ELECTRON MICROSCOPY OF THE AMMONIACAL

SILVER REACTION FOR HISTONES IN

THE ERYTHROPOIETIC CELLS OF THE CHICK

EDITH K . MAcRAE and GERALD D . MEETZ

From the Department of Anatomy, University of Illinois at the Medical Center, Chicago, Illinois60612. Dr. Meetz's present address is the Department of Molecular, Cellular, and DevelopmentalBiology, University of Colorado, Boulder, Colorado 80302

ABSTRACT

INTRODUCTION

Interest in histones has broadened since 1950when Stedman and Stedman (30) suggested thatthese basic proteins may function as gene modifiers .Since then, more specific biochemical evidencehas implicated the histones in possible repressionof DNA-dependent RNA synthesis . Investigationsof Huang and Bonner (16) and Allfrey, Littau,and Mirsky (2) have shown that arginine-richhistones differ from lysine-rich histones in theirrelative capacity to inhibit messenger RNA syn-thesis.A staining reaction using postformalin ammo-

niacal silver to demonstrate qualitative colordifferences for nucleohistones from several sourceswas developed by Black and Ansley (5) ; a yellowstain was reported as characteristic of lysine-richhistones and a brown-black stain, characteristic ofarginine-rich histones. Such staining differences

The product of the postformalin ammoniacal silver reaction, which has been claimed todistinguish lysine-rich from arginine-rich histones with the light microscope on the basis ofa color difference, was examined in developing erythroid cells of chick bone marrow withthe electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacalsilver reaction product, while small basophilic erythroblasts, polychromatophilic erythro-cytes, and reticulocytes exhibit an increasing amount of reaction product as maturationproceeds. The reaction product is in the form of discrete electron-opaque particles associ-ated with heterochromatin . The ammoniacal silver reaction in the erythroid cell series isinterpreted as reflecting either the accumulation of newly synthesized arginine-rich histonesor changes in the availability of reactive sites in preformed histones .

were detected in isolated histone extracts andwith the individual amino acids .

The presence of histones was investigated bythe authors (22) using the ammoniacal silverreaction in highly specialized cells, the developingerythroid cells of the chick bone marrow, whichare known to shut down their nuclear RNAsynthesis and the cytoplasmic synthetic machineryduring maturation (7, 13, 18, 28) . Observationson these cells showed the ammoniacal silver reac-tion as a yellow nuclear stain in early erythroblastcells and an increasing amount of brown nuclearstain in progressively more mature cell stages (22) .

To study this reaction further and to determinewhether the yellow coloration may be a dilutionof the brown stain, we examined the results of thereaction with the electron microscope.

The reaction product was observed as a deposit

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of electron-opaque particles in the nuclei ofdeveloping cells and correlated with the brownstain under the light microscope . A preliminaryreport of these observations has been presented(21) .

MATERIAL AND METHODS

Pieces of bone marrow removed from femurs of 3-12-day-old chicks were fixed for 3 hr at room tempera-ture in 10% formalin neutralized with sodium acetate(2 g/100 ml of formalin) at pH 6 .8-7.1 . The tissuewas thoroughly washed in distilled water for about 20min before immersion in the ammoniacal silver solu-tion (AS) . The latter is prepared just before use bygradual addition of 10% silver nitrate solution toconcentrated ammonium hydroxide until a slightturbidity persists. Tissues were immersed in the ASwith no further agitation for 5 min. Anotherthorough washing in distilled water followed the AStreatment. Tissues were then placed for 5 min in 3%

FIGURE 1 Low magnification of bone marrow cells after the ammoniacal silver reaction. Note theintracellular localization of ASR deposit in nuclear areas and a fine precipitate dispersed in the cyto-plasm. X 9,000 .

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formalin solution, at which point a yellow-to-browncoloration appeared . Tissues were washed in distilledwater again and dehydrated immediately or storedin buffer at 4 °C. (0 .1 M sodium cacodylate was usedbefore dehydration) . Subsequent treatment includedexposure to increasing concentrations of ethyl alcohol,propylene oxide, and final embedding in Epon 812 .Curing overnight was done at 60 °C. Thick sections(1-3 µ) were cut with glass knives and examined underthe light microscope without further staining . Thinsections were cut with a diamond knife on a LKBUltrotome III or a Porter-Blum MT-2 ; these sectionswere stained with uranyl acetate, alone or followedby lead citrate, or left unstained before viewing withthe Hitachi UH 11A electron microscope.The ammoniacal silver method, like most metallic-

impregnation methods, is capricious . Great care mustbe taken to use chemically pure reagents and cleanglassware, and to avoid metal instruments in handlingtissues. Ammoniacal silver solutions should be pre-pared immediately before use and the unused reagent

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should be inactivated by excess dilute hydrochloricacid or a solution of sodium chloride, and discarded .Attention must be made to the potentially explosiveproperties of ammoniacal silver solutions, especiallythose stored for some time, exposed to sunlight, orprepared in well silvered flasks (31) .

OBSERVATIONS

The results of the application of the ammoniacalsilver reaction (ASR) viewed with the electronmicroscope show a deposit of discrete electron-opaque particles localized in the nuclei of a varietyof cells examined in the chick bone marrow (Fig .1) . The deposit, presumably a complex of silver,consists of particles, 190-200 A in diameter,occurring individually or in aggregates up to500 A in diameter . Since it was necessary to stainthe sections with uranyl and lead ions to provide

sufficient contrast for cellular identification, sec-tions untreated with uranyl and lead ions servedas control to check that the observed depositwas, indeed, due to the AS reaction and not tosome other combination of metallic ions . Suchcontrols show a particulate deposit confined intra-cellularly (Fig . 1) .

Cells of the erythrocytic series in progressivelymaturing stages were identified by several charac-teristics, including an increase in nuclear densitywith clumping or condensation of chromatin(heterochromatin), a decrease in volume ratio ofnucleus to cytoplasm, a decrease in cell size, anda change in cell shape . After treatment withuranyl and lead ions, ribosomes become visibleas shown in Fig. 2. At higher magnifications, thedifference in the number and cluster size of cyto-

FIGURE 2 Low magnification of bone marrow cells of the erythroid series after ASR, uranyl, and leadion treatment . The visualization of cytoplasmic ribosomes after uranyl and lead ions allows for identifica-tion of cells at different stages of maturation in this and subsequent micrographs . Large basophilic erythro-blast (BI), a small basophilic erythroblast (B2), a polychromatophilic erythrocyte (P), and a reticulocyte(R) . X 8,000 .

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plasmic ribosomes and in the number of mito-chondria also serve as criteria for identificationof cell stages. All subsequent micrographs are ofcells treated with uranyl and lead ions after theASR. The following classification of cells of theerythrocytic series, modified after the terminologyof Lucas and Jamroz (20) is used : large and smallerythroblasts, early, mid-, and late polychromaticerythrocytes, reticulocyte, and mature erythrocyte .

Stem cells, antecedent to the erythroblasts,were occasionally observed ; they may be charac-terized as having a large, almost spherical nucleusfilled with uniformly dispersed or extended euchro-matin. Little or no evidence of chromatin con-densation or clumping is seen at this stage .

Nuclear pores and nucleoli consisting of granularand amorphous material may be observed .Numerous ribosomes are closely packed through-out the cytoplasm . The lack of osmium tetroxidepostfixation results in less contrast, particularlyof the membranous organelles ; nevertheless,mitochondria and sparse endoplasmic reticulumcan be recognized . Mitochondria whose paleappearance resembles negatively stained imagesare round or rod-shaped with slender cristaeextending the length of the mitochondria . Alarge cell identified as a possible stem cell or avery early basophilic erythroblast is seen in Fig . 3 .Such cells show essentially no deposit of ASR inthe nuclei.

FIGURE 3 A stem cell or early basophilic erythroblast after treatment as in previous micrograph . Thecytoplasm contains mitochondria (m) and closely packed ribosomes (r) . Note the large nucleus and itshomogeneous euchromatin, nuclear pores (np), and an absence of ASR particles . X 24,000 .

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The early large erythroblasts are distinguishedfrom stem cells primarily by an increase in nuclearcondensation indicated by areas of heterochro-matin. The cytoplasm contains closely packedribosomes and mitochondria similar to those ofstem cells . The granular endoplasmic reticulum issparse and occasional vacuoles appear in thecytoplasm. Some ASR deposit can be observedassociated with the chromatin areas of increaseddensity (Fig . 4). The nucleolus of such a cell isapparent and ASR particles appear primarilyassociated with the perinucleolar heterochromatinrather than with the nucleolus .

A small basophilic erythroblast is seen in Fig . 5 ;in this stage the cytoplasmic ribosomes are seenin clusters of polyribosomes (polysomes), andelectron-lucent areas presumably containinghemoglobin become apparent around the poly-

somes. The ASR deposit is associated with hetero-chromatin in the periphery of the nucleus andperinucleolar areas .

The polychromatophilic erythrocyte stages canbe distinguished from erythroblasts by severalcriteria . The volume ratio of nucleus to cytoplasmis decreased ; there may be a decrease in cell sizeand further elongation of the cell . The nuclei ofthe polychromes display further chromatincondensation, and nucleoli are still present . Thecytoplasmic ribosomes in the polysome arrange-ment are further dispersed and surrounded bymore extensive areas of electron-lucent cytoplasm .Mitochondria are smaller and fewer in number .Nuclear pores are evident but somewhat fewer innumber than in earlier stages . Bridges containingmaterial of the same density and continuous witheuchromatin extend from the nuclear pores into

FlcuRE 4 A part of a basophilic erythroblast with cytoplasmic ribosomes (r) and mitochondria (m) . Thenucleus has areas of heterochromatin (h) and associated ASR particles (ASR) . The nucleolus (nl) isrelatively devoid of ASR deposit as compared to the perinucleolar heterochromatin (ph) . X 68,000.

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FIGURE 5 A small basophilic erythroblast contains cytoplasmic ribosomes arranged in clusters aspolyribosomes (pr) and mitochondria (m) . Note the ASR deposit associated with heterochromatin ; thenuclear pores (np) and nucleolus (nl) are devoid of ASR deposit. X 23,000.

FIGURE 6 A polychromatophilic erythrocyte is shown with the increase in electron-lucent areas be-tween the individual polysomes (pr) due to cytoplasmic accumulations of hemoglobin . Mitochondria (m)are still evident . Note the nuclear pores (np) and the increased amount of ASR deposit associated withheterochromatin . X 26,000 .

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FIGURE 7 A reticulocyte is shown with a smaller nucleus and a decreased number of polysomes as com-pared with earlier stages . Note the fine ASR precipitate evident in the cytoplasm . The nucleus has areasof heterochromatin heavily covered with ASR deposit . X 18,000 .

FIGURE 8 A higher magnification of the cytoplasmicpolyribosomes of a late polychromatophilic erythro-cyte . At arrows are indicated ASR particles associatedwith ribosomes . X 100,000 .

the cytoplasm across a pale rim surrounding eachnucleus. This rim may be due to shrinkage . Amidstage polychrome is seen in Fig. 6 . Closelyassociated with the increased amount of hetero-chromatin is the ASR deposit, also increased inamount and somewhat coarser in particle size .The late polychromatophilic erythrocytes and

reticulocytes have smaller volume ratio of nucleusto cytoplasm and show further condensation ofchromatin into heterochromatin . Nucleoli are notobserved. The number of ribosomes is decreased,and all ribosomes appear in the form of polysomes .Electron-lucent areas surrounding the polysomesare more extensive . The usual cytoplasmic densityof hemoglobin-filled reticulocytes as seen afterosmium tetroxide fixation is not observed in theseformalin-fixed preparations . Few mitochondria areobserved . The cell contours in late stages arerelatively smooth . The clumped chromatin oc-cupies a larger area in a smaller nucleus, and mostof the heterochromatin is covered with ASRparticles . A reticulocyte is shown in Fig . 7 .Mature erythrocytes have no polysomes or

mitochondria in the cytoplasm. In comparisonwith reticulocyte stages, erythrocyte nuclei maybe decreased in size but the extent of any furtherchromatin condensation and of ASR deposit issmall.

In addition to the coarse ASR particles as-sociated with heterochromatin and correspondingto the brown-black stain seen with the lightmicroscope, there is present a fine precipitate ofparticles 40-80 A in diameter, observable in thecytoplasm . These particles seem to be present atall stages (see Fig. 1) . However, in stained sectionsof the later stages in which polysomes are dispersed,the small particles appear to be associated withthe ribosome clusters (Fig . 8) .

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FIGURE 9 A leukocyte of the chick bone marrow after the ASR, uranyl, and lead ions . The ASR coarseparticles are seen in the trilobed nucleus (N), and fine particles are seen in the larger and more electron-opaque secretory granules (gi) but are lacking in the other granules (g2) . X 18,000 .

Developing leukocytes present in the bonemarrow also have ASR particles . One such leuko-cyte (heterophil) is seen in Fig . 9 which showsASR particles associated with the heterochromatinof the polymorphic nucleus and a fine ASRdeposit of 40-50 A in the larger and more electron-opaque secretory accumulations .

DISCUSSION

These observations have two implications : oneregarding the ammoniacal silver reaction, and theother regarding the possible role of histones incell development.The ammoniacal silver reaction (ASR) particles

seen with the electron microscope appear tocorrespond to the brown-black nuclear stain seenwith the light microscope in the more maturestages of erythroid cell development. In contrast,the yellow nuclear stain seen with the lightmicroscope in the early erythroblast stages has noreaction product detectable with the electron

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microscope . The small number of ASR electron-opaque particles detectable in early basophilicerythroblasts are too few to be observable as abrown stain in the light microscope . The brownstain in localized nuclear areas of intermediate cellstages corresponds to deposit associated withheterochromatin in these cells . The chemical basisfor the reaction is not known. Black and Ansley(5) suggested that the yellow stain may be due toE-amino groups of lysine, while the brown-blackstain may be due to guanidino groups of arginine .

One possible interpretation of this stainingphenomenon as observed with the electron micro-scope is that the ASR is due to some specificinteraction of silver with reactive centers inarginine and is detectable only in proteins with ahigh content of arginine. This category wouldinclude the arginine-rich fractions III, IV, andfraction V present in avian erythrocytes (23, 24) .This interpretation assumes that a synthesis of

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these arginine-rich fractions begins at about theerythroblast stages .

Another interpretation is that some structuralrearrangement or reorientation of the histonesmakes previously masked reactive sites availableto the ASR . This rearrangement would be pre-sumed to occur at the erythroblast stages . Bio-synthetic changes within the histone molecule,having an influence on DNA-histone structure aswell as on DNA-dependent RNA synthesis, havebeen reported . Acetylation of certain residues inhistones produces such an influence in early stagesof cell differentiation in lymphocytes (25) . Thehigh peak of acetylation of the arginine-rich histoneshas been shown to occur before the maximalrates of RNA synthesis are reached in regener-ation of rat liver (26) . Methylation (1, 12) andphosphorylation (14, 17) also appear to alter thebinding of histones to DNA and to affect bothchromatin structure and function . Thus, suchmodifications of histone structure by group sub-stitution reactions may change the interaction ofhistones not only with DNA but with the silverions in the ammoniacal silver stain.

The observed data do not exclude either inter-pretation, but suggest that the ASR does detectreactive sites in either newly synthesized or inrearranged preformed arginine-rich histones.The method appears to detect nonhistone

arginine-rich proteins as well . Observations ongranulocytes in chick bone marrow show a fineparticulate ASR deposit in the larger secretorygranules of the heterophil. The presence of anarginine-rich protein in granules of the rabbitneutrophil has been reported from several typesof evidence (3, 29, 32) . It seems not unlikely thatchick leukocyte granules may also contain asimilar arginine-rich protein ; other sources ofarginine-rich proteins must be investigated further .The ASR product appears confined to the

nucleus and cytoplasmic secretory granules, inwhich arginine-rich proteins are likely or knownto occur. Mitochondria, endoplasmic reticulum,cell membranes, pancreatic zymogen granules,and extracellular spaces are all devoid of the ASRproduct. However, a very fine particulate depositof the ASR is visible in the cytoplasm of reticulo-cytes and mature erythrocytes .

The decreased density of the cytoplasm due to adispersal of ribosomal clusters in the hemoglobin-accumulating cytoplasm allows for the visualiza-tion of the ASR particles in their association with

polysomes . These particles may be either productsof interaction of silver with arginine-rich cyto-plasmic proteins or merely contaminants of themethod. Since extracellular space and mitochon-dria are consistently devoid of ASR particles, thepresence of a cytoplasmic basic protein similar oridentical to histones must be considered. The ASRparticles are often, though not always, associatedwith discernible ribosomes in the immediate areaof the individual particles, and may be interpretedas due to silver ion interaction with a basic proteinpresent in ribosomes (8) . Arginine in about thesame total proportion as in histones has beenreported in rabbit reticulocyte ribosomes (6) .The possibility remains that the cytoplasmic ASRparticles are indicative of silver reaction withnewly synthesized arginine-rich histones on theribosomes . Cytoplasmic synthesis of histones hasbeen reported in HeLa cells (27), and continuingsynthesis of histones in the absence of DNAsynthesis has been noted in maturing erythroidcells (10), particularly in the arginine-rich frac-tions III and IV (12) .

The ASR particle size is variable. However, theaverage size tends to increase with an increasingamount of deposit. The average particle size inerythrocyte nuclei appears larger than that in baso-philic erythroblast nuclei . The larger size is proba-bly due to some ionic attraction and coalescence ofsmaller silver particles with one another to form ag-gregates . When the relative amount of arginine-richprotein is low, the particle size may remain small,as in the reticulocyte cytoplasm and in heterophilsecretory granules.

If a valid assumption is that the ASR is detect-ing arginine-rich histones in the nuclei, one mayconclude that the ASR product for arginine-richhistones is first detectable during the erythroblaststage and increases in amount up to about the latepolychromatophilic erythrocyte or reticulocytestages .These data do not add further evidence for

histone as a repressor of gene action on DNA-dependent RNA synthesis. However, as indicatedby the ASR deposit, arginine-rich histones,fractions III and IV, or fraction V, or both, eitherare formed and accumulated in the maturingstages or preexisting histones are so changed intheir structure as to become reactive to AS. Thereactivity coincides in time with the decrease ofRNA synthesis, based on evidence of decreaseduptake of radioactively labeled uridine into RNA

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in duck erythrocytes (28) . The appearance orincreased reactivity of the histone also coincideswith the increased amount of heterochromatinwhich is known to be inactive in RNA synthesis(4, 9, 11, 15, 19) . These data are suggestive thathistone changes parallel gene inactivation inerythropoiesis of the chick .

The authors wish to thank Dr. J . P. Marbarger for theuse of the electron microscope facilities of the Medical

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Research Resources Laboratory, and Miss Irena

Kairys for technical assistance .

This work was supported in part by a GraduateResearch Grant from the University of Illinois to Dr .MacRae and a United States Public Health Service

predoctoral fellowship No . 5 F01-GM-37, 849-02 to

Dr. Meetz .

Received for publication 9 July 1969, and in revised form 18

December 1969 .

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