a b u n d a n t

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HUMAN I'ATIIOI.OGY--VOI,UME 13, NUMBER 4 April 1982

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acidic glycoconjugate with s'trong affinity for tile cationic reagent (Fig: 1). In the lainina propria o f mouse colon, tl~e stained materifll occupied space between collagen bundles and appeared concentrated on the collagen bundle surface, possibly as a result o f redistribution during fixhtion. ~,~ Although tumor matrices frequently show increased acidic mucosubstance in tile intercellular stroma by light microscopy, little knowledge exists concerning the nature, amount,, and distribution of complex carbohydrates in the stroma o f neoplastic or other pathologic lesions.at the ultrastructural level.

348

F i g u r e 1 (left). T h e lamina lucida ( a r r o w ) a n d l a m i n a diffusa (arrowhead) in b a s a l l a m i n a o f a rat p a n c r e a t i c d u c t cell s h o w densification d e m o n s t r a t i v e o f sulfated gl)'coconj u g a t e . Notice also the p e r i o d i c staining o f collagen fibers below t h e fibroblast (F). l l i g h - i r o n d i a m i n e stain, x 4 0 , 0 0 0 .

More recently, pre-embeclment staining with tile high-iron diamine method ~G'ar and post-embedntent staining with the periodic a c i d - t h i o c a r b o h y d r a zide-silver proteinate procedure zz have disclosed internaittent reactivity in the individual collagen fibers within a bundle. T h e latter stainiilg method shows periodicity reminiscent of that of bands seen in electron micrographs o f collagen fibers processed fill" routine morpholt~gic examination. These cytochemical resuhs testify to the presence of sulfated glycoprotein in collagen fibers. Cartilage matrix disclosed strong high-iron

LOCALIZATION OF COMPLEX CARBOHYDRATES--SwCER ANO SCHULTE diamine reactivity,22m which, because of its lability to testicular hyaluronidase, can be attributed to the chondroitin-4- or 6-sulfate known to be abundantly present in this site. The population of larger particles visualized by high-iron d i a m i n e intensified with thiocarbohydrazide-silver proteinate differs qualitatively from the smaller particles in being digestible with the hyaluronidase, suggesting that the smaller particles reflect the distribution in the cartilage matrix o f keratan sulfate. 4s Chondrocytes also have high-iron diamine affinity o f varying intensity in cytoplasmic vesicles, which appear to transport newly synthesized proteoglycan to the extracellular matrixY 2

CELL SURFACE COMPLEX CARBOHYDRATES

Among the early observations made on cells by electron microscopy was the recognition of a fuzzy coat on the cell surface. The term glycocalyx was proposed for this material, which was thought to contain abundant carbohydrate and to coat the surfaces of most cells.49 Evidence for glycocalyces on both connective tissue and epithelial cells has been s.upported by subsequent experience with cytochemical methods for visualizing glycoconjugates at the uhrastructural level. Connective tissue cells consistently show a uniform coat of carbohydrate-rich material on their external surfaces. This glycocalyx however, varies, qnalitatively and quantitatively on d i f f e r e n t cell types. Erythrocytes, for example, have a surface layer with sialidase-labile affinity for colloidal iron. 42 T h e abundance of the anionic sialic acid groups that account for the affinity for the cationic colloidal iron can be quantitated with a cationized preparation of ferritin3 ~ The relationship of this thin layer of surface staining to the plasmaleinma of the erythrocyte poses a question. Since glycophorin, a protein rich in carbohydrate, accounts for most of the protein in the red blood cell plasmalemma, it is assumed that the cationic reagents bind to the sialic acids terminating oligosaccharide side chains of glycophorin. Presumably, the iron binds to those acid groups, which are abundant in the hydrophilic N terminus of glycophorin extending from the cell surface? ~ T h e glycocalyx in this instance apparently represents an external extension of a protein embedded at its laydrophoblc end as an integral membrane protein. S t a i n i n g with the c o n c a n a v a l i n A - h o r s e r a d i s h peroxidase sequence further testifies to glycoprotein on the erythrocyte surfaceY Possibly this reactivity evidences m a n n o s e in the o l i g o s a c c h a r i d e s o f glycophorin. The prevalences of anionic groups as j u d g e d by dialyzed iron staining vary among different connective tissue cells. For example, in rat peritoneal wash cells, the intensity of staining of the glycocalyx with dialyzed iron increases in the order macrophage, mast cell, lymphocyte (Fig. 2). Whether the acidic moiety on these three cell surfaces is accounted for by

sialic acid as on tile erythrocyte has not been determined. It appears that the degree of negative charge on the surface of the cell correlates inversely with the biologic property o f adhesiveness of the cells to glass or other surfaces. Thus, macrophages that are readily separated from other cells in vitro on tile basis of their special capacity to adhere to a glass surface evidence the lowest concentration o f negative surface charges among these three cell types. The polyanion is not simply siaIoprotein on all cell surfaces, as the occurrence o f heparan sulfate has been documented on hepatoma ascites cells. 5~ In these cells, biochemical evidence for heparan sulfate as the only glycosaminoglycan present concurs with the high-iron diamine staining observed at the cell surface but not intracellularly. This occurrence of a glycosaminoglycan on the cell surface appears to distinguish the hepatoma t u m o r cells from tile cell of derivation, as hepatocytes in vivo at least lack such a cell coat. Whether lieparan sulfate exists as part of a proteoglycan and represents an external extension of an integral membrane protein of these cells remains a question. Tile failure o f heparin to displace the heparan sulfate, however, indicates the glycosaminoglycan itself is not bound to the plasmalemma by ionic forces3 2 Staining with Ricinus communis lectin conjugated to ferritin has provided evidence for a glycoprotein possessing terminal galactose residues on the surface of murine lymphoma cells. 3a A similar glycoprotein containing terminal galactose residues appears also to be present on normal rat lymphocytes, as these cells have been found to stain with Ricinus communis lectin conjugated to horseradish peroxidase? 3 Since the Ricin lectin is thought to bind selectively to terminal galactosefl4 its staining o f the lymphocyte surface in the rat indicates that at least one component of the glycocalyx is a glycoprotein with terminal galactose and no sialic acid, as the latter invariably occurs in a terminal location. Unless this glycoconjugate contains internal sulfate esters, it would appear to constitute an example o f a neutral glycoprotein in the rat lymphocyte glycocalyx. On the other hand, the.dialyzed iron staining on the surface o f rat peritoneal wash lymphocytes (Fig. 2) would attest to a second stainable component. Presumably either a heterogeneity of surface complex carbohydrates or a diversity o f oligosaccharide side chains in a single glycoprotein explainsthe contradictory basophilia and ricin lectin affinities observed on the surface of rat lymphocytes. Epithelial cells, unlike connective tissue cells, are polarized and accordingly capable of conducting net secretion or resorption o f ions and metabolites and unidirectional exocytosis or endocytosis o f macromolecules. Not surprisingly, therefore, the epithelial cell exhibits regional-variability in its glycocalyx, showing differences in the apical compared with the basolateral surface. Surface epithelia generally reveal. considerably stronger staining for complex carbohydrates on the apical than o n the basolatera! plasmalemma, but exceptions to this generalization are known, as for example in the gastric parietal cell.

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T h e pancreatic interlobular ducts have a much heavier glycocalyx on their apical c o m p a r e d with their basolateral plasmalemma (Fig. 3). In this instance, the lability o f the glycocalyx basophilia to sialidase digestion identifies the fixed surface anion as sialic acid in both the apical and basolateral plasmalemma. Notably, however, dialyzed iron staining in the lamina lucida and lamina diffilsa o f the basement m e m b r a n e is unaffected by prior sialidase treatment (Fig. 4). Glycoconjugates on epithelial cell surfaces vary qualitatively as well as quantitatively, and on some complex epithelial surfaces consisting o f several cell types, the glycocalyx differs qualitatively between neighboring cells o f d i f f e r e n t types. In the gastric glands o f the rat and guinea pig, for example, the isthmus cells contain a heavy luminal coat of an unusual, highly sulfated, asialoglycoprotein. This comp o n e n t is characterized by affinities for high-iron diamine and dialyzed iron, by reactivity with the periodic a c i d - t h i o c a r b o h y d r a z i d e - s i l v e r p r o t e i n a t e procedure, and by nonsusceptibility to sialidase digestion. 54'55 T h e l u m i n a l surface o f n e i g h b o r i n g parietal "cells, however, is unlike that o f . t h e isthmus cells in lacking affinity for dialyzed iron or high-iron diamine, but shows moderate periodic acid-tlfiocarb o h y d r a z i d e - s i l v e r proteinate reactivity. A neutral glycoprotein, as d e m o n s t r a t e d on the luminal surface o f the parietal cells, appears adapted to ttte secretion o f protons that occurs at this cell surface, whereas the unusual highly sulfated glycoprotein o f the isthmus cells could be viewed as an adaptation to the highly acidic e n v i r o n m e n t c r e a t e d by the n e i g h b o r i n g 3 5 0

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parietal cells a n d prevailing at the narrow isthmus o f the gastric glands. T h e collecting tubule in the guinea pig renal papilla exemplifies a n o t h e r epithelium with a heterog e n e o u s cell p o p u l a t i o n where n e i g h b o r i n g cells possess qualitatively different surface coats. 56 In this epithelium, the principal cells resemble the gastric isthmus cells in containing a hea~T, apparently periodic coat of sulfated complex carbohydrate with strong highiron diamine (Fig. 5) and dialyzed iron affinities. This surface material differs from that on the isthmus cell, however, since it is labile to digestion with testicular h y a l u r o n i d a s e a n d , thus, r e p r e s e n t s an u n u s u a l example of a glycosaminoglycan, specifically chondroitin 4- or 6-sulfate, on an epithelial cell surface, s6 Furthermore, the surface coat on the principal cell lacks periodic a c i d - t h i o c a r b o h y d r a z i d e - s i l v e r proteinate reactivity (Fig. 6), as expected for a site occupied by a glycosaminoglycan and no glycoprotein. T h e glycocalyx of the collecting tubule principal cell differs in a n o t h e r way from that of the gastric isthmus cell and, indeed most other epithelial cells, in that the basolateral surface stains nearly as strongly as the apical surface with the cationic reagents (Fig. 5). Apparently, Golgi-derived vesicles transport the newly synthesized chondroitin sulfate to the basal surface as well as the apical surface o f these collecting tubule light cells. Basolateral transport and secretion o f chondroitin sulfate by collecting tubule ceils could explain at least a part o f the source o f the glycosaminoglycan known to occur in abundance in the stroma of the renal papilla. T h e apical p l a s m a l e m m a o f the n e i g h b o r i n g

LOCALIZATION OF COMPLEX CARBOtlYDRATES--SPicER AN[)SCHUL'rE

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intercalated cells differs strikingly from that of the principal cells of the collecting tubule in lacking affinity for dialyzed iron or high-iron diamine and showing moderate periodic a c i d - thiocarbohydrazide- silver proteinate reactivity~6 (Figs. 5 and 6). The intercalated cell, like tlte gastric parietal cell, appears to contain a luminal glycocalyx of neutral glycoprotein. This cell also contains abtmdant carbonic anhydrase, 56 and is thought to function in regtdating urinary pH. The presence of a neutral glycoprotein on the luminal surface, therefore, constitutes a common property of two cells rich in carbonic anhydrase and exporting protons to the lumen, namely the gastric parietal cell 54'55 and the renal collecting tubule intercalated cell3 6 The prescnce of a highly sulfated glycoconjugate on the luminal surface is a conamon property of cells neighboring proton-sccrcting cells, as exemplified by the gastric isthmus cell and renal collccting tubule intercalated ceil. Epithelial cells characteristically produce a mnltilayercd basal lamina tlmt underlies the basal plasmalemma anti further evidences the polarization of tlte epithelial cells. The basal lamina materials under varions types of epithelial cells differ in ways that have not been fully characterized biochemically or cytochemically, but possibly relate to transport or

other biologic activities of the epithelial cell. T h e basal lamina underlying most epithelial cells, as well as endothelial cells and perhaps also smooth muscle cells, consistently has a s u l f a t e d glycoconjttgate with dialyzed iron and high-iron diamine affinities in the outermost stratum corresponding with the lamina diffusa. 46 Some, but not other, epithelial cells differ in also showing a concentration of fixed polyanionic substance in an inner layer corresponding with the lamina l u c i d a as defined morphologically in basement membranes. The latter layer is demonstrable by its dialyzed iron and high-iron diamine affinities in the basement membrane of duct cells of the pancreas, in contradistinction to the lack o f such a stainable stratum in the basement membrane of the pancreatic acinai" cells. 46 The latter stratum, interpreted as possibly playing a part in the concentration of cations in connection with ion transport by the duct cpitheliuna, could evidence morphochemically tlte fixed anionic laycr postulated to sequester specific ionic species and mediate ion fluxes during a secretory responseY A sulfated glycosaminoglycan identified by specific enzyme digestion as a heparan sulfate Itas been visualized in the lamina r a t a e x t e r n a and lamina t a r a interna of the renal glomerulus by its affinity fi)r cationized fcrritin or r u t h e n i u m red. 3'J'4~ Preembed-

351

tlUMAN I)ATIIOLOGY--VOLUME 13, NUMBER 4 APril 1982 ,

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Figure 7 (left). Secretory granules in a mucous duct cell in the rat trachea have a negative core surrounded by a moderately stained cortex. Cap-shaped areas in the periphery of the cortex are heavily stained, as is the luminal surface of the plasmalemma, lligh-iron diamine stain, x 2 1 , 0 0 0 . Figure 8 (right). Mucousduct cell in the rat trachea again shows a negative grannie core, moderately stain~3dcortex, and densely stained caps (see Fig. 7). Tile lunfinal surface coat appc,'irs heavily stained, l)ial)'zcd iron stain, x 18,000 m e n t staining o f guinea pig renal glomerulus with dialyzed iron o r ltigh-iron d i a m i n e d e m o n s t r a t e s these strata, wltich sintilarly show s t r o n g e r staining u n d e r the epithelial titan u n d e r the endothelial cells (Sato A, Spicer SS: u n p u b l i s h e d observations). INTRACELLULAR COMPLEX CARBOHYDRATES

Ultrastructural cytnchemical metltods d e m o n strate c a r b o h y d r a t e - r i c h c o m p o n e n t s in the stored cytoplasmic granules o f some, but not o t h e r , types o f cells secreting inacromolecules. ~4-~6''~s-6~ T h e cytochemical m e t h o d s thus s u p p l e m e n t fine-structural m o r p h o l o g y in differentiating anti characterizing cell types in epithelial surfaces c o m p o s e d o f a heterog e n e o u s cell p o p u l a t i o n . T h i s is e x e m p l i f i e d by periodic a c i d - t h i o c a r b o l w d r a z i d e - s i l v e r proteinate and dialyzed iron staining o f the rat tracheal surface, where several subtypes are distinguishable in both the serous and the mucous cell categories, ss T h e distinction between subtypes o f serous cells d e p e n d s on the absence o f neutral glycoprotein from the secretory granules or its presence selectively in a tlain r i m , a cap, o r a thick cortex in the granules o f the d i f f e r e n t cell types. Mucous cells in rat tracheal surface epithelium d i f f e r according to distributions o f nonsulfated, carboxylated glycoprotein in the granules and the presence o r absence o f a c a r b o h y d r a t e - f r e e core o f unknown composition. 58 Ultrastructural c a r b o h y d r a t e cytochemistry also provides a tneans o f distinguishing otherwise undetected zones within a single secretory granule and titus e v i d e n c i n g the p r e s e n c e w i t h i n i n d i v i d u a l granules o f several c o m p o n e n t s differing in carbohydrate contents? 8-6~ In rat tracheal m u c o u s tllbnles, 352

for example, lligh-iron diamine demonstrates, ill addition to a negative core, a t r a b e c u l a r zone with m o d e r a t e staining for sulfate and a p e r i p h e r a l zone with heavier staining. Dialyzed iron and high-iron d i a m i n e have disclosed similarly heavily stained crescentic zones in the m o d e r a t e l y stained c o r t e x s u r r o u n d i n g an u n r e a c t i v e core in t h e s e c r e t o r y granules o f mucous d u c t cells o f rat trachea (Figs. 7 and 8)Y ~ Evidence for the existence in a single cell prolile o f m o r e than o n e type o f secretory granule has been obtained using ultrastrnctural methods for staining c o m p l e x c a r b o h y d r a t e s . T h u s , in the g u i n e a pig stomach, granules resembling those o f istlunus cells and o t h e r granules m o r e like those in m u c o u s cells have been observed in the same cell profile. 5s This staining and the location o f these cells d e e p to the isthmus cells were i n t e r p r e t e d as indicative o f a stage o f transition fronl isthmus to mucous cells d u r i n g the d o w n w a r d migration a n d maturation o f the epithelial cells in the gastric gland. Intracellular sites o f biosynthesis o f c o m p l e x carbohydrates are d e m o n s t r a b l e by cytochenlical methods at the electron microscopic level. T h e periodic a c i d - t h i o c a r b o h y d r a z i d e - s i l v e r p r o t e i n a t e proced u r e consistently stains the Golgi cisternae o f cells storing glycoprotein in secretory granules, and the staining o f such cisternae generally increases in intensity from the c/s to the trans face 5t'55 (Fig. 9). S u c h staining is t h o u g h t to retlect sites o f glycosyl transferases, progressively a d d i n g sugar residues with vicinal glycols to nascent oligosacclmride side clmins o f glycoproteins. H i g h - i r o n diamine as a rule stains only the trans cisternae in cells storing sulfated glycoconjugate in secretory granules, such as the colonic goblet

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Figure 9 (left). The Golgi complex in a mouse duodenal goblet cell shows increased intensity of the staining m the cisternae from the c/s to the trans face. Periodic acid-thiocarbohydrazidc-silvcr proleinatc stain, • Figure 10 (center). In a Golgi complex from a mouse colonic goblet cell only the traris cisternae are stained. Forming mucous droplets are also densiticd. High-iron diamine stain, x28,000. Figure 11 (Hgl, t). Surface epithelial cell in the mouse stomach. Only the interlnediate Golgi cisternae appear stained. Peanut lectinhorseradish peroxidase procedure, x36,000. cell (Fig. 10). H i g h - i r o n d i a m i n e s t a i n i n g in o n l y t h e t r a n s c i s t e r n a e i n d i c a t e s t h a t s u l f a t i o n o c c u r s in t h e s e cells as a t e r m i n a l s t e p in t h e s y n t h e s i s o f t h e glycop r o t e i n . S e l e c t i v e s t a i n i n g o f t h e i n t e r m e d i a t e cisternae has been ol)served with a peanttt lectin-horseradish peroxidase conjugate for demonstrating t e r m i n a l g a l a c t o s e in tire g l y c o p r o t e i n s i d e c h a i n (Fig. 911)78 A l t l t o u g h i n t e r p r e t a t i o n o f this selective a f f i n i t y o f t h e i n t e r m e d i a t e c i s t e r n a e o f tire G o l g i c o m p l e x in mouse gastric surface epitltelinm and colonic goblet cells is c o n c e i v a b l y a t t r i b u t a b l e to d i f f e r e n c e s in p e r meability of these Golgi elements to the lectinp e r o x i d a s e c o n j u g a t e , t h e p o s s i b i l i t y also c a n b e e n tertained that the staining reflects the location of g a l a c t o s y l t r a n s f e r a s e a d d i n g p e n u h i m a t e g l a c t o s e to a g l y c o p r o t e i u . A c c o r d i n g to t h e l a t t e r i n t e r p r e t a t i o n , t h e c i s t e r n a e at t h e c/s f a c e l a c k r e a c t i v i t y b e c a u s e g a l a c t o s e h a s n o t b e e n a d d e d at tltat p o i n t to tire g r o w i n g o l i g o s a c c l m r i d e s ; a n d c i s t e r n a e at tire t r a n s face a n d s e c r e t o r y p r o d u c t s s t o r e d in tire g r a n u l e s are negative because a terntinal residue has been added, capping the galactose.

ACKNOWLEDGMENTS

T h e a u t h o r s a c k n o w l e d g e tire e x p e r t t e c h n i c a l a s s i s t a n c e o f Ms. Elite S e t s e r a n d Ms. M a r y B r i d g c s a n d t h e e x p e r t e d i t o r i a l a s s i s t a n c e o f Ms. F r a n Cameron.

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