Hepatic intranuclear glycogen inclusions in western barred bandicoots(Perameles bougainville)

Mark D. Bennett,1 Lucy Woolford, Philip K. Nicholls, Kristin S. Warren, Amanda J. O’Hara

Abstract. The western barred bandicoot, Perameles bougainville, is an endangered Australian marsupialspecies. Routine histology of liver samples collected at necropsy from 19 of 20 (95%) western barredbandicoots revealed the sporadic to common occurrence of abnormal hepatocyte nuclei characterized bymargination of chromatin and concomitant central pallor. Some abnormal hepatocyte nuclei were mildly tomarkedly enlarged and irregularly shaped. Periodic acid–Schiff reagent stained 131 of 142 (92%) of theseabnormal hepatocyte nuclei. Positive staining was completely eliminated by diastase pretreatment.Transmission electron microscopy revealed that abnormal hepatocyte nuclei with marginated chromatin didnot contain viral particles. Rather, glycogen b-particles and a-rosettes were identified within some abnormalhepatocyte nuclei. Glycogen intranuclear inclusions were an incidental finding in western barred bandicoothepatocytes.

Key words: Glycogen; intranuclear inclusion; marsupials; Perameles bougainville.

<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>Western barred bandicoots, Perameles bougainville (an

endangered Australian marsupial species), can be affectedby a papillomatosis and carcinomatosis syndrome.12,13 Thissyndrome is characterized by multicentric hyperplastic,dysplastic, and neoplastic lesions of cutaneous andmucocutaneous tissues and has been associated with anovel virus.12,13 Abnormal keratinocytes containing nucleiwith marginated chromatin and large amphophilic intra-nuclear inclusion bodies have been identified in the stratumgranulosum of conjunctival lesions examined by routinehistology.12 Crystalline arrays of spherical viral particles,45 nm in diameter, have been observed within the nuclei ofrare conjunctival keratinocytes within these lesions bytransmission electron microscopy.12

Routine histology of western barred bandicoot liverscollected at necropsy typically revealed the presence ofabnormal hepatocyte nuclei characterized by marginationof chromatin and concomitant central pallor with occa-sional hepatic nuclear enlargement and asymmetry (Fig. 1).The nature of these hepatic intranuclear inclusions was ofparticular interest, given the occurrence of intranuclearviral inclusions in keratinocytes from conjunctiva affectedby the western barred bandicoot papillomatosis andcarcinomatosis syndrome.

Archival hematoxylin and eosin–stained histology slidesfrom 20 western barred bandicoots necropsied between2000 and 2006 were examined for the occurrence ofabnormal hepatocyte nuclei. These individuals all hadlesions consistent with western barred bandicoot papillo-matosis and carcinomatosis syndrome, and some hadincidental Klossiella quimrensis infection of their kidneys.1

The frequency with which these abnormal nuclei occurredwas determined by examination of 10 randomly selectedhigh-power fields (403 objective) per slide. Of the 20 livers

assessed, 19 (95%) contained abnormal hepatic nucleicharacterized by intranuclear inclusions. On average, 7.8hepatocyte intranuclear inclusions were observed per 10high-power fields examined (range 0–56).

Sections (6-mm thick) of 4 formalin-fixed, paraffin-embedded western barred bandicoot liver samples weretreated with diastasea followed by periodic acid–Schiff(PAS) or with PAS alone. Examination of sections treatedwith PAS alone showed positive staining in 131 of 142(92%) intranuclear inclusions (Fig. 2). The intensity ofPAS-positive staining varied from mild to marked. ThePAS-positive staining of abnormal nuclei was completelyeliminated (0/143; 0%) in sections pretreated with diastase(Fig. 3), suggesting a glycogen content of the intranuclearinclusions. Glycogen intranuclear inclusions were mostprevalent in liver sections containing the highest levels ofcytoplasmic glycogen detected using the PAS/diastasemethod, both in terms of the number of glycogen-richhepatocytes and the amount of glycogen they contained.Glycogen-containing nuclei were scattered randomlythroughout the liver. Rare, profoundly PAS-positive,round intranuclear glycogen bodies were observed in thesesections (Fig. 2), as has been previously described.2

Approximately 8% of abnormal hepatocyte nuclei did notshow detectable PAS-positive staining. This result might bedue to leaching of glycogen from hepatocytes duringfixation and processing. Alternate fixation and processingtechniques to improve the retention of glycogen in liversections have been described4; however, the sections fromarchival specimens used in the present study were routinelyfixed in 10% neutral-buffered formalin and paraffinembedded.

One western barred bandicoot liver sample was fixed in5% glutaraldehyde; rinsed in Sorensen phosphate buffer;postfixed in Dalton chrome osmic acid; dehydratedthrough graded alcohols; transferred into propylene oxideor propylene oxide/Epon 812; and embedded in Epon 812.b

Ultrathin sections were cut, mounted on grids, and stainedwith lead citrate and uranyl acetate. These were examinedusing a Philips CM 100 BioTwin transmission electron

From the School of Veterinary and Biomedical Sciences,Murdoch University, Murdoch, Western Australia.

1 Corresponding Author: Mark D. Bennett, School of Veteri-nary and Biomedical Sciences, Murdoch University, South Street,Murdoch, Western Australia 6150, Australia. m.bennett@murdoch.edu.au

J Vet Diagn Invest 20:376–379 (2008)

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microscope at an accelerating voltage of 80 kV.c Abnormalnuclei had peripherally marginated chromatin and nucleoli.No virions were seen in any hepatic nuclei examined.Occasional abnormal nuclei contained a central zone ofglycogen b-particles, each with an average diameter of20 nm, typically arranged in a-rosettes. These glycogeninclusions were in direct contact with the nucleoplasm andappeared to have displaced the chromatin and nucleolus

peripherally. No contact was observed between the nuclearmembrane and the central glycogen inclusion, nor werecytoplasmic elements, such as mitochondria, ribosomes,and endoplasmic reticulum, observed within the glycogenmass (Figs. 4, 5).

Glycogen is a branched polymer of glucose that isnormally found in the cytoplasm of many cell types. It actsas a depot for intracellular glucose, which can be releasedfrom storage by the combined enzymatic activities ofphosphorylase, transferase, and a-1,6-glucosidase.3,11 Oc-casionally, glycogen may appear within hepatocyte nucleiand has been reported in humans with von Gierke disease5,9

(glucose-6-phosphatase deficiency), diabetes mellitus,4,5

Graves disease,5 Hodgkin disease,5 hepatitis,2,5 Wilsondisease,5 Gilbert disease,5 and carcinoma of the stomach5

and also after prednisolone treatment for disseminatedlupus erythematosus.5,10 Although intranuclear glycogen isan accompaniment to many disease processes, includinginflammation, neoplasia, and necrosis, there has beenspeculation on a possible association between the occur-rence of intranuclear glycogen inclusions and illnesses thatlead to relative hypoxia in humans.4 Therefore, it may benoteworthy that the western barred bandicoot with themost abundant intranuclear glycogen deposition in thepresent study was concurrently affected by numerouspulmonary metastases of a squamous cell carcinoma. Agedlactating and nonlactating clinically healthy ruminants8

and well-fed premetamorphic tadpoles5,6 commonly havehepatic intranuclear glycogen accumulations, highlightingthat intranuclear glycogen deposition is not necessarilyindicative of a pathologic process.

It is possible for glycogen to be synthesized in situ withinthe interchromatin regions of the nucleus.7,14 Some authorssuggest that glycogen may be translocated from thecytoplasm across the nuclear membrane and accumulate

Figure 2. Periodic acid–Schiff (PAS)-stained liver sectionfrom a western barred bandicoot (Perameles bougainville) showingpositive intranuclear staining of an abnormal hepatocyte nucleus.In this example, numerous PAS-positive, variably sized, roundbodies are present within the nucleus. The nuclear material hasbeen displaced peripherally by the presence of intranuclearglycogen. Bar 5 25 mm.

Figure 3. Diastase-pretreated, periodic acid–Schiff (PAS)-stained liver section from a western barred bandicoot (Peramelesbougainville) showing abolition of PAS-positive staining ofhepatocyte intranuclear inclusions (compared with Fig. 2). Bar5 25 mm.

Figure 1. Hematoxylin and eosin–stained liver section froma western barred bandicoot (Perameles bougainville) showing 2abnormal hepatocyte nuclei. Abnormal hepatocyte nuclei haveperipherally distributed chromatin surrounding a zone of centralpallor and are enlarged and asymmetrical. Bar 5 25 mm.

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in the nucleus; however, the experimental evidence tosupport this hypothesis is lacking.5 The illusion ofintranuclear glycogen inclusions may sometimes be givenby invagination of cytoplasmic elements (including glyco-gen) into the nucleus.5 These cytoplasmic invaginationstypically contain other cytoplasmic elements, such asribosomes, endoplasmic reticulum, and mitochondria, andare delimited by the nuclear membrane.5 The present studyclearly demonstrates that western barred bandicoot hepa-tocyte nuclei can contain genuine intranuclear glycogeninclusions. The occurrence of glycogen intranuclear inclu-sions in hepatocytes does not appear to be related to thepapillomatosis and carcinomatosis syndrome present in thisspecies but is, rather, an incidental finding of no apparentclinical significance.

Acknowledgements. Thanks to Michael Slaven andGerard Spoelstra for preparing the histology slides andPeter Fallon for assisting with electron microscopy. Thisproject is a collaboration between Murdoch University andthe Western Australian Department of Environment andConservation, funded through Australian Research Coun-cil linkage project LP0455050.

Sources and manufacturers

a. Diastase (a-amylase type VI-B from porcine pancreas), SigmaChemical Co., St. Louis, MO.

b. TAAB Laboratories Equipment Ltd., Reading, Berkshire, UK.c. Philips/FEI Corporation, Eindhoven, Holland.

References

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2. Caramia F, Ghergo FG, Branciari C, Menghini G: 1968, Newaspect of hepatic nuclear glycogenosis in diabetes. J ClinPathol 21:19–23.

3. Cheville NF: 1994, Ultrastructural pathology: an introductionto interpretation. Iowa State University Press, Ames, IA.

4. Chipps HD, Duff GL: 1942, Glycogen infiltration of the livercell nuclei. Am J Pathol 18:645–659.

5. Ghadially FN: 1988, Intranuclear glycogen inclusions. In:Ultrastructural pathology of the cell and matrix, ed. GhadiallyFN, 3rd ed., pp. 1–180. Butterworths, London, UK.

6. Himes MM, Pollister AW: 1962, Symposium: syntheticprocesses in the cell nucleus. V. Glycogen accumulation inthe nucleus. J Histochem Cytochem 10:175–185.

Figure 4. Transmission electron micrograph of a glycogenintranuclear inclusion from a western barred bandicoot (Pera-meles bougainville) hepatocyte (lead citrate and uranyl acetatestain). The nuclear material (N) has been displaced peripherally.The center of the nucleus is occupied by glycogen (G) arranged inb-particles ,20 nm in diameter and a-rosettes. The glycogeninclusion body is not part of a cytoplasmic invagination.

Figure 5. Higher magnification transmission electron mi-crograph of the glycogen intranuclear inclusion depicted inFigure 4. The fine structure of glycogen a-rosettes (G) is visiblewithin the nucleus. Chromatin (C) has been displaced to theperiphery of the nucleus. The nuclear membrane (M) iswell demarcated.

378 Brief Communication

7. Karasaki S: 1971, Cytoplasmic and nuclear glycogen synthesis inNovikoff ascites hepatoma cells. J Ultrastruct Res 35:181–196.

8. Reid IM: 1973, Hepatic nuclear glycogenosis in the cow. JPathol 110:267–270.

9. Sheldon H, Silverberg M, Kerner I: 1962, On the differingappearance of intranuclear and cytoplasmic glycogen in livercells in glycogen storage disease. J Cell Biol 13:468–473.

10. Sparrow WT, Ashworth CT: 1965, Electron microscopy ofnuclear glycogenosis. Arch Pathol 80:84–90.

11. Stryer L: 1995, Biochemistry, 4th ed. W.H. Freeman andCompany, New York, NY.

12. Woolford L, O’Hara AJ, Bennett MD, et al.: 2008, Cutaneouspapillomatosis and carcinomatosis in the western barredbandicoot (Perameles bougainville). Vet Pathol 45:95–103.

13. Woolford L, Rector A, Van Ranst M, et al.: 2007, A novelvirus detected in papillomas and carcinomas of the endan-gered western barred bandicoot (Perameles bougainville)exhibits genomic features of both the Papillomaviridae andPolyomaviridae. J Virol 81:13280–13290.

14. Zimmermann H-P, Granzow V, Granzow C: 1976, Nuclearglycogen synthesis in Ehrlich ascites cells. J Ultrastruct Res54:115–123.

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