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The distribution of succinic dehydrogenase and non-specific esterase in mouse skin throughout the hair growth cycle.

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THE DISTRIBUTION O F SUCCINIC
DEHYDROGENASE AND NON-SPECIFIC ESTERASE
I N MOUSE SKIN THROUGHOUT THE
HAIR GROWTH CYCLE
THOMAS S T E P H E N ARGPRIS
Department of Anatomy, Harvard Medical School,
Boston, Yassachusetts
EIGHT FIGURES
INTRODUCTION
Studies of enzyme localization and distribution in the skin
have increased considerably with the advent of new histochemical techniques. Among the enzymes which have received
attention are succinic dehydrogenase and esterase. The former enzyme has been noted in the skin of the rat (Padykula,
'52), guinea pig (Formisano and Montagna, '54), man (Montagna, '55b) and sheep (Rogers, '53), and the latter enzyme
in man (Montagna, '55a). However, in none of the above
studies has the distribution of these enzymes been followed
throughout the hair cycle. Since it has been clearly shown
that the morphology and chemistry of all the components of
the skin vary with different phases of the hair growth cycle
(Chase, '54), a study relating enzyme distribution with the
stage of follicular development appears pertinent.
MATERIALS AND METHODS
Approximately 30 young adult C57 black mice of both sexes
were used.
The growth of the hair follicles was initiated by plucking
the hairs from resting follicles on the dorsum of each mouse.
Post doctoral Fellow of the U.S.P.H.S.
105
106
THOMAS STEPHEN ARGYRIS
Plucking initiated hair growth only in the plucked areas.
Biopsy specimens were removed at intervals of 2, 4, 6, 7, 8,
9, 14, 16, 21 and 27 days after plucking. The skin was placed
on a piece of gelatin and frozen in a deep freeze. Sections
were cut in a cryostat a t 20 or 10 p, and stained for succinic
dehydrogenase with the neotetrazolium (NT) technique of
Seligman and Rutenberg (,51), as modified by Padykula
('53). Adjacent sections were treated to reveal esterase
activity using the method of Nachlas and Seligman ( '49).
I n both techniques appropriate controls were run. Approximately 45 biopsy specimens were studied in this manner.
OBSERVATIONS
For the histology of the phases of hair growth in mice the
present investigation will rely upon and quote from a detailed
account of this subject by Chase ('54).
Hair growth in mice occurs in waves. The events of hair
proliferation are known as anagen and require approximately
19 days for completion. Anagen is followed by a short transitional stage, called catagen, which leads to telogen, a stage
when the follicles are at rest.
Telogen: I n telogen, the epidermis has two layers of cells
covered by a thin line of keratin. The follicles, composed of
a club hair surrounded by a single sheath, the external root
sheath, lie within the dermis. Attached to each follicle is a
sebaceous gland, and below it a round ball of cells, the papilla.
Succinic dehydrogenase in the epidermis of skin possessing
resting follicles is negative except for a faint pink background
and occasional foci of purple staining granules (fig. 1). The
enzymic activity in the external sheath varies from very little
to moderate, with the highest activity in those cells immediately around the base of the club hair. The sebaceous glands
exhibit a diffuse red or reddish purple reaction and, at times,
there may appear superimposed on this diffuse background,
large blue crystals, indicative of intense succinic dehydrogenase activity. The papilla is usually negative or weakly positive.
The dermis is devoid of discrete formazan precipitation except
ENZYMES AND HAIR CYCLE I N MOUSE SKIN
107
for the arrecti pili muscles which are intensely positive. The
fat cells of the subcutaneous layer stain a diffuse pink. The
distribution of succinic dehydrogenase in the panniculus carnosus is characterized by many closely packed purple crystals,
oriented somewhat parallel to the myofibrils, whereas the
underlying connective tissue is usually negative. Controls
without succinate show no endogenoue neotetrazolium reduction except within the sebaceous glands and panniculus carnosus, in both of which the activity is merely a faint pink.
Esterase activity in the skin with resting follicles, in contrast to that of succinic dehydrogenase, is high in the epidermis and follicles (fig. 2 ) . The epidermal cells contain
purple staining granules which, in the basal layer, are usually
perinuclear. This intense epidermal activity occurs in all of
the cells of the follicle. The sebaceous glands exhibit a nongranular red o r reddish purple reaction, and occasionally
they contain dark blue granules superimposed upon the diffuse staining. If a plug of sebum is present in the hair canal,
it stains a dark reddish purple. The hair follicle papillae are
rarely positive for esterase. I n general the dermis is negative
except for the arrecti pili muscles which show intense activity.
The esterase reaction in the subcutaneous fatty layer is
diffuse except f o r some elements of the blood vessels and
nerves ; it is intense in the smooth muscle of the blood vessels
and in the perineurium. The panniculus carnosus shows a
high esterase activity similar to that seen f o r succinic dehydrogenase, and the loose connective tissue beneath it is usually negative.
Afiagew. One day after plucking the club hairs from the
resting follicles (anagen I), the germ region just below the
club hair begins to proliferate. Two to three days after plucking (anagen II), it envelopes the papilla and soon thereafter
an internal sheath arises, and keratinization of the ha6r
shaft begins.
Succinic dehydrogenase is still absent or scant in the epidermis but increases in the germ region where marked mitotic
activity is visible (fig. 3). I n the dermis, subcutis, panniculus
108
THOMAS STEPHEN ARGYRIS
carnosus and the connective tissue below the panniculus,
carnosus, succinic dehydrogenase activity is the same as in
resting skin and, furthermore, it does not change in these
sites throughout the rest of the growth cycle.
Esterase distribution in the epidermis and hair follicles
in anagen I1 is similar to that in skin with resting follicles
(telogen) except in the germ region of the hair follicles where
there is a decrease in esterase activity. In the dermis, subcutis, panniculus carnosus, and the connective tissue beneath
the panniculus carnosus, the pattern of esterase activity is
similar to the pattern seen in telogen, and it does not change
throughout the rest of the growth cycle.
About 3 to 4 days after plucking (anagen 111),the follicles
have penetrated considerably into the subcutis by marked
proliferation of both the germ region and the external root
sheath. These follicles are now approximately three times
their resting length. Concomitantly with these changes, the
epidermis also increases in thickness and becomes hyperplastic.
Succinic dehydrogenase activity increases in the hyperplastic epidermis, and remains intense in the follicles (fig. 4),
except in the region of increasing keratinization, where it
diminishes markedly. I n the matrix area, in addition to
keratinixation, melanin makes its appearance. Because of
the presence of this pigmentation, from now on it becomes
important to examine control sections carefully before attributing color to the reduction of the NT. The elongated
hair papillae and the sebaceous glands both show intense
staining.
Esterase activity in the epidermis decreases in anagen III
in areas which have enlarged. The upper part of the hair
follicle between the outlet of the sebaceous gland and the
basal layer of the epidermis is unchanged. However, below
the opening of the sebaceous gland the esterase activity is
diminished in the areas undergoing proliferation and in the
zone of keratinization (fig. 5). The sebaceous glands are still
very reactive f o r esterase.
ENZYMES AND HAIR CYCLE I N MOUSE SKIN
109
During anagen IV (6 days after plucking) the epidermis
recedes to one or two cell layers in thickness, proliferation
of the hair shaft is well under way and the internal sheath
and hair cuticles are evident. Thus, all of the structures of
the fully differentiated growing follicle are present.
The pattern of succinic dehydrogenase is similar to that
seen 4 days after plucking. The epidermis continues to show
increased dehydrogenase activity in areas of hyperplasia.
I n the hair follicle the bulb and matrix reveal the highest
activity, whereas, the external sheath is only moderately active. Scattered formazan precipitation is also seen in the
keratinizing internal sheath, upper hair shaft, and papilla.
Esterase activity is usually intense in the epidermis in
the areas in which proliferation has ceased, and moderate,
in foci of continued hyperplasia. The upper part of the hair
follicle above the level of the sebaceous gland stains intensely
purple, whereas in the bulb region and in the areas undergoing keratinization the reaction is decreased. The papilla
exhibits minimal esterase activity whereas the sebaceous
glands continue to show intense staining.
Anagen V, 8 days after plucking, is characterized by the
passage of the keratinized hair beyond the duct of the sebaceous gland and completion of the reduction of the epidermis
to one or two cell layers. Both the distribution of succinic
dehydrogenase and esterase are similar t o that observed in
the previous stage (figs. 6 and 7). At anagen VI, 9 days after
plucking, the growing hair erupts through the surface of the
skin and continues to grow for a period of days until it
reaches a length of approximately 7mm. During this stage
there are no changes in enzymatic activity.
Catagem. Catagen, a short transitional stage, intervenes
between anagen and telogen. During this transitional stage
of about two days, proliferation in the bulb ceases, and the
follicle shortens considerably by a process the mechanics of
which are poorly understood. A club hair is formed and the
elongated papilla rounds up into a ball of cells. Below the
shortened follicle a tab of cells remains which presumably
110
THOMAS STEPHEN ARGYRIS
degenerates. When these events are completed, the follicles
again enter the resting or telogen phase.
During catagen, succinic dehydrogenase activity is minimal or absent in the epidermis and may be decreased in the
upper part of the follicle (fig. S), from the outlet of the sebaceous gland to the basal layer of the epidermis. The long
tab of cells left below the hair follicle exhibits marked enzyme
activity (fig. 8).
Esterase activity is intense in the epidermis and upper
portion of the follicle. Below the duct of the sebaceous gland
the follicles stain faintly. The cells below the newly formed
resting hair, which are presumably undergoing degeneration,
show pronounced activity.
DISCUSSION
The distribution of succinic dehydrogenase varies within
the hair follicles during the course of the hair growth cycle.
I n the resting phase it is minimal, whereas with growth of
the follicles, it is increased in the areas undergoing proliferation. This is evident during the early stages when the follicular downgrowth still consists of a solid cord of cells. With
the further growth and differentiation of the f ollicle, succinic
dehydrogenase activity decreases in the external root sheath
where glycogen deposition is occurring (Chase, '54) and in
the cells undergoing keratinization. High activity persists
in the bulb in areas of mitosis and in the elongated papilla.
Similar activity and distribution of succinic dehydrogenase
have been reported in the fully differentiated growing hair
follicles of sheep (Rogers, ' 5 3 ) , man (Montagna, '55b) and
guinea pigs (Pormisano and Montagna, '54). I n resting follicles of the rat (Padykula, '52) and guinea pig (Formisano
and Montagna, '54), similarly to our findings, succinic dehydrogenase has been noted as minimal.
Similarly to growing follicles during anagen, the epidermis
shows an augmentation of succinic dehydrogenase activity
commensurate with the increased mitotic activity and thick-
ENZYMES AND HAIR CYCLE I N MOUSE SKIN
111
ness of the epidermis. Montagna (’55) has shown that, in
the multi-cell layered epidermis of man, there is a marked
succinic dehydrogenase reaction, especially in the stratum
basalis and spinosum, where proliferation is occurring. The
merocrine sweat glands are reported to have a high succinic
dehydrogenase activity, whereas the apocrine glands show
minimal activity.
The biochemical oxidative activity of the skin has been investigated, but the data are difficult to interpret because of
the lack of attention paid to the hair cycles. Barron (’48)
maintains that in the rat, oxygen consumption and pyruvate
utilization are higher in fetal than in adult skin. Moreover,
the R. Q. values for fetal skin are .95, and, for the adult .75
to -83. It is of interest to note that the skin utilized by Barron
which he called fetal was, in fact, from a two-day-old rat and
in the first phase of hair growth; whereas the adult skin was
probably in the resting phase. The high R.Q. value of .95
for “fetal” skin is in harmony with the fact that growing
hair follicles are loaded with glycogen, and the lowered R. Q.
in the resting follicles coincides with the absence of glycogen.
Similar results have been obtained by Adams ( ’36), studying
oxygen consumption in rat skin. I n these experiments, 36day-old rats showed a higher oxygen consumption than 105day-old rats. At 36 days the follicles are beginning to grow
and at 105 they are at rest, according to the estimates of
Butcher (’34). Within the limits of these fluctuations, the
metabolic activity of the skin is still much lower than that of
other organs. Rogers ( ’53) indicates that succinic dehydrogenase activity is 190 in kidney and 6 in skin, measured as
micrograms INT formazan/milligram dry weight of fat-free
tissue, and Carruthers and Suntzeff (’53) point out that the
succinic dehydrogenase activity of skin is respectively 29
and 9 times less than in liver and muscle.
Esterase distribution in the follicles is inverse to that of
succinic dehydrogenase, being highest in the resting follicles
and lowest in the growing ones. The decrease in esterase
is not due only to keratinization, since the enzymatic reaction
112
THOMAS STEPHEN ARGYRIS
diminishes in early anagen before the growing follicles undergo keratinization.
The proliferating epidermis also exhibits a decrease in
esterase activity in the early stages of hair cycle. With the
termination of mitotic activity and the reduction in the thickness of the epidermis, the enzyme pattern is again reversed.
Esterase has also been studied in the skin of other animals.
I n human skin from the axilla, Montagna ('%a) reports
moderate activity in the basal layer and minimal activity in
the rest of the stratum spinosum and stratum granulosum.
Just beneath the stratum corneum there is a thick band of
esterase activity. I n the palm, on the contrary, esterase activity in the epiderniis is high. The merocrine sweat glands
show no activity whereas the apocrine glands react intensely.
Thus, similarly to our findings in sites in which esterase
activity is high, succinic dehydrogenase is minimal, so that
the inverse correlation between these two enzymes is present
in many components of the skin. The epidermis of the palm
is excepted in which there is both high esterase and succinie
dehydrogenase activity (XIontagna, '55a, '55b).
The increased succinic dehydrogenase activity during the
growing phase of the hair cycle is understandable if one
remembers that, in the sites of increased oxidative activity,
mitosis is occurring. That oxidative energy is necessary for
cell division in the epithelium of skin, was first shown by
Illedawar ('47) and has been extended and elaborated by
Bullough ( ' 5 2 ) who has demonstrated that the epidermal
cells require the activity of the Krebs cycle to carry on mitosis.
Since the basal layer of cells of the epidermis, the cells of
the external sheath of the hair follicle, and the cells of the
matrix and the germ region are equipotential (Chase, ' S ) ,
it might be assumed that their energy requirements and
pathways are basically similar. Whether or not increased
succinic dehydrogenase is symptomatic of an increase in
the activity of the entire Krebs cycle remains to be shown.
ENZYMES A N D HAIR CYCLE IN MOUSE SKIN
113
According to Chase ('54) the resting hair follicle is the
permanent hair follicle. Perhaps esterase activity is a chemical index of the kind of metabolisA which characterizes the
permanent follicle, whereas its loss, plus the increase in the
succinic dehydrogenase, characterizes metabolism during the
regenerative phase of hair growth. If this is correct, then
the reversed enzyme pattern of the regenerating follicles
should simulate that occurring in the original follicular downgrowth during its fetal development. That this is so, has
been shown by Argyris ( '55).
SUMMARY
1. The changes in activity and localization of succinic dehydrogenase and esterase have been studied histochemically,
in the skin of the mouse throughout the hair growth cycle.
2. Fluctuations of these enzymes are limited to the epidermis and hair follicles; no cyclical changes are observed
in the dermis and subcutaneous tissue.
3. I n the resting phase of the cycle the epidermis and
hair follicles show little succinic dehydrogenase activity and
marked esterase activity.
4. After stimulation of the follicles by plucking, succinic
dehydrogenase activity increases in the growing follicles. The
epidermis also shows increased enzymatic activity in the
early part of the hair cycle when it is undergoing hyperplasia.
The esterase activity is decreased in both these sites.
5. I n fully differentiated growing follicles, succinic dehydrogenase and esterase activities are absent in the areas
undergoing keratinization and decreased somewhat in the
external hair sheaths. Maximal activity of the succinic dehydrogenase is maintained in the bulb and papilla, whereas,
esterase activity is markedly decreased in these sites.
6. The significance of the inverse correlation between the
presence of succinic dehydrogenase and esterase in mouse
skin is discussed.
114
THOMAS STEPHEN ARGYRIS
LITERATURE CITED
ADAMS,P. H. 1936 The oxygen uptake and composition of the skin of rats i n
vitamin G deficiency. J. Biol. Chem., 116: 641-651.
ARCYRIS,T. S. 1955 Unpublished data.
BURON, G. E. S., J. MEYERAND Z. B. MILLER 1948 The metabolism of skin.
Effects of vesicant agents. J. Invest. Dermat., 11: 97-118.
BULLOUGH,
W. S. 1952 The energy relations of mitotic activity. Biol. Rev.,
27: 133-168.
E. 0. 1934 The hair cycles in the albino rat. Anat. Rec., 62 :5-19.
BUTCHER,
CARRUTHERS,
C., AND V. SUNTZEFF 1953 Biochemistry and physiology of epidermis. Physiol. Rev., 3'3: 229-243.
CHASE, H. B. 1954 Growth of the hair. Physiol. Rev., 9 4 : 113-126.
FORMISANO,
V. R., AND W. MONTAGNA1954 Succinic dehydrogenase activity
in the skin of the guinea pig. Anat. Rec., 180: 893-906.
MEDAWAR,
P. B. 1947 The behaviour of mammalian skin epithelium under
strictly anaerobic conditions. Quart. J. Micr. Sci., 88: 27-37.
MONTAGNA,
W. 1955a Histology and cytoehemistry of human skin. I X : The
distribution of non-specific esterases. J. Biophy. and Biochem. Cytol.,
1: 13-16.
1955b Histology and cytochemistry o f human skin. VII: The
distribution of succinic dehydrogenase. Anat. Rec. (in press).
NACHLAS,M., AND A. M. SELIGMAN1949 The histochemical demonstration of
esterase. J. Nat. Cancer Inst., 9: 415-425.
PADYKULA,
H. A. 1952 The Iocalization of succinic dehydrogenase in tissue
sections of the rat. Am. J. Anat., 9 1 : 107-146.
1953 The localization of succinic dehydrogenase in tissue sections
of normal and hypophysectomized rats. Thesis, Radcliffe College,
Harvard University.
ROGERS,G. E. 1953 The localization of dehydrogenase activity and sulfhydryl
groups i n wool and hair follicles by the use of tetrazolium salts. Quart.
J. Micr. Sci., 9 4 : 253-268.
SELIGMAN,
A. M., AND A. M. RUTENRERG1951 The histochemical demonstration
of succinic dehydrogenase. Science, 123: 317-320.
PLATES
Non-specific esterase in resting skin.
TWOdays after plucking. Proliferation and downgrowth of germ region has begun. Note increased succinic dehydrogenase activity.
Succinic dehydrogenase activity 4 days after plucking. Note increased activity in hyperplastic epidermis with that
seen in figure 3.
3
3
4
1 Succinic dehydrogenase in skin with resting follicles. Note the intense reaction in the sebaceous gland, moderate
reaction in the follicle, and absence of enzymatic activity in the epidermis and dermis. The straight line indicates
the division between the epidermis and the dermis. The arrow delineates extent of the follicle below the outlet Of
the sebaceous gland.
All figures photographed at 100 X.
EXPLANATION OF FIGURES
PLATE 1
THOMAS S T E P H E N ARGYRIS
ENZI'MES A N D HAIR C Y C L E I N M O U S E SKIN
PLATE I
Non-specific esterase 4 days after plucking. Note decrease estcrase activity in areas undergoing proliferation.
Fully differentiated growing follicle, 8 days after plucking. Note intense snccinic dehydrogenase activity in the bulb
region and lower part of the external sheath.
Growing follicle same stage as that in figure 6 showing esterase activity. Note absence of esterase in the bulb region.
Growing follicle becoming transformed into resting one. Note club hair, area shortening, indie1tr.l by an arrow,
and below it the t a b of degenerating cells.
5
6
7
8
EXPLANATION O F FIGURES
PLATE 2
THONAS S T E P H E N ARGYRIS
E N Z Y M E S A N D HAIR C Y C L E I N MOUSE S K I S
PLATE 2
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distributions, growth, succinic, specific, mouse, esterase, non, dehydrogenase, skin, cycle, hair, throughout
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