electron microscopic classification of amine-producing endocrine cells by selective staining of...

Histochemie 27, 226 242 (1971) �9 by Springer-Verlag 1971

Electron Microscopic Classification of Amine-Producing Endo- crine Cells by Selective Staining of Ultra-Thin Sections

l~. HAKA~SON~ CH. OWMAN, B. SPORRONG, and F. SUNDLER Departments of Pharmacology, Anatomy and Histology,

University of Lund, Lund, Sweden

Received April 8, 1971

Summary. The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction ; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non- chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with L-3,4-dihydroxyphenylalanine or L-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accmnulated selectively over the secretory granules of the cells.

The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.

Introduction

Several systems of amine-producing cells with presumed endocrine funct ions occur diffusely dis t r ibuted in epithelial s tructures and organs th roughout the body (Feyrtcr, 1938, 1954; Falck and Owman, 1968; Pearse, 1969; I t~kanson, 1970). These cells, which are believed to synthesize and store various polypeptide hormones, have been referred to as paracrine by Feyr te r (1954) and as A P U D cells by Pearsc (1969). Several s ta ining reactions, par t icular ly modifications of chromium and silver impregnat ion techniques, have been used to classify such amine-producing endocrine cells in the l ight microscope (Hillarp and tISkfelt, 1953; Ratzenhofer and Lembeck, 1959; Singh, 1964; Coupland, 1965; Coupland and Hopwood, 1966; Vialli, 1966; Hopwood, 1967; Solcia, Capella and Vasallo, 1969; Grim61ius, 1969). The chromaffin and argentaff in reactions are believed to reveal the presence of reducing compounds in the cytoplasm of the cells (cf. Bar ter and Pearse, 1953; Ratzenhofer and Lembeck, 1959; Vialli, 1966); evidence was presented tha t the reducing compounds were indolamines and catecholamincs (cf. Erspamer and Asero, 1952; Bar ter and Pearse, 1953, 1955; Ratzenhofer

Electron Microscopic Staining of Endocrine Cells 227

and Lembeck, 1959; Vialli, 1966). Such amines have been identified in the cells by means of specific fluorescence histochemical methods (Falck, 1962; Falck0 Hillarp, Thieme and Torp, 1962; Falck and Owman, ]965; Corrodi and Jonsson, 1967). The argyrophi] reaction on the other hand, which involves the use of extraneous reducing agents and therefore does not require the presence of cytoplasmic reducing compounds (Grim~lius, 1969), is believed to reflect a high affinity for silver to certain cytoplasmic structures. The underlying mechanism of this staining reaction is unknown.

During the last few years, information has accumulated concerning the light microscopic (Pearse, 1966; Solcia, Vassallo and Sampietro, 1967; Capella, Solcia and Vassallo, 1969; Hs and Owman, 1969; Solcia, Capella and Vassallo, 1969) and fluorescence microscopic (Hs and Owman, 1966; Larson, Owman and Sundler, 1966; Hs Lilja and Owman, 1967; Cegrell, 1968; Falck and Owman, 1968; H~kanson, 1970) features of amine-producing endocrine cells. This has made it possible to demonst ra te a large number of cellular subtypes, which are morphologically indistinguishable with argyrophil staining (cf. Hs Owman, SjSberg and Sporrong, 1970). The histochemical criteria used to distinguish between the various types of endocrine cells by light and fluorescence microscopy have previously had no counterpar t at the ul t ras t ructural level. Therefore it has been difficult to correlate electron microscopic results with those obtained by optical microscopy.

Recently, both argentaffin and chromaffin reactions have been applied in ul t ras t ructural studies (Tramezzani, Chiocchio and Wassermann, 1964 ;Wood, 1965 ; Chang and Bencosme, 1968 ; J a im-Etcheve r ry and Zieher, 1968a, b, c ; Battaglia, 1969; Penttil~, 1969; Vassallo, Solcia and Capella, 1969). I n most of these proce- dures, staining was performed en bloc, thus preventing comparison between stained and unstained sections - or between differently stained sections - f rom the same specimens. Only in a few studies has the argentaffin reaction been carried out directly on ul t ra- thin sections (Chang and Bencosme, 1968 ; Battaglia, 1969).

The present report describes techniques by which the chromaffin, argentaffin and argyrophfl reactions can be performed directly on ul t ra- thin sections from one and the same tissue specimen. Prel iminary data on the electron microscopic staining properties of amine-producing endocrine cells in the adrenal medulla, gastric mucosa, and thyroid gland are presented.

Materials and Methods

Fluorescence Microscopy. Material was obtained from non-treated animals and from animals injected with L-3,4-dihydroxyphenylalanine (L-DOPA) or L-5-hydroxytryptophan (L-5-HTP) 100 mg/kg, intraperitoneally, and killed 11/2 hours after the injection. Specimens of the adrenal and the thyroid gland of the mouse and of the gastric mucosa (oxyntic gland area) of the mouse, rat and rabbit were quickly dissected out, frozen to the temperature of liquid nitrogen in a propane-propylene mixture, freeze-dried, exposed to formaldehyde gas for 1 hour at 80 ~ C, embedded in paraffin, sectioned at 6 ~ thickness and mounted in xylene for fluorescence microscopy as described in detail elsewhere (Falck and Owman, 1965).

Light Microscopy. Some of the sections treated as above for fluorescence microscopy were first photographed in the fluorescence microscope, after which the cover-slips were removed and the sections stained by one of the following methods: (a) Argyrophil Reaction (Grimdlius, 1969). The section was immersed in a 0.03 percent solution of silver nitrate in 0.02 M acetate

228 R. Hs Ch. Owman, ]~. Sporrong, and F. Sundler:

buffer, pH 5.6, at room temperature. After 24 hours, the silver solution was drained off the slides, which were immersed for 1-11/2 mln in a freshly prepared aqueous solution of 1 percent hydroquinone and 5 percent sodium sulphite. The slides were rinsed in re-distilled water, de- hydrated in graded ethanol solutions and mounted in Entellan (Merck) for examination in the light microscope. In one series of experiments, fresh specimens were fixed for 1-2 hours in a 10 percent neutralized formaldehyde solution or in a solution of 2.5 percent glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.3), or in various mixtures of these two fixation solutions. After dehydration in graded ethanol solutions, the specimens were embedded in paraffin, sectioned at 6 ~ and de-paraffinized before staining. (b) Argenta//in Reaction (Singh, 1964). The section was immersed in a solution of silver nitrate prepared in the following way : strong ammonia was added dropwise to a l0 percent solution of silver nitrate until the precipitate first formed was re-dissolved. Fresh silver nitrate solution (10 percent) was then added until a slight opalescence appeared and persisted. To each volume of this solution 9 volumes of re-distilled water was added. The slides were exposed to this ammoniacal silver nitrate solution in the dark at 60 ~ C until yellowing of the sections appeared (usually 30 to 60 rain). The slides were then rinsed in re-distilled water, transferred for fixation to a 1 percent sodium thiosulphate solution for 30 sec and mounted in glycerol. (c) Chroma//in Reaction (Jaim-Etcheverry and Zieher, 1968c). The sections were immersed for 2 14 hours at room temperature in a 2.5 percent solution of potassium bichromate, containing 1 percent sodium sulphite, in 0.2 M acetate buffer (pH 4.0). The slides were rinsed in re-distilled water and mounted in glycerol.

Conventional Electron Microscopy. Adult rats and mice were perfused for 5-10 rain with 2.5 percent glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.3, via the ascending aorta. Small tissue pieces (1 2 mm cubes), taken from the oxyntic gland area of mice and rats and from the adrenal medulla and thyroid of mice, were fixed in the same glutaraldehyde solution for 1 hour, postfixed for 1 hour in 1 percent osmium tetroxide in sodium cacodylate buffer, and contrasted for 1/2 hour en bloc in a mixture of 1 percent phosphotungstic acid and 0.5 percent uranylacetate in ethanol. Specimens from adult rabbits and from a few rats and mice were dissected out and fixed as above with glutaraldehyde except that the perfusion step was omitted. After dehydration and embedding in Vestopal-W the blocks were sectioned on a LKB Ultrotome. The sections were stained with lead citrate and uranylacetate, and then examined and photographed in a Philips EM 300 electron microscope.

Staining Reactions/or Electron Microscopy. The tissue specimens (as above) were fixed by perfusion and/or immersion using the conventional glutaraldehyde-cacodylate mixture or a 10 percent neutralized formaldehyde solution, sometimes followed by postfixation for 1 hour in I percent osmium tetroxide. They were rinsed in cacodylate buffer and sometimes contrasted for 1/,, hour en bloc in a mixture of t percent phosphotungstic acid and 0.5 percent uranylacetate in ethanol. After dehydration and embedding in Vestopal-W, ultra-thin sections were prepared and stained in one of the following ways: (a) Argyrophil Reaction. The sections were placed on nickel grids and left free-floating on a drop of silver nitrate solution prepared as described above (see also Grim61ius, 1969). After 6-12 hours at 60 ~ C in a humid chamber (Petri dish covered on the inside with wet filter paper) the grids were removed and developed in the hydroquinone-sodium sulphite solution, prepared as above (see Grim61ius, 1969). The grids were rinsed in re-distilled water and examined in the electron microscope after drying at room temperature. (b) Argent- a//in Reaction. The ultra-thin sections were placed on grids and treated in the same manner with an ammoniacal silver nitrate solution (see above and Singh, 1964) for 1~]: hours at 60 ~ C. The grids were rinsed in re-distilled water and air-dried before examination. (c) Chrqma]]in reaction. Ultra-thin sections were placed on grids and left floating on a drop of potassium bichromate solution (see above) for 2-8 hours at 60 ~ C. The grids were then rinsed in re- distilled water and dried at room temperature.

Results

Argyrophil Reaction. G l u t a r a l d e h y d e f i x a t i o n p r e v e n t e d t h e a r g y r o p h i l r e a c t i o n

in t h i n a n d u l t r a - t h i n s ec t ions f r o m a d r e n a l med u l l a , gas t r i c m u c o s a a n d t h y r o i d

g land . A m i x t u r e of f o r m a l d e h y d e a n d g l u t a r a l d e h y d e (equal p a r t s of t h e t w o

f i x a t i o n so lu t ions ) l ikewise p r e v e n t e d t h e s t a i n i n g r eac t ion . F i x a t i o n w i t h fo rma l -


:Fig. 1 a and b. R a t s tomach endocrine cells from oxyntic gland area. Elcctronmicrograph. • 19500. a Glutaraldehyde-osmium fixation. The most prominent feature is the presence of a

large number of electron-dense cytoplasmic granules, b Formaldehyde-osmium fixation followed by silver staining according to Grim~lius, demonstrat ing the argyrophilic character of the granules. The dense accumulation of silver over the granules contrasts with a less pro-

nounced precipitation of silver over the nucleus and a slight background precipitation

230 R. H~kanson, Ch. 0wman, B. Sporrong, and F. Sundler:

Fig. 2a and b. Rat adrenal medullary cells. • 22000. a Glutaraldehyde-osmium fixation. Two neighbouring eatecholamine cells, distinguishable by their differing types of osmiophilic, membrane-bound cytoplasmic granules, b Formaldehyde-osmium fixation followed by argyrophil staining, to demonstrate accumulation of silver grains on the content of the membrane-

bound granules. Silver grains are also diffusely distributed over the entire cytoplasm

dehyde alone, on the other hand, permit ted the demonst ra t ion of argyrophil cells in adrenal medulla, gastric mucosa and thyroid gland. After f ixation with formaldehyde alone or in combinat ion with osmium, the argyrophil reaction in u l t ra - th in sections resulted in a dense precipi tat ion of silver grains over the secretory granules


Fig. 3a and b. Mouse thyroid parafollicular cells, x 20000. a Glutaraldehyde-osmium fixation. Numerous membrane-bound cytoplasmic granules with varying electron density, b Formal- dehyde-osmium fixation followed by argyrophil staining. Silver grains are found all over the section with an accumulation over the content of some of the membrane-bound cytoplasmic

granules

of cells hav ing the e lect ron microscopic fea tures of endocr ine cells (Figs. 1-3). The reac t ion was posi t ive also af ter s ta in ing of the sect ioas wi th lead c i t ra te and u rany lace t a t e . Only a weak, non-select ive p rec ip i t a t ion of e lect ron-dense ma te r i a l a p p e a r e d over the nucleus and cy top la sm of all cells in the sect ion if the hydro- qu inonc-sodium sulphi te t r e a t m e n t was omi t ted . Once the cells had been ident i f ied as argyrophf l , the i r u l t r a s t ruc tu r a l character is t ics could be s tud ied in the be t t e r p reserved g lu t a r a ldchyde -osmium te t rox ide- f ixed mater ia l . Iden t i f i ca t ion a t the l ight microscopic level was achieved b y per forming the a rgyroph i l r eac t ion on sec- t ions f rom freeze-dr ied specimens t r e a t e d wi th gaseous fo rmaldehyde and pre- v ious ly examined for specific fo rma ldehyde- induced amine fluorescence (Fig. 4).

The following cells were found to be a rgyroph i l : Adrena l medu l l a ry cells and t h y r o i d parafo l l icu lar cells of the mouse, and enterochromaff in and enterochromaff in- l ike cells in the gastr ic mucosa of the mouse, r a t and rabb i t .

Argenta/]in Reaction. The argentaf f in reac t ion could be pe r fo rmed wi th bo th fo rma ldehyde- and g lu ta ra ldehyde- f ixed mater ia l . Usual ly , i ncuba t ion in the s i lver sal t solut ion for 1-2 hours a t 60 ~ C was needed for a s t rong s ta in ing reac t ion in u l t r a - th in sections. Pos t - f ixa t ion wi th osmium te t rox ide more or less abol i shed the argentaffin reaction. Phosphotungstic acid and uranyl acetate, which were used for contrast in en bloc staining, did not overtly affect the precipitation of silver. The argentaffin reaction resulted in heavy accumulation of electron-dense grains

232 R. Hs Ch. Owman, B. Sporrong, and F. Sundler:

Fig. 4a and b. Rabbit stomach, transverse section from oxyntic gland area. X 135. a Fluores- cence photomicrograph after formaldehyde condensation. Numerous 5-hydroxytryptamine- containing (yellow-fluorescent) enterochromaffin cells present basally in the mucosa, b Sub- sequent argyrophil staining of the same section to show that all enterochromaffin cells are

argyrophil and that a small number of the argyrophil cells are non-fluorescent

over the secretory granules of certain endocrine cells, i.e. all the adrenal medul lary cells and the gastric enterochromaffin (5-hydroxytryptamine-conta in ing) cells (Figs. 5 and 6). The cells were identified at the l ight microscopic level by a com- b ina t ion of the argentaff in reaction and fluorescence histochemical demonst ra t ion of catecholamines and indolamines (Fig. 7). Characteristically, short incuba t ion t ime (less t h a n 1 hour) resulted in almost selective silver impregnat ion of the secretory granules. Prolonged incubat ion t ime gave a more heavy staining of the granules, a t the same t ime result ing in a marked increase of the non-selective s ta ining of other cellular components.


Fig. 5a and b. Ra t adrenal medullary cells. Glutaraldehyde fixation, x 19000. a Argentaffin staining demonstrat ing a heavy deposit selectively located over numerous granules in the cytoplasm. A silver precipitation is seen also over nuclear structures. (b) Control section incubated

in the absence of silver

234 R. Hs Ch. Owman, B. Sporrong, and F. Sundler

Fig. 6a-d. Rabbi t stomach endocrine cells from oxyntic gland area. x 16000. a Glutaral- dehyde-osmium fixation. One type of endocrine cell containing a small number of strongly osmiophilic granules in the cytoplasm, b Glutaraldehyde fixation (without osmium) followed by argentaffin staining of the same cell type as in (a). A small number of strongly argentaffin granules in the cytoplasm. Silver precipitation is seen also over nuclear structures. In contro| section incubated without silver the cytoplasmic granules were barely visible (as in Fig. 5b).


Fig. 6c Glutaraldehyde-osmium fixation. Another type of endocrine cell containing a large number of osmiophilic granules in the cytoplasm, d Glutaraldehyde fixation (without osmium) followed by argentaffin staining. The cytoplasmic granules of this cell type (see Fig. 6c) were nonargentaffin in spite of prolonged incubation time reflected in the picture as a heavy

accumulation of silver over nuclear structures

236 R. Hs Ch. Owman, B. Sporrong, and F. Sundler:

Fig. 7a and b. Rabbit stomach, transverse section from oxyntic gland area. x 175. a Fluores- cence photomicrograph after formaldehyde condensation. Numerous 5-hydroxytryptamine- containing (yellow-fluorescent) enterochromaffin cells located basally in the mucosa, b Sub- sequent argentaffin staining of the same section showing a positive reaction in all fluorescent

cells

The specif ic i ty of the argentaf f in reac t ion was es tabl ished by inject ing ra ts and mice with L-DOPA or L-5-HTP, a t r e a t m e n t which is known to induce high levels of dopamine and 5-HT, respect ively , in cer ta in non-argentaff in endocrine cells (see below), otherwise devoid of h is tochemical ly de tec tab le monoamines (Larson, Owman, and Sundler , 1966; Hs Li l ja and Owman, 1967; Owman and Sundler , 1968). This t r e a t m e n t a p p a r e n t l y increased the reducing capac i ty of these cells (otherwise non-argentaf f in ; Fig. 8), since t hey now became argentaf f in (Fig. 8) as revealed a t both the l ight and electron microscopic level. An argentaf f in reac t ion was given by the following cells af ter inject ing ra ts and mice with L-DOPA or L-5-HTP: the parafol l icular cells of the mouse t hy ro id (Fig. 8), and the so- called enterochromaff in- l ikc cells in the oxynt ic gland area of the mouse and ra t s tomach. These cells required a prolonged incuba t ion t ime (2-3 hours) for a s t rong argentaf f in react ion. I t should be no ted t h a t also in these cells the electron-dense p rec ip i t a te accumula ted only over the secre tory granules.

Fig. 8a and b. Mouse thyroid parafollicular cells. Glutaraldehyde fixation followed by argentaffin staining, x 19000. a Untreated animal. Numerous cytoplasmic granules which were non- -argentaffin despite prolonged incubation time. Note the heavy deposit of silver grains over nuclear structures, b Animal treated by injection of L-5-hydroxytryptophan. A varying degree of argentaffinity now developed selectively in the granules. Insert: • Note the

selective accumulation of silver grains over cytoplasmic granules

Elec t ron Microscopic S ta in ing of Endocr ine Cells 237

17 Histochemie, Bd. 27

Fig. 8 ~ a n d b

238 R. H~kanson, Ch. Owman, B. Sporrong, and F. Sundler:

Fig. 9. Rat adrenal medullary cell (same type as in Fig. 5). x 12000. Glutaraldehyde fixation followed by chromaffin staining. Heavy chromaffin reaction selectively over cytoplasmic

granules. Control section incubated without chromium, see Fig. 5b

Chroma]/in Reaction. As with the argentaff in reaction, the chromaffin react ion could be induced with sections from both formaldehyde- and g lu ta ra ldehyde- f ixed mate r i a l ; pos t - f ixa t ion wi th osmium te t rox ide p reven ted the s ta ining react ion. W i t h the chromaff in react ion, the electron-dense prec ip i ta te accumula ted select ively over the secre tory granules {Fig. 9), and thus the nucleus was ent i re ly devoid of e lectron-dense mate r ia l wi th this method , in cont ras t to wha t was the case wi th the argentaf f in react ion. I t was observed t h a t all cells giving a s t rong argentaf f in reac t ion also gave a s t rong chromaff in reaction, p rovided an incuba t ion t ime of 2-6 hours was used.

A posi t ive chromaff in react ion could be induced also in otherwise non-chromaffin endocr ine cells (i.e. t hy ro id parafol l icular cells and gastr ic enterochromaff in- l ike cells af ter in ject ing ra t s and mice with L-DOPA or L-5-HTP. W i t h u l t r a - th in sections f rom such specimens the incuba t ion t ime needed for visible reac t ion was 8-14 hours. Also under these c i rcumstances , the reducing, chromaff in ma te r i a l was found only in the secre tory granules.

Discussion

I t could be demons t r a t ed t h a t the argyrophi l , a rgentaff in and chromaff in react ions, which are i m p o r t a n t tools in the l ight microscopic charac te r iza t ion of cer ta in amine-conta in ing , po lypep t ide -p roduc ing endocrine cell systems, can be


adapted for ultrastructural identification of such cell systems by direct staining of ultra-thin sections. In previous reports on the use of these techniques for electron microscopy, en bloc staining has usually been employed (Tramezzani, Chiocchio and Wassermann, 1964; Wood, 1965; Ja im-Etcheverry and Zieher, 1968a and b; Vassallo, Solcia and Capella, 1969). The main advantage of the techniques described in the present report is that consecutive ultra-thin sections can be stained differentially, thus permitting a more detailed s tudy of certain histochemical properties of one and the same cell. The use of staining techniques such as those described here will provide the information necessary for correlative studies of the stainable cells at the light microscopic and the electron microscopic levels. I t should be realized that the techniques are of cytodiagnostie value primarily, since morphological details are to some extent lost. This is due to the fact that the material is fixed in formaldehyde or glutaraldehyde but not postfixed in osmium-tetroxide ; such fixation, in combination with the staining proce- dures, does not provide opt imum electron density of the membranes.

The argyrophil reaction, which resulted in a finely granulated silver deposit over the secretory granules of certain endocrine cells, could be performed with formaldehyde-fixed but not with glutaraldehyde-fixed material. This was establish- ed both at the light microscopic and the electron microscopic level and with paraffin-embedded as well as plastic-embedded material. The reason for the failure of glutaraldchyde to permit the argyrophil reaction is unknown.

The argentaffin reaction, also giving a finely granulated, but usually heavy, silver deposit over the secretory granules of endocrine cells containing fluorogenic arylethylamines, could easily be obtained with sections from both formaldehyde- fixed and glutaraldehyde-fixed specimens. Post-fixation of the specimens with osmium tetroxide reduced the intensity of the argentaffin reaction. Staining en

bloc with phosphotungstic acid or uranylacetate, giving a weak background staining of all cellular components, did not overtly affect the argentaffin reaction.

The chromaffin reaction was performed with glutaraldehyde-fixed material and produced a smooth, extremely finely granulated (dust-like) electron dense precipitate over the secretory granules exclusively. I t is possible tha t fixation with osmium tetroxide prevents the chromaffin reaction. Osmium treatment, however, was unsuitable also since the resulting increase in electron density made it impos- sible to observe any additional staining of the secretory granules, following a superimposed chromaffin reaction. Staining with phosphotungstic acid or uranylacetate en bloc did not seem to affect the reaction.

The amine-storing granules of all adrenal medullary cells stained argyrophil as well as strongly argentaffin and ehromaffin. Different cell types could be recognized mainly on the basis of the ultrastructure of their storage granules (see also Tramezzani, Chiocchio and Wassermann, 1964; Chang and Bencosme, 1968). One of the cell types, believed to contain noradrenaline (Chang and Bencosme, 1968), remained strongly argentaffin also after post-fixation with osmium tetroxide. This may indicate a higher reducing capacity of the storage granules of noradrenaline-containing cells than of other adrenal medullary cells.

All endocrine cells of the rabbit oxyntie gland area were argyrophil. Using the argentaffin and chromaffin reaction this population of argyrophil cells was found

17"

240 R. H~kanson, Ch. Owman, B. Sporrong, and F. Sundler:

to consist of one strongly argentaffin (and chromaffin) and one non-argentaffin (and non-chromaffin) cell type (cf. Capella, Solcia and Vassallo, 1969; Hs Owman, Sj6berg and Sporrong, 1970). The argentaffin cell type was most numerous.

Also the endocrine cells of the rat and mouse oxyntic gland area were argyrophil (see also H~kanson and Owman, 1969) but in these species none of the cells were argentaffin or chromaffin. After pre t rea tment of the animals with L-DOPA or L-5-HTP, all the argyrophil cells became more or less argentaffin (and chromaffin). One cell type gave a strong argentaffin reaction under these conditions whereas another one exhibited a considerably weaker argentaffin reaction. The two cell types could be distinguished also on the basis of the morphology of their secretory granules. I t was noted tha t with the argentaffin reaction the staining intensi ty varied markedly between individual granules of one and the same cell.

The parafollieular cells of the mouse thyroid were argyrophil (see also Melander, Owman and Snndler, 1970) but neither argentaffin nor chromaffin. As with the endocrine cells in the rat and mouse oxyntie gland area, pre t rea tment of the animals with L-DOPA or L-5-HTP induced argentaffini ty and ehromaffinity of the secretory granules in the parafollicular cells ; the staining intensi ty varied between the cells in one and the same section. The staining intensi ty also of individual granules in one and the same cell varied markedly, probably reflecting a different content of the reducing compound.

The chemieM background of the argyrophil reaction is unknown, and the special affinity of the secretory granules for silver has never been explained. The argentaffin and ehromaffin reactions on the other hand, have been extensively studied in model experiments and require the presence of reducing compounds, capable of convert ing silver ions into metallic silver (see Introduction). Thus, it is generally accepted tha t the argentaffin and chromaffin reactions of certain neurons and certain endocrine cell systems reflect a high content of highly reducing compounds such as eatecholamines or 5-hydroxyt ryptamine . The demonstrat ion of the secre- to ry granules as the main locus of the argentaffin and ehromaffin reactions con- firms tha t these granules are the major storage sites for the eatecholamines and indolamines contained in these cells. Fur ther evidence for this view was obtained in the studies showing tha t non-argentaffin (and non-ehromaffin) polypeptide- producing endocrine cells can be converted into argentaffin (and ehromaffin) cells by pre t rea tment of the animals with the amine precursors, L-DOPA or L-5-HTP. Also after this t reatment , which is known to result in the accumulat ion of the corresponding amine in the endocrine cells ( tI~kanson and Owman, 1966; I-I~kanson, Lilja and Owman, 1967; Melander, Owman and Sundler, 1971), the reducing material was confined to the secretory granules as evidenced by the selective accumulat ion of the silver (and chromium) precipitates (el. Ericson, 1970).

Acknowledgements. This work was supported by grants from the Association for the Aid of Crippled Children, New York; The Swedish Medical Research Council (Project No. B71-14X- 1007-06C) ; and Albert PS~hlsson's Foundation and was carried out within a research organiza- tion sponsored by the Swedish Medical Research Council (Projects No. BTl-14X-56-07A and B71-14X-712-06A).


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Docent Ch. Owman Depar tment of Anatomy University of Lund Biskopsgatan 7 S-22362 Lund, Sweden

electron microscopic classification of amine-producing endocrine cells by selective staining of...

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