Occludin is concentrated at tight junctions of mouse/rat but
not human/guinea pig Sertoli cells in testes
Seiji
Moroi1,2,
Mitinori
Saitou1,
Kazushi
Fujimoto3,
Akira
Sakakibara1,
Mikio
Furuse1,
Osamu
Yoshida2, and
Shoichiro
Tsukita1
1 Departments of Cell Biology,
2 Urology, and
3 Anatomy, Faculty of
Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
Occludin is the
only integral membrane protein identified to date as a component of
tight junctions (TJs). Here, we examined the distribution and
expression of occludin in murine testis bearing well-developed TJ. In
the adult mouse testis, occludin was concentrated at TJ strands, which
are located at the most basal regions of lateral membranes of Sertoli
cells. In immunoblotting, occludin showed a characteristic multiple
banding pattern, suggesting that occludin is highly phosphorylated in
the testis. In 1-wk-old mouse testis, occludin was distributed
diffusely at the lateral membranes of Sertoli cells, and even at this
stage, highly phosphorylated occludin was detected. With development,
occludin gradually became concentrated at the most basal regions of
Sertoli cells. The same results were obtained in rat, but unexpectedly
occludin was not detected in human or guinea pig Sertoli cells by
immunofluorescence microscopy as well as by immunoblotting. Inasmuch as
TJs are also well developed in Sertoli cells of these species, we
concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of occludin or other TJ-associated integral membrane proteins that differ from occludin.
testis; blood-testis barrier; phosphorylation; cell adhesion; ZO-1
 |
INTRODUCTION |
THE TIGHT JUNCTION (TJ) is one mode of cell-to-cell
adhesion in epithelial or endothelial cell sheets. It constitutes
continuous, circumferential seals around cells, which serve as a
physical barrier preventing solutes and water from passing freely
through the paracellular spaces. TJs are also thought to play a role as a boundary between the apical and the basolateral plasma membrane domains to create and maintain cell polarity (for reviews, see Refs.
17, 18, 34). In thin-section electron microscopy, the TJ appears as a
series of discrete sites of apparent fusion, involving the outer
leaflet of the plasma membrane of adjacent cells (10). In
freeze-fracture electron microscopy, the TJ appears as a set of
continuous, anastomosing intramembranous strands or fibrils in the P
face (outwardly facing cytoplasmic leaflet) with complementary grooves
in the E face (inwardly facing extracytoplasmic leaflet) (35).
The testis has well-developed TJs between adjacent Sertoli cells in
seminiferous epithelia (9). These junctions have been reported to
constitute a major part of the so-called "blood-testis barrier"
(BTB), which is thought to be involved in the maintenance of a special
physiological milieu for spermatogenesis and in the protection of germ
cells, especially postmeiotic cells, from the immune system. Although
the morphological features of the BTB have been well described in
several species (for review, see Ref. 31), information regarding the
molecular architecture of the inter-Sertoli TJ is limited (3, 6, 23).
Recently, in addition to TJ-specific peripheral membrane proteins such
as ZO-1, ZO-2, cingulin, 7H6 antigen, and symplekin (1, 7, 19, 23, 36,
39), a novel TJ-associated integral membrane protein, occludin, has
been identified (2, 14). This molecule contains four transmembrane
domains, a long COOH-terminal cytoplasmic domain, a short
NH2-terminal domain, two
extracellular loops, and an intracellular turn (14). Immunolabeled
freeze-fracture replica analyses revealed that occludin is one of the
constituents of the TJ strand itself (12, 20, 32). Occludin is possibly associated with the cytoskeleton through a direct interaction with ZO-1
(15).
In immunoblotting, occludin shows a characteristic multiple banding
pattern (14, 32). We recently found that occludin is heavily
phosphorylated at its serine or threonine residues and that
phosphorylation shifts the occludin band upward, resulting in their
multiple banding pattern (33). Phosphorylated occludin was resistant to
NP-40 extraction and concentrated at the TJ proper.
The functional importance of occludin in TJs has been revealed in
several recent reports. When Madin-Darby canine kidney (MDCK) cells
were transfected with occludin cDNA, they showed elevated transepithelial resistance and an increased number of TJ strands (25).
Introduction of a COOH-terminal-truncated occludin into MDCK cells
caused the increased paracellular flux and perturbation of the
apicobasolateral intramembrane diffusion barrier (4). Furthermore, a
synthetic peptide corresponding to the second extracellular loop of
occludin disrupted the transepithelial permeability barrier (38).
In the present study, we demonstrated that occludin is present in TJ
strands between adjacent Sertoli cells of adult mice but not those of
humans or guinea pigs. Furthermore, we analyzed the developmental
changes of the expression level and subcellular distribution of
occludin in mouse testes.
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MATERIALS AND METHODS |
Antibodies.
Rat anti-mouse occludin monoclonal antibodies (MAbs; MOC37 and MOC60)
and rabbit anti-mouse occludin polyclonal antibodies (PAbs; F4 and F5)
were raised against the cytoplasmic domain of mouse occludin produced
in Esherichia coli (32). MOC37
recognizes occludin in various mammalian species, whereas MOC60 is
specific for mouse occludin. Rabbit anti-rat ZO-1 PAb and mouse
anti-rat ZO-1 MAb (T8-754) were raised and characterized
previously (22).
Testes samples.
Human testes were obtained from orchidectomies for patients with
testicular tumors or trauma. Microscopically normal regions of these
samples were used. Mice (ddY) and
guinea pigs (Hartley) of various ages were purchased from Japan SLC
(Shizuoka, Japan).
Immunofluorescence microscopy.
Mouse, human, and guinea pig testes were frozen in liquid nitrogen.
Sections ~10 µm in thickness were cut in a cryostat, mounted on
coverslips, and air dried. They were fixed with 95% ethanol for 30 min
on ice and then with 100% acetone for 1 min at room temperature. Some
frozen sections were fixed with 1% formaldehyde in PBS for 15 min and
then soaked in 0.2% Triton X-100-PBS for 15 min at room temperature.
Sections were washed three times with PBS, soaked in blocking solution
(PBS containing 1% BSA) for 15 min, and then incubated with the first
antibody for 1 h in a moist chamber. They were washed three times with
blocking solution and incubated with the second antibody for 1 h. As
second antibodies, FITC-conjugated goat anti-rat IgG (Tago, Burlingame,
CA), rhodamine-conjugated goat anti-mouse IgG, or rhodamine-conjugated
goat anti-rabbit IgG (Chemicon International, Temecula, CA) was used.
Sections were washed three times with PBS, mounted in PBS containing
1% p-phenylenediamine and 90%
glycerol, and examined under a fluorescence microscope (Axiophot
photomicroscope, Zeiss, Thornwood, NY).
SDS-digested freeze-fracture replica labeling technique.
The SDS-digested freeze-fracture replica labeling technique was
previously described in detail (12, 13). To remove collagenous tissues,
decapsulated testes were first incubated and stirred in Hanks'
balanced salt solution containing 0.5 mg/ml collagenase (types I and
II, Sigma-Aldrich Japan, Tokyo, Japan) at 37°C for 10 min, and
small fragments of seminiferous tubules were quickly frozen by contact
with a copper block cooled with liquid helium (13). The frozen samples
were fractured at
110°C and platinum-shadowed unidirectionally at an angle of 45° in Balzers Freeze Etching System (BAF 400T, Balzers, Hudson, NH). The samples were immersed in
sample lysis buffer containing 2.5% SDS, 10 mM
Tris · HCl (pH 8.2), and 0.6 M sucrose for 12 h at
room temperature, and replicas floating off the samples were washed
with PBS. Under these conditions, integral membrane proteins were
captured by replicas, and their cytoplasmic domain was accessible to
antibodies. Replicas were incubated with anti-occludin MAb (MOC37) for
60 min and then washed several times with PBS. They were incubated with
goat anti-rat IgG coupled to 10-nm gold (Amersham International,
Buckinghamshire, UK). Replicas were washed with PBS, picked up on
Formvar-coated grids, and examined in a Jeol 1200EX electron microscope
at an accelerating voltage of 80 kV.
Immunoprecipitation.
Occludin was recovered from testes by immunoprecipitation (IP) as
previously described (33) with a slight modification. After careful
removal of the epididymides, testes were minced and homogenized in 1 µl of ice-cold NP-40-IP buffer [25 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1% NP-40, 1 mM
Na3VO4, 1 mM 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF), 10 µg/ml leupeptin, and 10 µg/ml aprotinin] using a Kontes
homogenizer (Kontes, Vineland, NJ). The homogenate was gently rotated
for 30 min at 4°C and centrifuged at 10,000 g for 30 min. After collection of the
supernatant as the "NP-40-soluble fraction," the pellet was
resuspended by sonication in 100 µl of SDS-IP buffer [25 mM HEPES (pH 7.5), 4 mM EDTA, 25 mM NaF, 1% SDS, 1 mM
Na3VO4,
10 µg/ml leupeptin, and 10 µg/ml aprotinin]. Then 900 µl of
NP-40-IP buffer were added, followed by rotation for 30 min at 4°C.
After centrifugation at 10,000 g for
30 min, the supernatant was collected as the "NP-40-insoluble
fraction." Occludin was not detected in the pellet by
immunoblotting. The mixture of a half-volume of each fraction was
designated "total fraction."
Anti-occludin antiserum (4 µl; mixture of F4 and F5) and a 15-µl
bed volume of recombinant protein G-Sepharose 4B (Zymed Laboratories, South San Francisco, CA) were added to each fraction and rotated for 4 h at 4°C. Beads were washed five times with 1 ml of NP-40-IP buffer
and boiled in SDS-PAGE sample buffer containing 1 mM
Na3VO4 to elute the immunoprecipitates. Samples were separated by gel electrophoresis, followed by immunoblotting using anti-occludin MAb
MOC60.
Alkaline phosphatase treatment.
Alkaline phosphatase (AP) treatment was performed as previously
described (33). After IP, beads were washed three times with 1 ml of
NP-40-IP buffer and then three times with 1 ml of AP buffer [50
mM Tris · HCl (pH 8.2), 50 mM NaCl, 1 mM
MgCl2, 1 mM dithiothreitol, and 1 mM APMSF]. They were then resuspended in 200 µl of AP buffer
containing 20 U of calf intestine AP (Takara Shuzo, Ohtsu, Japan). To
some aliquots, phosphatase inhibitors (25 mM NaF, 100 mM
-glycerophosphate, 4 mM EDTA, and 1 mM
Na3VO4) were also added. After a 1-h incubation at 30°C, beads were washed and processed for SDS-PAGE as described above.
Immunoblotting.
Samples were resolved by one-dimensional SDS-PAGE according to the
method of Laemmli (24) and were electrophoretically transferred onto
nitrocellulose membranes (Protran, 0.45 mm pore size; Schleicher and
Schuell, Dassel, Germany). They were then incubated with the first
antibody. For antibody detection, a blotting detection kit (Amersham)
was used.
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RESULTS |
Occludin in adult mouse testis.
When frozen sections of adult mouse testis were immunofluorescently
stained with anti-occludin MAb, intense signals were detected in a
linear fashion from the most basal region of lateral membranes of
adjacent Sertoli cells, where TJs were reported to be well developed
(Fig. 1, A
and B). Endothelial cells of
microvessels were also intensely stained. The localization of occludin
in Sertoli cells was then analyzed at the electron microscopic level by
the SDS-digested freeze-fracture replica labeling technique (Fig. 1C). As reported previously in other
tissues, anti-occludin MAb MOC37 specifically labeled the TJ strand
itself in Sertoli cells.

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Fig. 1.
Subcellular localization of occludin in mouse testis.
A and
B: immunofluorescence and
phase-contrast microscopy of frozen sections of adult mouse testis with
anti-occludin MAb MOC37. Occludin was highly concentrated in a linear
pattern at basal regions of seminiferous tubules (arrows). Endothelial
cells of blood vessels in interstitial tissue were also intensely
stained (arrowheads). C:
immunolabeling of freeze-fracture replicas of Sertoli cells with
anti-occludin MAb MOC37. Tight junction (TJ) strands were exclusively
labeled. Bars, 25 µm (A and
B) and 200 nm
(C).
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Next, occludin expression in adult mouse testis was examined by
immunoblotting with anti-occludin PAb (Fig.
2). When the same amounts of total protein
from mouse kidney and testis were resolved by SDS-PAGE followed by
immunoblotting, a band of ~65 kDa was detected in both tissues, and
the level of expression of occludin in the testis was lower than that
in the kidney (Fig. 2A). Occludin was reported to be resolved as multiple bands in SDS-PAGE, and slowly
moving bands were concentrated in the NP-40-insoluble fraction in
cultured epithelial cells (33). Adult mouse testes were homogenized in
the presence of 1% NP-40, and both the NP-40-soluble and -insoluble fractions were immunoprecipitated with anti-occludin PAb followed by
immunoblotting with anti-occludin MAb MOC60 (Fig.
2B). Under these conditions, the
multiple banding pattern of occludin was clearly identified in mouse
adult testis, and slowly moving bands resistant to the NP-40 extraction
were also seen in the testis. AP analyses showed that phosphorylation
shifted the occludin bands upward and made occludin resistant to NP-40
extraction also in the testis (Fig.
2C).

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Fig. 2.
Occludin in adult mouse testis. A:
immunoblots of whole lysate of adult mouse kidney and testis with
anti-mouse occludin PAb F4 (30 µg proteins in each lane). Level of
~65 kDa occludin expression (arrow) in testis was rather lower than
that in kidney. B: NP-40-soluble and
-insoluble occludin in mouse adult testis. Anti-occludin PAbs (F4 and
F5) immunoprecipitates from total (T), NP-40-soluble (S), and
NP-40-insoluble fractions (I) of testis were immunoblotted with
anti-mouse occludin MAb MOC60. Note that occludin in total fraction was
resolved into multiple bands and that slowly moving bands were
specifically recovered in NP-40-insoluble fraction.
C: alkaline phosphatase (AP)
treatment. Immunoprecipitates in B
were incubated in presence (AP+) or absence (AP ) of AP and
specific phosphatase inhibitor (PI+). AP significantly decreased
apparent molecular masses of NP-40-soluble and -insoluble occludin
bands to level of lowest
Mr band, and its
inhibitor completely suppressed this effect.
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Developmental changes of occludin in mouse testis.
We next examined the developmental changes of occludin distribution in
mouse testis. Frozen sections of testis from 1-, 2-, 3-, and 7-wk-old
mice were stained with anti-mouse occludin MAb MOC37. In the testis of
1-wk-old mice, occludin was distributed discontinuously from apical to
basal regions of cell-cell borders of adjacent Sertoli cells (Fig.
3A).
With the development of testis, i.e., spermatogenesis, occludin was
gradually restricted to the basal part of the lateral membranes of the
epithelia, where TJs between Sertoli cells were reported to be located
in the adult testis (Fig. 3,
B-D). When these samples were
doubly stained with anti-ZO-1 PAb and MOC37, occludin was mostly
colocalized with ZO-1, but some ZO-1-positive and occludin-negative
structures were detected in the interstitium (Fig.
4).

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Fig. 3.
Developmental changes of occludin distribution in mouse testis. Frozen
sections of testes from 1 (A)-, 2 (B)-, 3 (C)-, and 7-wk-old
(D) mice were stained with
anti-mouse occludin MAb MOC37. With development of testes, seminiferous
tubules increased in diameter, and occludin in seminiferous epithelia
was gradually restricted to basal part of their lateral membranes.
Asterisks represent center of each tubule. Bar, 25 µm.
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Fig. 4.
Colocalization of occludin and ZO-1 in mouse testis. Frozen sections of
testis from 1 (A-C)- and
6-wk-old (D-F) mice were doubly
stained with anti-mouse occludin MAb MOC37
(A and
D) and anti-ZO-1 PAb
(B and
E).
C and
F: phase-contrast images. In both
cases, occludin was mostly colocalized with ZO-1 in seminiferous
tubules, but ZO-1 was also detected in occludin-negative sites in
interstitium. Asterisks represent center of each tubule. Bar, 25 µm.
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It was recently reported that the phosphorylation level of occludin
correlated well with the development of TJs in epithelial cells (33).
We then examined the developmental changes of the phosphorylation level
of occludin in mouse testis. As shown in Fig.
5A, even
in 1-wk-old mice, highly phosphorylated occludin was clearly detected
in the NP-40-insoluble fraction. As development of the testis
proceeded, an amount of both NP-40-soluble and -insoluble occludin
increased. Judged from the banding patterns of NP-40-insoluble occludin, the relative content of the most slowly moving band (i.e.,
most heavily phosphorylated occludin) gradually increased until 4 wk
and then decreased with development (Fig.
5B).

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Fig. 5.
Developmental changes of multiple banding patterns of occludin in mouse
testis. A: anti-occludin PAbs (F4 and
F5) immunoprecipitates from NP-40-soluble (S) and -insoluble fractions
(I) of a single testis of 1- to 7-wk-old mice were immunoblotted with
anti-occludin MAb MOC60. B:
densitometric scanning patterns of occludin bands from NP-40-insoluble
fractions. Relative content of most slowly moving band (arrows)
gradually increased until 4 wk and then decreased.
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Species differences of occludin in testis.
Unexpectedly, when guinea pig testes were immunofluorescently stained
with anti-occludin MAb MOC37 or with anti-occludin PAb, no signal was
detected from either the prepubertal (data not shown) or the adult
(Fig. 6, A
and D) seminiferous epithelia. The
ZO-1 signal was, however, intense at the inter-Sertoli junctions
similar to that of mouse testis (Fig. 6,
B and
E). We then examined the expression
and subcellular distribution of occludin in adult human testis using
anti-occludin MAb MOC37 (Fig. 7). Again,
occludin was not detected from the seminiferous epithelia, although
ZO-1 was concentrated at the inter-Sertoli junctions. This was not due
to the poor affinity of MAb MOC37 or PAb F4 to guinea pig (or human)
occludin, since they clearly stained microvessels in the testis
(arrowheads in Fig. 6, A and
D) as well as epithelial cells in
both guinea pig (Fig. 6, G and
H) and human kidneys (Fig. 7D). Other fixation methods and
other MAbs gave the same results (data not shown).

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Fig. 6.
Lack of occludin staining in guinea pig Sertoli cells. Frozen sections
of testes from adult guinea pig
(A-F) were doubly stained with
anti-ZO-1 MAb T8-754 (B and
E) and anti-mouse occludin MAb MOC37
(A) or anti-mouse occludin PAb F4
(D).
C and
F: phase-contrast images. As controls,
frozen sections of guinea pig kidney were singly stained with
anti-mouse occludin MAb MOC37 (G) or
anti-mouse occludin PAb F4 (H). In
testes, occludin was not detected from ZO-1-positive seminiferous
epithelia (arrows) and was detected only in ZO-1-positive structures in
interstitium (arrowheads). In kidney, TJs were clearly stained with MAb
MOC37 (G) as well as PAb F4
(H). G, glomerulus. Bar, 25 µm.
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Fig. 7.
Lack of occludin staining in human Sertoli cells. Frozen sections of
testes from adult humans (A-C)
were doubly stained with anti-mouse occludin MAb MOC37
(A) and anti-ZO-1 MAb T8-754
(B).
C: phase-contrast images. As controls,
frozen sections of human kidney were singly stained with anti-mouse
occludin MAb MOC37 (D). In testis,
occludin was not detected from ZO-1-positive seminiferous epithelia
(arrows). In kidney, TJs were clearly stained with MAb MOC37
(D). Asterisk represents center of
each tubule. Bar, 25 µm.
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Furthermore, we examined the expression level of occludin in guinea pig
testis by immunoblotting with anti-occludin PAb F4 (Fig.
8). When the same amounts of total protein
from mouse or guinea pig kidney and testis were resolved by SDS-PAGE
followed by immunoblotting, in mouse kidney and testis and guinea pig
kidney, an intense occludin band of ~65 kDa was detected. By
contrast, in guinea pig testis, only a weak occludin band that appeared to be derived from endothelial cells was detected. Among the several species examined, occludin was detected in the seminiferous epithelia of mouse and rat testes but not detected in those of guinea pig or
human testes.

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Fig. 8.
Immunoblots of whole lysate of adult mouse or guinea pig kidney (K) and
testis (T) with anti-occludin PAb F4 (30 µg proteins in each lane).
Compared with expression level of occludin in mouse kidney and testis
and guinea pig kidney, that in guinea pig testis was much lower. Note
that molecular mass of mouse occludin is slightly larger than guinea
pig occludin (see Ref. 32).
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 |
DISCUSSION |
When some substances, such as dyes, are administered via blood vessels,
they do not so readily reach the interior of the seminiferous tubules
(31), suggesting the existence of a unique barrier system in the
testes, which is now called the BTB. The BTB is believed to play a
crucial role in maintaining a favorable milieu for spermatogenesis. Many types of tracer studies have demonstrated that well-developed TJs
of Sertoli cells primarily constitute the BTB (5, 8, 16, 30, 31, 37),
although other structures, such as myoid cells around the seminiferous
tubules (9, 11) or some microvessels in the interstitium, were also
reported to be involved in the BTB to some extent.
In the present study, we demonstrated that occludin is present in the
TJ strands of murine Sertoli cells, and we followed the developmental
changes in its subcellular distribution and expression level. Occludin
in the early prepubertal period was located diffusely on the lateral
membranes of Sertoli cells and later gradually became concentrated in
their basal part. These findings were consistent with previous
observations of TJs in the developing testes of mice as well as other
species by freeze-fracture electron microscopy (26-29). In the
early prepubertal period, short and blind-ended TJ strands were
reported to be scattered around the lateral membranes of Sertoli cells,
and the continuous network of TJ strands was gradually developed in
their basal part. Interestingly, even in testes with poorly developed
TJs, the heavily phosphorylated and NP-40-insoluble occludin was
significantly detected, although in small amounts. With the development
of the testis, the de novo synthesis of occludin in Sertoli cells may
be upregulated, resulting in a relative increase of non- or
less-phosphorylated occludin in both NP-40-soluble and -insoluble
fractions. Of course, to correctly interpret the data on the
phosphorylation of occludin in testis, we must carefully evaluate the
contribution to the immunoblots of occludin coming from the endothelium
of vessels.
Another issue to be discussed here is the relatively high level of
occludin expression in testis endothelial cells. Although the
microvessels of the testis are not considered to be directly involved
in the BTB (11, 30), their permeability was reported to be lower than
that of blood vessels in nontestis and nonneuronal tissues (21).
Moreover, they express some of the marker molecules associated with
barrier properties detected in brain microvessels (21). Therefore our
results are consistent with the recent report by Hirase et al. (20)
that the level of occludin expression correlated well with the
tightness of endothelial sheets.
We conclude that occludin is an important structural and functional
component in well-developed TJ strands of the mouse testis, as
previously shown in other tissues. To our surprise, however, occludin
was immunofluorescently detected in mouse and rat testis, whereas it
was not detectable in human or guinea pig testis. Of course, in these
occludin-negative testes, ZO-1 was concentrated in inter-Sertoli
junctions, where TJ strands were reported to be well developed. The
possibility was excluded that our anti-occludin MAbs or PAbs did not
recognize human or guinea pig occludin, since these antibodies clearly
stained TJs in kidney epithelial cells of these species. Furthermore,
immunoblot analyses also suggested the absence of occludin in Sertoli
cells of guinea pig testis. It is generally accepted that the intensity
of immunofluorescent staining with anti-occludin antibodies is
correlated well with the number of TJ strands detected by
freeze-fracture electron microscopy (14, 32), but there were two
exceptions. First, in MDCK cells transfected with
COOH-terminal-truncated occludin, TJ strands were observed to
continuously surround each epithelial cell, but occludin was
concentrated at cell-cell borders in a discontinuous dotted pattern
(4). Second, although occludin was highly concentrated at cell-cell
borders of brain endothelial cells, where TJs were reported to be well
developed, it was hardly detected in nonbrain endothelial cells, where
TJ strands were observed (20). The identification of TJ strands lacking
occludin in human or guinea pig testis in the present study represents clearer exceptions. We were led to speculate that, in guinea pigs or
humans, there are some testis-specific isoforms of occludin. Inasmuch
as all MAbs and PAbs used here recognized the COOH-terminal cytoplasmic
tail of occludin, it is possible that some alternatively spliced form
of occludin specifically occurs in guinea pig or human testis.
Alternatively, it is also possible that there are some other
TJ-associated integral membrane proteins which differ from occludin.
These putative novel TJ-associated integral membrane proteins must
fulfill the following requirements:
1) they constitute TJ strands
without occludin; 2) they associate
with ZO-1 directly or indirectly; and
3) they may interact with occludin,
as seen in TJ strands of mouse or rat testis. Studies are currently
under way in our laboratory to evaluate these speculations.
 |
ACKNOWLEDGEMENTS |
We thank all the members of our laboratory (Dept. of Cell Biology,
Faculty of Medicine, Kyoto University) for helpful discussions throughout this study.
 |
FOOTNOTES |
This work was supported in part by a Grant-in-Aid for Cancer Research
and a Grant-in-Aid for Scientific Research (A) from the Ministry of
Education, Science and Culture of Japan (to S. Tsukita).
Address for reprint requests: S. Tsukita, Dept. of Cell Biology, Kyoto
University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan.
Received 10 November 1997; accepted in final form 12 February
1998.
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