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The junctional specializations of sertoli cells in the seminiferous epithelium.

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The Junctional Specializations of Sertoli Cells
in the Seminiferous Epithelium '
Department of Anatomy, Harvard Medical School, Boston, Massachusetts
Attention is directed to a n unusual type of junctional complex between
Sertoli cells in the seminiferous epithelium. The space bctween the membranes of adjoining cells is narrowed to 70-90 A over large areas of their contact surfaces. 111 the
superficial cytoplasm of each cell is an extensive cisterna of the endoplasmic rcticulum,
parallel to the membrane and 400-600 A from it. Spaced at more or less regular intervals in the thin layer of cytoplasm between the cisterna and the cell membrane are
periodic densities that appear to be band-like aggregations of fine filaments. The subsurface cisternae are smooth-contoured on the side toward the cell membrane but bear
ribosomes on the side facing the cytoplasm. The possible significance of these distinctive junctions is discussed in relation to the support of the germ cells and the coordination of the developmental events in the cycle of the seminiferous epithelium.
One of the most significant results of the
improved resolution afforded by the electron microscope has been the description
of an unexpected variety of local modifications of the contact surfaces of adjacent
cells - desmosomes, zonulae adherentes,
zonulae occludentes, etc. (Farquhar and
Palade, '63). The physiological implications of these junctional specializations are
just beginning to be explored but it is already clear that some provide for maintenance of cell cohesion; others are sites of
membrane fusion which deny access of
substances to the intercellular clefts at the
free surfaces of epithelia. Similar tight
junctions found at certain electrical synapses in the nervous system (Furshpan
and Potter, '59) and in smooth (Dewey
and Barr, '64) and cardiac muscle (Karrer,
'60) appear to be low resistance pathways
permitting the spread of electrical signals
from cell to cell. Low resistance has also
been demonstrated among epithelial cells
(Lowenstein et al., '65) and cells i n embryos (Potter, Furshpan and Lennox, '66)
that are connected by tight junctions
even though these cells generate no detectable electrical signals (Trelstad et al., '66;
Sheridan, '66 1.
I n view of the burgeoning interest in
cell junctions and their potential histophysiological importance it seems worthwhile to draw attention to another type of
junctional specialization which has hitherto received little attention. This is a distinctive specialization of the surface of
1 5 8 : 207-222.
contiguous Sertoli cells in the germinal
epithelium of the testis. It involves a narrowing of the intercellular space to 7595 A and the presence in each cell of a
cisterna of the endoplasmic reticulum disposed parallel to the cell membrane and
separated from it by a superficial layer of
cytoplasm 400 to 600 A thick containing
periodic densities. These dense areas at
high magnification are resolved as bundles
of fine filaments oriented parallel to each
other and to the cell surface.
The presence of junctions of this kind
has previously been noted by Nicander
('63) in the germinal epithelium of the
bull and the ram, and described more fully
by Brokelmann ('63) in the rat. The present paper records more detailed observations on these structures in the mouse, cat,
and chinchilla testis and offers some further speculations as to their possible significance.
Testes of two to six weeks old and adult
white mice were fixed by immersion for
two hours at room temperature in 5%
glutaraldehyde buffered at pH 7.6 with
s-collidine or in a modified Karnovsky's
fixative (Karnovsky, '65) containing 2.5%
glutaraldehyde, 2% paraformaldehyde,
ISupported by a research grant RG-06729 and
training grant G-406 from the Institute of General
Mcdical Sciences, Natiunal Institutes of Health.
2 Present address: University of Colorado, Institute
for Developmental Biology, PSRB no. 1, Boulder,
and 0.05% of calcium chloride in 0.1 M
cacodylate buffer at pH 7.3. Aldehyde fixed
blocks were subsequently fixed in 1% osmium. Portions of the testes of adult cats
and chinchillas were fixed in s-collidine
buffered glutaraldehyde. Blocks of tissue
were placed in cold buffered osmium tetroxide for one hour, dehydrated with ethanol or methanol, and embedded in Epon or
Araldite. Silver to pale gold sections were
cut with glass knives or diamond knives
on a Porter-Blum microtome, stained with
uranyl acetate and lead citrate (Venable
and Coggeshall, '65) or with lead citrate
alone and examined in an RCA EMU-3E
or -3G electron microscope.
In relatively low power electron micrographs of the seminiferous tubules, particularly near the base of the germinal epithelium, the boundaries between adjoining
Sertoli cells are readily distinguished from
those between germ cells and Sertoli cells,
because of the presence of conspicuous
junctional specializations on the one, and
their absence on the other (figs. 1 , 2 ) .
Where Sertoli cells are in contact with
spermatogonia, spermatocytes, or early
spermatids, the membranes run parallel at
the usual interval of 150 to 200 A and
there are no desmosomes or other junctional specializations (fig. 3 ) . But where
two Sertoli cells adjoin, the membranes are
only 70 to 90 A apart and the subjacent
cytoplasm appears dense owing to the presence of fine filaments. Deep to the layer of
filaments in each cell, cisternae of the
endoplasmic reticulum course parallel to
the pair of apposed membranes.
The filaments associated with the junction are not distributed in a continuous
layer but are aggregated into bundles that
are parallel to each other and to the cell
surface. In micrographs, the distribution
of densities in this superficial layer of cytoplasm therefore varies greatly depending
upon the angle of incidence of the section.
When the section is normal to the long axis
of the filament bundles, one observes discrete dense regions spaced at rather uniform intervals and separated by regions of
lower density. When the section is parallel
to the long axis of the filament bundles, a
continuous layer of uniform density is
seen, and in sections subtangential to the
membrane the filament bundles appear as
parallel dense stripes 400-600 A apart.
On the inner aspect of the layer of filaments are extensive membrane-limited flat
saccules. These run parallel to the cell
membrane at a uniform distance of 500 to
600 A. Their inner or cytoplasmic surface
bears occasional clusters of ribosomes
which identify these structures as cisternal
elements of the granular endoplasmic reticulum. However, their outer aspect, adjacent to the filamentous layer, is smoothcontoured and entirely devoid of ribosomes
(fig. 3 ) . I n some areas, particularly in very
young animals, a n array of vesicular or
tubular elements is found instead of the
cisternae, but as a rule, in well-preserved,
adult testis one characteristically finds a n
alignment of cisternae. These profiles
range up to three or four microns long.
Successive cisternae are separated by gaps
or fenestrations a few hundred A across.
In the mouse such junctional specializations appear to cover 70 to 80% of the area
of contact between Sertoli cells.
Complex junctions of this kind are never
observed, however, along the boundaries
of Sertoli cells with spermatogonia, with
spermatocytes, or with differentiating spermatids. But late in spermatid development
when they are nearing maturity and have
come to occupy recesses in the apical surface of the Sertoli cells, with their tails
projecting into the lumen of the tubule,
then the Sertoli cytoplasm immediately
surrounding each sperm head shows dense
parallel bundles of filaments interposed
between the plasma membrane and welldeveloped subsurface cisternae (figs. 4, 5).
Only the Sertoli cell shows this specialization - there is no corresponding differentiation of the surface of the maturing spermatozoon. Thus, only half of the junctional
complex is formed at the sites of attachment of Sertoli cells to the heads of maturing sperm, whereas at sites of union of
two Sertoli cells, both cells form complementary specializations. This is a rather
unusual circumstance, for in other epithelia wherever a desmosome or other specialization for cell attachment is formed
on the boundary between two cells, both
participate i n its formation. Although halfdesmosomes may occur on epithelial cell
surfaces exposed to the basement lamina,
unmatched half desmosomes are rarely, if
ever, encountered on surfaces of contact
between adjacent cells. It is as though the
formation of a half-desmosome on one cell
immediately induces the adjacent cell to
form the complementary half of the attachment device. In the exceptional case of the
Sertoli cell-sperm head junction, the sperm
may not be able to respond, for at this late
stage of its development there is essentially
no cytoplasm over the head - the plasmalemma being closely applied to the underlying acrosomal cap.
The Sertoli cell junction does not attain
the same degree of complexity in all species. The narrowing of the intercellular
space. and the presence of aggregations of
dense filaments immediately beneath the
cell membrane are features common to the
mouse, cat, guinea pig and chinchilla. Extensive subsurface cisternae are a prominent feature of the junctions in the mouse
and cat, but are poorly developed or entirely lacking in the guinea pig and chinchilla. In the latter species, if the endoplasmic reticulum is represented at all in
the junctional zone it is in the form of a
row of vesicular and elongate profiles disposed more or lcss parallel to the cell surface but some distance from it (figs. 6 , 7,
8). These discontinuous profiles may represent sections of a loose plexus of tubular
elements in the superficial cytoplasm of
the Sertoli cell in place of the broad Aat
cisternae found in the other species.
Septate desmosomes with associated superficial cisternae have been observed in
the gastroderm of Physalia and i n the salt
absorbing epithelium of Callinectes (Copeland, ' 6 6 ) . Junctions such as those described in this paper, however, have not
been observed to date in any vertebrate
tissue other than the testis. It is reasonable
therefore to turn to a consideration of the
unique features of Sertoli cell function for
clues to their possible physiological significance. Although still far from established,
the two functions commonly attributed to
the Sertoli cells are (1) a role in the nutrition of the developing germ cells, and ( 2 )
a possible role in the control and coordination of the cytological events of the cycle
of the germinal epithelium and of the spermatogenic wave.
If these distinctive surface specializations were concerned with the synthesis or
transfer of metabolites from cell-to-cell,
one would expect to find them on the
boundaries between Sertoli cells and the
developing germ cells. A specialized mechanism for transfer of small molecules,
polypeptides or even whole proteins across
these interfaces would enable the highly
specialized germ cells to depend upon the
sustentacular cells for much of the synthetic activity necessary to support their
differentiation. Actually, such specializations are never found on the boundary between Sertoli cells, spermatogonia, spermatocytes and early spermatids. Instead,
they occur at the junctions of neighboring
Sertoli cells where there is no reason to
expect a n exchange of metabolites. A specific function in the sustenance of the
germ cells therefore seems unlikely. Indeed, i n view of the fact that the intercellular path for diffusion from the bloodstream into the germinal epithelium is
narrowed over a large portion of the interface between Sertoli cells, this junctional
specialization would he more likely to restrict than to promote access of metabolites to the germ cells.
More appealing is the possibility that
special junctions between Sertoli cells
might favor the cell-to-cell communication
necessary for the regional synchronization
of developmental events which is characteristic of the spermatogenic wave. When
it was observed that all of the cells in
the clone of spermatids derived from the
same spermatogonium communicate freely
through sizeable intercellular bridges (Burgos and Fawcett, '55) i t was suggested that
this protoplasmic continuity was probably
the morphological basis for the precise
synchrony of their differentiation. While
this may well be true for the spermatids
within the same group, this does not offer
an adequate explanation of the fact that
the synchrony extends to many such
groups involving thousands of spermatids
in the same segment of the seminiferous
tubule (Roosen-Runge, ' 6 3 ) .
Numerous examples of connections providing for flow of ionic currents between
excitable cells have now been discovered
by electrophysiological methods (Furshpan
and Potter, ’59) and these have been correlated in most instances with the presence
of specialized cell junctions demonstrable
by electron microscopy, where the intercellular cleft is obliterated by the close
apposition of the membranes (Hama, ’59;
Karrer, ’60; Robertson, ’64; Dewey and
Barr, ’64). Recently it has been shown in
the squid embryo that sites some distance
apart are electrically coupled presumably
by way of low resistance interconnections
among the intervening embryonic cells
(Potter, Furshpan and Lennox, ’66). It has
been speculated that such connections may
play a role in the control of embryonic
differentiation. Electrical coupling clearly
offers no substitute for, and provides no
explanation of, classical embryonic induction but it could provide a n explanation of
the coordination and synchronization of
the behavior of large numbers of cells
during embryogenesis. Returning to the
testis, the only obvious connection between
widely separated groups of spermatids
within a synchronized segment of the seminiferous tubule would seem to reside in
their common relationship to a population
of supporting Sertoli cells. If i t could be
shown that the Sertoli cells were electrically coupled this might offer an explanation for synchronization of germ cell differentiation throughout sizeable segments
of the seminiferous tubule. The cell-tocell relationships of Sertoli cells are therefore of considerable potential interest. The
unique junctional specializations of Sertoli
cells involve a significant narrowing of the
intercellular cleft and could be described
as “close junctions” but the complete obliteration of the interspace and fusion of the
outer leaflets of the apposed membranes
that are the defining characteristics of
“tight junctions,” have not been observed
on the boundaries between Sertoli cells in
the adult testis. It is commonly assumed
that complete obliteration of the intercellular space is required for flow of ions between cells. The Sertoli junctions therefore
do not fulfill the currently accepted morphological criteria for a low resistance
electrical connection between cells. It is
fair to say, however, that the possibdity
that specializations other than tight junc-
tions may permit electrical coupling has
not been completely eliminated.
The functional significance of the cisterna of the endoplasmic reticulum associated with the Sertoli junction is particularly puzzling. It bears some resemblance
to the “subsurface cisterns” described i n
certain neurons of the central nervous system and peripheral ganglia (Rosenbluth,
’ 6 2 ) , in the synaptic region of the outer
hair cells of the organ of Corti (Engstrom,
’58; Smith and Sjostrand, ’61), and beneath the sarcolemma of invertebrate muscle cells. The occurrence of such cisternae
in sensory receptors, nerve and muscle led
Rosenbluth to speculate that they might be
concerned with the generation or conduction of changes of electrical potential. The
subsurface cisternae in these irritable cell
types are immediately beneath the plasma
membrane, separated from it by less than
100 A. The cisternae associated with the
Sertoli junctions on the other hand are
separated from the cell membrane by a
filamentous layer of cytoplasm 400-500 A
Although the two distinctive characteristics of the Sertoli cell junctions, namely,
the narrowing of the intercellular cleft and
the presence of a n associated cisterna of
the reticulum, leads one to suspect that
this type of junction has distinctive physiological attributes, it may well be that its
function is not fundamentally different
from that of desmosomes and zonulae adherentes. It must be borne in mind that
the seminiferous epithelium is unique i n
having two distinct populations of cells,
the Sertoli cells which are permanent and
relatively fixed and the germinal elements
which are transient - constantly being
renewed at the base and displaced toward
the lumen as they differentiate. The absence of attachment devices of any kind
between the Sertoli cells and the spermatogonia, spermatocytes and differentiating
spermatids permits the movement of the
transient cell population with respect to
the static cell population. The specialized
junctions of adjoining Sertoli cells may be
adaptations to the unusual mechanical
problem of maintaining the integrity of
the epithelium in the face of constantly
changing shape of the sustentacular cells
concurrent with the slow escalation of the
germinal cells.
Briikelmann, J. 1963 Fine structure of germ
cells and Sertoli cells during the cycle of the
seminiferous epithelium in the rat. Z. Zellforsch., 59: 820-850.
1961 Surface modifkations of Sertoli
cells at various stages of spermatogenesis in
the rat. Anat. Rec., 139: 211.
Copeland, E.
Septate desmosomes and juxtaposition membranes. Submitted for publication
to J. Cell Biol.
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structure and distribution of the nexus. J. Cell
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Engstrlim, H. 1958 On the double innervation
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21 1
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1965 Intercellular
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1963 Some ultrastructural features of mammalian Sertoli cells. J. Ultrastruct.
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Potter, D. D., E. J. Furshpan and E. S. Lennox
1966 Connections between cells of developing
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Smith, C. A., and F. S. Sjostrand 1961 Structure of the nerve endings on the external hair
cells of the guinea pig cochlea as studied by
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1 A low power electron micrograph of a n area of germinal epithelium
from the mouse testis showing a group of four spermatids surrounded
by irregular processes of the sustentacular cells. The arrows indicate
specialized junctions between neighboring Sertoli cells. The boundaries between the Sertoli cells and the spermatids have n o desmosomes or other specializations for cell-tocell attachment. X 7500.
Charles Flickinger and Don W. Fawcett
An electron micrograph of portions of three contiguous Sertoli cells
from mouse testis. The boundaries are marked by conspicuous junctional specializations of a kind that have not been reported to date in
any other tissue. The characteristic features of the junctional complex include a narrowing of the intercellular space; a condensation of
filamentous cytoplasm immediately subjacent to the apposed cell
membranes; and subsurface cisternae of the endoplasmic reticulum
in each cell oriented parallel to the cell surface. It is estimated that
70 to 80% of the surface of contact between Sertoli cells is specialized
in this way. 29,000 x.
Charles Flickinger and Don W. Fawcett
3 A higher magnification of a field permitting a comparison of the
simple Sertoli cell-spermatocyte junction with the complex Sertoli
cell-Sertoli cell junction. In the former, the membranes are unspecialized and separated by a n intercellular space about 200 A wide
with a content of appreciable density. In the Sertoli junction, the
intrecellular space is narrowed to 75-95 A. A filamentous superficial
layer of cytoplasm of uniform thickness is interposed between the
cell membrane and a subsurface cisterna of the endoplasmic reticulum. Ribosomes are found in limited numbers on the inner aspect of
the cisternae but never on the membrane exposed to the filamentous
layer. 70,000 X.
Charles Flickinger and Don W. Fawcett
Sections through the heads of nearly mature mouse sperm in the
apical cytoplasm of the Sertoli cell. The plasma membrane of the
sperm head is closely applied to the limiting membrane of the acrosome with no intervening layer of cytoplasm. The surface of the
Sertoli cell surrounding the sperm head is specialized in the same
manner as at junctions with other Sertoli cells. The intercellular
space is less than 100A wide. A layer of parallel bundles of fine
filaments appears in section as a row of periodic densities. Deep to
this are cisternae of the endoplasmic reticulum. Unlike those found
a t Sertoli junctions near the base of the epithelium, these have no
associated ribosomes. x 48,000.
Charles Flickinger and D o n W. Fawcett
The superficial filamentous layer of the Sertoli cell and narrowing
of the interspace with other Sertoli cells and sperm heads is found
i n many species, but the degree of development of the subsurface
cisternae varies. Around the heads of chinchilla spermatids shown
here there is a conspicuous layer of fine filaments i n the Sertoli cell
but instead of extensive cisternae closely associated with it there is
a row of discontinuous round and elongated profiles apparently representing a network of branching and anastomosing tubular elements of the reticulum disposed more or less parallel to the cell
surface, but some distance away from it. X 45,000.
Charles Flickinger and Don W. Fawcett
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epithelium, specialization, seminiferous, junction, sertoli, cells
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