Histochemie 29, 355--361 (1972)

9 by Springer-Verlag 1972
Histochemical Demonstration of Succinic Dehydrogenase
Activity during Sequential Molar Development
in the Swiss Albino Mouse*
D. Vi n c e n t Pr o v e n z a , Yu n g - F e n g Chang, Les l i e 1 ). Ga r t n e r , a n d Ri c h a r d M. Ga r l i t z
Depart ment of Anat omy, School of Dent i st ry
Uni versi t y of Maryland, Baltimore, Maryland, U.S.A.
Received December 22, 1971
Summary. Appl yi ng t he di t et razol i um sal t (Nitro-BT) method for succinic dehydro-
genase, murine molar t eet h were studied sequentially from the cap stage of development
through t he appositional stages of odontogenesis. React i on-di st ri but i on and i nt ensi t y varied
relative to t he developmental stage as well as t he zone of mat urat i on within a given stage.
The peripheral cells of t he parent dental lamina exhibited some act i vi t y, as di d t he outer
enamel epithelium of the bell stage. During t he period of mat r i x apposition, components of
t he st r at um intermedium, ameloblastic zone and odonteblastic layer region of t he dent al pa-
pilla demonst rat ed intense enzymatic act i vi t y. Cells act i vel y engaged in enamel mat ri x pro-
duction demonst rat ed act i vi t y in the basal and distal cell segments. High act i vi t y continued
in t he papi l l ary l ayer of t he enamel organ, as well as in t he cells of t he dental sac during the
postamelogenic period.
I nt r oduc t i on
Lo c a l i z a t i o n of s ucci ni c d e h y d r o g e n a s e ( SDH) a c t i v i t y ha s been r e p o r t e d i n
o d o n t o g e n i c t i s s ue of r o d e n t s ( Bur s t one, 1960; Swa r t z a n d Yeager , 1961; Bal ogh,
1963 ; Fu l l me r , 1963, 1964; Re i t h a n d Zus s ma n, 1964; Goggi ns a n d Fu l l me r , 1966)
a n d i n c a ni ne t o o t h g e r m (Mori , Mi z us hi ma a n d Os anai , 1961). The s e s t udi e s
h a v e i n v o l v e d t h e use of a d i t e t r a z o l i u m s a l t ( Ni t r o- BT) i n a p h o s p h a t e buf f e r e d
s o d i u m s uc c i na t e me d i u m, whe r e t h e Ni t r o - BT s er ves as t he f i na l e l e c t r on a c c e p t o r
a n d i t s r e d u c t i o n i s vi s ua l i z e d as de pos i t s of bl ue d i n i t r o f o r ma z a n . Re c e n t l y ,
Ke r p e l - F r o n i u s a n d Ha j o s (1968) d e v e l o p e d a me t h o d of e mp l o y i n g f e r r i c y a n i d e
as t he r e d u c i n g a ge nt .
The s t udi e s c i t e d a b o v e wer e c onc e r ne d wi t h speci f i c s t a ge s of odont oge ne s i s
a n d none i n c l u d e d mous e mol a r s . The p r e s e n t s t u d y pr opos e s t o e l u c i d a t e t h e
l oc a l i z a t i on of s ucci ni c d e h y d r o g e n a s e a c t i v i t y t h r o u g h o u t s e que nt i a l mo l a r deve-
l o p me n t i n t h e Swi ss a l b i n o mous e. I t f u r t h e r s eeks t o p r o v i d e a c o mp r e h e n s i v e
h i s t o r y of e n z y me a c t i v i t y p r e p a r a t o r y t o a n e l e c t r on mi c r os c ope c y t o c h e mi c a l
s t u d y .
Mat eri al s and Met hods
Randoml y bred Swiss albino mice of nonspecific strain, ranging in age from 15 days prena-
t al to 8 days post nat al , were employed in this study. The pregnant females were etherized,
t he uterine horns were opened and the excised fetuses were decapi t at ed. Whole heads were
t reat ed individually. I n post nat al animals, t he pups were etherized, and t he mandibles were
* Supported by PHS Grant No. 2800-02, National I nst i t ut e of Dental Research, National
Inst i t ut es of Health.
356 D.V. Provenza, Yung-Feng Chang, L. P. Gartner, and 1~. M. Garlitz:
removed. The specimens studied were immediately frozen in liquid nitrogen and oriented on
a microtome chuck to obtain buccolingual sections of developing molars. Frozen serial sections,
14-16 ~z thick, were placed on untreated glass slides and allowed to dry.
The sections were incubated for 10 minutes at 37 ~ C in substrate and phosphate buffered
sodium succinate, pH 7.6 to which an equal volume of an aqueous solution containing Nitro-
BT was added (Nachlas, Tsou, Souza, Chang and Seligman, 1957). Sections were then washed
in physiological saline, fixed in 10% neutral buffered formalin (10 minutes), dehydrated in
a graded series of alcohols, rinsed in xylene, and finally mounted with Permount.
Control sections were incubated in a substrate-free medium or dipped in the experimental
medium for five seconds. They were then treated as described above. Safranin-0 did not
significantly enhance visualization, thus its use was omitted.
Re s ul t s
The devel opment al stages i nvest i gat ed ranged from t he cap t hrough t he
apposi t i onal stage,
The basal l ayer of t he pr esumpt i ve oral epi t hel i um and t he par ent l ami na
of t he cap stage demonst r at ed a very slight degree of succinic dehydrogenase
act i vi t y, while t he t oot h germ per se showed no act i vi t y.
I n t he earl y devel opi ng enamel organ t he out er enamel epi t hel i um exhi bi t ed
moderat e act i vi t y which cont i nued i nt o t he peri pheral cells of t he dent al l ami na
and basal cells of t he oral epi t hel i um (Fig. 1). The component s of t he stellate reti-
cul um, st r at um i nt er medi um, t he i nner enamel epi t hel i um, dent al sac and papi l l a
were unreact i ve. Act i vi t y was great est i n t he cells of t he out er enamel epi t hel i nm
l ocat ed at t he apex of t he fut ure enamel organ. Fr om t hi s area act i vi t y gradual l y
di mi ni shed al ong t he slope, wi t h its nadi r at t he cervical loop (Fig. 1).
Wi t h i ni t i at i on of apposi t i onal stage t he cells of t he basal l ayers of t he oral
epi t hel i um, as well as those of t he peri phery of t he par ent l ami na were unreact i ve.
The r emnant s of t he out er enamal epi t hel i um, which formed i sl ands and st r ands
of cells showed no act i vi t y. The previ ousl y unr eact i ve cells of t he s t r at um i nt er-
medi um and amel obl ast s, however, cont ai ned react i on deposits. The i nt ensi t y
Fig. 1. Photomicrograph of the early bell stage of odontogenesis of a mouse embryo on the
17th day postconception. Note the enzyme activity (arrows) in the basal layer of the presump-
tive oral epithelium, the peripheral cells of the parent lamina, and in the outer enamel
epithelium. Original magnification x 16
Fig. 2. Section of early appositional stage of molar development from a one day old mouse.
Greatest activity is seen in the trough (T) and crest (C) areas of the enamel organ and decreases
along the slope. Note that the odonteblastic region (arrow) also demonstrates enzyme activity.
Dental papilla (D), ameloblast (A). Original magnification x 40
Fig. 3. High power photomicrograph of the ameloblastic region of a two day old mouse. The
activity is localized in the stratum intermedium (SI) and the infranuclear region of the
ameloblasts (A). Original magnifications x 160
Fig. 4. Appositional stage in a three day old mouse. Note that the highest activities are in
the crest (C) and trough (T) regions of the tooth germ and lowest at the cervical loop (arrow).
Original magnification x 16
Fig. 5. High power photomicrograph of the trough area of Fig. 4. Note activity in the distal
segments (arrows) of the ameloblasts. Original magnification X 160
Succinic Dehydrogenase Act i vi t y in Molar Development 357
Fig. 6. High power photomicrograph of the crest area of Fig. 4. Reaction deposits are present
in the distal cell segments of the ameloblasts (A) and in cell components of, and external to,
the st rat um intermedium (SI). Original magnification 160
Fig. 7. Slope area of the molar tooth germ of a six day old mouse. Note the intensity of the
act i vi t y along the slope (arrows). Minimal act i vi t y can be noted in Hertwig' s epithelial root
sheath (H). Original magnification 40
Fig. 8. Presumpt i ve crest and trough area of the tooth germ of Fig. 7. Note activity (arrows)
in the papillary pegs formed by the stratum intermedium. Original magnification 40
358 D.V. Provenza, u Chang, L. P. Gartner, and R. M. Garlitz:
of t he react i on vari ed wi t h t he t opographi cal location of t he cell. The cells of
f ut ur e cuspal and t r ough areas were most react i ve, while t hose of t he slope gra-
dual l y diminished in act i vi t y. The lowest level was not ed in t he cervical loop. The
odont obl ast i c zone paralleled t he pat t er n of t he ameloblastic act i vi t y regardi ng
di st ri but i on, however i t was considerably weaker (Fig. 2).
By t he second day of post nat al life t he amount of har d tissue (enamel and
dent i n) produced was appreciable. Succinic dehydrogenase act i vi t y in bot h stra-
t um i nt er medi nm and t he ameloblasts was considerable. The react i on deposits
in t he amelogenic cells were confined in t he i nfranucl ear region. Those cells which
were in t he i mmedi at e vi ci ni t y of t he st r at um i nt er medi um of t he prospect i ve
cuspal and t r ough areas also cont ai ned react i on pr oduct s (Fig. 3). Concurrent l y,
t he dent al papilla and especially t he odont obl ast i c region, exhi bi t ed great er
act i vi t y.
I n bot h odont obl ast i c and ameloblastic regions t he i nt ensi t y was sustained
at t he summi t s of t he euspal and t rough areas, but t he operat i onal t er r i t or y of
t he enzyme was spread over a broader, more cervically l ocat ed front .
The enzyme act i vi t y increased by t he t hi r d day of t he post nat al life bot h in
t he ameloblasts and st r at um i nt ermedi um. The dent al papilla, and especially
its odont obl ast i c l ayer showed strong succinic dehydrogenase act i vi t y. The
graded level of di formazan deposits, which was great est at t he cuspal and t hr ough
regions wi t h a nadi r at t he cervical loop, persi st ed t hr ough this stage (Fig. 4).
This peri od of post embryoni c devel opment reveal ed t ha t succinic dehydrogenase
act i vi t y was al t ered in location, specifically i t also appeared in t he distal cell
segment of t he amcloblast. The most act i ve region of t he ameloblast, however,
remai ned i nfranucl ear (Fig. 5). An increase of t he react i on pr oduct was not ed in
t he component s ext ernal t o t he st r at um i nt er medi um (Fig. 6). The presence of
t he enzyme was not ed onl y in t he component s of t he prospect i ve dent al sac imme-
di at el y adj acent t o t he most act i ve regions of t he s t r at um i nt ermedi um.
Wi t h f ur t her devel opment , enzyme act i vi t y remai ned const ant in developing
t oot h germ. Hert wi g' s epithelial r oot sheat h (former cervical loop) exhi bi t i ed
l east act i vi t y.
By t he si xt h day of post nat al devel opment , significant differences in t he
i nt ensi t y of enzyme act i vi t y bet ween t he occlusal surface and t he slopes cervical-
ward were not observed (Fig. 7). Enamel and t he dent i n mat ri ces were free of
act i vi t y. Act i vi t y in t he dent al sac was maxi mal by t he apposi t i onal stage of
devel opment as was t he case regardi ng t he r emnant of t he s t r at um i nt er medi um
which were t hr own i nt o folds, or papi l l ary pegs (Fig. 8).
Di scussi on
Succinic dehydrogenase, one of t he enzymes of t he citric acid cycle, was f ound
t o be ver y st rongl y i ncorporat ed i nt o t he fine st r uct ur e of t he mi t ochondri al
membr ane (Pet t e, Kl i ngenberg and Bucher, 1962). The presence of succinic de-
hydrogenase act i vi t y implicates an act i ve met abol i c state.
Studies of succinic dehydrogenase act i vi t y in odontogenesis have been limited
to i ndi vi dual devel opment al stages and specific t eet h (molars or incisors). Bur-
Succinic Dehydrogenase Activity in Molar Development 359
st one (1960) i nvest i gat ed hamst er molars pr obabl y in t he bell stage of odont o-
genesis. Mori et al. (1961) st udi ed rat , guinea pig and dog molars in t he bell and
earl y apposi t i onal stages, Swart z and Yaeger (1961) i nvest i gat ed mouse molars
in t he apposi t i onal stage while Balogh (1963) empl oyed growing incisors of mice.
Ful l mer (1963) and Rei t h and Zussman (1964) ut i l i zed t he enamel organ of r at
incisors. The s t udy of Rei t h and Zussman (1964) was concerned onl y wi t h t he
ameloblasts. Goggins and Ful l mer (1966) rest ri ct ed t hei rs t o dent al pul ps of
young rats.
The dat a concerning succinic dehydrogenase are in accord rel at i ve t o i t s
presence in t he s t r at um i nt crmedi um. Except for Bur st one (1960), all are in
agr eement t hat t he enzyme is present in ameloblasts. Mori e t al . (196]) and
Ful l mer (1963) have demonst r at ed t hat wi t h di fferent i at i on of t he ameloblasts,
act i vi t y is intensified. They addi t i onal l y have i ndi cat ed pol ari zat i on of act i vi t y
in t he amelogenic layer. Of t he studies conduct ed, onl y one has shown t ha t t he
enzyme is present in t he out er enamel epi t hel i um (Mori et al . , 1961). I n t he stellate
ret i cul um, Bur st one (1960), Mori et al. (1961) and Ful l mer (1963) r epor t ed slight
act i vi t y. All i nvest i gat i ons have shown succinic dehydrogenase t o be present in
t he odont obl ast s and dent al papillae. Rei t h and Zussman (1964) not ed t he pre-
sence of t he enzyme in all stages st udi ed (appositional t hr ough pi gment at i on).
Pol ari zat i on was not ed, however, with basal accumul at i ons in t he appositional
and pi gment stages. More i nt ense act i vi t y was seen in t he distal cell segment of
t he more gingivally l ocat ed amcloblasts.
The results of t he present s t udy demonst r at e t he di st ri but i on of succinic
dehydrogenase t hr oughout t he sequence of mol ar t oot h devel opment in mice.
These dat a establish t hat t he enzyme is not i ni t i al l y present in det ect abl e amount s
in t he dent al lamina, r at her i t appears in t he cap and disappears in t he bell stage.
I t was found, however, selectively t hr ough t he enamel organ and general l y t hr ough
t he reduced enamel epi t hel i um in var yi ng degrees of i nt ensi t y. The bud similarly
exhi bi t ed no act i vi t y. Wi t h hi st odi fferent i at i on, t he react i on pr oduct appear ed
in t he out er enamel epi t hel i um and gradual l y diminished as t he epi t hel i um was
reorgani zed wi t h st rands and cell nidi. The cells of t he stellate ret i cul um were
consi st ent l y nonreact i ve while t he s t r at um i nt er medi um and amel obl ast were
increasingly more act i ve wi t h t he acquisition of funct i onal compet ency. The
correl at i ve phenomenon of act i vi t y and degree of di fferent i at i on appear ed t o
be a funct i on of t he stage of devel opment . Pol ari zat i on of act i vi t y observed in
t he devel opi ng and funct i onal amel obl ast was in har mony wi t h t he dat a of Mori
et al (1961) and Ful l mer (1963). The odont obl ast i c region of t he dent al papilla
followed a similar pat h, but t he i nt ensi t y of react i on, as measured by t he deposits
of t he blue di formazan, did not reach t he level achi eved by t he act i ve regions
of t he enamel organ.
The present s t udy demonst r at ed t ha t t he devel opment al hi st or y of t he dent al
anlage is paralleled by funct i onal act i vi t y of its component s as i ndi cat ed by
succinic dehydrogenase act i vi t y. Accordingly, t he enzyme was not ed t hr oughout
t he apposi t i onal stage (amclogenesis) of t he enamel organ. The i nt ensi t y of locali-
zat i on vari ed in i ndi vi dual cells and cell layers and was commensurat e wi t h
met abol i c act i vi t y. This was similarly not ed by Mori et al. (1961), Balogh (1963)
360 D.V. Provenza, Yung-Feng Chang, L. P. Gartner, and R. M. Garlitz:
and Rei t h and Zussman (1964). Ul t r ast r uct ur al support for t hi s is i ndi cat ed by
t he pol ari zat i on of t he organelles, par t i cul ar l y t he mi t ochondr i a i n t he amelo-
bl ast s (Lenz, 1957; Nyl en and Scott, 1958, 1960; Rei t h, 1960, 1961, 1963, 1967,
1970; Pannese, 1962; Kal l enbach, 1966, 1968, 1970). The el egant i nvest i gat i on
of Rei t h (1970) which charact eri zed t he amel obl ast ul t r ast r uct ur al l y duri ng
vari ous stages of devel opment as well as those of Kal l enbach (1968, 1970) sub-
st ant i at e t he hi st ochemi cal dat a of t he present st udy regardi ng di st r i but i on of
mi t ochondr i a for t he amel obl ast . The studies of Pannese (1960) on t he stellate
r et i cul um and t he s t r at um i nt er medi um and Kal l enbach (1968) on t he papi l l ary
l ayer si mi l arl y suppor t t he fi ndi ngs of t hi s st udy.
Re f e r e nc e s
Balogh, K. : Histochemical study of oxidative enzyme systems in teeth and periodontal tissues.
J. dent. l~es. 42, 1457-1466 (1963).
Burstone, M. S. : Histochemical observations on enzymatic processes in bones and teeth.
Ann. N. Y. Acad. Sci. 85, 431444 (1960).
Fullmer, H. M. : Dehydrogenases in developing teeth of rats. J. Histochem. l l , 641-644 (1963).
Fullmer, H. M. : In: Oral Histology Inheritance and Development (D. V. Provenza), chapt.
18, Philadelphia: J. B. Lippincott 1964.
Goggins, J. F., Fullmer, H. M. : Dehydrogenase histochemistry or the rat molar pulp. Arch.
oral Biol. 11, 1365-1370 (1966).
Kallenbach, E. : Electron microscopy of the papillary layer of rat incisor enamel organ during
enamel maturation. J. Ultrastruct. Res. 14, 518-533 (1966).
Kallenbach, E. : Fine structure of rat incisor ameloblasts during enamel maturation. J. Ultra-
struct. Res. 22, 90-119 (1968).
Kallenbach, E. : Fine structure of rat incisor enamel organ during late pigmentation and
regression stages. J. Ultrastruct. Res. 80, 38-63 (1970).
Kerpel-Fronius, S., Hajos, F. : The use of ferricyanide for light and electron microscopic
demonstration of succinic dehydrogenase activity. Histochemie 14, 343-351 (1968).
Lenz, H.: Elektronenmikroskopische Untersuchungen an der Ameloblastenschicht. Dtsch.
zahn~rztl. Z. 12, 1568-1579 (1957).
Mori, M., Mizushima, T., Osanai, K. : The histochemical demonstration of succinic dehydro-
genase activity in developing teeth. Histochemie 2,348-355 (1961).
Nachlass, M.M., Tsou, K.C., Souza, E. de., Chang, C. S., Seligman, A.M.: Cytochemical
demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted
ditetrazole. J. Histochem. Cytochem. 5,420-436 (1957).
Nylen, M., Scott, D.: An electron microscopic study of the early stages of dentinogenesis.
PHS Pub. No. 613, Supt. Documents. Washington, D. C. : U. S. Govt. Printing Office 1958.
Nylen, M., Scott, D.: Electron microscopic studies of odontogenesis. J. Indiana dent.
Ass. 39,406-421 (1960).
Pannese, E. : Observations on the ultrastructure of the enamel organ. I. Stellate reticulum
and stratum intermedium. J. Ultrastruct. Res. 4, 372-400 (1960).
Pannese, E.: Observations on the ultrastructure of the enamel organ. III. Intgrnal and
external enamel epithelia. J. Ultrastruct. Res. 6, 186~204 (1962).
Pette, D., Klingenberg, M., Bucher, T.: Comparable and specific proportions in the mito-
chondrial enzyme activity pattern. Biochem. biophys. Res. Commun. 7, 425-429 (1962).
Reith, E. J. : The ultrastructure of ameloblasts from the growing end of rat incisors. Arch.
oral Biol. 2,253-262 (1960).
Reith, E. J. : The ultrastructure of ameloblasts during matrix formation and the maturation
of enamel. J. biophys, biochem. Cytol. 9, 825-840 (1961).
Reith, E. J.: The ultrastructure of ameloblasts during early stages of maturation of enamel.
J. Cell. Biol. 18, 691-708 (1963).
Succinic Dehydrogenase Activity in Molar Development 361
Reith, E. J. : The early stage of amelogenesis as observed in molar teeth of young rats.
J. Ultrastruct. l~es. 17, 503-526 (1967).
Reith, E. J. : The stages of amelogenesis as observed in molar teeth of young rats. J. Ultra-
struct. Res. 30, 111-151 (1970).
Reith, E. J., Zussman, W. : Snccinic dehydrogenase in ameloblasts during formation and
maturation of enamel. Arch. oral Biol. 9, 31-38 (1964).
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Res. 40, 1291 (1961).
Dr. D. Vincent Provenza
Department of Anatomy
School of Dentistry
University of Maryland
Baltimore, Maryland 21201, U.S.A.
Yung-Feng Chang
Department of Microbiology
University of Maryland
School of Dentistry
Baltimore, Maryland 21201, U.S.A.
24 Histochemie, Bd. 29