NEW OBSERVATIONS ON MICROBODIES

A Cytochemical Study on CPIB-Treated Rat Liver

PETER G. LEGG and RICHARD L. WOOD

From the Department of Anatomy, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT

The liver of male rats has been studied after CPIB stimulation by using the peroxidase reac-tion for localizing catalase in hepatic cells . CPIB administration leads to an increase in thenumber of microbodies, and it is suggested that one mechanism by which microbody pro-liferation occurs is a process of fragmentation or budding from preexisting microbodies .Reaction product was observed not only within the microbody matrix, but outside thelimiting membrane of the microbody and in association with ribosomes of adjacent roughendoplasmic reticulum . This localization of reaction product is interpreted as evidence thatcatalase after synthesis on rough endoplasmic reticulum may accumulate near microbodiesand may be transferred directly into these organelles without traversing the cisternae of theendoplasmic reticulum or Golgi apparatus .

1 18

INTRODUCTION

The existence of a distinct class of cytoplasmicorganelle, the microbody or peroxisonie, has beenincreasingly recognized since Rhodin's first de-scription in 1954 (see reviews of de Duve andBaudhuin, 1966 ; Hruban and Rechcigl, 1969) .The mechanism of formation of microbodies andtheir site of origin are poorly understood andcontroversial . Different cell organelles includingsmooth and rough endoplasmic reticulum, theGolgi apparatus, and multivesicular bodies havebeen implicated (5, 8, 9, 29, 40, 42, 43, 46, 47),but their definitive roles in microbody formationremain unclear.

Proliferation of microbodies in hepatic cells isknown to occur in three main circumstances :(1) during fetal and early postnatal development(8, 9, 23, 32, 39, 48), (2) during the recovery phasefollowing partial hepatectomy (36, 38), and (3) fol-lowing the administration of certain chemicalagents, notably salicylates and ethyl-cep-chloro-phenoxyisobutyrate (CPIB or Clofibrate, AyerstLaboratories, New York) (16, 22, 30, 34, 40-42) .Regular morphological observations on these sys-

tems have provided only limited information re-lated to microbody development and have led toapplication of ancillary technics .

Microbodies are known to contain several oxida-tive enzymes which yield hydrogen peroxide as areaction product and catalase, an enzyme capableof reducing hydrogen peroxide to water by twodistinct mechanisms, catalatic and peroxidatic (7,21, 24) . The application of the peroxidase stainingtechnic of Graham and Karnovsky (15), as modi-fied by Novikoff and Goldfischer (28), has beensuccessful in demonstrating microbodies at anultrastructural level, presumably through theperoxidatic activity of catalase (4, 11-13, 44) . Al-though the technic facilitates identification of mi-crobodies, it has provided little additional insightinto the mechanisms of microbody formation (4,10, 11,34) .

The present study combines an additional modi-fication of the cytochemical technic for demon-strating endogenous peroxidase activity with CPIBstimulation of microbody proliferation . The resultssuggest that (a) microbodies increase in number

through a fragmentation or budding phenomenon,

TABLE I

(b) catalase after synthesis on rough endoplasmic

Experimental Regimenreticulum may be inserted into microbodies with-out being transported through the cisternae of therough endoplasmic reticulum, and (c) smoothendoplasmic reticulum and the Golgi apparatusare apparently not involved directly in microbodyproliferation .

MATERIAL AND METHODS

Male Sprague-Dawley rats (Holtzman) 130-250 g inweight were used in this study . CPIB (Clofibrate,Ayerst) 1 was diluted with olive oil to provide 75mg/0.5 ml and injected intraperitoneally in a dosageof 375 mg/kg daily. Liver was sampled at intervalsfrom 12 hr to 5 days after the initial injection andexamined by electron microscopy with conventionaltechnics and after cytochemical reaction for perox-idase activity. Drug controls consisted of uninjectedanimals and animals injected with olive oil withoutCPIB. The regimen followed is summarized inTable I.

Following these procedures, animals were anes-thetized by intraperitoneal injection of sodium pen-tobarbital (Diabutal, Diamond Laboratories, Inc .,Des Moines, Iowa), and the liver was perfusedthrough the portal vein at room temperature with 3%glutaraldehyde containing 10 mm CaCI2 and bufferedto pH 7.4 with 0.1 M sodium cacodylate. Glutaralde-hyde was obtained from Fisher Scientific Co . (Pitts-burgh) and was vacuum distilled according to Fahimiand Drochmans (14), or the purified product waspurchased directly from Ladd Research Industries,Burlington, Vermont . Perfusion was continued for 2-5min after blanching of the liver, and then a portionwas removed, placed on dental wax in fresh fixative,and diced into small cubes with razor blades . Fixationwas continued at room temperature for a total time of30 min.

Routine PreparationsSpecimens were rinsed briefly in 0.1 M cacodylate

buffer, pH 7 .4, and postfixed in ice-cold 2 .57 Os04in the same buffer containing 5% sucrose . Subsequentdehydration in ethanol and embedding in Epon wereroutine (25) . Sections cut with a diamond knife werestained with uranyl acetate (45) and lead citrate (35)and examined in a Siemens Elmiskop I electronmicroscope .

Cytochemical PreparationsGlutaraldehyde-fixed tissue was rinsed several

times in cold 0 .1 M cacodylate buffer, pH 7.4, con-

1 We thank Dr . Jerome Noble, Ayerst Laboratories,

taining 5 0/0 sucrose, and selected specimens werechopped into 15- and 30-b sections with a SorvallTC-2 tissue sectioner (37) . These sections were storedin the refrigerator overnight in fresh rinsing solutionand stained the next day for peroxidase activity with amodification (48) of the procedure of Graham andKarnovsky (15) . Sections were rinsed briefly in 0 .05 MTris-HCI buffer, pH 9, and placed for I min in 0 .1CuSO4 dissolved in 0 .9% NaCl (17) . Following abrief rinse in Tris-HCl buffer, the tissues were in-cubated at room temperature for either 30 or 60 minin a medium composed of 10 ml of 0 .05 M Tris-HC1,pH 9, 0.025 g of 3 , 3'-diaminobenzidine tetrahydro-chloride (Sigma Chemical Co., St . Louis) and 0.1 mlof 3% H202. Cytochemical controls were run in theabsence of H202 or with 0 .02 M 3-amino-1,2,4-triazole (K & K Laboratories, Plainview, N.Y.)added to the complete medium (12) . The 30-µ sec-tions were rinsed briefly in distilled water and post-fixed, dehydrated, and embedded in Epon as de-scribed above . Most of the cytochemical material wasexamined in the electron microscope without subse-quent staining with heavy metal salts . The 15-µsections were mounted on glass slides in glycerol jelfor light microscopic study .

OBSERVATIONS

Light Microscopy

After the cytochemical reaction for detectingperoxidase activity, microbodies appeared in nor-mal hepatic cells as small, dense, circular particlessimilar in size to peribiliary dense bodies butdistributed evenly throughout the cytoplasm . Noevidence of a positive reaction could be localizedto other hepatic cell organelles, but red blood cellsand the granules of leukocytes stained intensely .

for supplying the CRIB .

Following the administration of CPIB, an increase

P. G. LEOG AND R. L . Woon New Observation on Microbodies

119

No . animalsTotal No .inj ./animal

Time of sacrificeafter inital inj .

Experimental 3 1 12 hr3 1 18 hr5 1 24 hr4 2 2 days4 3 3 days3 5 5 days

ControlsOlive oil

alone 2 1 24 hrUninjected 5

Scale marker on all figures equals 1 y .

FIGURES 1 and 2 Routine preparations of normal rat liver showing close apposition of endoplasmic reti-culum and microbodies . Ribosomes are absent where the reticulum membrane is closely applied to themicrobody membrane . Fig . 2 shows the same relationship of endoplasmic reticulum to a mitochondrion .Double stained. X 40,000.

in the number and heterogeneity of microbodieswas detected at 24 hr, and by 48 hr the changeswere marked . However, as in normal tissue, therewas no evidence of a positive reaction in otherorganelles . In the cytochemical controls incubatedwithout H 202 the staining of red blood cells andleukocyte granules remained intense, but only aweak reaction was observed in microbodies . Intissues incubated with aminotriazole in the me-dium, the microbody reaction was totally abolishedbut activity in blood cells remained .

Electron Microscopy

ROUTINE PREPARATIONS : CPIB treatmentcaused an increase in the number of microbodiesin hepatic cells clearly evident at 18 and 24 hr afteradministration of the drug, but more prominentafter longer treatment. The amount of tubular and

12 0

TEE JOURNAL OF CELL BIOLOGY • VOLUME 45, 1970

vesicular smooth endoplasmic reticulum increasedand there appeared to be a concomitant decreasein the amount of rough endoplasmic reticulum .Continuity between the membranes of rough andsmooth endoplasmic reticulum was common at theperiphery of areas comprised mainly of smoothendoplasmic reticulum (Fig. 4). By 3 and 5 days,mitochondria appeared more pleomorphic and thenumber of intramitochondrial matrix granules hadincreased noticeably. The nucleus, Golgi zone, andperibiliary dense bodies appeared normal in all ofthe experimental material .

Microbodies in stimulated cells resembled thosein normal cells and consisted of a moderatelyelectron-opaque, finely granular matrix sur-rounded by a single smooth membrane ; manymicrobodies possessed a distinct core or nucleoid .In normal cells and in hepatic cells under short

FIGURE 3 and 4 Routine preparations of rat liver 24 hr after CPIB treatment. Small vesicles (arrows)lie adjacent to microbodies . In Fig. 4, the small vesicle is attached to the larger microbody (arrow), andcontinuity of the rough and smooth endoplasmic reticulum is seen on the right . Double stained . Fig . 3, X21,500 ; Fig . 4, X 25,500.

bodies were about 0.2-0 .5 ,u in size and circular orovoid in outline, whereas at 2 days and later theirsize and shape varied widely.

A close spatial relationship between endoplasmicreticulum and microbodies was common . Manymicrobodies were situated between lamellae ofrough endoplasmic reticulum, and occasionally astrand of rough endoplasmic reticulum almostcompletely encircled individual microbodies (Fig .1) . Where microbodies were closely enshroudedwith endoplasmic reticulum, ribosomes were ab-sent from the membrane surface closely applied tothe microbody (Figs. 1, 2, 4) . Tubules of smoothendoplasmic reticulum also often surrounded mi-crobodies in a close spatial relationship . It wasnoteworthy that other particulate cell organellessuch as mitochondria and lipid droplets were foundin similar close relationships to both types of endo-plasmic reticulum (Fig . 2) . Despite careful exami-nation, a continuity between the limiting mem-brane of the microbody and membranes of eitherrough or smooth endoplasmic reticulum was notdemonstrated .

During the early phases (12-24 hr) of CPIBtreatment, many microbodies showed small ex-tensions of their limiting membrane. Smooth-

walled vesicles were often closely associated withmicrobodies and, although these vesicles could notbe positively identified as small microbodies, theycontained material similar in appearance to thatcomprising the microbody matrix (Fig. 3). Insome instances, it was possible to identify mem-brane continuity between the vesicle and theneighboring microbody (Fig . 4) .CYTOCHEMICAL PREPARATIONS : In tissue

incubated in the complete medium, all micro-bodies showed a uniform intense reaction of themicrobody matrix (Figs . 5, 6) . The small surfaceextensions and small vesicles associated with themicrobodies seen during the early phases of CPIBtreatment also contained material which stainedintensely (Figs . 5, 6) . The connections betweenmicrobodies and the adjacent vesicles were moreprominent in cytochemical preparations sincethese preparations were usually examined withoutfurther heavy metal contrasting (Figs . 6-8) . Inmaterial sampled 3 and 5 days after commencingCPIB treatment, relatively few microbodiesshowed protrusions or connections with adjacentvesicles which by these times appeared larger thanat earlier phases.Microbody extensions were occasionally iden-

P. G. LEGG AND R. L. WOOD New Observations on Microbodies 121

FIGURE 5 Peroxidase-stained rat liver 24 hr after CPIB treatment . There are numerous small peroxidase-positive vesicles (arrows), two of which appear connected to larger microbodies by delicate strands .Peribiliary dense bodies (Db) show some positive reaction, but no reaction occurs in the Golgi apparatus(Go) . Bile canaliculus, Be . No lead or uranyl staining . X 10,500 .

1 22

THE JOURNAL OF CELL BIOLOGY . VOLUME 45, 1970

FIGURE 6 Peroxidase-stained rat liver 24 hr after CPIB treatment . Small vesicles associated with micro-bodies are indicated at the single arrows . Evidence of ribosomal staining appears at the double arrows .The reaction product is sharply localized around other microbodies in the micrograph. No lead or uranylstaining . X 16,000 .

FIGURES 7-9 Rat liver 24 hr after CPIB treatment, showing microbodies in connection with smallvesicles . Figs. 7 and 8 show material reacted for peroxidase whereas Fig . 9 is a cytochemical control runwithout H202 . The preparations for Figs . 7 and 9 are double stained, and Fig. 8 shows material withoutlead or uranyl staining . X 32,000 .

P. G. LEGG AND R. L . WooD New Observations on Microbodies

123

FIGURE 10 Normal rat liver reacted for peroxidase . Ribosomal staining is clearly evident . No reactionproduct is present in cisternae of the rough endoplasmic reticulum . No lead or uranyl staining . X 30,000 .

FIGURE 11 Peroxidase-stained rat liver 18 hr after CPIB treatment. Ribosomal staining is similar tothat in the normal liver (Fig. 10) . No lead or uranyl staining . X 30,000 .

tified in untreated normal hepatic cells reacted forperoxidase activity . The continuities between ves-icles and microbodies also were seen clearly inCPIB-treated material used as cytochemical con-trols (Fig . 9) .

A preferential distribution of microbodies wasdetected after the cytochemical reaction . In nor-mal hepatic cells, microbodies appeared most

124

THE JOURNAL OF CELL BIOLOGY . VOLUME 45,1970

commonly in areas of rough endoplasmic reticu-lum. During the early phases of CPIB stimulation(12-18 hr), they tended to be located at theboundary between rough endoplasmic reticulumand areas containing smooth endoplasmic retic-ulum or "glycogen ." After 24 hr, the microbodieswere more numerous within the areas of smoothendoplasmic reticulum or "glycogen ."

An unusual distribution of staining was ob-served where microbodies were in close relation-ship to rough endoplasmic reticulum . Reactionproduct was present not only within the micro-body matrix, but outside the limiting membraneof the microbody and in association with ribo-somes of adjacent rough endoplasmic reticulum .This feature was most prominent at early inter-vals after CPIB administration (12-24 hr), butwas also present in normal hepatic cells (Figs . 6,

10, 11). In contrast, those microbodies not closelyrelated to rough endoplasmic reticulum but sur-rounded by tubules of smooth endoplasmicreticulum showed no reaction outside the micro-

body. At later phases of CPIB treatment, whenmicrobody numbers were greatly increased (2-5days), the ribosomal staining was markedly lessevident than in earlier phases . Ribosomal stainingwas not eliminated by variations in fixation time,variations in incubation time including shorterperiods, or by not utilizing preincubation inCuSO4 solution.

Under all conditions used, microbodies withsharp localization of reaction product werepresent in the same cells in which the diffusereaction was observed . Furthermore, there wasno evidence of a diffuse staining related to otherstructures with a positive reaction for peroxidase,including red blood cells, the azurophil granulesof leukocytes, and lysosomes .

At no stage was peroxidase activity demon-strated within cisternae of the rough or smoothendoplasmic reticulum or the Golgi apparatus ofhepatic cells.

DISCUSSION

Conclusions concerning the origin of microbodieswhich have arisen from previous morphologicalstudies (5, 8, 9, 29, 40, 42, 43, 46, 47) are basedon three main factors : (a) alleged demonstrationof continuity of membranes between the micro-body and a precursor organelle, (b) similarity inelectron opacity and texture of microbody con-tents and content of a precursor organelle, and(c) topographical relationships between themicrobody and a precursor organelle . For eachof these factors, the evidence on which it is basedremains somewhat equivocal . First, there is littledoubt that membranous extensions and protru-sions of the limiting membrane of the microbodyoccur, but evidence that these are actually in con-tinuity with endoplasmic reticulum is uncon-

vincing . Second, similarity in electron opacitybetween material within the microbody andwithin adjacent organelles is not conclusive evi-dence that the two structures are related . Thematerial observed within cisternae of the endo-plasmic reticulum by many investigators may wellrepresent totally unrelated substances . Third,proximity between microbodies and other or-ganelles provides only inferential evidence ofmicrobody origin .The present study with a cytochemical technic

for the localization of endogenous peroxidaseactivity was designed to explore microbody for-mation under conditions of rapid proliferationinduced experimentally. The staining method

involves oxidation of diaminobenzidine and de-position of a dense reaction product, the mecha-nism of which is not well understood . Neverthe-

less, many investigators have concluded that thedeposition of reaction product in microbodies isdue to the peroxidatic activity of catalase (4,11-13, 44) . It is possible that other unidentifiedperoxidases exist in microbodies and may beresponsible for part of the observed staining, butuse of this technic for studying microbody originappears valid regardless of the absolute specificityof the reaction .Though various mechanisms have been pro-

posed for microbody proliferation, the possibilitythat new microbodies arise from preexistingmicrobodies has not been advanced . In the pres-ent study, small smooth-walled vesicles containingmaterial similar in appearance to that comprisingthe microbody matrix were seen adjacent tomicrobodies, particularly during the early stagesof CPIB treatment (12-24 hr) . As already indi-cated, the morphological appearance of thismaterial does not establish the vesicles as smallmicrobodies, but the observation that this ma-terial possesses peroxidase activity suggests thatthe vesicles are indeed related to microbodies .

Proliferation of microbodies is known to occurduring CPIB administration (40-42), and thecontinuity seen between the membranes of thesmall vesicles and neighboring microbodies in thepresent study further suggests that these vesiclesare derived, at least in some instances, from micro-bodies. Since other evidence concerning the fateof the small vesicles is lacking and since micro-

bodies at later stages of CPIB treatment (2-5

days) are heterogeneous in size and shape, itseems reasonable to postulate that the small

P. G . LEGG AND R. L. WooD New Obsevrations on Microbodies 125

smooth-walled vesicles are immature microbodieswhich have the capacity to increase in size and toform mature microbodies . In this scheme, theprocess by which the vesicles form could be con-sidered either as fragmentation or as budding,but, whatever the exact mechanism, the endresult would be the formation of new microbodiesfrom preexisting microbodies.

Other evidence that catalase in newly formedmicrobodies may be derived from preexistingmicrobodies has recently been presented. Reddyet al. (34) found that when CPIB was adminis-tered to a strain of mice genetically deficient incatalase the number of peroxidase-positive micro-bodies increased but there was no measurableincrease in liver catalase activity. In an attemptto account for this inconsistency, they suggestedthat a redistribution of catalase occurs ; the meansby which this might be accomplished was notelaborated . The process of microbody fragmenta-tion or budding would readily explain their find-ings.

Aside from protrusions attributable to thisphenomenon, the present observations show noevidence of microbody membrane continuitywith other membranous organelles. It is possiblethat continuities do exist but, if so, they must beinfrequent. This finding does not agree with theobservations of Novikoff and Shin (29) and isdirectly opposed to the concept that microbodiesare always attached to smooth endoplasmicreticulum in vivo . The continuities betweenmicrobodies and rough endoplasmic reticulumreported by Essner (9) in fetal mouse liver and byTsukada et al . (43) in fetal rat liver were not con-

firmed in adult tissue . Different mechanisms may

be involved in initial microbody formation as dis-

tinct from microbody proliferation in the adult,

but it is noteworthy that such continuities were

not observed in fetal rat liver by Wood (48) .It is of interest that in the present study no

peroxidase reaction product was found within

cisternae of the endoplasmic reticulum or Golgi

apparatus. This observation has several possible

explanations. The most obvious is that catalase ispresent at these sites but at a concentration below

the sensitivity of the cytochemical technic (3, 4) .

Although this possibility cannot be refuted, theintensity of microbody staining and the fact that

intracisternal staining of peroxidase has been

observed in developing rat leukocytes with the 2 Wood, R. L., unpublished data.

126

THE JOURNAL OF CELL BIOLOGY . VOLUME 45, 1970

same technic2 suggest that the sensitivity of thetechnic may be sufficient to detect catalase atthese sites . However, it must be noted that theease of demonstration of peroxidase activitywithin cisternae of endoplasmic reticulum andGolgi apparatus of developing leukocytes is

species dependent (1-3, 27, 49) . Another explana-tion is that the enzyme is present in the endo-plasmic reticulum in an inactive form, a tenablehypothesis but difficult to test. A third possibilityis that catalase is not segregated into endoplasmicreticulum or Golgi cisternae after synthesis, but re-mains in the cytoplasmic matrix to be transferreddirectly into microbodies. The latter possibilitydeserves consideration in light of published bio-chemical evidence on catalase synthesis .

Biochemical studies by Higashi and Peters (18,19) have shown that catalase is synthesized bymembrane-associated ribosomes and transferredto particulate bodies, which can be assumed to bemicrobodies (their fractionation procedures didnot separate microbodies from mitochondria) .They found no evidence that the newly synthe-sized catalase is transferred into cisternae of roughor smooth endoplasmic reticulum, and they con-

catalase either becomesmicrobodies or is trans-reticulum membrane to

eluded tentatively thatsoluble and migrates toferred directly from theits definitive site .

In the present study, the ribosomal staining,the close apposition of strands of endoplasmicreticulum and microbodies, and the lack ofperoxidase activity within the cisternae of endo-plasmic reticulum are interpreted as morphologi-

cal evidence that catalase, after synthesis on theribosomes, may actually follow the pathways sug-gested by the biochemical studies of Higashi and

and Peters (18, 19). The possibility that the ribo-somal staining represents cytochemical diffusionartifact must be considered and cannot be totallydiscounted from the present evidence . Neverthe-less, it is thought to be unlikely for the followingreasons : (a) Ribosomal activity was not seen atall microbodies enwrapped with rough endoplas-mic reticulum. (b) Under all conditions used,microbodies with sharp localization of reactionproduct were present in the same sections thatshowed microbodies with adjacent ribosomalstaining. (c) Variations in fixation and stainingprocedures (shorter incubation times, lower con-

centrations of diaminobenzidine, no CuSO4 treat-ment) altered the intensity of staining, but hadno significant effect on localization of reactionproduct. (d) The effect was more pronounced inearly stages of CPIB stimulation, whereas at laterstages, when the number of microbodies had in-creased greatly, ribosomal staining was markedlyless evident than at early stages. (e) There was noevidence of a diffuse reaction in relation to redblood cells, peribiliary dense bodies, or phago-cytic vacuoles, all of which had peroxidatic ac-tivity .

The hypothesis that catalase may be synthe-sized at ribosomes and enter the microbodydirectly remains tentative, and confirmationobviously depends on further study . Such a routedoes not conform with the generally acceptedtenets concerning synthesis and sequestration ofproteins ; if the hypothesis is to be entertainedseriously, the mechanism has to be established bywhich catalase, a large molecular weight protein(mol wt 249,000 [33]), passes across the micro-body membrane. There is no direct evidencefrom this study to indicate by what means thismight be accomplished, but other studies providesome information relevant to the problem . First,it has been shown that the microbody membrane

REFERENCES

ACKERMAN, G . A. 1968. Contributions of thenuclear envelope, endoplasmic reticulum, andGolgi to the formation of peroxidase andlysosomal (azurophil) granules during neutro-phil granulopoiesis. Anat . Rec. 160:304 .

BAINTON, D. F., and M. G. FARQUHAR. 1967 .Segregation and packaging of granule enzymesin eosinophils. J . Cell Biol. 35:6 A. (Abstr .)

BAINTON, D. F., and M . G. FARQUHAR. 1968 .Differences in enzyme content of azurophil andspecific granules of polymorphonuclear leuko-cytes. II . Cytochemistry and electron micros-copy of bone marrow cells. J. Cell Biol.39:299 .

BEARD, M. E., and A. B. NoviKosa. 1969 . Dis-tribution of peroxisomes (microbodies) in thenephron of the rat. A cytochemical study . J.Cell Biol. 42:501 .

BRUNI, C., and K . R. PORTER . 1965 . The finestructure of the parenchymal cell of the normalrat liver . I. General observations . Amer. J.Pathol . 46:691 .

DE DUVE, C. 1965. The separation and charac-terization of subcellular particles . Harvey Lect.59:49.

1 .

2 .

3 .

4.

5.

6.

differs in osmotic and permeability propertiesfrom other cell membranes (6, 7) . Secondly, thereis biochemical evidence to indicate that smallerenzymatically active molecular forms of catalaseexist in the hepatic cell (20, 26, 31) . These lowermolecular weight configurations could conceivablybe the form of catalase at the time of incorpora-tion into the microbody .Thus, although additional knowledge is re-

quired before a definite conclusion can be reached,the possibility that catalase may be inserteddirectly into microbodies from ribosomes withoutinvolving transit through the endoplasmic reticu-luln or Golgi apparatus deserves consideration inspite of its being an unorthodox concept .

This study was supported by research grant HD-01337 from the Institute of Child Health and HumanDevelopment, United States Public Health Service .The authors acknowledge the technical assistance ofMrs. Barbara Mozayeny and Mrs. Judith Henrickson .We thank Dr . H . David Coulter for suggestions on themanuscript. Dr. Legg is at present on leave from theDepartment of Anatomy, Monash University, Mel-bourne, Australia.Received for publication 21 August 1969, and in revisedform 10 October 1969 .

7. DE DUVE, C., and P. BAUDHUIN. 1966. Perox-isomes (microbodies and related particles) .Physiol . Rev. 46:323 .

8. DvokkK, M., and K. MAZANEC. 1967 . Differ-enzierung der Feinstruktur der Leberzelle inder frühen postnatalen Periode . Z . Zellforsch .Mikrosk . Anat. 80:370 .

9. ESSNER, E. 1967. Endoplasmic reticulum and theorigin of microbodies in fetal mouse liver . Lab .Invest . 17 :71 .

10 . ESSNER, E. 1968 . Cytochemical studies of micro-bodies in developing hepatocytes . J. Cell Biol .39:42 A . (Abstr . )

11 . ESSNER, E . 1969 . Localization of peroxidase ac-tivity in microbodies of fetal mouse liver . J.Histochem . Cytochem . 17:454 .

12 . FAHIMI, H . D. 1968 . Cytochemical localization ofperoxidase activity in rat hepatic microbodies(peroxisomes) . J. Histochem . Cytochem . 16:547 .

13 . FAHIMI, H . D . 1968 . Peroxidase activity of micro-body catalase . J. Cell Biol. 39:42 A. (Abstr .)

14 . FAHIMI, H . D ., and P. DROCHMANS . 1965 . Essaisde standardisation de la fixation au glutaral-déhyde . I . Purification et détermination de la

P. G. LEGG AND R . L. WOOD New Observations on Microbodies

127

concentration du glutaraldéhyde . J. Microsc.4 :725 .

15 . GRAHAM, R. C ., and M. J. KARNOVSKY. 1966.The early stages of absorption of injectedhorseradish peroxidase in the proximal tubulesof mouse kidney : ultrastructural cytochemistryby a new technique . J. Histochem. Cytochem .14 :291 .

16 . HESS, R., W . STAUBLI, and W. RIESS . 1965 . Na-ture of the hepatomegalic effect produced byethyl -chlorophenoxy-isobutyrate in the rat .Nature (London) . 208:856 .

17 . HIGASHI, 0. 1966 . The shortest peroxidase staintime and the mean cellular peroxidase contentof blood leukocytes. Tohoku J. Exp . Med. 89 :1 .

18 . HIGASHI, T ., and T. PETERS, JR . 1963 . Studies onrat liver catalase. I . Combined immunochem-ical and enzymatic determination ofcatalase inliver cell fractions . J. Biol . Chem . 238 :3945 .

19 . HIGASHI, T ., and T. PETERS, JR . 1963 . Studies onrat liver catalase . II. Incorporation of 14 C-leucine into catalase of liver cell fractions invivo . J. Biol . Chem . 238 :3952 .

20 . HOLMES, R . S ., and C . J. MASTERS . 1964 . Catalaseheterogeneity. Arch . Biochem . Biophys . 109 :196

21 . HRUBAN, Z ., and M. RECHCIGL,JR. 1969 . Micro-bodies and related particles. Morphology,biochemistry and physiology . Int. Rev . Cytol.Suppl . I .

22 . HRUBAN, Z., H . SWIFT, and A . SLESERS . 1966 .Ultrastructural alterations of hepatic micro-bodies. Lab . Invest . 15:1884.

23 . JÉZÉQUEL, A.-M ., K . ARAKAWA, and J. W .STEINER . 1965 . The fine structure of the normalneonatal mouse liver . Lab. Invest . 14:1894 .

24 . LEIGHTON, F., B . POOLE, P . B. LAZAROW, and C .DE DuvE. 1969. The synthesis and turnover ofrat liver peroxisomes. 1 . Fractionation ofperoxisome proteins . J. Cell Biol . 41 :521 .

25 . LUFT, J . H . 1961 . Improvements in epoxy resinembedding methods. J. Biophys. Biochem . Cytol .9 :409.

26 . NISHIMURA, E. T., S . N. CARSON, and T. Y .KOBARA . 1964 . Isozymes of human and ratcatalases . Arch. Biochem. Biophys . 108 :452 .

27 . NOVIKOFF, A . B ., L . BIEMPICA, N. QUINTANA, A .ALBALA, and R. DOMINITZ . 1968 . Peroxidaticactivity in cell organelles. J. Cell Biol . 39 :100 A .(Abstr .)

28 . NOVIKOFF, A. B., and S . GOLDFISCHER . 1968 .Visualization of microbodies for light and elec-tron microscopy . J. Histochem. Cytochem . 16 :507 .

29 . NovIKOFF, A. B., and W.-Y. SHIN . 1964 . Theendoplasmic reticulum in the Golgi zone andits relations to microbodies, Golgi apparatusand autophagic vacuoles in rat liver cells . J.Microsc . 3 :187.

30. PAGET, G . E. 1963 . Experimental studies of the

toxicity of Atromid with particular reference tofine structural changes in the livers of rodents .J. Atheroscler. Res . 3 :729.

PATTON, G . W., and E . T. NISHIMURA . 1967 .Developmental changes of hepatic catalase inthe rat. Cancer Res . 27:117.

PETERS, V . B., G . W. KELLY, and H. M.DEMBITZER . 1963 . Cytologic changes in fetaland neonatal hepatic cells of the mouse. Ann .N.Y. Acad . Sci . 111 :87 .

PRICE, V. E ., and R. E . GREENFIELD . 1954. Livercatalase . II . Catalase fractions from normaland tumor-bearing rats . J. Biol . Chem . 209 :363 .

REDDY, J., S . BUNYARATVEJ, and D. SVOBODA .1969. Microbodies in experimentally alteredcells . IV . Acatalasemic (Csb) mice treated withCPIB . J. Cell Biol. 42 :587 .

REYNOLDS, E. S. 1963. The use of lead citrate athigh pH as an electron-opaque stain in electronmicroscopy . J. Cell Biol. 17:208.

ROUILLER, C ., and W. BERNHARD . 1956. "Micro-bodies" and the problem of mitochondrialregeneration in liver cells . J. Biophys . Biochem .Cytol. 2(4, Suppl .) :355 .

SMITH, R . E., and M. G. FARQUHAR . 1963 . Prep-arations of thick sections for cytochemistry andelectron microscopy by a non-freezing tech-nique . Nature. 200 :691 .

STENGER, R . J ., and D. B. CONFER . 1966 . Hepato-cellular ultrastructure during liver regenerationafter subtotal hepatectomy . Exp . Mol . Pathol.5:455.

STEPHENS, R . J ., and R . F . BILs . 1967 . Ultrastruc-tural changes in the developing chick liver . I .General cytology . J. Ultrastruct . Res . 18 :456 .

SVOBODA, D. J ., and D. L . AZARNOFF . 1966 .Response of hepatic microbodies to a hypo-lipidemic agent, ethyl chlorophenoxyisobutyr-ate (CPIB) . J. Cell Biol . 30 :442 .

SVOBODA, D., D . AZARNOFF, andJ . REDDY . 1969 .Microbodies in experimentally altered cells . II .The relationship of microbody proliferation toendocrine glands . J. Cell Biol . 40 :734 .

SVOBODA, D., H. GRADY, and D . AZARNOFF .1967. Microbodies in experimentally alteredcells. J. Cell Biol . 35 :127 .

TSUKADA, H., Y . MOCHIZUKI, and T . KONISHI .1968 . Morphogenesis and development ofmicrobodies of hepatocytes of rats during pre-and postnatal growth . J. Cell Biol . 37 :231 .

44. VENKATACHALAM, M. A., and H. D . FAHIMI .1969. The use of beef liver catalase as a proteintracer for electron microscopy . J. Cell Biol .42 :480 .

45 . WATSON, M . L . 1958 . Staining of tissue sectionsfor electron microscopy with heavy metals . J.Biophys . Biochem. Cytol . 4 :475 .

WOOD, R. L . 1965 . The fine structure of hepatic

31 .

32 .

33 .

34 .

35 .

36.

37.

38 .

39 .

40 .

41 .

42 .

43 .

46 .

128

THE JOURNAL OF CELL BIOLOGY • VOLUME 45, 1970

cells in chronic ethionine poisoning and during 48 . WOOD, R. L . 1969 . Studies on the origin of micro-recovery. Amer . J. Pathol . 46:307 .

bodies in embryonic liver . Anat . Rec . 163:287 .47 . WOOD, R. L . 1965. An electron microscope study 49 . YAMADA, E. 1966. Electron microscopy of the

of developing bile canaliculi in the rat . Anat . peroxidase in the granular leucocytes of ratRec . 151 :507 .

bone marrow. Arch. Histol. Jap. 27 :131 .

P. G. LEGG AND R. L. WOOD New Observations of Microbodies

129