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Testis |
Department of Anatomy and Cell Biology,3 Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Department of Molecular Biology and Biochemistry,4 Osaka University Medical School, Osaka 565-0871, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.
Sertoli cells, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ectoplasmic specializations occur only in Sertoli cells and are tripartite structures consisting of the plasma membrane, a layer of actin filament bundles, and an attached cistern of endoplasmic reticulum. In basal regions of attachment between Sertoli cells, other junction types (tight junctions, gap junctions, and desmosomes) occur within and around ectoplasmic specializations. In apical regions of attachment to spermatids, other junction types are less apparent or are absent. Among molecules identified as components of ectoplasmic specializations are actin [5–14], -actinin [6, 15], vinculin [16], espin [17], fimbrin [16], myosin VIIa [18, 19], gelsolin [3], rac1 [20], afadin [21], and Fyn tyrosine kinase [22]. Adhesion molecules identified at the ectoplasmic specializations include 6ß1 integrin [23, 24] and nectin 2 [21]. The binding partner for 6ß1 integrin on adjacent cells has not been identified. At basal junctions, the ligand for nectin 2 is likely another nectin 2 molecule in the ectoplasmic specialization in the adjacent Sertoli cell [21, 25]. At apical junctions, nectin 2 appears to bind heterotypically to nectin 3 in the plasma membrane of the adjacent spermatid heads [21, 25, 26].
Tubulobulbar complexes that develop between Sertoli cells and spermatids consist of tubular extensions from the plasma membrane of the spermatid head that protrude into corresponding plasma membrane invaginations of the adjacent Sertoli cell [27] (Fig. 2). Near the end of the tubular process is a bulblike swelling [27]. In the Sertoli cell, the tubular invagination is cuffed by a network of actin filaments and surrounding the bulbous region are elements of the endoplasmic reticulum, which are closely apposed to the plasma membrane. At distal ends of the invaginations are clusters of numerous vesicles, some of which have been identified as lysosomes [27].
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The function of tubulobulbar complexes is not entirely clear, although a number of possibilities have been suggested. One possibility is that they are an attachment device between Sertoli cells and spermatids. In the rat, tubulobulbar processes are the last structures to disappear at sperm release [27]. Another possibility is that they are a mechanism by which material from spermatids is endocytosed by Sertoli cells during spermatid maturation. In cases where tubulobulbar complexes do not develop, spermatids do not reduce their volume normally during maturation and release is delayed [28]. Another possibility is that they are part of the mechanism by which intercellular junctions, particularly ectoplasmic specializations, are disassembled during spermatogenesis [4, 28–30]. Pertinent to this possibility are three key observations made by Russell and Clermont in their original description of tubulobulbar complexes [27]. First, the structures occur in areas also occupied by ectoplasmic specializations, indicating that there may be a functional relationship between the two types of structures. Second, tubulobulbar complexes develop at basal junction complexes between adjacent Sertoli cells [27], indicating that the primary function of tubulobulbar complexes may not be related only to spermatid maturation. Third, ultrastructurally identifiable gap and tight junctions occur in the bulbous regions of basal tubulobulbar complexes, indicating that junction elements may occur in these structures [29].
In this study, we investigate the possibility that tubulobulbar complexes are involved in the disassembly of ectoplasmic specializations. We concentrate on the complexes that develop in association with apical junctions because they are much larger than those that occur basally, can be resolved at the light level using differential interference contrast (DIC) optics, have been better studied than those that occur at basal sites, can be isolated within apical Sertoli cell processes fragmented away from the epithelium, and can be located easily using late-step spermatid heads as morphological markers. We clearly demonstrate that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that tubulobulbar complexes contain molecular markers for ectoplasmic specializations. In addition, we demonstate that vesicular elements present in the Sertoli cell apical processes surrounding the spermatid heads, and related to the tubulobulbar complexes, stain positively for nectin 2 and nectin 3. Nectin 3 is not present on testicular spermatozoa released from the epithelium, and double membrane vesicles occur adjacent to tubulobulbar complexes. These results are consistent with the possibility that adhesion domains of spermatids are internalized together with related regions of the Sertoli cell membrane at tubulobulbar complexes. Significantly, we also demonstate lysosomal/endosomal markers are present in regions occupied by Sertoli cell vesicles and that protein kinase C (PKC), implicated as being involved with the endocytosis of adhesion molecules in other systems, is present at sites containing tubulobulbar complexes. Our results are consistent with the hypothesis that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of adhesion membrane domains both of the Sertoli cell and the adjacent spermatid at the time of sperm release.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals used in these studies were reproductively active male Sprague Dawley rats. They were obtained from the University of British Columbia animal care colony and were maintained according to the guidelines established by the Canadian Council on Animal Care. All experiments were performed at least in duplicate (most were performed more than three times) using separate animals.
Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents were obtained from Sigma-Aldrich Canada (Mississauga, ON). The paraformaldehyde and NaCl were obtained from Fisher Scientific (Vancouver, BC). All control immunoglobulins (IgGs) as well as all secondary antibodies conjugated to horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). All secondary antibodies conjugated to ALEXA fluorochromes were purchased from Molecular Probes (Eugene, OR). Polybed embedding resin was obtained from EM Sciences (Fort Washington, PA).
Immunofluorescence Tissue Preparation
Testes were removed from male rats under deep anesthesia. Warm (33°C) PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3) was perfused through the spermatic artery using a 26-gauge needle attached to a gravity-fed perfusion apparatus for 2 min to clear the organ of blood. Following this, warm 3% paraformaldehyde in PBS was perfused through the testis for 30 min. PBS was then reperfused through the organ to wash out any remaining fixative.
Frozen Sections
Fixed testes were frozen (using liquid nitrogen) while at the same time being attached to an aluminum stub by OCT compound (Sakura Finetek USA, Torrance, CA). Frozen testis sections were cut, attached to poly-L-lysine-coated glass slides, immediately plunged into –20°C acetone for 5 min, air dried, and then processed for immunofluorescence.
Fragmented Material
Fixed testes were decapsulated in PBS and the seminiferous tubule was mass minced into small pieces. The pieces were transferred into a 15-ml plastic Falcon tube along with about 5 ml of PBS. The material was gently passed through an 18-gauge, then 21-gauge needle for 2–5 gentle passes. This fragmented material was left to sediment by gravity at room temperature for 5 min and then the upper layer was transferred to another tube. The cells in suspension were pelleted using centrifugation, the pellet was resuspended in a small volume of PBS, and then the suspension was added to poly-L-lysine-coated slides and allowed to incubate in a humidity chamber for 10 min. All excess PBS was then removed and the slides were immediately treated with –20°C acetone for 5 min and allowed to air dry.
Immunofluorescence
Slides with attached tissue fragments or cryosections were rehydrated and blocked with 5% normal goat serum (NGS) in TPBS-BSA (PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin) for 20 min at room temperature. Primary antibodies consisted of rat anti-mouse nectin 2 antibodies (#502-57 [31]), rat anti-mouse nectin 3 antibodies (#103-A1 [32]), mouse anti-rat-l-afadin antibodies (#3 [33]), mouse anti-PKC antibodies (Transduction Labs, Lexington, KY), rabbit anti-rat espin antibodies (gift from Dr. Jim Bartles [17]), rabbit anti-human myosin VIIa antibodies (gift from Dr. Tama Hasson [18, 34]), rabbit anti-kelch/keap1 antibodies (gift from Dr. Tama Hasson [19]), rabbit anti-SGP1 antibodies (gift from Dr. Carlos Ramon Morales), and mouse anti-LAMP1 antibodies (developed by Dr. J. Thomas August and obtained from the Developmental Studies Hybridoma Bank, Univ. of Iowa, Iowa City, IA). Antibodies were added to the experimental slides, made up in TPBS-BSA with 1% NGS, and incubated overnight at 4°C in a humidity chamber. The material was washed extensively with the TPBS-BSA (wash buffer), then incubated for 60 min at 37°C with secondary antibody conjugated to a fluorochrome (goat anti-mouse ALEXA 488, goat anti-rabbit ALEXA 568, or goat anti-rat ALEXA 546). The slides were again washed and then coverslips were mounted using Vectashield (Vector Labs, Burlington, ON). The tissue was visualized using a Zeiss Axiophot microscope fitted with appropriate filter sets for detecting fluorescence and with the appropriate optics for DIC or phase microscopy.
Controls for immunofluorescence localization consisted of the following: 1) Primary antibodies were replaced with normal immunoglobulin (IgG) (in the case of affinity-purified antibodies) or normal serum (in the case of non-affinity-purified serum antibodies) from the host animal species at identical concentrations to the primary antibody (when the stock concentration of primary antibody was not known, it was assumed to be 1 mg/ml), 2) primary antibody was replaced with buffer alone, 3) both the primary and the secondary antibodies were replaced with buffer alone.
Phalloidin/Phallotoxins
Filamentous actin was labeled using ALEXA 488 or ALEXA 568 phalloidin (Molecular Probes). The stain was made up in PBS or in TPBS.
One-Dimensional Western Blotting
Seminiferous epithelium was isolated from the testis and extensively homogenized in RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA, 1% Nonidet P-40, 1% deoxychloic acid [sodium salt], 10% SDS) before being loaded into wells of 1-mm-thick 10% SDS-PAGE gels and run according to standard protocols [35]. Proteins were transferred onto Immobilon-P transfer membrane (Millipore, Billerica, MA), then washed for 5 min at room temperature with TBST (500 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). The blots were then blocked to decrease nonspecific antibody binding for 8 h at 4°C using 4% nonfat milk (Blotto, Santa Cruz Biotechnology, Santa Cruz, CA). Following blocking, membranes were washed three times, 10 min each, then incubated with primary antibody overnight at 4°C. The following day, blots were washed extensively with TBST followed by a 1-h secondary antibody (conjugated to horseradish peroxidase) incubation at room temperature. Upon further washing with TBST followed by TBS, blots were reacted with enhanced chemiluminescence (Pharmacia, Peapack, NJ) to visualize the reactive bands on X-OMAT film (Eastman Kodak, Rochester, NY).
Controls consisted of replacing the primary antibodies with IgG or serum at identical primary antibody concentrations or identical serum dilutions.
Electron Microscopy
Rat testes were removed and perfusion fixed for 30 min with 0.1 M sodium cacodylate, 1.5% paraformaldehyde, 1.5% glutaraldehyde. Each testis was then cut into small pieces and immersion fixed for an additional 90 min. The testis material was then washed with 0.1 M sodium cacodylate for three 10-min washes, then further fixed on ice for 60 min in a 1:1 mixture of 3% K4Fe(CN)6:2% osmium fixative. Following the incubation, the material was washed three times with ddH2O, 10 min each wash, then stained for 1 h with 0.1% uranyl acetate. The material was then washed another three times in ddH2O, then dehydrated in an ascending alcohol series (50%, 70%, 95%, 100%), for 10 min at each concentration. This was followed by two incubations of 15 min each in propylene oxide. The blocks then were left in a 1:1 solution of propylene oxide:Polybed overnight. The material was embedded in 100% Polybed and then incubated at 60°C for 24 h. Sections were viewed and photographed on a Philips 300 electron microscope operated at 60 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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If tubulobulbar complexes are involved in the disassembly of adhesion junctions, then the structures should develop in regions where ectoplasmic specializations previously occurred. This was confirmed in spermatid/junction complexes that had been mechanically dissociated from the epithelium and visualized with DIC or stained with fluorescent phallotoxins to label filamentous actin (Fig. 3). In early elongate spermatids (Fig. 3, A–F'), bundles of actin filaments in ectoplasmic specializations completely surround spermatid heads. As the spermatids continue to differentiate, tubulobulbar complexes clearly form adjacent to the concave surface of rat spermatid heads in areas previously occupied only by ectoplasmic specializations. Moreover, the intensity of actin bundle staining in ectoplasmic specializations appeared to qualitatively decrease in association with ectoplasmic specializations related to the dorsal aspect of the spermatid heads while tubulobulbar complexes became more distinct. The development of tubulobulbar complexes in regions previously occupied by ectoplasmic specializations was confirmed at the ultrastructural level (Fig. 3G). Here, regions that at earlier stages of spermatogenesis contained only ectoplasmic specializations now contained tubulobulbar complexes that were flanked on either side by ectoplasmic specializations. These fluorescence and ultrastructural observations are consistent with the conclusion that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations.
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Molecular Markers for Ectoplasmic Specializations Also Are Present at Tubulobulbar Complexes
If tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations, then molecules found at the adhesion junctions also should be found at tubulobulbar complexes. Antibodies to espin, myosin VIIa, and Keap 1, all previously shown to react at ectoplasmic specializations, also react with tubulobulbar complexes (Fig. 4). On the basis of these experiments, we conclude that tubulobulbar complexes and ectoplasmic specializations share similar molecular components.
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Nectin 2 and Nectin 3 Are Localized to Tubulobulbar Complexes
Once we had determined that markers for ectoplasmic specializations were present at tubulobulbar complexes, we were interested to determine if integral membrane adhesion molecules present at the adhesion junctions also were present at tubulobulbar complexes. For these studies, we treated spermatids, with attached regions of Sertoli cells that had been mechanically fragmented from fixed mouse seminiferous epithelium, with antibodies to nectin 2 and nectin 3 (Fig. 5, A–B'''). We chose to use mouse tissue because the nectin antibodies do not react with rat material. In addition to labeling ectoplasmic specializations, antibodies to nectin 2 specifically labeled, in a vesicular pattern, regions known from actin staining to contain tubulobulbar complexes. Surprisingly, antibodies to nectin 3 labeled the tissue in a similar vesicular pattern to the nectin 2 antibodies, in addition to labeling the spermatid head. We conclude that immunologically reactive nectin 2 and nectin 3 are present in tubulobulbar complexes and in vesicles associated with their ends.
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Afadin, an adaptor protein that binds nectin to actin filaments, had a similar yet distinct immunocytochemical localization. It too was found at ectoplasmic specializations as well as at tubulobulbar complexes (Fig. 5, C–E'). Interestingly, there was a distinct lack of staining along the dorsal part of the ectoplasmic specializations attached to the late-step spermatids (Fig. 5, C'–E'). When a step progression of afadin staining was investigated, staining of ectoplasmic specialization along the dorsal curvature of spermatids gradually appeared to decrease in intensity, whereas an increase in staining intensity was observed around tubulobulbar complexes where it formed a fingerlike staining pattern (Fig. 5, C'–E').
Profiles of Double Membrane Vesicles Are Found Associated with Tubulobulbar Complexes
The observation that antibodies both to nectin 2 and to nectin 3 labeled regions containing tubulobulbar complexes in a vesicular pattern suggested to us that plasma membrane adhesion domains of spermatids might be internalized together with similar domains of Sertoli cell ectoplasmic specializations. If this is true, then vesicles in the region should be surrounded by a double layer of membrane. This was confirmed by transmission electron microscopy. In sections of rat testis, double membrane-bound vesicles consistently were observed in association with the ends of tubulobulbar complexes (Fig. 6). These vesicles were distinguished from the bulbous region of tubulobulbar complexes by their lack of associated endoplasmic reticulum cisternae. At sperm release (Fig. 7A), similar profiles were observed in Sertoli cell stalks that support apical processes surrounding the spermatids being released (Fig. 7B). Also present in similar regions were multivesicular bodies (Fig. 7C). These results indicate that the appropriate morphological machinery is present in Sertoli cell apical processes for internalization of Sertoli cell/spermatid adhesion junction domains.
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Nectin 3 Is Present on Spermatids but Is Absent from Testicular and Epididymal Spermatozoa
If adhesion domains of spermatids are internalized by tubulobulbar complexes, then these domains should be absent from spermatozoa released from Sertoli cells. To verify this prediction, we stained mouse spermatids, testicular spermatozoa, and epididymal spermatozoa with antibodies to nectin 3. Antibodies to nectin 3 only reacted with spermatids (Fig. 8). We conclude that immunologically reactive nectin 3 is associated with spermatid heads but is absent from these regions once the cells are released from the epithelium.
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PKC Is Associated with Tubulobulbar Complexes
If tubulobulbar complexes are involved with internalizing adhesion junction domains, then PKC, a signaling molecule known to regulate endocytosis of junction proteins in other systems [36–38], may be present in regions of Sertoli cells associated with tubulobulbar complexes. To explore this possibility, we probed rat testis fragments with antibodies to PKC, which specifically labeled regions associated with the vesicular region at the terminal ends of tubulobulbar complexes in addition to labeling sperm tails. Similar staining was not present in controls (Fig. 9). The antibody labeled one band specifically on Western blots of rat seminiferous epithelium. This band was not present on normal IgG control blots. We conclude that at least one of the regulators of junction turnover in other systems is present at tubulobulbar complexes.
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Lysosomal and Endosomal Markers Are Present in Vesicles Associated with Tubulobulbar Complexes
We rationalized that, if tubulobulbar complexes internalize junction domains from regions occupied by ectoplasmic specializations, then at least some of the material has to be degraded (spermatid plasma membrane) while other components (Sertoli cell plasma membrane) can either be recycled and/or degraded. In either case, lysosomal markers should be present in vesicles associated with the ends of tubulobulbar complexes. To test this prediction, we labeled mouse and rat testicular fragments with antibodies to LAMP 1 (an endosomal and lysosomal marker generally in cells) and SGP1 (a marker for lysosomes in Sertoli cells), respectively. In both cases, the antibodies labeled structures consistent with the structures being vesicles associated with terminal ends of tubulobulbar complexes (Fig. 9). Controls were negative.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At sites of apical attachment between Sertoli cells and spermatids, tubulobulbar complexes consist of tubular extensions of spermatid heads that project into corresponding invaginations of Sertoli cells. Also considered part of the complexes are the surrounding cuffs of Sertoli cell cytoplasm that are rich in actin filaments in some regions and elements of the endoplasmic reticulum in others. The terminal ends of the tubulobulbar complexes are associated with vesicles, some of which have been identified previously as lysosomes based on positive staining for acid phosphatase [39]. The most popular hypothesis for the function of tubulobulbar complexes is that they function as a mechanism by which Sertoli cells phagocytose cytoplasm from spermatids as these cells mature. An alternative hypothesis that would account for the presence of tubulobulbar complexes at basal sites of attachment between adjacent Sertoli cells is that the structures are involved with the turnover or disassembly of intercellular adhesion junctions [4].
A number of observations are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations at apical sites of attachment to spermatids.
First, tubulobulbar complexes develop in regions occupied by ectoplasmic specializations and both structures share many of the same molecular components. In electron micrographs, networks of actin filaments that cuff the tubulobulbar complexes are contiguous with actin bundles in the ectoplasmic specializations from which the tubulobulbar complexes appear to emerge (this study, [40]). Espin, Myosin VIIa, and Keap 1 are present in both structures (this study), as is vinculin [41] and 1 integrin [42]. The finding that espin is localized in the actin networks of tubulobulbar complexes is particularly interesting because this protein is thought mainly to organize actin filaments into bundles [43], such as those in ectoplasmic specializations. The presence of espin at tubulobulbar complexes may simply be the result of a rearrangement of actin bundles in ectoplasmic specializations into actin networks without loosing the association with actin binding proteins previously present. The observations that the structures occur together and share molecular components indicate to us that they are related and that tubulobulbar complexes may develop from ectoplasmic specializations.
Second, the integral membrane adhesion proteins nectin 2, in the Sertoli cell, and nectin 3, in the spermatid, appear concentrated in vesicles near the ends of tubulobulbar complexes. Although labeling of the vesicles needs to be confirmed at the ultrastructural level, the fluorescence data raises the novel possibility that plasma membrane adhesion domains both of Sertoli cell ectoplasmic specializations and of the attached spermatid may be internalized together by Sertoli cells at tubulobulbar complexes. Consistent with this possibility is the observation that double membrane-bound vesicles occur among the mass of vesicles present at the ends of tubulobulbar complexes and that similar vesicles are observed deeper in the cytoplasm of Sertoli cells at sperm release. Also consistent with this possibility is the observation that nectin 3 antibodies label spermatids attached to Sertoli cells but do not label spermatozoa released from the epithelium. The implication is that adhesion domains in the spermatid plasma membrane are removed by tubulobulbar complexes as part of the sperm-release mechanism. An alternative possibility that we cannot rule out is that proteolytic processing of the protein on the sperm head may contribute to the lack of nectin labeling on spermatozoa.
Third, the adaptor protein afadin that links nectin to the actin cytoskeleton becomes less concentrated at ectoplasmic specializations and more concentrated in tubulobulbar complexes as spermatids mature. There is a striking shift in afadin localization from around the head in elongate rat spermatids to tubulobulbar processes in mature cells just before sperm release. This change is consistent with the movement of adhesion-related elements into tubulobulbar complexes.
Fourth, PKC is present in Sertoli cell regions surrounding tubulobulbar complexes and lysosomal/endosomal markers are present in vesicles associated with the ends of the complexes. PKC is a known regulator of junctional protein endocytosis. Previous studies have demonstrated that vesicles associated with tubulobulbar processes contain acid phosphatase [39], and we show here that they also contain SGP1 (sulphated glycoprotein 1/cathepsin) and LAMP1 (lysosome associated membrane protein 1). SGP1 is known to label Sertoli cell secondary lysosomes. Lamp1 is a transmembrane glycoprotein that labels lysosomes and late endosomes [44]. These results are consistent with the conclusion that tubulobulbar complexes are involved with endocytosis and that at least some of the internalized material likely enters the degradation pathway.
Adhesion domains that are internalized in apical regions of Sertoli cells during sperm release could either be degraded or recycled (Fig. 9). At sperm release in the seminiferous epithelium, large intercellular adhesion junctions must be disassembled and adhesion domains in the plasma membrane eliminated. Results presented here indicate to us that tubulobulbar complexes are involved with internalizing junction domains both of Sertoli cells and of the adjacent spermatids. In other systems that have been studied, adhesion molecules that are endocytosed predominantly enter a recyling pathway from which they can be reinserted into the plasma membrane [38]. In apical regions of Sertoli cells, it is unclear how much junction material enters the degradation and recycling pathways (Fig. 10), although internalized nectin 3 from spermatids is likely degraded because this adhesion molecule is not expressed by Sertoli cells. How much, if any, of the nectin 2 from the Sertoli cell plasma membrane enters the recycling pathway remains to be determined. Any material that is recycled would have to be inserted into the plasma membrane at locations in the cell distant from sites where it was internalized.
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At basal sites of attachment between neighboring Sertoli cells, tubulobulbar complexes also are present [27] and would be expected to function in a way similar to apical complexes. Preliminary studies we have done (unpublished data) indicate that tubulobulbar complexes are not nearly as abundant as one might predict if these complexes were the sole mechanism for junction turnover at these sites. At basal sites of attachment between Sertoli cells, junctions separate above translocating spermatocytes and are reassembled immediately below [1]. Although some internalization of junction elements may occur [29], it also is possible that many of the integral membrane junction molecules never leave the plane of the membrane but simply disengage from their ligands above the spermatocytes and reengage below. This movement of junction molecules in the plane of the membrane might account for the low number and small size of tubulobulbar complexes at basal sites relative to apical sites.
The internalization of intercellular adhesion domains by tubulobulbar complexes at sites of attachment between Sertoli cells and spermatids is potentially a significant component of the sperm-release mechanism in the seminiferous epithelium. The morphology of tubulobulbar complexes and the findings that lysosomal/endosomal markers and adhesion molecules are found in, or associated with, the structures are all consistent with the hypothesis that tubulobulbar complexes are involved with junction disassembly. These results do not rule out the possibility that the tubulobulbar complexes that form at sites of attachment to spermatids also may have secondary functions. The path of junction molecules through degradation and recycling pathways in Sertoli cells remains to be explored, as do alternative mechanisms for junction turnover at basal sites of attachment between Sertoli cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A. Wayne Vogl, 313-2177 Wesbrook Mall, University of British Columbia, Department of Anatomy and Cell Biology, Vancouver, BC, Canada V6T1Z3. FAX: 604 822 2316; vogl{at}interchange.ubc.ca
Received: 20 February 2004.
First decision: 22 March 2004.
Accepted: 1 April 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actinin and vinculin with actin: opposite effects in filament network formation. Proc Natl Acad Sci U S A 1981 78:3005-3009/Hvec with afadin for efficient cell-cell spread of herpes simplex virus type 1. J Virol 2001 75:4734-4743 regulates ß1 integrin-dependant cell motility through association and control of integrin traffic. EMBO J 1999; 18:3909-3923[CrossRef][Medline]6ß1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 1995 52:79-87[Abstract]This article has been cited by other articles:
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  L. M. Turner, E. B. Chuong, and H. E. Hoekstra Comparative Analysis of Testis Protein Evolution in Rodents Genetics, August 1, 2008; 179(4): 2075 - 2089. [Abstract] [Full Text] [PDF]
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 H. H. N. Yan, D. D. Mruk, W. M. Lee, and C. Y. Cheng Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells FASEB J, June 1, 2008; 22(6): 1945 - 1959. [Abstract] [Full Text] [PDF]
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 C. Q.F. Wang and C. Y. Cheng A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis J. Cell Biol., August 9, 2007; 178(4): 549 - 556. [Abstract] [Full Text] [PDF]
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 E. I. Surace, A. Strickland, R. A. Hess, D. H. Gutmann, and C. K. Naughton Tslc1 (Nectin-Like Molecule-2) Is Essential for Spermatozoa Motility and Male Fertility J Androl, November 1, 2006; 27(6): 816 - 825. [Abstract] [Full Text] [PDF]
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 A. Beardsley, D. M Robertson, and L. O'Donnell A complex containing {alpha}6{beta}1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium. J. Endocrinol., September 1, 2006; 190(3): 759 - 770. [Abstract] [Full Text] [PDF]
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