maturation of the reticulocyte in vitro · increased the reticulocyte count to approximately 60%...

J. Cell Sci. 71, 177-197 (1984) 177Printed in Great Britain © The Company of Biologists Limited 1984

MATURATION OF THE RETICULOCYTE IN VITRO

GLORIA GRONOWICZ*, HEWSON SWIFT AND THEODORE L.STECKDepartments of Biochemistry and Biology, The University of Chicago, IL 60637, U.SA.

SUMMARY

The maturation of reticulocytes into erythrocytes was demonstrated in vitro. Reticulocytosis wasinduced in rats by repeated bleeding or by phenylhydrazine injections. Whole blood samples werethen incubated for 2 days at 37 °C. Reticulocytes in culture changed from polylobulated, monocon-cave or triconcave forms to biconcave disks. During the first 12h in vitro, the average reticulocytecount decreased from 39% to 12%, and the membrane-bound organelles, ribosomes and exocyticfigures in the remaining reticulocytes were markedly diminished. In contrast, the number of redcells containing inclusions of denatured haemoglobin (Heinz bodies) in phenylhydrazine-treatedblood did not decline. The reduction in reticulocyte count was not the result of differential celldestruction, since little haemolysis occurred in vitro.

During red cell maturation three modes of organelle removal were observed particularly wellwhen mitochondria were followed by cytochrome oxidase cytochemistry. First, some mitochondriadegenerated, presumably through autolysis, by swelling, losing cristae and forming small singlemembrane-bound vesicles. Second, individual mitochondria became enclosed in vacuoles that fusedwith the plasma membrane and expelled their mitochondria by exocytosis. Third, autophagicvacuoles containing mitochondria, cytosol and membrane fragments fused with existing lysosomes.We conclude that all aspects of normal reticulocyte maturation occur in vitro, independent of thespleen, including the removal of organelles and the assumption of the mature biconcave disk shape.

INTRODUCTION

Reticulocytes become erythrocytes within 24-36 h after expulsion from the bonemarrow (Baldini & Pannacciuli, 1960; Stryckmans et al. 1968). Reticulocytes havean irregular polylobulated or deeply cupped shape and contain ribosomes and variousmembrane-bound organelles (Bessis, 1973). The mature mammalian erythrocyte issmaller than the reticulocyte, lacks organelles, and has the shape of a biconcave disk.During the maturation process reticulocytes lose organelles, membrane lipid,haemoglobin and water (Munn, 1958; Ganzoni, Hillman & Finch, 1969; Come,Shohet & Robinson, 1977a; Shattil & Cooper, 1972) and their polysomes separateinto monosomes, decrease in number and disappear (Marks, Rifkind & Danon, 1963;Rifkind, Danon & Marks, 1964; Glowacki & Millette, 1965).

Of primary interest is the mechanism by which organelles are removed from matur-ing reticulocytes. In vivo observations of exocytosis of organelles, autophagy,degeneration and 'pitting' by the spleen suggest various pathways for organelleremoval, but the maturation of reticulocytes into erythrocytes has never been fullydocumented in vitro under controlled conditions. Expulsion of mitochondria by

•Author for correspondence at: Department of Oral Biology, University of Connecticut HealthCenter, School of Dental Medicine, Farmington, CT 06032, U.S.A.

178 G. Gronowicz, H. Svnft and T. L. Steck

exocytosis is considered a rare event in vivo (Tooze & Davies, 1965; Simpson &Kling, 1968; Schnitzer, Rucknagel, Spencer & Aikawa, 1971). In the case ofautophagy, vacuoles containing organelles, haemoglobin and lysosomal enzymes havebeen found in vivo (Kent, Minick, Volini & Orfei, 1966; Kornfield & Gregory, 1969;Schnitzer et al. 1971). Ribosomes appear to deteriorate in situ (Bertles & Beck, 1962;Miller & Maunsbach, 1966) and the incidence of swollen mitochondria increases inolder reticulocytes (Gasko & Danon, 1972). That removal of unphysiological in-clusions in red cells (siderin granules, nuclear fragments, Heinz bodies, etc.) occursby a pitting mechanism in the spleen has been inferred from the increased numbersof inclusions observed in splenectomized patients. These inclusions disappear upontransfusion into normal subjects with spleens (Crosby, 1952, 1957, 1959), but thecytological details are unclear. It seemed important to us to develop a simple andcontrolled system for studying terminal maturation of the erythrocyte. Our in vitrosystem allowed us to assess the contribution of exocytosis, autophagy and disintegra-tion in the developmental process leading to the mature erythrocyte. We have foundthat reticulocytes mature in culture without the aid of the spleen or reticulo-endothelial system, shedding intracellular organelles and acquiring a mature bi-concave shape. A preliminary account of these findings has been published(Gronowicz, Greenwald & Steck, 1981).

MATERIALS AND METHODS

The following chemicals were purchased from Sigma Chemical Co.: phenylhydrazine hydro-chloride, 3,3'-diaminobenzidine tetrahydrochloride, catalase, cytochromec (type II), and cytidine5'-monophosphate. Penicillin-streptomycin was obtained from GIBCO. All other chemicals wereof reagent grade or better, from Fisher, Baker or Malincrodt.

Preparation of reticulocytes and erythroblasts

All procedures were performed on ice and centrifugation was in a Sorvall HSA rotor at 0 CC unlessotherwise indicated.

Wistar, Furth or Lewis rats weighing 200-300 g were used interchangeably with no observabledifferences. Rats were divided into three groups. The first was the untreated control. Reticulocytosiswas induced in the second group by bleeding approximately 1-2 ml from the tail on three successivedays. Rats in the third group were injected intraperitoneally with phenylhydrazine hydrochloride(10-15 mg/kg body weight) in saline on four successive days (Stryckmanset al. 1968). Three daysafter the end of the treatment, the animals were placed under light ether anaesthesia andapproximately 10 ml of blood was collected by sterile cardiac puncture in syringes containing 1 -0 mlof lOOmM-EDTA in saline. The haematocrit and reticulocyte counts of each sample were deter-mined. While phenylhydrazine produced more and younger reticulocytes, it has well-documenteddeleterious effects on erythrocytes (Rostorter & Cormier, 1957; Cohen & Hochstein, 1964; Rifkind& Danon, 1965; Beutler, 1969; Jain & Subrahymanyam, 1978; Jain & Hochstein, 1979, 1980).Therefore, the more benign technique of serial bleeding was studied in parallel.

Marrow cells were obtained from the femora and tibiae of rats according to the method ofGoldwasser, Eliason & Sikkema (1975).

Incubation of bloodBlood samples containing more than 30 % reticulocytes were selected for in vitro studies. Whole

blood samples were supplemented with 0-15M-NaHCO3 (pH7-4), lOmin-glucose, penicillin (100units/ml), and streptomycin (lOO^g/ml) and incubated without shaking in closed tubes in a 37°C

Maturation of the reticulocyte in vitro 179

water bath under room air for 2 days. At specified times, samples of blood were removed from thetubes by a sterile technique, for determination of reticulocyte number, morphology and haemolysis.Haemolysis was assessed on samples that were diluted 100-fold in 0-15 M-NaHCCb with and withoutO'l % saponin, incubated at room temperature for lOmin, and centrifuged at 3700 £ for Smin. Aportion of each supernatant was diluted with 0-15 M-NaHCC>3 and the absorbance of haemoglobinwas read at 415 nm in a Gilford 240 spectrophotometer.

Contamination of cultured blood samples was monitored by plating samples on medium at varioustimes (Miller, 1972).

Transmission electron microscopyPacked red cells were fixed in a 1: 5 volume of 1 % glutaraldehyde in 0-1 M-sodium cacodylate or

0-15M-NaHCC>3 at pH7-4 for 20min at 0°C. The samples were post-fixed with 1% osmiumtetroxide in 0-1 M-sodium cacodylate (pH 7-4) for 1 h on ice (Palade, 1952), dehydrated in a gradedseries of ethanol followed by propylene oxide, and embedded in Epon 812 (Luft, 1961). Silver togold sections were cut, stained with uranyl acetate (Watson, 1958) followed by lead citrate (Venable& Coggeshall, 1965) and examined with a Siemens 101 electron microscope.

Scanning electron microscopySamples of glutaraldehyde-fixed cells were absorbed to Millipore filters (13 mm, 0-45 jun) for

5-10 min, post-fixed with 1 % osmium tetroxide in 0-1 M-sodium cacodylate (pH7-4) for30minonice, dehydrated in a graded series of ethanol followed by critical-point drying. Ethanol was exchangedfor CO2 in a Denton critical-point drying apparatus. Specimens were coated for 2 min with Au/Pdin a Denton evaporator with a coldsputter module and examined in a Hitachi H300 equipped withan H-3010 scanning attachment.

Acid phosphatase cytochemistrySamples of blood were fixed as described above and rinsed three times with 0-1 M-sodium

cacodylate buffer (pH7-4) containing 7-5% (w/v) sucrose. The fixed cells were incubated for30 min at 37 °C in a modified Gomori medium with cytidine 5'-monophosphate as substrate(Novikoff, Essner & Quintana, 1964). Substrate-free medium served as a control. The cells werefinally washed with 0-1 M-cacodylate containing 7-5% sucrose, post-fixed, and processed asdescribed above for electron microscopy.

Cytochrome oxidase cytochemistrySamples of blood were fixed for 20 min in quarter-strength Karnovsky's fixative: 1 % formaldehyde

and 1-25 % glutaraldehyde in 0-1 M-sodium cacodylate buffer (pH7-4) (Karnovsky, 1965), thenwashed three times with 0-1 M-sodium cacodylate (pH 7 -4) and left overnight on ice in the same buffer.The samples were incubated at 37°C for 2h in freshly prepared medium (Seligman, Karnovsky,Wasserling & Hanker, 1968). The cells were pelleted and resuspended in fresh medium every 30 min.The same reaction mixture containing 10 mM-sodium azide served as a control; in no case was reactionproduct observed in the presence of this inhibitor. Samples were post-fixed with 2 % osmium tetroxidein 0"l M-NaPi (pH 7-4) for 1 h and processed for electron microscopy as described above.

AnalysisReticulocytes among at least 500 cells stained with Brilliant Cresyl Blue were counted by light

microscopy (Williams, Beutler, Ersler & Rundles, 1972). Reticulocytes were also determined bytransmission electron microscopy to be cells containing membrane-bound organelles (primarilymitochondria, Golgi apparatus and lysosomes), vesicles and vacuoles. We did not score asreticulocytes cells exhibiting ribosomes but lacking membrane-bound organelles in the electronmicroscope. In thin section, sparse ribosomes were difficult to identify; in contrast, membrane-bound organelles were easily distinguished. Therefore, the reticulocyte number obtained byelectron microscopy systematically underestimated by about one-third the reticulocyte count ob-tained by Brilliant Cresyl Blue staining for ribosomal material in the light microscope (Bessis &Breton-Gorius, 1964; Jensen, Moreno & Bessis, 1965).

180 G. Gronowicz, H. Swift and T. L. Steck

To avoid repeated sampling of the same cells only one section from each block was evaluated.Duplicate blocks were analysed for each time-point. Sections were selected for minimal occlusionof cells by grid bars or section defects and all visible cells were analysed.

Heinz bodies (denatured haemoglobin aggregates appearing as electron-dense masses) werescored using the same thin sections in which reticulocyte number was determined. Exocytic figuresin more than 200 reticulocytes from the blood of at least three rats each were counted at each time-point, except at 24 h when only 100 reticulocytes were analysed. Exocytosis was scored only if thevesicle had demonstrable contents and its membrane was in continuity with the plasma membraneon at least one side.

Cell shape was best appraised by dark-field microscopy of wet mounts where cell tumblingrevealed overall contour. The electron microscopic data provided below suffer from the non-randomsettling of cells on supports for scanning electron microscopy and from the hazards of interpretingthree-dimensional contour from thin sections; however, they correlated well with our results fromlight microscopy.

RESULTS

Elicitation of reticulocytosis

Two means for evoking reticulocytosis in rats were used: phenylhydrazine andbleeding, followed by a 3-day recovery period, which was required for optimalreticulocytosis. Phenylhydrazine reduced the haematocrit to approximately 30 % andincreased the reticulocyte count to approximately 60% (Fig. 1); for bleeding, thesevalues were 40 % and 30 % (Fig. 2). During this period, control rats had haematocritsof 45 % to 52 % and reticulocyte numbers of 1 % to 3 %. Prolonging either treatmentdid not significantly increase the reticulocyte number but greatly increased animalmortality.

Different anticoagulants for drawing blood and incubation mixtures were tried, tofind optimal conditions for studying reticulocyte maturation. Buffered EDTA, citratephosphate dextrose, heparin and acid citrate dextrose were used as anticoagulants;100 mM-EDTA in saline gave the least amount of haemolysis over 48 h of incubation(unpublished observation). Dulbecco's medium supplemented with 5 mM-adenosine,1 mM inorganic phosphate (Kim, Mullins & Zeidler, 1981) and 5 % (w/v) foetal calfserum or 5 % (w/v) horse serum did not improve red blood cell viability as deter-mined by haemolysis assay or quantitation of reticulocytes in vitro or by the morphol-ogy of cells in the electron microscope.

Morphology of freshly drawn and incubating blood

Control blood consisted of 1—2% reticulocytes, similar in appearance toreticulocytes from treated animals. Mature erythrocytes contained only haemoglobinand no organelles. The small percentage of reticulocytes in untreated animals declinedwith incubation and will not be further described.

In fresh blood from treated animals reticulocytes had irregular shapes andnumerous ribosomes and some membrane-bound organelles (Figs 3, 4, 5). Roughendoplasmic reticulum and clearly discernible Golgi cisternae arranged in parrallelwere infrequent. An occasional multivesicular body, numerous lysosomes andautophagic vacuoles, often staining for acid phosphatase (see Fig. 14, Table 1), wereseen in thin sections of reticulocytes. Small vesicles, single long cisternae and large


o00

EtoI

0 2 4 6Days of treatment (phenylhydrazine)

Fig. 1. Haematocrit (O O) and reticulocyte count ( • • ) of phenylhydrazine-treated rats. Four rats were injected with phenylhydrazine hydrochloride on days 0-3.Reticulocytes were counted by light microscopy. Error bars represent one standard devia-tion of the mean.

vacuoles were more prevalent in reticulocytes from blood freshly drawn fromphenylhydrazine-treated than from bled rats (Figs 4, 5). Thus, phenylhydrazineappears to increase vesiculation and cell destruction.

Mitochondria were the most numerous organelles seen in thin sections ofreticulocytes from freshly drawn blood from treated animals. Mitochondria wereoften confined to one region of the cell and varied in appearance. Some mitochondriadisplayed a dense matrix and extensive, well-defined cristae while others within thesame cell were swollen with a diffuse matrix and few cristae. Empty-appearing vesicleswere often seen clustered around mitochondria (Fig. 3).

182 G. Gronovncz, H. Swift and T. L. Steck

Reticulocytes matured rapidly in culture (Fig. 4 versus 6, Fig. 5 versus 7). Thereticulocyte number seen in thin section decreased to one-half the initial value after12 h and to 4% after 48 h of incubation (Fig. 8). Reticulocyte number, determinedby Brilliant Cresyl Blue staining in the light microscope, dropped from 56 % to 13 %.The number of organelles seen per cell in thin section also diminished with time.Single ribosomes, autophagic vacuoles and lysosomes often were the only organellesremaining at later times. The majority of mitochondria were swollen with few cristaeand less-dense matrix, and were bordered by small vesicles.

4 6Days of treatment (bleeding)

Fig. 2. Haematocrit (O O) and reticulocyte count ( • • ) of bled rats; 1-2 mlof blood was removed from 4 rats on days 0-2. Reticulocytes were counted by lightmicroscopy. Error bars represent one standard deviation of the mean.


Fig. 3. A representative reticulocyte in blood freshly drawn from a phenylhydrazine-treated rat. Mitochondria (m), vesicles (v) and lysosomes (/) are clustered in one regidnof the cell. Numerous ribosomes fill the cytoplasm. An irregular surface contour is alsoevident. X11 000. All sections were stained with uranyl acetate and lead citrate unlessspecified otherwise. Bar,

Table 1. Reticulocytes containing acid phosphatase-positive organelles

Rats

PIP2BlB2

0

20-622-612-532-4

Incubation time

6

37-747-427-040'2

(h)

12

49-045-540-855-5

24

36-832-332-350-0

At 0, 6, 12 and 24 h of incubation, samples of incubating blood from two phenylhydrazine-treated(PI, P2) and two bled rats (Bl, B2) were assayed for the percentage of reticulocytes in thin sectiondemonstrating acid phosphatase reaction product. At least 50 reticulocytes were analysed at eachtime.

Heinz bodies

In contrast to the disappearance of organelles, Heinz bodies did not decline in

number. Heinz bodies are produced by phenylhydrazine treatment and are aggregates

of denatured haemoglobin at the plasma membrane where they cause large surface

projections (Fig. 14). However, in reticulocytes Heinz bodies were found in small

G. Gronowicz, H. Swift and T. L. Steck

Fig. 4. An electron micrograph of blood freshly drawn from a phenylhydrazine-treatedrat. Reticulocytes with numerous mitochondria (m), cisternae (c), vesicles (u) andautophagic vacuoles (av) are seen. In reticulocytes small aggregates of denatured haemo-globin, Heinz bodies (//), are seen in the interior of the cell while in erythrocytes Heinzbodies (arrows) deform the cell at the plasma membrane. X38OO.

Fig. 5. An electron micrograph of blood freshly drawn from a bled rat. Reticulocytescontain mostly mitochondria (m), vesicles (D) and autophagic vacuoles (av). A monocyte(arrowhead) is also present. X380O.

Maturation of the rettculocyte in vitro 185

Fig. 6. Blood from the rat treated with phenylhydrazine, shown in Fig. 4, was incubatedfor 12 h. A few mitochondria (m) and vesicles (v) are found. Erythrocytes with Heinzbodies (arrows) remain. X3800.

Fig. 7. Blood from the bled rat, shown in Fig. 5, was incubated for 12 h. Few reticulocytesare seen and these contain mitochondria (m) and autophagic vacuoles (an). A leucocyteis apparent (arrowhead). X3800.


48

Incubation time (h)

Fig. 8. Reticulocyte count during the incubation in vitro of blood from phenylhydrazine-treated ( • • ) , bled (O O) and control (A A) rats. Assays were performedby transmission electron microscopy on samples from four rats in each group. Error barsare one standard deviation of the mean.

clusters and did not appear to be associated with the plasma membrane (Fig. 4). Infreshly drawn blood, 16 ± 11 % (mean ± standard deviation) of the red cells con-tained Heinz bodies. After 24 and 48 h, 20 ± 13 % and 25 ± 11 % of the red cells,respectively, from phenylhydrazine-treated rats, contained these inclusions. NoHeinz bodies were found in the blood from bled animals.

Cell shape

Phase, transmission and scanning electron microscopy showed clear differencesbetween reticulocytes and mature biconcave erythrocytes and confirmed the time-dependent decrease in reticulocyte number. In cell suspensions viewed under the


phase microscope, reticulocytes were large and lobulated, triconcave or monoconcave(resembling shallow bowls). In thin section the most irregularly shaped cells had themost organelles and thus were probably the youngest reticulocytes in the bloodstream(Fig. 4). In scanning micrographs multidimpled and triconcave forms were alsoevident (Fig. 9). Both scanning and transmission microscopy revealed numeroussmall surface indentations in reticulocytes in contrast to the smooth biconcave contourof erythrocytes (Fig. 4 versus 6, Fig. 9 versus 10).

Fig8 9, 10. Scanning electron micrographs. Samples were taken from a single bled rat atthe start of incubation (Fig. 9) and after 6 h of incubation (Fig. 10). Irregularly shaped andtriconcave reticulocytes (arrows) are visible in Fig. 9. In addition, reticulocytes havepitted and irregular surfaces (arrowheads). Smooth single dimple and/or biconcave formsof red cells predominate in Fig. 10 (arrows). X2600.


The number of biconcave erythrocytes increased in vitro (Figs 4, 6). Quantitationof biconcave (mature) profiles (500 cells) from thin sections of blood fromphenylhydrazine-treated rats yielded 29 % erythrocytes at the start of incubation and57 % after 12 h of incubation. Similar trends were seen in bled samples, particularlyin the phase microscope, but it was difficult to detect a similar significant changein thin sections of red cells from bled animals due to the more subtle differencesin shape betwen reticulocytes and erythrocytes (Figs 5,7). Specifically, many morepolylobulated cells, easily distinguished as reticulocytes, were seen inphenylhydrazine-treated blood than in blood from bled rats. These forms arebelieved to be younger reticulocytes, which are released prematurely from the bonemarrow due to phenylhydrazine treatment (Mel, Prenant & Mohandas, 1977).Polylobulated reticulocytes were rarely seen in thin sections after 12 h of incubation.There was a clear correlation between the smooth, regular contour of cells followingincubation and the absence of organelles (Figs 4, 6). Scanning electron microscopyrevealed more monoconcave and biconcave cells at 6h than at zero time (Figs 9,10).

By 48 h, 60 % of mature cells appeared spherical or spheroechinocytic, perhaps asa result of osmotic swelling following metabolic depletion (Bertles & Beck, 1962) orthe presence of lysolecithin (Deutcke, 1968) produced by plasma lecithin cholesterolacyltransferase. Therefore, quantitation of biconcave profiles became impossible.However, immature cells (i.e. those containing organelles) were not echinocytic andmost appeared monoconcave or biconcave in thin section.

Haemolysis and pH control during 48 h of incubation

The decrease in reticulocyte number in thin section was not associated withpreferential loss of reticulocytes by cell disruption. Assessment of haemolysis inincubating blood from all bled and phenylhydrazine-treated rats showed that only 6 %of cellular haemoglobin was released at 12 h and 21 % after 2 days of incubation. Sincethe percentage of cells haemolysed (6 %) is small compared to cells matured after 12 hof incubation (more than 50%, see Fig. 8), the decline in reticulocyte number mustbe associated with cell maturation and cannot be attributed solely to selectivehaemolysis of reticulocytes.

The pH of the medium was monitored at 3-h intervals for the first 12 h of incubationand at 12-h intervals thereafter. It remained stable at pH 7-3 ± 0-2; the range in allexperiments was 6-8 to 7-9.

Fig. 11. Expulsion of vesicles (arrow) by exocytosis from a multivesicular body of areticulocyte. Blood was from a rat made anaemic by phenylhydrazine treatment. The thinsection was stained with lead citrate only. X72 000. Bar, 0-5 /im.

Fig. 12. Expulsion of membrane fragments and vesicles (arrow) by exocytosis from anautophagic vacuole of a reticulocyte. The blood was freshly drawn from a rat madeanaemic by bleeding. X44 000.

Fig. 13. A mitochondrion (arrow) being expelled from a reticulocyte by exocytosis. Theblood was freshly drawn from a rat made anaemic by phenylhydrazine treatment.Mitochondria (m), and ribosomes (r) are seen in the cytoplasm. X38 000.


13Figs 11-13

190 G. Gtvnowicz, H. Surift and T. L. Steck

Exocytosis

Some organelles clearly were extruded from maturing reticulocytes. Three typesof exocytic figures were seen: (1) multivesicular bodies containing many vesicles ofuniform size (Fig. 11); (2) vacuoles enclosing membrane fragments, amorphousmaterial and vesicles of various sizes (Fig. 12); and (3) vacuoles encircling a singlemitochondrion (Fig. 13). The prevalence of these exocytic (omega) figures in thinsections of reticulocytes from bled rats after 0, 3, 6 and 12 h of incubation was 2, 5,6 and 3 %, respectively. For phenylhydrazine-treated rats, these values were 2, 9, 2and 2 %. No omega figures were found after 24 h of incubation or in four specimensof control blood.

Acid phosphatase cytochemistry

Autophagic lysosomes were also clearly responsible for further organelle elimina-tion. Lysosomes were identified cytochemically in reticulocytes by the presence ofacid phosphatase. Two types of acid phosphatase-containing bodies were seen: smallvesicles often completely filled with reaction product and autophagic vacuoles

-14

Fig. 14. Acid phosphatase-staining bodies in a reticulocyte from a phenylhydrazine-treated rat. Autophagic vacuoles (av) and lysosomes (/) demonstrate the electron-densereaction product. A neighbouring erythrocyte contains Heinz bodies (H). The thin sectionwas stained with lead citrate only. X17 000.


dm

Fig. 15. Cytochrome oxidase cytochemistry of a reticulocyte. A condensed mitochondrion(cm), a swollen mitochondrion (sm) and a mitochondrion with a double membrane remain-ing (dm) demonstrate reaction product within a reticulocyte from a phenylhydrazine-treated rat. A vesicle (v) near the degenerating mitochondrion also stains faintly for cyto-chrome oxidase activity. The thin section was stained with lead citrate. X 15 000.

Fig. 16. A double-membrane vacuole (dm) demonstrating cytochrome oxidase reactionproduct is seen in a reticulocyte from a bled rat. A condensed mitochondrion (cm) contain-ing stain and a lysosome (/) are also visible. The thin section was stained with lead citrateonly. X24000.

Fig. 17. Cytochrome oxidase staining of mitochondria (sm) that have begun to swell andlose matrix density. Vesicles (v) faintly stained for cytochrome oxidase activity are foundnear degenerating mitochondria while other vesicles are unstained (u). X26 000.

containing miochondria, haemoglobin and vesicles, with patches of reaction product(Fig. 14).

The number of reticulocytes in thin section demonstrating acid phosphatase-


positive structures varied from 13 % to 56% at different times of incubation (Table1). The greatest number of reticulocytes with lysosomes in thin sections was foundafter 12 h of incubation. A greater number of acid phosphatase-containing lysosomeswas seen in reticulocytes from phenylhydrazine-treated rats than in bled rats, especi-ally in reticulocytes that contained Heinz bodies. As with other organelles, lysosomesdisappeared with time. By 24 h of incubation, lysosomes were often the onlymembrane-bound organelles apparent in the few reticulocytes remaining.

Cytochrome oxidase cytochemistry

Mitochondria underwent degenerative changes, which were followed by cyto-chrome oxidase cytochemistry. Specifically, single-walled vesicles derived frommitochondria were able to be identified by their residual cytochrome oxidase activity.

As seen in Figs 15, 16 and 17 some mitochondria had a condensed matrix and manywell-defined cristae; the reaction product was confined to the intermembrane space.Other mitochondria were swollen with a diffuse matrix and few cristae that stillexhibited cytochrome oxidase activity. A third class of mitochondria appeared in thinsection as large, double-membrane vacuoles with diffuse, amorphous contents, oftencontaining a single small vesicle. Reaction product was confined to the space betweenthe double membranes. Additionally, single-walled vesicles near degenerating

Fig. 18. Cytochrome oxidase cytochemistry on an erythroblast from rat bone maiTow. Allmitochondria (m) within the erythroblast are condensed and contain reaction product.The nucleus (n) is also apparent. The thin section was stained with lead citrate only.X10000.


mitochondria often appeared to have reaction product associated with their mem-brane (Figs IS, 17).

In reticulocytes from fresh blood, condensed mitochondria prevailed, but swollenmitochondria were also apparent. After 12 and 24 h of incubation, condensedmitochondria were rare and the swollen and distended figures were most abundant.The aberrant forms of mitochondria and the pattern of cytochrome oxidase stainingwere not artifacts of our system; we observed that similarly incubated erythroblastsfrom rat bone marrow contained condensed mitochondria only, with numerous cristaedemonstrating cytochrome oxidase activity (Fig. 18).

DISCUSSION

Our results document reticulocyte maturation in vitro. Cells were transformedfrom an irregular shape to one resembling a biconcave disk. Membrane-boundorganelles were cleared by degeneration, autophagy and expulsion. The timing wascomparable to that seen in vivo: the reticulocyte count fell by half within the first 6 hof incubation and was less than 5 % at 48 h.

By using an in vitro system we were able to eliminate extrinsic factors, which someauthors have implicated in reticulocyte maturation (Crosby, 1957, 1977; Kent et al.1965; Nathan, 1969; Holroyde & Gardner, 1970; Zweig, Tokuyasu & Singer, 1981).It is evident that at the time of release from the bone marrow reticulocytes arebeginning or already involved in the process of losing organelles. Our in vitro systemsuggests that this process peaks at 3—12 h as demonstrated by the increase in thepercentage of reticulocytes in thin section that contain exocytic figures and lysosomes.Possibly, however, the processes of autophagy and expulsion of organelles arestimulated by in vitro incubation, especially since we have increased cell mortality ascompared to reticulocytes in vivo. Nonetheless, this in vitro system does show thatremoval of all organelles from reticulocytes can be an autonomous process requiringonly 24— 36 h for completion just as it does in vivo. Degeneration, autophagy andexpulsion of organelles are extensive and can account for the disappearance of allorganelles within reticulocytes.

We were able to delineate organelle elimination most clearly in vitro by followingthe fate of mitochondria. Mitochondrial degeneration involved the conversion froma normal condensed form to a swollen, double-membrane vacuole, identifiable by itsresidual cytochrome oxidase activity. Our finding reaction product in some single-walled vesicles near degenerating mitochondria indicates that mitochondria can breakdown into vesicles. Degeneration did not appear to be an artifact, since similarly fixederythroblasts from bone marrow contained only norrnal appearing mitochondria, andmitochondria at all stages could be found within a single reticulocyte.

A precedent for organelle destruction without lysosomal involvement is found inthe disappearance of ribosomes from the cytosol (Bertles & Beck, 1962). In our systemafter 24 and 48 h of incubation, numerous ribosomes and few lysosomes were foundin thin section; therefore, the possibility that the major route for ribosomal destruc-tion involves autophagic vacuoles is unlikely.


A non-lysosomal ATP-dependent pathway for degradation of proteins has beenfound in rabbit reticulocytes and an ATP-dependent endoprotease was localized in ratliver mitochondria (Etlinger & Goldberg, 1977; Muller, Dubiel, Rathmann &Rapaport, 1980; Bodies & Goldberg, 1982; Desautels & Goldberg, 1982). Therefore,our morphological data support a non-lysosomal route for degeneration of somemitochondria in reticulocytes. However, we have also found autophagy and expulsionof mitochondria to be operating simultaneously in the removal of organelles fromreticulocytes.

Another route for disposal of mitochondria was by autophagy. Numerousautophagic vacuoles, which often contained other cytoplasmic material and stainedfor acid phosphatase, were frequently seen. In these cells, as in other systems whereautophagic vacuoles arise, the source of the enclosing membranes was unclear (Holtz-man, 1976). The paucity of the Golgi apparatus and endoplasmic reticulum in moremature reticulocytes makes it unlikely that they are the source of vacuolar membranein these cells. The vacuole membrane might arise from some of the vesicles clusteredin the vicinity of mitochondria, possibly coalescing to form membrane sacs similar tothose thought to be involved in the expulsion of the nucleus from the erythroblast andbelieved to be derived from the plasma membrane (Simpson & Kling, 1967, 1968;Skutelsky & Danon, 1967).

Exocytic vesicles containing membrane fragments and debris demonstrate thatextrusion of autophagic vacuoles occurs in reticulocytes (Figs 11, 12). However,exocytic figures containing mitochondria were never seen to include other organellesor particulate matter and these mitochondria appeared intact (Fig. 13). Thus, theremoval of some mitochondria by exocytosis might be a separate route that bypasseslysosomal digestion. Since 2 % of the reticulocytes in thin section from freshly drawnblood released mitochondria by exocytosis, expulsion of organelles is not an artifactof this in vitro system. Exocytosis was quantitated in two dimensions; therefore, theactual proportion of reticulocytes involved in exocytosis is considerably higher than2%.

Our observations indicate that Heinz bodies were not removed during reticulocytematuration in vitro. On the other hand, in rats treated with phenylhydrazine, thenumber of Heinz bodies has been shown to decrease over a period of 4 days tonegligible levels (our unpublished observations). Clearly, mechanisms must exist inthe intact animal for the clearance of these inclusions. It has been shown that splenicmacrophages do remove the entire cell containing Heinz bodies (Azen & Schilling,1963; Rifkind, 1965). The process of 'pitting' whereby organelles are mechanicallyremoved from red cells when they pass through splenic sinusoids has also been pos-tulated by several workers, although the cytological mechanisms for this process arestill largely obscure (Miller, Singer & Damashek, 1942; Lorber, 1958; Berendes,1959; Weed & Weiss, 1966; Lawson, 1969; Comeetal. I977a,b; Lux & John, 1977).

Our studies have so far been unable to define the processes involved in the develop-ment of the red cell's mature biconcave shape, except to emphasize that it can beacquired in vitro. A conventional cytoskeleton responsible for cell shape has not beenidentified in typical reticulocytes and erythrocytes. A submembrane reticulum of


spectrin and actin is present but does not extend into the cytoplasm (Yu, Fischman& Steck, 1973). Erythroblasts and very young reticulocytes are motile, which doessuggest that a contractile system exists at early stages (Mel et al. 1977; Bessis, 1973).Additionally, bulk membrane movement to and from the cell surface is occurring inreticulocytes (Gasko & Danon, 1974; ZweigeJ al. 1981), without the involvement ofknown cytoskeletal elements.

We conclude that, except for the splenic destruction of whole abnormal cells, andpossibly also the selective pitting of Heinz bodies, the process of reticulocyte matura-tion is essentially an autonomous process. What we observe in culture is the com-pletion of organelle clearance by mechanisms initiated many hours earlier in thenucleated erythroblast. In vitro cultivation offers a system for denning the complexprocesses of organelle clearance and the acquisition of mature red cell shape in bothmorphological and biological terms.

We are grateful for the excellent technical assistance of Robert Greenwald, Sagami Paul and DrChris Chou. We thank Dr Allen Labrecque for the rats and Dr T. J. McAllister for use of hisscanning electron microscope.

The work was supported by a U.S. Public Health Service Training Grant CA 90267 (to G.G.),and American Cancer Grant BC-95 (to T.L.S.) and CA 14599 (to H.S.).

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(Received 30 January 1984-Accepted, in revised form, 10 May 1984)

maturation of the reticulocyte in vitro · increased the reticulocyte count to approximately 60%...

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