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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 Children’s 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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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). 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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). 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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, 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, A–E). There appeared to be no differences in immunostaining between
the different stages of the cycle of the seminiferous epithelium (Fig. 4, A–E). 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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). 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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, 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 15–18 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 18–20 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.
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Footnotes |
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2 Joint first authors. Both authors contributed equally to all parts
of the work.
Received October 14, 1998.
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References |
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4-5-reductase and 3--hydroxysteroid dehydrogenase in the rat
epididymis during development. Endocrinology 107:948–953[Abstract]
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