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Fine structure of the interstitial cells of leydig in the squirrel monkey during seasonal regression.

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Fine Structure of the Interstitial Cells of Leydig in the
Squirrel Monkey during Seasonal Regression '
W. D. BELT AND L. F. CAVAZQS
Department o f A n a t o m y , T u f t s University School o f Medicine,
Boston, Massachusetts 031 11
The fine structure of the interstitial cells of Leydig has been
examined in the squirrel monkey, Saimiri sciureus, during the non breeding portion of the year when the testes are essentially azoospermic. Interstitial cells are
present in abundance as a layer, several cells thick, on the inner aspect of the
tunica albuginea. Cytologically, the cells possess the characteristics usually associated with cells which synthesize and secrete steroid hormones: an abundant
agranular endoplasmic reticulum in the form of anastomotic tubules; mitochonchondria with both lamellar and tubular cristae; only a few strands of granular
endoplasmic reticulum; a Golgi apparatus and associated vesicles, some of which
are of the coated variety; a varying population of lipid droplets and dense bodies.
Some of the dense bodies appeared to be lipofuscin pigment. A previously undescribed inclusion was frequently encountered which consisted of arrays of a
honeycomb like structure. Most frequently these structures were present near the
cell periphery or in blunt protrusions of the cytoplasm.
ABSTRACT
The fine structure of the interstitial cell *
has been reported for a number of species
(reviewed by Christensen and Gillim, '69).
Among primates, the interstitial cell of the
human has received considerable attention
(e.g., Fawcett and Burgos, '60; de Kretser,
'67); sub-human experimental primates
have not been studied. The squirrel monkey, Saimiri sciureus, is becoming increasingly widely used as a laboratory primate
and interestingly, the male exhibits seasonal breeding activity correlated with
spermatogenic activity (Du Mond, '68).
Mating in the native habitat takes place
in July, August and September. Following
this, the testis undergoes spermatogenic
arrest until the approach of the next annual breeding season. It is the purpose
of this report to present a fine structural
analysis of the interstitial cell of the
squirrel monkey during the sexual regression that accompanies the non-breeding
portion of the year. It will be shown that
in addition to the usual attributes of interstitial cells, those of the squirrel monkey
are unusual in number and positional distribution, and the cells possess a unique
fine structural feature.
ANAT. REC., 169: 115-128.
MATERIALS AND METHODS
Material for this study was provided by
testes from three squirrel monkeys, Saiiniri sciureus (Peruvian), which were in
the quiescent period of the reproductive
cycle. The testicular material used in the
present study was collected in December
through February. The animals were sexually mature as indicated by the presence
of residual spermatozoa in the cauda epididymidis and in the ductus deferens. Only
one monkey displayed any spermatogenesis
and that a t a low level. Testicular material
was procured during anesthesia resulting
from intraperitoneal administration of sodium pentobarbitol. Usually one testis was
removed, cut into blocks and fixed, while
a portion of the other testis was fixed in.
situ by inserting a fine gauge needle about
2 m m into the testis, incising the tunica
albuginea several millimeters away to prevent a buildup of hydrostatic pressure
within the testis and then infusing fixative
into the testis with a pressure of about
Received April 27, '70. Accepted July 15, '70.
1 Supported in part by USPHS grant GM 01451.
2 In a strict sense, any cell of the interstitium is an
interstitial cell; however, the term is applied only to
the epithelioid cell which bears the full designatlon of
the interstitial cell o f Leydig.
115
116
W. D. BELT AND L. F. CAVAZOS
$5 cm of water. The primary fixative used
was 6% glutaraldehyde in 0.1 M phosphate
buffer. Following infusion for about 20
minutes, blocks were cut of tissue which
appeared grossly to be fixed. Fixation was
continued in the same fixative for one
hour and followed by a series of short
rinses in buffered sucrose. Post-fixation
was carried out with unbuffered 2% osmium tetroxide for one hour. Blocks were
then treated for one hour with 5% aqueous
uranyl nitrate, transferred to 50% alcohol,
rapidly dehydrated and embedded in epoxy
resin (Luft, '61). Sections were obtained
with a Porter-Blum MT-2 microtome and
LKB Ultratome with diamond knives, and
stained with uranyl acetate (Watson, '58)
followed by lead citrate (Venable and Coggeshall, '65). Examination and photography
of the tissue was accomplished with Philips EM-200 and RCA EMU 3H electron
microscopes.
RESULTS
Interstitial cells are rather more abundant in the testis of the squirrel monkey
than in the testis of laboratory rodents.
In the squirrel monkey, interstitial cells
form a layer several cells thick just beneath the tunica albuginea (fig. 1). Sometimes cell nests are located within the
tunica albuginea and surrounded by it,
quite probably in continuity with the subtunical mass in the other dimension.
The interstitial cells of all the specimens
examined were generally similar in their
fine structure, except for the numbers of
lipid droplets and residual bodies or pigment (figs. 1, 2). The testes of all animals
showed inactive or greatly reduced spermatogenesis; one showed no maturing
sperm in the seminiferous tubules, indicating that the animal was in the fully
quiescent period of the male reproductive
cycle.
In those sites where interstitial cells are
most tightly packed together (fig. 2), the
gap space between adjacent cells is reduced to an irregular space of 200-300 A
with occasional dilations. Poorly developed
desmosomes are sometimes present. More
frequently, the cells are separated by quantities of flocculent and fibrillar matrix (fig.
3); only some of the fibrils are identifiable
as collagen. In such areas an incomplete
basal lamina is often found and microvilli
and surface folds are frequent and extensive (fig. 3 ) . Coated vesicles (about 1200
A ) are frequently observed in continuity
with the plasmalemma or just beneath it.
The nucleus of the interstitial cell tends
to be oval, but may be irregularly contoured, especially when impressed by adjacent lipid droplets (fig. 1). Heavily
clumped heterochrornatin is present adjacent to the inner membrane of the nuclear
envelope and centrally in the nucleus. Nucleoli are well developed. Nuclear pores
are frequent.
Within the cytoplasm, mitochondria are
present in abundance as rod-like profiles
(figs. 2, 3 , 4). The mitochondria1 matrix
is quite dense; both plate-like and tubular
cristae are present in a most random arrangement and orientation. The Golgi apparatus consists of groups of stacks of relatively short saccules which are flattened
or somewhat dilated, associated with numerous small vesicles (figs. 2, 6). Frequently the small vesicles possess a content
of moderate density. Occasional vesicles
are of the coated variety, about 600-750 A
in diameter. A few stacks of granular endoplasmic reticulum are evident in most cells
(fig. 3 ) . They tend to be preferentially
oriented in a juxtanuclear and/or subplasmalemmal location. Free ribosomes
may be found throughout the cytoplasm,
except in the area of the Golgi apparatus.
The most abundant organoid is, however,
the agranular endoplasmic reticulum. It
fills most of the space between the other
formed elements of the cytoplasm (figs. 2,
3, 4). It consists of membranous tubules
in anastomotic assemblage. Since it is so
abundant and pervasive, it cannot always
be delimited from elements of the Golgi
apparatus, with which it seems to communicate freely. Frequently the agranular
endoplasmic reticulum is related intimately
to mitochondria and to lipid droplets (figs.
4, 6). Sometimes concentric arrays are
formed about lipid droplets. Complicated
regular arrays of agranular endoplasmic
reticulum sometimes occur (see inset, fig.
4 ) , and in such areas other organelles are
not present. This is usually seen at one end
of the cell or in a blunt process.
Lipid droplets are present in all specimens examined. The quantity of droplets
INTERSTITIAL CELLS OF THE MONKEY
varies widely between specimens, but tends
to be fairly constant for cells from any
one animal. The specimen which had the
greatest abundance of lipid droplets also
had the greatest abundance of large dense
bodies, considered to be pigment. The
fresh specimen itself was darkly colored.
At least two varieties of dense bodies
can be identified, both of which are surrounded by a limiting membrane (fig. 2).
Those simplest in form are smoothly contoured, round or oval, with a uniformly
granular and moderately dense content.
Another variety of dense body is strikingly
pleomorphic, frequently irregular in profile, and contains granular material of high
density. In some instances a small lipid
droplet appears to be contained as well.
These appear to be residual bodies which
result from lysosomal activity. In massive
accumulation of larger forms, these large
dense bodies correspond to lipofuchsin pigment (fig. 3 ) . Structures intermediate in
form between the two varieties of dense
bodies are present.
Both microtubules and fine filaments are
sometimes present in interstitial cell cytoplasm. Microtubules are found singly, with
no specific location of orientation to other
cell structures. In all specimens, some cells
contain structures in remarkable array
(fig. 3, 4). Frequently these structures
were present in blunt pseudopods. When
viewed in oblique or nearly longitudinal
section, these elements appear as aggregates of more or less parallel coursing
bundles of fine fibrils, with the fibrils in
each bundle also arranged in parallel. However, cross sections of such apparent bundles demonstrate in this plane an appearance of a section through an array of
packed prisms, like a honey-comb (fig. 5).
Although precise regularity is not present,
so that four to seven sides are present, the
prisms tend toward six-sidedness. The
wall or septa1 thickness is 40-50 A; the
space enclosed varies from 100-200 A.
DISCUSSION
In most respects the interstitial cells of
Leydig observed in this study are typical
of steroid secretory cells in general (reviewed by Christensen and Gillim, '69).
These include a variable population of lipid
droplets, mitochondria with some degree of
117
tubularization of the internal membranes,
a paucity of granular endoplasmic reticulum which, when present, is perinuclear or
subplasmalemmal in location, a tendency
toward dispersion of the Golgi apparatus,
lysosomes and lipofuscin pigment, and an
extensively developed agranular endoplasmic reticulum, distinctly tubular, and in
tortuously anastomotic form. Frequently
the agranular endoplasmic reticulum is
seen in intimate association with lipid droplets and mitochondria.
Amongst the higher mammals that one
might house for laboratory usage, none
has the abundance of interstitial cells
possessed by the squirrel monkey. Unlike
most other primates, the squirrel monkey
is a seasonal breeder, having one period
of sexual activity per year. This period of
sexual activity is mainly dependent upon
the gonadal function of the male, insofar
as the female undergoes cyclic periods of
estrous throughout much of the year
(Rosenblum, '68). It might be considered
as unusual, then, that the interstitial cells
during the season of non-breeding appear
to be as morphologically competent as they
do. A generally similar situation exists,
however, in immature male pigs (Cavazos
and Belt, '66) where at six weeks of age
the interstitial cells were present with a
fine structure similar to those from mature
boars (Belt and Cavazos, '67).
In addition to the usual complement of
organelles and inclusion possessed by most
cells, interstitial cells of man are known to
have elongate crystalloids (Reinke, 1896;
Winiwarter, '12). Although Duesberg ('18)
reported Reinke crystals in the interstitial
cells of the opossum, recent electron microscopic observations (Christensen and
Fawcett, '61) do not bear this out. The fine
structure of the Reinke crystal was first
described by Fawcett and Burgos ('55,
'61). A later and detailed analysis of the
Reinke crystal (de Kretser, '67) showed a
precisely ordered hexagonal array when
observed in cross-section. The central hollow space measured about 150-200 A; the
septa between the adjacent enclosed spaces
measured about 150 A. The structures reported here in the Leydig cell of the squirrel
monkey do not possess near the ordered
regularity of the Reinke crystal, nor is
there enough similarity to suggest that
118
W. D. BELT AND L. F. CAVAZOS
these honeycomb structures are related to
Reinke crystals. Furthermore, they do not
appear to be similar to the fibrillar strncture of Charcot-Bottcher crystals of Sertoli
cells (Nagano, '66). It will be of interest
to learn whether alterations of this honeycombed structure in the squirrel monkey
occur in the interstitial cell in the period
associated with sexual activity.
ACKNOWLEDGMENT
Acknowledgment is made of the technical assistance of Miss Ann Campbell, Tufts
University School of Medicine.
LITERATURE CITED
Belt, W. D., and L. F. Cavazos 1967 Fine structure of the interstitial cells of Leydig in the
boar. Anat. Rec., 158: 333-350.
Cavazos, L. F., and W. D. Belt 1966 Observations sobre l a estructura fina de las celulas de
Leidig en el jabale inmaduro. Archiv. Mexicanos de Anat., 7 : 19-20 (Abstr.)
Christensen, A. K., and D. W. Fawcett 1961
The normal fine structure of opossum testicular
interstitial cells, J. Biaphys. and Biochem. Cytol., 9: 653-670.
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. Kenneth W.
McKerns, ed. Appleton-Century-Crofts, New
York, pp. 415488.
de Kretser, D. M. 1967 The fine structure of
the testicular interstitial cells i n men of normal androgenic status. Z. Zellforsch., 80: 594609.
Duesberg, J. 1918 On the interstitial cells of
the testicle of Didelphys. Biol. Bull., 35: 175199.
DuMond, F. V. 1968 The squirrel monkey in a
seminatural environment. In: The Squirrel
Monkey. L. Rosenblum and R. Cooper, eds.
Academic Press, New York. pp. 87-145.
Fawcett, D. W., and M. H. Burgos 1955 Observations on the cytomorphosis of the germinal
and interstitial cells of the human testis. In:
Ciba Foundation Colloquia on Ageing. Vol. 2,
Ageing i n transient tissues. G. E. W. Wolstenholm and E. C. P. Miller, eds. Little, Brown and
Co., Boston, pp. 86-96.
1960 Studies on the fine structure of
the mammalian testis. 11. The human interstitial
tissue. Am. J. Anat., 107: 245-269.
Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. and Biochem.
Cytol., 9: 409414.
Nagano, T. 1966 Some observations on the fine
structure of the Sertoli cell i n the human testis.
Z. Zellforsch., 73: 89-106.
Reinke, F. 1896 Beitrage zur Histologie des
Menschen. I. Uber Krystalloidbildungen in den
interstitiellen Zellen des Menschlichen Hodens.
Arch. Micr. Anat., 47: 34-44.
Rosenblum, L. A. 1968 Some aspects of female
reproductive physiology in the squirrel monkey.
In: The Squirrel Monkey. L. Rosenblum and R.
Cooper, eds. Academic Press. New York, pp.
147-169.
Venable, J. H., and R. Coggeshall 1965 A simple lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408.
Watson, M. L. 1958 Staining of tissue sections
for electron microscopy with heavy metals. J.
Biophys. and Biochem. Cytol., 4: 475-478.
Winiwarter, H. 1912 Observations cytologiques
sur les cellules interstitielles du testicule humain. Anat. Anz., 41: 309-320.
-
Abbreviations
AER, agranular endoplasmic
reticulum
C, centriole
CL, capillary lumen
D, desmosome
G, Golgi apparatus
GC, glycocalyx
GER, granular endoplasmic
reticulum
L, lipid droplet, extracted
LS, longitudinal section
M, mitochondria
N, nucleus
T, tunica albuginea
XS, transverse section
PLATE 1
EXPLANATION OF FIGURE
1 This micrograph shows the concentration of interstitial cells that occurs just deep to the collagen bundles of the tunica albuginea ( T ) .
This specimen contained the most large lipid droplets of those examined, shown here as extracted vacuoles. Wispy connective tissue
and an occasional blood vessel are present between the cells. x 3,200.
INTERSTITIAL CELLS OF THE MONKEY
W. D. Belt and L. F. Cavazos
PLATE 1
119
PLATE 2
EXPLANATION O F F I GU R E
2
120
Portions of three interstitial cells are shown in a n area where little
connective tissue is present between the cells. The plasmaleinma of
adjacent cells in some areas approaches the 200 A gap (arrows)
commonly seen in epithelial cells but without that regularity. Thus,
dilations of the intercellular space exist into which microvilli or folds
protrude. Contact areas resembling rudimentary desmosomes ( D )
occur infrequently. Within the cytoplasm, a well developed Golgi
apparatus ( G ) often has membrane bound granules of moderate
density associated with it. Other structures of higher density, some
with an enclosed lipid droplet, are considered to be pigment ( P ) . This
section is from a specimen with relatively few large lipid droplets
x 21,000.
INTERSTITIAL CELLS OF THE MONKEY
W. D. Belt and L. F. Cavazos
PLATE 2
121
PLATE 3
EXPLANATION OF FIGURE
3
122
A portion of two interstitial cells is shown in relation to a blood
vessel (lumen at CL) and intervening perivascular cell (PC).In areas
of separation of cells by a quantity of connective tissue, some degree
of glycocalyx formation (GC) occurs. Pigment ( P ) , a longitudinally
sectioned centriole ( C ) and mitochondria ( M ) are evident. The en.
doplasmic reticulum is primarily agranular tubules; occasional cisternae of granular endoplasmic reticulum (GER) occur, often as
seen here just deep to the plasma membrane. A blunt pseudopod
contains a structure with a patterning which appears fibrous.
X 27,000
INTERSTITIAL CELLS OF THE MONKEY
W. D. Belt and L. F. Cavazos
PLATE 3
123
PLATE 4
EXPLANATION OF FIGURE
4 A complex pattern, again fibrillar in appearance, is present amidst
abundant agranular endoplasmic reticulum (AER) and lipid droplets
(L). Intimate association of agranular endoplasmic reticulum with
lipid droplets is indicated (arrows). X 30,000.
Inset. Complicated regular arrays of agranular endoplasmic reticulum. Areas such as this are occasionally present. Neither their three
dimensional organization nor their significance is apparent. X 42,600.
124
INTERSTITIAL CELLS OF THE MONKEY
W. D. Belt and L. F. Cavazos
PLATE 4
125
PLATE 5
EXPLANATION OF FIGURES
126
5
Here the fibrillar patterned structure is shown in transverse section
(XS) and near longitudinal section ( L S ) . Although i n near longitudinal section, the structure appears to be fibrous, the honeycomb
array is obvious when observed transversely. x 122,000.
6
Adjacent to the nucleus ( N ) , a well developed Golgi apparatus (G)
is present. Several stacks of short saccules are present, along with a
variety of small vesicles. Adjacent is a centriole ( C ) and several
lipid droplets ( L ) . Note that agranular endoplasmic reticulum is in
intimate relation (arrows) to the lipid droplets. x 29,000.
INTERSTITIAL CELLS OF THE MONKEY
W. D. Belt and L. F. Cavazos
PLATE 5
127
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