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a Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have focused on the identification of tight junctional proteins and how they interact to maintain the structure of tight junctions in various epithelia [4, 5]. However, few have focussed on the tight junctions of the blood-epididymis barrier. Occludin, a ~65-kDa integral membrane protein, has been identified as being responsible for sealing the adjacent plasma membranes of tight junctions together [6]. At the molecular level, ZO-1, a peripheral membrane protein, has been found to be directly associated with the carboxyl terminus of occludin in the tight junction [7]. Together, occludin and ZO-1 have been postulated to maintain the structural integrity of the tight junction [4, 5, 7]. While there are several other tight junction-associated proteins such as ZO-2 [8], cingulin [9], and 7H6 [10], ZO-1 binding to occludin has been shown to be important in targeting occludin to the tight junction and anchoring occludin at the extracellular seal [7, 11].
The cadherins, a family of calcium-dependent cell adhesion glycoproteins joining together adjacent cells within tissues [12–15], have been implicated in the formation and maintenance of tight junctions between epithelial cells of many tissues, including the kidney [16], intestine [17], and liver [18]. In the adult Sprague-Dawley rat, Cyr et al. [19] have shown that E-cadherin is present in the cytoplasm of principal cells of the epididymal epithelium, and that its expression is dependent on serum androgen levels. Studies by Suzuki and Nagano [20] have also suggested that the formation and maintenance of tight junctions in the caput epididymidis relies on gonadal hormones.
The epididymal tight junctions are among the most highly developed contacts between mammalian epithelial cells [21]. Tight junctions, adherens junctions, and gap junctions are present in the epididymal junctional complex [22]. However, studies using electron-opaque tracers, including lanthanum nitrate, have shown that in the adult rat, the tight junction is the only one that does not allow the passage of the tracer [1]. Thus, in situations in which the function of the blood-epididymis barrier is altered, such as in immature rats before the development of the barrier is complete [23], lanthanum nitrate easily permeates the tight junction and enters the lumen.
To date, the blood-epididymis barrier has been investigated during development [23] and in the adult rat [1], but the effects of aging on this barrier are unknown. In addition, the effects of age on tight junction structure or function have not been investigated previously in any tissues, except the testis [24]. In the aging Brown Norway rat testis, the typical Sertoli-Sertoli junctions were rarely seen and were replaced by focal contact points [24]. Lanthanum nitrate was able to penetrate both the basal and adluminal compartments of the seminiferous epithelium of the aging testis [24]. The Brown Norway rat has become a valuable model for the study of aging. It does not exhibit many of the age-related pathologies seen in other rat strains, nor does it become obese during its long life span [25]. The male reproductive system of the Brown Norway rat undergoes dramatic changes with age while the rat remains completely healthy. There is a decrease in spermatogenesis and steroidogenesis [25–27] accompanied by marked histological alterations [28]. There is also an apparent decrease during aging in the quality of sperm with respect to progeny outcome [29]. Importantly, these changes are similar to those observed in the human male reproductive system with age [30, 31].
There are striking transformations in the epididymis of the Brown Norway rat with age [32]. The presence of a large number of halo cells, which may be lymphocytes [33] or monocytes [34], suggests that the immune system is activated in the aging epididymis of these animals. In the present study, we determined whether there were changes in the structural integrity of the blood-epididymis barrier with age by analyzing the distribution of the tight junctional proteins occludin and ZO-1 in the adult and aging Brown Norway rat epididymis. As there is a decrease in androgens with age in the Brown Norway rat, the localization of E-cadherin, a possible regulator of tight junctions, was also compared in the epididymis during aging. Finally, to examine the functional integrity of the blood-epididymis barrier in the aging Brown Norway rat, resistance to penetration by lanthanum nitrate was assessed. We report segment-specific changes in the localization and expression of occludin, ZO-1, and E-cadherin in the aging Brown Norway rat epididymis consistent with the changes seen in lanthanum nitrate penetration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brown Norway rats aged 3, 18, and 24 mo were purchased from the National Institute on Aging, Bethesda, MD, and supplied by Charles River Breeding Laboratories (Wilmington, MA). Animals were housed at the McGill University McIntyre Animal Centre in a temperature- (22°C) and light- (14L:10D) controlled room, with rat food and water available ad libitum. We followed the policies set forth by the facility animal care committee at McGill University as well as those described in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care.
Immunocytochemistry
Brown Norway rats aged 3 (n = 6), 18 (n = 6), and 24 (n = 6) mo were perfused with Bouin's solution for 10 min. While the group of 24-mo-old animals contained regressed and nonregressed testes, the immunostaining patterns were consistent in all of these aged animals with the three antibodies used. Retrograde perfusions through the abdominal aorta were used to fix the initial segment and caput epididymidis, and prograde perfusions were used to fix the corpus and cauda epididymidis. The epididymides were removed, cut along their longitudinal axis, and immersed in Bouin's solution for 24 h. The tissues were then dehydrated and embedded in paraffin. Tissues were cut into 5-µm-thick sections and mounted on glass slides. Subsequently, tissues were rehydrated through graded concentrations of ethanol, including 70% alcohol with 1% hydrogen peroxide for 10 min to remove endogenous peroxidase activity, and 70% alcohol with 1% lithium carbonate for 5 min to remove residual picric acid. The sections were then incubated in 300 mM glycine for 5 min to block free aldehydes, and washed in 1 M PBS at pH 7.4.
Three antibodies were used in this study: a rabbit polyclonal anti-occludin antibody from Zymed Laboratories, Inc. (San Francisco, CA), a rat monoclonal anti-ZO-1 antibody from Chemicon Laboratories Inc. (Temecula, CA), and a mouse monoclonal anti-E-cadherin antibody from Transduction Laboratories (Lexington, KY). Each antibody was used in conjunction with the appropriate Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Incubations with each of the primary antibodies were done for 18 h at 4°C. The working dilutions were 1:540 for occludin and 1:100 for ZO-1 and E-cadherin. Normal appropriate animal sera and omission of the primary antibody served as negative controls. Subsequently, the sections were incubated with a peroxidase diaminobenzidine (DAB) substrate kit (Vector Laboratories). Epididymal sections were then washed in 1 M PBS, counterstained for 30 sec with 0.1% methylene blue dye, dehydrated in solutions containing graded concentrations of ethanol, immersed in xylene, and mounted in Permount (Fisher Scientific, Pittsburgh, PA). Sections were then examined using the light microscope (Leitz Wetzlar, Laborlux D, Montreal, Canada) for immunoperoxidase activity.
To assess the intensity of staining in occludin, ZO-1, and E-cadherin at 3, 18, and 24 mo (Table 1), four animals were studied for each age group. Four different slides per animal were examined for each antibody. Slides were coded and examined without knowledge of the identity of the sample in order to eliminate any potential observer bias. At least 150 tubules were examined for each segment in every age group.
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Lanthanum Nitrate Tracer Study
Brown Norway rats aged 3 (n = 6) and 24 mo (n = 6) were anesthetized with an i.m. injection of a cocktail of ketamine hydrochloride (Ketalean; MTC Pharmaceuticals, Cambridge, ON, Canada), xylazine (Rompun; Bayer Inc., Etobicoke, ON, Canada), acepromazine maleate (Atravet; Ayerst Laboratories, Montreal, PQ, Canada), and 0.9% sodium chloride (Baxter Corporation, Toronto, ON, Canada). This cocktail is often used because it decreases the risk of respiratory depression that can occur with sodium pentobarbital, especially in aged animals. While the group of 24-mo-old animals contained rats having both regressed and nonregressed testes, the results of the lanthanum nitrate study were consistent in all of these aged animals. Retrograde and prograde perfusions were used for the reasons cited above. The fixative consisted of 5% glutaraldehyde buffered with 0.16 M collidine buffer (pH 7.4) containing a final concentration of 2% lanthanum nitrate (Marivac, Nova Scotia, Canada) and 2.5% polyvinylpyrrolidone (PVP), at a final pH of 7.3. The solution was filtered through a Millipore filter using a 5-micron membrane (MicronSep Membrane Filters, Westborough, MA) before use. After perfusion, the epididymides were removed, cut into 1-mm3 pieces, and left either in the same fixative or in the same fixative without lanthanum for 2 h at 4°C. The tissues were washed quickly 3 times in a 0.16 M collidine buffer with or without 2% lanthanum nitrate and 2% sucrose, pH 7.4, and were subsequently washed 3 times in 0.1 M sodium cacodylate buffer with or without 2% lanthanum nitrate and 2% sucrose, pH 7.4 (this last wash was done to remove any toxic collidine that remained). The reason lanthanum was omitted from the immersion solutions in some cases was to confirm that the observed path of tracer was due exclusively to extravasated lanthanum. Tissues were postfixed in 1% osmium tetroxide containing the same cacodylate buffer mixture used for washing, stained en bloc with uranyl acetate, and embedded in epoxy resin. Thin (75-nm) sections were cut using an ultramicrotome; some sections were unstained, and others were routinely stained with uranyl acetate and lead citrate, and examined with a Philips (Mahwah, NJ) 410 electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial segment Occludin (Fig. 1A), ZO-1 (Fig. 1B), and E-cadherin (Fig. 1C) immunostaining was observed at the apico-lateral junction between adjacent principal cells in the entire initial segment of the 3-mo-old animals. While ZO-1 (Fig. 1B) and E-cadherin (Fig. 1C) were present as a punctate reaction at the apico-lateral junction of adjacent principal cells, occludin was localized to the same region but was more extensively distributed, spanning part of the apico-lateral membrane between the principal cells (Fig. 1A). Occludin and ZO-1 immunostaining was not seen in any other cell types in this segment, while narrow cells and apical cells were immunoreactive to E-cadherin (not shown).
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With age, the immunostaining pattern of occludin and ZO-1 differed from that of E-cadherin. At 18 mo, occludin immunostaining became more diffuse around the apico-lateral junction and decreased in intensity (Table 1). The punctate reaction of ZO-1 was no longer seen at every junction of principal cells at 18 mo (Table 1). By 24 mo, occludin immunostaining was dramatically reduced, with a faint reaction at the apex between adjacent principal cells (Fig. 1D); ZO-1 punctate reactivity between principal cells was virtually absent (Fig. 1E and Table 1). While occludin and ZO-1 reactivity decreased with age in the entire initial segment of the Brown Norway rat, E-cadherin immunostaining increased in intensity (Table 1) and was observed along the entire lateral membrane between principal cells at 24 mo (Fig. 1F). No other cell types showed occludin or ZO-1 reactivity, while only apical cells continued to show E-cadherin reactivity at 24 mo (not shown). For all three markers, the initial segment exhibited a unique staining pattern; cytoplasmic staining was never seen in this segment at any age for any of the junctional proteins examined.
Caput epididymidis In contrast to the initial segment at 3 mo, in which occludin, ZO-1, and E-cadherin exhibited a similar staining pattern, the reactivity to these junctional proteins differed in the caput epididymidis for all three markers. At 3 mo, the occludin immunostaining was dramatic in the caput epididymidis (Fig. 2A). Occludin immunostaining intensity in the caput epididymidis was the highest of all the epididymal segments (Table 1). There was a surprising reactivity with a grainy appearance in the entire cytoplasm of the principal cells, and it appeared stronger at the base of the epithelium (Fig. 2A). An intense, concentrated staining was observed at the apical margin of the principal cells (Fig. 2A). ZO-1 was not abundantly expressed in the caput epididymidis at 3 mo (Fig. 2B and Table 1). There was a uniform staining of the cytoplasm of the principal cells (Fig. 2B). In contrast to the ZO-1 punctate reaction seen at the apico-lateral junction between all principal cells in the initial segment at 3 mo (Fig. 1B), ZO-1 punctate staining was only observed at the apico-lateral junction of few principal cells (Fig. 2B and Table 1). E-cadherin immunostaining at 3 mo in the caput epididymidis consisted of both cytoplasmic staining of principal cells and a punctate reactivity at their apico-lateral margins (Fig. 2C); the punctate reactivity was greater in the proximal caput epididymidis than in the distal region. There was no ZO-1 or occludin immunoreactivity in any other cell types in the caput epididymidis at 3 mo; however, the tops of clear cells were immunoreactive to E-cadherin (not shown).
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As in the initial segment, in the caput epididymidis there was a decreased reactivity of occludin and ZO-1 at 18 mo along with an increase in E-cadherin immunostaining (Table 1). By 24 mo, occludin immunostaining had greatly diminished in the cytoplasm of principal cells (Fig. 2D and Table 1). The occludin immunostaining at the apex of the principal cells appeared more intense than in the rest of the cytoplasm but was diffuse in appearance (Fig. 2D). At 24 mo, ZO-1 reactivity was absent; a rare punctate reaction at the apico-lateral junction of principal cells was observed only a few times throughout the entire segment (Fig. 2E and Table 1). Again, E-cadherin exhibited a change in its staining pattern. The cytoplasmic staining of principal cells was absent at 24 mo while immunoreactivity was observed as a punctate stain at the margin of principal cells at their apex (Fig. 2F). The E-cadherin reactivity spread to the entire lateral membrane of principal cells in addition to the punctate reactivity (Fig. 2F). The staining of the lateral membranes was most intense in the proximal caput epididymidis. This E-cadherin immunostaining pattern at 24 mo is similar to that seen for this marker in the initial segment of the aged animals (Fig. 1F). With age, there was a gradual decrease in the E-cadherin immunoreactivity of clear cells at their apex such that, at 24 mo, the majority of clear cells were unreactive (not shown); a few clear cells still showed intense reactivity. No other cell types showed any immunostaining for occludin, ZO-1, or E-cadherin.
Corpus epididymidis The corpus epididymidis showed the most dramatic alterations in immunostaining for occludin, ZO-1, and E-cadherin with age. The staining patterns were identical in the proximal and distal corpus epididymidis. However, the intensity of staining was greater in the distal corpus epididymidis than in the proximal region for all the markers studied. The occludin reactivity was the same as the staining pattern seen in the caput epididymidis at 3 mo but with a much lower intensity (Figs. 2A and 3A). A grainy cytoplasmic staining and an intense band at the apex of the principal cells was seen in the corpus epididymidis (Fig. 3A). The most intense cytoplasmic staining for ZO-1 and E-cadherin was observed in the corpus epididymidis at 3 mo (Fig. 3, B and C). However, ZO-1 also showed a punctate reactivity at the apico-lateral margin of some principal cells (Fig. 3B), which was not seen for E-cadherin at 3 mo (Fig. 3C). As in the caput epididymidis, immunostaining for occludin and ZO-1 was not seen in any other cell types; the clear cells, however, exhibited an intense E-cadherin immunoreactivity along the apex of these cells (not shown).
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The most striking change seen in the corpus epididymidis was that occludin, ZO-1, and E-cadherin reactivity all decreased progressively with age (Table 1). This was the only segment of the epididymis in which E-cadherin immunostaining was absent at 24 mo (Fig. 3F); the intense cytoplasmic staining seen at 3 mo had disappeared. It should be noted, though, that an occasional punctate reactivity at the apico-lateral junction of principal cells was observed. In addition, the majority of clear cells no longer exhibited E-cadherin immunoreactivity at 24 mo (not shown).
Cauda epididymidis The cauda epididymidis showed yet another staining pattern for occludin, ZO-1, and E-cadherin. At 3 mo, occludin immunoreactivity in the cauda epididymidis (Fig. 4A) exhibited a staining pattern similar to that seen in the caput (Fig. 2A) and corpus (Fig. 3A) epididymidis. The grainy cytoplasmic staining was observed, and, while the apical band at the top of the principal cells was more intense than in the rest of the cytoplasm (Fig. 4A), it was not as distinctive as in the other segments. A cytoplasmic reactivity to ZO-1 was seen in the cauda epididymidis at 3 mo (Fig. 4B). The ZO-1 immunostaining seen in the cytoplasm (Fig. 4B) was much more intense than that seen for E-cadherin (Fig. 4C and Table 1). E-cadherin immunostaining was the only one that differed in the proximal and distal cauda epididymidis at 3 mo. In the proximal cauda epididymidis, E-cadherin reactivity was only cytoplasmic (not shown). In contrast to the staining seen for E-cadherin in the other segments of the epididymis at 3 mo, E-cadherin immunostaining was observed at the basolateral membrane between adjacent principal cells in the distal cauda epididymidis (Fig. 4C). An immunoreactivity to occludin or ZO-1 was not seen for any other cell types in the cauda epididymidis. However, as in the caput, corpus, and cauda epididymidis, clear cells continued to show E-cadherin immunostaining at their apical border, most intensely in the proximal cauda epididymidis (not shown).
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With age, each junctional protein exhibited a different change in its immunostaining pattern. By 24 mo, the occludin immunoreactivity progressively increased in intensity (Fig. 4D and Table 1). However, ZO-1 immunostaining decreased to become absent by 24 mo (Fig. 4E). This is the only segment in which occludin and ZO-1 did not change in a coordinate manner. Finally, E-cadherin immunoreactivity was now seen at the basolateral membrane of principal cells of the entire cauda epididymidis but had spread to stain the entire lateral membrane between principal cells (Fig. 4F and Table 1). At 24 mo, the clear cell E-cadherin immunoreactivity was absent (not shown).
Lanthanum Nitrate Tracer Study
As reported in previous studies [1, 23], lanthanum nitrate entered the capillaries and subsequently crossed into the epididymal epithelium, where it was seen between adjacent principal cells. In the initial segment, the electron-opaque tracer was consistently stopped at the tight junction at 3 (Fig. 5A) and 24 mo (Fig. 5B). While the tight junction obstructed the passage of lanthanum nitrate into the lumen in the caput epididymidis at 3 mo (Fig. 5C), the tracer was occasionally seen entering the first fusion points of the tight junction. At 24 mo, lanthanum nitrate often entered the tight junction leaflets of the caput epididymidis and, in addition, the tracer could be seen at times in the junction as well as within the lumen as dark granules (Fig. 5D).
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While lanthanum nitrate did not penetrate the tight junction in the corpus epididymidis at 3 mo (Fig. 6A), there was a striking amount of tracer that was able to cross the blood-epididymis barrier and enter the lumen in this segment at 24 mo; this permeability was observed consistently (Fig. 6B). In contrast, lanthanum nitrate was blocked at the tight junction of both 3- (Fig. 6C) and 24-mo-old (Fig. 6D) animals in the cauda epididymidis, in a manner similar to what was seen in the initial segment at both ages (Fig. 5, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the initial segment, occludin, ZO-1, and E-cadherin colocalized to the apico-lateral junction of adjacent principal cells. While the immunoreactivity to occludin and ZO-1 decreased with age in the initial segment, the lateral membrane between adjacent principal cells stained intensely for E-cadherin with increasing age. The initial segment exhibited a unique staining pattern in that cytoplasmic staining was not seen in this segment as in the caput, corpus, and cauda epididymidis for any of these junctional proteins. The junctional complex in the initial segment has been previously shown to span a considerable length of the apical plasma membranes with few desmosomes [22]. In contrast, in the rest of the epididymis the span of merging plasma membranes is considerably reduced, and there are numerous desmosomes in the apical region [22]. This difference in the junctional complex may be one of the reasons for the differing distribution of occludin, ZO-1, and E-cadherin in the initial segment.
The blood-epididymis barrier maintains the ability to block the passage of lanthanum nitrate in the initial segment of the aging animals despite the decrease in occludin and ZO-1 reactivity. Interestingly, Saitou et al. [35] have recently shown that tight junctions in occludin-deficient epithelial cells are able to function as a primary barrier to the diffusion of a low-molecular-mass tracer through the paracellular pathway. These findings indicate that there may be unidentified tight junction integral membrane proteins that can form strand structures, recruit ZO-1, and function as a barrier without occludin [35]. Furuse et al. [36] have recently discovered two novel integral membrane proteins, termed claudin-1 and -2, that localize at tight junctions but bear no sequence similarities to occludin. Claudin-1 or -2 can reconstitute tight junctions and recruit occludin in fibroblasts [37]. The possibility therefore exists that there may be other junctional proteins that enable the tight junction to block the passage of lanthanum nitrate despite the decrease in occludin and ZO-1. Alternatively, the amount of occludin and ZO-1 remaining in the aged animals may be sufficient to inhibit the passage of the tracer, especially since E-cadherin is still present and shows a greater intensity in the initial segment of the aged animals.
In contrast to the initial segment, occludin, ZO-1, and E-cadherin all exhibited some cytoplasmic staining in the caput, corpus, and cauda epididymidis of the adult Brown Norway rats. E-Cadherin cytoplasmic reactivity has previously been seen in the epididymis of adult male Sprague-Dawley rats [19]. It has been suggested that the cytoplasmic localization of E-cadherin may reflect the synthesis and processing of this protein in the endoplasmic reticulum and Golgi apparatus, and its transport via vesicles within the cytoplasm toward the lateral membranes [19]. Recently, however, a study on the mechanisms of epithelial cell-cell adhesion reported that insertion of preassembled E-cadherin membrane-insertion structures from the cytoplasm into the membrane at cell-cell contacts was never seen [38]. It was suggested that E-cadherin originates by de novo aggregation at sites of cell-cell contact [38]. Cadherins are known to associate with the actin-based cytoskeleton via the catenin family [39, 40], and occludin has also been shown to bind to the cytoskeleton via ZO-1 [41]. It is likely, therefore, that the cytoplasmic staining we are seeing for E-cadherin and ZO-1 is a result of the binding of these proteins to the cytoskeleton in the cytoplasm.
The most dramatic age-dependent changes seen in the present study occurred in the corpus epididymidis. The loss of occludin, ZO-1, and E-cadherin with age was most pronounced in this segment of the epididymis and was reflected by the loss of the functional integrity of the blood-epididymis barrier. Thus, it appears that the major decrease in occludin, ZO-1, and E-cadherin leads to the change in permeability of the blood-epididymis barrier of the Brown Norway rat with age. Interestingly, a study by Kimura et al. [42], which looked at the expression of occludin, ZO-1, and E-cadherin in cancers of the human digestive tract, suggested that expression of occludin and ZO-1 may not be enough to form a tight junction. The authors postulated that perhaps both intercellular adhesion, mediated by E-cadherin, and expression of occludin and ZO-1 may be required for normal tight junction formation [42]. It appears that maintenance of tight junctions is also dependent on the presence of E-cadherin, occludin, and ZO-1 in the epididymis. Cyr et al. [19] have shown that bilateral orchidectomy followed by low-dose testosterone replacement is able to maintain mRNA concentrations of E-cadherin in all regions of the epididymis except in the corpus epididymidis region of adult Sprague-Dawley rats. As androgen levels are decreased with age in Brown Norway rats [25, 26], our data support the possibility that the corpus epididymidis requires more androgen than other segments of the epididymis to maintain E-cadherin expression.
The most striking changes that have been reported in aging Brown Norway rats seem to occur in the corpus epididymidis, especially in the distal region. There is a large increase in the size and number of lysosomes with age in this segment. By 24 mo, principal cells show a few giant lysosomes that are usually filled with translucent small vacuoles [32], suggesting that there is extensive degradation and breakdown of substances in this segment. The activation of the immune system is most pronounced in the aging corpus epididymidis [32]. In addition to an increase in halo cells, Serre et al. [32] have seen eosinophils in the interstitial tissue and within the epithelium of the corpus epididymidis of rats aged 18 mo and older. Finally, the expression of certain subunits of the glutathione-S-transferases, involved in protecting cellular constituents from electrophilic and oxidative attack, were found to change their immunoreactivity patterns only in the distal regions of the epididymis [43]. Together these data point to a loss in the ability to handle oxidative stress in the corpus epididymidis of the aging Brown Norway rats.
The segment-specific alterations in the expression of occludin, ZO-1, and E-cadherin and the changes in the penetration of lanthanum nitrate into the blood-epididymis barrier with age indicate that there are both structural and functional changes taking place. The fact that the blood-epididymis barrier is responsible for creating the unique luminal microenvironment present in each segment of the epididymis [2] raises questions as to the ability of the barrier to maintain this function with age. The penetration of lanthanum nitrate into the lumen of the aged animals in the caput and corpus epididymidis suggests that there is a greater permeability of the blood-epididymis barrier in these segments with age.
The regulation of junctional proteins is currently under extensive investigation. Phosphorylation has been suggested to be a possible mechanism by which occludin and ZO-1 localization and function are regulated [44–46]. Tyrosine phosphorylation may also cause an increase in tight junction permeability [45]. The Rac subfamily of small G proteins has been shown to regulate the cadherin-based cell-cell adhesion but not the formation of tight junctions [47]; the rho subfamily has been shown to be necessary for both functions [47, 48]. It is, therefore, tempting to speculate that the changes with age in the expression of E-cadherin, occludin, and ZO-1 in the epididymis are due in part to a change in the phosphorylation of these proteins.
In summary, we have demonstrated that there are segment-specific changes in the localization and distribution of occludin, ZO-1, and E-cadherin in the aging Brown Norway epididymis, and that the permeability of the blood-epididymis barrier is also affected in a region-specific manner with age. The permeability of the blood-epididymis barrier increases with age in the caput and corpus epididymidis. The present study is one of the first to provide information about the effects of age on the structure and function of tight junctions in any tissue.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Bernard Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, PQ, Canada H3G 1Y6. FAX: 514 398 7120; brobaire{at}pharma.mcgill.ca
Accepted: January 26, 1999.
Received: November 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin is a vinculin homologue. Proc Natl Acad Sci USA 1991; 88:9156–9160.This article has been cited by other articles:
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  S. Skrovanek, M. C. Valenzano, and J. M. Mullin Restriction of sulfur-containing amino acids alters claudin composition and improves tight junction barrier function Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1046 - R1055. [Abstract] [Full Text] [PDF]
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J. Dufresne and D. G. Cyr Activation of an SP Binding Site Is Crucial for the Expression of Claudin 1 in Rat Epididymal Principal Cells Biol Reprod, May 1, 2007; 76(5): 825 - 832. [Abstract] [Full Text] [PDF]
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