Journal of Histochemistry and Cytochemistry, Vol. 49, 285-292, March 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Immunochemical Characterization of a Chicken Egg Yolk Antibody to Secretory Forms of Rat Incisor Amelogenin

Giovanna Orsinia, Patrice Lavoiea, Charles E. Smithb, and Antonio Nancia
a Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry, Université de Montréal, Montréal, Québec, Canada
b Division of Oral Biology, Faculty of Dentistry, McGill University, Montréal, Québec, Canada

Correspondence to: Antonio Nanci, Université de Montréal, Faculty of Dentistry/Stomatology, PO Box 6128, Station Centre-Ville, Montréal, QC, Canada H3C 3J7. E-mail: antonio.nanci@umontreal.ca


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino–enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.

(J Histochem Cytochem 49:285–292, 2001)

Key Words: polyclonal antibody, amelogenin, secretory forms, immunoblotting, immunocytochemistry


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

During enamel formation, ameloblasts produce a number of matrix proteins that are believed to promote and regulate mineral ion deposition into unique and extremely long apatite crystals. Amelogenins represent the major secretory product of these epithelium-derived cells (reviewed in Fincham et al. 1999 ; Nanci and Smith 2000 ). It is generally accepted that all mammalian ameloblasts produce several amelogenin variants from a single gene (reviewed in Simmer and Snead 1995 ). Most of these amelogenin isoforms result from differential splicing of the mRNA (Lau et al. 1992 ). Molecular, metabolic, and mass analyses have helped to clarify possible derivative relationships between newly secreted, intact amelogenins and their degradation products (Smith and Nanci 1996 ; Fincham et al. 1999 ; Chen et al. 2000 ). However, there is still uncertainty about identifying these components in gels or immunocytochemical preparations. Despite the widespread availability of several polyclonal, monoclonal, and anti-peptide antibodies, none of these can selectively reveal the intact versions of nascent amelogenin.Over the past few years, we have successfully used the chicken egg yolk system (Gassmann et al. 1990 ; Losch et al. 1986 ) to produce polyclonal antibodies to enamel proteins and other calcified tissue matrix proteins (Chen et al. 1995 ; Nanci et al. 1996 ). One advantage of this system is that chickens can yield high-titer antibodies against conserved mammalian antigens (Gassmann et al. 1990 ).

The aim of this study was to determine whether this system could produce an antibody selective for secretory forms of amelogenin. Although such an antibody is beneficial for biochemical characterizations, it would be particularly useful for immunocytochemical mapping of the temporospatial distribution of the protein. This information is essential to understanding of its function and its implication in pathological alterations.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

All animal handling and experimental procedures were approved by the Comité de Déontologie de l'expérimentation sur les Animaux of the Université de Montréal.

Preparation of Chicken Egg Yolk Polyclonal Antibody
A mouse recombinant amelogenin [M179, lacking the N-terminal methionine and the Ser16-phosphate group found on the main native mouse amelogenin M180 isoform (Simmer et al. 1994 )] was purified, resuspended in 10 mM PBS, and emulsified in Quil A saponin adjuvant. A polyclonal antibody was raised in chickens and purified from egg yolks using the procedure of Gassmann et al. 1990 . Briefly, 50 µg of the purified protein was injected into the pectoral muscle of egg-laying hens, followed by a second injection of the same amount 15 days later. Eggs were collected before injections (preimmune controls) and for 30 days after the first injection of antigen. This antibody is referred to as M179y.

Sample Preparation for Immunoblotting
Male Wistar rats weighing 100–150 g (Charles Rivers Canada; St-Constant, QC, Canada) were anesthetized with Metofane (methoxyfluorane; Janssen Pharmaceutica, North York, ON, Canada) and decapitated. The hemimandibles were dissected out and the enamel organ was partially exposed by cracking off some of the covering alveolar bone. They were immediately plunged into liquid nitrogen and maintained in it for a minimum of 5 hr before freeze-drying for at least 48 hr at -80C on a 12-liter cascade lyophilizer system (Labconco; Kansas City, MO). The enamel organ, with adhering labial connective tissue and enamel, was then transected on each incisor into a series of five sequential strips relative to the secretory (S) and maturation (M) stages of amelogenesis, using a molar reference line (Smith and Nanci 1989 ). The two secretory stage strips were 2.5 mm long and the three maturation stage strips were about 2 mm long. Each strip was placed in a separate sterile 1.5-ml screw-top microfuge vial and proteins were extracted directly into 100 µl of a sample preparation buffer containing 62.5 mM Tris (pH 6.8), 2% SDS, 15% glycerol, 5% {beta}-mercaptoethanol, and 0.005% bromophenol blue. The vials were immersed in a boiling water bath for 5 min, cooled, and stored at 4C.

Immunoblotting
Twenty µl of extraction fluid from each vial was applied to separate lanes of standard format (16 cm x 14 cm x 1 mm) 12% polyacrylamide slab gels. Broad-range molecular weight marker proteins (Bio-Rad; Mississauga, ON, Canada) were also loaded in one lane of each gel. Proteins were separated by electrophoresis at 20 mA per gel constant current using a discontinuous buffer system (Laemmli 1970 ). They were then electrotransferred from the gels onto 0.45-µm pore size nitrocellulose membrane and probed with M179y, followed by alkaline phosphatase-labeled anti-chicken IgG antibody (Cappel Research Products; Scarborough, ON, Canada), as described previously (Chen et al. 1995 ).

Tissue Processing for Immunohistochemistry
Male Wistar rats weighing 100 ± 10 g (Charles Rivers Canada) were anesthetized with chloral hydrate (0.4 mg/g bw) and sacrificed by intravascular perfusion with a fixative solution consisting of 1% glutaraldehyde in 0.1 M sodium phosphate (PB), pH 7.2. The hemimandibles were removed and immersed in the fixative overnight at 4C. They were then washed in 0.1 M PB, pH 7.2, and decalcified for 21 days in 4.13% EDTA at 4C (Warshawsky and Moore 1967 ). Segments of incisors from the secretory and early to midmaturation stages were prepared using a molar reference line (Smith and Nanci 1989 ). They were then dehydrated in graded alcohols and processed for embedding in LR White resin (London Resin; Berkshire, UK). Thin sections were cut with a diamond knife, mounted on Formvar–carbon-coated nickel grids, and processed for postembedding protein A–gold immunocytochemistry (reviewed in Bendayan 1995 ).

Immunocytochemistry
Sections were floated for 15 min on a drop of 0.01 M PBS containing 1% ovalbumin (Oval; Sigma Chemical, St Louis, MO). They were transferred for 3 hr onto a drop of M179y diluted 1:100, washed with PBS, refloated on PBS–Oval, and then incubated for 1 hr with a rabbit anti-chicken IgG antibody (diluted 1:2000) (Cappel Research Products). Finally, they were washed again with PBS, refloated on PBS–Oval, and incubated with protein A–gold complex for 30 min. After immunolabeling, the grids were extensively rinsed with PBS, followed by distilled water. Controls consisted of incubations with preimmune antibody followed by the secondary antibody and protein A–gold, secondary antibody and protein A–gold, or protein A–gold alone. All incubations were carried out at room temperature. Grids were stained with 4% aqueous uranyl acetate and lead citrate for examination in a JEOL JEM-1200EX-II transmission electron microscope operated at 60 kV.

Quantitative Analysis of Immunocytochemical Labeling
Sections from secretory, early, and mid-maturation stage of amelogenesis (see Warshawsky and Smith 1974 ) from a minimum of two rats were examined. For each stage, the enamel layer was partitioned into three regions. Region 1 was a randomly selected area close to the apical surface of ameloblasts (n = 478); Region 2 was situated near the middle of the enamel layer (n = 396); Region 3 was at the dentino–enamel junction (n = 528). For examination of labeling density, the numbers of gold particles were counted in all three regions within a window precalibrated to 150.4 µm2. ANOVA and post-hoc comparisons of means, including the Tukey HSD test for unequal n, were performed with {alpha} = 0.05, using version 5.5A of Statistica for Windows (Statsoft; Tulsa, OK). Power tests of differences between means were done using version 1.01 I of GraphPad StatMate (GraphPad Software; San Diego, CA). The lowest sampling number overall (n = 66) was in the middle region of the enamel layer in early maturation (see Fig 8, EMAT, Mdl).



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Figure 1. Immunoblot of whole enamel organ cells and enamel extracts probed with chicken egg yolk M179y antibody. Lanes S1–M3 represent contiguous strips dissected from the same incisor. Standard broad-range molecular weight marker proteins stained with Ponceau S are shown at left. Cell extracts show only one major immunoreactive band, whereas five bands are revealed in the enamel samples. The most intensely stained band is at 27 kD and corresponds to the major amelogenin secreted by rat incisor ameloblasts. S, secretory stage; M, maturation stage.



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Figure 2. Immunocytochemical preparation illustrating the labeling detected over the Golgi apparatus of a secretory stage ameloblast with the M179y antibody. The absence of gold particles over mitochondria (m) and the nucleus (N) indicates a very low level of background labeling. rER, rough endoplasmic reticulum.



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Figure 3. Cross-cut view of the distal portion of a Tomes' process (Tomes) and its associated forming enamel rod. Secretory granules (sg) in the process are labeled by the M179y antibody. Both the rod and the surrounding inter-rod enamel show intense reactivity. Note the paucity of gold particles near the cell surface in the area (arrows) corresponding to the rod growth site (RGS).



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Figure 4. Immunocytochemical preparation illustrating the increasing gradient of labeling over a forming inter-rod enamel prong. The area (arrows) at the extremity of the prong at which enamel crystallites actively elongate, the inter-rod growth site (IRGS), shows substantially fewer gold particles. dpTP, distal portion of Tomes' process; ppTP, proximal portion of Tomes' process.



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Figure 5. Micrographs from early maturation stage specimens labeled with M179y antibody. Ameloblasts at this stage still exhibit immunoreactivity over the Golgi apparatus (Golgi) and secretory granules (sg). A significant amount of labeling for secretory forms of amelogenin is still observed over enamel. ly, lysosome-like element.



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Figure 6. Preparations from the secretory (A) and mid-maturation (B) stage comparing the labeling obtained with M179y antibody over enamel in the region near the dentino–enamel junction (arrows). During the secretory stage, there is a concentration of gold particles over this region. By mid-maturation, the labeling is significantly reduced and the accumulation of particles at this site is no longer readily apparent visually but can still be detected by quantitative analyses (see Fig 7). There are almost no gold particles over dentin, confirming the specificity of the antibody.



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Figure 7. Box plots of mean density of immunolabeling (± SD) by stage (top) and by sampling location within the enamel layer (bottom). There is a significant decrease in labeling density for secretory forms of amelogenins as the developing enamel ages (matures) (p<0.0000 for SEC to MMAT). Regional analyses indicate that secretory forms are in higher concentration in the surface portion of the enamel layer than in deeper areas of the enamel (p<0.0000 for Near AM to Middle). They also appear to slightly accumulate near the dentino–enamel junction (DEJ) (the difference is not statistically significant in these data compared to Middle). AM, ameloblasts; SEC, secretory stage; EMAT, early maturation stage; MMAT, mid-maturation stage.



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Figure 8. Composite box plots of mean density of immunolabeling (± SD) for all stages and the three regional locations across the thickness of the enamel layer. All stages show the same basic pattern in distribution of labeling, with counts being highest over the surface portion of the enamel layer. Both secretory stage enamel (SEC) and mid-maturation stage enamel (MMAT) show a trend for higher counts over the portion of the enamel layer near the dentino–enamel junction (DEJ) than in the middle (Mdl) of the enamel layer (only the difference between Mdl and DEJ is significant at p<0.0000 in SEC). AM, ameloblasts; EMAT, early maturation stage.


  Results
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Immunoblotting
M179y revealed a band near 27 kD in the S1 enamel organ cell extracts and an additional weakly stained band near 29 kD in the M1 sample (Fig 1). No immunoreactive proteins were discernible in the M2 and M3 cell extracts. Enamel extracts, on the other hand, showed three bands at 23, 27, and 29 kD from S1 to M1 (Fig 1). Additional faint bands were present near 30 kD in S2 and M1 and near 31 kD only in S2 samples.

Immunolabeling
Many gold particles were observed over the saccules of the Golgi apparatus (Fig 2) and over secretory granules in Tomes' processes (Fig 3) of secretory stage ameloblasts. Enamel was intensely immunoreactive except at rod (Fig 3) and inter-rod (Fig 4) enamel growth sites, at which few particles were present. Early to mid-maturation stage ameloblasts still showed immunoreactivity over the Golgi apparatus and occasional secretory granules were found in these cells (Fig 5). The overall density of labeling over enamel gradually decreased towards late maturation. However, the general distribution was similar throughout (compare Fig 6A and Fig 6B). In the regions sampled, no immunoreactivity was seen in other cells of the enamel organ or in odontoblasts.

Statistical analyses of the three stages of amelogenesis confirmed that there was a general decline in the density of M179y labeling from secretory to early and mid-maturation stages (Fig 7, SEC to EMAT, p< 0.0000; EMAT to MMAT, p<0.0152). When the three regions in which the enamel layer was partitioned were considered, all stages showed a higher density of labeling over the surface portion of the enamel layer (Region 1) than over the middle part of the enamel layer (Region 2) (Fig 7, Near AM to Middle, p<0.0000; Fig 8, AM to Mdl for all stages, p<0.0000). An increase in the density of labeling near the DEJ (Region 3) was detected in secretory and mid-maturation stage samples (Fig 8; SEC, Mdl to DEJ, p<0.0000; MMAT, Mdl to DEJ is not significant). Early maturation stage samples, in contrast, showed fairly uniform density of labeling for most of the thickness of the enamel layer except near the surface, where the density of labeling was higher (Fig 8; Mmat, AM to Mdl or DEJ, p<0.0000).

In all cases, control incubations resulted in a major reduction of the labeling and in the presence of few randomly distributed gold particles throughout the tissue sections.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Using the chicken egg yolk system (Losch et al. 1986 ; Gassmann et al. 1990 ; Schmidt et al. 1993 ), we have produced a polyclonal antibody that reveals an intensely reactive protein band at 27 kD and a very faint one near 29 kD in rat incisor enamel organ extracts, and five bands (23, 27, 29, 30, and 31 kD) in enamel extracts. Immunocytochemistry showed reactivity only in the protein synthetic organelles of ameloblasts and enamel matrix, demonstrating that the epitopes recognized by the M179y antibody are present only on secretory products produced by these cells. The proteins revealed by immunoblots correspond very closely to the secretory products identified previously by direct metabolic radiolabeling in the rat (Smith and Nanci 1996 ). In addition, their relative staining intensities by immunoblotting are remarkably similar to the signals obtained on fluorographs at 1 hr after injection of [35S]-methionine (Smith and Nanci 1996 ). The predominance of the 27-kD band in cell extracts is consistent with this being the major secretory form of amelogenin in rat incisors (see Smith and Nanci 1996 ; Chen et al. 2000 ). The other secretory forms are most probably present in too small quantities in cell extracts to be resolved by the alkaline phosphatase blotting method we have used. Indeed, even with high-energy [35S]-methionine, radiolabeling is mostly associated with the 27-kD protein. This interpretation is further supported by the appearance of a very faint band near 29 kD in the M1 cell extract only at the time when it stains most intensely in enamel samples. The absence of staining of fragments derived from postsecretory degradation further suggests that the antibody is directed against intact forms or forms that have undergone little C-terminal processing.

One past interpretation given to autoradiographic findings of a sharp increase of labeling over areas of enamel that initially show almost no radioactivity is that protein fragments cleaved from parent amelogenins diffuse into deeper regions of the enamel layer (discussed in Smith et al. 1989 ). This view predicts that amelogenins in deeper (older) regions of enamel essentially consist of fragments that are becoming smaller over time. The presence of substantial amounts of M179y immunoreactivity throughout the entire thickness of the enamel layer suggests that this is not necessarily the case and that intact and/or relatively intact amelogenins are found even in the oldest enamel, at least in the case of rat incisors. In secretory stage enamel, about 53% of sampled gold particles were found over the surface portion of the enamel layer, and 47% were accounted for by counts over regions near the middle of the layer and near the dentino–enamel junction. The latter percentage is surprisingly close to the estimated 40% of total counts in radiolabeling studies originally released as secretory forms by ameloblasts and that appear to move deeper into the enamel layer (Smith et al. 1989 ). Hence, rather than protein fragments "moving in the wrong direction," this study suggests that intact amelogenins move continuously toward the DEJ.

It has long been assumed that amelogenins are uniformly distributed throughout the enamel layer, a belief consistent with the notion that enamel proteins are arranged in a thixotropic gel that allows free mixing of all components (Eastoe 1979 ). Immunocytochemical results with the present anti-amelogenin antibody do not support this assumption, and suggest that the distribution of amelogenins is more complex than was expected. Two patterns of immunolabeling were observed over enamel. First, as has been shown previously with other anti-amelogenin antibodies (Nanci et al. 1996 , Nanci et al. 1998 ), there is a paucity of gold particles both at rod and inter-rod growth sites at which enamel crystals actively elongate. Second, there is a difference in density of labeling across the enamel layer among the three representative regions of the enamel layer sampled, which is most dramatic during the secretory stage (Fig 8, SEC, p<0.0000 among all locations analyzed). Although such a difference has not been detected in pulse-label autoradiographic studies (see Nanci et al. 1989 ; Smith et al. 1989 ; Smith and Nanci 1996 ), it could be inferred from the reported randomization behavior of the main wave of radiolabeled enamel proteins that accumulation over time could potentially lead to its establishment. Indeed, judging from the approximate thickness (40–60 µm) of the secretory stage enamel layer sampled, the immunolabeling gradient observed could have taken up to 4.5 days to establish itself (in the rat incisor it takes 7.5 days to form the full 100-µm enamel thickness) (Smith and Nanci 1996 ).

The persistence of secretory forms of amelogenin throughout the enamel layer has some important functional implications. Because amelogenin fragments adsorb less efficiently to enamel crystals (Ryu et al. 1998 ), one would expect that the presence of relatively large amounts of apparently intact amelogenin in the deeper regions of forming enamel might have an impact on the rate at which the crystals will grow. This may not be the case in certain species in which there is no well-defined temporal segregation of formative and degradative events. In the pig, for example, biochemical analyses have shown that forming enamel near the dentino-enamel junction comprises mostly fragments (Bartlett and Simmer 1999 ).

In conclusion, we have prepared a chicken egg yolk antibody that appears to recognize only secretory forms of amelogenins. This antibody has revealed a more complex distribution of parent amelogenins than had been previously suspected and represents a potentially useful tool for studying their functional relationship with enamel crystals.


  Acknowledgments

Supported by a grant from the Canadian Institute of Health Research (CIHR).

We thank Dr J.P. Simmer (University of Texas at San Antonio) for providing the recombinant mouse M179 amelogenin used for injection into chickens, Mireille Fyfe and Line Lespérance for their technical help with production of the antibody, and Sylvia Zalzal for advice on immunolabeling.

Received for publication September 14, 2000; accepted October 5, 2000.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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