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The fine structure of the interstitial tissue of the testis of the nine-banded armadillo.

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The Fine Structure of the Interstitial Tissue of the
Testis of the Nine-banded Armadillo 1
Department of Anatomy, Louisiana State University Medical Center,
New Orleans, Louisiana 70119
The interstitial tissue of the testis of the nine-banded armadillo is
composed of blood vessels, clusters of Leydig cells, the usual connective tissue elements, and a network of lymphatic sinusoids. The endothelial walls of the sinusoids
are separated from the peritubular contractile cell layer surrounding the seminiferous tubules by a thin layer of collagen. The peritubular contractile cell is
characterized by filaments and dense bodies within the cytoplasm, whereas the endothelial cells lack these structures. Within each cluster, several Leydig cells surround one or more blood vessels. Adjacent Leydig cells are joined by 2- to 3-nm
wide gap junctions and desmosome-like specializations. The Leydig cell is polygonal in shape with an ovoid nucleus. The cell is characterized by an abundance of'
smooth endoplasmic reticulum which appears as sheets of membranes, concentric
whorls around vacuoles, and a random tubular network. Only a few short cisternae
of rough endoplasmic reticulum are observed. Centrioles are closely associated
with the Golgi apparatus. Rod-like mitochondria with tubular cristae are scattered
throughout the cytoplasm. In addition, the cells contain vacuoles resulting from
lipid extraction, filaments, microtubules, and glycogen. The surfaces of the cell exposed to the intercellular spaces exhibit numerous pinocytotic vesicles and cell
processes which indicate active movement of material across the plasma membrane. In comparison to other mammalian species, the ultrastructural organization
of the interstitium and the fine structure of the Leydig cell of the armadillo resemble those of the guinea pig.
T h e ultrastructure of the interstitial tissue of mammalian testis has been described in numerous animals (for example,
Christensen and Fawcett, '61, '66; Leeson,
'63; Christensen, '65, '70; Belt and Cavazos, '67; Sinha and Seal, '69; Ichiara, '70;
Fawcett et al., '73; Connell and Christensen, '75). However, the structure of the interstitium of the testis of the nine-banded
armadillo has not been studied. In recent
years, the armadillo has been introduced
as a model in leprosy research because of
its natural susceptibility to leprosy. In addition, since t h e a r m a d i l l o p r o d u c e s
monozygous quadruplets, it is possible to
replicate experiments with genetically
identical animals. This is relevant t o
leprosy research because the mechanism of
resistance of human beings to leprosy is assumed at this time t o be of a genetic origin.
AMT. REC.. 187: 11-28.
This necessitates the colonization of pure
strains of armadillos (Kirchheimer and
Storrs, '711; however, the armadillo rarely
reproduces in captivity. Only a few investigations regarding the structure and function of the testis of the animal are noted in
the literature (Nagy and Edmonds, '73a;b;
Weaker, '75). This investigation on the ultrastructure of the interstitial tissue is part
of a study to increase our understanding of
the male reproductive system.
TesteS used in this study were obtained
f r o m a d u l t n i n e - b a n d e d armadillos,
Received May 11, '76. Accepted July 15, '76.
1 This research was supported in part by USPHS Grant
2 Current address: Department of Anatomy, The University of Texas Health Sciencc Center, San Antonio, Texas
Dasypm novemcinctus, which were captured in the wild in southern Louisiana.
The testes were fixed by whole body vascular perfusion with a mixture of 2%
paraformaldehyde and 4% glutaraldehyde
in 0.1 M cacodylate buffer (pH 7.2).Tissues
were rinsed in 0.1 M cacodylate buffer (pH
7.2) containing 0.2 M sucrose and were
placed in 1% osmium tetroxide in 0.1 M
cacodylate buffer (pH 7.2). Following fixation, tissues were stained overnight en
bloc with aqueous 0.5% uranyl acetate, dehydrated in alcohol, and embedded in
Epon-Araldite (Mollenhauer, '641. One
micron plastic sections, cut for light microscopy, were stained with toluidine blue.
Light micrographs were taken with a Leitz
Dialux optical microscope. Tissues for
electron microscopy were sectioned on a
Reichert OM-U2 ultramicrotome. Sections
were mounted on uncoated grids and
stained with lead citrate (Reynolds, '63).
Electron micrographs were taken with a
Philips EM 300 electron microscope.
The testis of the nine-banded armadillo
is composed of numerous tightly packed
seminiferous tubules approximately 200 pm
in diameter, separated by angular intertubular spaces (@. 1) containing the interstitial tissue which is composed of a
labyrinthine network of lymphatic sinusoids, blood vessels, fibrocytes, Leydig cells,
and a sparsity of connective tissue matrix
(figs. 1, 2). The lymphatic sinusoids, which
conform in shape to the contour of the intertubular spaces, are large irregular
cavities lined with endothelium. The walls
of the sinusoids are continuous and are
formed by the overlapping of attenuated
endothelial cell processes (figs. 3, 4). A
basal lamina and a smooth muscular wall
are lacking (&s. 3,4);junctional specializations between adjacent cell processes are
rarely observed. Thus sinusoids are easily
distinguished from the blood vessels after
perfusion because the lumen of the sinusoids contains a homogeneous floccular preof
lvmnh DrOteinS. whereas the
- r
blood vessels are generally devoid of cells
or coagulated plasma proteins (fig. 7 ) .
The walls of the sinusoids are closely
associated with the peritubular contractile
cell layer surrounding the seminiferous
epithelium and are separated from the
contractile cell layer by only a thin layer of
collagen (fig. 3 ) . The peritubular contractile cell, like the endothelid cell, is a flattened cell which contains an elongated
nucleus (fig. 4). Unlike the endothelial cell,
the peritubular contractile cell is surrounded by a basal lamina (fig. 3 ) .The nucleus is characterized by dense condensations of chromatin along the nuclear
membrane and small eccentrically placed
nucleolus. The organelles are generally concentrated in the perinuclear area (&. 4).
The cell contains a small Golgi apparatus
composed of a parallel stack of saccules
with a few associated vesicles. Short cisternae of rough endoplasmic reticulum are
scattered in the cytoplasm of the perinuclear region and often appear dilated. The
mitochondria are small and dense with
tubular cristae (fig.5).The most characteristic features of the cell are the numerous
dense areas adjacent to the plasma membrane and the abundance of fine filaments
which a r e oriented parallel t o t h e
plasmalemma (fig. 4). Other than the fine
filaments and an occasional mitochondrion,
the processes of the peritubular contractile
cell are free of organelles. Frequently, the
overlapping processes are joined b y desmosome-like junctions. In addition, the surfaces of the processes demonstrate numerous pinocytotic vesicles (fig. 6). On the
other hand, the processes of the endothelial cell are rarely connected by intercellular junctions (fig.41, and these structures
are devoid of filaments and dense bodies
(figs. 3, 4). Additionally, the endothelial
cell processes are attenuated and are characteristically smaller in diameter than the
processes of the peritubular contractile
cell (fig. 3). Frequently, pinocytotic vesicles are observed on the surfaces of the
Drocesses, while larger cytoplasmic vesi;hes are noted within the process (fig. 3).
The Leydig cells are organized into cell
clusters which are located either between
the walls of adjacent lymphatic sinusoids or
between the sinusoids and the seminiferous tubules. The cells surround one or
more blood vessels, either venules or nonfenestrated capillaries, depending on the
number of cells within the cluster (figs. 2,
7). The clusters of Leydig cells are usually
surrounded by a sparse network of collagen fibers and fibrocytes which separate
the cell clusters from the walls of the sinusoids (fig. 7 ) ;however, frequently no connective tissue is observed between the
clusters and the sinusoids (fig. 12).A floccular precipitate, similar to that contained in
the lymphatics, is observed (fig.12) in the
intercellular space between Leydig cells
and the spaces separating the cells from
either the blood vessels or the sinusoids
(fig.7 ) .Adjacent Leydig cells are joined by
two surface specializations. One is a 2- to 3nm wide gap junction, which is randomly
distributed along the plasma membrane
and is often associated with relatively large
projections from one cell which adjoins the
body of another cell (fig. 8).The other type
resembles a desmosome and is characterized by a dense amorphous layer closely
applied to the cytoplasmic side of the
membrane but lacks tonofilaments within
this dense matrix. This specialization is also
randomly distributed on the cell surface.
The cells are generally separated by a 20nm intercellular space; however, dilations
of the intercellular space are noted at
various points between two adjacent cells
(fig. 10). In addition, angular spaces are observed among three or more adjoining
cells. The surfaces of the cell exposed to
these intercellular spaces exhibit numerous
pinocytotic vesicles and thin cell processes
or fdopodia (fig.11).
The Leydig cells are generally polyhedral in shape and contain an ovoid nucleus with large amounts of heterochromatin adjacent to the nuclear membrane
(figs.7, 12).A pair of centrioles is generally
closely associated with a well-developed
juxtanuclear Golgi apparatus (figs. 12, 13).
The cytoplasm also contains rod-like mitochondria with tubular cristae which are
embedded in a dense matrix (figs. 16, 17).
Only a few short cisternae of rough endoplasmic reticulum are observed. These
cisternae, which are located both adjacent
to the nucleus and scattered in the peripheral cytoplasm, freely anastomose with the
smooth endoplasmic reticulum (fig. 17).
The most characteristic feature of the
cell is an abundance of smooth endoplasmic reticulum which is unusually
well-developed in the armadillo and appears variable in form. This membranous
organelle is observed as isolated mass of
membranes in one portion of the cell (figs.
7, 121, concentric rings around lipid
vacuoles, (fig. 15) and a random tubular
network in other areas of the cell (fig. 16).
The mass of membranes appear to be composed of a highly organized, integrated
system of branched, anastomosing tubules
(fig. 14).The center of this tubular network
is devoid of other organelles (fig. 7, 12). At
the periphery, the smooth endoplasmic reticulum becomes less organized and either
becomes confluent with the random tubular network in the remainder of the cell or
forms concentric lamella around lipid
vacuoles. The lamellated layers, which are
composed of fenestrated cisternae, appear
to be further encircled by a discontinuous
layer of mitochondria (fig. 15).
In addition to being encircled by smooth
endoplasmic reticulum, the lipid vacuoles
are associated with spherical, dense granular structures which are probably lysosoma1in nature, as indicated by the myelin
figures located within the spheres (figs. 16,
17). These myelinated bodies are also observed as separate entities in the cytoplasm
and presumably form residual pigment
bodies (fig. 7 ) .
The cell also contains numerous filaments, as well as glycogen, coated vesicles,
and microtubules. The filaments are scattered randomly, either singly or in bundles,
in the cytoplasm (fig. 17).No structural relationships are observed between the filaments and other organelles. Coated vesi-
cles are noted budding off the plasma
membrane, scattered in the peripheral cytoplasm (fig. 161, and adjacent to the Golgi
apparatus (fig. 13). The microtubules are
sparse and are observed singly, never in
bundles as the filaments.
In addition to Leydig cells, several other
cell types are noted in the interstitium such
as plasma cells, macrophages, lymphocytes
and fibrocytes (not shown). However, in
general, the connective tissue is very scanty with minimal connective tissue stroma.
and relatively few Leydig cells as compared to the interstitium of the zebra, boar,
and mole rat (Fawcett et al., '73). The
Leydig cells are organized into cell clusters
which surround one or more blood vessels,
varying on the number of cells within the
cluster. The cell clusters are located between thc walls of the sinusoids and the
seminiferous tubules and are partially surrounded by the cytoplasmic processes of
either fibrocytes or endothelial cells of the
sinusoid. As in the guinea pig, the endothelid walls of the sinusoids are continuous.
The Leydig cells of the armadillo conSince there have been no previous tain the various organelles observed in this
studies on the morphology of the in- cell type in other mammals (Christensen,
terstitium of the testis of the armadillo, this '75). The most striking feature of the
investigation was undertaken in order to Leydig cell in the armadillo is the regular
describe the ultrastructure of the tissue arrangerncnt of smooth endoplasmic reticfrom animals living in their natural habitat. ulum into large masses which occupy a maUsing this study as a guideline, further in- jor portion of the cytoplasm in one area of
vestigations will be conducted in order to the cell. These masses are composed of a
determine if any morphological changes highly organized system of anastomosing
occur in the testis of armadillos maintained tubules that are interconnected to a more
in captivity for lengthy time periods, a con- random network of tubules in the cytodition necessary in leprosy research.
plasm and t o concentric lamellae of
According to a comparative study of fenestrated cisternae which surround lipid
mammalian testes, the interstitial tissue can droplets. Although the smooth endoplasbe classified into three categories depend- mic reticulum is recognized as the site of
ing on the size, number, and organization of testosterone synthesis, the functional sigLeydig cells, lymphatic vessels, and ground nificance of the complex organization of
substance (Fawcett et a]., '73). The in- this organelle in the armadillo has not been
terstitium of the first category contains rel- determined. Christensen and Gillim ('69)
atively few Leydig cells, negligible ground indicate t h e amount of smooth ensubstance, and an extensive system of doplasmic reticulum is related to the
lymphatic sinusoids. The second category amount of cholesterol that is produced by
is characterized by clusters of Leydig cells the cell, rather than taken up from the
scattered in an abundance of edematous plasma. Perhaps, the Leydig cells in the arconnective tissue which is drained by madillo must synthesize large amounts of
various size lymphatic vessels that are cen- cholesterol for steroid biosynthesis. If this
trally or eccentrically located in the inter- assumption is correct, the orderly arrangetubular spaces. The third category is ment of tubules in the armadillo offers a
characterized by numerous Leydig cells large surface area of membranes for adecompacted into an enlarged intertubular quate synthesis of cholesterol, as well as
space. Using this classification, the ar- steroids.
madillo belongs to the first category that
The plasma membrane of the Leydig
includes guinea pig, chinchilla, rat, and cells demonstrates numerous pinocytotic
mouse. The intertubular spaces contain an vesicles, coated as well as non-coated,
extensive network of large lymphatic sinus- which are associated with slender cell
oids, very little connective tissue stroma, processes. This indicates that the cells are
actively engulfing material contained in
the intercellular spaces. Dilations of the intercellular spaces are frequently observed
between adjacent Leydig cells. These
spaces, which contain interdigitating processes of neighboring cells, may represent a
means of circulating the interstitial fluid
among the cells and channeling secretory
products to the blood vascular and
lymphatic systems.
Two types of junctional specializations
are noted in the armadillo. The more frequently observed specialization is the 2- to
3-nm wide gap junction, whereas the other
resembles desmosomes but lacks tonofilaments. Gap junctions have been observed
in a variety of somatic cell types (Staehelin,
'74) and have been noted in Leydig cells of
the dog (Connell and Christensen, '75).
These junctions ionically and metabolically
couple adjacent cells (Staehelin, '74).
Sheridan ('71) suggests that in sparsely innervated tissues such as adipose tissue, low
molecular weight molecules may pass
through the gap junction to insure a
uniform response to circulating hormones.
Such a function may also exist in the testis
which would insure a uniform response of
Leydig cells within the cluster to the circulating interstitial cell stimulating hormone. The desmosome-like junction has
also been observed in Leydig cells of othe r species (Christensen, '65; Belt and
Cavazos, '71). These juctions appear to be
supportive in nature, and together with the
gap junction, they may render structural
stability to the Leydig cell cluster.
Filaments are present in the Leydig cells
of several mammalian species such as rats
(Lesson, '63; Belt and Cavazos, '671, squirrel monkeys (Belt and Cavazos, '711, and
dogs (Connell and Christensen, '75). Although the function of filaments in the
Leydig cell is not known, Connell and
Christensen ('75) suggested that in the dog
the filaments are involved in the movement
of lipid and other steroid precursors to the
mitochondria and smooth endoplasmic reticulum. In the armadillo, there appears to
be no functional relationships between the
filaments and other cellular components as
observed in the dog. It is suggested that in
the armadillo, the filaments may render
structural stability to the cell.
Since the armadillo is a seasonal breeder,
attempts were made to detect seasonal
regressions and fluctuations of the interstitial tissue. Preliminary observations
indicate that there are no major changes in
the morphology of the interstitial tissue;
however, a more thorough investigation is
currently in progress.
Belt, W. D., and L. F. Cavazos 1967 Fine structure of
the interstitial cells of Leydig in the hoar. Anat.
Rec., 1,58:333-350.
1971 Fine structure of the interstitial cells of
Leydig in the squirrel monkey during seasonal
regression. Anat. Rec., 169: 115-128.
Christensen, A. K. 1965 The fine structure of testicular cell in the guinea pig. J. Cell Biol., 26: 911-935.
1970 Fine structure of testicular interstitial
cells in humans. Adv. Exp. Med. Biol., 10: 75-89.
1975 Leydig cells. In: Handbook of Physiology. Vol. V, Endocrinology. Section 7. D. W.
Hamilton and R. Greep, eds., American Physiological Society, Washington, D.C.. pp. 57-94.
Christensen, A. K., and D. W. Fawcett 1961 The normal fine structure of opossum testicular interstitial
cell. J. Biophysic. Biochem. Cyt., 9: 653-670.
1966 The fine structure of testicular interstitial cells in mice. Am. J. Anat., 118: 551-572.
Christensen, A. K., and S. Gillim 1969 The correlation of fine structure and function in steroid-secreting cells with emphasis on those of the gonads. In:
The Gonads. K. W. McKerns, ed. Appelton-Century-Crofts, New York, pp. 415-488.
Connell, C. J., and A. K. Christensen 1975 The ultrastructure of the canine testicular interstitial tissue.
Biol. Reprod., 12; 368-382.
Fawcett, D. W., W. B. Neaves and M. N. Flores 1973
Comparative observations on intertuhlar lymphatics and the organization of the interstitial tissue of
the mammalian testis. Biol. Reprod., 9: 500-532.
Ichihara, I. 1970 The fine structure of testicular interstitial cells of mice during postnatai development. Z. Zellforsch., 108: 475-486.
Kirchheimer, W., and E. Storrs 1971 Attempts to establish armadillo fDasyp2csnocerncinctus linn.1as a
model for the study of leprosy. Int. J. Leprosy, 39:
Leeson, C. R. 1963 Observations on the fine structure of rat interstitial tissue. Acta Anat., 52: 34-48.
Mollenhauer, D. 1964 Plastic embedding mixtures
for use in electron microscopy. Stain Technol., 39:
Nagy, F., and R. Ednionds 1973a Morphology of the
reproductive system of the armadillo, the spermatogonia. J. Morph., 140: 307-320.
-1973b Some observations on the fine structure of the armadillo spermatozoa. J. Reprod. Fert.,
34: 551-553.
Reynolds, F. 1963 The use of lead citrate at high pH
as an electron opaque stain in the electron microscopy. J. Cell Biol., 17: 208-213.
Sheridan, J. D. 1971 Electrical coupling between fat
cells in the new fat body and mouse brown fat. J.
Cell Biol., 50: 795-803.
Sinha, A., and U. Seal 1969 The testicular interstitial
cells of a lion and a three-toed sloth. Anat. Rec.,
164: 35-46.
Staehelin, L. A. 1974 Structure and function of intercellular junctions. Int. Fi-v. Cyt., 39: 191-283.
Weaker, F. J. 1975 Spermiogenesisin the nine-banded armadillo. In: Electron Microscopic Concepts of
Secretion. M. Hess, ed. John Wiley & Sons, New
York, pp. 169-188.
BL, Basal lamina
C , Centriole
CAP, Capillary
D, Dilation
EC, Fmdothelial
F, Filament
FA, Filopodia
FC, Fibrocyte
G, Golgi apparatus
LC, Leydig cell
M, Mitochondria
P, Pigment body
PC, Peritubular
RER, Rough endoplasmic
S, Sinrisoid
SE, Seminiferous epithelium
SEX, Smooth endoplasmic
ST, Seminiferous tubule
VA, Lipid vacuole
1 A light-micrograph of the testis demonstrating blood vessels (arrows),
and Leydig cell clusters (LC) in the angular
lymphatic sinusoids (3,
spaces between the seminiferous tubules. Note the sparsity of connective
tissue. x 250.
2 A light micrograph of a large Leydig cell cluster containing blood vessels
(arrows) located between three seminiferous tubules. x 1,000.
Frank J. Weaker
3 The limiting membrane of a seminiferous tubiile (ST) composed of
peritubular contractile cells (PC) and three layers of collagen (X,Y, ZI,
basal lamina of seminiferous epithelium (BL),basal lamnia of contractile
cells (arrows),endothelial wall (EC) of lymphatic sinusoid. The sinusoid
in this electron micrograph does not contain a floccular precipitate hccause the tissue was fixed by immersion. x 14,000.
4 The nuclear region of a peritubular contractile cell (PQ. Endothelial cell
(arrow), seminiferous epithelium (SE), lymphatic sinusoid (3.
x 11,900.
5 A high magnification of the perinuclear area demonstrating Golgi apparatus (GI, mitochondria (MI, rough mdoplasmic reticulum (RER),
filaments (F),dense areas (arrows). x 29,800.
6 An electron micrograph demonstrating junctions (arrows) between contractile cells. Note pinocytotic vesicles. x 33,900.
Frank J. Weaker
7 An electron micrograph of a Leydig cell cluster containing a capillary
(CAP). The cell cluster is bordered by processes of fibrocytes (FC).Note
the floccular precipitate in sinusoid (S) and the intercellular spaces; mllagen (C). The Leydig cells contain a large mass of smooth endoplasmic
reticulum (SEX),mitochondria (MI, centrioles (arrow), Golgi apparatus
(GI. x 8,800.
Frank J. Weaker
8 A gap junction found between the process of one Leydig cell and the
cell body of another. x 53,300.
9 A second type of junction found between adjacent cells which resembles a desmosome without filaments. x 139,700.
10 The intercellular space between Leydig cell is approximately 20 nm
with dilations of space at various points between the cells (D).Note the
gap junctions (arrows) adjoining the cells. x 19,200.
11 An electron micrograph demonstrating pinocytotic activity (arrows) and
filopodia (FA)at the surface of a Leydig cell. x 28,200.
12 A typical Leydig cell containing a large mass of smooth endoplasmic reticulum (SEX), Golgi apparatus (GI, centriole (arrow).Note the abscnce
of connective tissue in the space between the Leydig cell and the wall
of the lymphatic sinusoid (S). x 8,800.
Frank J. Weaker
13 A pair of centrioles (C) and Golgi apparatus (GI adjacent to the nucleus
of a Leydig cell. Coated vesicles (arrows) are frequently noted adjacent
to the Golgi. Mitochondria (M), pigment bodies (PI. x 22,300.
14 The smooth endoplasmic reticulum is composed of a highly organized,
complex system of anastomosing tubules (arrows). x 31,300.
Frank J. Weaker
15 A lipid vacuole (VA) surrounded by whorls of smooth endoplasmic reticulum (SER) and mitochondria (M).
x 18,200.
16 A high magnification of the cytoplasm of a Leydig cell containing a random tubular network of smooth endoplasmic reticulum (SER), rough
endoplasmic reticulum (RER), coated vesicles (arrows), mitochondria
with tubular cristae (M), and myelin figures (MY) associated with
vacuoles, and vacuole (VA). x 29,800.
Frank J. Weaker
Frank J. W-eakcr
17 A high magnification of' a Leydig cell demonstrating tubular cristae of
mitochondria (M),
rough endoplasmic reticulum (RER), filaments (F),
glycogen (arrow), myelin figure
x 43,900.
(MY)associated with a vacuole IVA).
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bander, structure, interstitial, testis, armadillo, nine, tissue, fine
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