Endocrinology Vol. 140, No. 8 3815-3825
Copyright © 1999 by The Endocrine Society
Cellular Immunolocalization of Occludin during Embryonic and Postnatal Development of the Mouse Testis and Epididymis1
Daniel G. Cyr2,
Louis Hermo2,
Nicole Egenberger,
Carmen Mertineit,
Jacquetta M. Trasler and
Dale W. Laird
Centre de Recherche en Santé Humaine (D.G.C.), Institut
National de la Recherche Scientifique-Institut Armand Frappier,
Université du Québec, Pointe Claire, Québec, H9R 1G6
Canada; Department of Anatomy and Cell Biology (L.H., N.E.), McGill
University, Montréal, Québec, H3A 2B2 Canada;
Montréal Childrens Hospital Research Institute and Departments
of Pediatrics, Pharmacology and Therapeutics and Human Genetics (C.M.,
J.M.T.), McGill University, Montréal, Québec, H3H 1P3
Canada; and Department of Anatomy and Cell Biology (D.W.L.), University
of Western Ontario, London, Ontario, N6A 5B8 Canada
Address all correspondence and requests for reprints to: Dr. Daniel Cyr INRS-Institut Armand Frappier, Université du Québec, 245 Hymus Boulevard, Pointe Claire, Québec, H9R 1G6 Canada. E-mail: daniel.cyr{at}inrs-sante.uquebec.ca
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Abstract
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Cellular junctions in the testis and epididymis play crucial roles for
the development and maturation of spermatozoa. In the testis, tight
junctions between Sertoli cells form a functional blood testis barrier
between 10 and 16 days of age, whereas the tight junctional blood
epididymal barrier between adjacent epithelial cells is formed between
days 18 and 21. In the present study, occludin, a constituent integral
membrane protein of tight junctions, was localized by immunofluorescent
confocal microscopy in embryonic (days 13.518.5), postnatal (days
523) and adult (day 70) mouse testes and epididymides to correlate
its expression with the onset of tight junctions and eventual formation
of these barriers. At embryonic days 13.5 and 16.5, low diffuse
cytoplasmic levels of occludin were observed in cells of the testicular
cords. By embryonic day 18.5, the level of occludin was still low but
appeared as a filiform-like network streaming toward the center of the
cord. At postnatal days 5 and 7 immunostaining became more intense and
appeared to outline the periphery of Sertoli cells of seminiferous
tubules. Postnatal day 14 marked the appearance of an intense, focal
band-like localization of occludin at the base of the tubules,
correlating with the appearance of a functional blood-testis barrier.
By day 23 and in adults, expression of occludin was noted at the base
of the tubule appearing as intense, wavy, discontinuous bands similar
in appearance irrespective of the stage of the seminiferous epithelium
cycle. In the developing epididymis, intense cytoplasmic immunostaining
was present in epithelial cells of many epididymal tubules at embryonic
day 13.5. By embryonic day 16.5, intense occludin immunostaining
appeared along the lateral plasma membranes of epithelial cells,
whereas at embryonic day 18.5, immunostaining was punctate and apically
located, suggesting the presence of tight junctions by this age;
similar immunostaining was noted at postnatal days 5 and 7. In the
adult epididymis, distinct punctate apical staining was observed
between adjacent principal cells of all epididymal regions except the
proximal initial segment, where occludin was found only in association
with narrow cells. These results indicate that in the epididymis, the
appearance of occludin at apical sites between adjacent epithelial
cells occurs during embryonic development suggesting that tight
junctions form earlier than in the testis. While occludin was expressed
in a similar pattern between Sertoli cells at all stages of the cycle
in the adult testis, its expression in the adult epididymis was cell-
and region-specific. Taken together these data suggest that different
factors regulate occludin expression in the testis and epididymis.
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Introduction
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THE ULTRASTRUCTURAL properties of tight and
gap junctional complexes in the male reproductive tract have been well
characterized particularly in the seminiferous epithelium where tight
junctions between adjacent Sertoli cells are responsible for the
formation of the blood-testis barrier (1, 2, 3) and gap junctions allow
for the intercellular passage of molecules (2, 3, 4, 5, 6). Tight junctions
create two distinct compartments within the epithelium, namely a basal
compartment containing spermatogonia and preleptotene spermatocytes
which have access to circulating blood-borne substances and an
adluminal compartment containing all other germ cells (1, 3, 6, 7). The
adluminal compartment is a structurally defined environment created by
the secretory and endocytic activities of Sertoli cells. This
compartment is essential for spermatogenesis and creates an immune-free
environment (8, 9, 10). Interestingly, gap junctions have been reported to
assemble among the strands of tight junctions both in thyroid
epithelial cells and in the seminiferous epithelium, suggesting that
the adherence provided by tight junctions may facilitate gap junction
formation (1, 6, 7, 11).
There have been a number of studies on the testis of animals at
different embryonic and postnatal ages dealing with the structural
features of Sertoli and germ cells (12, 13). In addition, studies on
occluding junctions have revealed that linear strands of such junctions
appear during the second postnatal week, whereupon they progressively
increase and develop at the base of the cell with advancing age (14).
Between 10 and 16 days of age in the mouse, there is the establishment
of an impermeable blood-testis barrier preventing the entry of electron
dense tracers into the lumen (15).
In the epididymis, the final events of sperm maturation occur whereby
they acquire motility and ability to fertilize (16, 17, 18). While all the
intricacies by which spermatozoa mature in the epididymis have not been
resolved, a number of factors are thought to contribute to the
maturation of spermatozoa. These include proteins secreted by the
epididymal principal cells that interact with the sperm surface,
substances endocytosed by principal and clear cells and ionic changes
along the epididymal length, all of which create a favorable luminal
environment (17, 18, 19). In the epididymis, the luminal environment is
restricted to circulating substances by tight junctions interposed
apically between adjacent principal cells which form the
blood-epididymal barrier (20, 21, 22, 23, 24). Studies of the epididymis of
embryonic and postnatal animals have revealed that tight junctions
appear during embryonic development but increase in number during
postnatal development up until day 21 when an impermeable barrier to
tracers is established (20, 23). Clearly, specific environments in the
seminiferous epithelium and epididymal epithelium play crucial roles in
the formation and maturation of spermatozoa.
Tight junctions are composed of a variety of peripheral membrane
proteins (zona occludens 1, 2, and 3 (ZO-1, 2, 3) symplekin, cingulin,
7H6 antigen, cytoskeletal elements (fodrin, actin) and an integral
transmembrane protein termed occludin (25, 26, 27, 28). One of the most well
characterized proteins associated with occludin, ZO-1, has recently
been demonstrated to also bind the gap junction protein, connexin 43,
(Cx43) (29) suggesting that ZO-1 may regulate the intracellular
assembly of both occludin and connexins.
Occludin has been noted in a number of different tissues and shown to
be a constituent of tight junctional strands and associated with the
cytoskeleton through a direct interaction with various proteins (26, 29, 30, 31). Occludin, a phosphoprotein, contains four membrane spanning
domains and two extracellular loops (26, 32, 33). Occludin binds in the
extracellular space to occludin from an adjacent cell (34). Recently,
in seminiferous tubules of the adult rat and mouse testis, occludin was
localized to tight junctions between adjacent Sertoli cells at the base
of the seminiferous epithelium (33).
The objectives of the present study were to determine, by confocal
immunofluorescent microscopy, the embryonic and postnatal developmental
pattern of occludin expression in the developing testis and epididymis
of the mouse. Also, it was our objective to examine the spatial
localization of occludin with respect to the gap junction protein,
connexin 43 (Cx43), in Sertoli-Sertoli junctional complexes. Together
the data generated from this study will allow us to establish the
developmental pattern of expression and regulation of occludin in the
testis and epididymis.
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Materials and Methods
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Isolation and preparation of tissue
CD-1 mice were obtained from Charles River Canada
(St. Constant, Québec, Canada). Noon of the day on which a
vaginal plug was observed, was considered to be embryonic day 0.5 and
the day of delivery as postnatal day 1. Mice were obtained at embryonic
days 13.5, 16.5, and 18.5, and postnatal days 5, 7, 14, 23, and 70
(n = 4 at each age). These ages were chosen because they
represented time points before, during and after the blood testis and
blood epididymal barriers are fully functional, and in this way we
could determine when occludin localizes to the site of tight junctions
in these different tissues during development. After the pregnant
females were euthanized by cervical dislocation, the embryos were
dissected out of the uterine horns and freed from extraembryonic
membranes. Embryos were fixed by perfusion through the heart under a
dissecting microscope with 100% saline, a 1:1 mixture of saline and
Sainte-Maries fixative (95% ethanol/glacial acetic acid, 99:1),
followed by 100% Sainte-Maries fixative. Post-natal mice were
anesthetized with 4% chloral hydrate and fixed by perfusion through
the heart with a physiological saline solution followed by
Sainte-Maries fixative. The testes and epididymides were dissected,
postfixed in the same fixative for 1 h on ice, dehydrated through
graded ethanols, cleared in xylene, and embedded in paraffin. All of
the procedures used on animals in this study were approved by the
University Animal Care Committee.
Immunocytochemistry
Immunofluorescent localization of occludin was done as
previously described (35) on 5 µm sections mounted on slides coated
with 5% gelatin, baked at 60 C for 1 h, and conserved at 4 C
until used. All of the procedures for the avidin-biotin-fluorescent
staining of occludin were carried out at room temperature with
solutions prepared in PBS, pH 7.2, unless specified otherwise. Sections
were blocked for 30 min in blocking buffer (0.2% cold-water fish skin
gelatin; Sigma Chemical Co. Chemicals, Mississauga,
Ontario, Canada), 5% goat serum (Life Technologies, Inc.,
Mississauga, Ontario, Canada) and 0.2% Tween 20 (Sigma Chemical Co.) incubated overnight at 4 C, in a humidified chamber, with
affinity-purified polyclonal rabbit antioccludin antisera (Zymed Laboratories, Inc., Seattle, WA) at a concentration of 10
µg/ml. Following the washing in Tween-20 PBS, the sections were
blocked in 5% goat serum, washed, and incubated for 30 min with a
biotinylated goat antirabbit IgG (1:200 dilution; Vector Laboratories, Inc., Burlingame, CA). The sections were then
washed and incubated for 1 h with fluorescein (FITC)-avidin D
(1:125 dilution in HEPES buffer; Vector Laboratories, Inc.) and counterstained with 0.5 µg/ml propidium iodide
(Molecular Probes, Inc., Eugene, OR) to visualize the
nuclei. Stained sections were then mounted with an antifade solution
containing 0.025% 1,4-diazobicyclo-(2,2,2)-octane (DABCO; Sigma Chemical Co.) in 90% glycerol.
Colocalization experiments
Sections were blocked for 30 min in blocking buffer and
incubated overnight at 4 C in a humidified chamber with both a
polyclonal affinity purified rabbit antioccludin antibody at a
concentration of 10 µg/ml and a monoclonal IgG mouse anti-Cx43
antibody at a dilution of 1:50 (Chemicon). After washing in Tween-20
PBS, the sections were blocked in 5% goat serum, washed, and incubated
for 30 min with biotinylated goat antirabbit IgG (1:200) and a rabbit
antimouse IgG conjugated to Texas Red (1:200). The sections were then
washed and incubated for 1 h with FITC-avidin D (1:200). Mounting
was performed as described above.
Fluorescence of the fluorochromes FITC, Texas Red, and propidium iodide
was visualized with a Zeiss LSM410 confocal microscope as described by
Laird et al. (36) and digitalized images were printed on a
Tektronix high resolution printer.
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Results
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Embryonic testis
At embryonic day 13.5, a weak diffuse staining was observed in the
cytoplasm of cells distributed throughout the testicular cords (Fig. 1A
). By embryonic day 16.5, weak staining
persisted in cells of the testicular cords (Fig. 1C
). By embryonic day
18.5, the reaction appeared as a weak filiform-like network radiating
from the periphery of the cord, where the small nuclei of Sertoli cells
were located, to the central area where germ cells were present (Fig. 1E
, arrows).

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Figure 1. Testicular cords at embryonic days 13.5 (A), 16.5
(C), and 18.5 (E) immunostained with antioccludin antibody. At day
13.5, diffuse cytoplasmic staining is noted over epithelial cells,
presumably gonocytes (open arrows), randomly scattered
in the testicular cords (outlined in white). At day
16.5, small nuclei, intensely stained with propidium iodide and
corresponding to Sertoli cells, begin to be distributed at the
periphery of the cords (circled in white), while
gonocytes, scattered within the cord, present a large round pale
nucleus and diffuse cytoplasmic staining (open arrows).
By day 18.5, the testicular cords (circled in white)
reveal small peripheral intensely stained nuclei corresponding to
Sertoli cells. These cells are weakly immunostained and show a
filiform-like network extending toward the center of the tubule
(arrows). The cells with a large pale nucleus,
corresponding to germ cells, appear to be unlabeled. The developing
epididymis at embryonic days 13.5 (B), 16.5 (D), and 18.5 (F)
immunostained with antioccludin antibody. At embryonic day 13.5,
intense immunostaining is noted over the cytoplasm of epithelial cells
of the epididymal tubules (curved arrows). By embryonic
day 16.5, occludin appears to be localized to the apical and lateral
plasma membranes of the epithelial cells of epididymal tubules
(curved arrow), whereas at day 18.5, intense
immunostaining is distributed along the apical plasma membrane of the
epithelial cells (curved arrows). Bar, 10
µm.
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Embryonic epididymis
In the epididymis at embryonic day 13.5, an intense cytoplasmic
reaction was observed in epithelial cells of many tubules that at this
age already showed a lumen (Fig. 1B
). By embryonic day 16.5, the
reaction in epithelial cells was observed along the lateral plasma
membranes (Fig. 1D
). By embryonic day 18.5, the reaction became intense
and localized to the apical cell surface of the epithelial cells, which
in appropriate planes of section appeared as punctate dots (Fig. 1F
).
Postnatal development of testis
By postnatal day 5, a more pronounced filiform-like network was
observed in the testis (Fig. 2A
), and
this was also noted at postnatal day 7 (Fig. 2C
). At both these ages,
occludin appeared to be localized to the periphery of Sertoli cells
(Fig. 2C
). At postnatal day 14, the seminiferous tubules of the testis
were still small in diameter and contained both mitotic and meiotic
germ cells and Sertoli cells. Coincident with the presence of more
advanced germ cells in the epithelium, distinct immunostaining for
occludin in Sertoli cells was noted near the base of the tubule and
appeared as intense focal wavy bands, whereas a weak filiform-like
network radiated toward the center of the tubule (Fig. 3
, A and B). By postnatal day 23, the
diameter of the seminiferous tubules increased in size and contained
Sertoli cells, mitotic, meiotic, and haploid germs cells and a distinct
lumen (Fig. 3
, C and D). The most intense immunostaining was noted in
Sertoli cells at the base of the tubules as discontinuous, wavy
reactive bands, whereas some staining was still noted at higher levels
of the epithelium (Fig. 3
, C and D).

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Figure 2. Testis at postnatal days 5 (A) and 7 (C)
immunostained with antioccludin antibody. At postnatal day 5,
filiform-like immunostaining is noted over the cytoplasm of Sertoli
cells (arrows), whereas in postnatal day 7 tubules at
higher magnification and in grazing sections reveal intense
immunostaining along the base of the seminiferous epithelium
(arrows) corresponding mainly to Sertoli cells. A
section of a postnatal day 7 testis treated with normal goat serum
shows an absence of reaction (E). Epididymis at postnatal days 5 (B)
and 7 (D) and rete testis at postnatal day 7 (F) immunostained with
antioccludin antibody. At postnatal days 5 and 7, labeling is seen
along the apical plasma membrane of the epithelial cells of epididymal
tubules (curved arrows). Intense immunostaining for
occludin is also found in epithelial cells of the rete testis at
postnatal day 7 (horizontal arrows). Bar,
10 µm.
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Figure 3. Testes at low and high magnifications of postnatal
days 14 (A, B) and 23 (C, D). At postnatal day 14, the seminiferous
tubules containing several germ cells, are immunostained for occludin
mainly over the basal region of the epithelium (arrows),
whereas a weak filiform-like network of occludin extends toward the
center of the tubule. At higher magnification and in grazing sections
(B), these immunolabeled bands of occludin appear extensive and often
extend over considerable distances of the basal region of the
epithelium (arrows). At day 23, when germ cells are
abundant and the tubule size is enlarged, the most pronounced
immunostaining appears as discontinuous, wavy bands occupying the basal
region of the epithelium and corresponding to areas of Sertoli-Sertoli
junctional complexes (arrows). Bar, 10
µm.
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Postnatal development of the epididymis
At postnatal days 5 and 7, an intense reaction was observed at the
apical surface of epithelial cells of the epididymis (Fig. 2
, B and D).
The rete testis also expressed occludin at postnatal day 7 (Fig. 2F
).
Sections treated with either normal goat serum or blocking buffer
failed to show any significant background immunostaining over cells of
the testicular cords (Fig. 2E
), testis, or developing epididymis (data
not shown).
Adult testis and epididymis
In the adult testis, intense immunolocalization of occludin was
observed in Sertoli cells and seen at the periphery of the tubules as
discontinuous wavy reactive bands, whereas weak staining was noted at
higher levels of the epithelium (Fig. 4
, AE). There appeared to be no differences in immunostaining between
the different stages of the cycle of the seminiferous epithelium (Fig. 4
, AE). The endothelial cells of blood vessels showed numerous areas
of intense immunostaining along their surface (Fig. 4E
, double
arrows). Treatment of sections with normal goat serum or blocking
buffer showed weak or absence of reaction over cells of the
seminiferous tubules (Fig. 4F
). In some areas of the interstitial
space, autofluorescence was observed over Leydig cells, but this was
consistent with background levels also noted over some of these cells
of negative control sections (Fig. 4
).

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Figure 4. Seminiferous tubules of adult mice immunostained
with antioccludin antibody. Low magnification of tubules at early
(top tubule) and mid (bottom tubule)
stages of the cycle (A), reveal occludin immunostaining at the base of
the seminiferous epithelium (arrows). High magnification
of tubules (B) at various stages of the cycle present intense
immunostaining observed as discontinuous wavy bands at the base of the
epithelium (arrows). A tubule at stage XI of the cycle
is noted at low (C) and high (D) magnifications and reveals intense
discontinuous wavy bands of immunostaining at the base of the
epithelium (arrows). The endothelium of a blood vessel
(E) shows intense areas of immunostaining (large double
arrows) and an adjacent tubule reveals a basal reaction
(arrows). Control sections of the testis (F) treated
with normal goat serum fail to show any specific immunostaining over
seminiferous tubules. Bar, 10 µm.
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In adult mice, distinct punctate labeling was observed over the apical
cell surface of epithelial cells of the efferent ducts (Fig. 5A
). In the epididymis, region-specific
differences were noted. In the proximal initial segment, the only
reactive sites were found on narrow cells of the epithelium (Fig. 5B
, curved arrows), while immunostaining between adjacent
principal cells was absent. From the distal initial segment (Fig. 5C
),
through the caput (Fig. 5D
), corpus (Fig. 5E
), and cauda regions (data
not shown) of the epididymis, reactive sites were noted at the apical
cell surface of principal cells where they were seen in side views as
punctate dots or in oblique sections as short bands. While
immunostaining was noted between principal cells, there was little
immunostaining associated with clear cells (Fig. 5E
, asterisk). Basal
cells at the periphery of the tubules did not show any evidence of
immunostaining for occludin. Absence or weak staining with normal goat
serum or blocking buffer indicated the specificity of the
immunostaining procedure (Fig. 5F
).

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Figure 5. Efferent ducts and epididymis of adult
animals immunostained with antioccludin antibody. A, Intense, punctate,
immunostaining, is observed at the apical plasma membrane of the
epithelial cells of the efferent ducts (curved arrows).
B, Proximal initial segment of the epididymis reveals immunostaining
exclusively in the area of narrow cells (curved arrows).
C, Distal initial segment of the epididymis displays staggered punctate
immunostaining over the apical plasma membrane of principal cells
(curved arrows). D, Epithelium of the caput
epididymidis, as seen in side and oblique views, reveals intense
punctate or band-like staining, respectively, over the apical cell
surface of principal cells (curved arrows). E,
Epithelium of the corpus epididymidis cut slightly obliquely presents
intense band-like staining over the apical surface of principal cells
(curved arrows). No staining is noted over clear cells
(asterisk). F, Adult mice treated with normal goat serum
serving as controls show absence of staining. Bar, 10
µm.
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Colocalization of occludin and Cx43 in adult testis
Immunofluorescent colocalization studies were performed to
determine whether or not occludin and Cx43 were localized to the same
sites in the seminiferous epithelium. Both occludin (Fig. 6
, A and B) and Cx43 (Fig. 6
, C and D)
were localized to the periphery of the seminiferous epithelium in
relation to Sertoli cells, with occludin being far more abundant.
Overlays of these images revealed that Cx43 colocalized with occludin
(Fig. 6
, E and F, arrows) at the base of the epithelium, but
Cx43 was also localized to focal sites higher up in the epithelium
devoid of occludin (Fig. 6
, E and F, double arrows). In some
areas of the interstitial space, autofluorescence was observed over
Leydig cells, but this was consistent with background levels also noted
over some of these cells of negative control sections (Fig. 6
).

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Figure 6. Colocalization of occludin and Cx43. Low (A, C, E)
and high (B, D, F) magnifications of mouse testis immunostained for
occludin (A, B) and Cx43 (C, D). Overlays of antioccludin and anti-Cx43
images (E, F) reveal that Cx43 at the base of the epithelium
colocalized with occludin (arrows). However, Cx43
localized to sites higher in the epithelium does not overlap with
occludin (small double arrows). Bar, 10
µm.
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Discussion
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In testicular cords at embryonic days 13.5 and 16.5, occludin was
expressed in all cells, randomly distributed at these ages, suggesting
that occludin was already synthesized during early embryonic
development. By embryonic day 18.5, Sertoli and germ cells
redistributed themselves in the cords. Sertoli nuclei, smaller in size,
occupied the periphery of the tubule, whereas the few germ cells were
more centrally distributed. At this age, a filiform-like staining
pattern extended toward the center of the cord, suggesting that it was
localized in Sertoli cells but not yet in areas of tight junctions.
Sertoli cells have been shown to possess cell processes that radiate
toward the center of the cord among germ cells (12). Germ cells at this
age appeared unreactive and further work would be required to know why
they would express occludin for such a short period during embryonic
development.
At postnatal days 5 and 7, the reaction became more intense but was
distributed along the lateral plasma membranes of Sertoli cells. By
postnatal day 14, the reaction changed dramatically and for the first
time appeared as intense, focal bands distributed close to the base of
the epithelium, suggesting that occludin was localized to areas of
tight junctions between adjacent Sertoli cells. These findings
correlate with those of others who reported that Sertoli-Sertoli tight
junctions are almost completely formed with sites of membrane
apposition clearly defined and with the formation of an impermeable
blood-testis barrier between 10 and 16 days of age in the mouse and
1518 days in the rat (15, 37, 38, 39). Immunolocalization of ZO-1 in the
developing mouse testis becomes progressively restricted to the tight
junctional region of Sertoli cells between 7 and 14 days of age.
Because ZO-1 is associated with occludin in the formation of tight
junctions, these observations, as well as those from the present study,
support the proposal that ZO-1 and occludin are involved in the
formation of the blood-testis barrier (37).
The finding of occludin expression early during embryonic and postnatal
development suggests that it appears before the formation of a more
fully patent seminiferous tubular lumen that occurs between days 1820
in the rat (14, 40), and at a time when not all generations of germ
cells are present. Expression and localization to tight junctions also
takes place before androgens are at high levels which occurs by
postnatal day 39 (41). Thus for the time being, it is undetermined what
factor(s) regulates occludin expression during early embryonic and
postnatal development.
The assembly of tight junctions is thought to involve calcium-dependent
cell adhesion molecules (cadherins) that, via intracellular G proteins
results in an increase in intracellular calcium and acts as a trigger
to target the junctional proteins to the tight junctions (27). Previous
studies have identified N-cadherin mRNA in the testis and N-cadherin
was localized to the contact points between adjacent Sertoli cells (42, 43). However, N-cadherin mRNA levels during postnatal testicular
development did not correlate with the formation of the blood-testis
barrier suggesting the involvement of another member of the cadherin
family (42). Recently, Munro and Blaschuk (44) have identified several
testicular cadherins including two novel cadherins. There is no
information, however, whether these may be involved in the formation of
Sertoli cell tight junctions.
At postnatal day 23, occludin expression was intense at the base of the
epithelium. This coincides with an increase in size of the tubular
diameter, number of germ cells and the presence of a distinct lumen.
However, at postnatal days 23 as well as 14, a filiform-like network
still persisted and radiated toward the lumen, suggesting that occludin
was not strictly targeted to tight junctional sites at these ages. In
the adult testis, now showing a complete complement of germ cells,
intense occludin expression was noted at the base of the seminiferous
epithelium, where its ultrastructural localization was restricted to
Sertoli-Sertoli cell tight junctional strands (33). Occludin has been
localized to tight junctions in other tissues (45). While it has been
observed that tight junctions appear apically as focal sites in the
seminiferous epithelium between adjacent Sertoli cells as well as
between Sertoli and germ cells of some species (46), the present data
do not allow us to determine whether or not occludin is present at
these sites. This is due to the fact that immunostaining apically is
weak and diffuse and would require electron microscopic
immunocytochemical analysis for confirmation.
In the present study, close analysis of the various stages of
spermatogenesis revealed no major qualitative differences in the
expression of occludin between Sertoli cells. This is particularly
interesting as preleptotene spermatocytes penetrate through tight
junctions and displace from the basal to the adluminal compartment of
the tubule at mid stages of the cycle (7, 47), suggesting that, at
these stages, occludin expression would be absent or weak. Furthermore,
while it has been reported that an intermediate compartment exists at
specific stages of the cycle (47), future electron microscopic studies
would be required to determine whether or not occludin is localized on
both sides of spermatocytes that are migrating upward. Also while the
structural features of Sertoli cells and many of their functions show
stage-specific distribution (9, 48), the present results indicate no
such differences for occludin. As may be predicted, ZO-1, which binds
occludin, was also previously shown not to be stage-specific (7). Thus,
it would appear that both these proteins are important to the
blood-testis barrier and must be present at all stages of the
cycle.
In the developing epididymis, apical punctate reactive sites were noted
between adjacent epithelial cells of many tubules by day 18.5.
Thereafter, during postnatal development and into adulthood, the
reaction persisted at the apical cell surface. The formation of tight
junctions in the epididymis has been identified as early as embryonic
day 12 by freeze fracture electron microscopy (20). At this age a mesh
network of tight junctions was noted surrounding the entire
circumference of the epithelial epididymal cells at the juxtaluminal
position and this was maintained throughout embryonic development (20).
However, during postnatal development, the number of apically localized
tight junctional strands continued to increase up until 21 days of age
resulting in the formation of a lanthanum impermeable blood-epididymal
barrier (24). Thus, while we noted that occludin was localized at
apical sites in the developing epididymis as early as embryonic day
18.5, there may be a quantitative increase in occludin during
development which cannot be assessed by the immunofluorescent approach
used in our study. It should be pointed out that during development,
not all regions of the epididymis were investigated as serial sections
were not performed. As a result, it is not possible to determine if
there are region-specific differences in occludin expression at the
different ages of development.
The expression of occludin in the area of the tight junctions in the
developing epididymis (embryonic day 18.5) occurs much earlier than
noted in the seminiferous epithelium (postnatal day 14). Clearly the
results of these experiments suggest that the regulation of occludin
expression in testicular tight junctions is different from that in
epididymal tight junctions. Similar conclusions have also been made in
the mink (49, 50, 51). While there is little information on the regulation
of tight junctions of the blood-testis and blood-epididymal barriers,
Suzuki and Nagano (22) have suggested that there is a loss of tight
junctional strands associated with orchidectomy in the epididymis. The
localization of occludin to the apical cell surface by embryonic day
18.5 at an age when androgen levels are low and germ cells are not
abundant would suggest that they do not regulate its expression. Thus
at present, data concerning factors regulating the expression of
junctional proteins of the blood testis barrier are not known.
In the adult, occludin was noted between adjacent epithelial cells of
the efferent ducts. In the epididymis, occludin was noted apically
between adjacent principal cells except in the proximal initial
segment. In the latter region, occludin was seen only in association
with narrow cells. These data would suggest that other tight junctional
proteins must be present between principal cells in this region, as
tight junctions have been shown to exist between these cells (23), but
the nature of these proteins is at present unknown. Recently, two new
integral transmembrane proteins, claudin-1 and claudin-2, have been
isolated from junctional complexes of chicken hepatocytes, which do not
share homology with occludin and may be localized to this region (52).
The manner by which occludin in narrow cells establishes itself with
tight junctions of adjacent principal cells lacking occludin is
undefined.
Oblique sections of the apical cell surface of the epididymis often
revealed linear strands of occludin over the apical cell surface of
principal cells; however, clear cells appeared devoid of occludin. How
adjacent principal cells interact with the neighboring clear cell to
form tight junctions in the absence of occludin is also undetermined.
In other respects, the appearance of occludin between adjacent
principal cells correlates with the presence of tight junctions known
to be present along the epididymal length (23, 50).
In the present study we noted the presence of occludin between
endothelial cells of blood vessels, similar to what has been reported
by Moroi et al. (33), suggesting that occludin forms tight
junctions between these cells. However, endothelial cells of testicular
blood vessels are not involved in the blood testis barrier, although
their permeability is lower than that of blood vessels of other tissues
(47, 53, 54).
The junctional complex between Sertoli cells in the seminiferous
epithelium is composed of tight and gap junctions that are present in
the area of the Sertoli-Sertoli junctional complex (1, 3, 7). Previous
studies have indicated that Cx43 gap junctions are present in the
testis between adjacent Sertoli cells at the base of the seminiferous
epithelium (55). Our present data indicate that occludin and Cx43
colocalize together at many sites near the base of the seminiferous
epithelium. However, Cx43 was also localized at sites higher in the
epithelium, where it has been shown to be present between germ cells
and Sertoli cells (Cyr, D. G., L. Hermo, and D. W. Laird,
unpublished observations), and where gap-like junctions have been
reported (2, 3), but this was not the case for occludin. It has been
shown that occludin is associated with ZO-1 and that the latter may be
involved in the localization of occludin at tight junctions (31). Moroi
et al. (33) also noted that occludin and ZO-1 colocalized to
interSertoli junctions. It has recently been reported that ZO-1 links
Cx43 to
-spectrin in cardiac myocytes and may be involved in
regulating the intracellular distribution of Cx43 (29). The
localization of occludin, ZO-1 and Cx43 to the same junctional
complexes suggests that Sertoli cell-cell communication and structural
adherens occurs at the same cell-cell interfaces, and ZO-1 may serve a
mechanistic role in regulating both of these junctional complexes.
In summary, occludin is expressed in Sertoli cells during early
embryonic and postnatal development and becomes well localized to the
Sertoli-Sertoli tight junctions by postnatal day 14 coinciding with the
formation of a functional blood-testis barrier. In the epididymis,
expression of occludin at apical tight junctions between epithelial
cells occurs by embryonic day 18.5 and is maintained throughout
postnatal development. The early appearance of occludin in the
embryonic epididymis occurs well before the formation of a functional
blood-epididymal barrier taking place at about day 21. Together, these
data suggest that different factors regulate occludin expression in the
testis and epididymis.
 |
Footnotes
|
|---|
1 This work was supported by the Medical Research Council of Canada and
INRS. 
2 Joint first authors. Both authors contributed equally to all parts
of the work. 
Received October 14, 1998.
 |
References
|
|---|
-
Dym M, Fawcett DW 1970 The blood-testis
barrier in the rat and the physiological compartmentation of the
seminiferous epithelium. Biol Reprod 3:308326[Abstract]
-
Russell LD 1993 Morphological and functional
evidence for Sertoli-germ cel relationships. In: Russell LD, Griswold
MD (eds) The Sertoli Cell. Cache River Press, Clearwater, FL, pp
365390
-
Enders GC 1993 Sertoli-Sertoli and Sertoli-germ
cell communications. In: Russell LD, Griswold MD (eds) The Sertoli
Cell. Cache River Press, Clearwater, FL, pp 447460
-
Gilula NB, Fawcett DW, Aoki A 1976 The Sertoli
cell occluding junctions and gap junctions in mature and developing
mammalian testis. Dev Biol 50:142168[CrossRef][Medline]
-
Russell LD, Peterson RN 1985 Sertoli cell
junction: morphological and functional correlates. Int Rev Cytol 94:177211[Medline]
-
Pelletier R-M 1998 Blood-tissue barriers in the
male reproductive system. In: Martínez-García F,
Regarda J (eds) Male Reproduction. Churchill Communications Europress
España, Lisbon, pp 183195
-
Byers SW, Pelletier R-M, Suarez-Quian C 1993 Sertoli-Sertoli cell junctions and the seminiferous cell barrier. In:
Russell LD, Griswold MD (eds) The Sertoli Cell. Cache River Press,
Clearwater, FL, pp 431446
-
Hermo L, Oko R, Morales CR 1994 Secretion and
endocytosis in the male reproductive tract: a role in sperm maturation.
Int Rev Cytol 154:105189
-
Morales CR, Clermont Y 1993 Structural changes of
the Setoli cell during the cycle of the seminiferous epithelium. In:
Russell LD, Griswold MD (eds) The Sertoli Cell. Cache River Press,
Clearwater FL, pp 305330
-
Fawcett DW 1975 Ultrastructure and function of the
Sertoli cell. In: Hamilton DW, Greep RO (eds) Handbook of Physiology,
Section 7, Volume 5, Male Reproductive System. American Physiological
Society, Washington, DC, pp 2175
-
Guerrier A, Fonlupt R, Morand I, Rabilloud R, Audebet C,
Krutovskikh, Gros D, Rousset B, Munari-Silem Y 1995 Gap junctions
and cell polarity: connexin32 and connexin43 expressed in polarized
thyroid epithelial cells assemble into separate gap junctions, which
are located in distinct regions of the lateral plasma membrane domain.
J Cell Sci 108:26092617[Abstract]
-
Pelliniemi LJ, Fröjdman K, Paranko J 1993 Embryological and prenatal development and function of Sertoli cells.
In: Russell LD, Griswold MD (eds) The Sertoli Cell. Cache River Press,
Clearwater, FL, pp 87114
-
Gondos B, Berndtson WE 1993 Postnatal and pubertal
development. In: Russell LD, Griswold MD (eds) The Sertoli Cell. Cache
River Press, Clearwater, FL, pp 115154
-
Russell LD, Bartke A, Goh JC 1989 Postnatal
development of the Sertoli cell barrier, tubular lumen, and
cytoskeleton of Sertoli and myoid cells in the rat, and their
relationship to tubular fluid secretion and flow. Am J Anat 184:179189[CrossRef][Medline]
-
Nagano T, Suzuki F 1976 The postnatal development
of junctional complexes of the mouse Sertoli cells as revealed by
freeze fracture. Anat Rec 185:403417[CrossRef][Medline]
-
Orgebin-Crist M-C, Danzo BJ, Davies J 1975 Endocrine control of the development and maintenance of sperm
fertilizing ability in the epididymis. In: Greep RO, Astwood EB (eds)
Handbook of Physiology, Section 7, Volume 5. American Physiological
Society, Washington, DC, pp 319338
-
Robaire B, Hermo L 1988 Efferent ducts, epididymis,
and vas deferens: structure, functions, and their regulation. In:
Knobil E, Neill J (eds) The Physiology of Reproduction. Raven Press,
New York, pp 9991080
-
Cooper TG 1986 Function of the epididymis and its
secretory products (Part III). In: The Epididymis, Sperm
Maturation and Fertilization. Springer Verlag, Berlin, pp 117230
-
Hinton BT, Palladino MA 1995 Epididymal epithelium:
its contribution to the formation of a luminal fluid microenvironment.
Microsc Res Tech 301:6781
-
Suzuki F, Nagano T 1978 Development of tight
junctions in the caput epididymal epithelium of the mouse. Dev Biol 63:321334[CrossRef][Medline]
-
Friend DS, Gilula NB 1972 Variations in tight
junctions and gap junctions in mammalian tissues. J Cell Biol 53:758776[Abstract/Free Full Text]
-
Suzuki F, Nagano T 1976 Changes in occluding tight
junctions of epididymal epithelium in the developing and gonadectomized
mammals. J Cell Biol 70:101a
-
Cyr DG, Robaire B, Hermo L 1995 Structure and
turnover of junctional complexes between principal cells of the rat
epididymis. Microsc Res Tech 30:5466[CrossRef][Medline]
-
Agarwal A, Hoffer AP 1989 Ultrastructural studies
on the development of the blood-epididymis barrier in immature rats. J
Androl 10:425431[Abstract/Free Full Text]
-
Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara
A, Itoh M, Yonemura S, Furuse M, Tsukita S 1996 Interspecies
diversity of the occludin sequence: cDNA cloning of human, mouse, dog,
and rat-kangaroo homologues. J Cell Biol 133:4347[Abstract/Free Full Text]
-
Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S,
Tsukita Sa, Tsukita Sh 1993 Occludin: a novel integral membrane
protein localizing at tight junctions. J Cell Biol 123:16171626
-
Denker BM, Nigam SK 1998 Molecular structure and
assembly of the tight junction. Am J Physiol 274:F1F9
-
Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson
BR 1998 ZO-3, a novel member of the MAGUK protein family found at
the tight junction, interacts with ZO-1 and occludin. J Cell Biol 141:199208[Abstract/Free Full Text]
-
Toyofuku T, Yabuki M, Otsu K, Kuzuya T, Hori M, Tada
M 1998 Direct association of the gap junction protein connexin-43
with ZO-1 in cardiac myocytes. J Biol Chem 273:1272512731[Abstract/Free Full Text]
-
Staddon JM, Rubin LL 1996 Cell adhesion, cell
junctions and the blood-brain barrier. Curr Opin Neurobiol 6:622627[CrossRef][Medline]
-
Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S,
Tsukita Sa, Tsukita Sh 1994 Direct association of occludin with
ZO-1 and its possible involvement in the localization of occludin at
tight junctions. J Cell Biol 127:16171626[Abstract/Free Full Text]
-
Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y,
Tsukita S 1997 Possible involvement of phosphorylation of occludin
in tight junction formation. J Cell Biol 137:13931401[Abstract/Free Full Text]
-
Moroi S, Saitou M, Fujimoto K, Sakakibara A, Furuse M,
Yoshida O, Tsukita S 1998 Occludin is concentrated at tight
junctions of mouse/rat but not human/guinea pig Sertoli cells in
testis. Am J Physiol 274:C1708C1717
-
Wong V, Gumbiner BM 1997 A synthetic peptide
corresponding to the extracellular domain of occludin perturbs the
tight junction permeability barrier. J Cell Biol 136:399409[Abstract/Free Full Text]
-
Mertineit C, Yoder JA, Taketo T, Laird DW, Trasler JM,
Bestor TH 1998 Sex-specific exons control DNA methyltransferase in
mammalian germ cells. Development 125:889897[Abstract]
-
Laird DW, Castillo M, Kasprzak L 1995 Gap junction
turnover, intracellular trafficking, and phosphorylation of connexin43
in brefeldin A-treated rat mammary tumor cells. J Cell Biol 131:11931203[Abstract/Free Full Text]
-
Byers S, Graham R, Dai HN, Hoxter B 1991 Development of Sertoli cell junctional specializations and the
distribution of the tight-junction-associated protein ZO-1 in the mouse
testis. Am J Anat 191:3547[CrossRef][Medline]
-
Meyer R, Polsalky Z, McGinely D 1977 Intercellular
junction development in maturing rat seminiferous tubules. J
Ultrastruct Res 61:271283[CrossRef][Medline]
-
Vitale RD, Fawcett DW, Dym M 1973 The normal
development of the blood-testis barrier and the effects of clomiphene
and estrogen treatment. Anat Rec 176:333344[CrossRef]
-
Tindall DJ, Vitale R, Means AR 1975 Androgen
binding protein as a marker of formation of the blood-testis barrier.
Endocrinology 97:636648[Abstract]
-
Scheer H, Robaire B 1980 Steroid
4-5
-reductase and 3-
-hydroxysteroid dehydrogenase in the rat
epididymis during development. Endocrinology 107:948953[Abstract]
-
Cyr DG, Blaschuk OW, Robaire B 1992 Identification
and developmental regulation of cadherin messenger ribonucleic acids in
the rat testis. Endocrinology 131:139145[Abstract]
-
Byers S, Jégou B, MacCalman C, Blaschuk O 1993 Sertoli cell adhesion molecules and the collective organization of
the testis. In: Russell LD, Griswold MD (eds) The Sertoli Cell. Cache
River Press, Clearwater, FL, pp 461476
-
Munro SB, Blaschuk OW 1996 A comprehensive survey
of the cadherins expressed in the testes of fetal, immature, adult mice
utilizing the polymerase chain reaction. Biol Reprod 55:822827[Abstract]
-
Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J,
Fujimoto K, Tsukita S 1997 Mammalain occludin in epithelial cells:
its expression and subcellular distribution. Eur J Cell Biol 73:222231[Medline]
-
Pelletier R-M, Byers SW 1992 The blood-testis
barrier and Sertoli cell junctions: structural considerations. Micros
Res Tech 20:333[CrossRef][Medline]
-
Russell LD 1978 The blood-testis barrier and its
formation relative to spermatocyte maturation in the adult rat: a
lanthanum tracer study. Anat Rec 190:99112[CrossRef][Medline]
-
Parvinen M 1993 Cyclic function of Sertoli cells.
In: Russell LD, Griswold MD (eds) The Sertoli Cell. Cache River Press,
Clearwater, FL, pp 331348
-
Bardin CW, Cheng CY, Musto NA, Gunsalus GL 1988 The
Sertoli Cell. In Knobil E, Neill JD (eds) The Physiology of
Reproduction. Raven Press, New York, pp 933974
-
Pelletier R-M 1995 Freeze-fracture study of cell
junctions in the epididymis and vas deferens of a seasonal breeder: the
mink (Mustela vison). Microsc Res Tech 30:3753[CrossRef][Medline]
-
Pelletier R-M 1994 Blood barriers of the epididymis
and vas deferens act asynchronous with the blood barrier of the testis
in the mink (Mustela vison). Micros Res Tech 27:333349[CrossRef][Medline]
-
Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse
M, Takano H, Noda T, Tsukita S 1998 Occludin-deficient embryonic
stem cells differentiate into polarized epithelial cells bearing tight
junctions. J Cell Biol 141:397408[Abstract/Free Full Text]
-
Fawcett DW, Leak LV, Heidger PM 1970 Electron
microscopic observations on the structural components of the
blood-testis barrier. J Reprod Fertil [Suppl] 10:105122[Medline]
-
Holash JA, Harik SI, Perry G, Stewart PA 1993 Barrier properties of testis microvessels. Proc Natl Acad Sci USA 90:1106911073[Abstract/Free Full Text]
-
Risely MS, Tan IP, Roy C, Sáez JC 1992 Cell-,
age- and stage-dependent distribution of connexin43 gap junctions in
testes. J Cell Sci 103:8196[Abstract/Free Full Text]
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