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Further observations on tubulobulbar complexes formed by late spermatids and sertoli cells in the rat testis.

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Further Observations on Tubulobul bar Complexes Formed by
Late Spermatids and Sertoli Cells in the Rat Testis
Department of Physiology and School of Medicine, Southern Illinois University at
Carbondale, Carbondale, Illinois 62901
The present study extends earlier findings (Russell and Clermont, '76) by describing additional details of the structure, relationships, distribution, origin, and fate of tubulobulbar complexes. Freeze-fracture and thin
section observations reveal few membrane associated particles and no appreciable cell coat in the membranes forming the tubulobulbar complex. The opposing
plasma membranes of the complex do not appear to form a junction, although
junctional formation might be expected by the close proximity (4.0 nm apart)
of the Sertoli and spermatid plasma membranes. Sertoli filaments of the network which encircle the tubular portion of the complex measure 5.0-7.0 nm
across and appear to insert into the Sertoli plasma membrane. Tubulobulbar
complexes initially form in association with a cell surface modification (bristlecoated pit) of the Sertoli plasma membrane. The first tubulobulbar complexes
develop large bulbous components (up to 2.5 p m across), which soon lose connection with the spermatid and become incorporated into large phagocytic vacuoles (secondary lysosomes). As bulbs undergo dissolution, newly formed
tubulobulbar complexes are observed to replace these structures. Thus, the data
indicate that more than one generation of tubulobulbar complexes develop.
Moreover, the tubular and bulbous portions of most dissociating complexes (as
well as neighboring Sertoli lysosomes) show acid phosphatase activity. Near the
time of sperm release, all complexes a t the concave aspect of the spermatid
head are resorbed. New tubulobulbar complexes, many lacking terminal bulbous dilations, form a t the dorsal and lateral aspects of the spermatid head.
These persist even after all ectoplasmic specializations and most Sertoli cytoplasm have been withdrawn from a position facing the spermatid head. The
presence of tubulobulbar complexes just prior to the time of sperm release is in
support of previous findings indicating that tubulobulbar complexes participate
in anchoring the head of the late spermatid prior to sperm release. Past the
zone of sperm release a few abnormally shaped cells, which had not been
released with other spermatids of the same generation, display intact tubulobulbar complexes. The persistence of these and other structures may be the
means by which abnormally shaped spermatids are retained and prevented
from traversing the male duct system.
The tubulobulbar complex has been described as a structural relationship between
Sertoli cells and late spermatids as the latter
cells approached release into the tubular
lumen (Russell and Clermont, '76; also fig. 1).
The postulated function of tubulobulbar complexes is to act as anchoring devices which
retain the heads of the spermatids a t the surface of the seminiferous epithelium. DissoluANAT. REC. (1979)194: 213-232.
tion of these structures would contribute to
the release of the spermatozoan from the
seminiferous epithelium into the tubular lumen (spermiation). The present study provides new data concerning tubulobulbar complexes and the interrelationships of Sertoli
cells. It emphasizes the progressive turnover
Received June 8, '78. Accepted Nov. 21,'78.
of tubulobulbar complexes in the period prior
to sperm release and also the role of the Sertoli
cell in this process.
Preparation of freeze-cleave replicas
Four Sprague-Dawley rats were anesthetized with sodium pentobarbital and the
testes fixed by vascular perfusion (Vitale et
al., ’73) with a 3.5%solution of glutaraldehyde
in 0.2 M sodium cacodylate buffer (pH 7.4) preceded by a washout of 0.9% saline. After the
testes hardened (-20 minutes) they were removed, diced into 1.0-mm blocks and placed in
three changes of a 30%glycerol solution. The
tissue was frozen in freon, cooled with liquid
nitrogen, and fractured in a Balzers freeze-
fracture apparatus. Platinum replicas coated
with carbon were prepared, isolated and
viewed under a Phillips 201 electron microscope. The terminology employed to describe
membrane faces was that proposed by Branton et al. (‘75).
Standard tissue preparation
Testes from two Sherman and four SpragueDawley rats were fixed by vascular perfusion
(see above) of 5.0%glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.4). Testes were removed, cut into small pieces and fixed in perfusate for an additional hour. Tissue was
washed in buffer and post-fixed either with
2.0% osmium tetroxide, or with a 2.0%
osmium:2.5% potassium ferrocyanide mixture
A b breuiations
b, Sertoli plasma membrane
b , spermatid plasma membrane
a, acrosome
b, bulbous portion of the tubulobulbar complex
cp, bristle-coated pit
es, ectoplasmic specialization
Es, extracellular face of the Sertoli
plasma membrane
Et, extracellular face of the spermatid
plasma membrane
f, filaments
1, lysosome
n, nucleus
Ps, protoplasmic face of the Sertoli
plasma membrane
Pt, protoplasmic face of the spermatid
plasma membrane
pv, phagocytic vacuole
S, Sertoli cell
t, tubular portion of the tubulobulbar
ser, smooth endoplasmic reticulum
Fig. 1 The relationship of the late (step 19) spermatid to the Sertoli cell apical expansion a t the luminal aspect of
the seminiferous tubule is depicted. In this micrograph, a tubulobulbar complex, showing both tubular and bulbous portions, is seen in longitudinal profile. The perinuclear cytoplasm (pc) of the spermatid head is continuous with the cytoplasm of the tubular portion of the complex. In this particular micrograph this body of cytoplasm appears somewhat
denser than is usually seen. The intercellular space between spermatid and Sertoli cell (S) is considerably reduced in the
region where these two cells form the tubulobulbar complex. Facing the head of the spermatid is a subsurface modification of the Sertoli cell, consisting of filaments and more deeply placed saccules of endoplasmic reticulum, termed the
ectoplasmic specialization (Russell, ’7%; es). Tissue treated with a ferrocyanide:osmium mixture. x 39,000.
Figs. 2-4 Tissues treated with an osmium:ferrocyanide mixture. Mid-Stage VII.
Fig. 2 Longitudinal section of a tubulobulbar complex measuring approximately 4.0 wm in length. The diameter of
the tubular portion of the complex is remarkably uniform. In this particular micrograph, the bulb appears to be
unusually small. X 50,000.
Fig. 3 Cross section of the tubular portion of the complex showing the trilaminar appearance of both spermatid and
Sertoli plasma membranes. The intercellular space is approximately one half the width of one of the individual plasma
membranes. No evidence of a glycocalyx is seen on the external aspect of either membrane. Filaments around the tube
are poorly visualized. x 165,000.
Fig. 4 Longitudinal section of a portion of tubulobulbar complex showing nearby Sertoli filaments. In several areas
(indicated by arrows) these filaments appear to make contact with the Sertoli plasma membrane. x 83,000.
Figs. 5-7 Tissues secondarily treated with osmium. Mid- and late-Stage VII.
Fig. 5 Longitudinal section of the tubular portion of a tubulobulbar complex of late-stage VII showing the “wavy”
course which the complex often takes. Sertoli filaments are prominent and extend about 0.2-0.3p m into the Sertoli cell.
These filaments are packed most tightly near the Sertoli plasma membrane. In a few areas (arrow) the spermatid tube
is discontinuous. The Sertoli cytoplasm near the tube (also fig. 2) is virtually free of organelles. x 36,000.
Fig. 6 Cross section of a spermatid showing only the ventro-lateral aspect of the head. Continuity of the plasma
membranes of the Sertoli cell and spermatid with their respective membranes forming the tubular portion of the complex is demonstrated. The plasma membrane of spermatid head proper appears thicker than it is within the tubular portion of the complex. This is the result of the presence of an appreciable cell-coat on the spermatid plasma membrane of
the head, whereas this feature is not evident in the tube. X 65,000.
Fig. 7 Cross-section of the tubular portion of the tubulobulbar complex. Although filaments are prominent, the
trilaminar structure of membranes is not evident. The intercellular space in tissues treated in this manner appears
slightly wider than that shown in figure 3. x 90,000.
(Russell and Burguet, ’77; modified from Karnovsky, ’71). Subsequent dehydration, infiltration and embedding in Epon were according to standard techniques. At the time of
embedding, seminiferous tubules were oriented to produce either longitudinal or crosssectional profiles upon sectioning. Gray, silver
and silver-gold appearing thin sections were
prepared with an ultramicrotome, stained
with uranyl acetate and lead citrate and examined with a Phillips 201 transmission electron microscope.
tissues incubated in medium lacking substrate or with the addition of sodium fluoride.
Descriptive data
All membranes participating in the formation of the tubular or bulbous portions of the
tubulobulbar complex measured about 7.5 nm
across. The spermatid plasma membrane over
most other regions of the head appeared
thicker (about 14 nm; figs. 1, 6, 7; Nicander,
’67), but this appearance was due to the presence of a substantial glycocalyx which was not
Timing of spermatogenesis
observed in the membrane of the tubulobulbar
The classification of Leblond and Clermont complex. The evaginated process of the sper(’52) was used to select tubules a t the desired matid which forms the tube appeared much
stages of the spermatogenic cycle. Since it closer to the Sertoli plasma membrane (fig. 3)
was necessary to follow sequential events re- than reported for osmium treated tissues
lating to the formation and degradation of t u - (Russell and Clermont, ’76). Thus, in ferrobulobulbar complexes, it was critical that cyanide-osmium treated tissues the interStages VII and VIII be divided into recogniza- cellular space is recorded to be about 4.0 nm in
ble “substages” which could be used to “time” width whereas by osmium-only treatment it
related events. The term substage is used to was about 6.0 nm (compare figs. 3, 7). Sertoli
designate a particular cross-sectional profile filaments surrounding the tubular portion of
of a seminiferous tubular subsegment. These the complex were not as conspicuous after fersubstages, when placed in a logical time rocyanide:osmium treatment (figs. 2, 3) but
sequence order, could then be selected for ob- were more prominent following standard gluservation a t the electron microscope level. The taraldehyde-osmium treatment (figs. 5, 7).
classification of Perey et al. (‘61) based on The degree of packing of the network of filastudies of longitudinal sections of seminifer- ments was tightest just deep to the Sertoli
ous tubules and previously defined subseg- plasma membrane (figs. 5, 7, 8). Isolated filaments was used in the designation of Stage ments measured 5.0-7.0 nm and in many secVII into early, middle and late substages. Our
own observations and those of Perey et al.
Fig. 8 Fortuitous longitudinal section through a
were used to divide Stage VIII into three tubulobulbar complex showing both the proximal (long
substages. In early Stage VIII the apical Ser- arrow) and distal (short arrow) tube and an intervening
The distal tube faces a Sertoli bristle-coated pit.
toli expansion was seen to engulf the whole bulb.
X 30,000.
head region where as in mid-Stage VIII it was
Fig. 9 The distal aspect of a tubulobulbar complex,
only related to a small area of t h e head portion similar to t h a t shown in figure 8, is seen a t high magof this cell (Russell, ’77b).Late Stage VIII was nification. A bristle-coated pit is present a t this site and
can be identified by t h e fuzzy substance projecting from
designated as the particular substage in t h e internal aspect of t h e Sertoli plasma membrane. The
which sperm release had already taken place spermatid tube is slightly expanded a t its distal tip.
Fibrils are seen extending from i t to the Sertoli bristle(Perey et al., ’61).
Acid phosphatase cytochemistry
Testes were perfused with a 2.0% buffered
glutaraldehyde, washed in buffer and slices of
tissue (75-100 p m in thickness) were incubated at 37°C in a medium containing sodium-/3-glycerophosphate a t pH 5.0 for 90
minutes (Barka and Anderson, ’63). Tissues
were washed in buffer containing sucrose and
post-fixed in 1.0%osmium tetroxide. Dehydration, infiltration and embedding were similar
to that described above. Controls consisted of
coated pit. X 200,000,
Fig. 10 This large bulbous component of a tubulobulbar complex is typical of those seen in early Stage VII.
I t measures approximately 2.0 p m by 1.0 pm. Dispersed
flocculent material is observed inside t h e bulb and smooth
endoplasmic reticulum of the Sertoli cell is closely apposed to it. X 70,000.
Fig. 11 High magnification micrograph of the limiting
aspect of a bulbous portion of the tubulobulhar complex.
The spermatid and Sertoli plasma membranes are about
4.0 nm apart and neither exhibit a glycocalyx. In thin sections of this type, there is no evidence of junctional formation between the two cells. Smooth endoplasmic reticulum is positioned about 15.0 nm from t h e Sertoli plasma
membrane. X 185,000.
tions appeared to make contact with the Sertoli plasma membrane (fig. 4). In longitudinal
section the tubular portion of the complex was
rarely linear, but usually was seen to describe
a wavy course which took it in and out of the
plane of section (figs. 2, 5). The length of the
entire complex was variable, most commonly
measuring 3.0-4.0p m and in some cases up to
5.0 p m (fig. 5). In all areas of the tubular portion of the complex, the diameter of the spermatid tube and width of the intercellular
space were remarkably uniform (figs. 2-81.
The bulbous portion of the tubulobulbar
complex also displayed a 4.0 nm intercellular
space (fig. 11).Individual bulbs measured up
to 2.5 p m in diameter although the majority
were less than 1 p m across. Most bulbs contained evenly dispersed flocculent material
(figs. 8, 101, and a few bulbs showed a concentration of dense homogeneous substance (fig.
23). In some sections it was noted that the distal portion of bulb narrowed down to form
tubular structure similar to t h e tube connecting the bulb to the spermatid head proper (fig.
8). This distal spermatid tube extended only a
short distance (0.1-0.5 pm), and at its termination the facing Sertoli cytoplasm invariably
displayed a bristle-coated pit (figs. 8, 9).
Freeze-cleaue observations
Replicas from Stage VII and VIII tubules
(Stages in which tubulobulbar complexes are
present) could be recognized by identifying
the early generation of spermatids (steps 7
and 8) as well as the large fully-formed flagella of the late generation of spermatids.
Tubulobulbar complexes were encountered
within a recognizable apical Sertoli expansion. The characteristic form of the head of
the late spermatid was also evident (figs. 12,
13). Only the Sertoli plasma membranes
which surrounded the spermatid evagination
was fractured in the plane of the membrane;
however, in the cluster of bulbs an occasional area where the fracture plane coursed
through the spermatid plasma membrane
could be detected (figs. 12-14). In all of the
membrane faces studied, it was apparent that
these membranes displayed only an occasional
membrane associated particle. In fact, none
were seen in the tubular portion of the
complex. For comparison, figure 16 shows intracytoplasmic vesicles within a nearby step 7
spermatid of the same stage, all of which
display numerous membrane associated
A time-course study of tubulobulbar
Early stage VII
Formation of tubulobulbar complexes in
large numbers takes place a t the beginning of
Stage VII. Prior to this time (in Stage VI) the
heads of the late spermatids are deeply embedded within Sertoli recesses and only rarely
during this period were these complexes observed. The newly formed complexes could be
described as short (0.1-0.2p m ) tubular projections of the spermatid which face a corresponding indentation of the Sertoli cell (fig.
17). In every profile of this type observed, the
spermatid projection was seen to oppose a
bristle-coated pit in the depth of the Sertoli recess. (Coated pits are frequently seen a t the
surface of the Sertoli cell including that area
of plasma membrane which faces the spermatid head.) Sertoli filaments were usually
found around the invaginated Sertoli plasma
membrane (fig. 17).
Figs. 12-16 Freeze-fracture replicas of step 19 spermatids, Sertoli cells, and tubulobulbar complexes formed
by the two cells. All figures have been oriented such that
the direction of platinum deposition is from the bottom to
the top of the figure (with the exception of fig. 14 which
has been shadowed from the top).
Figs. 12, 13 The spermatid head (sh) is cross-fractured and the apical Sertoli expansion occupies most of
the area in the figure. Fracture faces indicated are as follows: E face of the Sertoli plasma membrane around the
head; P and E faces of the Sertoli plasma membrane of
the bulb; and P and E faces of the spermatid plasma membrane of the bulb. The tubular portion of the complex can
be identified (isolated arrow) as well as the ectoplasmic
specialization, and smooth endoplasmic reticulum which
faces the bulb. Numerous membrane associated particles
are present in the Sertoli plasma membrane facing the
head (fig. 12), yet few are seen in the bulbous portion of
the complex (figs. 13, 14). X 29,000;X 42,000.
Fig. 14 Replica showing the apical Sertoli expansion
in the region of the clustered bulbs. Indicated are likely
areas of the P and E fracture faces of the Sertoli plasma
membrane in both the tube and bulb; and the P and E
face of the spermatid plasma membrane. Membrane associated particles are not seen on the membranes forming
the bulb. x 37,000.
Fig. 15 Longitudinal fracture of the tubular portion of
the complex showing predominantly the P and E faces of
the Sertoli plasma membrane. The spermatid tube is also
visible (isolated arrow). The membranes display a slightly
“lumpy” appearance (as do all membranes of this area)
but there are no particles intercalated within them.
X 60,000.
Fig. 16 This cross-fracture of a step 7 spermatid
shows both nuclear and cytoplasmic areas of the cell.
Unlike tubulobulbar complexes, vesicles of the cell demonstrate numerous membrane associated particles. The chromatoid body (c) is also fractured, and it is shown to be
composed of closely aggregated cytoplasmic particles associated with small cytoplasmic vesicles. x 39,000.
A bulbous structure was evident as part of
complexes which were slightly larger than
those described above. This dilated component
apparently developed somewhere in the midregion of the spermatid tube since constricted
spermatid tubes were seen both proximal and
distal to the dilation (fig. 18). At this time,
Sertoli endoplasmic reticulum of the smooth
variety was generally observed facing the bulb
(fig. 18). In slightly more developed profiles,
the bulbs were quite large, measuring up to
2.5 p m across (fig. 10). The proximal tubular
portion of the complex elongated and by early
Stage VII attained a length of 3.0-5.0 p m
(fig. 2).
Concomitant with the formation of tubulobulbar complexes in early Stage VII, a remodeling of the Sertoli cell cytoplasm took place.
An apical Sertoli expansion circumscribing
the spermatid head was formed (figs. 19;
Russell and Clermont, ’76) and the head was
noticeably more sickle-shaped. Thus, tubulobulbar complexes emanating from the concave
aspect of the spermatid ended in bulbous
expansions which were clustered together
among lysosomes and lipid droplets. Tubulobulbar complexes also formed a t the lateral or
the dorsal convex aspect of the spermatid (fig.
171, although a t this time they were not commonly found to emanate from these sites.
Near the completion of early Stage VII, it
was observed that not all bulbous endings appeared identical. One type of bulb appeared
similar to those which initially formed in early Stage VII (fig. 19). In another type, the
spermatid plasma membrane was found to be
lacking a connection with the spermatid head,
i.e., in numerous sections and profiles surveyed, no connection of the bulb to the tubular
portion of the complex could be demonstrated.
Flocculent material was in both types of
bulbs, yet only in connected bulbs was it evenly dispersed. Here its appearance closely resembled the cytoplasm of the head region
proper. Because the flocculent material was
clumped in the disconnected bulbs, areas
devoid of this substance were especially electron translucent (figs. 19, 21). In sections of
“disconnected” bulbs, the Sertoli endoplasmic
reticulum was either not as extensive or was
absent from its characteristic position facing
the bulbs. It should be emphasized that although serial section of this type of bulb was
not undertaken to conclusively demonstrate
this discontinuity, extensive examination of
many hundreds of this type of bulb never
showed it to be connected to the spermatid
head proper.
Mid-Stage VII
By mid-Stage VII very large vacuoles were
seen in juxtaposition to the clustered bulbs
(figs. 20-24). Within these vacuoles were two
or more smaller, membrane-bound vesicles
and a dense substance which filled in the gap
between these vesicles (figs. 20-23). Lysosomes displaying dense material of a similar
nature were numerous in the area of the vacuoles (figs. 20, 241, and in several instances
the bounding membranes of the lysosome
encircled or partially encircled bulbous endings (figs. 20-23). Upon closer examination of
the vesicular structure contained within vacuoles, i t was found t h a t many displayed aggregated flocculent material of the type seen
within the bulbous portions (disconnected
bulbs) of the complex (figs. 20, 23). The space
between the membranes of the vesicle and
t h a t of the bounding membrane of the vacuole
(in regions where they came into close apposition) was about 4.0 nm (figs. 21, 22). Based
upon size, contents, appearance and morphological relationships, it was assumed that the
structures within the phagocytic vacuoles (lysosomes containing bulbs) were bulbous endings which were freed from their connection to
the spermatid tube and were in the process of
undergoing dissolution.
During mid-Stage VII the vesicles contained within the phagocytic vacuoles apFigs. 17-19 Early Stage VII.
Fig. 17 Forming tubulobulbar complex at the dorsal
convex aspect of a step 19 spermatid. A bristle-coated pit,
seen at the depth of a Sertoli invagination, is associated
with a short evagination of the spermatid plasma membrane. The spermatid plasma membrane “thins” down
considerably (i.e., the glycocalylx is lost) in the region of
the evagination. x 75,000.
Fig. 18 Newly formed tubulobulbar complex at the
ventral (concave) aspect of the spermatid. The tube remains constricted in both its proximal and distal portions;
however, a central bulb has developed which is flanked by
smooth endoplasmic reticulum of the Sertoli cell. A bristle-coated pit is present at the depth of the Sertoli recess.
X 46,000.
Fig. 19 Section of the Sertoli apical expansion showing the spermatid head and several clustered bulbs. Two
types of bulbs are present; smaller bulbs with evenly distributed flocculent material, and larger ones with a
patchy distribution of this same type of material. The
smaller ones demonstrate association with Sertoli smooth
endoplasmic reticulum and a connection with the tubular
portion of the complex, whereas the larger ones have little
or no association with this reticulum and do not demonstrate a connection with the tubular portion of the complex. X 38,800.
peared smaller, developed irregular outlines,
and thus were barely recognizable as vesicular
structures. Several large phagocytic vacuoles
of the type just described may be seen within
each apical Sertoli expansion, although the
contents of each may be in different stages of
dissolution (fig. 20).
During the period in which the large bulbous endings (those formed in early Stage VII)
were being resorbed, attention was directed
toward determining the influences of those
events on the status of the remaining tubulobulbar elements in the concavity of the sickleshaped head. The following morphological observations were recorded: (1) tubulobulbar
complexes were about as numerous, or more
so, in mid-Stage VII as in early Stage VII; (2)
the general arrangement of these structures
within the apical Sertoli expansion remained
unchanged from that of early Stage VII; (3)
tubulobulbar complexes in an early phase of
development were occasionally seen a t this
time (fig. 23); (4) bulbous endings of tubulobulbar complexes were smaller (0.2-0.5
pm across) in mid-Stage VII and more elongated than those seen in early Stage VII (like
that shown in fig. 25).
Late Stage VII and early Stage VIII
In late Stage VII and early Stage VIII the
ectoplasmic specializations (filaments and
more deeply placed endoplasmic reticulum)
were displaced from their position facing the
head of the spermatid (figs. 23, 24). Large
phagocytic vacuoles and the smaller primary
lysosomes were also a conspicuous feature in
the apical Sertoli droplet in late Stage VII and
early Stage VIII (figs. 23,241. The contents of
some vacuoles, because of distortion of their
internal membranes, were in an advanced
stage of degradation whereas others appeared
in the initial stages of resorption.
Sections which reveal the tubular portion of
the complex to best advantage showed that
many of these tubular structures were also in
the process of being resorbed. In cross sections,
the spermatid tube was often absent, or when
present, its plasma membrane appeared discontinuous (fig. 27). When sectioned longitudinally, the spermatid component of the tube
was represented by stacked cylinders along
the tube with “empty” areas seen between
each cylindrical fragment (figs. 25, 26). Some
fragmented tubes of the type just described
could be traced to bulbous structures whereas
others terminated blindly facing a Sertoli
bristle-coated pit (fig. 26). With time, further
regressive changes were observed in the tubular portions of the complexes, which led t o the
eventual resorption of these structures.
At the same time these regressive phenomena were noted, it was found that new tubulobulbar complexes were being formed. This was
evidenced by bristle-coated pits in the depth
of Sertoli invaginations and short opposing
evaginations of the spermatid plasma membrane, all of which were found along the dorsal
convex and lateral aspects of the spermatid
(also seen in mid-Stage VIII; fig. 28, inset).
When fully formed, the evaginated spermatid
tube usually extended a variable distance to
end blindly facing a Sertoli bristle-coated pit.
A bulbous component was usually not part of
this complex.
Mid-Stage VIII
The apical expansion in which the head of
the late spermatid is embedded was gradually
withdrawn (Russell and Clermont, ’76;
Russell, ’77b). The mass of Sertoli cytoplasm
occupying the concavity of the sickle-shaped
head was first to be withdrawn leaving
smaller processes of Sertoli cytoplasm in contact with the dorsal convex and the lateral
surfaces of the head (figs. 28,291. Appropriate
sections revealed that both intact and fragmented tubulobulbar complexes extended into
Figs. 20-22 Mid Stage VII: ferrocyanide:osmium
treated tissues.
Fig. 20 Cross section of the spermatid head and the
apical Sertoli expansion. Lysosomes and lipid droplets (li)
and phagocytic vacuoles are seen in the region where
bulbs are clustered. Evenly dispersed flocculent material
lies within some of the bulbs; in others this material appears clumped. Only in the former does the Sertoli smooth
endoplasmic reticulum face the bulb. One bulb (isolated
arrow) appears to be engulfed (or surrounded) by a phagocytic vacuole. X 34,000.
Fig. 21 Apical Sertoli expansion showing the bulbous
portions of two tubulobulbar complexes. Flocculent material within these bulbs appears clumped. Note the absence
of associated smooth endoplasmic reticulum. In most
areas the Sertoli and spermatid membranes are very close
together (about 4.0 nm), however, in one region, small vesicles and an electron dense lysosome-like material lie between the spermatid and Sertoli plasma membranes.
X 46,000.
Fig. 22 A small bulb is shown a t the upper right portion of this figure within a n apical Sertoli expansion. It
displays evenly dispersed flocculent material and facing
Sertoli endoplasmic reticulum. At the lower left is a large
phagocytic vacuole containing numerous internally p i tioned bulbs. These bulbs display aggregates of flocculent
material and are separated from the Sertoli plasma membrane by a 4.0 nm space (arrow). A dense substance and
small vesicles (v) fills the gap between phagocytised bulbs
and the Sertoli plasma membrane. x 67,000.
these Sertoli processes. Tubulobulbar complexes were in evidence through mid-Stage
VIII and up until near the time of sperm
release; however, as the sperm release zone
was approached, these complexes generally
appeared fragmented (fig. 29).
Late Stage VIII
In longitudinal sections of seminiferous
tubules during Stage VIII it has been shown
that all but a few sperm are released a t a specific point along the tubule (Perey et al., '61;
Russell, '77a). After release, residual bodies
line the surface of the seminiferous epithelium. Of the few remaining spermatids encountered, some were abnormal in appearance
(fig. 30). In cells of this type that were malformed, the defect generally involved a malformation in the acrosomal region. Especially
interesting was the relationship of these
abnormally shaped cells to the Sertoli cell. It
was similar to that noted for mid and late
Stage VII, i.e.: (1)the Sertoli apical expansion
circumscribed the head of the spermatid, (2)
ectoplasmic specializations were present facing the spermatid head, and (3) intact tubulobulbar complexes were in evidence (fig. 30,
Acid phosphatase cytochemistry
In late Stage VII and early Stage VIII acid
phosphatase activity could be demonstrated
within saccules of the Golgi of all germ cells
as well as within boundaries of lysosomes
throughout the testis, including the large,
clear vacuolar structures of the cytoplasmic
lobe.' As evidenced by an electron dense lead
precipitate, the tubular portions of many complexes were found to be reactive (figs. 32, 33)
as were nearby phagocytic vacuoles (fig. 31).
Control tissues were without reactivity.
The tubulobulbar complex is observed in
two widely separated areas of the seminiferous epithelium (Russell, '75; Russell and
Clermont, '76; Russell, '79). The present report details further observations on tubulobulbar complexes formed between Sertoli cells
and late spermatids.
Structure and relationships of the
tubulobulbar complex
me freeze-cleave technique was employed
to d&ermine if tJlbulobulbar plasma membranes were in any way different from those
plasma membranes of the spermatid head
proper or of the Sertoli cell. Frozen-cleaved
Sertoli membranes of the tubulobulbar complex were shown to possess few membrane associated particles. It was thought that the E
face of the Sertoli plasma membrane around
the spermatid tube might reveal membrane
particles associated with attachment sites of
subsurface filaments or associated with junctional proteins. These features, however, could
not be demonstrated with the freeze-cleave
technique. Most fracture faces identified were
those of the Sertoli cell, although there were
sufficient fractures of spermatid plasma
membranes to show that the internal aspects
of these membranes were likewise nearly particle-free.
In thin-sectioned material the extracellular
aspects of the membranes of the complex were
examined for evidence of a cell coat. Although
a substantial glycocalyx was noted on the
spermatid plasma membrane of the head
proper, this feature was lost as the membrane
became continuous with that of the spermatid
tube of the tubulobulbar complex. Nicander
('67) has also observed a "thinning out" of the
spermatid plasma membrane of the head as it
became the membrane of the spermatid tube.
The Sertoli plasma membrane of the tubulobulbar complex also lacked a visible glycocalyx. Thus, both Sertoli and spermatid
plasma membranes of the complex appeared
quite thin, measuring about 7.5 nm in thickness; and each was deficient of internal mem' The term cytoplasmic lobe ("lobe protoplasmique"; Regaud, '01)
is used to designate that excess cytoplasm of the spermatid which
extends from this cell toward the base of the tubule. Once the cytoplasmic lobe is freed from the spermatid it is termed the resrdual
Fig. 23 Apical Sertoli expansion showing the head of
the step 19 spermatid in mid-Stage VII and portions of
several tubulobulbar complexes. A large phagocytic vesicle containing two bulbous endings is seen at the bottom
of the micrograph. In this same micrograph a developing
tubulobulbar complex is present. All bulbs containing aggregations of dense material. x 42,000.
Fig. 24 Several spermatids circumscribed by apical
Sertoli expansions are shown in this late Stage VII
tubule. The bold arrow at the top of the figure points to
the basal aspect of the same Sertoli cells which reach to
the lumen to surround these spermatids. Both tubular and
bulbous portions of the complex are indicated as well as
lysosomes and a phagocytic vacuole. Sertoli ectoplasmic
specializations have shifted from a position facing the
heads of the late swrmatids to one considerablv closer to
the base of the tubile. Here they are seen alongthe apical
Sertoli stalk, either at its free edge or, in some cases, internalized within the cell. Tissue treated with ferrocyanide:osmium. x 11,000.
brane associated particles as well as an external cell coat.
An especially narrow intercellular space
(4.0 nm) was recorded between the two plasma
membranes forming the tubulobulbar complex, which, at first, was thought to be indicative of some type of junctional relationship between the two cells. The opposite point of view
was taken after the freeze-cleave technique
showed no significant number or particular
arrangement of membrane-associated particles. Lacking a glycocalyx, these plasma membranes may be allowed t o reside much closer to
one another, t o a degree not seen in other
areas where the same two cells are adjoining.
Since membranes of both the tube and the
bulb exhibit this close spacing, it can be
assumed that Sertoli filaments. which are
only present around the tube, are not responsible for drawing the two cells this close
The type of subsurface filamentous condensation around the tube appears similar in appearance t o the contractile ring of dividing
cells. Further studies are needed t o determine
if these filaments are actin or actin-like in
Formation and resorption of
tubulobulbar complexes
To minimize possible error in interpretation
of numerous sequential events, it was found to
be advantageous t o subdivide (based on morphology) known stages of the cycle into several substages (MATERIALS AND METHODS). During these time periods, a likely series of events
can be constructed from micrographs which
depict the formation and dissolution of tubulobulbar complexes. The sequence given in
figure 34 is based on the observations presented herein.
The smallest profiles of tubulobulbar complexes, observed in early Stage VII, consist of
short tubular evaginations of the spermatid
which are found within corresponding Sertoli
invaginations (fig. 34B). A bristle-coated pit is
always present in the depth of the recess
formed by the Sertoli cell. Bristle-coated pits
were not uncommon along the plasma membrane which faces the head of the spermatid
(fig. 34A) and i t is, therefore, tempting to suggest that since these are the first structures
visible that they are responsible for initiating
the formation of this complex. Sertoli cells are
well equipped, by virture of possessing an
abundance of microfilaments and micro-
tubules, t o accomplish the shape changes necessary in the development of tubulobulbar
complexes, whereas the spermatid head visiibly lacks these cytoplasmic organelles. Christensen ('76) has reported that small processes
of a Leydig cell may protrude for a short distance into neighboring macrophages. Although the structures described by Christensen show little resemblance to tubulobulbar
complexes, the two cell-to-cell relationships
share one feature in common. A bristle-coated
pit is always seen in the depth of the recess
formed by the invaginated cell. Available evidence indicates that bristle-coated pits, of the
type seen in this study, are important in the
uptake of materials from outside the cell
(Roth and Porter, '64; Lagunoff and Curran,
'72). To this author's knowledge, they have not
been shown to be involved in the uptake of portions of another cell.
As the tube elongates (figs. 34C,D) and develops a bulbous expansion (figs. 34C,D), appropriate sections show that the bulb develops
in the mid-region of the complex. Thus, the
distal portion of the complex remains narrow
and faces a bristle-coated pit in the deepest
portion of the Sertoli recess. This observation
necessitates a slight modification of our conFigs. 25-27 Late Stage VII or Early Stage VIII; secondarily treated with a ferrocyanide:osmium mixture.
Fig. 25 Apical Sertoli expansion showing a tubulobulbar complex in longitudinal section and a nearby bulbous component. The spermatid tube is fragmented and has
separated from the bulb. X 25,000.
Fig. 26 Longitudinal sections of tulbulobulbar complexes showing that these structures lack a bulbous component. The spermatid tube is discontinuous in many
areas and is represented by stacked cylindrical masses
within the invaginated Sertoli cell. At the depth of the recess, a coated pit opposes the terminal endings of the spermatid tube. x 14,000.
Fig. 27 This section through the apical Sertoli expansion shows several tubes in cross sectional profile. The
spermatid plasma membrane which forms the tubular
portion of the complex is either absent or poorly represented. Sertoli filaments remain a conspicuous structure
in a position surrounding the Sertoli plasma membrane.
X 45,000.
Fig. 28 Survey micrograph showing the relationship
of the late spermatids to the Sertoli cell in mid-Stage
VIII. The direction of the basal aspect of the Sertoli cell,
since only the apical processes of these cells are seen in
this micrograph, is indicated by the bold arrow. These apical processes extend toward the lumen to make contact
with the spermatid a t its dorsal convex and lateral surfaces. Numerous tubulobulbar complexes are seen to extend from the spermatid into these processes. The excess
spermatid cytoplasm or cytoplasmic lobes are also indicated (cl). The inset shows an enlargement of one of these
spermatids and the profile of a developing tubulobulbar
complex. Tissue post-treated with osmium. X 5,200.
cept of the general shape of the tubulobulbar
complex as shown in figure 34D.
The data strongly suggest t h a t more than
one generation of tubulobulbar complexes
form during late spermiogenesis. Observations
to support this suggestion are as follows: (1)
Many bulbous components of the complex undergo dissolution in mid-Stage VII (figs.
34E,F), yet as many intact tubulobulbar complexes (displaying both tube and bulb) remain; (2) Tubulobulbar complexes of early
Stage VII primarily emanate from the ventral
aspect of the spermatid whereas those in
Stage VIII protrude from dorsal convex and
lateral aspects of the cell (fig. 34G); (3) Bulbous components of the complex in mid-Stage
VII are considerably smaller than those initially formed in early Stage VII (figs. 34E-GI;
(4) Newly formed tubulobulbar complexes are
seen throughout Stage VII and Stage VIII
(fig. 34F). The preceding observations are consistent with what might be considered aprocess of active formation and resorption o f
tubulobulbar complexes in the period prior to
sperm release. Assuming that more than one
generation of tubulobulbar complexes is
formed, it is soon realized that a substantial
portion of the spermatid membrane and cytoplasm is resorbed in the 4-day period prior to
sperm release. Considering the resorption of
just one large complex, the tubular portion
measuring 4.0 p m in length and the bulbous
portion measuring 2.0 p m across, the loss of
surface membrane would be approximately
12.0 p m ' and the volume of cytoplasm resorbed would be approximately 4.0 pm3. Up to
24 tubulobulbar complexes associated with
one spermatid may be detected in a single section (Russell and Clermont, '76).
Acid phosphatase cytochemistry
The results from acid phosphatase cytochemistry confirm that it is the phagocytic activity of the Sertoli cell which is important i n
resorbtion of tubulobulbar complexes. Acid
phosphatase is a well known hydrolytic enzyme of lysosomes, which, together with other
lysosomal enzymes, acts to digest material
taken up by cells. Lysosomes are present within the Sertoli cell, in and around the clustered
bulbous portions of the complex, and become
more prevalent as time passes. Both the
phagocytic vacuoles and the tubular portions
of the tubulobulbar complex displayed dense
lead deposits which demonstrate that acid
phosphatase activity is present a t these sites.
The precise manner in which enzymes are
transferred from within the Sertoli cell into
the tubular and bulbous portions of the complex remains to be elucidated; however, the
nearly particle-free nature of the membrane
of the bulb may facilitate fusion between bulb
and lysosome as has been shown for other
membrane systems (Lucy, '70; Ahkong et al.,
Tubulobulbar complexes and spermiation
Relatively little information is available
about the spermiation process. Most investigators, including this author, are in general
agreement with the work of Fawcett and
Phillips ('69) which indicates that the Sertoli
cell, by actively changing its configuration,
influences the release of late spermatids. It is
difficult to envision that the spermatid might
be the sole active agent in this process, for
during spermiogenesis, the spermatid had
been transformed into a highly specialized
gamete, which, in its near final form, hardly
seems capable of engineering its own release.
Fig. 29 Figure shows the relationship of the head of
t h e late spermatid to the apical Sertoli cell process. The
micrograph was taken from a specific area of a longitudinal section of a seminiferous tubule in which t h e sperm
release zone was about 0.2 millimeter further along t h e
tubule. Apical Sertoli processes have withdrawn from t h e
ventral concave aspect of t h e spermatid and are related
only to t h e dorsal convex and lateral aspects of the spermatid. Tubulohulbar complexes are seen extending from
the dorsal aspect of t h e spermatid into this apical process.
Microtubules (mt) are numerous within this apical Sertoli
process and extend along its long axis. Post-treated with
osmium. X 12,000.
Fig. 30 Survey micrograph of late Stage VIII showing
the surface of the seminiferous epithelium. Residual
bodies (rb) are essentially the most superficial structures
since the apical Sertoli processes have been withdrawn
toward the base of t h e tubule. All spermatids have been
released except the two shown a t the upper left of the
figure. In one of these (see inset) the acrosome displays a
bizarre configuration. Here, a n apical Sertoli expansion is
present circumscribing the head. Also, ectoplasmic specializations of the Sertoli cell face the head and the presence of intact tubulobulbar complexes is indicated by
arrows. X 4,300; x 20,000.
Figs. 31-33 Acid phosphatase preparations of l a t e
Stage VII and early Stage VIII.
Fig. 31 Dense reaction product is present within a
large phagocytic vacuole, yet this precipitate lies external
to phagocytosed bulbs. x 27,000.
Fig. 32 Longitudinal section of a n apical Sertoli expansion showing reaction product within cross-sectional
profiles of t h e tubulobulbar complex (bold arrows). Some
tubes show little or no reaction product (small arrows).
x 12,000.
Fig. 33 This longitudinal section of t h e tubular portion of a tubulobulbar complex shows a heavy disposition
of reaction product. Sertoli filaments are seen a t the periphery of the reaction product. x 39,000.
Recent reports have indicated that ectoplasmic specializations are important in the spermiation process. Ross and colleagues have
shown experimentally that the subsurface
bundles of filaments and associated endoplasmic reticulum, which comprise the ectoplasmic specialization, are sites associated with
cell adhesion between the spermatid and the
Sertoli cell (Ross, '76; Romrell and Ross, '78).
Others have suggested that the ectoplasmic
specialization maintains a grasp on the head
of the spermatid (Toyama, '76; Russell, '77b)
andlor acts as a motile apparatus which may
transport the late spermatid (Russell, '77b;
Gravis, '78). All investigators agree that this
surface specialization is important in preventFigs. 34A-H Diagram summarizing the steps in the
formation and dissolution of tubulobulbar complexes in
Stages VII and VIII. This drawing depicts a likely series
of events which is based on numerous electron micrographs from these stages. The Sertoli cytoplasm is shown
in yellow.
A Cross section of a spermatid head showing
the nucleus (n), perinuclear cytoplasm (pc), acrosome (a),
Sertoli cell apical expansion ( 8 ) and Sertoli ectoplasmic
specialization (es). A bristle-coated pit is seen in the Sertoli plasma membrane facing the head of the spermatid
(arrow). Early Stage VII.
B An evagination of the spermatid is seen
within a corresponding recess of the Sertoli cell. The spermatid tube thus formed faces a bristle-coated pit. Filaments are seen within the area of the Sertoli cytoplasm
which faces this spermatid projection. early Stage VII.
C The spermatid tube elongates, and its midportion dilates to form the bulbous portion of the complex.
Sertoli filaments are present, facing both the proximal
and distal narrowings forming the tube. Early Stage VII.
D Further elongation of the proximal spermatid tube takes place such that the complex now extends
3.0-5.0 p m into the apical Sertoli expansion. A short distal
spermatid tube is present which ends facing a Sertoli bristle-coated pit. Lysosomes are shown in the region of the
clustered bulbs. Early Stage VII.
E In early and mid-Stage VII many bulbs
appear to lose their connection to the spermatid proper.
The dense material within disconnected bulbs appears
F In mid-Stage VII new tubulobulbar complexes form. Meanwhile large vacuoles containing phagocytized bulbs are present in the vicinity of intact bulbs.
G In late Stage VII and Early Stage VIII, ectoplasmic specializations facing the spermatid head become displaced and are then seen mainly along the peripheral aspect of the Sertoli cell. Fragmentation of the spermatid tube. takes place. The bulbous portions of many
tubulobulbar complexes are seen facing a Sertoli bristlecoated pit.
H By mid-Stage VIII the apical Sertoli expansion faces only the dorsal convex andlor lateral aspects of
the spermatid head. Tubulobulbar complexes (without
lulbs) form and extend into this Sertoli cell process. Prior
t ) sperm release these complexes also dissociate leaving
1.3 trace of tubulobulbar complexes on the mature spermatozoan.
ing premature sloughing of germ cells, and
that the loss of the relationship of the germ
cells with this surface specialization of the
Sertoli cell facilitates sperm release. The present study has shown that tubulobulbar complexes are present long after Sertoli ectoplasmic specializations have left their position
facing the spermatid (fig. 34g). From the configuration of these complexes it appears that
they must make some contribution to the
stability of the relationship between the spermatid head and the Sertoli cell. This relationship persists until near the time of release
when their dissolution from the dorsal region
of the head takes place. The relative importance of tubulobulbar complexes as anchoring
devices when compared with other possible
stabilizing factors has yet to be ascertained.
Beyond the zone in which most spermatids
had been released (in longitudinal sections of
Stage VIII) was an area in which residual
bodies, and on occasion a few abnormally
shaped sperm, were seen near or bordering the
tubular lumen. The relationship between the
Sertoli cell and these late spermatids had not
progressed as was described for the normally
shaped spermatids but had remained a t the
same developmental stage as seen in midStage VII. In evidence were: (1) The apical
Sertoli expansion around the head; (2) opposing Sertoli ectoplasmic specializations and (3)
intact tubulobulbar complexes. Since these
few sperm were not released, as were others of
the same generation, it might be assumed that
one or more of these features was important in
preventing this action. The persistence of a
type of relationship between the two cells like
that just described may be a means by which
abnormal spermatids are retained and prevented from traversing the male excurrent
duct system.
The generosity of Doctor Donald Caspary
in providing an electron microscope facility
a t the Springfield Campus is acknowledged.
Many thanks are due Doctors David Smith
and Marilyn Cayer and the Papanicolau
Cancer Research Institute in Miami, Florida,
for providing help and allowing the use of
equipment for the freeze-cleave studies. The
help of Mr. J. Malone and Mr. J. Ostenburg in
the preparation of the manuscript is also appreciated. This work was supported by a Grant
from the Institute of Child Health and Human
Development (NIH-11823).
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1979 Observations on the inter,relationships of
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Note a d d e d in proof.. The author is grateful to Dan Friend for recently pointing out the morphological similarities in the relationship of the Sertoli cell and late spermatid (as
demonstrated in this study) to that of the rod cells of the retina and the covering pigment epithelium. Pigment cells of the retina cap the rod outer segments and are
involved in phagocytosis of detached disks (Young, R. W., ’76, Invest. Ophth., 15: 700725). In this manner a portion of a living cell is degraded by a neighboring cell. The
Sertoli cell performs a similar task.
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observations, spermatids, testis, latex, rat, former, complexes, sertoli, cells, tubulobulbar
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