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The testicular interstitial cells of a lion and a three-toed sloth.

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The Testicular Interstitial Cells of
a Lion and a Three-toed Sloth'
AKHOURI A. SINHA AND ULYSSES S. SEAL
Metabolic Research Laboratory, Veterans Administration Hospital, and
Department of Biochemistry, University of Minnesota, Minneapolis,
Minnesota
ABSTRACT
The fine structure of testicular interstitial cells of lion and three-toed
sloth showed an abundance of agranular endoplasmic reticulum, mitochondria and
lipid droplets. Mitochondria were usually small and rod to rounded in shape, but large
bizarre forms, such as triangular, oval and rounded with tubular and lamellar cristae
were also observed. Mitochondria with lamellar cristae occurred in lion, but not in
three-toed sloth. These cristae were highly developed and appeared unique for the
testicular interstitial cells. Both small and large mitochondria closely adhered to lipid
droplets which were found singly or in clusters. The stored lipid droplets probably
serve as a major source of substrate in androgen biosynthesis. The possible role of
agranular endoplasmic reticulum in androgen biosynthesis is suggested.
MATERIALS AND METHODS
The fine structure of testicular interstitial cells has been described in the oposThe lion, Panthera leo L, was sacrificed
sum (Christensen and Fawcett, '61), the on December 7, 1967 because of deterioratguinea pig (Christensen, '65), the mouse ing health and the three-toed sloth, Brady(Christensen and Fawcett, '66), the rat pus tridactylus L, on November 4, 1967.
(Leeson, '63; Murakami, '66), the rabbit Testes from these animals were sliced into
(Crabo, '63) and man (Fawcett and Bur- small pieces of about 1 mm in thickness
gos, '56, '60; deKretser, '67). The cyto- and fixed in cold 3% glutarldehyde in 0.1
plasm of these cells contained varying M phosphate buffer at pH 7.3 for two to
amounts of agranular endoplasmic retic- three hours. Three-toed sloth testis was also
ulum, mitochondria with tubular cristae, fixed in cold Karnovsky's ('65) iixative for
and osmiophilc lipid droplets. Christensen
and Fawcett ('66) have summarized the two hours. They were washed four to six
evidence which indicates that the abundant times in cold 0.1 M phosphate buffer, then
agranular endoplasmic reticulum of these post-fixed in 2% osmium tetroxide in phoscells is the site of most of the enzymes phate buffer for one and one-half hours.
that mediate the synthesis of testosterone Lion's testis was also fixed in 2% osmium
from acetate. The morphology of inter- tetroxide in cacodylate buffer for three
stitial cells as seen with light microscope hours. Following dehydration in graded
cold ethyl alcohol, tissues were embedded
was surveyed by Rasmussen ('32).
in
Epon 812 (Luft, '61) and sectioned
Wislocki ('28) said that the testicular
interstitial cells were more abundant in with an LKB ultratome 111. Thick sections
two-toed than three-toed sloths. These cells from these blocks embedded for electron
were large, polygonal and contained yellow- microscopy were stained with toluidine
ish pigment in their cytoplasm. Apparently blue or azure B and examined with a
nothing has been described on the testic- phase contrast microscope for orientation.
Thin sections were stained with the lead
ular interstitial cells of lion.
The purpose of this study is to report citrate method of Reynolds ('63) and also
the ultrastructure of interstitial cells of in uranyl acetate, and examined in an
lion and three-toed sloth testes and to comReceived June 27, '68. Accepted Dec. 20. '68.
pare the results with those of previously
1This research was supported by USPHS grant
described species.
AM CA 11376-07.
ANAT.REC.,164: 35-46.
35
36
AKHOURI A. SINHA AND ULYSSES S . SEAL
RCA EMU-3G electron microscope. Unstained sections were also examined.
OBSERVATIONS
The Zion. This approximately 14-yearold male lion had an excellent breeding
record at the zoo since his arrival on
August 30, 1966. He bred a lioness who
had stillborn cubs on December 9, 1966
(first pregnancy), and subsequent live cubs
(two cubs on June 14, 1967, three
cubs on October 9, 1967, and four cubs
on January 28, 1968). Asdell ('64) said
that the gestation period in the lion varies
between 105 and 113 days. Our observation supports earlier reports regarding the
year-around breeding season in lion (Asdell, '64).
The interstitial cells found between the
seminiferous tubules of testis were large,
polygonal in shape and often occurred in
clumps. The nucleus was usually oval but
varied in shape, and it frequently contained a nucleolus. Occasionally binucleate
interstitial cells were observed. The cytoplasm showed abundant agranular endoplasmic reticulum, mitochondria and lipid
droplets (fig. 1).
The agranular endoplasmic reticulum
was dilated into isolated vesicles of various
sizes (cf. fig. 16; Christensen, '65) (fig. 1 ) .
Apparently such vesicles were due to inadequate preservation of the agranular
membranes as was suggested for the
guinea pig (Christensen, '65). They were
found throughout the cytoplasm but were
relatively sparse in the area of other organelles and lipid droplets. In favorable
sections some of these vesicles were interconnected. Occasionally agranular endoplasmic reticulum paralleled the surf ace of
a mitochondrion much as in the interstitial
cells of mouse (Christensen and Fawcett,
'66). Rough or granular endoplasmic reticulum was sparse but free ribosomes were
distributed randomly in the cytoplasm.
Two types of mitochondria were observed in the interstitial cells lion; namely,
the most common small rounded to rod
shaped and the unusually large bizarre
shaped. The size of small mitochondria
vaned and their cristae were tubular and
of variable length (figs. 1, 3 ) . The large
mitochondria were triangular. oval and
round in shape. These mitochondria mea-
sured 2 p or more in diameter (figs. 2 ,
3). The large mitochondrion had either
lamellar or tubular cristae but occasionally
both types were observed in a single organelle (fig. 2 ) . The lamellar cristae frequently extended nearly all the way across
the organelle (fig. 2 ) . Sometimes these
cristae were arranged at right angles in a
mitochondrion (fig. 3). In favorable sections the lamellar cristae of a large mitochondrion appeared amorphous in tangential section and in parallel arrays in
longitudinal plane (fig. 2 ) . The tubular
cristae in these mitochondria were variously arranged (fig. 3). In both small
and large mitochondria the matrix was
more electron dense than the ground
substance of the cytoplasm. Intramitochondrial dense granules were more frequently observed in the matrix of large
mitochondria than the small ones. Their
size and number varied in each organelle
(figs. 1, 2 , 3). Similar observations have
been made for the intramitochondrial granules of the mouse (Christensen and Fawcett, '66), but their possible function remains obscure.
Both small and large mitochondria were
found to adhere closely to the lipid droplets (figs. 1, 3). Occasionally only the
large mitochondria partially or completely
encircled a lipid droplet. Similar observations have been made in the interstitial
cells of man (Fawcett and Burgos, '56,
'60) and rabbit (Crabo, '63). Since the
lipid droplets in the interstitial cells appeared moderately osmiophilic, they probably contained a mixture of saturated and
unsaturated fats much as in other steroidsecreting cells (Deane, '58; Baillie, '64).
These droplets varied in size and were
found singly or in clusters.
The Golgi complex consisted of several
closely packed cisternae and associated
small vesicles. Its location in the cytoplasm
varied from the juxta-nuclear position to
near the surface of interstitial cells. The
Golgi complex in the interstitial cells of
the lion was essentially like that described
for the guinea pig, mouse and man. Occasionally pigment granules and microtubules were observed in the cytoplasm
(fig. 3 ) . The microtubules appeared singly
and randomly much as in the guinea pig
(Christensen, '65). Crystals of Reinke and
INTERSTITIAL CELLS OF LION AND THREE-TOED SLOTH
membranous whorls of agranular endoplasmic reticulum were not observed in
these cells.
The three-toed sloth. Our observation
supports Wislocki's ('28) description of the
interstitial cells in the three-toed sloth. In
fine structure these cells showed a large
number of lipid droplets which were closely
associated with mitochondria and agranular endoplasmic reticulum (fig. 4). Most
of the mitochondria were rod-shaped and
their cristae were tubular and of variable
length. Mitochondria with lamellar cristae
were not observed in this animal. Some
mitochondria were also oval, branched
and rounded, but unusually large mitochondria were not observed in these
cells. Intramitochondria dense granules of
various sizes occurred in the mitochondria1
matrix (fig. 4). The agranular endoplasmic
reticulum was dilated into isolated vesicles
like that of the lion. Occasionally a membranous whorl of agranular endoplasmic
reticulum was observed (fig. 4). The Golgi
complex, granular endoplasmic reticulum,
and pigment granules were essentially like
those of the lion.
DISCUSSION
The present report is limited to material
from one lion and one three-toed sloth.
The condition of the seminiferous tubules,
epididymis and the fine structure of the
testicular interstitial cells indicated that
these animals were reproductively active.
These animals have been reported to breed
throughout the year (Asdell, '64; Wislocki,
'27). The reproductive history of this lion
supports the above observation.
In general, the testicular interstitial cells
of the lion and three-toed sloth were comparable to the interstitial cells of the rabbit
(Crabo, '63), man (Fawcett and Burgos,
'56, '60; deKretser, '67), and rat (Leeson,
'63; Murakami, '66). The so-called "dark
cells as reported in rabbit, rat and man
were not observed in the lion and threetoed sloth.
The interstitial cells of mammals are considered to be the main source of androgen
(Rasmussen, '32;Wattenberg, '58; Deane
and Rubin, '65; Christensen and Mason,
'65). The interstitial cells of the lion and
three-toed sloth predominantly contained
37
agranular endoplasmic reticulum, a feature common with other animals studied
so far. In guinea pigs, rats and mice, the
abundant agranular endoplasmic reticulum of the testicular interstitial cells
plays an important role in androgen biosynthesis (Christensen, '65; Christensen
and Fawcett, '66). Our observation in the
lion and three-toed sloth belonging to two
different orders of mammals also shows
predominance of agranular endoplasmic
reticulum, which can be presumed to have
a role similar to that of the guinea pig,
rat and mouse.
The close association of mitochondria
with lipid droplets in the interstitial cells
of the lion and three-toed sloth suggests
an important role of this organelle in lipid
utilization much as in the rat testis (Toren,
Menon, Forchielli and Dorfman, '64) and
the cardiac muscle of the cat (Fawcett,
'66). Furthermore, the abundance of lipid
droplets in the interstitial cells of the lion
and three-toed sloth indicates that these
are their storage products and probably
the major source of substrate in androgen
biosynthesis. On the contrary, Christensen
and Fawcett ('66) have suggested that the
agranular endoplasmic reticulum of interstitial cells may also serve as a reservoir for
the storage of cholesterol in the mouse and
some other animals. However, these two
storage sites are not mutually exclusive,
and both could be important in the same
cell.
The interstitial cells of the lion contained
both small and large mitochondria but the
latter were densely packed with lamellar
or tubular cristae. Mitochondria with tubular, vesicular and lamellar cristae have
been described in a variety of steroid-secreting cells (Belt and Pease, '56; Fawcett
and Burgos, '56; Enders, '62; Crabo, '63;
Sheridan and Belt, '64; Zelander, '64; Brenner, '66; Christensen and Fawcett, '66;
Long and Jones, '66). But very large mitochondria with highly developed lamellar
cristae, as reported in the lion (fig. 2),
have not been described in the testicular
interstitial cells of other species studied so
far. Such large mitochondria usually show
tubular cristae in other species. The significance of these modifications of the cristae
in steroid-secreting cells is unknown.
38
AKHOURI A. SINHA AND ULYSSES S. SEAL
LITERATURE CITED
Asdell, S. A. 1964 Patterns of mammalian reproduction. 2nd Ed. Cornell Univ. Press, 425508.
Baillie, A. H. 1964 Further observations on the
growth and histochemistry of the Leydig tissue
i n the postnatal prepubertal mouse testis. J.
Anat., 98: 402-419.
Belt, W. D., and D. C. Pease 1956 Mitochondrial structure i n sites of steroid secretion. J.
Biophys. Biochem. Cytol., 2: ( No. 4, suppl.)
369-3 74.
Brenner, R. M. 1966 Fine structure of adrenal
cortical cells in adult male rhesus monkeys.
Am. J. Anat., 119: 429-454.
Christensen, A. K. 1965 The fine structure of
testicular interstitial cells in guinea pigs. J.
Cell. Biol., 26: 911-935.
Christensen, A. K., and D. W. Fawcett 1961
The normal fine structure of opossum testicular
interstitial cells. J. Biophys. Biochem. Cytol.,
9: 653-670.
1966 The fine structure of testicular
interstitial cells i n mice. Am. J. Anat., 118:
551-572.
Christensen, A. K., and N. R. Mason 1965 The
comparative ability of seminiferous tubules and
interstitial tissue of rat testes to synthesize
androgens from progesterone -4-I4C i n vitro.
Endocrinology, 76: 646-656.
Crabo, B. 1963 Fne structure of the interstitial
cells of the rabbit testes. Z. Zellforsch., 61:
58 7-604.
Deane, H. W. 1958 Intracellular lipides: Their
detection and significance. In Frontiers in Cytology (S. L. Palay, ed.), New Haven, Yale
Univ. Press, Chap. 8: 227-263.
Deane, H. W., and B. L. Rubin 1965 Identification and control of cells that synthsize steroid
hormones in the adrenal glands, gonads and
placentae of various mammalian species. Arch.
Anat. Microsc. et Morph. Exper., 54: 49-66.
deKretser, D. M. 1967 The fine structure of the
testicular interstitial cells in men of normal
androgenic status. Z. Zellforsch., 80: 594-609.
Enders, A. C. 1962 Observations on the fine
structure of lutein cells. J. Cell. Biol., 12: 101113.
Fawcett, D. W. 1966 A n Atlas of Fine structure. The Cell, its Organelles and Inclusions.
W. B. Saunders Co., Philadelphia and London,
1-448.
Fawcett, D. W., and M. H. Burgos 1956 Observations on the cytomorphosis of the germinal and interstitial cells of the human testis.
Ciba Foundation, Colloquia on Ageing: Ageing
in Transient Tissues, 2: 86-99.
___
1960 Studies on the fine structure of
the mammalian testis. I. The human interstitial tissue. Am. J. Anat., 107: 245-269.
Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in
electron microscopy. J. Cell. Biol., 27: 137A.
Leeson, C. R. 1963 Observations on the fine
structure of rat interstitial tissue. Acta Anat.,
52: 34-48.
Long, J. A., and A. L. Jones 1966 The fine
structure of the zona glomerulosa and the zona
fasciculata of the opossum. Anat. Rec., 154:
378-379.
Luft, J. H: 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.
Murakami, M. 1966 Electron microscopic studies on the interstitial tissue of rat testis with a
special reference to the Leydig interstitial cells.
Z. Zellforsch., 72: 139-156.
Rasmussen, A. T. 1932 Interstitial cells of the
testis. I n : Special Cytology (E. V. Cowdry, ed.),
Paul Hoeber, New York, 1675-1725.
Reynolds, E. S. 1963 The use of lead citrate
at high pH as a n electron-opaque stain i n electron microscopy. J. Cell. Biol., 17: 208-212.
Sheridan, M. N., and W. D. Belt 1964 Fine
structure of the guinea pig adrenal cortex.
Anat. Rec., 149: 73-98.
Toren, D., K. M. J. Menon, E. Forchielli and R. I.
Dorfman 1964 In vitro enzymatic cleavage
of the cholesterol side chain i n rat testis preparations. Steroids, 3: 381-390.
Wattenberg, L. W. 1958 Microscopic histochemical demonstration of steroid-3-p-ol-dehydrogenase in tissue sections. J. Histochem.
Cytochem., 6: 225-232.
Wislocki, G. B. 1927 On the placentation of
the rridactyl sloth. Contribs. Embryol. Carneg.
Inst. Wash., 19: 209-228.
1928 Observations on the gross and
microscopic anatomy of the sloths (Bradypus
griseus griseus, Gray and Cloloepus hoffmanni
Peters). J. Morph., 46: 317-398.
Zelander, T.
1964
Endocrine organs: The
adrenal gland. In Electron Microscopic Anatomy ( S . M. Kurtz, Ed.), Academic Press, New
York and London, Chap, 8: 199-220.
PLATES
PLATE 1
EXPLANATION O F FIGURE
1
40
Electron micrograph of portions of interstitial cells and a fibroblast
cell (Fbc) from lion testis. Note the agranular endoplasmic reticulum
vesicles (Aer), small mitochondria ( M ) with tubular cristae and
lipid droplets ( L ) which show some extraction. These cells are also
showing Golgi complex (G), nucleus ( N ) , ribosomes (R), filopodia
( F ) and poorly developed desmosomes ( D ) . X 21,500.
INTERSTITIAL CELLS OF LION AND THREE-TOED SLOTH
Akhouri A. Sinha and Ulysses S. Seal
PLATE 1
41
PLATE 2
EXPLANATION OF FIGURES
2
A portion of the interstitial cell of lion showing nucleus ( N ) and
unusually large mitochondria with lamellar cristae which extend
nearly all the way across the organelle. The lamellar cristae appear
in parallel arrays (P) i n longitudinal plane and amorphous ( A ) in
tangential section. The tubular cristae occur as tubes (T). Note the
tips of lamellar cristae (arrows) and continuity of matrix i n the
organelle. Also note the intramitochondrial dense granules. X 32,760.
3 An area from the interstitial cell of lion showing a mitochondrion (M)
in which most of the cristae extend at various lengths across the
diameter while others are arranged at right angles. Note the microtubules (arrow) and lipid droplets (L). X 29,200.
42
INTERSTITIAL CELLS OF LION AND THREE-TOED SLOTH
Akhouri A. Sinha and Ulysses S. Seal
PLATE 2
43
PLATE 3
EXPLANATION OF F I G U R E
4
44
Portions of two interstitial cells from three-toed sloth testis. Note the
agranular endoplasmic reticulum vesicles (Aer), lipid droplets ( L ) ,
variously shaped mitochondria ( M ) with their cristae and dense
granules, Golgi complex ( G ) , ribosomes (R), nucleus ( N ) , granular
endoplasmic reticulum (Ger), filopodia ( F ) agranular membranous
whorl (W), micropinocytotic vesicles (arrows) and pigment ( P ) . Also
note the collagen fibers ( C ) . X 16,200.
INTERSTITIAL CELLS OF LION AND THREE-TOED SLOTH
Akhouri A. Sinha and Ulysses S. Seal
PLATE 3
45
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