Int. J. Exp. Path. (1994), 75, 313-320

Expression and localization of sulphated glycoprotein-2 mRNA inthe rat incisor tooth ameloblasts: relationships with apoptosis

B.K. JOSEPH*, G.C. GOBEt, N.W. SAVAGE* AND W.G. YOUNG**Department of Dentistry, Division of Oral Biology and Pathology, The University of Queensland,

Brisbane, Queensland, and tDepartment of Pathology, The University of Queensland Medical

School, Herston Road, Herston, Brisbane, Queensland 4006, Australia

Received for publication 18 January 1994Accepted for publication 6 June 1994

Summary. The expression of sulphated glycoprotein-2 (SGP-2) is associatedwith the onset of cellular atrophy and death in many rodent tissues. Thisgene has a multifunctional involvement that includes apoptosis, sperma-togenesis, promotion of cell-cell interactions, modulation of complementsystems and tissue regeneration and remodelling. Using decalcified mand-ibles, mRNA for SGP-2 in rat incisor tooth ameloblasts was examined by insitu hybridization using 35S riboprobes. The rat incisor is unique in that, atone time, all stages of the complex life cycle of the ameloblasts arerepresented along the length of the enamel forming aspect of the tooth.The pre-ameloblasts only secrete enamel matrix after mitosis. When the fullthickness of the enamel has been formed, a remarkable transition inphenotype takes place in the ameloblast. This transition is accompaniedby apoptosis or programmed cell death of approximately 25% of amelo-blasts. An additional 25% of ameloblasts undergo apoptosis when matura-tion of enamel matrix takes place with removal of water and protein from theincreasingly mineralized matrix. In the present study, expression of SGP-2was localized most often in the post-secretory transition and maturationameloblasts. In contrast, the presecretory and secretory ameloblasts did notdemonstrate specific hybridization signals. Consistently, neither the odonto-blasts nor the pulp demonstrated hybridization signals. Hence our resultssupport other published results which show that increased expression ofSGP-2 is associated with apoptosis. The exact function of the SGP-2 geneand its products is not fully defined. However, the results of our study showthat expression of the SGP-2 gene may provide an early indication ofpresence of apoptosis in rat incisor ameloblasts.

Keywords: apoptosis, sulphated glycoprotein-2 gene, in-situ hybridization,ameloblasts, odontogenesis, rat incisor, transitionalameloblasts, autoradiography

Correspondence: B.K. Joseph, Department of Dentistry, Divi- Apoptosis is a basic cellular phenomenon whereby cells

sion of Oral Biology and Pathology, The University of Queens- are deleted from living tissues in a physiologicalland, Brisbane, Queensland 4072, Australia. manner (Kerr et al. 1972). It occurs as programmed

313

314 B.K. Joseph et at.

death of cells during embryonic development of palate(Goldman et a/. 1983), normal tissue turnover, organinvolution and pathological atrophy of tissues (Buttyanet a/. 1988; Gob6 & Axelsen 1987; Wyllie 1992), hormone-induced atrophy (Tenniswood et at. 1992), normal (andneoplastic) cell turnover (Wyllie 1992) and occurs

naturally at the end of the life span of differentiatedcells (Benedetti et a/. 1988). Apoptosis is also triggeredby ionizing radiation (Savage et a/. 1985) and hyper-thermia (Harmon et a/. 1991), as well as followingdeprivation of growth factors (Duke & Cohen 1986).Morphologically the process is characterized by cellsundergoing both nuclear and cytoplasmic condensationresulting in the formation of apoptotic bodies which are

phagocytosed rapidly by neighbouring cells or bymacrophages (Kerr & Harmon 1991). At the molecularlevel, the key role in programmed cell death is played byendogenous endonucleases, which are directly or in-directly activated by a variety of stimuli and cut DNA intofragments of nucleosomal or oligonucleosomal size(Gerchenson & Rotello 1992). A distinctive feature ofapoptosis is that, notwithstanding the dramatic morpho-logical changes, the cells are removed without any

leakage of intracellular components and without theinduction of the inflammatory response and scar for-mation. The dead cells are recognized and ingestedwhile intact, protecting tissues from the potentiallyharmful consequences of exposure to the contents ofthe dying cell (Fesus 1993). Apoptosis differs funda-mentally from necrosis, the pathological form of celldeath, in which cells swell and lyse (Kerr & Harmon1991).

Transitional ameloblasts are the enamel forming cellsassociated with a brief reorganizational stage betweenenamel secretion and maturation (Salama et a/. 1991).During this stage, the ameloblasts undergo substantialstructural modification to accommodate a functionalshift from mainly secretion during early enamel forma-tion (Warshawsky 1985) to mainly ion transport (Bawden1989) and putative absorption (Smith 1979) during thematuration stage. Programmed cell death has beenreported in the transitional ameloblast during itsnormal developmental cycle (Smith & Warshawsky1977; Smith 1979; Warshawsky 1985).

Induction or inhibition of the apoptotic process isunder gene control in many instances (Owen 1992).One of the genes known to be associated with apoptosisin several tissues is sulphated glycoprotein-2 (SGP-2)(Buttyan et a/. 1989). To date, its expression has notbeen studied in odontogenesis (tooth formation) inwhich apoptosis is known to occur. SGP-2 is referredto under a variety of names: clusterin (Cheng et a/.

1988); testosterone repressed prostate message 2(TRPM-2) (Buttyan et a/. 1989); serum protein-40,40 (SP40,40) (Kirszbaum et at. 1989); complement lysis inhibi-tor (Jenne & Tschopp 1989); and apolipoprotein J (deSilva et a/. 1990). The protein, usually termed 'clusterin',has been used as a marker of apoptosis and is thoughtto be involved in the onset of apoptosis (Kyprianou et at.1990). SGP-2 expression is induced in a variety ofmodels of apoptosis: the prostate after castration(Montpetit et a/. 1986); the kidney after chronic ob-struction of the ureter (Buttyan et at. 1989) and theinterdigital regions of developing embryos (Buttyan eta/. 1989). However, SGP-2 has functions which are notassociated with apoptosis; it is a major protein which issecreted from normal Sertoli cells in the testis (Collard& Griswold 1987), and is known to be associated in manycases with inhibition or modulation of complementmediated cell lysis (Tenniswood et at. 1992). Recentstudies (Joseph et at. 1994) from our laboratory havemapped the distribution of Insulin-like growth factor-I(IGF-I) receptor in the odontogenic cells by immuno-histochemistry. The absence of staining over thetransitional ameloblasts may require the non-expres-sion of IGF-I which may be a prerequisite or even atrigger for apoptosis. Accordingly, in this study thepattern of expression of the SGP-2 gene was examinedduring different stages of odontogenesis in the ratincisor. The study was planned to give further insightinto the role of SGP-2 in apoptosis in the rat incisorameloblasts.

Materials and methods

Animals

Ten 4-week-old male Lewis rats were obtained from theCentral Animal Breeding House, University of Queens-land. Animals were caged in a room and were fed foodand water ad libitum. The guidelines for animal experi-mentation prescribed by the National Health andMedical Research Council of Australia were followedand the project had the approval of the InstitutionalEthics Committee.

Tissue preparation

Animals were anaesthetized by an intraperitoneal injec-tion of pentobarbitone and perfused intracardially withphosphate buffered saline (PBS, pH 7.4) until blanching,followed by Bouin's solution (0.9%(v/v) picric acid,9%(v/v) formaldehyde and 5%(v/v) acetic acid). Mand-

Sulphated glycoprotein-2 mRNA expression in ameloblasts 315

I*bial surfac

Figure 1. Diagramatic representation of the mandibular incisor showing different stages in odontogenesis. The boxed areasrepresent positive hybridization signals for SGP-2. D, Dentine; E, enamel; G, gingiva; MA, maturation ameloblasts; 0, odontoblasts;PA, pre-ameloblasts; PD, predentine; PO, preodontoblasts; SA, secretory ameloblasts; TA, transitional ameloblasts.

ibles were dissected and post-fixed in Bouin's solution.Following two changes of buffer, mandibles were de-calcified in neutral EDTA for 2 weeks. This method ofprocessing has been shown to preserve adequateamounts of mRNA within the cells (Walsh et a/. 1993).Tissues were then embedded in paraffin using standardhistological procedures.

Preparation of probes

RNA probes were prepared from a plasmid vector(pGEM vector, Promega) (Buttyan et a/. 1989) contain-

Figure 2. This haematoxylin and eosinstained section shows the topography ofthe principal cells studied in the dentalorgan. The ameloblasts (A) in the zoneof transition are illustrated here. Theameloblasts (A) are reduced to abouthalf their original length and apoptoticbodies (AP) (arrow heads) are foundwithin the cytoplasm of the ameloblasts.x160.

ing cDNA homologous to SGP-2 inserted between SP6and T7 RNA polymerase (Promega) promoter sites, andcut for sense and antisense.

In situ hybridization

Paraffin sections were cut at 4 tm thickness ontoVectabond coated (Vector, California, USA) anti-RNasetreated slides. Essentially, de-waxed sections wererehydrated, equilibrated in prehybridization buffer,then saturated with prehybridization buffer understerile plastic squares for 4 hours at 500C. Sections

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316 B.K. Joseph et al.

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Sulphated glycoprotein-2 mRNA expression in ameloblasts 317

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Figure 3. a, Bright field and b, dark field micrographs showing the post-secretory ameloblasts (A) treated for in situ hybridizationwith the SGP-2 antisense probe. Autoradiographic silver grains representing the signal for SGP-2 mRNA are densely accumulatedover the ameloblasts (A) and sparsely distributed over the odontoblasts (0) and pulpal cells (P). c, Bright field and d, dark fieldmicrographs of the rat incisor treated for in situ hybridization with the SGP-2 antisense probe. The maturation ameloblasts (A) areintensely labelled. In contrast, only background hybridization signals are identified from cells located in the papillary layer (PL). e,Bright field and f, dark field micrographs of the rat incisor treated for in situ hybridization with the control (sense SGP-2 probe).There was no positive hybridization signal over the different cell types. xlOO.

were pretreated with proteinase to better expose theRNA targets and then treated with hybridizationbuffer containing 35S-labelled SGP-2 riboprobes pre-pared from the plasmid vector. After hybridization,slides were washed to remove unhybridized probe,dehydrated in graded ethanol solutions and air-driedovernight.

Autoradiography

Slides were dipped in Kodak NTB-2 photographicemulsion melted at 420C and diluted 1: 1 with ultrapurewater. Exposure was in light-tight boxes at 40C forvarious periods of time for best labelling. Slides weredeveloped in Kodak D-19, fixed with Kodak RapidFixer and stained with haematoxylin and eosin forviewing and photography using light and dark fieldmicroscopy.

Results

Photomicrographs were taken from the boxed areas(Figure 1) to illustrate the location of SGP-2 mRNA inthe ontogenic sequence of ameloblasts during odonto-genesis. Our results show that differential accumulationof mRNAs coding for SGP-2 occurs during rat incisortooth development. In all animals, a similar pattern ofdifferential expression was seen in odontogenic celltypes at various identifiable stages of differentiation.Figure 2 shows a high magnification photomicrograph ofthe transitional ameloblasts in odontogenesis. Theameloblasts decrease in height, and the zone is char-acterized by the presence of large, round darkly stainedbodies in the proximal ends of the ameloblasts and inthe adjacent papillary cells. These cytologically identifi-able bodies are the apoptotic bodies. It is noteworthythat apoptotic bodies were most discernible in thistransitional zone.

318 B.K. Joseph et a/.

Expression of SGP-2 mRNA was usually localized inthe post-secretory transition and maturation amelo-blasts. Light and dark field photomicrographs demon-strate this location (Figure 3a-f). With the antisenseprobe, SGP-2 mRNA was detected after enamel secre-tion, in the transitional stage, where the hybridizationsignal was intense over the ameloblasts in contrast tothe odontoblasts and pulp, which did not demonstratespecific hybridization signals (Figure 3a and b). Therewas no specific hybridization signal over the amelo-blasts in the earlier phase of odontogenesis. With theonset of enamel maturation, positive hybridizationsignal was visible over the early and mid maturationameloblasts (Figure 3c and d). Consistently, neither theodontoblasts nor the pulp demonstrated hybridizationsignals. Sections prepared for sense and antisensehybridization techniques had equivalent background,but only sections prepared for antisense hybridizationshowed definitive labelling over ameloblasts. Nospecific labelling was observed after hybridization ofconsecutive sections with the sense probes used as acontrol for SGP-2 (Figure 3e and f).

Discussion

In the present study, we have examined serial sectionsfrom the developmental stages of forming rat incisorusing high-resolution in situ hybridization. This tech-nique permits the examination, on a cell-cell basis, ofthe transcription of specific genes. Utilizing a radio-labelled riboprobe specific to the rat SGP-2 mRNA, wewere able to localize SGP-2 transcripts in the post-secretory transition and maturation ameloblasts. Thisis in agreement with the known preferential expressionof SGP-2 in tissues undergoing apoptosis (Buttyan et a/.1989).The first report of cell death in the ameloblasts came

from quantitative analysis of cell turnover in the ratincisor enamel organ, which suggested that about one-half of the total cell loss occurs within the transitionalstage (Smith & Warshawsky 1977). The lightly stained,large, diversely shaped bodies seen by light microscopyin the transitional ameloblasts are thought to be relatedto the massive cell death reported to occur during thisstage (Salama et a/. 1991). This occurs chiefly in theregion of post-secretory transition and occupies aperiod of approximately 19 hours in the 30-day life-cycle of the ameloblast population (Smith &Warshawsky 1977). Apoptotic ameloblasts can be recog-nized readily at the light microscopic level as pyknoticnuclear fragments within and between adjacent amelo-blasts or in the stratum intermedium (Smith 1979). The

fine structural features of transitional ameloblasts havebeen described in rat molars (Reith 1970) and in ratincisors (Kallenbach 1974; Smid et a/. 1992). These cellswere characterized by the presence of abundant phago-somes as well as diverse structures labelled as lyso-somes on a morphological basis. Previous histo-chemical and cytochemical studies have demonstratedthe activities of lysosomal enzymes such as dipeptidylpeptidase 11 (Smid et a/. 1992), trimetaphosphate andcytidine-5'-monophosphate (Salama et a/. 1991) in thiszone. Apoptotic changes have been reported in thedental papilla of rat incisor after the administration ofactinomycin D (Moule et a/. 1993). The cellular changesfound in the dental papilla were dominated by apoptosisand heterophagy. These studies establish a definite roleof apoptosis in normal tooth development and in oraldisease processes.

Apoptosis occurs in rapidly proliferating populationssuch as the intestinal crypt epithelium (Potten 1992) anddifferentiating spermatogonia (Alan et a/. 1987). How-ever, ameloblasts are post-mitotic during their dif-ferentiation. There is evidence that the homeostaticregulation of normal tissue mass is affected by thecyclic production of growth factors, which induce mito-sis and apoptosis. Moreover, some growth factors andhormones have been shown to induce or suppressapoptosis, as well as to stimulate mitosis (Williams eta/. 1990). Recent immunohistochemical studies (Josephet a/. 1994) have demonstrated the presence of insulin-like growth factor-I receptor in the ameloblast layer atdifferent stages of its development with non-expressionof the receptor in the transitional stage. This correlatedwith our previous findings that growth hormone receptorand insulin-like growth factor-I expression was absent inthe transitional zone (Joseph et a/. 1993). Perhaps non-expression of the receptor is a prerequisite or even atrigger for the onset of apoposis.

Positive hybridization signals for SGP-2 over themature ameloblasts occur at a time when apoptosishas been found morphologically in the ameloblastpopulation. Influx of calcium, which is known to occurin maturation ameloblasts (Borke et a/. 1993), is alsoconsistent with the appearance of apoptosis: anincrease in intracellular calcium and activation ofcalcium-dependent endonuclease, which cleaveschromatin at internucleosomal sites, are features ofapoptosis (Wyllie et a/. 1984). While the function ofSGP-2 is not fully understood, its presence in a varietyof tissues during apoptosis indicates a role for the geneand its protein in this basic process. The study of otherapoptotic genes is promising and further work is neededto determine the role and interaction of these genes and

Sulphated glycoprotein-2 mRNA expression in ameloblasts 319

their association with 'survival' genes in odontogenesis.Our results suggest that SGP-2 expression of the ame-loblasts in the continuously erupting rat incisor may berelated to apoptosis, and that rat incisor tooth develop-ment may consistute a suitable biological systemto investigate expression of the genes coding for apo-ptosis.

Acknowledgements

This work was done in the Pathology Department,University of Queensland Medical School, and wassupported by The National Health and MedicalResearch Council of Australia and the AustralianDental Research Fund, Inc.

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