Adherens Junction Dynamics in the Testis and Spermatogenesis

Anchoring or adhering junctions are points at which cells attach to neighboring cells. They play an important role in determining and maintaining tissue organization (for reviews, see Yap et al, 1997; Tsukita et al, 2001; Cheng and Mruk, 2002). In the testis, unique cell-cell actin-based adherens junctions (AJs) between Sertoli cells, as well as between Sertoli and germ cells, and cell-cell intermediate filament-based desmosome-like junctions between Sertoli and germ cells (for reviews, see Russell and Peterson, 1985; Russell et al, 1990; Byers et al, 1993; Russell, 1993) not only provide mechanical adhesion of germ cells onto Sertoli cells, they also play a crucial role in germ cell morphogenesis and differentiation (Ozaki-Kuroda et al, 2002). In addition, the turnover of these junctions in the testis is important for permitting germ cell translocation from the basal compartment to the adluminal compartment of the seminiferous epithelium to complete spermatogenesis. In this review we limit our discussion to the recent and crucial development of the study of AJs instead of desmosome-like anchoring junctions because the latter type has been reviewed elsewhere (Russell and Peterson, 1985; Russell et al, 1990; Byers et al, 1993; Russell, 1993). Four structurally and functionally different forms of anchoring junctions exist: 1) AJs between cells, 2) focal contacts between cells and the extracellular matrix (ECM), 3) desmosomes between cells, and 4) hemidesmosomes between cells and the ECM (for a review, see Alberts et al, 2002). Anchoring junctions are subdivided into two categories based on their connection sites. Both cell-cell AJs and cell-matrix focal contacts are connected to actin filaments, whereas desmosomes and hemidesmosomes are connected to intermediate filaments (for a review, see Alberts et al, 2002). The cell-cell, actin-based AJ is by far the best studied adhering junction type in the testis, and includes ectoplasmic specialization (ES), a testis-specific AJ between Sertoli and germ cells. In the testis, AJs confer adhesion between cells, which is known to be mediated by three AJ-integral membrane protein complexes; namely, cadherin/catenin, nectin/afadin, and integrin/laminin (for reviews, see Taga and Suginami, 1998; Rowlands et al, 2000; Vogl et al, 2000; Cheng and Mruk, 2002) (Figure 1 and Table 1). These three complexes in turn are connected to the actin cytoskeleton (for reviews, see Kemler, 1993; Gumbiner, 1996; Yap et al, 1997; Miyahara et al, 2000; Tachibana et al, 2000) (Figure 1; Table 2). For instance, E- or N-cadherin/β-catenin complex interacts with the actin network via α-catenin, whereas afadin is a putative F-actin-binding protein that also links the nectin/afadin complex to the actin cytoskeleton network (Figure 1). However, recent studies have shown that afadin can also interact with ZO-1, ponsin (an afadin- and vinculin-binding protein), and α-catenin in the cytoplasm at the site of AJs (Yokoyama et al, 2001), suggesting that the nectin/afadin complex can also link to the cytoskeleton network via α-catenin, a putative actin-binding protein, in addition to its interaction with actin via afadin. Furthermore, α-catenin also provides a structural bond that links together the cadherin/catenin and nectin/afadin complexes (Pokutta et al, 2002) indicating that cross-talk exists between the two complexes. In the testis, another AJ complex based on α6β1 integrin has been shown to be the major cell adhesion constituent protein complex of the ES (Vogl et al, 2000). Because epithelial cells are not likely to use integrin-integrin interaction to mediate cell adhesion (Weitzman et al, 1995), and because α6β1 integrin receptors are largely restricted to Sertoli cells (for a review, see Vogl et al, 2000) (we also failed to detect integrin β1 in germ cells isolated from adult rat testes; Siu et al, 2002), it is apparent that laminin, the binding partner of integrin in other epithelia (for a review, see Dym, 1994), is the putative binding partner of α6β1 integrin and should reside in germ cells. Yet this possibility remains to be studied. Recent studies have shown that laminin γ3, a potential nonbasement membrane binding partner of integrin, is localized to AJ sites between round and elongated spermatids, and Sertoli cells in the testis (Koch et al, 1999), which is consistent with its localization at the apical ES. Its pattern of localization is also in sharp contrast to α1/β1/γ1 laminin, which is restricted to the basement membrane in the seminiferous epithelium (Koch et al, 1999). These results collectively seem to suggest that the binding partner of α6β1 integrin in the testis at the site of the ES is composed of at least laminin γ3, and possibly may be laminin 12 (Koch et al, 1999). . A diagrammatic drawing that illustrates the current structural units of adherens junctions in the testis. Adherens junctions are cell-cell actin-based anchoring junctions and are found between Sertoli cells as well as between Sertoli and germ cells. The cadherin-catenin and nectin-afadin complexes are the basic structural units of adherens junctions between Sertoli cells as well as between Sertoli and germ cells. The integrin-laminin based AJ complex is largely restricted between Sertoli cells and elongated spermatids at the site of ectoplasmic specialization. SG indicates spermatogonia; pSP, preleptotene/leptotene spermatocyte; SP, pachytene spermatocyte; rSp, round spermatid; Sp, elongated spermatid; SC, Sertoli cell; AJ, adherens junction; ILK, integrin-linked kinase; FAK, focal adhesion kinase. Cadherin—Classical cadherins, such as epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), and placental cadherin (P-cadherin) are AJ-integral membrane proteins (for reviews, see Takeichi, 1990; Takeichi et al, 2000). Each cadherin molecule consists of a highly conserved cytoplasmic domain, followed by a single-pass transmembrane region, and one extracellular domain of approximately 550 residues (for reviews see Takeichi, 1990, 1995; Miyatani et al, 1992; Kemler, 1993; Herrenknecht, 1996; Pötter et al, 1999) (Figure 1). Intracellularly, each cadherin molecule interacts with β- or γ-catenin and p120ctn to form the cadherin/catenin complex, which is the most extensively studied AJ functional unit. p120ctn binds to the juxtamembrane domain of cadherin (Finnemann et al, 1997; Yap et al, 1998), whereas β- or γ-catenins associate with the catenin-binding domain of cadherin (Nagafuchi and Takeichi, 1989; Ozawa et al, 1989; Stappert and Kemler, 1994). Recent studies have confirmed the presence of this complex in the testis (Chung et al, 1998a; Wine and Chapin, 1999; Chapin et al, 2001; Johnson and Boekelheide, 2002a,b; Lee et al, in press). Studies using immunoprecipitation and immunoblotting techniques with and without cross-linkers have also demonstrated the presence of cadherin and catenin in isolated Sertoli and germ cells in vitro (Lee et al, in press). For instance, germ cells isolated from adult rat testes with negligible contaminations of Sertoli, Leydig, or peritubular myoid cells were shown to express E-cadherin and β-catenin at an almost equimolar ratio of 1:1 with Sertoli cells (Lee et al, in press). This latest finding thus suggests that both Sertoli and germ cells possess the functional cadherin/catenin complexes and interact with each other in a homotypic fashion, thereby conferring cell adhesion function in much the same way as in other epithelia (see Figure 1). These results are also consistent with a recent immunofluorescent microscopy study that localized N-cadherin to the site between spermatocytes and Sertoli cells (Johnson and Boekelheide, 2002b). These results also strongly illustrate that the testis uses the N-cadherin/catenin complex to regulate cell adhesive function (Lee et al, in press) in addition to the desmosome-like junctions found between Sertoli cells and spermatogonia, spermatocytes, and round spermatids (for reviews, see Russell and Peterson, 1985; Russell, 1993). Furthermore, studies using seminiferous tubules isolated from adult rat testes and Sertoli-germ cell cocultures for cross-linking and immunoprecipitation have shown that this N-cadherin/catenin complex links to the actin network rather than to the intermediate filament-based (eg, vimentin) or microtubule-based (eg, tubulin) cytoskeleton in the testis (Lee et al, in press). This latest finding via a biochemical approach contrasts somewhat with two earlier studies with immunofluorescent microscopy in which cadherin was found to colocalize with the intermediate filament-based cytoskeleton (Mulholland et al, 2001; Johnson and Boekelheide, 2002b). These apparently conflicting observations can be explained as follows. First, the anti-pancadherin antibodies used in immunoelectron microscopy (Mulholland et al, 2001) with at least different that have been in the testis (Johnson et al, 2000). Yet a was used for the cross-linking and immunoprecipitation other of the cadherin other than N-cadherin use the intermediate as sites for the desmosome-like junctions in the testis, they be from the biochemical an to suggest that the of or their used by different can different results by or microscopy is a highly that can detect a of complex in a Yet the cross-linking and immunoprecipitation an to detect a with the use of a much of for its with apparently and it is also that an filament-based cadherin/catenin complex is in the testis, it exists only and in the seminiferous This is by the that N-cadherin is found at the apical ES only at (Johnson and Boekelheide, 2002b). In addition, a of cell adhesion are also and in the seminiferous epithelium and Chapin, 1999). In this it is to that earlier studies have shown that desmosome-like junctions cell-cell intermediate filament-based adhering junction are used to spermatogonia, spermatocytes, and spermatids onto Sertoli cells (Russell and Russell, 1993; Russell and Peterson, see Figure 2). However, it be that the constituent proteins of desmosomes in other epithelia are and (for a review, see Alberts et al, 2002), of these proteins have been or are studied in the testis. is that a biochemical study to the of desmosome-like junctions in the testis is . A that illustrates the current of AJ in the testis. The cell adhesion function apparently is by the of and that the of the three AJ structural Furthermore, and also play a role in the and of such as between Sertoli and germ cells, as well as between Sertoli cells. with based on and at least were in the rat testis, and (Johnson et al, 2000). Furthermore, each cadherin unique and in the seminiferous For instance, N-cadherin is localized at basal and junctions between and (Johnson and Boekelheide, 2002b). However, in studies to the of E-cadherin and N-cadherin in AJ in the testis are not to because of or E- cadherin in results in development at and with at et al, et al, 1995; et al, binds to the cadherin cytoplasmic and as a to α-catenin, an actin-binding protein, which in turn the cadherin/catenin complex to the actin binds to the cadherin cytoplasmic via its with its to α-catenin 1993; et al, et al, et al, 1995; et al, 1995; Yap et al, (Figure 1). γ-catenin is highly to suggesting that both proteins function have shown that β-catenin be by γ-catenin or For instance, of the γ-catenin in in et al, β-catenin epithelial cell adhesive et al, These results thus that whereas both β- and γ-catenins each be functionally by the Furthermore, also at the it to the role of β-catenin in et al, is for cadherin adhesive For instance, cells α-catenin have to each other the presence of cadherin et al, 1992; et al, 1994). studies have shown that α-catenin links the cadherin-catenin complex to actin or via an actin-binding protein, its in adhesion et al, et al, et al, et al, 1995; et al, 1995; et al, Recent studies have also demonstrated that α-catenin is used by the nectin/afadin adhesion complex in afadin to the actin network via a putative site on α-catenin at residues which also links the nectin/afadin complex to the cadherin/catenin complex (Pokutta et al, 2002), permitting cross-talk between the two complexes. α-catenin, and p120ctn have been in the testis et al, Wine and Chapin, 1999; Chapin et al, 2001; et al, 2001; Johnson and Boekelheide, Lee et al, in press). For instance, p120ctn is in junctions between Sertoli cells as well as at the sites of Sertoli and Sertoli spermatids (Johnson and Boekelheide, consistent with its localization at the ES. p120ctn with β-catenin and at the sites between Sertoli cells and spermatocytes (Johnson and Boekelheide, Furthermore, an of cadherin and β-catenin was AJ between Sertoli cells (Chung et al, 1999) as well as between Sertoli cells and germ cells (Lee et al, in press). is another cell-cell adhesion at AJs (Figure 1) that both homotypic and between cells. is a cell-cell membrane adhesion molecule to the et al, 1999). has three extracellular a transmembrane region, and a cytoplasmic The cytoplasmic domain of has a conserved of that interacts with the domain of which links to the actin cytoskeleton et al, 1997; et al, 1999) or via α-catenin (Pokutta et al, 2002) (Figure 1). of are and and 1992; et al, 1998; et al, 2000; et al, 2002). is the most form in the testis, is in the testis, whereas is largely in the et al, 2000). In is with germ cells the of round spermatids into spermatids et al, 2000). the localization of is largely restricted between elongated spermatids and Sertoli cells, in et al, 2000). Studies of have shown that these are and have tubules germ cells, that spermatids from to they have and are found in the of spermatids instead of in the et al, 2000). are in most cell such as spermatids, Sertoli cells, and cells, is almost by spermatids, suggesting that the adhesion between Sertoli cells and spermatids may be mediated via (Ozaki-Kuroda et al, 2002). using techniques to and to and testis, of between Sertoli cells and spermatids at the site of ES are only via between in Sertoli cells and in spermatids (Ozaki-Kuroda et al, 2002). of afadin are known to and is and is also found in the testis, whereas is in the et al, 1997; et al, 1999). is the a domain and three and can to the actin cytoskeleton et al, 1997; et al, 1999). has one domain the F-actin-binding domain et al, 1997; et al, 1999; et al, of has shown that afadin is for structural organization of AJs and junctions in epithelia et al, 1999) because and such as of and of the to et al, 1999). is an afadin- and vinculin-binding ponsin binds to it with to the AJ et al, 1999; Tachibana et al, 2000). ponsin binds to it is to both the AJs and focal et al, 1999). ponsin can interact with afadin and these three not form a structural complex et al, 1999). Studies using have both afadin and ponsin in the biochemical and functional studies to in the testis are recent study with microscopy and microscopy (Ozaki-Kuroda et al, 2002) has also the that the nectin/afadin complex is another actin-based cell adhesion functional between Sertoli and germ cells in the seminiferous epithelium (see Figure 1) in addition to the cadherin/catenin complex For instance, both and were found in spermatids in at namely, both round spermatids, and spermatids, as well as in Sertoli cells not and were found to interact with each other (Ozaki-Kuroda et al, 2002). These collectively et al, 2000; et al, Lee et al, in press) that it is that functional complexes for the anchoring of germ cells onto Sertoli cells, which are and are found in the seminiferous epithelium in a For instance, was localized at the of spermatids and Sertoli cells also with between spermatocytes and Sertoli at its at to the of spermatids at (Ozaki-Kuroda et al, 2002). are transmembrane cell adhesion with putative and (Figure 1) (for reviews see and 1995; and 1999). Each integrin molecule is composed of an and a which are to form a functional The integrin also in cells. integrin is a functional molecule that ECM proteins with the cytoskeleton network and neighboring cells to form an epithelium (for reviews see et al, 1998; et al, 2002). The extracellular domain of an integrin by the and can the of that can the in a known as (for reviews see 1999; 2002). is that the domain can also a cell via the integrin a of integrin receptors on the cell and can its in a known as (for reviews see et al, 2000; 2002). Intracellularly, the cytoplasmic of both interact with different actin-binding and In the testis, and and and (an are known to interact with the cytoplasmic of integrin receptors and Chapin, 1999; et al, 2000; et al, In and are known to to which can to form at least different integrin receptors (for reviews see 1999; 2002). In the testis, and β1 and have been et al, 1992; et al, 1993; et al, 1995; et al, 1998; 2001; et al, 2002). Yet the most extensively studied integrin in the testis is α6β1 (for a review, see Vogl et al, which has been to be a of the major protein complex in the apical ES (Mulholland et al, is found both in the basal and the adluminal of the seminiferous epithelium and is a at et al, 1992; et al, In the known binding partner for α6β1 or integrin is laminin and 1993; and most of the laminin to in the testis are to the basement membrane (Koch et al, 1999), a form of ECM in the testis 1994). A recent study using has localized the laminin in the testis, that it is almost restricted to the adluminal compartment of the seminiferous epithelium (Koch et al, 1999). These results suggest that laminin is one of the putative that the binding partner for α6β1 Because a functional laminin binding protein is composed of three laminin the in the apical ES to be in the testis. The of α6β1 integrin has been by studies using For instance, in integrin and et al, In integrin at to et al, 1995; et al, Because of these their in the ES is not Furthermore, it is not known integrin α6β1 to regulate the actin-based cytoskeleton network that integrin-linked is in these (Mulholland et al, In this it is important to that and integrin are also localized to the same site of α6β1 in the testis et al, 1993). both and have been in Sertoli cells by et al, 1998), and a recent study found integrin in cells et al, 2002). In the integrin/laminin complex is an functional in the testis, in at the site of the ES. is to this the cytoskeleton is a AJ-integral membrane protein that is at the site of AJs in such as and it is known is in the testis et al, 2000). interacts with both and the cadherin-catenin complex et al, 2000). is a cytoskeleton protein found at the site of cell-cell contacts and is of binding to actin and and and 1989; et al, 1994). In the testis, is with ES at the adluminal and basal with actin filaments and 1989; et al, 1990; and molecule that is of for Sertoli-germ cell AJ is because a in its was at the of AJ in Sertoli-germ cell cocultures and 1999). Recent studies have a of at the sites of AJs such as and et al, and 1996; and 1997; et al, 1999; and 2000; 2001) (see Table 2). For instance, and have been demonstrated in rat testis by and et al, 1989; et al, et al, 1995; et al, 1999; Wine and Chapin, 1999; Chapin et al, the localization of and in the testis and their and localization have their role in the of and Chapin, 1999; et al, For instance, is and is in pachytene spermatocytes et al, Recent studies of have shown that these apparently strongly suggesting that the function of be by other in the testis et al, 2001) such as is localized in both Sertoli cells and spermatids and is a AJ molecule in the testis, at to et al, These can the cadherin/catenin or the nectin/afadin complexes via of or cadherins, which in cell adhesive function at the site of AJs and 1996; and 1997; and 2000; Yet this remains an of in the testis. are cell adhesion complexes found at the apical of the seminiferous epithelium in which spermatids onto Sertoli cells their into the at and at the basal of the seminiferous epithelium between Sertoli cells (Figure 1; Table 2). recent studies using immunofluorescent microscopy suggest that in the testis are they may use intermediate filaments as sites (Mulholland et al, 2001; Johnson and Boekelheide, 2002b). Yet a recent study by cross-linking using and immunoprecipitation using antibodies or have demonstrated that the cadherin/catenin between Sertoli cells and between Sertoli and germ cells are actin-based (Lee et al, in press). was that the turnover of between Sertoli cells at the basal of the seminiferous epithelium the of preleptotene/leptotene spermatocytes the at and of the whereas the of spermatids at the adluminal compartment is by the of in the apical (Vogl et al, 2000). are by the membrane of Sertoli cells with a of actin and a of et al, 2002). α6β1 and integrin-linked were shown to be the major of ES et al, 1992; et al, 2). and are also found at the site of and are likely the putative constituent proteins of (see Table 2) et al, 1989; and 1989; et al, 1990; et al, 1996; et al, 1997; et al, et al, 2002) 2). the function of the that the ES in the testis remains to be complex is another testis-specific form of actin-based AJs the of spermatids at and into the of Sertoli cell membrane (Russell and Russell, consists of two structural a and a (Russell and The constituent of is not have been the function of 1993). For instance, it was that as an anchoring to spermatids in the seminiferous epithelium also be used to the between Sertoli cells and spermatids to the of spermatids into the at recent studies have shown that an is into the in the of the suggesting that the may function as a protein at the site of AJs to et al, 1999). this junction type has been for almost three its and biochemical to be The and of AJs is one of the because germ cells from the basal to the adluminal compartment of the seminiferous epithelium to complete (for reviews see Russell and Peterson, 1985; Russell et al, 1990; Byers et al, 1993; and 2000). However, was to and Sertoli-germ AJs are studies have shown that AJ are by a of extracellular that and (Figure 2). AJ function largely by the of the two known AJ functional complexes; namely, cadherin/catenin and For instance, and can of α-catenin, and which in of the cadherin-catenin complex et al, and 1998), thereby Furthermore, not only of and p120ctn et al, 1998), it also the of p120ctn and at the sites of these et al, 2000). These results thus suggest that adhesive between cells can be by both of cadherin and β-catenin and of which are by et al, 1998; et al, 2000). recent studies have shown that such as may play a crucial role in the of junction in the testis et al, 2002), can be found in the that has the of in AJ in the testis. The of the cadherin-catenin complex is on the of cadherin and catenin 1993). cadherin-catenin adhesive function via of AJ protein such as and also play an important role in the of the cadherin-catenin complex (see Table 1). For instance, a protein can of the of thus cell adhesion function et al, Ozawa and Kemler, 1998; et al, 1999). protein have been shown to interact with the cadherin-catenin complex at the site of and et al, 1995; et al, 1996; 1996; et al, 1996; Cheng et al, a of can regulate the between and to provide an for AJ a putative protein has been in rat testis et al, study has shown that with both round and elongated spermatids and Sertoli cells at the site of AJs in the seminiferous epithelium et al, 2000). was by Sertoli and germ cells et al, and Sertoli cell can be by germ or Sertoli-germ cell et al, and seem to suggest that the of AJ in the testis are at least in via protein The study in the to illustrate that and are in the of Sertoli-germ cell AJ is the by et This study demonstrated that the of Sertoli-germ cell AJs in vitro was with a in as well as a in the of and at the germ cells to the Sertoli cell epithelium that the of This study is different from other of germ cells on Sertoli cell because Sertoli cells were for in to form an epithelium with complete AJs and 1997; et al, isolated germ cells were to this Sertoli cell epithelium to Sertoli-germ cell AJ and thereby the in Furthermore, such an in vitro has been in two earlier studies and and indicating that junctions to found in such as desmosome-like junctions and in this which was also in our et al, A recent study with microscopy also N-cadherin and β-catenin to the same sites between Sertoli cells as well as between Sertoli cells and germ cells these cells were in vitro (Lee et al, in indicating the presence of functional AJs in this this is the only in vitro to study the and of Sertoli-germ cell AJ these earlier studies using this in vitro the role of and on Sertoli-germ AJ they are in and the of and in AJ is not this in vitro with to cell adhesion it was shown that the presence of putative Sertoli cell et al, or the binding of germ cells onto the Sertoli cell epithelium et al, in cell adhesion is a of the Sertoli-germ cell AJ the presence of an not the also germ cell binding to the Sertoli cell epithelium et al, in This recent study thus provides of the of and in Sertoli-germ cell AJ In this it is of to that Sertoli, and peritubular myoid cells are also known to a of and the seminiferous (for a review see et al, of which are in a For instance, the or localization of et al, et al, and (Chung et al, and are et al, These studies collectively also illustrate the of in AJ such as is our that and in a to regulate the of junction and which in turn the translocation of germ cells from the basal to the adluminal compartment of the seminiferous epithelium et al, 1993). it was shown that the of and by cocultures of Sertoli-germ cells AJ are et al, their role in AJ on in the on the testis as reviewed it is apparent the AJ between Sertoli and germ cells in the testis are by and different of as shown in Figure For instance, the and of AJ are by the of the cadherin/catenin and nectin/afadin and possibly the integrin/laminin an in is likely to the of AJ whereas an in at the site of AJs their Furthermore, to be to the biochemical of and the constituent proteins interact with each other at the site of both basal and to regulate AJ and germ cell the seminiferous epithelium spermatogenesis.