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Regular Article |
a Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, Sertoli cells, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three morphologically distinguishable junctions have been described in the rat testis: desmosome-like junctions [1], ectoplasmic specializations [6], and tubulobulbar complexes [7]. Desmosome-like junctions are spot-like, intermediate filament-based junctions located between Sertoli cells and all non-elongate spermatid germ cells [1]. Once germ cells differentiate into elongate spermatids, the adhesive function of desmosome-like junctions is replaced by ectoplasmic specializations. Actin filaments, endoplasmic reticulum, and microtubules subjacent to the Sertoli cell plasma membrane characterize ectoplasmic specializations [6]. In the basal portion of the Sertoli cell, ectoplasmic specializations are found between neighboring Sertoli cells. As ectoplasmic specializations between Sertoli cells and elongate spermatids dissociate prior to spermiation, tubulobulbar complexes form between these two cell types. Long processes of elongate spermatid membrane that project into Sertoli cell cytoplasm identify these junctions.
Like the junctional protein ß-catenin, p120 catenin (p120) is a protein found at classic cadherin-based adhesion/signaling junctions that serves dual, perhaps related, functions within the cell [8, 9]. At cadherin-based junctions, p120 directly binds to a juxtamembrane cytoplasmic domain of classic cadherins, and this interaction, along with p120 phosphorylation status, modulates the adhesive strength of such junctions. In addition, p120 is found within the nucleus of some cell types where it may bind transcription factors and modulate gene expression [10]. Due to alternative start site usage and mRNA splicing, p120 exists in multiple isoforms [11]. The armadillo repeat, which mediates interaction with classic cadherins, is present in all isoforms. Although p120 isoform expression is tissue specific, the functional significance of differential isoform expression is not known [11].
Although the morphology of testicular junctions has been thoroughly described, the molecular composition of these junctions is poorly understood. Because a large number of classic cadherins are expressed in testis [12, 13], it was hypothesized that p120 is a component of junctions between most, if not all, cells in the seminiferous epithelium. Through Northern and Western blot analysis, p120 has been found to be expressed in adult testis [14, 15]. In addition, p120 immunostaining at the basal aspect of seminiferous tubules has been noted [15], but the junctional location or cell type association of p120 immunostaining is not known. In this paper, the localization of p120 in rat testis using three monoclonal antibodies is described. The results show that p120 is a prominent constituent of inter-Sertoli and Sertoli-germ cell desmosome-like junctions. In addition, differential p120 immunostaining suggests that the various testicular desmosome-like junctions are molecularly unique and dynamic complexes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
For all studies, Fisher 344 rats (Charles River Laboratories, Inc., Wilmington, MA) were used. Rats were treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals; they were housed in wire cages at a constant room temperature (21 ± 2°C) with 35%–70% humidity and a 12L:12D schedule, and given water and chow (Pro-Lab Rat, Mouse, and Hamster chow 3000) ad libitum. The rats were killed by CO2 asphyxiation.
Antibodies
Primary antibodies and working dilutions included 5A7 (IgG1 subtype), 8D11 (IgG2a subtype), 15D2 (IgG1 subtype), 12F4, and pp120 (BD Transduction Laboratories, San Diego, CA) p120 mouse monoclonal antibodies at 1 µg/ml [16]; mouse monoclonal antibody against ß-catenin (clone 6F9; IgG1 subtype) at 1 µg/ml; and mouse monoclonal antibody against plectin (clone 7A8; IgG1 subtype) diluted 1:1000. Based upon Western blotting and immunoprecipitation experiments [16] the 5A7 p120 antibody recognizes only the largest isoforms using the two most 5' start sites; all the other antibodies recognize all p120 isoforms. The 5A7, 8D11, 15D2, and 12F4 antibodies were supplied as protein G-purified immunoglobulin G (IgG) fractions from hybridoma supernatants.
Secondary antibodies and working dilutions included horseradish peroxidase-conjugated sheep anti-mouse IgG (NA931; Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:2000, rabbit anti-mouse IgG-RITC diluted 1:400 (610-4020; Rockland, Gilbertsville, PA), rabbit anti-mouse IgG1-fluorescein isothiocyanate (FITC) diluted 1:500 (Rockland), and rabbit anti-mouse IgG2a-RITC diluted 1:500 (Rockland).
Western Blotting and Immunoprecipitation
Testes from Postnatal Days 7, 21, 31, and 43, and from adults were detunicated, weighed, and homogenized in three volumes of ice-cold RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (P2714; Sigma) by 10 strokes in a Dounce homogenizer. The numbers of animals used at each time point (Day 7 through adult) were 7, 4, 3, 2, and 2, respectively. Protein levels in each homogenate were similar on the basis of Coomassie blue staining of polyacrylamide gels. The homogenate was centrifuged at 14 000 x g for 30 min at 4°C. For immunoprecipitation, supernatants were precleared with 2 µg/ml of normal mouse IgG and 20 µl/ml of protein G-agarose for 30 min at 4°C. Protein G-agarose was pelleted by centrifugation at 2000 x g for 5 min. To 200 µl of cleared supernatant, 2 µg of p120 antibody was added and the sample was incubated for 1 h at 4°C with rotation. Protein G-agarose (6 µl) was added and the sample was incubated overnight at 4°C with rotation. Protein G-agarose beads were washed four times with ice-cold RIPA buffer, and bound proteins were eluted from the beads by adding 20 µl of 1x electrophoresis sample buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, and 0.001% bromophenol blue) and boiling for 5 min. For Western blotting, 2 µl of testis supernatant or 10 µl of immunoprecipitate was separated by 7% SDS-PAGE and transferred to Immobilon-P membrane. Blocking solution (20 mM Tris pH 7.4, 137 mM NaCl, 5% nonfat dry milk) was added to the membranes for 30 min. Primary antibodies were diluted in blocking solution and added to the membranes for 2 h at room temperature. After washing three times with 20 mM Tris (pH 7.4), 137 mM NaCl, and 0.1% Tween-20 (TBS-Tween), horseradish peroxidase-coupled secondary antibody diluted in blocking solution was incubated with the membranes for 1 h at room temperature. Membranes were washed three times with TBS-Tween and secondary antibody was detected by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech). As negative controls, either normal mouse IgG was used as the precipitating antibody or the primary antibody was omitted during Western blotting; in both cases, no bands were seen following enhanced chemiluminescent detection (data not shown). Experiments were performed twice with similar results.
Immunostaining
For cryosections, testes were submerged in OCT compound (Sekura Finetek Inc., Torrance, CA) and snap-frozen by immersion in liquid nitrogen. Frozen sections (8 µm) were air dried onto positively charged glass slides (VWR Scientific, West Chester, PA). For touch preparations, testes were cut in cross-section with a razor blade, and the exposed seminiferous tubules were briefly touched to a glass slide. After air drying, slides were treated exactly as they were for cryosection immunostaining. For all nonactin localizations, slides were fixed for 5 min in -20°C methanol. For actin localizations, cryosections were dried and fixed for 10 min with 4% paraformaldehyde in PBS; after washing three times for 5 min with PBS, cryosections were treated with -20°C acetone for 10 min. All subsequent incubations were performed at room temperature in a humidified chamber. After blocking for 30 min in 5% normal goat serum (or 5% normal rabbit serum), 0.1% BSA in PBS (pH 7.4; PBS+), sections were incubated for 1 h in primary antibody diluted in PBS+. After three washes for 5 min each, sections were incubated for 45 min in secondary antibody diluted in PBS+. For f-actin localization, FITC-phalloidin (0.2 µg/ml) was included in the secondary antibody solution. After washing as before, sections were mounted in Gel/Mount (Biomeda Corp., Foster City, CA) and viewed with a 63x objective using a Zeiss Axiovert 35 microscope (Carl Zeiss, New York, NY) or a 100x objective using a Nikon Eclipse E800 microscope, both equipped with epifluorescence. For colocalization of p120 with f-actin or plectin, images were taken with a dual wavelength filter set. As negative controls, slides were processed for immunostaining except that the primary antibody was omitted. In all experiments involving colocalization, sections were examined with a dual wavelength filter set to determine those areas showing colocalization.
For all antibodies, following cryosectioning of fresh frozen testes, one of three fixation techniques was employed; either paraformaldehyde, acetone, or methanol. Except where noted, the immunostaining patterns were similar irrespective of fixation technique. However, methanol fixation best preserved seminiferous epithelial morphology and routinely gave the most intense immunostaining signal; therefore, all images shown here used methanol fixation, except when noted.
For the color figure, images were obtained using a Spot RT camera (Diagnostic Instruments Inc., Sterling Heights, MI) connected to the microscope, downloaded into Photoshop 6.0 software (Adobe Systems Inc., San Jose, CA), and assembled with Canvas 5.0 software (Deneba Systems Inc., Miami, FL). For black and white figures, photographs were taken using Tri-X pan film (Kodak, Rochester, NY) and digital images of the developed photographs were made with an Epson Expression 1600 scanner (Epson America Inc., Long Beach, CA). Scanned images were assembled into figures using Canvas 5.0. Corresponding differential interference contrast (DIC) images were used to stage seminiferous tubules. The staging criteria we used were based upon the position and morphology of elongate spermatid heads within the seminiferous epithelium [17].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Through differential start site usage and RNA splicing, many cells express multiple p120 isoforms [18]. To determine whether testis expresses multiple p120 isoforms and whether their expression is developmentally regulated, detunicated testis homogenates from Postnatal Days 7, 21, 31, 43 and adult were immunoprecipitated with the 5A7, 8D11, and 15D2 antibodies (Fig. 1). As described in Materials and Methods, the 5A7 antibody recognizes only the large p120 isoforms that utilize the two most 5' start sites, whereas both 8D11 and 15D2 potentially recognize all isoforms [16]. With both 8D11 and 15D2, four major p120 isoforms were immunoprecipitated throughout postnatal testis development and had apparent molecular weights of 120, 104, 72, and 65 kDa. Additional specific bands in the 80- to 100-kDa range were detected with both 8D11 and 15D2 antibodies. Whether these bands represent expression of additional p120 isoforms or the phosphorylation or proteolysis of existing isoforms requires additional experimentation. At Day 31 and Day 43, 99-kDa and 95-kDa proteins were immunoprecipitated with either 8D11 or 15D2 antibodies. Because the appearance of these bands is not correlated with changes in p120 immunostaining (see below), their significance is unclear. Unlike the 8D11 and 15D2 antibodies, the 5A7 antibody immunoprecipitated only the 120-kDa isoform at all time points. These data suggest that testis expresses at least one p120 isoform using start site 1 or 2 (120 kDa), one isoform using start site 3 (104 kDa), and two isoforms using start site 4 (72 and 65 kDa).
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In addition to p120, the expression of ß-catenin protein was determined during postnatal development by Western blotting of detunicated testis homogenates (Fig. 1). Like p120, ß-catenin was expressed throughout postnatal testis development.
p120 Localization to Inter-Sertoli, Sertoli-Spermatocyte, and Sertoli-Elongate Spermatid Junctions
Using the 8D11 antibody, p120 colocalized with ß-catenin throughout postnatal testis development. At Day 7, punctate p120 and ß-catenin immunostaining was observed at the periphery of seminiferous tubules (dashed arrows in Fig. 2, A and A'). This immunostaining colocalized with antibodies against cadherin-11 and represented junctions between peritubular cells [19]. A diffuse immunostaining pattern of p120 and ß-catenin was seen throughout the developing epithelium at Day 7, in addition to numerous punctate structures (Fig. 2, A and A'). At Day 21, inter-Sertoli junctions and punctate elements associated with spermatocytes were stained brightly with p120 and ß-catenin antibodies (Fig. 2, B and B'). At this time point, the seminiferous epithelium maintained diffuse p120 and ß-catenin immunostaining, but with additional postnatal development this immunostaining diminished. Inter-Sertoli p120 and ß-catenin immunostaining was observed during subsequent postnatal development through adulthood (Fig. 2, C, C', D, and D'). Large, plaque-like and small punctate structures were observed at the spermatocyte level at Day 43; however, only the small structures were observed in the adult testis. At all time points, the 5A7 p120 antibody colocalized with both the 8D11 antibody and antibodies against ß-catenin (data not shown), indicating that the large p120 isoform was present at the described junctions. In addition, p120 and ß-catenin localized to junctions between endothelial cells but not other cells of the interstitium (data not shown). Two other p120 monoclonals (12F4 [16] and pp120) that recognize all isoforms gave identical immunostaining patterns to the 5A7 and 8D11 antibodies (data not shown).
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Additional p120 immunostaining in adult testis using the 8D11 antibody is shown in Figure 3. Spermatocyte-associated p120 colocalized with an antibody against plectin using acetone fixation (arrows in Fig. 3, A and A'). At this location, plectin is a component of desmosome-like junctions between Sertoli cells and spermatocytes [20]. Within the occasional tangential cryosection of the basal epithelium, circular p120 immunostaining was evident, representing inter-Sertoli junctions (arrowhead in Fig. 3B). These inter-Sertoli junctions appeared as multiple curvilinear structures completely enveloping the Sertoli cells. In addition, punctate immunostaining was observed in tangential cryosections, which may represent additional inter-Sertoli junctions, Sertoli-germ cell junctions, or both. Similar to postnatal development, ß-catenin and 8D11 immunostaining colocalized at inter-Sertoli junctions and basal Sertoli-germ cell junctions (data not shown), which is in agreement with ß-catenin immunostaining observed by others [21, 22]. Using acetone fixation and the 8D11 antibody, stage-dependent p120 immunostaining was seen associated with the heads of elongating spermatids (arrows in Fig. 3C). Such p120 immunostaining was most intense at the caudal aspect of the spermatid head and was seen only in late stage tubules (approximately stages XII–XIII). ß-Catenin was never observed in association with elongate spermatid-associated junctions (data not shown).
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Inter-Sertoli junctions are complex structures including intermediate filament-based desmosome-like and actin-based ectoplasmic specialization junctions in juxtaposition. To determine whether p120 was localized to inter-Sertoli ectoplasmic specializations, f-actin and p120 were colocalized in Day 31 and adult testis cryosections fixed with acetone. At both time points, p120 and f-actin were juxtaposed but showed different immunostaining patterns (Fig. 4, A and B). On the other hand, p120 colocalized with plectin in adult (Fig. 4C) and developing testis (data not shown). In the basal epithelium, p120 and plectin did not always colocalize (Fig. 3, A and A'); that plectin is also localized to the Sertoli cell perinuclear area while p120 is a component of peritubular cell junctions likely explains this observation. Because plectin is a component of inter-Sertoli desmosome-like junctions [20], these data indicate that p120 is present at desmosome-like but not at ectoplasmic specialization junctions between Sertoli cells.
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p120 Localization to Sertoli-Round Spermatid Junctions
Using the 15D2 antibody against p120, an immunostaining pattern that was different from that seen with the 8D11 antibody was observed. At all time points, the interstitium and seminiferous tubule basement membrane showed nonspecific fluorescence, as seen in cryosections that were immunostained without the primary antibody (data not shown). No specific immunostaining was seen at Day 7 with the 15D2 antibody (Fig. 5A). Like the 8D11 antibody, the 15D2 antibody immunostained basal inter-Sertoli junctions from Day 21 through Day 43 (arrows in Fig. 5, B–D). As postnatal testis development progressed, 15D2 immunostaining of basal inter-Sertoli junctions diminished. Such immunostaining was intense at Day 31 and was observed in all tubules. At Day 43, however, most tubules showed no basal inter-Sertoli junctional 15D2 immunostaining (data not shown), and adult cryosections exhibited no inter-Sertoli junctional 15D2 immunostaining at all stages (Fig. 6).
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Unlike testes of Days 7 and 21, Day 31 and Day 43 testes contained abundant, punctate 15D2 immunostaining at the level of round spermatids (arrowheads in Fig. 5, C and D). This round spermatid-associated immunostaining exhibited a cyclic, stage-dependent pattern in adult testis. Beginning at approximately stage I, the time of round spermatid formation, masses of 15D2 immunostaining appeared in the middle of the seminiferous epithelium (Fig. 6A). From subsequent stages through approximately stage VII, such immunostaining was very intense and compact (Fig. 6B). During stage VIII, the compact 15D2 immunostaining appeared to unfold into linear elements showing occasional bulbous areas (Fig. 6C). In stages IX through approximately XI, the 15D2 immunostaining eroded into multiple punctate structures of various sizes (Fig. 6D), which was often seen throughout the seminiferous epithelium. Tubules at approximately stages XII–XIII showed no specific immunostaining with the 15D2 antibody (Fig. 6E). No specific interstitial, spermatocyte-associated, or elongate spermatid-associated immunostaining was observed with the 15D2 antibody.
The timing and spatial localization of the 15D2 immunostaining in postnatal and adult testes suggests that Sertoli-round spermatid junctions are being detected with this p120 antibody. Such a conclusion is supported by 15D2 immunostaining of adult testis touch preparations. In these samples, each round spermatid was associated with a single 15D2-positive structure (Fig. 7). Other cell types showed no specific immunostaining with the 15D2 antibody in touch preparations (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to being molecularly unique, testicular junctions are dynamic structures. During postnatal development, inter-Sertoli junctions of Day 21 and Day 31 testes were immunostained with the 15D2 p120 antibody. By Day 43, however, inter-Sertoli junctions of most tubules did not react with the 15D2 antibody, and no immunostaining at inter-Sertoli junctions was observed in adult testis. These data contrast with the other p120 antibodies and ß-catenin antibodies, which immunostained inter-Sertoli junctions at all time points beginning at Postnatal Day 21. One conclusion from these data is that as postnatal development proceeds, transient protein-protein interactions occur at inter-Sertoli junctions, which is reflected in differential 15D2 immunostaining. Such a maturation process is observed during epithelial differentiation of the preimplantation embryo and between endothelial cells comprising the blood-brain barrier [23–25]. In these systems, cadherin-based junctions show transient localization of ZO-1
- and late expression of plakoglobin, respectively. The factors responsible for differential 15D2 inter-Sertoli immunostaining remain to be determined.
Like inter-Sertoli junctions during development, junctions between Sertoli cells and spermatids showed dynamic p120 immunostaining. Using the 15D2 antibody on adult cryosections, the pattern of immunostaining associated with round spermatids was stage-dependent, as seen in Figure 6. This pattern coincides with two processes: the life span of a round spermatid, and the stage VIII turnover of adhesive Sertoli-spermatid junctions from desmosome-like to ectoplasmic specialization [26]. Unlike junctions between Sertoli cells or Sertoli cells and earlier germ cell types, the Sertoli-round spermatid junction has a unique composition, which is reflected in specific immunostaining with the 15D2 antibody, as well as in classic cadherin composition [19]. In addition, Sertoli-elongate spermatid junctional p120 immunostaining using the 8D11 antibody was stage-dependent. Like cadherin-6 and L4+cadherin immunostaining [19], elongate spermatid-associated p120 immunostaining was limited to a subset of stages (approximately XII–XIII). These results suggest that the molecular properties and function of Sertoli-elongate spermatid junctions change during spermiogenesis. Because data are accumulating showing that cadherin-based junctions are signaling complexes [9, 27], the differential molecular composition of Sertoli-round spermatid desmosome-like and Sertoli-elongate spermatid junctions suggest that these junctions convey unique signals that are necessary for germ cell maturation.
In testis, p120 is likely a component of intermediate-filament based desmosome-like junctions, but not actin-based junctions [1]. At basal inter-Sertoli junctions, desmosome-like junctions adjoin actin-based ectoplasmic specializations [28]. Colocalization of 8D11-positive p120 immunostaining with f-actin at inter-Sertoli cell junctions showed that the two proteins are juxtaposed, but clearly within distinct structures. On the other hand, plectin and p120 immunostaining showed extensive colocalization. This pattern was also seen for antibodies against N-cadherin [19]. Together with electron microscopy data showing classic cadherin immunostaining at inter-Sertoli desmosome-like junctions [21], these data indicate that testicular classic cadherin junctions are associated with intermediate filaments, not actin filaments. These data contrast with data for other tissues in which classic cadherin junctions are associated with the actin cytoskeleton [29]. Any functional significance of this distinction awaits further experimentation. The proteins that link classic cadherins to underlying vimentin intermediate filaments at testicular junctions are not known, but the classic cadherin and intermediate filament binding protein plakoglobin is one candidate [22].
At this time, the cell types that express p120 in the seminiferous epithelium are not entirely known. Immunostaining for p120 at inter-Sertoli junctions shows that Sertoli cells express at least the large p120 isoform. The resolution of light microscopy is not sufficient to assign expression of p120 found at Sertoli-germ cell junctions to either Sertoli or germ cells. Within Sertoli-round spermatid junctions, however, p120 is likely found on the Sertoli cell aspect, because the p120 immunostaining pattern during the dissolution of these junctions showed punctate elements scattered throughout the seminiferous epithelium. Because Sertoli-germ cell junctions are morphologically asymmetric [1, 6], the molecular components present on the Sertoli or germ cell aspects are likely unique, at least partially. Therefore, p120 may not be expressed in germ cells. Immunoprecipitation data of p120 showing that all isoforms are expressed throughout postnatal development also are consistent with p120 being a Sertoli cell product.
The role of p120 in testis junction biology remains to be determined, but attractive functions for p120 are modulation of junction adhesiveness and recruitment of junction-specific components. As described above, Sertoli-germ cell junctions are dynamic and molecularly unique. In addition, a relaxation of inter-Sertoli cell junctions is required for the transit of preleptotene spermatocytes from basal to adluminal compartments [30]. In cell culture systems, p120 appears to regulate the adhesive strength of cadherin junctions [8]. Although the mechanism in not known in detail, p120 appears to act as a molecular switch to modulate junction adhesive strength. Thus, p120 may regulate the passage of preleptotene spermatocytes through inter-Sertoli cell junctions during stage VIII and the formation and dissolution of Sertoli-round spermatid junctions depicted in Figure 6. Testicular junctions contain junction-specific cadherins as well as other components (catenins and plectin) [19–22]. We propose that recruitment of junction-specific proteins is the result of differential localization of p120 isoforms. Within the seminiferous epithelium, the large p120 isoform (recognized by the 5A7 antibody) is invariably associated with junctions containing N-cadherin, ß-catenin, and plectin. On the other hand, Sertoli-round spermatid junctions (which are not immunostained by 5A7 antibody) contain different classic cadherins and do not immunostain with antibodies against ß-catenin or plectin [19]. Because testis expresses multiple p120 isoforms, these data suggest that each testicular junction contains a unique p120 isoform. Differential protein binding to p120 isoforms has been described [31], and the affinity of different cadherins for p120 (and possibly p120 isoforms) may result in junction-specific cadherin recruitment [32]. These data support the concept that the existence of unique molecular components at testicular junctions is related to the function of p120.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Rhode Island Foundation to K.J.J. and by PHS NIEHS grant R01 ES08956 to K.B. 
2 Correspondence: Kamin J. Johnson, Department of Pathology and Laboratory Medicine, 175 Meeting Street, Brown University, Providence, RI 02912. FAX: 401 863 9008; kamin_johnson{at}brown.edu
Accepted: November 1, 2001.
Received: August 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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