J. Cell Set. 12, 491-523 (i973) 491Printed in Great Britain

ERYTHROPOIESIS IN THE NEWT, TRITURUS

CRISTATUS LAUR.

II. CHARACTERISTICS OF THE ERYTHROPOIETICPROCESS

J. A. GRASSODepartment of Anatomy, Boston University School of Medicine,80 East Concord Street, Boston, Massachusetts 02118, U.S.A.

SUMMARY

In splenectomized newts (Triturus cristatus Laur.) rendered anaemic by acetylphenylhydrazine(APH), an erythropoietic response is delayed so that the animals are completely devoid of theerythron, including erythrocytes. At 11-14 days after APH, 'erythroid precursor cells' (EPC)in the blood signal the occurrence of an erythropoietic response. Ultrastructural studies haveshown few or no ribosomes in EPCs, but well developed nucleoli and intense RNA synthesisare seen in these cells. Correlated morphological and cytochemical data indicate the productionof ribosomes in EPCs, a process culminating in the formation of basophilic erythroblasts (BE).Microphotometric studies show the accumulation of haem during this interval. Thus, in EPCsand BEs, both ribosomal (rRNA) and messenger (m) RNA are synthesized, making possiblethe early synthesis of haemoglobin. In subsequent stages, nucleoli exhibit a size decrease, mostevident in the particulate component, while all RNA synthesis ceases during the mid-poly-chromatophilic erythroblast (MPE) stage. Coupled with the gradual loss of ribosomes charac-teristic of this developmental period, the results suggest that rRNA synthesis occurs in EPCs,BEs, and in early polychromatophilic erythroblasts, where it is completed. Haemoglobin mRNAis also formed in these stages since ultrastructural and microphotometric data show the accumu-lation of haemoglobin. Beyond the MPE, haemoglobin production is dependent upon stablemessenger RNA since no RNA synthesis is detected in this period. Ultrastructural studiesdemonstrate that autophagy may play an important role in the loss of cytoplasmic organellescharacteristic of the erythropoietic process. The occurrence of swollen cristae and outer com-partment in mitochondria coupled with the presence of myelin-like membrane whorls inassociation with mitochondria is seen in all erythroid cells except EPCs. Membrane profilesderived from endoplasmic reticulum are frequently encountered in the vicinity of mitochondriaand appear to encircle these organelles and adjacent cytoplasm to form autophagic vacuoles.In the process, the limiting membranes assume a dense laminar appearance. Elements of theGolgi complex also display morphological alterations suggestive of degeneration. Cytoplasmicbodies of varying appearance and content, some of which are similar to lysosomes in other celltypes, are seen in all erythroid cells. Ferritin is seen in some bodies, in vacuoles, or in aggre-gates, but does not appear in micropinocytotic vesicles.

INTRODUCTION

Administration of acetylphenylhydrazine (APH) to salamanders (Triturus sp.)results in destruction of mature erythrocytes with concomitant loss of haemoglobin(Grasso & Shephard, 1968). Despite the complete anaemia, no immediate erythro-poietic response occurs in treated animals which, therefore, display a total absence ofthe erythron. Within 2 weeks after the initial APH injection, erythropoiesis takesplace in the peripheral circulation, such activity marked by the appearance of manylymphocyte-like cells described tentatively as 'erythroid precursor cells' (Grasso,

32-2

492 J. A. Grasso

1973). These cells have been shown to give rise to basophilic erythroblasts, a cell typewhich forms 80-90% of the total erythroid cell population during the 15-18 dayinterval after APH. In this paper, the ultrastructural and cytochemical features of theerythropoietic response in totally anaemic newts (Triturus cristatus) are described.

For biochemical analyses of sequential events in RBC differentiation, it is desirableto possess a system in which (1) the earliest progenitor cell, especially the uncom-mitted stem cell, can be identified and obtained in abundance, and (2) homogeneouspopulations of the respective erythropoietic stages can be obtained for study in theirproper developmental sequence. The erythropoietic system encountered in totallyanaemic newts (T. viridescens, T. cristatus) after APH injection closely approximatesthese requirements and offers a potentially valuable system for diverse studies ofRBC development. The present studies were undertaken to define the system and toserve as a basis for further cytological and biochemical analyses of the RBC differ-entiative process.

MATERIALS AND METHODS

The procedure used to produce total anaemia with acetylphenylhydrazine (APH) has beendescribed previously (Grasso, 1973). For electron microscopy, peripheral blood was collectedat various times after APH injection by either cutting the tail or decapitation and allowing bloodto flow directly into fixative contained in 12-15 n^ conical centrifuge tubes. The fixatives usedwere: (a) 1% OsO4 in 0-2 M phosphate buffer (pH 7-2-7-5); (b) paraformaldehyde-glutar-aldehyde mixture in 005 M cacodylate buffer at pH 6-9 (Karnovsky, 1965); (c) paraformalde-hyde-glutaraldehyde-acrolein mixture in 0-05 M cacodylate buffer containing 1 % CaCl, and2 % dimethyl sulphoxide (Kalt & Tandler, 1971). For routine light microscopy, blood smearswere stained with Wright's stain.

For microphotometric measurements of cytoplasmic RNA, methanol-fixed smears werestained in 0025 % azure B in Mcllvaine's buffer at pH4-o. Measurements were made at 540 nm.Haem absorption was measured on similarly prepared smears at a wavelength of 414 nm. Therelative total amount of absorbing substance was calculated by the formula M = EA, whereM = relative total amount, E = extinction or absorbance, and A = area of cytoplasm deter-mined with a polar planimeter on camera lucida drawings.

Total RNA synthesis in respective erythropoietic stages was measured by radioautographicprocedures. Animals received 40-50 /tCi of [*H]cytidine (specific activity 6 Ci/mmol) by intra-peritoneal injection. After intervals of 10 min-7 days, smears were prepared, fixed in methanolfor 10—15 niin, and processed for radioautography. Extractions were performed in DNase,RNase, or ice-cold 5 % trichloroacetic acid (TCA) to demonstrate specific incorporation of thelabelled precursor. Smears were then coated with Kodak NTB-2 emulsion, stored at 4 °C, anddeveloped in D19 for 2-5 min. Grain counts were made over individual cells on DNase-extracted smears which were stained briefly (3-5 min) in 0-025 % azure B or Giemsa.

The nomenclature and sequence of erythropoietic stages used in these studies is: erythroidprecursor cell (EPC) ->• proerythroblast (PrE) -> basophilic erythroblast (BE) -*• early, mid-,and late polychromatophilic erythroblasts (EPE, MPE, and LPE) -*- reticulocyte (RETIC) ->erythrocyte (RBC).

RESULTS

Effects of acetylphenylhydrazine (APH)

The injection of acetylphenylhydrazine in T. cristatus or T. viridescens resulted indegeneration of erythrocytes within 12-24 h, a process initially recognized by ex-

Newt erythropoietic process 493

tensive vacuolization of the nucleus and cytoplasm and subsequently by fragmentationof the entire cell body. By 24-36 h after injection, RBC fragments were found lyingfree in the plasma or engulfed by free macrophages. The latter constituted the largestfraction of peripheral blood cells at this time and were the only blood elementsobserved to be phagocytic. Within 48-72 h, all erythrocytes were destroyed ordamaged and the colour of the blood had changed from a deep red to a rust hue.Phagocytic free macrophages were most abundant, though their origin was unclear.Many cells similar in appearance to erythroid precursor cells (Grasso, 1973) wereseen in the blood at this time. By 7 days after injection, the erythrocytic fragmentshad been cleared from the plasma and the free macrophages were greatly reduced innumber. The blood was a clear transparent fluid containing granulocytes, thrombo-cytes, small lymphocytes, and some macrophages. However, there was no evidenceof erythrocytes nor of erythroid regeneration, despite the total anaemia. Spectro-photometric analysis of blood plasma revealed the absence of a Soret peak or ofoxyhaemoglobin peaks (Grasso & Shephard, 1968).

In splenectomized newts of both species, erythropoiesis occurred in the peripheralblood and was recognized by the appearance of numerous EPCs at 11-14 days afterinitial APH injection (Grasso, 1973). The morphology and behaviour of these cellshave been described in a previous report (Grasso, 1973). During the 11- to 14-dayperiod, EPCs were the most abundant cellular element in the blood, consisting of aheterogeneous population of cells characterized by variable size and variable cyto-plasmic basophilia (Grasso, 1973). Mainly on day 14, though occasionally earlier,basophilic erythroblasts made their appearance and rapidly became the predominantblood cell type in the 15-18 day interval after APH. During this latter period, BEsformed 80—90% of the total erythroid cell population, the remaining erythroid ele-ments represented by EPCs initially and later by polychromatophilic erythroblasts.Thus, in 2 time intervals, it was possible to obtain nearly homogeneous populationsof early erythroid cells: EPCs during the 11- to 14-day period and BEs from days15 to 18.

On day 17, early polychromatophilic erythroblasts (EPE) were seen and, by19-20 days after APH, most of the erythroid cells consisted of mid-polychromato-philic erythroblasts (MPE). Some BEs and an occasional EPC were noted at this time.On days 22-25 a^er APH, the blood contained a variety of mid- and late polychro-matophilic erythroblasts (M, LPE). Frequently, during this period, EPCs reappearedin abundance, an observation interpreted to signal the beginning of a second erythro-poietic wave since BEs subsequently increased. By days 32-35, some mature erythro-cytes could be found but their occurrence was variable; sometimes they did notbecome visible until day 40. With the appearance of RBCs, the erythroid system wasessentially similar to that seen in usual erythropoietic responses, i.e. a heterogeneoussystem of erythroid cells at different levels of development.

In unsplenectomized newts treated with APH, the initial erythropoietic responseoccurred generally at 7-8 days after injection, visible as an accumulation of numerousbasophilic erythroblasts in the blood. However, at 11-12 days after APH, erythroidprecursor cells also appeared in abundance, an occurrence which coincides temporally

494 7- A. Grasso

with their appearance in APH-treated, splenectomized newts. These results suggestedthat the spleen and peripheral circulation are separately involved in an erythropoieticfunction. The presence of BEs in the blood at 7-8 days probably represents theirrelease from the spleen, but their accumulation does not prevent the appearance ofEPCs in the blood at 11-12 days. While these results were obtained consistently, nostudies were made of erythropoiesis in the spleen since our objective was to definethe blood erythropoietic system for subsequent studies.

There was no indication of RBC development in the liver, heart, kidneys, orintestinal wall in splenectomized, anaemic newts. In all experiments, erythropoiesiswas endemic in the peripheral circulation.

Phenylhydrazine seemed to have no effect on erythroblasts or non-erythroid cellsat the dosages used in these studies. With higher doses, thrombocytes and granulo-cytes were damaged.

Erythroblasts

Light microscopy. Basophilic erythroblasts were the prevalent component of theblood at 15-18 days after APH and exhibited intense cytoplasmic basophilia (Figs. 2,3)which often obscured nuclear detail. Their nuclei were identical to those describedfor EPCs (Grasso, 1973), consisting of loosely woven chromatin blocks or massesseparated by thin interchromatin areas and containing one or more nucleoli.

Beyond the BE stage, the light-microscopic features of the various erythropoieticstages were largely similar to those described in the normal blood of T. viridescens(Grasso & Woodard, 1966) and in the spleen of T. cristatus (Tooze & Davies, 1967).Briefly, the erythropoietic process subsequent to the BE was characterized by: (1) acontinual decrease in cytoplasmic basophilia, (2) an increase in cytoplasmic acido-philia, (3) a decrease in nuclear size accompanied by increased density of the chromatinblocks or masses, reflecting heterochromatinization of the chromatin, (4) decrease insize and visibility of the nucleoli, and (5) a change in cell shape from a round topredominantly oblong form. Some of these aspects are illustrated in Figs. 2-4.

In the erythropoietic response following production of total anaemia, the erythroidcells varied greatly in size within each given developmental stage (Figs. 5-7). In theearlier (BE, EPE) and intermediate to late (MPE, LPE, RETIC) stages, cell division,measured by pHJthymidine labelling and microphotometric quantitation of Feulgen-DNA values, was present, although occurring most frequently in earlier stages anddecreasing in the later stages (Walker, 1971). Thus, many of the smaller cells seen atdifferent developmental stages probably represented recently formed daughter cellswhile the larger cells were probably late interphase cells or cells already withdrawnfrom the cell cycle. (Go). Thus, one reason for size variation was obviously the occur-rence of asynchronous division which was especially prevalent in the early erythroidstages.

Alternatively, variation in cell size could reflect the establishment of several erythro-poietic lines in response to total anaemia, each synthesizing different haemoglobinssuch as has been reported in sheep in response to anaemia (Van Vliet & Huisman,1964; Blunt, 1965; Gabuzda, Schuman, Silver & Lewis, 1968) or in a fashion similar

Newt erythropoietic process 495

to the metamorphosing effects of thyroxine in frog tadpoles (Moss & Ingram, 1968;DeWitt, 1968). In preliminary experiments in which haemoglobin from late erythroidstages was electrophoresed, no evidence for haemoglobin other than the adult typehas been noted (Grasso & Troxler, work in progress). Thus, the cause of size variationin individual developmental stages appeared to be mainly due to mitosis within asyn-chronous cell populations, but contributions from other sources cannot be ruledout.

Electron microscopy. In most respects, the ultrastructural appearance of basophilicerythroblasts was similar to the EPC. The main difference, and the basis for the dis-tinction, was the presence of rich ribosomal concentrations in the cytoplasm of theBE (Figs. 8, 14, 20). The ribosomes were arranged mainly as long chains or clusters,often surrounded by a low to moderately dense amorphous component (Fig. 20)presumed to represent haemoglobin. The occurrence of this component was highlyvariable and, in many cells, its presence was not impressive. The nuclei of BEs con-tained distinct masses or blocks of loosely woven chromatin between which the inter-chromatin areas were enclosed (Fig. 8). The occurrence of numerous dense granulesidentical to those seen in EPCs (Grasso, 1973) was impressive in the nuclear inter-chromatin zones (Figs. 8, 14). As in EPCs, the granules fell into 3 size classes(25—37-5 nm, 40-55 nm, and > 60 nm), with the 40-55 nm components most abundant.One or more nucleoli were present, always containing prominent fibrillar andparticulate zones (Figs. 8, 16).

Mitochondria occurred as single entities or clustered in groups which were scatteredthroughout the cytoplasm (Fig. 8). With osmium fixation, many mitochondria re-vealed rather aberrant forms in that the cristae or the outer compartment were swollen(Fig. 8). This phenomenon was noted in all erythroid cells and is considered later inmore detail.

The Golgi complex was usually small, consisting of a few elements of lamellae andvesicles. Endoplasmic reticulum was limited to a few scattered granular profiles.Occasional lipid droplets were present.

A variety of vesicles of the smooth-surfaced and coated types (Grasso, 1973)occurred in all BEs. As in EPCs, they lacked any apparent content. Ferritin wasusually contained within specific structures and was not clearly defined as dispersedor free ferritin. These ferritin-containing structures are considered below.

With the onset of the polychromatophilic stages of erythroblast development, theelectron-microscopic appearance of the cells was unequivocally recognizable aserythroid. None of the characteristics to be described were specific for any givenpolychromatophilic stage or reticulocyte but instead occurred as part of a continualprocess terminating in the production of a mature erythrocyte. The following descrip-tions of erythropoietic morphology use the basophilic erythroblast as a referencepoint for comparison.

The chromatin of early polychromatophilic erythroblasts (EPE) was increased indensity and had assumed a coarser texture than in BEs (Figs. 9, 10). Indeed, exceptfor its more dense appearance, the distribution of the chromatin masses in basophilicand polychromatophilic erythroblasts was similar. Nucleoli in EPEs were smaller,

496 J- A. Grasso

especially in the extent of the granular component (Figs. 10, 17). The fibrillar com-ponent often exhibited a very dense appearance which clearly distinguished it fromthe granular component and the nucleolus-associated chromatin (Figs. 9, 10). Theinterchromatin regions remained extensive and contained numerous nuclear granules(Figs. 9, 10), although a decrease in the concentration of the particles was alreadyevident in many EPEs. In addition to the granules, the interchromatin regions con-tained distinct fibrillar areas which were denuded of nuclear granules (Fig. 9). Theseareas have been described previously in splenic erythroid cells and were called'nuclear light-staining zones' (Tooze & Davies, 1967). In some respects, they weresimilar to the larger, loose-textured chromatin masses of EPCs and basophilic erythro-blasts. It is possible that these zones represent areas of euchromatin rendered morevisible by the disappearance of the dense granules.

In subsequent stages (MPE, LPE, RETIC), the proportion of the dense chromatinmasses increased so that, in reticulocytes and mature RBCs, the bulk of the nucleusconsisted of dense chromatin clumps (Fig. 13). In MPEs and LPEs, the nucleoli weresharply decreased in size, appearing as small, sharply circumscribed nubbins of thefibrillar zone with little or no particulate component (Figs. 18, 19). The interchromatinregions exhibited an increased density (Figs. 11-13, 15) as a result of haemoglobinaccumulation (Davies, 1961; Tooze & Davies, 1963; Small & Davies, 1970). Thenuclear granules were greatly reduced in number (Figs. 9—13, 15). Size measurementsof granules in all 3 polychromatophilic stages gave a single class whose size (37*5—55 nm) and appearance was similar to perichromatin granules (Watson, 1962; Swift,1962; Monneron & Bernhard, 1969). Few particles in the 25-37-5 nm or > 60 nmrange were observed. In several non-dividing cells, granules of similar appearancewere observed in the cytoplasm (Fig. 21).

The major nuclear modifications can be summarized as: (a) increased density andproportion of chromatin clumps, (b) decrease in nucleolar size especially evident inthe decrease of the particulate zone, (c) haemoglobin infiltration of the interchromatinregions, and (d) decline in number of nuclear particles with enrichment of the peri-chromatin granule population. The total concentration of all nuclear granules, how-ever, was decreased considerably with respect to earlier stages.

Within the cytoplasm, the most obvious feature was the accumulation of haemo-globin which increased in density concomitantly with the level of maturation(Figs. 20-23). Haemoglobin accumulation occurred also within the nuclear inter-chromatin regions, the 2 compartments (nuclear and cytoplasm) being in apparentcontact at the nuclear pores. Embedded within the haemoglobin were single ribosomesor multiple clusters consisting of varying numbers of ribosomes, in contrast to thepredominant long-chain aggregates of ribosomes seen in BEs. In MPEs and later stagesa decrease in ribosomal concentration was evident (Figs. 14, 15, 20-23), a pheno-menon described in numerous erythropoietic systems (Grasso, Swift & Ackerman,1962; Rifkind, Danon & Marks, 1964; Tooze & Davies, 1967). Scattered profiles ofgranular endoplasmic reticulum continued to be present, especially in proximity tomitochondria (see below).

Microtubules were present chiefly in the form of a marginal bundle subjacent to

Newt erythropoittic process 497

the plasma membrane (Fig. 32). Although best preservation of these elements wasobtained with aldehydes, microtubules were seen even after primary fixation inosmium. In sections passing transversely through the cell poles, the marginal bundleappeared to consist of 80-140 individual microtubules whose diameter was approxi-mately 21-25 nm (Fig. 32).

Micropinocytotic vesicles and vacuoles persisted throughout the intermediate andlate erythroid stages although they were not as abundant as in earlier stages. Signifi-cantly, micropinocytotic vesicles irrespective of their location or stage of formationnever contained ferritin particles (Figs. 27, 28) nor was any evidence of ferritinadsorbed to the plasma membrane ever observed. However, dispersed ferritin lyingfree within the cytoplasm was often seen in the polychromatophilic stages andreticulocytes (Figs. 24, 25, 32) but was absent in mature erythrocytes. Ferritinaggregates of varying concentrations were frequent, both in membrane-limitedvacuoles or bodies (Figs. 33, 35) and in clusters in which a limiting membrane wasdifficult to discern (Fig. 34).

The mitochondria of most polychromatophilic erythroblasts and reticulocytesdisplayed an unusual appearance, presumably as a consequence of autolytic pheno-mena involving these organelles. Some displayed the presence of a dense infiltratewithin the matrix (Fig. 29). In osmium-fixed cells, the mitochondrial matrix containedconspicuous dense granules and the cristae were often swollen (Figs. 29, 31). Fre-quently, in EPEs and subsequent stages, the outer compartment was dilated, forminglocalized blister-like protrusions of various sizes on the mitochondrial surface (Figs. 29,31). These protrusions seemed to arise by a contraction of the inner membrane or bydilatation of the outer membrane. The high frequency of the surface dilatationsimparted a vacuolated appearance to some erythroblasts at low magnification.

In aldehyde-fixed cells, similar surface dilatations were seen but were always associ-ated with unusual dense structures (Figs. 9, 12, 24, 25) which seemed to consist ofwhorls of membranes (Figs. 24, 25) similar to the myelin-like figures described byTooze & Davies (1967). The membrane whorls or myelin-like figures occurred any-where on the mitochondrial surface and often formed spherical or elliptical cap-likeprotrusions at the mitochondrial poles (Figs. 24, 25). The protrusions usually weredevoid of content but occasionally contained material of varying density. Densemembrane whorls also occurred in the cristae independently of surface dilatations butless frequently. Often, these internal, myelin-like whorls abutted against, or projectedthrough, the surface.

Single profiles of membranes, sometimes clearly recognizable as, or derived from,the granular endoplasmic reticulum, were found close to or surrounding individual,or groups of, mitochondria (Fig. 24). Occasionally, autophagic vacuoles were seen inwhich mitochondria and adjacent cytoplasmic structures were contained (Figs. 26, 30).Most of the autophagic vacuoles were limited by a dense laminar wall (Figs. 26, 30)with a similar thickening occurring in the endoplasmic reticulum near the point ofcontact with the mitochondrion or its myelin-like figure (Fig. 24). This finding sug-gests that the membranes of the endoplasmic reticulum are involved in walling offthe autophagic vacuole, undergoing a thickening in the process. Apparent fusion of

498 J. A. Grasso

EPC BE EPE MPE LPE

Cell stage

RETIC RBC

Fig. i. Results of microspectrophotometric measurements of cytoplasmic azureB-RNA and cytoplasmic haem, performed at 540 and 414 nm respectively. • #,RNA concentration; 0 #, relative total amount of RNA; 9 • , haem con-centration; # 9, relative total haem amount.

mitochondria with heterogeneous bodies presumed to be lysosomes was sometimesencountered (Fig. 25).

A number of dense bodies and vacuoles occupied the cytoplasm of erythroblastsand reticulocytes, all of which varied greatly in size, content, and appearance. Theseincluded: (a) dense bodies, 0-5-1-2 fim in width, containing crystalline arrays (Fig. 37),(b) dense bodies containing a moderately dense substance against which was super-imposed a varying number of ferritin particles (Fig. 33), (c) tightly packed ferritinaggregates contained within a membrane-limited vacuole (Fig. 35), (d) dense bodiessimilar to 'b' but which contained irregular whorls and amounts of membrane(Figs. 38, 39), (e) multivesicular bodies (Fig. 40), (/) vacuoles with a finely granularcontent (Fig. 36) similar to cytolysomes or vacuolar polar bodies in amphibianerythrocytes (Tooze & Davies, 1965), and (g) dense membrane, myelin-like whorls ofvarying shape. While most of the latter were probably associated with mitochondrianot evident in the plane of section, dense membrane whorls, not associated withmitochondria, were located occasionally at the plasma membrane, a finding which wasrarely encountered in OsO4-fixed material. Thus, it was not clear whether suchmembrane formations were aldehyde-fixation artifacts.

The Golgi complex, regardless of fixation, was greatly dilated, often displayingmembrane whorls in association with the lamellae (Figs. 27, 28). Given the similaroccurrence of such structures in mitochondria and the aberrant appearance of theGolgi elements in both types of fixatives (Figs. 27, 28), these findings were consideredprobably to reflect the presence of degenerative phenomena within the Golgi complex.

Newt erythropoietic process 499

Table 1. Concentrations and relative total amount of azure B bound tocytoplasmic RNA in erythroid cells

Cell type

EPC (12)BE (38)EPE (25)MPE (45)LPE (15)RETICRBC

Average absorbance(Extinction) + S.D.

0-069 ±00320-449 ± 0-020

0-288 ±0-0670193 ±0-0330-151 ±0-020

• •• •

Relative total amount(Extinction x area) ± S.D.

•

41-4 ±9-736-1 ±8-736-1 ±9-227-7 ±5-7

•*• •

• Not determined due to inability to measure cytoplasmic area.• • Not available for measurement in these preparations.Figures in parentheses indicate number of cells.

Cell

No.

Table 2.

stage

grains//tm'

?H\cyt

EPC

0341

idine

BE

0-305

incorporation in

EPE MPE

0284 0-141

erythroid cells

LPE RETIC

0 0

RBC

0

Cytochemical studies

RNA. Microphotometric measurements of azure B binding to cytoplasmic RNArevealed low absorbancy (concentration) in EPCs with values slightly higher thanbackground levels (Fig. 1; Table 1). Cytoplasmic RNA was at its highest concentra-tion in basophilic erythroblasts where it was increased 6- to 7-fold with respect to theEPC. Subsequently, the concentration of azure B/RNA decreased sharply to theEPE stage, then declined more gradually through successive maturation stages (Fig. 1).Although no mature erythrocytes were present in these preparations, measurements ofRBCs in other instances have demonstrated sufficiently the absence of cytoplasmicRNA in mature cells.

When expressed as relative total amount of cytoplasmic azure B/RNA the curve hada significantly different shape (Fig. 1). Again, the highest level occurred in basophilicerythroblasts but the subsequent decline was not dramatic, decreasing only by 13%during the BE-MPE interval. Beyond the MPE, the decrease was more rapid and pre-sumably reached zero in the mature RBC. Due to the impossibility of determiningthe cytoplasmic area in EPCs the total amount of azure B/RNA in these cells couldnot be expressed.

In radioautographs of blood smears prepared 1-1-5 h ̂ sr injection of ^HJcytidine,the highest level of incorporation was observed in the nuclei of EPCs, basophilicerythroblasts, and EPEs (Table 2). Cytoplasmic grain counts were negligible at thistime. In MPEs, incorporation was decreased to 35-50% of the activity seen in earlierstages (Table 2). Moreover, while the labelling index (% cells labelled) in earlierstages was high (60-100%), the frequency of labelling in MPEs was 40% or less.No labelling was observed in LPEs, reticulocytes, or mature RBCs.

500 J. A. Grasso

Table 3. Concentration and relative total amount of cytoplasmic haemin erythroid cells

Cell type

EPC (8)BE (17)EPE (s)MPE (22)LPE (20)RETIC (15)RBC (12)

• Not determined due toindicate number of cells.

Average absorbance±S.D.

0-0080073 ±0-0140-068 ± 0-0070-084 ± O - O I I0102 ±0-0080-123 ±0-0240-162 ±0-027

inability to measure cytoplasmic

Average relativetotal amount

±S.D.

•

io-8±3-s11-5 ±2-1

16-5 ±4-724-S ±5'330-9 ±7-443-6 ± IO-I

area. Figures in parenthese;

Haem. Cytoplasmic haem absorbance at 414 nm was absent in most EPCs withvalues equivalent to background or to extinctions in cells known not to containhaemoglobin. In one experiment, however, 20% (3 out of 14) of the EPCs were clearlyhigher than background, suggestive of the presence of cytoplasmic haem. Haemabsorbance was recorded in the cytoplasm of all basophilic erythroblasts measuredand continued to increase steadily in successive maturation stages (Fig. 1, Table 3).The relative total amount of cytoplasmic haem essentially paralleled the increase inconcentration (Fig. 1). Again, this parameter could not be determined in EPCs dueto the inability to measure accurately the cytoplasmic area of these cells.

DISCUSSION

In splenectomized newts rendered anaemic by APH, erythropoiesis occurs only inthe blood, a result observed in both T. cristatus and T. viridescens. This finding con-firms the observations of Jordan & Speidel (1930), who reported that splenectomy inT. viridescens was followed by erythropoietic activity in the peripheral circulation.These authors found no evidence of RBC formation in the lymphogranulocytopoieticcapsule of the liver, the intertubular regions of the kidneys, the intestinal wall, or theheart wall, a result confirmed in the present study. Indeed, the blood appears to be anerythropoietic locus in its own right, as indicated by our observations that unsplen-ectomized newts, made anaemic by APH, exhibited 2 overlapping erythropoieticwaves at approximately 8 and 11 days after APH, respectively. The initial burst at8 days featured many basophilic erythroblasts (BE) in the blood, the BEs presumablyreleased from the spleen. The second wave at 11-12 days was represented mainly byEPCs and was confluent in time with the single burst seen in anaemic splenectomizedanimals.

Presumably, T. cristatus survives the absence of erythrocytes in a way similar to thatpostulated in totally anaemic T. viridescens (Grasso & Shephard, 1968). In the latter,the plasma volume was found to be critical, the loss of only 5-10 /tl often being fatal.

Newt erythropoietic process 501

This finding, combined with oxygen consumption rate measurements in totally anaemicnewts, suggested that these animals were meeting their requirements by solution ofoxygen in the plasma. Such a mechanism has an advantage in this genus due to theextensive vascularity of the skin which is responsible for 70-75 % of respiration innormal animals (Czopek, 1959). Yet the fact remains that APH-treated newts of bothspecies are completely devoid of all RBCs and haemoglobin but do not respond to thissupposed stress state for about 2 weeks. Recently we obtained similar results in theJapanese newt, T. pyrrhogaster, and, to some extent, in adult Necturus maculosus. Thereasons for the delayed erythropoietic response in anaemic newts and the mechanismsunderlying its initiation and control are unknown.

Renewed erythropoiesis in anaemic animals is always associated with the occurrencein the blood of numerous lymphocyte-like cells termed 'erythroid precursor cells'(EPC). In their earliest recognizable form, EPCs demonstrate variability in cell sizeand, most significantly, a cytoplasm which is weakly basophilic or unstained (Grasso,1973). Evidence that these cells are involved in an erythropoietic function has beenpresented in a previous report (Grasso, 1973). Based upon our experience with thissystem, it is our opinion that EPCs represent one of the earliest erythroid elements,not far removed from the haemopoietic stem cell. Indeed, it is quite possible thatEPCs may be identical morphologically to pluripotential stem cells, i.e. that, althoughfunctional evidence indicates their commitment to RBC production, the morphologyof these cells has not yet been affected by this commitment.

As demonstrated by radioautographic analysis of pHJcytidine incorporation, RNAsynthesis is pronounced in EPCs and BEs but declines rapidly in early and mid-polychromatophilic erythroblasts (E, MPE). Not only is a lower level of incorporationseen in the latter, but fewer cells are labelled. No RNA synthesis can be detected insubsequent stages, i.e. late polychromatophilic erythroblasts (LPE), reticulocytes(RETIC), or mature erythrocytes (RBC). These results are similar to those reportedin rabbit foetal (Grasso, Woodard & Swift, 1963) and adult newt erythropoiesis(Grasso & Woodard, 1966), results that served as the basis for our conclusions thathaemoglobin messenger RNA (Hb mRNA) was synthesized and established duringthe early stages of RBC differentiation, prior to the bulk of haemoglobin productionand accumulation in subsequent developmental stages. Although some RNA synthesishas been reported in later developmental stages (Scherrer et al. 1966), especially inanaemic animals, prolonged synthesis of RNA can be explained largely on the basisof differences between erythropoiesis in normal and anaemic animals (Borsook,Lingrel, Scaro & Millette, 1962; Borsook, 1964). The anaemic newt does not seem topresent the type of stress state seen in birds and mammals where the response tophenylhydrazine-induced haemolysis is an immediate erythropoietic burst in thehaemopoietic organs.

Although the results of the present study were obtained from morphological andcytochemical methods and lack the specificity of biochemical characterization, the datanevertheless allow us to reach certain conclusions concerning the types of RNA formedwith respect to erythroid developmental stages. First, ribosomal RNA synthesis andits conversion to cytoplasmic ribosomes occurs during the development of the EPCs

502 Jf. A. Grasso

into BEs and is largely completed during the EPE stage. This conclusion is supportedby the observations that: (i) EPCs initially show a lack or paucity of cytoplasmicribosomes which accounts for the faint basophilic reaction of these cells (Grasso, 1973);(2) EPCs are intensely engaged in RNA synthesis at a level approximately equal tothat in BEs; (3) nucleoli, now accepted as the site of ribosomal production (Birnstiel,Chipchase & Hyde, 1963; Brown & Gurdon, 1964; Brown, 1967; Perry, 1967), aremost prominent and best developed in EPCs and BEs but the nucleolar particulatecomponent begins to decrease in EPEs and is largely absent in MPEs and subsequentstages; and (4) the EPC population is characterized by an increase in ribosomalnumber, culminating in the formation of the intensely basophilic BE, an increasewhich corresponds to the increase in azure B-RNA concentration measured in thisinterval. These observations demonstrating increased ribosomal presence and majornucleolar modifications suggest that ribosomal production occurs in the earliestrecognizable stages of RBC differentiation and ceases in the EPE. Both the decreasein ribosomal concentration seen in ultrastructural studies and the measured decreasein azure B-RNA absorbance beyond the EPE support this conclusion.

Several biochemical studies of different erythropoietic systems also revealed theearly occurrence and cessation of rRNA production. In anaemic ducks, rRNA wasmore evident in erythroid cell preparations rich in early developmental stages (Attardi,Parnas, Hwang & Attardi, 1966; Scherrer et al. 1966; Attardi, Parnas & Attardi, 1970).Similarly, in mouse bone marrow, rRNA synthesis was found to have kinetic featuresconsistent with its limitation to earlier developmental stages (Evans & Lingrel, 1969),the authors suggesting that the bulk of rRNA production took place in basophilicerythroblasts.

In addition to rRNA, Hb mRNA must also be synthesized and transferred to thecytoplasm in EPCs since: (a) EPCs actively incorporate labelled <J-aminolaevulinicacid (ALA) which preliminary studies have shown as representing incorporation intohaemoglobin (Grasso, 1973), and (b) an increase in haem absorbance, suggestinghaemoglobin accumulation, is noted in the interval EPC-BE (Fig. 1). Both theseobservations support the contention that Hb mRNA is already present in the cyto-plasm of EPCs and further suggest that the initial event of haemoglobin synthesismay be largely under transcriptional control, i.e. that translation of Hb mRNA occursat the time of, or soon after, its initial appearance in the cytoplasm of EPCs. This doesnot preclude the possibility of translational control at later stages such as has beensuggested by the effects of exogenous haem on globin synthesis (Bruns & London,1965; Waxman & Rabinowitz, 1966; Maxwell & Rabinowitz, 1969; Ponka, Neuwirt,Sperl & Brezfk, 1970).

Again, results in other erythropoietic systems can be correlated with our data. Thus,in chick blastoderms, haemoglobin synthesis was detectable in early stages (Granick &Levere, 1965; Levere & Granick, 1967) and was sensitive to actinomycin D treatment("Wilt, 1965; Levere & Granick, 1967). In humans, haemoglobin was found in pro-erythroblasts by cytophotometric determinations (Yataganas, Gahrton & Thorell,1970). In mouse foetal hepatic erythroblasts, haemoglobin production was observedin cell populations containing many proerythroblasts on day 13 of gestation and was

Newt erythropoietic process 503

found to be sensitive to actinomycin D (Fantoni, De La Chapelle, Rifkind & Marks,1968). In untreated newts, measurable haem absorbance was already detectable inbasophilic erythroblasts (Grasso & Woodard, 1966). Thus, in a variety of erythro-poietic systems, there exists substantial support for the early synthesis and accumula-tion of haemoglobin.

From our data, it is not possible to determine where Hb mRNA synthesis stopswith respect to specific developmental stage nor can we determine the relative pro-portion of Hb mRNA to other RNA in different erythropoietic cells. Biochemicalcharacterization of RNA in the various erythroid stages is currently in progress.

The results of the present study essentially confirm previous reports of erythro-poiesis in newts (Ceresa-Castellani & Lanzavecchia, 1966; Grasso & Woodard, 1966;Tooze & Davies, 1967). In ultrastructural examination, erythropoietic cells show noevidence of ferritin uptake by rhopheocytosis (Bessis & Breton-Gorius, 1962), afinding that has been described not only in newts (Ceresa-Castellani & Lanzavecchia,1966; Tooze & Davies, 1967) but also in the rabbit (Grasso, 1966). However, in inter-mediate and later developmental stages (EPE -> RETIC), dispersed ferritin has beenobserved in the cytoplasm while all erythroid cells, including EPCs but not RBCs,contained ferritin in various bodies which probably correspond to 'siderosomes'. Theabsence of ferritin uptake by micropinocytosis indicates that iron for haemoglobinsynthesis is obtained through other mechanisms, most probably the plasma transferrinpathway (Jandl, Inman, Simmons & Allen, 1959; Jandl & Katz, 1963). Under theseconditions, erythroid ferritin could represent a storage form of excess iron (Zamboni,1965) which can be metabolized to produce iron usable for haemoglobin synthesis bya mechanism similar to that suggested by Matioli & Baker (1963).

Heterogeneous bodies of diverse content have been described in several erythro-poietic systems (Bessis & Breton-Gorius, 1961; Grasso et al. 1962; Tooze & Davies,1965, 1967; Grasso & Hines, 1969). These bodies have been interpreted to representlysosomes in the form of autophagic vacuoles, cytolysomes, or residual bodies (Tooze &Davies, 1965, 1967; Grasso & Hines, 1969). In addition, vacuolar segregation of cyto-plasmic organelles has also been described in mammalian reticulocytes (Kent, Minick,Volini & Orfei, 1966; Simpson & Kling, 1968) and erythrocytes (Holroyde & Gardner,1970). Both the heterogeneous bodies and vacuoles comprise a mechanism by whichthe loss of cytoplasmic organelles, so characteristic of the normal erythropoieticprocess, could be effected. In the present studies, the Golgi elements to some extentbut especially the mitochondria exhibited morphological alterations consistent withthe notion of organelle degeneration through autolytic phenomena, an interpretationbuttressed by the findings showing close proximity and contact of heterogeneousbodies and mitochondria, the frequent association of smooth-surfaced membraneprofiles with mitochondria and the occasional containment of cytoplasmic organellesin segregated areas. However, it must be pointed out that the myelin-like figures inmitochondria and elsewhere were most pronounced in aldehyde-fixed cells, thoughoccasionally present after osmium fixation (cf. also Tooze & Davies, 1967). While it isclear, from an examination of cells after both types of fixation, that the mitochondriado appear to be altered in structure, it is not clear whether the myelin-like figures

504 J. A. Grasso

represent an artifact of aldehyde fixation or betray in vivo structural alterations bestpreserved by aldehydes. Although the latter alternative is preferred, no conclusiveevidence supports this preference.

In various cells including newt polychromatophilic erythroblasts, Davies and hiscolleagues have described an unusual tripartite structure in heterochromatin which isoptimally seen near the nuclear envelope (Davies, 1968; Everid, Small & Davies, 1970).Similar structures have been observed in the present studies (Fig. 10) but not enoughto make significant measurements. Most interesting is our finding that in EPCs andbasophilic erythroblasts the chromatin occurs in sizable discrete blocks or masses(Grasso, 1973; also this report), the major difference between these 2 cell types andlater erythroid stages then being mainly the coarseness and density of the chromatinthreads and granules. The light microscopic appearance of chromatin in EPCs andBEs is also different from that of subsequent stages in that, while chromatin blockscharacterize all stages, they are most densely stained in polychromatophilic erythro-blasts and successive stages. Whether this staining difference can be attributed todifferences in chromatin concentration in each block or to changes in the structure ofthe unit chromatin threads cannot be resolved by our studies. In measurements ofchromatin granules and threads in EPCs and BEs, we have observed a mean thicknessof 11-15 nm (range 7-3-20 nm, 23 measurements) while in PEs, their mean thicknesswas 22-23-9 n m (range 8-7-29-8 nm, 32 measurements). However, these measure-ments were made on cells fixed in osmium and aldehyde mixtures, respectively, sothat the differences resulting from fixation cannot be determined.

In EPCs and BEs, the nuclear interchromatin regions contained numerous granuleswhich, in subsequent stages, were decreased. This decrease corresponds to the noteddecrease and cessation of RNA synthesis, given that the granules appear to containRNA (Grasso, unpublished results). Tooze & Davies (1967) also reported the de-creasing frequency of nuclear granules in splenic erythropoiesis in T. cristatus. Theseauthors described the majority of such granules as 'interchromatin granules' with adiameter of 15-40 nm. However, our studies do not support this conclusion in thatmost of the granules were in the range of 40-55 nm and practically all were abolishedby ribonuclease extraction (Grasso, unpublished results). Their size and RNaselability is more characteristic of 'perichromatin granules'.

In previous reports of newt erythropoiesis, it has been customary to designate the'lymphoid haemoblast' as the stem cell of origin (Jordan & Speidel, 1930; Jordan,1938; Grasso & Woodard, 1966; Tooze & Davies, 1967). This cell has been des-cribed as giving rise to proerythroblasts (Grasso & Woodard, 1966; Tooze & Davies,1967) and hence to basophilic erythroblasts. Grasso & Woodard (1966) describednewt proerythroblasts as intensely basophilic cells. Our present results do not supportthis viewpoint. The earliest recognizable erythropoietic cell is the EPC which presentsa variable, weakly basophilic cytoplasm. The most basophilic cell appears at 14-18 daysafter phenylhydrazine and corresponds to the basophilic erythroblast which comprisesabout 80% of the total erythroid population at this time. Proerythroblasts as anintermediate between EPCs and BEs, never form more than 5-10% of the erythroidpopulation at a given time and display a moderate rather than intensive cytoplasmic

Newt erythropoietic process 505

basophilia (Grasso, 1973, see fig. 3). If an intensely basophilic cell such as thehaemoblasts depicted in previous reports (Grasso & Woodard, 1966; Tooze & Davies,1967) gives rise to EPCs, no evidence for this origin has been found in our studies(cf. also Grasso, 1973). However, preliminary studies in anaemic newts suggest theorigin of EPCs from circulating lymphocytes (Chromey & Grasso, studies in progress)although the correspondence of these lymphocytes to the classical lymphoid haemo-blast (Jordan & Speidel, 1930; Dawson, 1933; Jordan, 1938) is at present unclear.

This work was supported in part by grants from the National Institute of Arthritis andMetabolic Diseases (AM 15403) and from General Research Support funds granted to BostonUniversity School of Medicine (PHS-5-SO1RR 05380). The author is a Developmental CareerAwardee of the National Heart and Lung Institute, Grant No. 7 KO 4-HE-17572-06. Theauthor extends his sincere gratitude to Prof. Nicol6 Miani, Director; Dott. Corrado Olivier -Sangiacomo; Dottoressa G. De Renzis; Sig. Vincenzo Panetta; and Sig. Attilio Caniglia,Istituto di Anatomia Normale Umana, Universita Cattolica di Sacro Cuore, Rome, Italy. Then-kindness and friendship is deeply appreciated.

The author thanks Dottoressa Chiara Campanella Trautteur of the Universita di Napoli forsupplying many of the animals used in these studies.

REFERENCESATTARDI, G., PARNAS, H. & ATTARDI, B. (1970). Pattern of RNA synthesis in duck erythrocytes

in relationship to the stage of cell differentiation. Expl Cell Res. 62, 11—31.ATTARDI, G., PARNAS, H., HWANG, M-I. H. & ATTARDI, B. (1966). Giant-size rapidly labeled

nuclear ribonucleic acid and cytoplasmic messenger ribonucleic acid in immature duckerythrocytes. J. molec. Biol. 20, 145—182.

BESSIS, M. & BRETON-GORIUS, J. (1961). Ultrastructure du pro-erythroblaste. Nouv. Revue fr.Hhnat. 1, 529-533.

BESSIS, M. & BRETON-GORIUS, J. (1962). Iron metabolism in the bone marrow as seen byelectron microscopy: a critical review. Blood 19, 635-663.

BIRNSTIEL, M. L., CHIPCHASE, M. H. L. & HYDE, B. B. (1963). The nucleolus, a source ofribosomes. Biochim. biophys. Ada 76, 454-462.

BLUNT, M. H. (1965). Changes in type of hemoglobin during experimental hemorrhagicanemias in sheep. Am. J. Physiol. 209, 986-990.

BORSOOK, H. (1964). DNA, RNA, and protein synthesis after acute severe blood loss: a pictureof erythropoiesis at the combined morphological and molecular levels. Arm. N. Y. Acad. Sci." 9 . 523-539-

BORSOOK, H., LINGREL, J. B., SCARO, J. L. &MILLETTE, R. L. (1962). Synthesis of haemoglobinin relation to the maturation of erythroid cells. Nature, Lond. 196, 347-350.

BROWN, D. D. (1967). The genes for ribosomal RNA and their transcription during amphibiandevelopment. In Current Topics in Developmental Biology, vol. 2 (ed. A. A. Moscona &A. Monroy), pp. 47-73. New York: Academic Press.

BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolatemutant of Xenopus laevis. Proc. natn. Acad. Set. U.S.A. 51, 139-146.

BRUNS, G. & LONDON, I. M. (1965). The effect of hemin on the synthesis of globin. Biochem.biophys. Res. Commun. 18, 236—242.

CERESA-CASTELLANI, L. & LANZAVECCHIA, G. (1966). Recherches sur le cycle du fer dans larate de Triton (Triturus cristatus Laur.). J. Microscopie 5, 65-80.

CZOPEK, J. (1959). Skin and lung capillaries in European common newts. Copeia, No. 2,91-96.

DAVIES, H. G. (1961). Structure in nucleated erythrocytes. J. biophys. biochem. Cytol. g,671-687.

DAVIES, H. G. (1968). Electron-microscope observations on the organization of heterochro-matin in certain cells. J. Cell Set. 3, 129—150.

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DAWSON, A. B. (1933). An experimental study of hemopoiesis in Nccturus: effects of lead poison-ing on normal and splenectomized animals. ,7. Morph. 55, 349-385.

D E W I T T , W. (1968). Microcytic response to thyroxine administration. J. molec. Biol. 32,502-504.

EVANS, M. J. & LINGREL, J. B. (1969). Hemoglobin messenger ribonucleic acid. Synthesis of9 s and ribosomal ribonucleic acid during erythxoid cell development. Biochemistry, N. Y. 8,3000-3005.

EVERID, A. C , SMALL, J. V. & DAVIES, H. G. (1970). Electron-microscope observations on thestructure of condensed chromatin: evidence for orderly arrays of unit threads on the surfaceof chicken erythrocyte nuclei. J. Cell Sci. 7, 35-48.

FANTONI, A., D E LA CHAPELLE, A., RIFKIND, R. & MARKS, P. A. (1968). Erythroid cell de-velopment in fetal mice: synthetic capacity for different proteins. J. molec. Biol. 33, 79-91.

GABUZDA, T. G., SCHUMAN, M. A., SILVER, R. K. & LEWIS, H. B. (1968). Erythropoietic

kinetics in sheep studied by means of induced changes in hemoglobin phenotype. J. clin.Invest. 47, 1895-1904.

GRANICK, S. & LEVERE, R. D. (1965). The intracellular localization of heme by a fluorescencetechnique. J. Cell Biol. 26, 167-176.

GRASSO, J. A. (1966). Cytoplasmic microtubules in mammalian erythropoietic cells. Anat.Rec. 156, 397-414.

GRASSO, J. A. (1973). Erythropoiesis in the newt, Triturus cristatus Laur. I. Identification of the'erythroid precursor cell'. J. Cell Sci. 12, 463-489.

GRASSO, J. A. & HINES, J. D. (1969). A comparative electron microscopic study of refractoryand sideroblastic anaemia. Br. J. Haemat. 17, 34-44.

GRASSO, J. A. & SHEPHARD, D. C. (1968). Experimental production of totally anaemic newts.Nature, Lond. 218, 1274-1276.

GRASSO, J. A., SWIFT, H. & ACKERMAN, G. A. (1962). Observations on the development oferythrocytes in mammalian fetal liver. J. Cell Biol. 14, 235-254.

GRASSO, J .A. & WOODARD, J. W. (1966). The relationship between RNA synthesis andhemoglobin synthesis in amphibian erythropoiesis. J. Cell Biol. 31, 279-294.

GRASSO, J. A., WOODARD, J. W. & SWIFT, H . (1963). Cytochemical studies of nucleic acids andproteins in erythrocytic development. Proc. natn. Acad. Sci. U.S.A. 50, 134-140.

HOLROYDE, C. P. & GARDNER, F. H. (1970). Acquisition of autophagic vacuoles by humanerythrocytes: physiological role of the spleen. Blood 36, 566—575.

JANDL, J. H., INMAN, J. K., SIMMONS, R. L. & ALLEN, D. W. (1959). Transfer of iron fromserum iron-binding protein to human reticulocytes. jf. clin. Invest. 38, 161-185.

JANDL, J. H. & KATZ, J. H. (1963). The plasma-to-cell cycle of transferrin in iron utilization.J. clin. Invest. 42, 314-326.

JORDAN, H. E. (1938). Comparative hematology. In Handbook of Hematology, vol. 2 (ed. H.Downey), pp. 700—862. New York: Hoeber.

JORDAN, H. E. & SPEIDEL, C. C. (1930). The hemocytopoietic effect of splenectomy in thesalamander, Triturus viridescens. Am. J. Anat. 46, 55-90.

KALT, M. & TANDLER, B. (1971). A study of fixation of early amphibian embryos for electronmicroscopy. J. Ultrastruct. Res. 36, 633—645.

KARNOVSKY, M. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use inelectron microscopy. J. Cell Biol. 27, 137 A-138 A.

KENT, G., MINICK, O. T., VOLLNI, F. I. & ORFEI, F. (1966). Autophagic vacuoles in humanred cells. Am. J. Path. 48, 831-858.

LEVERE, R. D. & GRANICK, S. (1967). Control of hemoglobin synthesis in the cultured chickblastoderm. J. biol. Chem. 242, 1903-1911.

MATIOLI, G. T. & BAKER, R. F. (1963). Denaturation of ferritin and its relationship withhemosiderin. J. Ultrastruct. Res. 8, 477—490.

MAXWELL, C. R. & RABINOWITZ, M. (1969). Evidence for an inhibitor in the control of globinsynthesis by hemin in a reticulocyte lysate. Biochem. biophys. Res. Commun. 35, 79—85.

MONNERON, A. & BERNHARD, W. (1969). Fine structural organization of the interphase nucleusin some mammalian cells. J. Ultrastruct. Res. 27, 266-288.

Moss, B. & INGRAM, V. (1968). Hemoglobin synthesis during amphibian metamorphosis. II .Syndiesis of adult hemoglobin following uayroxine administration. J. molec. Biol. 32, 493-504.

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PERRY, R. P. (1967). The nucleolus and the synthesis of ribosomes. In Progress in NucleicAcid Research and Molecular Biology, vol. 6 (ed. J. N. Davidson & W. E. Cohn), pp. 219—257. New York: Academic Press.

PONKA, P., NEUWLRT, J., SPERL, M. & BREzfK, Z. (1970). The ability of exogenous heme torestore globin synthesis in reticulocytes with impaired heme formation. Biochem. biophys.Res. Commun. 38, 817-824.

RIFKIND, R., DANON, D. & MARKS, P. A. (1964). Alterations in polyribosom.es during erythroidcell maturation. J. Cell Biol. 22, 599-611.

SCHERRER, K., MARCAUD, L., ZAJDELA, F., LONDON, I. M. & GROS, F. (1966). Patterns of RNAmetabolism in a differentiated cell: a rapidly labeled, unstable 60s RNA with messengerproperties in duck erythroblasts. Proc. natn. Acad. Sci. U.S.A. 56, 1571-1578.

SIMPSON, C. F. & KLLNG, J. M. (1968). The mechanism of mitochondrial extrusion fromphenylhydrazine-induced reticulocytes in the circulating blood. J. Cell Biol. 36, 103-109.

SMALL, J. V. & DAVIES, H. G. (1970). The haemoglobin in the condensed chromatin of matureamphibian erythrocytes: a further study. J. Cell Set. 7, 15-33.

SWIFT, H. (1962). Nucleoprotein localization in electron micrographs: metal binding andradioautography. Proc. int. Soc. Cell Biol. 1, 213-232.

TOOZE, J. & DAVIES, H. G. (1963). The occurrence and possible significance of haemoglobinin the chromosomal regions of mature erythrocyte nuclei of the newt, Triturus cristatuscristatus. J. Cell Biol. 16, 501-511.

TOOZE, J. & DAVIES, H. G. (1965). Cytolysomes in amphibian erythrocytes. J. Cell Biol. 24,146-150.

TOOZE, J. & DAVIES, H. G. (1967). Light- and electron-microscope studies on the spleen of thenewt Triturus cristatus: the fine structure of erythropoietic cells. J. Cell Sci. 2, 617—640.

VAN VLLET, G. & HUISMAN, T. H. J. (1964). Changes in the haemoglobin types of sheep as aresponse to anaemia. Biochem. J. 93, 401-409.

WALKER, C. C. (1971). An Analysis of Cell Cycle Kinetics in Erythropoiesis. Doctoral Dissertation,Case Western Reserve University, Cleveland, Ohio, pp. i - m .

WATSON, M. L. (1962). Observations on a granule associated with chromatin in the nuclei ofcells of rats and mouse. J. Cell Biol. 13, 162-167.

WAXMAN, H. S. & RABLNOWITZ, M. (1966). Control of reticulocyte polyribosome content andhemoglobin synthesis by heme. Biochim. biophys. Acta 129, 369-379.

WILT, F. H. (1965). Regulation of the initiation of chick embryo hemoglobin synthesis.J. molec. Biol. 12, 331-341.

YATAGANAS, X., GAHRTON, G. & THORELL, B. (1970). RNA and hemoglobin during erythro-blast maturation. A cytophotometric study. Expl Cell Res. 62, 254-261.

ZAMBONI, L. (1965). Electron microscopic studies of blood embryogenesis in humans. II. Thehemopoietic activity in the fetal liver. J. Ultrastruct. Res. 12, 525-541.

(Received 3 July 1972)

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Fig. 2. Bright-field view of blood erythropoietic cells from a newt exhibiting erythro-poietic activity during the spring breeding season. This field is used to demonstratethe various stages of development: EPC, 'erythroid precursor cell'; BE, basophilicerythroblast; EPE, early polychromatophilic erythroblast; MPE, mid-polychromato-philic erythroblast; M-LPE, mid- to late polychromatophilic erydiroblast. Arrowindicates an MPE in mitosis. Wright's stained smear, x 1780.

Fig. 3. Same preparation as Fig. 2 showing an intensely basophilic erythroblast (BE)and a late polychromatophilic erythroblast (LPE). The small cell at the arrow isprobably a recently formed daughter cell. Wright's stain, x 1630.

Fig. 4. Same preparation as Fig. 2 showing early-, mid-, and late polychromatophilicerythroblasts (E, M, LPE). Wright's stain, x 1630.

Figs. 5—7. Three MPEs showing variation in cell size. Taken from blood of newt 22 daysafter APH. Wright's stain, x 1560.

Newt erythropoietic process 5°9

510 J. A. Grasso

Fig. 8. Electron micrograph of a basophilic erythroblast 15 days after APH. Thechromatin blocks (c) border on distinct interchromatin regions (»'e) containing manynuclear granules. A nucleolus {nc) with a prominent particulate component is visible.In the cytoplasm, ribosomes can be seen as well as a small Golgi complex (go) andmitochondria (m). OsO4 fixation, x 10600.Fig. 9. Electron micrograph of an EPE in blood taken from newt 19 days after APH.The chromatin blocks (c) are very dense, unlike their counterparts in BEs and EPCs.The interchromatin regions (ic) are conspicuous and contain nuclear granules andlight-staining zones (arrow). A nucleolus (nc) with a prominent fibrillar component isindicated. Within the cytoplasm, mitochondria (m) with swollen cristae and myelin-like figures are visible. The cytoplasm presents a moderately dense background asa result of haemoglobin accumulation. Paraformaldehyde—glutaraldehyde—acroleinfixation, x 5500.

Newt erythropoietic process

512 J. A. Grasso

Fig. 10. An EPE from the same preparation as Fig. 9. Distinct dense chromatin blocks(c) pervade the nucleus but interchromatin regions (ic) are abundant. While nucleargranules are present, their concentration appears decreased in comparison to BEs.A nucleolus (tic) with a dense fibrillar component and paniculate component isshown. Beneath the nuclear envelope, a unit thread in the heterochromatin is indicated(arrows). ParaformaJdehyde—glutaraldehyde-acrolein fixation, x 11 200.Fig. 11. Electron micrograph of an M-LPE from blood of newt 21-22 days after APH.The chromatin (c) appears as dense blocks enclosing a substantial interchromatinregion (ic). Within the latter, nuclear granules (arrows) can be seen. Note the decreasein granule concentration compared with interchromatin regions (ic) of BEs (Fig. 2).Also note the increased density within the interchromatin regions as a probable resultof haemoglobin accumulation. The cytoplasm presents a very dense appearance dueto the presence of haemoglobin. Note oval contours of the cell. Karnovsky fixation,x 11 700.

Newt erythropoietic process 5*3

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514 J- -A- Grasso

Fig. 12. A late erythroid stage, probably an LPE, from the same preparation as Fig. 11.The interchromatin regions (ic) contain nuclear granules whose concentration isgreatly decreased as compared to earlier stages. A dense component, probably haemo-globin, occupies the entire ic region. The cytoplasm is well haemoglobinized andcontains several mitochondria (m), most of which display swollen cristae and/or densemyelin-like figures. At b, a body probably representing a cytolysome is shown. This cellclearly displays an elongated shape. Karnovsky fixation, x 9700.

Fig. 13. A reticulocyte (RETIC) from newt blood 29 days after APH. The nucleusis very dense as a result of haemoglobin infiltration of the interchromatin regions(areas surrounding arrows). The chromatin (c) composes a large proportion of thenucleus. There are substantially fewer nuclear granules within the interchromatinregions and they are difficult to resolve. The cytoplasm of this elongate cell presentsdense accumulation of haemoglobin in which some ribosomes and several mito-chondria can be seen. The clear or less-dense region immediately surrounding thenucleus occurs in many RETICs and RBCs after OsO4 fixation and probably repre-sents a fixation or sectioning artifact. OsO* fixation, x 11000.

Newt erythropoietic process

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516 J. A. Grasso

Fig. 14. Segment of a BE displaying typical appearance of the nuclear chromatin (c)and interchxomatin regions. The chromatin consists of finely woven componentsarranged in blocks or masses. The interchromatin regions are occupied by numerousgranules (arrows) whose size and RNase sensitivity are suggestive of perichromatingranules. At the upper left, rich concentrations of ribosomes can be seen in thecytoplasm. OsO4 fixation, x 21000.Fig. 15. Segment of an MPE showing the alterations in nuclear appearance character-istic of the maturation process. The chromatin blocks (c) are of increased density whilethe interchromatin regions (ic) exhibit the presence of haemoglobin, judged on thesimilarity of this component to the cytoplasmic haemoglobin. Although nucleargranules are still present (arrows), they are decreased in concentration and appear tocorrespond to perichromatin granules on the basis of their size and RNase lability.The cytoplasm (in the lower section of the micrograph) exhibits presence of haemo-globin in which ribosomes are embedded. The latter appear to be in lower concentra-tion than in the BE of Fig. 14. OsO4 fixation, x 40000.

Figs. 16—19. Electron micrographs depicting changes in nucleolar structure witherythroid maturation.

Fig. 16. A nucleolus from a basophilic erythroblast. The fibrillar component (/) isrelatively inconspicuous due to the expansive particulate component (j>). Surroundingthe nucleolus is the nucleolus-associated chromatin (c). OsO4 fixation, x 24000.

Fig. 17. Nucleolus in an EPE. The fibrillar component (/) is dense and very prominentwhile the particulate component (p) appears limited to a small zone near one pole of thenucleolus. Most of the remaining material surrounding the fibrillar zone appears to bechromatin. Paraformaldehyde-glutaraldehyde—acrolein fixation, x 40000.

Fig. 18. A nucleolus from an MPE. The particulate component is relativelyinconspicuous although the fibrillar component (/) still persists. Karnovsky fixation,x 40000.

Fig. 19. Nucleolus of an MPE consisting mainly of the fibrillar zone (/). The less-dense region (arrows) may be the remnant of a particulate component but nucleolarparticles are not readily seen. Note the decrease in total nucleolar size compared topreceding stages. OsO4 fixation, x 41000.

Newt erythropoietic process 517

518 J. A. Grasso

Figs. 20—23. Electron micrographs of successive erythropoietic stages to demonstratethe major changes in cytoplasmic appearance. OsO4 fixation.

Fig. 20. A BE displaying chain-like aggregations and clusters of ribosomes. Theregion between the ribosomes is occupied by a low-density, thread-like component(surrounding arrows) which may represent early accumulations of haemoglobin.A ferritin-containing heterogeneous body (hb) is at far left, x 42 500.

Fig. 21. An MPE. Note increased density of cytoplasm as haemoglobin is accumu-lated. Single ribosomes and clusters appear embedded in the haemoglobin, althougha ribosomal decrease is already evident (compare with Fig. 20). The clear space (peri-ribosomal space) surrounding the ribosomes is probably a fixation artifact. Severalgranules (arrows) similar to the nuclear granules are present in the cytoplasm of thisnon-dividing cell, x 57500.

Fig. 22. An LPE or RETIC exhibiting further cytoplasmic density increase anddecrease in ribosomal concentration. Several ribosomes are indicated by arrows,x 50000.

Fig. 23. An RBC. The field consists entirely of haemoglobin and few, if any,ribosomes are to be seen, x 50000.

Newt erythropoietic process 519

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520 J. A. Grasso

Figs. 24-31. Electron micrographs of various erythropoietic cells showing evidencesuggestive of organelle alterations and degeneration.

Fig. 24. A mitochondrion (m) with swollen cristae and outer compartment capped by aconspicuous myelin-like dense figure (upper arrows). A profile of endoplasmic reticulum(er) appears to increase in density as it nears the myelin-like figure (lower arrow).Within the cytoplasm, some particles reminiscent of ferritin are dispersed. Para-formaldehyde-glutaraldehyde-acrolein fixation, x 52750.

Fig. 25. A mitochondrion (m) exhibiting a myelin-like figure and in apparent contactwith a dense body (fib). Nucleus (n) is at left. Paraformaldehyde—glutaraldehyde—acrolein fixation, x 45 000.

Fig. 26. Autophagic vacuoles (arrows) limited by thick dense walls (arrows). Thelower body contains a mitochondrion (m) and a region of cytoplasm consisting ofribosomes and haemoglobin. The body at the upper right is also limited by athickened membrane but appears to be formed widiin the indented region of themitochondrion. It contains mainly ribosomes and haemoglobin. The thickened wallsor membranes probably form as a result of alterations in the membranes of the ER.A body (b) similar to cytolysomes of Tooze & Davies (1965) is indicated. Note thedense intramitochondrial granules. O8O4 fixation, x 36000.

Fig. 27. Elements of the Golgi complex in an EPE. Note the dilated lamellae orsaccules, the thickened membranes and/or myelin-like figures (arrows). A smooth-surfaced vesicle (v) is devoid of apparent content. Paraformaldehyde—glutaraldehyde—acrolein fixation, x 30000.

Fig. 28. Golgi elements {go) in an MPE. The lamellae or saccules are very dilated,especially in the portion to the right. Several smooth-surfaced vesicles (y) withoutapparent content are shown. OsO4 fixation, x 46000.

Fig. 29. A mitochondrion exhibiting swollen cristae, dense granules, and a massivedense infiltrate (arrow). A smooth-surfaced vesicle is indicated at (v). OsO4 fixation,x 34500.

Fig. 30. An autophagic vacuole (arrows) containing 2 mitochondria and portions ofhaemoglobin-containing cytoplasm in which ribosomes are embedded. The bodyis walled off from the surrounding cytoplasm by a thick, dense layer (at arrows).The similarity of this limiting layer to that seen in association with the endoplasmicreticulum and mitochondrion in Fig. 24 suggests the origin of the dense layer from theER. A heterogeneous dense body is seen at the lower left. OsO* fixation, x 27000.

Fig. 31. Several mitochondria (m) in an MPE showing swollen cristae, densegranules, and the association of a myelin-like figure (arrow) with the mitochondria.OsO4 fixation, x 34000.

Newt erythropoietic process 521

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522 J. A. Grasso

Fig. 32. Segment of the cytoplasm of an MPE showing microtubules in transversesection (left side of micrograph). Dispersed ferritin particles can also be seen. Para-formaldehyde—glutaraldehyde-acrolein fixation, x 60000.Figs. 33-40. Various bodies seen in different erythroid cells.

Fig. 33. A ferritin-containing dense body in a BE. OsO4 fixation, x 61000.Fig. 34. A ferritin aggregate widiout an apparent limiting membrane in an EPE.

Paraformaldehyde-glutaraldehyde-acrolein fixation, x 51 500.Fig. 35. A ferritin-containing vacuole in an EPE. Paraformaldehyde-glutaralde-

hyde-acrolein fixation, x 65 000.Fig. 36. A cytolysome or polar body in a reticulocyte. Karnovsky fixation, x 60000.Fig. 37. A crystalline heterogeneous dense body in an EPE. Paraformaldehyde—

glutaraldehyde-acrolein fixation, x 35 000.Fig. 38. A heterogeneous dense body in an MPE, containing dense myelin-like

arrays. OsO4 fixation, x 60000.Fig. 39. A heterogeneous body with enclosed myelin-like arrays of membranes and

ill-defined material. OsO4 fixation, x 50000.Fig. 40. Two multivesicular bodies in the cytoplasm of an MPE. OsO4 fixation,

x 50000.

Netot erythropoietic process 523

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