Regional changes in nitrogen and nucleic acid levels in bovine incisors during development

Archs oral Biol. Vol.12, pp.683493,

1967. Pergamon Press Ltd. Printed in Gt. Britain.

REGIONAL CHANGES IN NITROGEN AND NUCLEIC ACID LEVELS IN BOVINE INCISORS DURING DEVELOPMENT S. SAKAMOTO, S. SASAKIand S. ARAYA

Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Summary-The dry weight of calcifiedmaterial and the quantities of nitrogen and of nucleic acids in bovine incisor tooth germs were determined at various stages of development to provide a basis for quantitative analysis of the growth pattern of the tooth germ. The wet weightof the fresh tooth-germ was used to express the degree of development. In the enamel organ, the peak of the ratio of ribonucleicacid to deoxyribonucleic acid, which is generally considered to indicate protein biosynthesis,was observed for germs of approximately 0.5 g, when the calcified tip appeared. Values of this ratio for the inner layer of the enamel organ revealed a more rapid decrease than those for the outer layer, suggesting that the former may contribute to the enamel matrix formation more than the latter. These marked changes in the content of nucleic acids quantitatively support the histochemical and electronmicroscopical findings of others. INTRODUCTION

MANY morphological investigations of tooth development have been reported. Few, however, have utilized quantitative analytical methods. Remarkable progress in the field of tooth development has resulted from application of the electron microscope, which has revealed dynamic changes in the ultrastructure of the tissues during development. However, these morphological investigations require support from quantitative analysis for the interpretation of the relationship between function and structure. DEAKINS(1942) reported changes in the ash, water and organic content of pig enamel during calcification. SASAKI(1959) studied the respiration of the dog tooth germ at different stages of development and reported a positive correlation between respiration and degree of calcification. ARAYAand SASAKI(1963) showed that the enamel organ has a higher glycolytic activity than the dental papilla in bovine tooth germs. The present study was undertaken to obtain quantitative chemical data relating to the mechanism of tooth development. As an approach to the problem, the dry weight of the calcified material, and the quantities of nitrogen and of nucleic acids were determined using bovine tooth germs at various stages of development. Ribonucleic acid (RNA) is involved in protein biosynthesis ; therefore the ratio of RNA to deoxyribonucleic acid (DNA), which represents the RNA content per cell, is generally considered to be a suitable index for expressing the rate of protein biosynthesis in a tissue. In the present study, this ratio was determined primarily in order to follow changes in the activity of enamel matrix formation at various developmental stages. 683 A

684

S. SAKAMOTO, S. SASAKIAND S. ARAYA MATERIALS

AND

METHODS

Dissection of the tooth germ Bovine mandibles obtained fresh from the slaughterhouse were kept cold in vacuum jars. The germs of the lower third permanent incisors were extracted by breaking the alveolar bone, care being taken to avoid injury to the dental sac. Sixtyseven tooth germs were collected, forty-six of which were used to determine nitrogen and nucleic acid content, twenty-one were used to calculate calcification coefficient. Each tooth germ was weighed rapidly on an analytical balance and transferred to cold saline solution. The tooth germ was separated with tweezers into the following four parts : (i) the outer layer of the enamel organ consisting of the dental sac and the greater part of the stellate reticulum; (ii) the inner layer of the enamel organ consisting of the remainder of the stellate reticulum, the stratum intermedium, and ameloblasts; (iii) the calcified portion which was separated from (ii) by sharp dissection and (iv) the dental papilla which histologically contains the odontoblasts. Part (i) was obtained by taking off the dental sac from the germ leaving a portion of the inner layer of the enamel organ, subsequently separated as (ii), behind on the surface of the calcified portion. This separation seems to depend on the existence of the zone of stellate reticulum, in which intercellular connection is very loose. However, a constant proportion of the stellate reticulum could not be expected to reside in both layers at various stages of development. Each of these parts except the calcified one, after being kept in cold saline solution, was individually blotted, weighed on a micro-balance, and transferred to a glass homogenizer which contained 15 ml of icecold ethanol. Aliquots of the homogenate were used for determination of nucleic acids and nitrogen. Determination of nucleic acids The extraction and determination of tissue RNA and DNA were undertaken chiefly according to the method of KATSURA (1960), a modification of the original OGUR and ROSEN (1950) extraction, and the CERIOTTI(1952, 1955) micro-determination. These methods were found to be suitable for the analysis of nucleic acids in oral tissues. Each 2 ml of homogenate, after being kept at 4°C for 30 mm, was centrifuged in the cold. The precipitate was washed with 2 ml of cold 70 % ethanol containing O-1% perchloric acid (PCA) and defatted with 2 ml of ether-ethanol (1:3 v/v) at room temperature for 30 min. After drying, the defatted residue was washed again with 2 ml of cold 2% PCA, centrifuged in the cold as quickly as possible, and the supernatant discarded. The residue was then extracted with 0.5 ml of 10% PCA at 70°C for 20 min, centrifuged, the supernatant fluid saved, and the residue extracted again with 1 ml of 5 % PCA at 70°C for 20 min. The combined solutions were diluted to provide optimal concentrations for the determination. DNA was determined by colour reaction with indole and RNA by colour reaction with orcinol. The optical densities being read at 490 and 670 rnp, respectively, in a Beckman’s spectrophotometer.

REGIONAL

CHANGES

IN NITRGGEN

AND

NUCLEIC

ACID

LEVELS

685

Determination of nitrogen The nitrogen contents of the tissue parts were determined by the micro-Kjeldahl Parnas method as modified by HILLER, PLAZIN and VAN SLYKE (1948). Determination of the degree of tooth development It is important to choose a suitable index to express the degree of tooth development. In order to express quantitatively the degree of calcification, SASAKI (1959) used a “calcification coefficient”, which is the ratio of the dry weight of the calcified portions to the dry weight of the whole tooth germs, including calcified portions. This value is zero until calcification begins, when it rises and approaches 1.0 in the later stages. Since a linear correlation was observed between the fresh weight of the tooth germ and the calcification coefficient, in the present study the fresh weight of the tooth germs was used as an index of development. RESULTS

Correlation between the fresh weight of the tooth germs and “calciJcation coefficient” A linear correlation was observed between the two factors throughout the period from 0.5 g to 2.5 g of the total fresh weight (Fig. 1) with the equation of y= 0*41x-O-14 (v= calcification coefficient, x= fresh weight of tooth germ). The correlation coefficient was +0*91. ko-

0

a

I

I

I

I

I

0.5

IO

I5

2.0

2.5

3.0

Fresh weight of tooth germ,

g

FIG. 1. Correlation between fresh weight of the tooth germ and the “calcification coefficient” for 21 specimens. Correlation coefficient was + 0.91, calculated by product moment formula. Equation of regression line was y= 0.41x- 0.14where, y = calcification coefficient, x = fresh weight of the tooth germ, calculated by the method of least squares.

Correlation between the fresh weight of the tooth germ and the dry weight of the calcified portion The dry weight of the calcified portion increases along a sigmoid curve as is usual with growth curves (Fig. 2). Calcification begins when the fresh weight is approximately 0.4 g. So far as the shape is concerned, the crown is half-formed when the tooth germ weighs 1.0 g and the whole crown weight does not quite attain 2-O g.

686

S. SAKAMOTO,S. SASAKI AND S. ARAYA

Fresh

weiqht

of

tooth

germ,

g

FIG. 2. Correlation between the fresh weight of the tooth germ and the dry weight of the calcified portion for sixty-seven specimens. Arrows indicate the morphological changes of the calcified portion.

Changes in total nitrogen content of the enamel organ and dental papilla

In Fig. 3 the nitrogen content is plotted against the total wet-weight of tissue, both cellular and intercellular. The total weight of nitrogen in the enamel organ (part i plus ii) increased, reaching a peak value of approximately 7 mg when the total fresh 10 -

0

,

0

I

1

#

1

1

1

05

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lb5

20

2-5

3.0

Fresh

weight

of

tooth

germ,

g

FIG. 3. Changes in total nitrogen content of enamel organ (part i plus ii) and dental papilla. Twenty-four specimens. 0, enamel organ; 0, dental papilla; 0, unseparated whole germ.

lW3IONAL CHANGFS IN NITROGEN AND NUCLEIC ACID LEVELS

687

weight was l-7 g and decreased rather rapidly thereafter. The decrease can be ascribed to degeneration of the enamel organ after crown formation. On the other hand, the total nitrogen content of the dental papilla increased more gradually than that of the enamel organ and continued to rise slowly throughout the period under investigation. Changes in total DNA and RNA of the tooth germ

Total DNA content of the tooth germ was determined in order to have an insight into the changes in numbers of cells. It was observed that the value had a tendency to decrease gradually beyond the stage when the tooth germ weighed approximately 1.7 g, as shown in Fig. 4. This decrease can be ascribed to degeneration of the enamel . .

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Fresh weight of tooth germ,

c

30 g

FIG. 4. Change in total DNA content of the tooth germ. Twenty-five specimens. Each point represents an average of four determinations with an error range of f 6.8 per cent.

organ. More rapid increase and decrease was observed in the total RNA content, the peak value corresponding tooth germ weight of approximately 1.3 g (Fig. 5). The ratio of total DNA in the enamel organ (part i plus ii) to that in the dental papilla (part iv) showed a pattern of gradual fall between 1.95 and 0.61 with the mean value of 1.38 f 0.36, and the corresponding ratio for RNA fell rather rapidly between 1.95 and 050 with the mean value of 1.23 f 0.41. The change of the RNA/DNA

ratio of the enamel organ

The above observation led us to analyse the changing pattern of RNA content per cell in the enamel organ by calculating the RNA/DNA ratio. As shown in Fig. 6,

S. SAKAMOTO,

688

S. SASAKI

.

.

.

S. ARAVA

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AND

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20

25

30

Fresh

weight

of tooth

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FIG. 5. Change in total RNA content of the tooth germ. The specimens were the same as in Fig. 4. Each point represents an average of four determinations with an error range of * 5.3 per cent.

.

. ’

.

l*

0

05

IO

Fresh

weight

I.5

2.0

of toothgerm,

3.0

25

g

FIG. 6. Change in the RNA/DNA ratio of the enamei organ (part i plus ii). Each point represents a ratio calculated with the values of enamel organ used in Figs. 4 and 5. 0, unseparated whole germ.

the values for this ratio fell more rapidly than those described above. The peak value seemed to be located when the tooth germ weight reaches approximately 0.5 g; at this stage enamel matrix formation is considered to be most active and some part of the matrix begins to be calcified.

REGlONAL

CHANGJS IN NITROGEN

Comparison of the RNA/DNA organ

AND

NUCLEIC

ACID

689

LEVELS

ratios for the inner and outer layers of the enamel

As described in “Materials and Methods”, the enamel organ was separated into two layers and the changes in their RNA/ DNA ratios were compared (Table 1 and TABLE 1. COMPARISONOFTHE CHANGESINTHE RNA/DNA FOR BOTH LAYERSOFTHEENAMEL~RGAN~

Outer layer of enamel organ

Inner layerof

enamel organ Mean RNA/DNA

SD.

Mean

1.93 f 0.73

Correlation coefficient7 Equation:

RATIOS

SD.

2.22 +I 0.52

- 0.70

- 0.63

y = - 044x + 3.05

y = - 0.23x + 2.75

*Number of specimens: twenty-one. tcalculated

by product moment formula.

:Calculated by method of least squares, x: fresh weight of tooth germ. SD., Standard deviation.

y :

the RNA/DNA

ratio,

Figs. 7a and 7b). On the assumption made for convenience of calculation, that these values decreased proportionately as tooth weight increased, the equations of regression line for RNA/DNA ratios for both layers were determined in an attempt to 4

.

:

i

i lx

I

0

.

I

1

05

I

I

IO

15

I 20

I

I

25

1 30

05

Fresh weight of tooth germ,

FIG. 7. Comparison

IO

15

20

25

g

of the RNA/DNA ratios for the inner layer (Fig. 7a) and the outer layer (Fig. 7b). Each point represents a ratio calculated with the values used in Figs. 4, 5 and 6. Correlation coefficients and equations of regression line were listed in the Table.

30

690

s.

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s.

%SAKl

AND

S. hAYA

clarify the difference between them. The ratios for the inner layer revealed a significantly more rapid decrease (y= -0*44x+ 3.05) than those for the outer layer (y= - 0-23x + 2.75). The change in the RNA/DNA

ratio of the dental papilla

The ratio for the dental papilla appeared to rise gradually through all the stages investigated in the present study with the equation of y= 0.06x + 2.30 where, y= the RNA/DNA ratio, x= the fresh weight of tooth germ. DISCUSSION In order to have tooth germs of suitable size for biochemical determinations in sufficient numbers, bovine tooth germs were used. Considerable variability of observations was therefore expected. In order to lessen the expected deviation as much as possible, only mandibular third permanent incisor germs were used. In preliminary experiments, the tissue contents of the enamel organ layers were examined under a phase-contrast microscope and in histological specimens produced by standard methods. The separated layers contained various histological elements and were mixtures of the components at different developmental stages. Therefore, the results obtained in this study represent mean values for integrated quantities. It is pointed out that the cattle teeth used in this study are quite different in their mode of development from rodent incisors, which have been widely used in morphological investigations, and where the entire sequence of tooth development from the earliest beginnings to maturation can be studied in the same tooth. It proved very practical to use the fresh weight of tooth germ as an index to express the degree of tooth development. Although differing from SASAKI’S calcification coefficient, the fresh weight index was shown to be proportional to it (Fig. 1). The sigmoid curve obtained in the present study is similar to those obtained by KUIDA, NAKANISHIand ARAYA(1960). The curve remained sigmoid because the root continued its development within the period covered by the present study. Similar changes were recognized in the total nitrogen content of the dental papilla. It was of interest to observe that the degeneration of the enamel organ after crown formation was recognized by chemical determinations of the content of nitrogen and of DNA. It has been noted (VENKATARAMAN and LOWE, 1959; HUTCHINSONand MUNRO, 1961; HALLINAN,FLECKand MUNRO,1963) that, in the SCHMIDTand THANNHAUSER (1945) and the SCHNEIDER (1945) procedures of extraction, a considerable amount of tissue RNA is transferred to lipid solvents after acid precipitation. However, there seems to be no report concerning possible adverse effects of lipid solvent treatment on the recoveries of nucleic acids by the method of OGUR and ROSEN.The authors checked this problem in separate experiments with tooth germs at various stages and found that a fairly constant loss of some 25 per cent of tissue RNA did occur under the conditions of the present extraction procedure. No loss of tissue DNA was observed, however. Although the RNA values are not absolute ones, this does not appear to affect the interpretation of the changes occurring during development, the present

REGIONAL

CHANGES

IN NITROGEN

AND NUCLEIC

ACID

LEVELS

691

study being undertaken primarily to clarify those variations in the activity of the tooth germ which concern matrix formation. The rapid change in RNA contents and the RNA/DNA ratio obtained are considered to indicate dynamic changes in matrix formation activity. From recent electron microscopical studies on amelogenesis, it appears that abundant rough-surfaced endoplasmic reticulum and free ribonucleoprotein particles may be observed in ameloblasts when the enamel organ is most active in matrix formation (LENZ, 1958; REITH, 1960; WATSON, 1960; NYLEN and SCOTT, 1960; REITH,1961; DECKER,1963; ICHIJO, 1964). Those findings suggest the presence of considerable amounts of RNA in the ameloblasts. Localization of RNA in the enamel organ has been demonstrated histochemically with pyronin-methyl green stain (JOHNSONand BEVELANDER, 1954; SYMONS,1956; SUGA, 1956, 1959). The problem was investigated by quantitative methods in the present study, by establishing the RNA/DNA ratio, which is generally considered to be a measure of protein biosynthesis. Since it was impossible to separate the enamel organ from the dental papilla in a small tooth germ without including a calcified region, an exact value for the earlier stages could not be obtained. Considering the values for unseparated germs (Fig. 6), however, there seems to be a peak ratio corresponding to a weight of 0.5 g. To discover which layer of the enamel organ makes the larger contribution to matrix formation, a comparison of the two layers was attempted. There was a significant difference between the rates of fall in RNA/DNA ratios of these layers. The results led us to the conclusion that the inner layer is more directly concerned with enamel matrix formation than the outer layer. However, it is impossible to clarify what kind of cells-ameloblasts, cells of stratum intermedium, or both-have a role in matrix formation, since our inner layer contained both ameloblasts and stratum intermedium. The persistence of a relatively high ratio of RNA to DNA in the outer layer throughout amelogenesis was of interest and seemed to suggest that the outer layer was more concerned with functions other than matrix formation, e.g. maturation. When these aspects are taken into consideration, cells of the outer layer and the stratum intermedium would appear to play an important part in amelogenesis. Although the ameloblasts, matrix and inorganic crystallites have received much attention in electron microscope studies on amelogenesis, the other cells associated with enamel have been comparatively neglected. More extensive investigations are still desirable to clarify their roles. Acknowledgements-The authors wish to express their appreciation to Dr. H. ICHIJOfor his constant interest and encouragement, to Dr. S. SUGAfor his kind advice and constructive criticism. Thanks are also due to Dr. R. P. KORF for his valued help in preparing the manuscript.

R&mr&Le poids set de matkriel c&if?6 et les quantitks d’azote et d’acides nuclkiques des germes d’incisives de boeufs sont dkterminks B divers stades de dkveloppement pour servir de base 31une analyse quantitative du mode de dkveloppcment d’un germe dentaire. L.e poids humide du germe dentaire frais sert Bexprimer le degrk de dkveloppement.

S.

692

SAKAMOTO, S. SASAKI AND S. ARAYA

Dans I’organe de l’kmail, le rapport maximum acide ribonuclCique--acide dCsoxyribonu&ique, consideri g&&alement comme indiquant unc biosynthtse prottique, est not& pour des genes pesant 0,s g, dont I’extr&it& est calcifiee. Les valeurs de ce rapport pour les couches internes de I’organe de l’email montrent une dkrossiance plus rapide que celle de la couche externe, suggtrant que la premitre est plus active dans la formation de la matrice de I’Cmail que la dernitre. Ces changements quantitatifs marques dans le contenu en acides nucl&ques confirment les rtsultats obtenus par I’histochimie et la microscopic Clectronique. Zusammenfassung-In Schneidezahnkeimen verschiedener Entwicklungsstadien von Rindern wurde das Trockengewicht des verkalkten Materials und die Mengen Stickstoff und Nukleinsauren bestimmt, urn eine Grundlage fi.ir die quantitative Analyse des Zahnkeimwachstums vorzubereiten. Das NaBgewicht des frischen Zahnkeims wurde als Ausdruck des Entwicklungsgrades beniitzt. Im Schmelzorgan wurde das maximale Verhlltnis von Ribonukleinslure zu Desoxyribonukleinslure, das im allgemeinen auf die Proteinbiosynthese hinweist, fiir Keime von ungefghr 0,5 g beobachtet, wenn die verkalkte Hijckerspitze erschien. Die Werte dieser Eeziehung fiir die innere Schicht des Schmelzorgans zeigten einen schnelleren Abfall als diejenigen fiir die luBere Schicht, was darauf hinweist, daB ersterc mehr als letztere zur Schmelzmatrixbildung beitrlgt. Diese bemerkenswerten Verinderungen im Gehalt der Nukleinsluren unterstiitzen quantitativ die histochemischen und elektronenmikroskopischen Befunde anderer.

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HALLINAN,T., FLECK, A. and MUNRO, H. N. 1963. Loss of ribonucleic acid into lipid solvents after acid precipitation. Biochim. biophys. Acta (Amst.) 68, 131-133. HILLER,A., PLAZIN,J. and VAN SLYKE,D. D. 1948. A study of conditions for Kjeldahl determination of nitrogen in proteins. J. bin/. Chem. 176, 144X-1420. HUTCHINSON,W. C. and Mu&no, H. N. 1961. The determination of nucleic acids in biological materials. Analyst 86, 768-813. ICHIJO, H. 1964. An electronmicroscopic study of the rat incisor ameloblasts. Jup. J. oral Biol. 6, 17-18. JOHNSON,P. L. and BEVELANDER,G. 1954. The localization and interrelation of nucleic acids and alkaline phosphatase in the developing tooth. J. dent. Res. 33, 128-135. KATSURA,N. 1960. Nucleic acids content of oral tissues. J. Japan sfonzato/. Sot. 27, 58-67. KUIDA, H., NAKANISHI,S. and ARAYA,S. 1960. Growth of dog tooth germ and change in contents of P, Ca and Mg. Bull. Tokyo med. dent. Univ. 7, 169-178. LENZ, H. 1958. Elektronenmikroskopische Untersuchungen der Schmelzgenese. Dt. zuhniirztl. Z. 13,991-1005. NYLEN,

M. U. and Scorr,

denr. Ass.

D. B. 1960. Electron microscopic

studies of odontogenesis.

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and ROSEN,G. 1950. The nucleic acids of plant tissue. I. The extraction and estimation of desoxypentose nucleic acid and pentose nucieic acid. .4rchs Biochem. 25, 262-276. REITH, E. J. 1960. The ultrastructure of ameloblasts from the growing end of rat incisors. Archs oral Biol. 2, 253-262.

REITH, E. J. 1961. The ultrastructure of ameloblasts during matrix formation and the maturation of enamel. J. biophys. b&hem. Cytol. 9, 825-840. SASAKI, S. 1959. Studies on the respiration of the tooth germ. J. Biochc*m. (Tokyo) 46, 269-279.

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G. and THANNHAUSER, S. J. 1945. A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. biul. Chem. 161, 83-89. SCHNEIDER,W. C. 1945. Phosphorous compounds in animal tissues. I. Extraction and estimation of desoxypentose acid and of pentose nucleic acid. J. biol. Gem. 161, 293-303. SUGA, S. 1956. Histochemical studies on the distribution of alkaline phosphatase and ribonucleic acid in amelogenesis of rat. .Z. Nippon dent. Coil. 43, 1-13. SUGA, S. 1959. Amelogenesis. Some histological and histochemical observations. Znt. dent. .Z. 9, 394420. SYMONS,N. B. B. 1956. Ribonucleic acid-alkaline phosphatase distribution in the developing teeth of the rat. J. Anut. (Land.) 90, 117-122. VENKATARAMAN, P. R. and LOWE, C. U. 1959. Effect of ethanol on rat-liver ribonucleoprotein previously exposed to cold trichloroacetic acid. Biochem. J. 72,43@-435. WATSON,M. L. 1960. The extracellular nature of enamel in the rat. J. biophys. biochem. Cytol. 7, 489492.

SCHMIDT,