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The fine structure of the monkey (Macaca) sertoli cell and its role in maintaining the blood-testis barrier.

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The Fine Structure of the Monkey (Macaca) Sertoli
Cell and Its Role in Maintaining the
Blood-Testis Barrier ’
Department of Anatomy, and Laboratory of Human Reproduction and
Reproductive Biology, Harvard Medical School, Boston,
Massachusetts 02115
The monkey Sertoli cell, a tall columnar cell, extends from the
basement membrane of the seminiferous epithelium to the tubule lumen. Its
nucleus occupies a basal position and reveals extensive nuclear envelope infoldings. A zone of fine filaments, approximately 0.5 irc in thickness, invests the nucleus and appears to prevent other cell organelles from approaching it. The basal
cytoplasm is characterized by numerous mitochondria and abundant smooth
endoplasmic reticulum. Lipid droplets, 3 to 4 P in diameter, membrane-limited
dense bodies of various shapes and densities, Golgi cisternae, scattered free ribosomes and parallel profiles of rough endoplasmic reticulum are common. The
more apical portions of the cell contain longitudinally oriented microtubules and
rod-shaped mitochondria, but other organelles are rare.
The seminiferous tubules of monkeys are surrounded by three to five circumferentially arranged cells that overlap each other but are separated by intercellular spaces of a t least 300 to 400 A. Tracers such as horseradish peroxidase and
lanthanum nitrate injected intravascularly readily pass between the peritubular
cells and enter the germinal epithelium. Within the epithelium the tracers outline
the spermatogonia and early spermatocytes by permeating the surrounding intercellular spaces. Further penetration toward the tubule lumen is effectively prevented by the occluding tight junctions joining adjacent Sertoli cells. Thus, in
monkeys the peritubular epithelioid cells do not impede vascularly introduced
tracers from penetrating into the germinal epithelium. The only morphological
component of the blood-testis barrier in the macaque appears to be the SertoliSertoli occluding junction.
The original work of Sertoli (1865) and
a number of articles since that time
(Brown, 1885; Regaud, ’00; Winiwarter,
’12; Rolshoven, ’44; Elftman, ’50, ’63;
Nishida, ’54) established that the transformations taking place within the germ
cell line occur in close association with the
Sertoli cell. No doubt the spermatogenic
process is very largely dependent upon this
ramifying cell type, yet evidence of the
way in which its control over the germ
cells is exerted remains elusive. In mammals the Sertoli cell is found evenly distributed in the seminiferous epithelium extending from the basement membrane to
the tubule lumen. The general cytological
characteristics observed with the light miANAT. REC.. 175: E39-656.
croscope suggest that this non-germinal
element is functionally very active. Electron microscopic studies provided additional morphological data but added little
conclusive experimental evidence which
could assist investigators in understanding
its overall role in spermatogenesis. The
idea that remains prevalent today was
summed up in the following way by
Nishida (’54) : “diffusion currents of nutritive substances occurred in the Sertoli
cell from base toward lumen, which nourished the germ cells.” More recently experimental evidence in rats and guinea
Received Sept. 6, ‘72. Accepted Nov. 15, ’72.
1 Supported by contract NIH 69-2107 from the National Institute of Child Health and Human Development.
pigs has suggested a new role for the mammalian Sertoli cell; i.e., the maintenance of
the blood-testis barrier and the partitioning
of the seminiferous epithelium into a basal
compartment containing the spermatogonia and early spermatocytes and a n
adluminul compartmepnt containing the
more advanced germ cells (Dym and
Fawcett, '70; Fawcett et al., '70; Dym, '72).
Vascularly injected electron-opaque markers such as lanthanum nitrate readily fill
the lymphatic channels and interstitial
spaces in the testis and enter the basal
compartment of the seminiferous epithelium but are prevented from reaching the
tubule lumen mainly by the occluding
junctions between adjacent Sertoli cells
(Dym and Fawcett, '70). Elegant physiological studies have established that
plasma proteins are found in very low
concentrations in the fluid within the
lumina of rat seminferous tubules (Tuck
et al.. ' 7 0 ) , and it is likely that the proteins
are excluded by the junctional complexes
between Sertoli cells. On the other hand,
Reddy and Svoboda ('67), in abstract form,
and Aragon, Lustig and Mancini ('72) reported that in mice intravenously injected
peroxidase was localized within the cytoplasm of the Sertoli cells and germ cells
and did penetrate the blood-testis barrier
to reach the tubule lumen.
The purpose of this study was to determine whether there is a blood-testis
barrier to vascularly infused horseradish
peroxidase and lanthanum nitrate in the
monkey and, if so, to localize its morphological site. In view of the contradictory
report by Aragon, Lustig and Mancini
('72) on mice, the blood-testis barrier in
this species was also investigated using
horseradish peroxidase. In addition, the
fine structure of the monkey Sertoli cell
was examined in the different stages of the
cycle of the seminiferous epithelium in a n
attempt to correlate, if possible, Sertoli cell
organelle modifications with specific developmental events of the germ cells.
A total of six adult male monkeys
(Macaca mulatta and Macaca n e m e s t r i n a )
were used in these experiments. The testes
were fixed by vascular perfusion using a
technique first described by Christensen
('65). Under deep anesthesia the processus
vaginalis was opened, the remnant of the
gubernaculum cut, and the testis and
spermatic cord mobilized. Two ligatures
(no. 0 surgical silk) were tied around the
cord approximately two inches proximal to
the testis. The spermatic cord was cut between the ligatures and the testis was removed. A 22 gauge needle at the end of
Intramedic polyethylene tubing PE190, attached to a reservoir 130 cm high, was inserted under the tunica albuginea and into
the testicular artery at the upper pole of the
testis. The pampiniform plexus of veins
was incised to permit egress of blood and
perfusate. Ten cm" of .9% saline followed
by 200 cm" of 5 % glutaraldehyde buffered
with 0.2 M s-collidine were permitted to
flow through the testis over a 30 minute
period. One millimeter cubes of tissue were
then cut with a razor blade and immersed
for a n additional 60 minutes in the same
fixative. Secondary fixation was carried
out in a solution containing two parts of
2% osmium tetroxide and one part of 0.2
M s-collidine. The tissue was dehydrated
in alcohol and embedded in Epon. One
micron sections stained with toluidine blue
were used to identify the stages of the
cycle of the seminiferous epithelium as
described by Clermont ('69). Thin sections
exhibiting silver to pale gold interference
colors were cut with a diamond knife and
examined on a Siemens Elmiskop I or RCA
EMU 3G electron microscope.
One major source of variability in descriptions of the fine structure of the
seminiferous epithelium has been the lack
of uniformity in methods of fixation. When
pieces of testicular tissue are immersed in
the fixative the ultrastructural preservation of the Sertoli cell is often inadequate.
However, fixation by perfusion has markedly improved the quality of preservation
of the monkey seminiferous epithelium.
Tracer studies
The lanthanum technique employed in
these experiments was similar to that described previously (Dym and Fawcett,
'70) : A solution of 4% lanthanum nitrate
was adjusted to pH 7.7, using 0.1 N NaOH,
and added to a n equal volume of glutaraldehyde buffered collidine (.2 M ) . The
final concentration of the lanthanum was
2% and that of the glutaraldehyde 5 % . In
monkeys this mixture was perfused into
the testicular artery as described above.
In order to use peroxidase as an intercellular tracer, one 10 kg monkey, while
lightly tranquilized was injected in the
femoral vein with 2 gm of Sigma type I1
horseradish peroxidase dissolved in 35 cm3
of distilled HzO. In mice 20 mg of horseradish dissolved in .5 cm3 of H,O was
slowly injected into the tail vein. Fifteen
minutes after infusion of peroxidase the
animals were anesthetized, the testes were
removed, and small pieces were fixed for
three hours in Karnovsky's fixative ('65).
Following an overnight wash in cacodylate
buffer, 50 to 100 p sections were prepared
on a Smith and Farquhar Tissue Sectioner
and reacted in diaminobenzidine tetrachloride and hydrogen peroxide using the
method of Graham and Karnovsky ('66).
Subsequently the tissue was osmicated in
1.3% osmium tetroxide, dehydrated in
graded alcohols, and embedded in Epon.
Architecture of the monkey
Sertoli cell
Sections of seminiferous tubules embedded in paraffin, stained with routine
histological dyes, and examined with the
light microscope reveal much information concerning the developmental patterns
of the germ cells. However, using these
same techniques the Sertoli cell cytoplasm
often remains inconspicuous, crowded in
between the more numerous germ cells;
therefore, it has proved difficult both to
examine the finer morphology of this cell
type under normal conditions and to study
its response to experimental treatments.
Examination of favorably oriented one
micron sections of Epon embedded tissue
permits greater resolution and reveals that
the Sertoli cell is a tall columnar cell extending from the base of the seminiferous
epithelium to the tubule lumen. It consists of a stem resting on the basal lamina,
an intermediate portion which provides
lateral processes around which the spermatocytes and spermatids are arranged,
and apical projections which enclose the
late spermatids just prior to their release
into the tubule lumen. Stages of the cycle
as defined by Clermont ('69) are readily
identifiable in these 1 I.1 toluidine blue
stained sections (figs. 1, 2).
In Sertoli cells of monkeys the basal portion of the cytoplasm is voluminous and a
large variety of the common cell organelles
is evident (figs. 3, 4). Lipid droplets, 3 to 5
;FL in diameter, are common and their size
and number do not appear to vary in the
different stages of the cycle of the seminiferous epithelium. Numerous spherical and
elongated mitochondria exhibiting transverse cristae of orthodox configuration are
present in the basal portion of the cell,
while elongated mitochondria predominate
in the more apical portions.
Perhaps the most characteristic feature
of the basal Sertoli cell cytoplasm is the
abundant profiles of smooth endoplasmic
reticulum. Often these are aligned in parallel arrays near the nucleus. Isolated
patches of flattened profiles of rough endoplasmic reticulum are present, and occasionally several such cisternae can be
found arranged circularly around a single
lipid droplet. Mitochondria and abundant
smooth ER are frequently observed in the
immediate vicinity of the lipid. Free ribosomes are scattered throughout the Sertoli
cell cytoplasm. Membrane limited bodies
of various sizes, shapes, and densities are
characteristic features. Their origin, turnover rate, enzymatic content and ultimate
fate all remain obscure. But it is likely
that many of these figures are products of
the degenerating germ cells and residual
bodies in the seminiferous epithelium.
Each Sertoli cell possesses a well developed Golgi apparatus, and often stacks of
Golgi cisternae are visible in a single thin
section at varying distances from each
other. This suggests that a single Sertoli
cell may contain several non-connected
Golgi profiles. In other cell types the Golgi
apparatus is the main building site for a
variety of large carbohydrates. The role of
the extensive Golgi in the Sertoli cell still
remains to be elucidated. Electron microscopic radioautographic studies using
labeled sugars such as galactose, mannose, or fucose may provide clues concerning the substances which are elaborated or
packaged by this cell type.
The intermediate and apical portions of
Figs. 1 and 2 Light micrographs of 1 + sections of monkey seminiferous tubules showing germ
cells in various stages of development and Sertoli cells. The schematic drawings on the left (Clermont, '69) depict the stage of the spermatogenic cycle. x 637.
the columnar Sertoli cells contain rodshaped mitochondria and numerous longitudinally oriented microtubules. Other
organelles are rarely observed in large
numbers in these regions.
The Sertoli cell nucleus is large and is
characterized by a homogenous nucleoplasm and a distinctive nucleolus when
viewed with the light microscope. It occupies a basal position in the cell. In mon-
Fig. 3 A n electron micrograph of the basal portion of a monkey Sertoli cell. Note the filamentous zone surrounding the nucleus and the abundant agranular reticulum and mitochondria. Stage
111. x 8190.
Fig. 4 A n electron micrograph of a monkey Sertoli cell demonstrating deep infoldings of the
nuclear envelope. A prominent Golgi apparatus and membrane-bounded dense bodies are apparent.
Note the filaments in ihe inner cell layer of the tunica propria. Stage VII. x 8190.
keys the nuclear envelope is elaborately
infolded and fine structural analysis often
reveals bizarre shapes (fig. 5). Nuclear
pores are very numerous, fairly evenly distributed, and usually separated by a distance of approximately 300 A. Neither the
position of the nucleus in the epithelium
nor the architecture of the nuclear lobulations in the monkey appears to change in
the various stages of the cycle of the seminiferous epithelium. Similarly the number
of nuclear pores and their distribution
over the Sertoli cell nuclear surface seem
to be relatively constant along the length
of the seminiferous tubules. A sheath of
70 A filaments, 0.5 cL in thickness, completely surrounds the Sertoli cell nucleus
and prevents other cellular organelles from
approaching it (fig. 3). The function of
this filamentous zone still remains obscure
but it may provide rigidity to the pleomorphic nucleus. Similar sized filaments
are also scattered throughout the Sertoli
cell cytoplasm, and frequently a basal web
is preserved adjacent to the basal lamina
(fig. 3 ) .
Residual bodies (fig. 6 ) and degenerating germ cells (fig. 7 ) are identifiable with
the electron microscope, and they appear
to be membrane-bounded inside the cytoplasm of the Sertoli cell. An early sign of
degeneration of the germ cells is the disappearance of all its membranes including
the plasmalemma. Careful examination of
the neighboring Sertoli cell cytoplasm fails
to reveal any local changes in population
of organelles, such as an increase in concentration of lysosomes, which could be
correlated with the germ cell degeneration
or residual body phagocytosis.
Near the base of the seminiferous epithelium three types of junctional complexes join adjacent Sertoli cells to each
other. These are essentially identical to the
Sertoli cell junctions described previously
in the rat by Dym and Fawcett ('70) and
include: 1. A series o f tight occluding
junctions which extend for a considerable
length in either direction perpendicular to
the plane of a section. 2. Nexuses or gap
junctions with a 20 A interspace. 3. Narrowing of t h e intercellular space t o 70 A
without local specialization of cell surfaces.
The junctional complexes between Sertoli
cells are characterized by subsurface filaments hexagonally packed in bundles
which run parallel both to the cell surface
and to cisternae of endoplasmic reticulum
subjacent to the filaments.
T h e tunica propria of monkey
seminiferous tubules
The tunica propria of the monkeys is
markedly different from that found in
rodents and more closely resembles the
pattern in humans. Whereas in the rodents
two layers of flattened cells surround the
seminiferous tubules, monkeys exhibit
three to five circumferentially arrayed cell
layers each one overlapping the other but
separated by intercellular spaces of at least
300 to 400 A (fig. 8). On no occasion are
occluding junctions observed between
neighboring cells. The innermost cell layer
immediately subjacent to the seminiferous
tubules of subhuman primates contains
abundant filaments and is probably contractile, whereas the more peripherally
placed cells exhibit fewer filaments and
are fibrocyte-like. This is in contrast to
rodents where the outer cell layer is the
endothelium of large interstitially placed
Tracer experiments
The horseradish peroxidase injected intravenously in vivo and the lanthanum
nitrate perfused agonally with the fixative,
exhibited a distribution which was essentially identical in all respects. Both of these
electron opaque markers are intercellular
tracers and normally do not enter into the
cytoplasm of cells except in small quantities via pinocytotic vesicles. It is generally
agreed that if vascularly infused peroxidase is found within the cytoplasm of cells
in large amounts, this is an artifact. Although such artif acts occasionally occur
with the recommended dose, artifacts are
produced consistently when ten times the
recommended dose of peroxidase is used.
Rodent seminiferous tubules are easily
teased apart in the fresh native condition
(Christensen and Mason, '65; Dym and
Clermont, '70), and indeed the intertubular areas consist of a very loose connective tissue and large pervasive lymphatic
sinusoids. This may account for the rela-
Fig. 5 A n electron micrograph of a monkey Sertoli cell nucleus demonstrating the bizarre nuclear
pattern. Note the filaments surrounding the nucleus. Stage X. x 22,295.
Fig. 6 An electron micrograph of a portion of a monkey seminiferous tubule showing four residual bodies of Regaud within the Sertoli cell cytoplasm. Note the parallel profiles of granular
endoplasmic reticulum in the upper left. Stage VII. x 10,920.
tive ease with which exogenously added
proteins such as horseradish peroxidase
reach the seminiferous tubules. On the
other hand, monkey and human tubules
(Barn, '67) are tightly packed together and
do not permit such an easy separation. In
primates morphological examination of
the interstitial tissue reveals abundant
dense collagen fibrils. The intertubular
lymph channels, more typical of such vessels in general (Fawcett et al., '73), are
lined by a single layer of flattened endothelial cells and contain a fine protein precipitate. Nevertheless, in the monkey experiments the vascularly introduced tracers
leave the capillaries, fill the interstitial
spaces and easily enter the seminiferous
epithelium by passing between the circumferential peritubular cells. In the seminiferous epithelium the tracers demarcate the
spermatogonia and early spermatocytes by
occupying the interspaces between these
cells and the Sertoli cells, as in rodents
(figs. 10, 1 1 ) . Further penetration toward
the tubule lumen is effectively prevented
by the occluding junctions between the
SertoIi cells. The tunica propria in monkeys does not appear to prevent or retard
the penetration of the tracer molecules
into the seminiferous epithelium. Examination of numerous sections from one
testis reveals the same pattern of penetration indicating that there is no variation in
permeability along the length of a seminiferous tubule. Consequently there is no variation in the different stages of the seminiferous epithelium cycle. This confirms the
suggestion (Fawcett and Ito, '72) that in
monkeys the tunica propria offers no barrier to penetration of vascularly introduced
substances. The only component of the
blood-testis barrier in the monkey appears
to be the Sertoli-Sertoli occluding junction.
On rare occasions, peroxidase and lanthanum are observed in large quantities
inside the cytoplasm of scattered germ cells
and Sertoli cells but this is interpreted as
artifact since these areas of the seminiferous tubules seem to be poorly fixed, as
manifested by membrane discontinuities
and organelle disruption.
The results in mice confirmed our previous work on the rat concerning the location of the blood-testis barrier in rodents
and the distribution of the markers (Dym
and Fawcett, '70). Over large areas of the
seminiferous tubules the tracers are excluded from the seminiferous epithelium
by the peritubular myoid layer and their
edge-to-edge tight junctions (fig. 9). In
other areas of the rodent tubules where
open junctions separate adjacent myoid
cells the tracers penetrate between the
cells of the tunica propria (fig. 10) and
fill the interspaces surrounding the spermatogonia and early spermatocytes. As in
the monkey, further penetrating toward
the tubule lumen is blocked by the more
effective component of the blood-testis barrier, the Sertoli-Sertoli occluding junction.
On no accasion do tracers penetrate these
junctions to reach the more advanced germ
cells or the tubule lumen.
Spermatogenesis is a long complex process originating with the renewal and differentiation of the type A spermatogonia
and concluding with the release of spermatozoa into the lumen of the seminiferous tubules. The microenvironment in
which these events occur is provided by
the Sertoli cell; therefore, it is reasonable
to suggest that the production of sperm is
largely dependent upon the normal functioning of this cell type. Yet surprisingly
little is known concerning the role played
by the Sertoli cell in spermatogenesis. On
the basis of its shape and strategic position
in the seminiferous tubules the functions
of support for the germ cells and provision
of bloodborne nutrients have been suggested. Recent evidence has implicated the
Sertoli cell in the release of late spermatids
into the tubule lumen and the retention of
the residual bodies within the seminiferous
epithelium (Fawcett and Phillips, '69).
Lacy and coworkers (Lacy et al., '68; Lacy
and Pettitt, '70) have suggested that this
Fig. 7 A n electron micrograph of a degenerating spermatogonium within the cytoplasm of a
Sertoli cell. Note the lack of germ cell membranes including the nuclear envelope and pIasmalemma. The limiting membrane investing the
degenerating figure (small arrowheads) is believed to belong to the Sertoli cell. Compare the
thickness of this membrane with the nearby germ
cell-Sertoli cell membranes to the right of the
small arrowheads. For an enlargement of this
region see "inset." The large arrowheads depict
the junctional complexes between adjacent Sertoli
cells. Stage VII. x 7735.
Figure 7
Fig. 8 A n electrom micrograph of the tunica propria of a monkey seminiferous tubule.
Wide intercellular spaces (at least 300 to 400 A ) separate the peritubular cells of the seminiferous tubules (arrowheads). x 7280.
cell type produces a steroid hormone involved in local control of the cycle of the
seminiferous epithelium. This latter function remains controversial. Hall et al. ('69)
rejected a steroidogenic function for the
Sertoli cells because the seminiferous
tubules are unable to convert cholesterol7a-TI to androgens in vitro. Numerous profiles of smooth endoplasmic reticulum and
mitochondria are common in Sertoli cells
and in steroidogenic cells, but this similarity does not in itself substantiate a claim
that the Sertoli cell produces steroids.
The role of the Sertoli cell in phagocytizing residual bodies has been suggested
(Rolshoven, '44; Roosen-Runge, '55; Sapsford et al, '69) and correlated with lysosomes (Dietert, '68) and acid phosphatase
activity in the seminiferous tubules (Niemi
and Kormano, '65), but little experimental
evidence has been provided to demonstrate
conclusively its phagocytic ability (Carr
et al., '68). The fate of the numerous degenerating germ cells (Oaltberg, '56; Cler-
mont, '62) during normal spermatogenesis
has also received sparse attention. It may
be recalled that one-third to one-half of
the total germ cells in the testis degenerate
(Clermont, '62; Clermont and BustosObregon, '68) and are presumably continuously digested by the Sertoli cell. However, the ultrastructure of the Sertoli cell
cytoplasm containing these degenerating
figures is not altered in any way to suggest
an active phagocytosis. No doubt more experimentation will be necessary in order
to determine the ultimate fate of the vast
numbers of degenerating figures and the
role played by the Sertoli cell.
The fine structural morphology of the
major cellular organelles in the monkey
Sertoli cell appears identical in all stages
of the cycle of the seminiferous epithelium,
aDparently both in quantity and quality.
This was somewhat disappointing since we
had hoped to find Sertoli cell organelle differences in various stages which could be
correlated with the dramatic morphological
65 1
Fig. 9 A light micrograph of several mouse seminiferous tubules showing the distribution of vascularly injected horseradish peroxidase. The black reaction product is present in the interstitial tissue
and surrounds the seminiferous tubules. No peroxidase is found inside the lumen of the seminiferous
tubules. x 182.
Fig. 10 An electron micrograph of a mouse seminiferous tubule showing the distribution of
peroxidase. The tracer penetrated the peritubular myoid cell layer via the intercellular spaces (see
anow a t bottom left) and entered the germinal epithelium. Within the epithelium the tracer delineated a spermatogonium by filling the intercellular space between this germ cell and the neighboring
Sertoli cells. Further penetration toward the tubule lumen is blocked by the Sertoli-Sertoli occluding
junctions (arrowheads). x 9,555. (Micrograph courtesy of Dr. Roberto Vitale-Calpe).
Fig. 11 An electron micrograph of a monkey spermatogonium surrounded by lanthanum nitrate.
The lanthanum was perfused into the monkey’s testis with the fixative. The junctional complexes between Sertoli cells (arrowheads) above the spermatogonium prevent the electron-opaque tracer from
deeper penetration into the seminiferous epithelium. x 10,920.
changes in germ cells during differentiation. We do not wish to imply that biochemical events along the length of the
seminiferous tubules are similar. Major
enzymatic alterations are well documented
for various germ cell types (Blackshaw,
'70; Mills and Means, '72) and both nuclear and cytoplasmic structural proteins
alter quantitatively in different areas of
the testis (Davis and Langford, '70). These
modifications have not been assigned to
specific stages of the cycle in the majority
of the studies. Recently, a technique was
described which permits visual identification of spermatogenic stages in long segments of freshly teased seminiferous tubules (Parvinen and Vanha-Perttula, '72)
and three enzymes were correlated with
certain stages of the cycle of the seminiferous epithelium.
A significant step forward in understanding the physiology of the seminiferous tubules was the discovery of a bloodtestis barrier by Kormano ('67) and
Setchell ('67). By cannulating the rete
testis and individual seminiferous tubules
of rams and rats (Waites and Setchell, '69;
Tuck et al., '70), it was revealed that the
composition of a variety of substances in
the fluid within the lumina of the tubules
is markedly different from that found in
blood plasma. Proteins and certain amino
acids that are abundant in in peripheral
blood, were either absent or present in very
low concentrations in the rete testis fluid.
Certain sugars and ions also exhibited significant differences in concentration. This
led Setchell ('67) to conclude that there is
a barrier in or around the seminiferous
epithelium which prevents many bloodborne substances including proteins from
reaching the tubule lumen.
Using silver nitrate staining methods,
Regaud ('01) described two flattened,
plate-like layers of cells surrounding the
seminiferous tubules in rodents. This
original observation has been confirmed
repeatedly, and more recently with the advent of the electron microscope a better
definition of the tunica propria was possible (Clermont, '58; Ross, '67; Dym and
Fawcett, '70). The spermatogonia, early
spermatocytes, and the Sertoli cells rest on
a thin basal lamina separated from the
innermost epithelioid layer of cells by a
clear zone containing sparsely distributed
collagen fibrils. Outside of this is a second
layer of cells described by Fawcett, I-Ieidger
and Leak ('70) as lymphatic endothelial
cells. The innermost cell layer contains
numerous filaments, and evidence suggests
that these are responsible for the contractions of the seminiferous tubules observed
in vitro. In rodents adjacent myoid cells
are frequently joined by tight junctions,
but occasionally they are found separated
by a continuous interspace of 200 A. Electron microscopic studies using lanthanum
nitrate have indicated that the peritubular
myoid cell layer in rodents prevents the
vascularly introduced tracers from reaching the seminiferous epithlium over large
areas of the tubules (Dym and Fawcett,
Our results have demonstrated that
there is also a blood-testis barrier in monkeys. Since the peritubular cells in monkeys overlap and do not meet edge-to-edge,
it was expected that the only component
of the primate barrier would be an intraepithelial portion, namely the SertoliSertoli occluding junctions. This hypothesis was borne out by the tracer studies.
It is likely that the location of the bloodtestis barrier in humans is similar to that
observed in monkeys, since both primates
have much the same organization of the
tunica propria.
Experiments in this laboratory have confirmed the presence of a blood-testis barrier in monkeys, rats, mice, hamsters, and
guinea pigs. Other studies indicate that the
barrier is present in rams, sheep, and bulls
(Waites and Setchell, '69). Since the phenomenon of the blood-testis barrier is of
such general occurrence in the animal
kingdom it probably has a fundamental
importance in the control of spermatogenesis, but the full biological significance
of this relationship still remains unclear.
The integrity of the barrier depends upon
the normal functioning of the occluding
junctions between adjacent Sertoli cells.
Therefore, we can now add to the accepted
functions of support and nutrition for the
Sertoli cell the role of the maintenance of
the blood-testis barrier and the compartmentalization of the seminiferous epithelium.
The significance in rodents of a partial
barrier in the peritubular layer of cells to
vascularly injected tracers remains obscure. Since the tracers used in these experiments are available for only very short
periods of time prior to fixation (horseradish peroxidase) or are added with the
fixative (lanthanum), this does not simulate the in vivo physiological condition.
If it were possible to permit exogenous
peroxidase or other markers to circulate
in the blood for many hours or days, it is
likely that the tracers would penetrate
through the permeable sites in the myoid
layer and then equilibrize in all directions
along the length and width of the seminiferous tubules (Bennett, '71). Thus it is
probable that, in rodents as in monkeys,
circulating plasma constituents of a similar or smaller size to horseradish peroxidase and lanthanum nitrate have continuous access to the germ cells in the basal
compartment of the seminiferous epithelium and the Sertoli cells. It is not expected
that longer availability of marker substances would breach the intra-epithelial
portion of the blood-testis barrier since on
no occasion was the tracer observed deeper
in the seminiferous epithelium.
The subdivision of the seminiferous epithelium into a basal compartment, between
the occluding Sertoli cell junctions and the
basal lamina and an adluminal compartment between the Sertoli cell junctions and
the tubule lumen may be important for
a number of reasons. The germ cells in the
basal compartment are the spermatogonia
and preleptotene spermatocytes and bloodborne substances have direct access to
these cells via the interstitial tissue and the
intercellular spaces in the tunica propria.
Biological processes associated with stem
cell renewal and differentiation, such as
DNA synthesis, which occur in the basal
compartment of the epithelium may be
directly under the control of circulating
plasma constituents. Other renewal systems in the body such as the intestinal
epithelium, stratified squamous epithelium
of skin, and blood forming elements are
not sequestrated behind a barrier but exposed directly to circulating substances in
the plasma. It would be surprising if
DNA synthesis and mitotic division in the
testes occur in a different environment.
Since circulating blood-borne substances
can reach the spermatogonia directly without passing through the Sertoli cells, stem
cell renewal in the testis may occur independently of Sertoli cell function.
Stem cell renewal is not a specialized
function of the testis but similar to other
renewal systems in the body. On the other
hand when the germ cells of the testis
enter the long meiotic prophase of the first
maturation division they leave the basal
compartment of the seminiferous epithelium and enter the specialized environment
of the adluminal compartment. Germ cells
in this area of the seminiferous tubules are
not exposed directly to circulating plasma
substances. To reach these advancing
germ cells all such materials must pass
through the cytoplasm of the Sertoli cells
and are subject to modification. Meiotic
prophase, the two maturation divisions and
spermiogenesis are processes unique to the
gonads which may require the special
environment provided by the Sertoli cell
junctions and the blood-testis barrier. It is
reasonable to suggest that these specialized
biological functions of the seminiferous
tubules are dependent upon the Sertoli
cell. Therefore, it is not surprising that
attempts to culture isolated germ cells beyond meiosis have failed (Eddy and Kahri,
'71). Perhaps when we learn to simulate
the environment produced by the normal
Sertoli cell, further germ cell differentiation in vitro will be achieved.
The results obtained by Reddy and
Svoboda ('67) and Aragon, Lustig and
Mancini ('72) indicated that there is penetration of peroxidase into the cytoplasm
of cells of the seminiferous epithelium and
into the tubule lumen. This contradicts
our results. We have not observed horseradish peroxidase inside the cytoplasm of
the seminiferous epithelial cells nor in the
tubule lumen. Their studies were carried
out with the light microscopy, and frequently the photographs were too low in
magnification to determine cellular outlines. Furthermore, the fixation was not
optimal and identification of germ cell
types was not possible.
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