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Myoid cells in the capsule of the adrenal gland and in monolayers derived from cultured adrenal capsules.

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Myoid Cells in the Capsule of the Adrenal Gland
and in Monolayers Derived from Cultured
Adrenal Capsules
ROBERT S. BRESSLER
Department of Anatomy, Mount Sinai School of Medicine of the
City University of New York, Fifth Avenue and 100th Street,
New York, New York 10029
Previous studies have demonstrated the presence of myoid cells
in monolayers derived from cultured testicular fragments. In order to determine
if monolayers of myoid cells can also be produced by other types of tissue cultures, monolayers derived from fragments of mouse adrenal capsules, cultured
in McCoys modified 5A medium, were examined for the presence of myoid cells.
The cells of the resultant monolayers contained numerous free ribosomes and
abundant profiles of granular endoplasmic reticulum, as well as 40-60 A thick
filaments and associated dense bodies. In these respects they are identical to
myoid cells found in monolayers in testis cultures. Contrary to earlier reports
indicating that the adrenal capsule is composed of fibroblasts, this study demonstrates that myoid cells are a major component of the capsule of the rat and
mouse adrenal gland.
ABSTRACT
Recent investigations have disclosed the
presence of a cell type in a variety of
organs (reviewed in Bressler and Ross,
'73) that exhibits certain ultrastructural
features characteristic of smooth muscle
cells and others usually associated with
fibroblasts. On the basis of their fine
structure these cells have been referred
to a s "myofibroblasts" (Gabbiani and
Majno, '72; Majno et al., '71) and their
existence h a s been interpreted as a n indication that fibroblasts and smooth muscle
cells are more closely related than previously assumed. From this point of view
it is interesting that in the developing tunica media of the chick aorta (Karrer, '60)
and in the peritubular tissue of the differentiating testis (Bressler and Ross, '72;
Ross, '67) fibroblasts apparently differentiate into contractile cells. The findings of a n
earlier investigation indicate that myoid
cells in monolayers derived from cultured
testicular tissue are the progeny of fibroblasts in the tunica propria of the
seminiferous tubules (Bressler and ROSS,
'73). In order to determine if the formation
of monolayers of myoid cells is unique to
testis cultures, similar cultures were estabANAT. REC., 177: 525-532.
lished using fragments of adrenal capsule
and the resultant monolayers were examined for the presence of myoid cells.
The adrenal capsule has previously been
reported to be composed exclusively of
fibroblasts (Brenner, '66; Fujita, '72;
Kahri, '66; Mackay, '69; Nishikawa et al.,
'63; Penny e t al., '63; Yamori et al., '61).
While the present study was in progress,
Bloodworth and Powers ('68) reported the
presence of smooth muscle-like cells in the
adrenal capsule of the dog. On the basis
of that observation a re-examination of the
structure of the rodent adrenal capsule
was undertaken to determine if it is indeed
devoid of myoid cells. The present report
describes the presence of myoid cells in
the capsules of adult rat and mouse
adrenal glands and in monolayers derived
from cultured adrenal capsules.
MATERIALS AND METHODS
Adrenal glands obtained from adult,
seven or ten day old Swiss albino mice
(Cd-1, Charles River Laboratory) were removed from anesthetized animals, bisected,
the glandular tissue squeezed out from the
capsule and the latter placed in a plastic
Received M a y 15, '73. Accepted July 11, '73.
525
526
ROBERT S. BRESSLER
tissue culture bottle (Falcon Plastic Co.).
Five milliliters of McCoys modified 5a
medium containing 30% fetal calf serum
and antibiotics (GIBCO) were added to
each bottle. The cultures were maintained
at 37°C for 11 days after which the culture
medium was poured off and processing of
the tissue for electran microscopy was performed in the culture bottles as described
below for blocks of adrenal glands.
Adrenal glands of adult rats and mice
were exposed and bathed in situ in the
ether-anesthetized animal in 0.1 M phosphate-buffered, 4 % glutaraldehyde fixative
(pH 7.4) for five minutes. The adrenal
glands were then removed, cut in 1 mm3
pieces and fixed for five hours in glutaraldehyde fixative at 4°C. The tissues were
transferred to the buffer solution and left
at 4°C overnight. The following morning
they were further fixed for one hour in 1%
osmium tetroxide, also in phosphate buffer
(pH 7.4). The tissue blocks were dehydrated through a graded series of ethanols
and propylene oxide and embedded in
Epon. Monolayers were prepared in the
same manner but the time allowed for
each of the steps was reduced. Glutaraldehyde fixation was for one hour, followed
by a five minute wash in buffer and half
hour of osmium tetroxide fixation. Propylene oxide was omitted from the dehydration scheme since it dissolves the plastic bottles. BEEM capsules filled with Epon
were inverted over the monolayers to provide Epon blocks for sectioning.
Thin sections of the monolayer outgrowths and tissue blocks were cut, stained
with uranyl acetate and lead citrate (Venable and Coggeshall, ’65) examined and
photographed in a Hitachi HS-8 electron
microscope.
OBSERVATIONS
The cells of the monolayers derived from
the explanted adrenal capsule were similar
to those previously described in monolayers
in testis cultures (Bressler and Ross, ’73).
Most of the cells were spindle shaped (fig.
1) and were arranged in a radial manner
with respect to the explant. As in testis culture monolayers, these cells displayed a
myoid appearance in that parallel arrays
of 40-60 A filaments were found in the
peripheral cytoplasm. Dense bodies similar
to those of smooth muscle cells were associated with the filaments (figs. 1, 2).
The capsules of the adrenal glands of
the rat and mouse are basically of similar
structure. Both are composed of layers of
elongated cells and intervening collagen
fibers. Adjacent cells come into close opposition to each other but their parallel
plasma membranes usually are separated
by a narrow electron lucent space. Most
of the cells are surrounded by a moderately electron dense layer that resembles
the basement lamina of epithelial cells.
The cells of the capsule contain varying
amounts of 40-60 A filaments in their peripheral cytoplasm and consequently exhibit a greater or lesser electron density.
The filaments appear to terminate at the
plasma membrane. Some capsule cells exhibit periodic dense regions along the
plasma membrane, but pronounced dense
bodies comparable to those seen in monolayer cells are not frequently found (figs.
3 , 4). Occasionally typical smooth muscle
cells were observed. These cells were usually arranged side by side so as to produce
a strip of smooth muscle tissue. On the
basis of this arrangement they were considered to be the muscle cells of a tangentially sectioned arteriole.
DISCUSSION
Numerous investigators have studied
the ultrastructure of the adrenal cortex
(reviewed by Luse, ’67) but little attention
has been directed to the capsule. This is
surprising in view of the importance
ascribed to the capsule by some authors
for adrenal regeneration (reviewed by
Skelton, ’59). Contrary to earlier reports
that the capsule is composed of fibroblasts
(Brenner, ’66; Fujita, ’72; Kahri, ’66;
Mackay, ’69; Nishikawa et al., ’63; Penney
et al., ’63; Yamori et al., ’61) the present
investigation demonstrates that cells in the
adrenal capsule of the adult rat and mouse
display certain ultras truc tural features
resembling those of smooth muscle cells.
In these respects they are similar to “myofibroblasts” or “myoid’ cells that have been
observed in a variety of situations and are
comparable in appearance to the cells resembling smooth muscle that have been
described in the adrenal capsule of the dog
MYOID CELLS IN ADRENAL CAPSULES AND CULTURES
(Bloodworth and Powers, '68). The functional significance of these myoid cells in
the adrenal capsule is not readily apparent.
The failure of previous investigators to
note the contractile features of these cells
may be due to the use of osmium tetroxide
as the only fixative. Microfilaments are not
evident in published electron micrographs
of adrenal capsule cells fixed in this way
except i n the case of the dog (Bloodworth
and Powers, '68). In a more recent investigation, employing glutaraldehyde fixation,
cells with microfilaments can be seen
adjacent to the zona glomerulosa but were
not noted as such by the authors (Nickerson et al., '69, fig. 15).
The role of the capsule in adrenal regeneration has not been studied with the electron microscope. I n his review article
Skelton ('59) recounts reports indicating
that adrenal regeneration is dependent on
the presence of the capsule and is influenced by sex hormones. The latter finding
is interesting in view of reports that myoid
cells in testis culture monolayers metabolize steroids (de Kretser et al., '71; Dufau
et al., '71) and are identical in appearance
(Bressler and Ross, '73) to the myoid
cells presently found i n adrenal culture
monolayers. It would be interesting to see
if myoid cells in adrenal capsules and
monolayers derived from them exhibit any
capacity lo synthesize steroids.
ACKNOWLEDGMENTS
This work is a n extension of studies
begun in collaboration with Dr. Michael H.
Ross. I would like to thank Dr. Ross for
his constructive comments concerning the
nature of this investigation.
LITERATURE CITED
Bloodworth. J. M. B., Jr., and K. L. Powers 1968
The ultrastructure of the normal dog adrenal.
J. Anat., 102: 457-476.
Brenner, R. M. 1966 Fine structure of adrenal
cortical cells in adult male rhesus monkeys.
Am. J. Anat., 119: 4 2 9 4 5 4 .
Bressler, R. S., and M. H. Ross 1972 Differentiation of peritubular myoid cells o€ the
testis: Effects of intratesticular implantation
of newborn mouse testes into normal and
hypophysectomized adults. Biol. Reprod., 6:
148-159.
527
1973 On the character o f the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis. Expt.
Cell Res., 78: 295-302.
de Kretser, D. M., K. J. Catt, M. L. Dufau and
B. Hudson 1971 Studies on rat testicular
cells in tissue culture. J. Reprod. Fert., 24:
3 11-3 18.
Dufau, M. L., D. M. de Kretser and B. Hudson
1971
Steroid metabolism by isolated rat
seminiferous tubules in tissue culture. Endocrinol., 88: 825-832.
Fujita, H. 1972 O n the fine structure of alteration of the adrenal cortex in hypophysectomized rats. 2. Zellforsch., 125: 4 8 0 4 9 6 .
Gabbiani, G., and G. Majno 1972 Dupuytren's
contracture: Fibroblast contraction? Am. J.
Pathol., 66: 131-138.
Kahri, A. 1966 Histochemical and electron
microscopic studies on the cells of the rat
adrenal cortex in tissue culture. Acta Endocrinol., 52: Suppl. 108.
Karrer, H. E. 1960 Electron microscope study
of developing chick embryo aorta. J. Ultrastruct.
Res., 4: 420-454.
Luse, S. 1967 Fine structure of the adrenal
cortex. In: The Adrenal Cortex. A. B. Eisenstein, ed. Little, Brown and Co., Boston Massachusetts, pp. 1-60.
Mackay, A. 1969 Atlas of human adrenal
ultrastructure. In: Functional Pathology o f the
Human Adrenal Gland. T. Symington, ed.
E. and S. Livingston Ltd., Edinburgh and
London, pp. 345-384.
Majno, G., G. Gabbiani, B. J. Hirschel, G . B.
Ryan and P. R. Statkov 1971 Contraction of
granulation tissue in vitro: Similarity to smooth
muscle. Science, 173: 548-550.
Nickerson, P. A., A. C. Brownie and F. R. Skelton
1969 A n electron microscopic study o f the
regenerating adrenal gland during development
of adrenal regeneration hypertension. Am. J.
Pathol., 57: 335-364.
Nishikawa, M., I. Murone and T. Sato 1963
Electron microscopic investigations of the
adrenal cortex. Endocrinol., 72: 197-209.
Penney, D. P., D. I. Patt and W. C. Dixon, Jr.
1963 The fine structure of regenerating
adrenocortical autotransplants. Anat. Rec., 146:
319-335.
Ross, M. H. 1967 The fine structure and development of the peritubular contractile cell
component i n the seminiferous tubules of the
mouse. Am. J. Anat., 121: 523-558.
Skelton, F. R.
1959 Adrenal regeneration
and adrenal-regeneration hypertension. Physiol.
Revs., 39: 162-182.
Venable, J. H., and R. Coggeshall 1965 A
simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408.
Yamori, T., S. Matsuura and S. Sakamoto 1961
A n electron microscopic study of the normal
and stimulated adrenal cortex in the rat.
Z. Zellforsch., 55: 179-199.
PLATE 1
EXPLANATION O F FIGURES
1 Low power electron micrograph of a section parallel to and just
within the contact surface of cells in a portion of the monolayer outgrowth derived from a fragment of enucleated adrenal capsule taken
from a ten day old mouse and after 11 days in culture. Parallel
arrays of filaments and associated dense bodies are found in the
peripheral cytoplasm (arrowheads ). Membrane bound bodies, autophagic in appearance, are abundant in the cells (A). The area i n
the upper right corner of this micrograph is shown at higher magnification in figure 2 . X 3,900.
2
528
Higher magnification of the area in the upper right corner of figure
1 illustrating the aggregates of filaments and associated dense bodies
usually found in the peripheral portions of the monolayer cells.
x 10,000.
MYOID CELLS IN ADRENAL CAPSULES AND CULTURES
PLATE 1
Robert S. Bressler
529
PLATE 2
EXPLANATION OF FIGURES
530
3
Electronmicrograph of a portion of the adrenal capsule of a normal
adult mouse. The cells are surrounded by a pronounced basement
lamina-like material ( B ) and usually come into close apposition to
each other. Parallel arrays of filaments (arrowheads) are found in the
cytoplasm. The ends of the filaments appear to be associated with
thickened regions of the cell membrane (arrow). X 36,000.
4
Electronmicrograph of cells in the adrenal capsule of an adult rat.
An abundance of filaments are found in the peripheral cytoplasm
(arrowheads) and the cells are surrounded by a conspicuous layer
of basement lamina-like material. The microfilaments of the portion
of the myoid cell at the lower arrowhead are shown at higher
magnification in the inset at the lower lefthand corner. X 17,500.
Inset: X 32,500.
MYOID CELLS I N ADRENAL CAPSULES AND CULTURES
Robert S. Bressler
PLATE 2
531
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monolayer, myoid, gland, capsules, culture, adrenal, derived, cells
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