548

BIOLOGY OF REPRODUCTION 71, 548559 (2004)

Published online before print 14 April 2004.
DOI 10.1095/biolreprod.104.028803
Evidence That Tubulobulbar Complexes in the Seminiferous Epithelium Are Involved
with Internalization of Adhesion Junctions
1
Julian A. Guttman,
3
Yoshimi Takai,
4
and A. Wayne Vogl
2,3
Department of Anatomy and Cell Biology,
3
Faculty of Medicine, The University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z3
Department of Molecular Biology and Biochemistry,
4
Osaka University Medical School, Osaka 565-0871, Japan
ABSTRACT
Tubulobulbar complexes may be part of the mechanism by
which intercellular adhesion junctions are internalized by Ser-
toli cells during sperm release. These complexes develop in re-
gions where Sertoli cells are attached to adjacent cells by inter-
cellular adhesion junctions termed ectoplasmic specializations.
At sites where Sertoli cells are attached to spermatid heads, tu-
bulobulbar complexes consist of ngerlike processes of the sper-
matid plasma membrane, corresponding invaginations of the
Sertoli cell plasma membrane, and a surrounding cuff of modi-
ed Sertoli cell cytoplasm. At the terminal ends of the com-
plexes occur clusters of vesicles. Here we show that tubulo-
bulbar complexes develop in regions previously occupied by ec-
toplasmic specializations and that the structures share similar
molecular components. In addition, the adhesion molecules
nectin 2 and nectin 3, found in the Sertoli cell and spermatid
plasma membranes, respectively, are concentrated at the distal
ends of tubulobulbar complexes. We also demonstrate that dou-
ble membrane bounded vesicles are associated with the ends of
tubulobulbar complexes and nectin 3 is present on spermatids,
but is absent from spermatozoa released from the epithelium.
These results are consistent with the conclusion that Sertoli cell
and spermatid membrane adhesion domains are internalized to-
gether by tubulobulbar complexes. PKC, a kinase associated
with endocytosis of adhesion domains in other systems, is con-
centrated at tubulobulbar complexes, and antibodies to endo-
somal and lysosomal (LAMP1, SGP1) markers label the cluster
of vesicles associated with the ends of tubulobulbar complexes.
Our results are consistent with the conclusion that tubulobulbar
complexes are involved with the disassembly of ectoplasmic spe-
cializations and with the internalization of intercellular mem-
brane adhesion domains during sperm release.
Sertoli cells, spermatid, spermatogenesis, testis
INTRODUCTION
Turnover of unique actin-related intercellular adhesion
junctions in the seminiferous epithelium of the testis is fun-
damental to fertility in men. These massive junctions,
termed ectoplasmic specializations, occur near the base of
the epithelium as part of the junction complex between ad-
1
Supported by CIHR MOP 62728 awarded to A.W.V. and by a CIHR doc-
toral research award awarded to J.A.G.
2
Correspondence: A. Wayne Vogl, 313-2177 Wesbrook Mall, University
of British Columbia, Department of Anatomy and Cell Biology, Vancouver,
BC, Canada V6T1Z3. FAX: 604 822 2316;
e-mail: vogl@interchange.ubc.ca
Received: 20 February 2004.
First decision: 22 March 2004.
Accepted: 1 April 2004.
2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
jacent Sertoli cells and in more apical regions between Ser-
toli cells and spermatids as the major form of intercellular
attachment (Fig. 1). Disassembly of the basal junction com-
plexes ahead of translocating preleptotene spermatocytes
and the simultaneous assembly of similar complexes behind
these cells allows the next generation of spermatogenic
cells to move into the adluminal compartment of the sem-
iniferous epithelium without compromising the integrity of
the blood/testis barrier [1] (Fig. 1). In apical regions, as-
sembly of ectoplasmic specializations is responsible for an-
choring spermatids to Sertoli cells and disassembly of these
junctions is part of the mechanism of sperm release [2]
(Fig. 1). Little is known about the mechanism by which
ectoplasmic specializations are assembled and disassembled
[3]. In this study, we explore the hypothesis, originally pre-
sented in preliminary form elsewhere [4], that structures
termed tubulobulbar complexes may be involved with the
disassembly of ectoplasmic specializations and the inter-
nalization of membrane adhesion elements during sperm
release.
Ectoplasmic specializations occur only in Sertoli cells
and are tripartite structures consisting of the plasma mem-
brane, a layer of actin lament bundles, and an attached
cistern of endoplasmic reticulum. In basal regions of at-
tachment between Sertoli cells, other junction types (tight
junctions, gap junctions, and desmosomes) occur within
and around ectoplasmic specializations. In apical regions of
attachment to spermatids, other junction types are less ap-
parent or are absent. Among molecules identied as com-
ponents of ectoplasmic specializations are actin [514], -
actinin [6, 15], vinculin [16], espin [17], mbrin [16], my-
osin VIIa [18, 19], gelsolin [3], rac1 [20], afadin [21], and
Fyn tyrosine kinase [22]. Adhesion molecules identied at
the ectoplasmic specializations include 61 integrin [23,
24] and nectin 2 [21]. The binding partner for 61 integrin
on adjacent cells has not been identied. At basal junctions,
the ligand for nectin 2 is likely another nectin 2 molecule
in the ectoplasmic specialization in the adjacent Sertoli cell
[21, 25]. At apical junctions, nectin 2 appears to bind het-
erotypically to nectin 3 in the plasma membrane of the
adjacent spermatid heads [21, 25, 26].
Tubulobulbar complexes that develop between Sertoli
cells and spermatids consist of tubular extensions from the
plasma membrane of the spermatid head that protrude into
corresponding plasma membrane invaginations of the ad-
jacent Sertoli cell [27] (Fig. 2). Near the end of the tubular
process is a bulblike swelling [27]. In the Sertoli cell, the
tubular invagination is cuffed by a network of actin la-
ments and surrounding the bulbous region are elements of
the endoplasmic reticulum, which are closely apposed to
the plasma membrane. At distal ends of the invaginations
549 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
FIG. 1. Diagrammatic representation of
the seminiferous epithelium illustrating the
location of basal and apical ectoplasmic
specializations and tubulobulbar complex-
es. Assembly and disassembly of ectoplas-
mic specializations occur at apical and at
basal sites in the epithelium.
FIG. 2. The ultrastructure of tubulobulbar complexes. A) Cross-section through a mature spermatid head and adjacent Sertoli cell cytoplasm. Ecto-
plasmic specializations (es) and tubulobulbar complexes (tc) are visible. Bar 200 nm. B) Diagram of the structure of a single tubulobulbar complex
and its associated vesicles distal to the complex. Actin laments surround the proximal tube and endoplasmic reticulum surrounds the bulbous region.
C) Electron micrographs of vesicles found in the region of tubulobulbar complexes. The inset shows an example of a coated pit (cp) distal to the bulbous
region of a tubulobulbar complex. The bulbous region is associated with cisternae of the endoplasmic reticulum (er). The spermatid tubulobulbar process
(tp) also is indicated in the inset. Bar 200 nm, bar (inset) 100 nm. D) Tubulobulbar complexes viewed in cross section. Dashed line marked by
(tc) demarcates the actin surrounding the proximal tube of the complex. The center of the complex is the tubulobulbar process of the spermatid. An
ectoplasmic specialization (es) is present around the spermatid head. Bar 500 nm.
550 GUTTMAN ET AL.
are clusters of numerous vesicles, some of which have been
identied as lysosomes [27].
The function of tubulobulbar complexes is not entirely
clear, although a number of possibilities have been sug-
gested. One possibility is that they are an attachment device
between Sertoli cells and spermatids. In the rat, tubulobul-
bar processes are the last structures to disappear at sperm
release [27]. Another possibility is that they are a mecha-
nism by which material from spermatids is endocytosed by
Sertoli cells during spermatid maturation. In cases where
tubulobulbar complexes do not develop, spermatids do not
reduce their volume normally during maturation and release
is delayed [28]. Another possibility is that they are part of
the mechanism by which intercellular junctions, particularly
ectoplasmic specializations, are disassembled during sper-
matogenesis [4, 2830]. Pertinent to this possibility are
three key observations made by Russell and Clermont in
their original description of tubulobulbar complexes [27].
First, the structures occur in areas also occupied by ecto-
plasmic specializations, indicating that there may be a func-
tional relationship between the two types of structures. Sec-
ond, tubulobulbar complexes develop at basal junction
complexes between adjacent Sertoli cells [27], indicating
that the primary function of tubulobulbar complexes may
not be related only to spermatid maturation. Third, ultra-
structurally identiable gap and tight junctions occur in the
bulbous regions of basal tubulobulbar complexes, indicat-
ing that junction elements may occur in these structures
[29].
In this study, we investigate the possibility that tubulo-
bulbar complexes are involved in the disassembly of ecto-
plasmic specializations. We concentrate on the complexes
that develop in association with apical junctions because
they are much larger than those that occur basally, can be
resolved at the light level using differential interference
contrast (DIC) optics, have been better studied than those
that occur at basal sites, can be isolated within apical Sertoli
cell processes fragmented away from the epithelium, and
can be located easily using late-step spermatid heads as
morphological markers. We clearly demonstrate that tubu-
lobulbar complexes develop in regions previously occupied
by ectoplasmic specializations and that tubulobulbar com-
plexes contain molecular markers for ectoplasmic special-
izations. In addition, we demonstate that vesicular elements
present in the Sertoli cell apical processes surrounding the
spermatid heads, and related to the tubulobulbar complexes,
stain positively for nectin 2 and nectin 3. Nectin 3 is not
present on testicular spermatozoa released from the epithe-
lium, and double membrane vesicles occur adjacent to tu-
bulobulbar complexes. These results are consistent with the
possibility that adhesion domains of spermatids are inter-
nalized together with related regions of the Sertoli cell
membrane at tubulobulbar complexes. Signicantly, we
also demonstate lysosomal/endosomal markers are present
in regions occupied by Sertoli cell vesicles and that protein
kinase C (PKC), implicated as being involved with the
endocytosis of adhesion molecules in other systems, is pre-
sent at sites containing tubulobulbar complexes. Our results
are consistent with the hypothesis that tubulobulbar com-
plexes are involved with the disassembly of ectoplasmic
specializations and with the internalization of adhesion
membrane domains both of the Sertoli cell and the adjacent
spermatid at the time of sperm release.
MATERIALS AND METHODS
Animals
All animals used in these studies were reproductively active male
Sprague Dawley rats. They were obtained from the University of British
Columbia animal care colony and were maintained according to the guide-
lines established by the Canadian Council on Animal Care. All experi-
ments were performed at least in duplicate (most were performed more
than three times) using separate animals.
Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents were obtained
from Sigma-Aldrich Canada (Mississauga, ON). The paraformaldehyde
and NaCl were obtained from Fisher Scientic (Vancouver, BC). All con-
trol immunoglobulins (IgGs) as well as all secondary antibodies conju-
gated to horseradish peroxidase were purchased from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA). All secondary an-
tibodies conjugated to ALEXA uorochromes were purchased from Mo-
lecular Probes (Eugene, OR). Polybed embedding resin was obtained from
EM Sciences (Fort Washington, PA).
Immunouorescence Tissue Preparation
Testes were removed from male rats under deep anesthesia. Warm
(33C) PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH
2
PO
4
, 3.2 mM
Na
2
HPO
4
, pH 7.3) was perfused through the spermatic artery using a 26-
gauge needle attached to a gravity-fed perfusion apparatus for 2 min to
clear the organ of blood. Following this, warm 3% paraformaldehyde in
PBS was perfused through the testis for 30 min. PBS was then reperfused
through the organ to wash out any remaining xative.
Frozen Sections
Fixed testes were frozen (using liquid nitrogen) while at the same time
being attached to an aluminum stub by OCT compound (Sakura Finetek
USA, Torrance, CA). Frozen testis sections were cut, attached to poly-L-
lysine-coated glass slides, immediately plunged into 20C acetone for 5
min, air dried, and then processed for immunouorescence.
Fragmented Material
Fixed testes were decapsulated in PBS and the seminiferous tubule
was mass minced into small pieces. The pieces were transferred into a 15-
ml plastic Falcon tube along with about 5 ml of PBS. The material was
gently passed through an 18-gauge, then 21-gauge needle for 25 gentle
passes. This fragmented material was left to sediment by gravity at room
temperature for 5 min and then the upper layer was transferred to another
tube. The cells in suspension were pelleted using centrifugation, the pellet
was resuspended in a small volume of PBS, and then the suspension was
added to poly-L-lysine-coated slides and allowed to incubate in a humidity
chamber for 10 min. All excess PBS was then removed and the slides
were immediately treated with 20C acetone for 5 min and allowed to
air dry.
Immunouorescence
Slides with attached tissue fragments or cryosections were rehydrated
and blocked with 5% normal goat serum (NGS) in TPBS-BSA (PBS con-
taining 0.05% Tween-20 and 0.1% bovine serum albumin) for 20 min at
room temperature. Primary antibodies consisted of rat anti-mouse nectin
2 antibodies (#502-57 [31]), rat anti-mouse nectin 3 antibodies (#103-A1
[32]), mouse anti-rat-l-afadin antibodies (#3 [33]), mouse anti-PKC an-
tibodies (Transduction Labs, Lexington, KY), rabbit anti-rat espin anti-
bodies (gift from Dr. Jim Bartles [17]), rabbit anti-human myosin VIIa
antibodies (gift from Dr. Tama Hasson [18, 34]), rabbit anti-kelch/keap1
antibodies (gift from Dr. Tama Hasson [19]), rabbit anti-SGP1 antibodies
(gift from Dr. Carlos Ramon Morales), and mouse anti-LAMP1 antibodies
(developed by Dr. J. Thomas August and obtained from the Developmental
Studies Hybridoma Bank, Univ. of Iowa, Iowa City, IA). Antibodies were
added to the experimental slides, made up in TPBS-BSA with 1% NGS,
and incubated overnight at 4C in a humidity chamber. The material was
washed extensively with the TPBS-BSA (wash buffer), then incubated for
60 min at 37C with secondary antibody conjugated to a uorochrome
(goat anti-mouse ALEXA 488, goat anti-rabbit ALEXA 568, or goat anti-
rat ALEXA 546). The slides were again washed and then coverslips were
mounted using Vectashield (Vector Labs, Burlington, ON). The tissue was
visualized using a Zeiss Axiophot microscope tted with appropriate lter
sets for detecting uorescence and with the appropriate optics for DIC or
phase microscopy.
Controls for immunouorescence localization consisted of the follow-
ing: 1) Primary antibodies were replaced with normal immunoglobulin
551 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
(IgG) (in the case of afnity-puried antibodies) or normal serum (in the
case of non-afnity-puried serum antibodies) from the host animal spe-
cies at identical concentrations to the primary antibody (when the stock
concentration of primary antibody was not known, it was assumed to be
1 mg/ml), 2) primary antibody was replaced with buffer alone, 3) both the
primary and the secondary antibodies were replaced with buffer alone.
Phalloidin/Phallotoxins
Filamentous actin was labeled using ALEXA 488 or ALEXA 568 phal-
loidin (Molecular Probes). The stain was made up in PBS or in TPBS.
One-Dimensional Western Blotting
Seminiferous epithelium was isolated from the testis and extensively
homogenized in RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.4,
5 mM EDTA, 1% Nonidet P-40, 1% deoxychloic acid [sodium salt], 10%
SDS) before being loaded into wells of 1-mm-thick 10% SDS-PAGE gels
and run according to standard protocols [35]. Proteins were transferred
onto Immobilon-P transfer membrane (Millipore, Billerica, MA), then
washed for 5 min at room temperature with TBST (500 mM Tris, pH 7.5,
150 mM NaCl, 0.1% Tween-20). The blots were then blocked to decrease
nonspecic antibody binding for 8 h at 4C using 4% nonfat milk (Blotto,
Santa Cruz Biotechnology, Santa Cruz, CA). Following blocking, mem-
branes were washed three times, 10 min each, then incubated with primary
antibody overnight at 4C. The following day, blots were washed exten-
sively with TBST followed by a 1-h secondary antibody (conjugated to
horseradish peroxidase) incubation at room temperature. Upon further
washing with TBST followed by TBS, blots were reacted with enhanced
chemiluminescence (Pharmacia, Peapack, NJ) to visualize the reactive
bands on X-OMAT lm (Eastman Kodak, Rochester, NY).
Controls consisted of replacing the primary antibodies with IgG or
serum at identical primary antibody concentrations or identical serum di-
lutions.
Electron Microscopy
Rat testes were removed and perfusion xed for 30 min with 0.1 M
sodium cacodylate, 1.5% paraformaldehyde, 1.5% glutaraldehyde. Each
testis was then cut into small pieces and immersion xed for an additional
90 min. The testis material was then washed with 0.1 M sodium cacodylate
for three 10-min washes, then further xed on ice for 60 min in a 1:1
mixture of 3% K
4
Fe(CN)
6
:2% osmium xative. Following the incubation,
the material was washed three times with ddH
2
O, 10 min each wash, then
stained for 1 h with 0.1% uranyl acetate. The material was then washed
another three times in ddH
2
O, then dehydrated in an ascending alcohol
series (50%, 70%, 95%, 100%), for 10 min at each concentration. This
was followed by two incubations of 15 min each in propylene oxide. The
blocks then were left in a 1:1 solution of propylene oxide:Polybed over-
night. The material was embedded in 100% Polybed and then incubated
at 60C for 24 h. Sections were viewed and photographed on a Philips
300 electron microscope operated at 60 kV.
RESULTS
Tubulobulbar Complexes Form in Areas Previously
Occupied by Ectoplasmic Specializations
If tubulobulbar complexes are involved in the disassem-
bly of adhesion junctions, then the structures should de-
velop in regions where ectoplasmic specializations previ-
ously occurred. This was conrmed in spermatid/junction
complexes that had been mechanically dissociated from the
epithelium and visualized with DIC or stained with uo-
rescent phallotoxins to label lamentous actin (Fig. 3). In
early elongate spermatids (Fig. 3, AF), bundles of actin
laments in ectoplasmic specializations completely sur-
round spermatid heads. As the spermatids continue to dif-
ferentiate, tubulobulbar complexes clearly form adjacent to
the concave surface of rat spermatid heads in areas previ-
ously occupied only by ectoplasmic specializations. More-
over, the intensity of actin bundle staining in ectoplasmic
specializations appeared to qualitatively decrease in asso-
ciation with ectoplasmic specializations related to the dorsal
aspect of the spermatid heads while tubulobulbar complex-
es became more distinct. The development of tubulobulbar
complexes in regions previously occupied by ectoplasmic
specializations was conrmed at the ultrastructural level
(Fig. 3G). Here, regions that at earlier stages of spermato-
genesis contained only ectoplasmic specializations now
contained tubulobulbar complexes that were anked on ei-
ther side by ectoplasmic specializations. These uorescence
and ultrastructural observations are consistent with the con-
clusion that tubulobulbar complexes develop in regions pre-
viously occupied by ectoplasmic specializations.
Molecular Markers for Ectoplasmic Specializations Also
Are Present at Tubulobulbar Complexes
If tubulobulbar complexes are involved with the disas-
sembly of ectoplasmic specializations, then molecules
found at the adhesion junctions also should be found at
tubulobulbar complexes. Antibodies to espin, myosin VIIa,
and Keap 1, all previously shown to react at ectoplasmic
specializations, also react with tubulobulbar complexes
(Fig. 4). On the basis of these experiments, we conclude
that tubulobulbar complexes and ectoplasmic specializa-
tions share similar molecular components.
Nectin 2 and Nectin 3 Are Localized to Tubulobulbar
Complexes
Once we had determined that markers for ectoplasmic
specializations were present at tubulobulbar complexes, we
were interested to determine if integral membrane adhesion
molecules present at the adhesion junctions also were pre-
sent at tubulobulbar complexes. For these studies, we treat-
ed spermatids, with attached regions of Sertoli cells that
had been mechanically fragmented from xed mouse sem-
iniferous epithelium, with antibodies to nectin 2 and nectin
3 (Fig. 5, AB). We chose to use mouse tissue because
the nectin antibodies do not react with rat material. In ad-
dition to labeling ectoplasmic specializations, antibodies to
nectin 2 specically labeled, in a vesicular pattern, regions
known from actin staining to contain tubulobulbar com-
plexes. Surprisingly, antibodies to nectin 3 labeled the tis-
sue in a similar vesicular pattern to the nectin 2 antibodies,
in addition to labeling the spermatid head. We conclude that
immunologically reactive nectin 2 and nectin 3 are present
in tubulobulbar complexes and in vesicles associated with
their ends.
Afadin, an adaptor protein that binds nectin to actin l-
aments, had a similar yet distinct immunocytochemical lo-
calization. It too was found at ectoplasmic specializations
as well as at tubulobulbar complexes (Fig. 5, CE). Inter-
estingly, there was a distinct lack of staining along the dor-
sal part of the ectoplasmic specializations attached to the
late-step spermatids (Fig. 5, CE). When a step progres-
sion of afadin staining was investigated, staining of ecto-
plasmic specialization along the dorsal curvature of sper-
matids gradually appeared to decrease in intensity, whereas
an increase in staining intensity was observed around tubu-
lobulbar complexes where it formed a ngerlike staining
pattern (Fig. 5, CE).
Proles of Double Membrane Vesicles Are Found
Associated with Tubulobulbar Complexes
The observation that antibodies both to nectin 2 and to
nectin 3 labeled regions containing tubulobulbar complexes
in a vesicular pattern suggested to us that plasma membrane
552 GUTTMAN ET AL.
FIG. 3. Tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations. AF) Paired DIC and phalloidin-stained
images of spermatid (st)/junction complexes that were xed and mechanically dissociated from the seminiferous epithelium of rats. The series of images
is a developmental one, with the least mature spermatid shown in (A) and the most mature spermatid shown in (F). Ectoplasmic specialization (es) and
tubulobulbar complexes (tc) are labeled, as are vesicles (v). Note that the tubulobulbar complexes and associated vesicles are visible with DIC mi-
croscopy. Bar 5 m. In the electron micrograph in (G), a tubulobulbar complex is anked on either side by an ectoplasmic specialization. The
components of an ectoplasmic specialization (es) are labeled as is a tubulobulbar process (tp) of the tubulobulbar complex (tc). The endoplasmic
reticulum is labeled as (er). Bar 200 nm.
adhesion domains of spermatids might be internalized to-
gether with similar domains of Sertoli cell ectoplasmic spe-
cializations. If this is true, then vesicles in the region should
be surrounded by a double layer of membrane. This was
conrmed by transmission electron microscopy. In sections
of rat testis, double membrane-bound vesicles consistently
were observed in association with the ends of tubulobulbar
complexes (Fig. 6). These vesicles were distinguished from
the bulbous region of tubulobulbar complexes by their lack
of associated endoplasmic reticulum cisternae. At sperm
553 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
FIG. 4. Actin-associated molecules found
at tubulobulbar complexes. AC) Paired
phase and uorescent micrographs of
spermatid/tubulobulbar complexes labeled
with antibodies to espin (A), keap1 (B),
and myosin VIIa (C). Bar in A 5 m.
release (Fig. 7A), similar proles were observed in Sertoli
cell stalks that support apical processes surrounding the
spermatids being released (Fig. 7B). Also present in similar
regions were multivesicular bodies (Fig. 7C). These results
indicate that the appropriate morphological machinery is
present in Sertoli cell apical processes for internalization of
Sertoli cell/spermatid adhesion junction domains.
Nectin 3 Is Present on Spermatids but Is Absent from
Testicular and Epididymal Spermatozoa
If adhesion domains of spermatids are internalized by
tubulobulbar complexes, then these domains should be ab-
sent from spermatozoa released from Sertoli cells. To verify
this prediction, we stained mouse spermatids, testicular
spermatozoa, and epididymal spermatozoa with antibodies
to nectin 3. Antibodies to nectin 3 only reacted with sper-
matids (Fig. 8). We conclude that immunologically reactive
nectin 3 is associated with spermatid heads but is absent
from these regions once the cells are released from the ep-
ithelium.
PKC Is Associated with Tubulobulbar Complexes
If tubulobulbar complexes are involved with internaliz-
ing adhesion junction domains, then PKC, a signaling
molecule known to regulate endocytosis of junction pro-
teins in other systems [3638], may be present in regions
of Sertoli cells associated with tubulobulbar complexes. To
explore this possibility, we probed rat testis fragments with
antibodies to PKC, which specically labeled regions as-
sociated with the vesicular region at the terminal ends of
tubulobulbar complexes in addition to labeling sperm tails.
Similar staining was not present in controls (Fig. 9). The
antibody labeled one band specically on Western blots of
rat seminiferous epithelium. This band was not present on
normal IgG control blots. We conclude that at least one of
the regulators of junction turnover in other systems is pre-
sent at tubulobulbar complexes.
Lysosomal and Endosomal Markers Are Present
in Vesicles Associated with Tubulobulbar Complexes
We rationalized that, if tubulobulbar complexes inter-
nalize junction domains from regions occupied by ecto-
plasmic specializations, then at least some of the material
has to be degraded (spermatid plasma membrane) while
other components (Sertoli cell plasma membrane) can ei-
ther be recycled and/or degraded. In either case, lysosomal
markers should be present in vesicles associated with the
ends of tubulobulbar complexes. To test this prediction, we
labeled mouse and rat testicular fragments with antibodies
to LAMP 1 (an endosomal and lysosomal marker generally
in cells) and SGP1 (a marker for lysosomes in Sertoli cells),
respectively. In both cases, the antibodies labeled structures
consistent with the structures being vesicles associated with
terminal ends of tubulobulbar complexes (Fig. 9). Controls
were negative.
DISCUSSION
The loss of intercellular adhesion between cells in the
seminiferous epithelium is essential for sperm release and
for the movement of spermatocytes from basal to adluminal
compartments of the epithelium. Ectoplasmic specializa-
tions are large intercellular adhesion plaques formed in Ser-
toli cells at certain sites of intercellular attachment. In this
study, we present evidence consistent with the conclusion
that tubulobulbar complexes are part of the mechanism by
which ectoplasmic specializations are disassembled and ad-
hesion domains in the membranes both of Sertoli cells and
spermatids are internalized by Sertoli cells at the time of
sperm release.
At sites of apical attachment between Sertoli cells and
554 GUTTMAN ET AL.
FIG. 5. Intercellular adhesion elements are found at ectoplasmic specializations, tubulobulbar complexes, and in vesicular regions associated with
tubulobulbar complexes. AA) Grouped phase (A), nectin 2 (A), lamentous actin (A), and merged images of mouse spermatids with associated
tubulobulbar complexes (A). Bar 5 m. (BB). Grouped phase (B), nectin 3 (B), lamentous actin (B), and merged images of mouse spermatids
with associated tubulobulbar complexes (B). Bar 5 m. Note in A and B the presence of a vesicular staining pattern (v) and labeling at ectoplasmic
specializations (es). Phalotoxin-labeled lamentous actin identies the location of ectoplasmic specializations and of tubulobulbar complexes (tc).
(CE) A stage progression of paired phase and uorescent images of anti-afadin-labeled rat ectoplasmic specializations and/or tubulobulbar complexes.
Prior to tubulobulbar complex formation, afadin is present at ectoplasmic specializations (C). As tubulobulbar complexes form, afadin appears at these
sites and labeling at ectoplasmic specializations decreases (D). Afadin is barely detectable at ectoplasmic specializations associated with late spermatids
and is concentrated almost entirely at tubulobulbar complexes (E). Bar 5 m.
spermatids, tubulobulbar complexes consist of tubular ex-
tensions of spermatid heads that project into corresponding
invaginations of Sertoli cells. Also considered part of the
complexes are the surrounding cuffs of Sertoli cell cyto-
plasm that are rich in actin laments in some regions and
elements of the endoplasmic reticulum in others. The ter-
minal ends of the tubulobulbar complexes are associated
with vesicles, some of which have been identied previ-
ously as lysosomes based on positive staining for acid phos-
phatase [39]. The most popular hypothesis for the function
555 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
FIG. 6. Vesicles found associated with
tubulobulbar complexes. Electron micro-
graphs showing numerous single and dou-
ble membrane (arrowheads) bound vesi-
cles, as well as large vesicles (*) contain-
ing double membranes proles (arrows). A
tubulobulbar complex is indicated by the
bracket in A. Bar (A) 200 nm, bar (B)
250 nm.
FIG. 7. Electron micrographs of apical regions of Sertoli cells at sperm
release. A) Electron micrograph of a late-stage (VIII) spermatid at sperm
release. Note the absence both of tubulobulbar complexes and of ecto-
plasmic specializations. Bar 250 nm. B) Apical stalk supporting another
spermatid at a similar maturation step as in (A) and containing a double
membrane-bound vesicle (arrowhead) similar to those associated with
tubulobulbar complexes at an earlier stage of spermatogenesis. Residual
lobes (rl) of spermatids are labeled (rl). Bar 200 nm. C) The apical
Sertoli cell stalk supporting the spermatid in (A) containing structures re-
sembling multivesicular bodies (mvb). Bar 200 nm.
of tubulobulbar complexes is that they function as a mech-
anism by which Sertoli cells phagocytose cytoplasm from
spermatids as these cells mature. An alternative hypothesis
that would account for the presence of tubulobulbar com-
plexes at basal sites of attachment between adjacent Sertoli
cells is that the structures are involved with the turnover or
disassembly of intercellular adhesion junctions [4].
A number of observations are consistent with the con-
clusion that tubulobulbar complexes are involved with the
disassembly of ectoplasmic specializations at apical sites of
attachment to spermatids.
First, tubulobulbar complexes develop in regions occu-
pied by ectoplasmic specializations and both structures
share many of the same molecular components. In electron
micrographs, networks of actin laments that cuff the tubu-
lobulbar complexes are contiguous with actin bundles in
the ectoplasmic specializations from which the tubulobul-
bar complexes appear to emerge (this study, [40]). Espin,
Myosin VIIa, and Keap 1 are present in both structures (this
study), as is vinculin [41] and 1 integrin [42]. The nding
that espin is localized in the actin networks of tubulobulbar
complexes is particularly interesting because this protein is
thought mainly to organize actin laments into bundles
[43], such as those in ectoplasmic specializations. The pres-
ence of espin at tubulobulbar complexes may simply be the
result of a rearrangement of actin bundles in ectoplasmic
specializations into actin networks without loosing the as-
sociation with actin binding proteins previously present.
The observations that the structures occur together and
share molecular components indicate to us that they are
related and that tubulobulbar complexes may develop from
ectoplasmic specializations.
Second, the integral membrane adhesion proteins nectin
2, in the Sertoli cell, and nectin 3, in the spermatid, appear
concentrated in vesicles near the ends of tubulobulbar com-
plexes. Although labeling of the vesicles needs to be con-
rmed at the ultrastructural level, the uorescence data rais-
es the novel possibility that plasma membrane adhesion do-
mains both of Sertoli cell ectoplasmic specializations and
of the attached spermatid may be internalized together by
Sertoli cells at tubulobulbar complexes. Consistent with this
possibility is the observation that double membrane-bound
vesicles occur among the mass of vesicles present at the
ends of tubulobulbar complexes and that similar vesicles
are observed deeper in the cytoplasm of Sertoli cells at
sperm release. Also consistent with this possibility is the
observation that nectin 3 antibodies label spermatids at-
tached to Sertoli cells but do not label spermatozoa released
from the epithelium. The implication is that adhesion do-
mains in the spermatid plasma membrane are removed by
tubulobulbar complexes as part of the sperm-release mech-
anism. An alternative possibility that we cannot rule out is
that proteolytic processing of the protein on the sperm head
may contribute to the lack of nectin labeling on sperma-
tozoa.
Third, the adaptor protein afadin that links nectin to the
556 GUTTMAN ET AL.
FIG. 8. Nectin 3 localization in sperma-
tids and spermatozoa. Paired phase and
uorescent images of nectin 3 and la-
mentous actin labeled spermatid/tubulo-
bulbar complexes (AC) of a mouse. Fila-
mentous actin labeling (A) conrms the
presence of both ectoplasmic specializa-
tions (es) and tubulobulbar complexes (tc)
associated with the spermatid in (A). Nec-
tin 3 also is present (A) in these struc-
tures. On both testicular spermatozoa (B)
and spermatozoa in the epididymis (C),
nectin 3 is absent (B, C). Labeling of la-
mentous actin with phalloidin conrms
that spermatozoa are not associated with
ectoplasmic specializations or tubulobul-
bar complexes (B). Bar in A 5 m.
actin cytoskeleton becomes less concentrated at ectoplas-
mic specializations and more concentrated in tubulobulbar
complexes as spermatids mature. There is a striking shift
in afadin localization from around the head in elongate rat
spermatids to tubulobulbar processes in mature cells just
before sperm release. This change is consistent with the
movement of adhesion-related elements into tubulobulbar
complexes.
Fourth, PKC is present in Sertoli cell regions surround-
ing tubulobulbar complexes and lysosomal/endosomal
markers are present in vesicles associated with the ends of
the complexes. PKC is a known regulator of junctional
protein endocytosis. Previous studies have demonstrated
that vesicles associated with tubulobulbar processes contain
acid phosphatase [39], and we show here that they also
contain SGP1 (sulphated glycoprotein 1/cathepsin) and
LAMP1 (lysosome associated membrane protein 1). SGP1
is known to label Sertoli cell secondary lysosomes. Lamp1
is a transmembrane glycoprotein that labels lysosomes and
late endosomes [44]. These results are consistent with the
conclusion that tubulobulbar complexes are involved with
endocytosis and that at least some of the internalized ma-
terial likely enters the degradation pathway.
Adhesion domains that are internalized in apical regions
of Sertoli cells during sperm release could either be de-
graded or recycled (Fig. 9). At sperm release in the semi-
niferous epithelium, large intercellular adhesion junctions
must be disassembled and adhesion domains in the plasma
membrane eliminated. Results presented here indicate to us
that tubulobulbar complexes are involved with internalizing
junction domains both of Sertoli cells and of the adjacent
spermatids. In other systems that have been studied, adhe-
sion molecules that are endocytosed predominantly enter a
recyling pathway from which they can be reinserted into
the plasma membrane [38]. In apical regions of Sertoli
cells, it is unclear how much junction material enters the
degradation and recycling pathways (Fig. 10), although in-
ternalized nectin 3 from spermatids is likely degraded be-
557 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
FIG. 10. Diagram of apical Sertoli cell regions indicating that vesicles
internalized by tubulobulbar complexes may be degraded or recycled.
FIG. 9. Paired phase and uorescent micrographs of spermatids with
associated Sertoli cell regions labeled for endocytosis and lysosomal
markers. AC) Paired phase and uorescent images of mouse testis ma-
terial stained with anti-PKC antibodies and controls. The anti-PKC an-
tibody labeled areas known to contain tubulobulbar complexes (tc) in the
mouse (A). Both primary antibody NMIgG controls (B) and secondary
antibody controls (C) showed no specic staining. A) Western blot of rat
seminiferous epithelium probed with the anti-PKC antibody. A band at
the appropriate molecular weight, 82 kDa, is present. The top and bottom
of the gel are represented by bars. DD) Phase (D), anti-PKC (D), and
lamentous actin (D) labeled and merged (D) micrographs of mouse
spermatids with associated Sertoli cell regions. Filamentous actin staining
was used to ensure the presence of the ectoplasmic specialization. EE)
Phase (E) and double-labeled anti-LAMP1 (E) and lamentous actin (E)
images. Note that the LAMP1 antibodies label circular structures that are
distal to the lamentous actin of the tubulobulbar complexes. FI) Anti-
SGP1 labeling of rat spermatids with associated tubulobulbar complexes
and controls. F) Antibodies to SGP1 specically label punctate structures
(arrowheads) in the vesicular region associated with the ends of tubulo-

bulbar complexes. Some nonspecic staining occurs in the normal rabbit

serum control micrographs (G), but focal vesicular staining is absent.
Staining also is absent from secondary antibody (H) and autouorescent
(I) controls. Bars on all micrographs 5 m.
cause this adhesion molecule is not expressed by Sertoli
cells. How much, if any, of the nectin 2 from the Sertoli
cell plasma membrane enters the recycling pathway re-
mains to be determined. Any material that is recycled
would have to be inserted into the plasma membrane at
locations in the cell distant from sites where it was inter-
nalized.
At basal sites of attachment between neighboring Sertoli
cells, tubulobulbar complexes also are present [27] and
would be expected to function in a way similar to apical
complexes. Preliminary studies we have done (unpublished
data) indicate that tubulobulbar complexes are not nearly
as abundant as one might predict if these complexes were
the sole mechanism for junction turnover at these sites. At
basal sites of attachment between Sertoli cells, junctions
separate above translocating spermatocytes and are reas-
sembled immediately below [1]. Although some internali-
zation of junction elements may occur [29], it also is pos-
sible that many of the integral membrane junction mole-
cules never leave the plane of the membrane but simply
disengage from their ligands above the spermatocytes and
reengage below. This movement of junction molecules in
the plane of the membrane might account for the low num-
558 GUTTMAN ET AL.
ber and small size of tubulobulbar complexes at basal sites
relative to apical sites.
The internalization of intercellular adhesion domains by
tubulobulbar complexes at sites of attachment between Ser-
toli cells and spermatids is potentially a signicant com-
ponent of the sperm-release mechanism in the seminiferous
epithelium. The morphology of tubulobulbar complexes
and the ndings that lysosomal/endosomal markers and ad-
hesion molecules are found in, or associated with, the struc-
tures are all consistent with the hypothesis that tubulobulbar
complexes are involved with junction disassembly. These
results do not rule out the possibility that the tubulobulbar
complexes that form at sites of attachment to spermatids
also may have secondary functions. The path of junction
molecules through degradation and recycling pathways in
Sertoli cells remains to be explored, as do alternative mech-
anisms for junction turnover at basal sites of attachment
between Sertoli cells.
ACKNOWLEDGMENTS
We would like to thank Dr. Tama Hasson for the antibodies to myosin
VIIa and Keap 1, Dr. Carlos Morales for the antibody to SGP1, and Dr.
James Bartles for the antibody to espin. The anti-LAMP1 antibody was
developed by Dr. J. Thomas August and was obtained from the Devel-
opmental Studies Hybridoma Bank, developed under the auspices of the
NICHD, maintained at the University of Iowa, Department of Biological
Sciences.
REFERENCES
1. Russell LD. Movement of spermatocytes from the basal to the adlu-
minal compartment of the rat testis. Am J Anat 1977; 148:313328.
2. Russell LD. Spermiationthe sperm release process: ultrastructural
observations and unresolved problems. In: Van Blerkom J, Modda PM
(eds.), Ultrastructure of Reproduction. Boston: Martinus Nijhoff Pub-
lishers; 1984:4666.
3. Guttman JA, Janmey P, Vogl AW. Gelsolinevidence for a role in
turnover of junction-related actin laments in Sertoli cells. J Cell Sci
2002; 115:499505.
4. Russell LD, Goh JC, Rashed RMA, Vogl AW. The consequences of
actin disruption at Sertoli ectoplasmic specialization sites facing sper-
matids after in vivo exposure of rat testis to cytochalasin D. Biol
Reprod 1988; 39:105118.
5. Toyama Y. Actin-like laments in the Sertoli cell junctional speciali-
zations in the swine and mouse testis. Anat Rec 1976; 186:477491.
6. Franke WW, Grund C, Fink A, Weber K, Jokusch BM, Zentgraf H,
Osborn M. Location of actin in the microlament bundles associated
with junctional specializations between Sertoli cells and spermatids.
Biol Cell 1978; 31:714.
7. Vogl AW, Linck RW, Dym M. Colchicine-induced changes in the
cytoskeleton of the golden-mantled ground squirrel (Spermophilus la-
teralis) Sertoli cells. Am J Anat 1983; 168:99108.
8. Suarez-Quian CA, Dym M. Detection of microlaments in rat Sertoli
cell ectoplasmic specializations with NBD-phallicidin. Int J Androl
1988; 11:301312.
9. Vogl AW, Soucy LJ. Arrangement and possible function of actin l-
ament bundles in ectoplasmic specializations of ground squirrel Sertoli
cells. J Cell Biol 1985; 100:814825.
10. Vogl AW, Grove BD, Lew GJ. Distribution of actin in Sertoli cell
ectoplasmic specializations and associated spermatids in the ground
squirrel testis. Anat Rec 1986; 215:331341.
11. Camatini M, Anelli G, Casale A. Identication of actin in boar sper-
matids and spermatozoa by immunoelectron microscopy. Eur J Cell
Biol 1986; 42:311318.
12. Camatini M, Casale A. Actin and calmodulin coexist in the equatorial
segment of ejaculated boar sperm. Gamete Res 1987; 17:97105.
13. Masri BA, Russell LD, Vogl AW. Distribution of actin in spermatids
and adjacent Sertoli cell regions of the rat. Anat Rec 1987; 218:20
26.
14. Fouquet JP, Kann ML, Dadoune JP. Immunogold distribution of actin
during spermiogenesis in the rat, hamster, monkey, and human. Anat
Rec 1989; 223:3542.
15. Jockush BM, Isenberg G. Interaction of -actinin and vinculin with
actin: opposite effects in lament network formation. Proc Natl Acad
Sci U S A 1981; 78:30053009.
16. Grove BD, Vogl AW. Sertoli cell ectoplasmic specializations: a type
of actin-associated adherens junction? J Cell Sci 1989; 93:309323.
17. Bartles JR, Wierda A, Zheng L. Identication and characterization of
espin, an actin-binding protein localized to the F-actin-rich junctional
plaques of Sertoli cell ectoplasmic specializations. J Cell Sci 1996;
109:12291239.
18. Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker
MS. Expression in cochlea and retina of myosin VIIa, the gene prod-
uct defective in Usher syndrome type 1B. Proc Natl Acad Sci U S A
1995; 92:98159819.
19. Velichkova M, Guttman J, Warren C, Eng L, Kline K, Vogl AW,
Hasson T. A human homologue of Drosophila kelch associates with
myosin-VIIa in specialized adherens junctions. Cell Mot Cytoskel
2002; 51:147164.
20. Guttman JA, Vaid KS, Vogl AW. Rac1 is present in Sertoli cell struc-
tures (ectoplasmic specializations) associated with intercellular adhe-
sion. FASEB J 2002; 16:A1100.
21. Ozaki-Kuroda K, Nakanishi H, Ohta H, Tanaka H, Kurihara H, Muell-
er S, Irie K, Ikeda W, Sakai T, Wimmer E, Nishimune Y, Takai Y.
Nectin couples cell-cell adhesion and the actin scaffold at heterotypic
testicular junctions. Curr Biol 2002; 12:11451150.
22. Maekawa M, Toyama Y, Yasuda M, Yagi T, Yuasa S. Fyn tyrosine
kinase in Sertoli cells is involved in mouse spermatogenesis. Biol
Reprod 2002; 66:211221.
23. Pfeiffer DC, Vogl AW. Evidence that vinculin is co-distributed with
actin bundles in ectoplasmic (junctional) specializations of mam-
malian Sertoli cells. Anat Rec 1991; 231:89100.
24. Palombi F, Salanova M, Tarone G, Farini D, Stefanini M. Distribution
of 1 integrin subunit in rat seminiferous epithelium. Biol Reprod
1992; 47:11731182.
25. Takai Y, Nakanishi H. Nectin and afadin: novel organizers of inter-
cellular junctions. J Cell Sci 2003; 116:1727.
26. Mueller S, Rosenquist TA, Takai Y, Bronson RA, Wimmer E. Loss
of nectin-2 at Sertoli-spermatid junctions leads to male infertility and
correlates with severe spermatozoan head and midpiece malformation,
impaired binding to the zona pellucida, and oocyte penetration. Biol
Reprod 2003; 69:13301340.
27. Russell L, Clermont Y. Anchoring device between Sertoli cells and
late spermatids in rat seminiferous tubules. Anat Rec 1976; 185:259
278.
28. Russell LD. Deformities in the head region of late spermatids of hy-
pophysectomized-hormone-treated rats. Anat Rec 1980; 197:2131.
29. Russell LD. Observations on the inter-relationship of Sertoli cells at
the level of the blood-testis barrier: evidence for formation and re-
sorption of Sertoli-Sertoli tubulobulbar complexes during the sper-
matogenic cycle of the rat. Am J Anat 1979; 155:259280.
30. Russell LD, Saxena NK, Turner TT. Cytoskeletal involvement in sper-
miation and sperm transport. Tissue Cell 1989; 21:361379.
31. Takahashi K, Nakanishi H, Miyahara M, Mandai K, Satoh K, Satoh
A, Nishioka H, Aoki J, Nomoto A, Mizoguchi A, et al. Nectin/PRRR:
an immunoglobulin-like cell adhesion molecule recruited to cadherin-
based adherens junctions through interaction with afadin, a PDZ do-
main-containing protein. J Cell Biol 1999; 145:539549.
32. Satoh-Hirokawa K, Nakanishi H, Takahashi K, Miyahara M, Nishi-
mura M, Tachibana K, Mizoguchi A, Takai Y. Nectin-3, a new mem-
ber of immunoglobulin-like cell adhesion molecules that shows hom-
ophilic and heterophilic cell-cell adhesion activities. J Biol Chem
2000; 275:1029110299.
33. Sakisaka T, Taniguchi T, Nakanishi H, Takahashi K, Miyahara M,
Ikeda W, Yokoyama S, Peng YF, Yamanishi K, Takai Y. Requirement
of interaction of nectin-1/Hvec with afadin for efcient cell-cell
spread of herpes simplex virus type 1. J Virol 2001; 75:47344743.
34. Hasson T, Walsh J, Cable J, Mooseker MS, Brown SDM, Steel KP.
Effects of shaker-1 mutations on myosin-VIIa protein and mRNA ex-
pression. Cell Mot Cytoskel 1997; 37:127138.
35. Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 1970; 227:680685.
36. Ng T, Shima D, Squire A, Bastiaens PIH, Gschemeissner S, Hum-
phries MJ, Parker P. PKC regulates 1 integrin-dependant cell mo-
tility through association and control of integrin trafc. EMBO J 1999;
18:39093923.
37. Le TL, Yap AS, Stow JL. Recycling of E-cadherin: a potential mech-
anism for regulating cadherin dynamics. J Cell Biol 1999; 146:219
232.
38. Le TL, Joseph SR, Yap AS, Stow JL. Protein kinase C regulates en-
559 ADHESION JUNCTION INTERNALIZATION IN THE TESTIS
docytosis and recycling of E-cadherin. Am J Physiol Cell Physiol
2002; 283:C489C499.
39. Russell LD. Further observations on tubulobulbar complexes formed
by late spermatids and Sertoli cells in the rat testis. Anat Rec 1979;
19:213232.
40. Russell LD, Malone JP. A study of Sertoli-spermatid tubulobulbar
complexes in selected mammals. Tissue Cell 1980; 12:263285.
41. Grove BD, Pfeiffer DC, Allen S, Vogl AW. Immunouorescence lo-
calization of vinculin in ectoplasmic (junctional) specializations of
rat Sertoli cells. Am J Anat 1990; 188:4456.
42. Salanova M, Stefannini M, De Curtis I, Palombi F. Integrin receptor
61 is localized at specic sites of cell-to-cell contact in rat semi-
niferous epithelium. Biol Reprod 1995; 52:7987.
43. Chen B, Li A, Wang D, Wang M, Zheng L, Bartles JR. Espin contains
an additional actin-binding site in its N terminus and is a major actin-
bundling protein of the Sertoli cell-spermatid ectoplasmic specializa-
tion junctional plaque. Mol Biol Cell 1999; 10:43274339.
44. Rhorer J, Schwizer A, Russell D, Kornfeld S. The target of Lamp1
to lysosomes is dependent on the spacing of its cytoplasmic tail ty-
rosine sorting motif relative to the membrane. J Cell Biol 1996; 132:
565576.