j. cell set. a, 359-37° (1967 35) 9 printed in great britain · j. cell set. a, 359-37° (1967 35)...

J. Cell Set. a, 359-37° (1967) 359Printed in Great Britain

AN ELECTRON-MICROSCOPE STUDY OF THE

IN VITRO TRANSFORMATION OF HUMAN

LEUCOCYTES

I. TRANSFORMATION OF LYMPHOCYTES TO

BLASTOID CELLS IN THE PRESENCE OF

PHYTOHAEMAGGLUTININ

J. A. CHAPMAN, M. W. ELVES* AND J. GOUGHfRheumatism Research Centre, University of Manchester, andDepartment of Clinical Haematology, The Royal Infirmary, Manchester

SUMMARY

Electron-microscope studies of cultured small lymphocytes from human peripheral bloodtransforming into larger blastoid cells in the presence of phytohaemagglutinin (PHA) show thatthe transformed cell possesses the preliminary stages of development of a protein-synthesizingsystem. The transformed blastoid cell has abundant ribosomes, although, in contrast within vivo protein-secreting cells, many of these occur as single particles with only a small pro-portion linked in polysomal clusters. Endoplasmic reticulum membranes occur to a very limitedextent and with a marked paucity of attached ribosomal particles; the few attached particlesare usually located in groups. Some endoplasmic reticulum membranes revealed degenerativechanges in otherwise normal cells. A moderately well-developed Golgi apparatus was a charac-teristic feature of the cells. Apart from the relatively low proportion of polysomes, in vitroPHA-transformed blastoid cells are identical in fine structure to in vivo blast cells (otherwiseknown as immunoblasts, haemocytoblasts, etc.) occurring in the immune response. It is sug-gested that messenger-RNA production in PHA-stimulated transformed cells may be reducedand that this could explain the limited number of polysomes and the restricted development ofthe endoplasmic reticulum.

INTRODUCTION

There is now ample evidence that the small lymphocyte of peripheral blood cantransform in vitro into at least two other cell types. One type of transformation occursin the presence of phytohaemagglutinin (PHA) (MacKinney, Stohlman & Brecher,1962; Carstairs, 1962; Elves & Wilkinson, 1963; Cooper, Barkhan & Hale, 1963), asa result of antigenic stimulation in a secondary immune response (Pearmain, Lycette& Fitzgerald, 1963; Elves, Roath & Israels, 1963; Schrek, 1963) or when lymphocytesfrom two individuals are mixed (Bain, Vas & Lowenstein, 1964; Elves & Israels,1965). This transformation leads to the development and proliferation of largeblastoid (blast-like) cells similar to cells found in vivo which have been variously

• Present address: The Charles Salt Research Centre, Robert Jones and Agnes Hunt Ortho-paedic Hospital, Oswestry, Salop.

f Present address: Department of Pathology,' University of Manchester.23-2

360 J. A. Chapman, M. W. Elves andj. Gough

described by others as transitional cells (Fagraeus, 1948), large lymphocytes (Trowell,1958; Nossal & Makela, 1962), activated reticular cells (Marshall & White, 1950),large pyroninophilic cells (Gowans, McGregor, Cowen & Ford, 1962), haemocyto-blasts (Fagraeus, i960; Andre"-Schwartz, 1964; Wellensiek & Coons, 1964), immuno-blasts (Dameshek, 1963; Movat & Fernando, 1965) and blasts (Nossal & Mitchell,1963; de Petris & Karlsbad, 1965). In this paper the large cells which develop incultured peripheral blood in the presence of PHA will be referred to simply as'PHA-transformed cells' or, more generally, as 'blastoid cells'.

The other type of in vitro transformation occurs in the absence of stimulating agents(such as PHA) and results in the development of macrophages. A cytochemical studyof this transformation has recently been made by Gough & Elves (1966, 1967).

The purpose of this paper is to describe the salient ultrastructural features of blastoidcells proliferating in cultures of human peripheral blood in the presence of PHA.A separate paper (Chapman, Gough & Elves, 1967) is devoted to a similar study ofmacrophages developing in cultures in the absence of PHA.

MATERIALS AND METHODS

Leucocytes were obtained from the peripheral blood of normal healthy humansubjects by dextran sedimentation, using 2 ml of 6% dextran in isotonic saline ('Dex-traven' from Benger Laboratories, Holmes Chapel, Cheshire, England) per 20 mlof heparinized blood. For the study of the blastoid transformation, o-1 ml of Difcophytohaemagglutinin P (PHA) was added to the separated leucocytes, which were thendivided into 2-3 ml aliquots; these were made up to 10 ml each by the addition ofTC 199 (Glaxo). The cultures were placed in universal containers and to some of them500 fig of hydrocortisone were added not more than 10 min after the addition of PHA.Cultures were sacrificed after 10-30 min, 24, 48 and 72 h by centrifugation at 800-1000 rev/min for 10 min. After withdrawal of the supernatant medium the cells werewashed with isotonic saline, collected again by centrifugation and fixed for electronmicroscopy.

Fixation was in 1 % ice-cold osmium tetroxide in veronal-acetate buffer at pH 7-2-7-4, made isotonic with sodium chloride, and the duration of fixation was 15-20 min.Two methods were used for the treatment of cells. In the first method the cells werefixed in suspension and pre-embedded in agar on a microscope slide as previouslydescribed (Elves, Gough, Chapman & Israels, 1964); in the second method a centri-fuged button of cells was fixed for 5 min, cut into small fragments with a cataractknife and then fixed for a further 15 min. Both methods gave satisfactory results butthe second method was more convenient and yielded a greater concentration of cellsin the sections.

Specimens were dehydrated in increasing concentrations of ethanol and embeddedin Araldite. Thin sections cut with a Huxley ultramicrotome were mounted on carbon-filmed grids and examined at instrumental magnifications ranging from x 5 000 tox 80000 in a Siemens Elmiskop I electron microscope operating at 60 or 80 kV. Mostsections were stained with aqueous lead hydroxide, aqueous lead citrate, or methanolic

Transformation of lymphocytes to blastoids 361

uranyl acetate (Stempak & Ward, 1964). Sometimes a combination of lead anduranyl staining was used.

RESULTS

Initial cultures

In cultures sacrificed at the start of the culture period (10-30 min) neutrophils andsmall lymphocytes were predominant; occasional monocytes and eosinophils were alsoseen. The ultrastructural features of these cell types were as described by others(Grey & Biesele, 1955; Low & Freeman, 1958; Paegle, 1961; Bessis & Thiery, 1961;Bernhard & Leplus, 1964) and will not be described again here.

PHA cultures

Those ultrastructural features of PHA-transformed lymphocytes that have alreadybeen considered by others (Tanaka, Epstein, Brecher & Stohlman, 1963; Inman &Cooper, 1963; Johnson & Roberts, 1964) will be described only briefly.

In 24-h PHA cultures, neutrophils showed signs of degeneration, evidenced bypyknosis and vacuolation. The predominant lymphocytes were, in many instances,increased in size and the enlarged cytoplasm in such cells contained many ribosomalparticles, identifiable by their size, electron-optical staining properties and distribu-tion. Other cytoplasmic features included mitochondria, centrioles and occasionalcompound vacuoles (Low & Freeman, 1958). In addition there were small numbersof vesicles, 500-1000 A across, occurring as membrane-bounded structures withelectron-transparent contents, and distributed in isolated form, in chains of vesiclesor as short cisternae. Although, in 24-h cultures, these vesicles were only rarely associ-ated with ribosomal particles, observations on later cultures suggest that they may beregarded as developing vesicles of a limited endoplasmic reticulum.

By 48 h these larger lymphoid cells (which may now be described as blastoid cells)were present in greater numbers and in a greater size range, many of them withdiameters up to 15 fi. Cytoplasmic organelles were similar to those described for the24-h cells but ribosomal particles and endoplasmic reticulum vesicles were moreabundant. Some of the vesicles of the endoplasmic reticulum appeared to containmaterial of increased electron opacity and a poorly developed Golgi apparatus wassometimes present. In addition, large electron-opaque granules were commonlyobserved; these granules, up to 1 /i in diameter and membrane-bounded, occurred inrounded form or as irregular stellate structures. In a few instances the membranesurrounding these granules was seen to be continuous with the membrane of anendoplasmic reticulum or Golgi vesicle (Elves et al. 1964). Similar granules observedby others have been described as lipid droplets (Johnson & Roberts, 1964). Some ofthe smaller granules are probably lysosomes (Parker, Wakasa & Lukes, 1965). Evi-dence of mitotic division was occasionally noted and in a few cells the nuclear mem-brane seemed to be shedding vesicles into the cytoplasm. In these 48-h culturestypical small lymphocytes were still present although in reduced numbers.

362 J. A. Chapman, M. W. Elves and J. Gough

In 72-h PHA cultures the blastoid cells, now very much larger than the few remain-ing small lymphocytes, were the predominant cell type in almost all specimens.Nucleus and cytoplasm were both increased in volume, compared with earlier cultures,although the increase in volume of cytoplasm was significantly greater than that ofthe nucleus. Nuclei possessed finely-divided chromatin, in marked contrast to theclumped pattern present in the nuclei of small lymphocytes; one or two well-developednucleoli were usually visible in a sectioned nucleus. In suitably-oriented sections thenucleus displayed a deep indentation, frequently branched. Fig. 2 shows a typicalblastoid cell in which many of these characteristics are apparent. At this magnificationthe most notable feature of the cytoplasm of these cells is the paucity of the morehighly organized cytoplasmic organelles. Mitochondria were always present but weremostly small and round in section, with elongated forms occurring infrequently; inmost cells the mitochondria possessed numerous well-defined cristae, although evi-dence of mitochondrial swelling and disruption was commonly noted. In many cellsround or stellate electron-opaque granules were prominent; several of these granulesoccur in the cell of Fig. 9; some stellate granules are present in Fig. 8 but granules areabsent from Fig. 2. No' crystalline structures' showing regular periodicity, as describedby Inman & Cooper (1963), were observed. Compound vacuoles and multivesicularbodies (Low & Freeman, 1958) were noted in both blastoid cells and small lymphocytes.

At higher magnification it is immediately apparent that the bulk of the cytoplasmof the larger blastoid cells (such as that shown in Fig. 2) is occupied by ribosomalparticles. A rough calculation shows that a typical cell may contain from io6 to io6

ribosomes, comparable with the number found in haemoglobin-synthesizing rabbitreticulocytes (Mathias, Williamson, Huxley & Page, 1964). The ribosomes, illustratedhere in Figs. 4-7, occurred singly as isolated particles or in clusters containing up to11 ribosomal particles per cluster; in many of these the ribosomes were arranged inrosettes having the typical appearance of polysomes (Slayter, Warner, Rich & Hall,1963; Mathias et al. 1964). In a few instances the ribosomes of a polysomal clustercould be seen to be connected by a thin strand, not more than about 20 A in diameterand usually located on the inner side of the rosette (Figs. 4, 6).

In an attempt to obtain a rough quantitative estimate of the fraction of the totalribosomal population occurring as polysomes, a histogram was plotted to show thedistribution of ribosomal particles in clusters of various sizes. In Fig. 1 this distributionin PHA-transformed blastoid cells in vitro is compared with the corresponding riboso-mal distribution observed by dePetris, Karlsbad, Pernis & Turk (1966) in immunoblastsin vivo. Although only a small number of cells was used to obtain the distribution itwas clear from visual inspection of many other cells that the results were typical.The two distributions show that a far higher proportion of ribosomes occurs as singleparticles in PHA-cultured cells, compared with in vivo immunoblasts; the proportionis also very much greater than that found in haemoglobin-synthesizing rabbit reti-culocytes (Rifkind, Danon & Marks, 1964). To a rough approximation, when certaincorrecting factors are taken into account (see legend to Fig. 1), it would seem thatlittle more than half the total ribosomal population of PHA-transformed cells aregrouped together into polysomal aggregates, compared with not less than 90% in


immunoblasts. Nor is it certain that all the multiple clusters of ribosomes are, in fact,polysomes for many of the clusters consisted only of irregular aggregates of particles,unrecognizable as typical polysomal rosettes (see Fig. 4).

Although endoplasmic reticulum could be detected in most 72-h blastoid cells, itwas severely limited in extent and usually restricted to isolated, elongated cisternae(Figs. 2, 3) or strings of smaller vesicles. It was rare to find more than 4 or 5 easilyrecognizable elongated profiles in a single cell in section, and the field of view ofFig. 3 contains an unusually high density of profiles. A conspicuous and puzzling

_ 40 r

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M 25

20

15

I 10

^ 1 2 3 4 5 6 7 8 9 10 11 12 13 14No. of ribosomes per cluster

Fig. 1. Distribution of ribosomes as single particles and as clusters of various sizes inPHA-transformed blastoid cells in cultured human peripheral blood (solid line). Hydro-cortisone was present in the culture medium. The total number of ribosomes countedwas 6620 in six different cells; the cells were selected at random and were all typical72-h blastoid cells. The ribosomal particles were considered to belong to the samecluster (polysome) when the gap between adjacent particles was not greater than thediameter of a single particle. The dotted line shows the corresponding distribution ofribosomes in guinea-pig lymph-node immunoblasts occurring in vivo in a delayedhypersensitivity response (de Petris et al. 1966). Values are not corrected for shiftingof the distribution towards lower values through transection of clusters by the sectioningplane. The high proportion of clusters of 2 or 3 ribosomes in the m vitro situation maybe partly due to chance association of single ribosomes.

feature of most of these endoplasmic reticulum profiles was the paucity of attachedribosomes. Compared with the dense packing of ribosomes on the endoplasmicreticulum membranes in normal protein-synthesizing and -secreting cells, the mem-branes of the blastoid cell endoplasmic reticulum were relatively devoid of particles,despite the occurrence of ribosomes in large numbers in the surrounding cytoplasm.


Some attachment of particles to membranes did occur, usually with several ribosomesin close proximity (Figs. 4, 5, 7), suggesting an attachment of polysomes to themembrane. No trace of the thin connecting strand was visible in such situations,implying that the strand, if present, was not far removed from the membrane. Ribo-somal particles, singly and in groups, were occasionally observed attached to the outersurface of the perinuclear membrane.

Most 72-h blastoid cells had one or more regions containing a moderately well-developed Golgi apparatus; this was frequently located in proximity to the nuclearcleft and consisted of numbers of flattened sacs and rounded vesicles, all with smooth-surfaced membranes (Figs. 2, 8).

A peculiar, but not uncommon, characteristic of blastoid cells was the presence ofsingle elongated membranes adjacent on one side only to an electron-transparent zone;several of these structures are apparent in Fig. 2. Although probably degenerative orartifactual in origin, they are not immediately interpretable as embedding or sectioningartifacts, as the relative orientation of the membrane component and the clear zonewas independent of the cutting direction; ribosomes or polysomes were sometimesattached to one side of a membrane (away from the clear zone), suggesting that themembrane was derived from a degenerating endoplasmic reticulum sac. In some cellsthese peculiar structures took the form of complex whorls.

Hydrocortisone was added to PHA cultures in an endeavour to bring about astabilization of intracytoplasmic membranes but few significant effects resulted.Electron-opaque granules tended to be fewer and it seemed that polysomal rosetteswere slightly more abundant in hydrocortisone-treated cultures; the histogram ofFig. 1 was obtained from cell cultures to which hydrocortisone had been added.Hydrocortisone is known to depress the incorporation of labelled amino acids and toreduce the numbers of polysomes in thymus cells in rats pre-exposed to the steroid6-12 h before sacrifice (Gabourel & Fox, 1965), but the effect does not occur if hydro-cortisone is added directly to the incubation vessel during the amino acid incorpora-tion period (Gabourel & Comstock, 1964).

The plasma membrane of blastoid cells showed occasional pseudopodial extensionsand a tendency for adjacent cells to be in close apposition. No well-developed filopodiawere seen and there was no evidence of phagocytic activity; pinocytotic vesicles were rare.

A few older PHA cultures were examined. After 5-6 days, extensive necrosis hadoccurred and the few remaining non-degenerate blastoid cells did not differ signifi-cantly in appearance from 72-h blasts.

DISCUSSION

It is now generally accepted that the small lymphocyte is the precursor of theblastoid type of cell in PHA-cultured human blood (Carstairs, 1962; MacKinneyet al. 1962; Elves & Wilkinson, 1963; Schrek, 1963). The salient ultrastructuralfeatures of the PHA-transformed blastoid cell as described by others (Tanaka et al.1963; Inman & Cooper, 1963; Johnson & Roberts, 1964) are in general agreementwith the observations made in the present investigation.

Transformation of lymphocytes to blastotds 365

The main object of this paper is to derive, from a study of blastoid cells at higherelectron-optical magnifications, further information about the significance of thisin vitro transformation. It is evident from our observations that the PHA-transformedcell is one showing the preliminary stages of development of a protein-synthesizingsystem. The concentration of ribosomes in a typical PHA-transformed cell is of thesame order of magnitude as that encountered in rabbit reticulocytes, known to besynthesizing haemoglobin in large amount (Mathias et al. 1964). It has been shown,however, that, in contrast to reticulocytes and immunoblasts, little more than abouthalf the total population of ribosomes in PHA-transformed cells is present as polysomes('polyribosomes'). In such polysomal clusters a thin connecting strand is sometimesresolvable and it seems reasonable to posulate that this strand is messenger RNA(Penman, Scherrer, Becker & Darnell, 1963; Gierer, 1963; Slayter et al. 1963;Mathias et al. 1964).

The restricted development of the endoplasmic reticulum is a characteristic featureof PHA-transformed cells. Although protein can be synthesized in the absence ofintracytoplasmic membranes (as in reticulocytes), a well-developed endoplasmicreticulum does appear to be a functional requirement for the synthesis of proteinsintended for subsequent secretion, as in pancreatic exocrine cells (Caro & Palade,1964), fibroblasts (Chapman, 1961, 1962; Ross & Benditt, 1961) and plasma cells(Braunsteiner & Pakesh, 1955; Thiery, 1955, 1957, 1958; Bernhard & Granboulan,1960; Movat & Fernando, 1962). The limited numbers of isolated cisternae occurringin PHA-transformed cells are recognizable as endoplasmic reticulum sacs by theoccasional attachment of ribosomal particles, but extensive parallel arrays of elongatedprofiles are never found. The attachment of ribosomal particles to the few existingmembranes is sporadic and some membranes are almost entirely devoid of particles.Moreover attached particles tend to occur in groups, indicating the attachment ofpolysomes rather than individual ribosomes; this could mean that messenger RNAis necessary before attachment can occur.

These results are consistent with light-microscope studies in which PHA-trans-formed cells have been shown to synthesize RNA (Mclntyre & Ebaugh, 1962; Epstein& Stohlman, 1964; Hayhoe & Quaglino, 1965; Darzynkiewicz, Krassowski & Sko-pfnska, 1965; Rubin & Cooper, 1965; Gough & Elves, 1966) and protein (Sell, Rowe& Gell, 1965).

In a recent electron-microscopic study of lymphoid tissue during antibody forma-tion Movat & Fernando (1965) have described the fine-structural changes occurringduring maturation of plasma cells following primary and secondary antigenic stimula-tion and have shown that the large pyroninophilic blast cells (designated by theseauthors as 'immunoblasts') become plasmablasts by the development of a polysome-studded endoplasmic reticulum; plasmablasts, in turn, develop into plasma cells. It issignificant that the ' immunoblast' of Movat & Fernando (1965) bears a strikingmorphological resemblance to the in vitro PHA-transformed cell of the presentinvestigation (figs. 21 and 30 of Movat & Fernando's paper should be compared withFig. 2 of the present work). Like the PHA-transformed cell the immunoblast possessesabundant ribosomal material, with variable but restricted amounts of endoplasmic


reticulum, moderate numbers of mitochondria and a complex nucleolus. Moreoverthese investigators present ultrastructural evidence indicating that the immunoblastdevelops from the small lymphocyte, both in the diffuse lymphoid tissue of lymph nodesand periarteriolar lymphoid tissue of the spleen and also in the germinal centres. Theonly significant ultrastructural difference between the in vivo immunoblast of Movat &Fernando and the in vitro blastoid cell of the present study is that the ribosomes of theformer occur almost entirely in polysomal clusters; these migrate to the endoplasmicreticulum membranes as the immunoblast develops into a plasmablast. Similarly,abundant polysomes and few single ribosomes are seen in lymph-node cells involvedin antibody synthesis (de Petris & Karlsbad, 1965), and during the development ofcontact hypersensitivity (de Petris et al. 1966). In other respects these cells are similarto PHA-transformed blastoid cells. These experiments strongly support the view thatin vivo blast cells are derived from lymphocytes, and that a proportion of them candevelop into plasmablasts, and then into plasma cells, in the immune response. Inthis sense the blast cell can be regarded as transitional in character. It is thereforepertinent to enquire why the PHA-transformed cell develops in vitro only as far asthe blast ('blastoid') stage.

The morphological evidence alone is insufficient to provide a clear-cut answer tothis question. It is possible that a simple nutritional deficiency in the culture mediumcould result, for example, in defective or inadequate membrane production. Theapparently degenerate endoplasmic reticulum membranes noted in many blastoidcells lend some support to this possibility, although other membrane systems, suchas that of the Golgi apparatus, show little evidence of degeneration or retardeddevelopment.

An alternative explanation is that the limiting factor is the supply of messengerRNA to the ribosomes. An essential feature of the transformation, both in vitro andin vivo, is the increase in ribosomal content. The present investigation has shown,however, that cultured cells contain a significantly smaller proportion of ribosomesarranged in polysomal clusters, compared with the high proportion of polysomespresent in blast cells in vivo. The observed ribosomal distribution in vitro is entirelyconsistent with a substantially reduced production of messenger RNA. That some ofthe RNA which is produced in PHA-stimulated lymphocytes is messenger RNA isindicated by the pulse-labelling studies of Rubin & Cooper (1965) and Darzynkiewiczet al. (1965), and this is in accord with the observation that some polysomes do occur.Moreover, it has already been noted that ribosomes tend to occur in groups on endo-plasmic reticulum membranes, suggesting an attachment of polysomes rather than in-dividual ribosomes to membranes. If development of the endoplasmic reticulummembranes were dependent on the attachment of polysomes, then the limited productionof messenger RNA could also explain the restricted growth of the endoplasmic reti-culum and the failure of the cultured cells to develop further in the direction of matureprotein-synthesizing cells.

The authors are grateful to Professor Kellgren and Dr Israels for their interest and encourage-ment and to Mrs H. C. Jeffery and Mr S. Grundy for technical assistance. This investigationhas been rendered possible by the financial assistance of the Nuffield Foundation.


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SELL, S., ROWE, D. S. & GELL, P. G. H. (1965). Studies on rabbit lymphocytes in vitro. III.Protein, RNA and DNA synthesis by lymphocyte cultures after stimulation with phyto-haemagglutinin, with staphylococcal nitrate, with antiallotype serum, and with heterologousantiserum to rabbit whole serum. J. exp. Med. 122, 823-839.

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{Received 9 September 1966—Revised 10 February 1967)

370 J. A. Chapman, M. W. Elves andj. Gough

Figs. 2-9 are electron micrographs of blastoid cells in cultures of human peripheralblood, grown for 72 h in the presence of phytohaemagglutinin (PHA). With the excep-tion of Fig. 2, all micrographs are of cells from cultures to which hydrocortisone hadbeen added.

Fig. 2. Typical PHA-transformed blastoid cell, about 13 x 9/* in section. The largenucleus (n) is characterized by finely-divided chromatin, two prominent nucleoli anda deep, branched invagination of the nuclear membrane. The cytoplasm contains feworganelles apart from numbers of mitochondria (m), a Golgi zone (g) and infrequentendoplasmic reticulum cistemae (er); some membranes, presumed to be derivedfrom the endoplasmic reticulum, show a clear zone on one side (arrows). The bulk ofthe cytoplasm is occupied by ribosomes. x 16000.

Journal of Cell Science, Vol. 2, No. 3

J. A. CHAPMAN, M. W. ELVES AND J. COUGH (I) (Facing p. 370)

Fig. 3. Endoplasmic reticulum cisternae (er) in the cytoplasm of a blastoid cell.This field of view shows an unusually high density of profiles, x 30000.Fig. 4. Ribosomes as single particles (r) and linked in polysomes (p) in a blastoidcell. In some polysomes the ribosomes can be seen to be connected by a thin strand(mr), presumably messenger RNA. Other groups of ribosomes consist merely of irregu-lar aggregates of particles (a), unrecognizable as polysomes; these were, however,counted as polysomal clusters in the ribosomal distribution of Fig. 1. Some attach-ment of ribosomes to membranes occurs (arrows), usually with several ribosomes inclose proximity, x 100000.


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J. A. CHAPMAN, M. W. ELVES AND J. GOUGH (I)

Fig. 5. Single ribosomes (r), polysomes (p), irregular aggregates of ribosomes (a)and an endoplasmic reticulum membrane (er) with some attached ribosomes.x 160000.

Fig. 6. A field of view showing a high density of polysomes. In some instances thethin connecting strand (mr) is detectable. It is possible that this section is grazing thesurface of an endoplasmic reticulum cisterna; there is evidence of a membranesystem in the lower right-hand corner, x 160000.


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Fig. 7. Attachment of ribosomal particles in groups (arrows) to an endoplasmicreticulum membrane (er). Also shown is the cytoplasmic membrane (cm) and thenuclear membrane (nm). x 160000.


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Fig. 8. Golgi apparatus (g), stellate granules (sg), mitochondria (PI) and centrioles(c) in a blastoid eel), x 25000.Fig. 9. Round, electron-opaque granules (rg) and irregular stellate granules (sg) ina blastoid cell. Also shown are a number of incomplete endoplasmic reticulummembranes associated with a clear zone on one side (arrows), x 25000.


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J. A. CHAPMAN, M. W. ELVES AND J. COUGH (I)

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