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Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021, USA
1 Department of Zoology, University of Hong Kong, Hong Kong, China
(Requests for offprints should be addressed to C Yan Cheng; Email: y-cheng{at}popcbr.rockefeller.edu)
* (P P Y Lie, W Xia, C Q F Wang and D D Mruk contributed equally to the completion of this work) 
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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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Dynamin is a family of large GTPases implicated in the formation of nascent vesicles during both endocytosis and the secretory process (for reviews, see McNiven et al. 2000, Sever et al. 2000b, Hinshaw 2006, Kruchten & McNiven 2006, Robinet et al. 2006). Dynamin is known to play an important role in the internalization of integral membrane proteins (e.g. occludin and N-cadherin) in multiple epithelia (Orth & McNiven 2003) and serves as pinchase-like mechanoenzyme to facilitate the formation of endocytic vesicles by severing nascent endocytic pits from the plasma membrane (Thompson & McNiven 2001, Cao et al. 2003). The best studied function of the dynamin family of proteins is the promotion of vesicle fission during clathrin-mediated endocytosis (Sever et al. 2000a, Hinshaw 2006, Kruchten & McNiven 2006). Other functions, such as membrane tubulation and phagosome formation, have also been ascribed to dynamin (Orth & McNiven 2003). Members of the dynamin family include three classical dynamins, namely dynamins I, II, and III, and also dynamin-like proteins. Dynamin I is a neural specific isoform pertinent to synaptic vesicle recycling. Dynamin II is ubiquitously expressed in all eukaryotic cells, and it has recently been identified in the testis (Iguchi et al. 2002). It is crucial for the endocytosis of integral membrane proteins (McNiven et al. 2000, Sever et al. 2000b). Dynamin III is a testis-specific isoform (Kamitani et al. 2002). A recent study has reported that dynamin I is absent in the testis, while the other two isoforms are highly expressed (Kamitani et al. 2002). Nonetheless, the functions of dynamins in the testis remain to be elucidated. It was postulated that dynamins may play a role in nutrient provision to germ cells via endocytosis (Kamitani et al. 2002). In this report, we have examined the possible role of dynamin II in BTB dynamics via its specific interactions with the occludin/ZO-1 and the N-cadherin/ß-catenin protein complexes at the BTB.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Sprague–Dawley rats (~270–300 g body weight) were purchased from Charles River Laboratories (Kingston, NY, USA). The use of animals in this study was approved by the Rockefeller University Animal Care and Use Committee with Protocol Numbers 03017 and 06018.
Primary Sertoli and germ cell cultures
Sertoli and germ cells were isolated from the testes of 20- and 90-day-old rats respectively, as detailed elsewhere (Mruk et al. 1997, 2003). In short, Sertoli cells were cultured at a density of 5 x 105 cells/cm2 on 100 mm dishes in F12/DMEM (Ham’s F12 Nutrient Mixture: Dulbecco’s Modified Eagle’s Medium, V/V, 1:1) at 35 °C in a humidified atmosphere of 95% air/5% CO2 with supplements (Mruk et al. 1997). On day 2, these cultures were subjected to a hypotonic treatment (10 mM Tris, pH 7.4 at 22 °C for 2 min; Galdieri et al. 1981) to lyse residual germ cells, and cultures were terminated on day 4 for lysate preparation and RNA extraction as described (Mruk et al. 2003). On the other hand, total germ cells were isolated from adult rat testes and used for lysate preparation and RNA extraction within 6 h as described (Aravindan et al. 1996). Both primary cultures had negligible contamination of other cell types, and this has been vigorously characterized by RT-PCR, immunoblotting, light and electron microscopy as reported earlier from this laboratory (Siu et al. 2005).
Isolation of seminiferous tubules
Seminiferous tubules were isolated from adult rat testes (~300–350 g body weight) as described (Zwain & Cheng 1994). Tubules were incubated in F12/DMEM containing insulin (20 µg/ml), human transferrin (20 µg/ml), gentamicin (100 µg/ml), and penicillin (100 IU/ml) at 35 °C for about 6 h before they were harvested for lysate preparation. The tubules used in this report had negligible contamination of Leydig cells since these tubules failed to respond to hCG (10 ng/ml) treatment when the level of testosterone was quantified as described (Zwain & Cheng 1994).
Treatment of rats with adjudin to induce junction restructuring
Adult rats (~270–300 g body weight, b.w.; n = 3–5 for each time point) were treated with adjudin (1-(2,4-dichloroben-zyl)-1H-indazole-3-carbohydrazide, formerly called AF-2364, a molecule that induces adherens junction disruption in testis) via gavage at 50 mg/kg b.w. at time 0. The treated rats were killed after 1, 4, 8 h and 1, 4, 7, and 14 days thereafter. Adjudin was suspended in methylcellulose (0.25% (w/v) in sterile water) as a stock solution of 20 mg/ml. This treatment is known to induce extensive AJ restructuring in the seminiferous epithelium at the Sertoli–germ cell interface, most notably between Sertoli cells and elongating/elongate spermatids, followed by round spermatids and spermatocytes, but not spermatogonia (Chen et al. 2003, Cheng et al. 2005). However, the BTB integrity has been observed to be unaffected by day 15 following adjudin treatment (Mruk & Cheng 2004, Cheng et al. 2005).
Sample preparation
Lysates were obtained by treating Sertoli cells, germ cells, testes, or seminiferous tubules with a lysis buffer (50 mM Tris–HCl, 0.15 M NaCl, 1% Nonidet P-40 (v/v), 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM sodium orthovanadate), followed by sonication and centrifugation at 13 000 g for 10 min to obtain the clear supernatant. The use of EGTA instead of EDTA in the lysis buffer as a chelating agent to block metalloprotease activity was important. Since EDTA was shown to chelatewith vanadate (Crans et al. 1989, Huyer et al. 1997, Siu et al. 2005), it would reduce the level of free vanadate included in the buffer that blocked the activity of protein-tyrosine phosphatases (PTP) in lysates. If PTP activity remained unchecked, this would affect the phosphorylation status of component proteins at the BTB, adversely affecting protein–protein interactions in the samples to be analyzed. Protein concentration was estimated by Coomassie blue dye-binding assay using BSA as a standard (Bradford 1976).
Antibodies
Commercially obtained antibodies listed in Table 1
were used for immunoblot analysis, immunohistochemistry, fluorescent microscopy, and co-immunoprecipitation. All three anti-dynamin antibodies used in our study cross-reacted with dynamins I and II, but since dynamin I is absent in the testis (Kamitani et al. 2002), dynamin signals reported herein were only from dynamin II.
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One-hundred micrograms protein from each sample within an experimental group were resolved by SDS-PAGE using 7.5, 10, or 12.5% T SDS-polyacrylamide gels under reducing conditions, depending on the relative molecular mass of the target proteins. Proteins were then transferred onto nitro-cellulose membranes for immunoblot analysis as described earlier (Mruk et al. 2003). Commercially obtained antibodies against different target proteins are listed in Table 1
. All immunoblots were densitometrically scanned using SPSS SigmaGel software (version 1.05) from SPSS Inc. (Chicago, IL, USA) and normalized against the level of ß-actin to confirm equal protein loading.
Immunohistochemistry and fluorescent microscopy
For both immunohistochemistry and fluorescent microscopy experiments, the rabbit anti-dynamin antibodies from Cell Signaling Technology (Beverly, CA, USA) and Abcam, Inc. (Cambridge, MA, USA; see Table 1) were used, whereas the mouse antibody from BD Transduction Laboratories (San Jose CA, USA) failed to work in these studies. While both rabbit antibodies yielded specific staining in the seminiferous epithelium in preliminary studies, the antibody from Abcam produced much better resolution and at a significantly lower working dilution (1:300 vs 1:75, see Table 1). As such, all reported results regarding dynamin were from experiments carried out with the antibody from Abcam. Immunohistochemistry was performed as described earlier (Siu et al. 2005). In short, frozen testes were embedded in optimal cutting temperature (OCT; Sakura Finetek USA Inc., Torrance, CA, USA) compound, and sectioned to ~6–8 µm thickness in a cryostat at –20 °C. Cross-sections from different testes within an experimental group were mounted onto one to two poly-L-lysine-coated slides (Polysciences Inc., Warrington, PA, USA; with approximately two to three sections per glass slide) and processed simultaneously in an experimental session to avoid inter-experimental variations. Color development was carried out with Histostain-SP kit (Zymed, South San Francisco, CA, USA). In preliminary experiments, paraffin sections were also tested for immunohistochemistry studies. However, none of the three anti-dynamin antibodies from different vendors (see Table 1) yielded satisfactory results. Therefore, data presented here were all obtained from the use of frozen sections. Immunofluorescent microscopy was performed as described earlier (Lee et al. 2003, Siu et al. 2005) using either Cy3 or FITC-conjugated secondary antibodies (see Table 1). All micrographs were acquired using an Olympus BX40 microscope equipped with UPlanF1 fluorescent optics and an Olympus DP70 Digital Camera. Images were captured using QCapture Suite Imaging Software (version 2.56; Quantitative Imaging Corp., Burnaby, BC, Canada). Results from morphology studies reported in this paper were representative data from four to six different experiments performed by two different investigators, which yielded similar observations.
Co-immunoprecipitation (co-IP)
Co-IP was performed essentially as described earlier (Wong et al. 2005, Yan & Cheng 2005). In brief, sample lysates containing 400 µg protein were pre-cleared as follows to eliminate non-specific interactions between IgG and proteins in lysates. Samples were incubated with normal rabbit (or mouse) IgG (~5 µg), followed by an incubation with Protein A/G PLUS Agarose (10 µl), and then centrifuged to obtain the clear supernatant for subsequent Co-IP. The pre-cleared samples were then incubated with corresponding primary antibodies overnight. The immunocomplexes were immuno-precipitated by Protein A/G PLUS agarose (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), extracted in SDS sample buffer, and resolved by SDS-PAGE for immunoblotting. Negative controls were included such that the precipitating antibodies were replaced by either rabbit or mouse IgG. All samples within an experimental group were processed simultaneously in a single experimental session to eliminate interexperimental variations. Each Co-IP experiment was repeated at least thrice using different batches of cell and tubule lysates.
Statistical analysis
All experiments reported herein were repeated for three to five times using different batches of cells. For in vivo experiments, at least six rats, including controls, were used for each time point. Data within an experimental group were analyzed by ANOVA, and statistical significance was estimated by Tukey’s honest significant test using GBSTAT (version 7.0) software package from Dynamic Microsystem Inc. (Silver Spring, MD, USA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1A–F illustrates the representative results of an immunohistochemistry experiment performed on frozen sections of adult rat testes by incubating with the rabbit anti-dynamin antibody. Specific signals from dynamin II were detected, as explained in Materials and Methods. Dynamin II appears as reddish-brown precipitate localized mostly to the apical ES with strongest staining in stages VII–VIII tubules (see Fig. 1A). Figure 1B shows a normal rat testis section incubated with rabbit IgG in place of the primary antibody, illustrating the antibody specificity. Since only very weak background staining was found in the interstitium. The strongest dynamin II staining was detected in an early stage VIII tubule as shown in Fig. 1C. Figure 1D is a magnified view of the boxed area in Fig. 1C, demonstrating the dynamin II staining on the convex surface of the heads of elongating spermatids, consistent with its localization at the apical ES. Weak but still discernible dynamin II staining was found at the apical ES site of elongating spermatids in stages Vand X tubules (see Fig. 1E and F vs C and D). Furthermore, dynamin II was detected in the basal compartment of the seminiferous epithelium, localizing at the BTB in virtually all tubules examined (see Fig. 1A and C–F). Dynamin II was also found to be associated with round spermatids, spermatocytes, and spermatogonia in stages VII–VIII tubules, though signals attenuated in stages V and X (see Fig. 1C–F). This was suggestive of its localization at the desmosome-like junctions, since apical ES is absent at the interface between these germ and Sertoli cells in the epithelium. The specificity of this antibody from Abcam (Table 1) was further verified by immunoblotting as shown in Fig. 1G. Only one prominent band with an electrophoretic mobility corresponding to the apparent relative molecular mass of dynamin II (100 kDa) was detected in the lysates of seminiferous tubules and germ cells from adult rats, and Sertoli cells from 20-day-old rats. The relative expression level of dynamin II in different cellular fractions of the testis was also investigated (Fig. 1H and I). It was shown that germ cells expressed approximately three times more dynamin II than Sertoli cells (Fig. 1I). This result is consistent with an earlier report describing the relative abundance of dynamin in germ vs Sertoli cells (Kamitani et al. 2002).
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We next used fluorescent microscopy to examine the co-localization of dynamin II with proteins at the BTB. Figure 2A–D, E–H illustrate the co-localization of occludin and JAM-A (both are putative integral membrane proteins at the BTB) with their adaptor ZO-1 in the seminiferous epithelium, consistent with their localization at the BTB in adult rat testes. It is worthy to note that dynamin II was found to be localized at the apical ES as well as the BTB (see Fig. 2I and M), which is consistent with the results of the immunohistochemistry as shown in Fig. 1A–D. Dynamin II partially co-localized with ZO-1 at the BTB (Fig. 2I–L). At the same time, its co-localization with N-cadherin was also observed, but the merged signal was much weaker (Fig. 2M–P). To further verify these observations, specific antibodies against different target proteins (see Table 1) were used for co-immunoprecipitation (Co-IP), and precipitable immunocomplexes were then probed for dynamin II by immunoblotting as shown in Fig. 2Q. It was shown that dynamin II indeed was associated with occludin, N-cadherin, ZO-1, ß-catenin, JAM-A, p130Cas, and ß-actin, but not nectin-3 (Fig. 2Q). These results are thus in agreement with the current knowledge that dynamins are involved in trafficking events between cell membrane and the actin network in particular internalization of integral membrane proteins (e.g. occludin and N-cadherin; Orth & McNiven 2003).
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Adjudin is known to induce germ cell depletion from the seminiferous epithelium without compromising the BTB integrity at the time of extensive anchoring junction restructuring (Mruk & Cheng 2004, Cheng et al. 2005). The drug apparently exerts its effects at the Sertoli–germ cell interface, for instance, by disrupting the integrin/laminin protein complex at the apical ES (Siu et al. 2005). In previous mating studies, the anti-fertility effect of adjudin was not visible until the sperm reserve at the epididymides was exhausted, 3–4 weeks after treatment (Cheng et al. 2005). This observation illustrated that adjudin has no apparent toxicity to germ cells. Otherwise, the infertility effect would have been detected within 1–2 weeks since elongate/elongating spermatids were virtually depleted from the epithelium from days 4 to 14 (see Fig. 3). Therefore, this model was used to examine changes in the dynamin II protein level and its cellular association during extensive junction restructuring in the seminiferous epithelium. As shown in Fig. 3A and B, there was a trend of time-dependent decline in dynamin II protein level in testes when germ cells began to detach from the epithelium from day 1 onwards except for a mild and statistically insignificant increase in hours 1–8 post-treatment. This trend of declining dynamin II in the testis was not entirely unexpected since dynamin II is largelyexpressed by elongating/elongate spermatids (see Fig. 1). The data shown in Fig. 3A and B were validated by immunohistochemistry studies to visualize the distribution of dynamin II in the seminiferous epithelium during adjudin-induced germ cell loss (see Fig. 3C–F). For instance, a decline in dynamin II staining was detected in the epithelium from days 4 to 14 when elongating/elongate spermatids were being depleted from the epithelium. This event was accompanied by a loss of dynamin II at the Sertoli cell–spermatid apical ES site (Fig. 3D–F vs A). It is also worthy to note that dynamin II was still seen to be surrounding the heads of the elongate spermatids remaining in the tubule lumen (see black arrowheads in Fig. 3D–F). Interestingly, while the overall dynamin II staining in the epithelium was weak, its signals at the BTB did not appear to be diminished by day 14 when the tubules were virtually devoid of spermatids and spermatocytes. Instead, the staining intensity at the BTB was considerably higher as compared with normal testes (see Fig. 3F vs C–E).
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In the same experiment during which the level of dynamin II was quantified (see Fig. 3), the steady-state levels of two known protein complexes at the BTB, namely N-cadherin/ß-catenin and occludin/ZO-1, were also measured (Fig. 4A and B). It was found that levels of these proteins increased significantly. As some of these proteins are restricted to (e.g. occludin) or predominantly expressed by (e.g. ZO-1) Sertoli cells, their increase in protein levels shown by immunoblots might reflect changes in cellular contribution instead of an increase in de novo synthesis. For instance, as shown in Fig. 3E and F, the lysates obtained from these testes were largely contributed by Sertoli cells, whereas the lysates from testes on day 4 and earlier time points including normal rats (Ctrl) had more proteins contributed by germ cells. As such, the data shown in Fig. 4B were corrected against the declining testicular weight (see Fig. 4C), and re-plotted as the relative target protein level per pair testes (see Fig. 4D). Consistent with the data shown in Fig. 4B on day 1, most of the target proteins (except ß-catenin) were induced at the beginning of germ cell depletion (see Fig. 3). However, on days 7 and 14, when most germ cells, in particular elongating/elongate and round spermatids and most spermatocytes, were depleted from the epithelium, only N-cadherin was still induced. This observation is consistent with an earlier report (Chen et al. 2003). Yet the levels of ß-catenin, occludin, and ZO-1, did not appear to be significantly different from the controls (Fig. 4D vs B). In fact, a mild inhibition was detected for ß-catenin on day 14 (Fig. 4D vs B).
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We next used samples from testes of adjudin-treated rats to examine changes in the association of the BTB target proteins with dynamin II by Co-IP as shown in Fig. 5. When these testes lysates were examined for dynamin II protein level, a significant decline in overall dynamin II level was detected (see the sixth panel in Fig. 5A, the seventh panel is ß-actin that serves as a protein loading control; and the lower panel in Fig. 5B). This was consistent with results shown in Fig. 3. The drop in total protein level of dynamin II in the testis on day 7 was accompanied by a loss of association between dynamin II and occludin as well as N-cadherin, which might be explained by the reduced dynamin II protein level at that time point (see also Fig. 2). Interestingly, at the same time, there was a significant increase in the association of dynamin II with ZO-1 and ß-catenin, the corresponding adaptors of occludin and N-cadherin (Fig. 5A and B). Although the levels of dynamin II in the testis on days 7 and 14 remained low (vs Ctrl and day 1) and were not statistically significantly different from each other (see Fig. 5B), the binding between dynamin II and the two BTB protein complexes rebound by day 14 (Fig. 5A and B). These observations are physiologically important, as they suggest that when the tubules were almost devoid of germ cells on days 7 and 14 following adjudin treatment (see Fig. 3E and F vs C and D), the association between dynamin II and the two BTB junctional complexes, namely occludin/ZO-1 and N-cadherin/ß-catenin, become tighter. These biochemical results are also supported by immunohistochemistry studies, showing that by day 14 when the tubules were virtually devoid of spermatids and spermatocytes (see Fig. 3F), dynamin II staining in the basal compartment of the seminiferous tubule and at the BTB did not diminish vs control rats (see Fig. 3F vs C). In short, the changes in protein–protein association between dynamin II and the adaptors ZO-1 and ß-catenin on days 7 and 14 vs controls and day 1 (see Fig. 5) could not be explained simply by an alteration of steady-state protein levels. Instead, there was a shift in affinity between dynamin II and the adaptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dynamin II is most abundantly found in the testis vs otherorgans (Iguchi et al. 2002). However, its physiological role in the testis remains unknown. Studies in other epithelia have demonstrated that dynamins are crucial for endocytosis of integral membrane proteins at cell junctions, such as occludin, and they are involved in protein-trafficking events between cell membrane and the actin network (Oh et al. 1998, Schmid et al. 1998, Sever et al. 2000a, Orth & McNiven 2003, Shen & Turner 2005). Moreover, recent studies have shown that membrane protein internalization and its recycling are important for regulating junction dynamics in multiple epithelia (for a review, see Maxfield & McGraw (2004)). In general, this process of protein internalization is regulated by clathrin- or caveolin-mediated pathways (Le et al. 1999, Ivanov et al. 2004, Shen & Turner 2005), or alternatively by a clathrin- and caveolin-independent mechanism such as pinocytosis (Utech et al. 2005). Dynamins are known to be involved in both clathrin- and caveolin-mediated protein trafficking (Oh et al. 1998, Shen & Turner 2005), facilitating internalization, and recycling of integral membrane proteins. However, it remains to be investigated if dynamin II indeed is involved in promoting internalization and recycling of occludins, cadherins, JAM-A, and claudins at the BTB; but since dynamin II was found to co-localize with clathrin in Sertoli cells (Kamitani et al. 2002), its role in cellular protein trafficking was implicated. Furthermore, recent studies have provided strong support for the model in which dynamins function as pinchase-like mechanoenzymes, to sever nascent endocytic pits from the plasma membrane to form endocytic vesicles, rather than acting as molecular switches like small GTPases (Thompson & McNiven 2001, Cao et al. 2003). Thus, it is highly likely that dynamin II acts as a ‘pinchase-like mechanoenzyme’ in Sertoli cells, similar to other epithelial cells, to facilitate internalization of occludin and/or N-cadherin.
It is obvious that the best approach to assess the function of dynamins at the BTB is to examine the testes of dynamin knockout mice. Yet, dynamin knockout mice are presently unavailable, thus it is not known if the deletion of any one of the three classical dynamins would affect spermatogenesis and/or BTB function. Nonetheless, in light of the extensive junction restructuring events at the BTB during spermatogenesis at stage VIII of the epithelial cycle to facilitate preleptotene spermatocyte migration across the barrier (Russell 1977), we sought to examine the possible involvement of dynamin II in the protein complexes at the BTB using a different approach. Studies were carried out by a model of junction restructuring in which adult rats were treated with adjudin (Mruk & Cheng 2004). In normal testes, it was shown that dynamin II was associated with the two most extensively studied protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/ß-catenin. It is also worthy of note that the association between dynamin II and the adaptors in these two complexes, namely ZO-1 and ß-catenin, significantly increased during germ cell depletion. In other epithelia, TJ fibrils are restricted to the apical region of epithelial cells, beneath these are the AJ plaques and followed by the desmosomes, forming the junction complexes (Alberts et al. 2002). In light of such intimate association between TJ and AJ, a disruption of AJ is known to perturb the TJ barrier invirtually all the epithelia and endothelia examined to date (Man et al. 2000, West et al. 2002, Guo et al. 2003). However, this general cell physiological response is not applicable to the seminiferous epithelium. Otherwise, the BTB cannot maintain its ‘fence’ and ‘immunological barrier’ functions during spermatogenesis when developing preleptotene spermatocytes are traversing the BTB at stage VIII of the epithelial cycle. Additionally, TJ and AJ are even more intimately associated at the BTB vs other epithelia and endothelia. This is because at the BTB, TJs ‘co-exist’ side-by-side with desmosome-like junctions and two types of testis-specific AJ, namely basal ES and basal tubulobulbar complex (basal TBC). Hence, it is conceivable that although the disruption of AJ usually leads to TJ disassembly, a unique mechanism is in place in the testis to ‘disengage’the events of TJ and AJ disassembly pertinent to spermatogenesis. Indeed, recent studies have shown that the testis apparently utilizes a ‘disengagement’ and ‘engagement’ mechanism to ensure the integrity of TJs during AJ restructuring (Yan & Cheng 2005). For instance, it was shown that the co-existing occludin/ZO-1 and N-cadherin/ß-catenin complexes were structurally ‘engaged’ in normal testes via their adaptors, ZO-1 and ß-catenin, to reinforce BTB integrity, creating the tightest barrier in the mammalian body (Yan & Cheng 2005).
However, when the testis undergoes restructuring during spermatogenesis or when AJ restructuring is induced by adjudin treatment, ZO-1 is dissociated from ß-catenin, i.e. in a ‘disengagement’ state. As such, the occludin/ZO-1 and N-cadherin/ß-catenin protein complexes are not structurally ‘engaged’ but rather ‘disengaged’. This permits AJ restructuring to facilitate germ cell movement without compromising the occludin-ZO-1 interactions at the BTB. As reported herein, dynamin II became more significantly ‘engaged’ with ZO-1 and ß-catenin during extensive AJ restructuring induced byadjudin, which may explain the lack of damage to the BTB at the time of germ cell loss during adjudin-induced AJ restructuring (Mruk & Cheng 2004). This phenomenon seemingly suggests that dynamin II may play a role in assisting the ‘disengagement’ of ZO-1 and ß-catenin by pulling them away from each other during adjudin-induced junction restructuring so that these adaptors can associate primarily with their corresponding integral membrane proteins, occludin and N-cadherin respectively. In this way, the occludin–ZO-1 complex can continue to maintain the TJ barrier. This postulation is also supported by the protein–protein interaction data. By day 14 when virtually all the tubules were devoid of germ cells with only Sertoli cells and spermatogonia found in the basal compartment, there was an increase in the association between dynamin II and the two major junctional protein complexes at the BTB.
Obviously, future investigations should be expanded to examine the changes in the kinetics of internalization and recycling of BTB integral membrane proteins (e.g. occludin, N-cadherin, and/or JAM-A) following adjudin treatment, using seminiferous tubule cultures and the techniques of biotinylation, immunoblotting, and co-immunoprecipitation. In addition, the functional significance of the changes in association between dynamin II and ZO-1/ß-catenin, as presented in this report, should be carefully evaluated.
In this context, it is of interest to note that while dynamin II is found in the BTB and may be crucial to BTB dynamics, it is the most abundantly detected at the apical ES in a stage specific manner (i.e. at early stage VIII). This seemingly suggests that dynamin II may also facilitate protein internalization at the apical ES during spermiation, which should be investigated in future studies.
Is the adjudin-induced AJ restructuring and germ cell loss a reliable model to probe the function of dynamin II?
It is obvious that the results reported herein were based on the use of the adjudin model and comparison with testes from normal rats. As such, one would argue if the observed changes in protein–protein interactions reported herein were simply the result of drug toxicity, and they might be irrelevant to normal testicular physiology. We offer several explanations to support our conclusion. First, recently completed acute toxicity studies in mice and rats, as well as pertinent mutagenicity and genotoxicity conducted by licensed toxicologists according to FDA guidelines, have shown that adjudin is not toxic at doses effective to induce transient infertility in these animals (Mruk et al. 2006). However, as illustrated in a 29-day subchronic toxicity study, a narrow margin between adjudin’s safety and efficacy was detected, making it unlikely to become a male contraceptive unless it can be targeted specifically to the testis to improve its efficacy and selectivity (Mruk et al. 2006). These toxicity studies thus illustrate that the data reported herein were not likely to be the manifestation of drug toxicity. Secondly, if adjudin is indeed acutely toxic to Sertoli and/or germ cells, its infertility effects would have been more rapid when it was administered to adult rats at 50 mg/kg b.w. (once a week for 2–4 weeks) by gavage, without requiring a ~20-day wait period for the sperm reserve in the epididymides to be exhausted (Cheng et al. 2001, 2005). Furthermore, the anti-fertility effect of adjudin was highly reversiblewhen ratswere given just two to six doses at 50 mg/kg b.w. (once a week for 2–6 weeks), suggesting that at these dosings, not all Sertoli cells and spermatogonia were killed by the adjudin treatment (Cheng et al. 2005). Nonetheless, these findings do not rule out the possibility that adjudin is a Sertoli and/or germ cell toxicant, in particular if it is given chronically. Thirdly, recent studies from our laboratory using the adjudin model have identified the signaling molecules and pathways, such as focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) which is one of the mitogen-activated protein kinases, crucial for regulating cell adhesion in the epithelium particularly between Sertoli cells and elongating/elongate spermatids at the apical ES (Siu et al. 2005, Xia & Cheng 2005). These findings have largely been confirmed using a well-established and thoroughly characterized in vivo model for studying AJ dynamics in the testis by testosterone/estradiol implants to suppress the intratesticular androgen level (O’Donnell et al. 1996, 2000, Saito et al. 2000, Beardsley & O’Donnell 2003); (for reviews, see O’Donnell et al. 2001, McLachlan et al. 2002). In this model, the suppression of endogenous androgen level resulted in a selective disruption of apical ES at the Sertoli cell–spermatid (step 8 and beyond) interface without apparent toxic effects to testicular cells since germ cells eventually repopulated the epithelium during recovery, and the BTB integrity was also not compromised (O’Donnell et al. 2000, Beardsley & O’Donnell 2003). For instance, a study using this androgen suppression model has shown that cSrc, FAK, and ERK are indeed the crucial kinases in the ERK-signaling pathway that regulates apical ES dynamics (Wong et al. 2005), which is consistent with the results obtained from the adjudin model (Siu et al. 2005, Xia & Cheng 2005). Lastly and perhaps the most important of all, as shown in studies using the androgen suppression model, the observed changes in cadherins–catenins interactions in the epithelium as a result of increasing tyrosine phosphorylation in ß-catenin, ultimately led to germ cell loss from the epithelium (Xia et al. 2005b, Zhang et al. 2005); and these results were consistent with data obtained from the adjudin model (Xia & Cheng 2005, Yan & Cheng 2005). Collectively, the above arguments illustrate that even if adjudin is a Sertoli cell toxicant, it produces changes in the seminiferous epithelium including the BTB and induces junction restructuring at the cell–cell interface, which can be used to learn something about the normal system. Thus, the results presented in this report are physiologically relevant to events that occur in the seminiferous epithelium during spermatogenesis.
What is the physiological significance regarding changes in protein–protein interactions at the BTB pertinent to preleptotene spermatocyte migration across the BTB at stage VIII of the epithelial cycle?
Recent studies have shown that at least five different structural protein complexes (excluding their isoforms) are found at the BTB, conferring barrier and anchoring functions in adult rat testes. These include claudins/ZO-1, JAMs/ZO-1, and nectins/afadins, in addition to the previously mentioned occludin/ZO-1 and N-cadherin/ß-catenin (Wong & Cheng 2005, Xia et al. 2005a). Undoubtedly, we will see this list grow in the years to come. At the BTB, these co-existing protein complexes and the different junction types serve to maintain not only the BTB integrity but also the cell polarity. More importantly, they ensure the timely BTB restructuring that facilitates preleptotene spermatocyte movement during stage VIII of the epithelial cycle in adult rat testes. It would be physiologically ‘difficult’, perhaps even ‘uneconomical’, to have de novo synthesis of all these proteins simultaneously at the BTB each time the preleptotene spermatocytes traverse the BTB. For this reason, protein complexes at the BTB may employ an efficient mechanism to allow rapid BTB restructuring. As illustrated by recent studies using different models, the changes in protein–protein interactions at the BTB or apical ES are indeed being used to facilitate AJ and/or TJ restructuring in the seminiferous epithelium, so as to elicit rapid changes in cell adhesion (Xia et al. 2005b, Yan & Cheng 2005). Studies from other epithelia have shown that cadherins can be shuffled rapidly to the cytoplasm via internalization, and re-shuffled back to the plasma membrane with the help of GTPases, kinases, and adaptors (e.g. Rap1, PKC, p120ctn; Le et al. 1999, 2002, Balzac et al. 2005, Mruk et al. 2005, Xiao et al. 2005). In addition, GTPases (e.g. dynamin II) are likely to be involved in these protein-trafficking events, and some of them may act as molecular switches for trafficking between cell membrane and cytoskeletal networks in various epithelia, including the seminiferous epithelium in adult rat testes (Olkkonen & Stenmark 1997, Takai et al. 2001, Deneka et al. 2003, Mruk et al. 2005). Work is now in progress in our laboratory to assess internalization and recycling of integral membrane proteins at the Sertoli–Sertoli and the Sertoli–germ cell interfaces, and the factor(s) and/or mechanism(s) that regulate these events.
In summary, we have demonstrated that dynamin II is a potentially important regulator for the protein–protein interactions between different adaptors (e.g. catenins and ZO-1) and their corresponding integral membrane proteins (e.g. cadherins and occludins) in rat testes. Based on these recent data including the results reported herein, we have provided a hypothetical model (see Fig. 6) illustrating the dynamic interactions between GTPases (e.g. dynamin II) and the corresponding BTB-associated protein complexes (e.g. occludin/ZO-1 and N-cadherin/ß-catenin), to facilitate preleptotene spermatocyte migration across the BTB without compromising its integrity.
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P < 0.01. (C–F) These micrographs are the results of an immunohistochemistry experiment using an anti-dynamin antibody (see Table 1
