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BOR - Papers in Press, published online ahead of print March 31, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027268
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BIOLOGY OF REPRODUCTION 71, 392–404 (2004)
DOI: 10.1095/biolreprod.104.027268
© 2004 by the Society for the Study of Reproduction, Inc.

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Adaptors, Junction Dynamics, and Spermatogenesis1

Nikki P.Y. Lee, and C. Yan Cheng2

Population Council, New York, New York 10021


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
Adaptors are component proteins of junctional complexes in all epithelia, including the seminiferous epithelium of the mammalian testis. They recruit other regulatory and structural proteins to the site of both anchoring junctions (such as cell-cell actin-based adherens junctions [AJs], e.g., ectoplasmic specialization [ES] and tubulobulbar complex, which are both testis-specific cell-cell actin-based AJ types, and cell-cell intermediate filament-based desmosome-like junctions) and tight junctions (TJ). Furthermore, adaptors per se can be substrates and/or activators of kinases or phosphatases. As such, the integrity of cell junctions and the regulation of junction dynamics during spermatogenesis rely on adaptors for their ability to recruit and link different junctional components to the same site and to tether transmembrane proteins at both anchoring and TJ sites to the underlying cytoskeletons, such as the actin filaments, intermediate filaments, and microtubules. These protein-protein interactions are possible because adaptors are composed of conserved protein binding domains, which allow them to link to more than one structural or signaling protein, recruiting multi-protein complexes to the same site. Herein, we provide a timely review of adaptors recently found at the sites of AJ (e.g., ES) and TJ. In addition, several in vivo models that can be used to delineate the function of adaptors in the testis are described, and the role of adaptors in regulating junction dynamics pertinent to spermatogenesis is discussed.

spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
In most epithelia, tight junctions (TJs) between two adjacent cells, such as keratinocytes, are found at the cellular apex, furthest away from the extracellular matrix (ECM) (Fig. 1). Underneath TJs are the cell-cell actin-based adherens junctions (AJs) and cell-cell intermediate filament-based desmosomes. Collectively, TJs, AJs, and desmosomes are referred to as the junctional complex (Fig. 1) [1]. Behind the junctional complex lie the communicating gap junctions (GJs) and cell-matrix actin-based focal contacts and cell-matrix intermediate filament-based hemidesmosomes (for reviews, see [1, 2]) (Fig. 1). In the seminiferous epithelium, however, the relative locations of TJs, anchoring junctions (such as ectoplasmic specializations [ESs] and desmosome-like junctions), and GJs between two adjacent Sertoli cells in the seminiferous epithelium are different from their locations in other epithelia (see Fig. 2). For instance, Sertoli cell TJs are closest to the basement membrane, a modified form of ECM in the testis [3], whereas ES, a testis-specific AJ type, and desmosome-like junctions are present side by side with TJs (for a review, see [4]), and these junctions in turn constitute the blood-testis barrier (BTB, also known as the seminiferous epithelium barrier) in the testis (for reviews, see [2, 4, 5, 6]) (Fig. 2).



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  		FIG. 1. A schematic drawing that illustrates the relative locations of tight junction (TJ), adherens junction (AJ), desmosome, gap junction, focal contact, and hemidesmosome in virtually all epithelia, such as those found in skin, small intestine, and the collecting tubule in kidney. The three classes of junctions in epithelia are: occluding junctions (e.g., TJs), anchoring junctions (e.g., cell-cell actin-based AJs, cell-matrix actin-based focal contacts, cell-cell intermediate filament-based desmosomes, and cell-matrix intermediate filament-based hemidesmosomes), and communicating gap junctions. TJs are found at the apical portion between two adjacent epithelial cells (such as keratinocytes); behind these are the AJs that form the adhesion belt, followed by desmosomes. Collectively, these structures form the junctional complex. Behind the junctional complex lie the gap junctions. Epithelial cells in turn rest on basal lamina, which is composed of extracellular matrix. The two junction types between cell and matrix are focal contacts and hemidesmosomes. This figure was prepared based on earlier reviews [1–4]. When compared to Figure 2, it is obvious that the relative locations of different junction types found in the seminiferous epithelium between two adjacent Sertoli cells and between Sertoli cells and the different classes of developing germ cells are quite different



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  		FIG. 2. This figure depicts adaptors that are currently found at the site of AJs and TJs in the seminiferous epithelium of the testis and their association with different structural protein complexes at these sites. Also shown are the currently known regulatory pathways that, based on recent findings, regulate AJ dynamics in the testis, and the relative locations of TJ, AJ (e.g., apical ES, basal ES, and tubulobulbar complex), desmosome-like junctions, and gap junctions. The BTB found between adjacent Sertoli cells has physically divided the seminiferous epithelium into the basal and adluminal compartment. In the testis, Sertoli cell TJ is constituted of three different classes of transmembrane proteins, namely JAMs, occludins, and claudins. These proteins in turn associate with the underlying actin cytoskeletons via different adaptors, such as ZO-1, ZO-2, and afadin (for reviews, see [2, 5, 18, 19]). Nectins, cadherins, and integrins are integral membrane proteins found at the site of ES in the testis (for reviews, see [2, 18]). These integral membrane proteins are also associated with the underlying cytoskeletons via several adaptors, such as afadin, catenins, and actinin (for reviews, see [2, 34]). The Sertoli-germ cell AJ dynamics have recently been shown to be regulated by at least four putative signaling pathways. These include Pathway I, integrin/pFAK/PI 3-kinase/p130Cas/ERK pathway [14]; Pathway II, integrin/RhoB/ROCK/LIMK/ cofilin pathway [15]; Pathway III, Rab8B-mediated pathway [16]; and Pathway IV, NO/sGC/cGMP/PKG/ß-catenin pathway (unpublished results). The downstream action of these pathways, at least in part, alters the stability and functionality of adaptors at the AJ site, thereby perturbing Sertoli-germ cell adhesion function in the seminiferous epithelium. Also shown are results of recent studies demonstrating that many molecules and proteins can also regulate the opening and closing of AJs. For instance, the closing of AJ requires calcium ions, and an increase in cGMP can induce AJ disruption. Also, high concentrations of testosterone also favor AJ closing, whereas cytokines (e.g., TGFß3) can induce AJ disruption (for reviews, see [2, 10, 71, 79]). Furthermore, recent studies using AF-2364 have shown that it affects AJ dynamics via its effects on the integrin- and cadherin-mediated protein complexes (see text for explanation)

ES is the best-studied testis-specific cell-cell actin-based AJ type in the seminiferous epithelium of the rat testis. In the rat testis, ES can be further subdivided into basal and apical ES (see Fig. 2), which are confined between Sertoli cells at the site of the BTB, and between Sertoli cells and round/elongating/elongate spermatids, respectively, in the seminiferous epithelium (Fig. 2) [2, 6–10]. Apical ES is composed of a layer of hexagonally packed actin bundles sandwiched between the plasma membrane of a Sertoli cell and a cistern of endoplasmic reticulum (ER) on the Sertoli cell side between an apposing Sertoli cell and an elongating/elongate spermatid (or round spermatid) (see Fig. 2) (for reviews, see [7–9]). The basic ultrastructural features of basal and apical ES are similar, except that the intercellular space between two apposing Sertoli cells in basal ES at the BTB site is sealed by TJs (Fig. 2) (for reviews, see [2, 7–9]). Also, the actin bundles and ER at apical ES are confined to the Sertoli cell side of the plasma membrane and TJs are not present. Recent studies have shown that nectin-2, nectin-3, N-cadherin, E-cadherin, laminin {gamma}3, RhoB GTPase, Rab8B, zyxin, axin, and other proteins are found at the apical ES; some of these are also associated with elongating/elongate spermatids [11–17]. The primary function of ES is to facilitate germ cell movement and to anchor germ cells in the epithelium until spermiation (for reviews, see [7–9]). Apical ES is associated with spermatids up to step 19 in the mouse before the tubulobulbar complex appears, which is also a testis-specific AJ type (see Fig. 2) (for reviews, see [4, 6–9]).

Both TJs and AJs (e.g., ES) in the seminiferous epithelium of the testis are composed of integral-membrane proteins, adaptors, and signaling molecules. Although the biochemical composition and molecular architecture of desmosome-like junctions in the seminiferous epithelium are virtually unknown, three putative protein complexes that regulate AJ dynamics, as at the site of ES, have recently been characterized in the testis. These include the cadherin/catenin-, the nectin/ afadin/ponsin-, and the integrin/laminin-based complexes (Fig. 2) (for reviews, see [2, 7, 10, 18]). For TJs, they are composed of occludins, claudins, and junctional adhesion molecules (JAMs) (Fig. 2) (for reviews, see [2, 5, 18, 19]). The integral membrane proteins at the sites of AJ (e.g., cadherins, nectins, and integrins) and TJ (e.g., occludins, claudins, and JAMs) use adaptors, such as ß-catenin, {alpha}-actinin, afadin, vinculin, and cortactin, as linkers for tethering to the underlying cytoskeletal networks (actin filaments, intermediate filaments, and microtubules) and for recruiting additional signaling molecules, such as p130Cas (a 130-kDa adaptor encoded by Crkas gene also called Crk-associated protein, a substrate of src family kinases; upon its phosphorylation, it acts as an adaptor for proteins with SH2 domains), focal adhesion kinase (FAK), and integrin-linked kinase (ILK) to the cell adhesion sites. The activation of these proteins, such as p130Cas, via phosphorylation can recruit additional signaling molecules to the AJ and TJ site to regulate the association of integral membrane proteins with the underlying cytoskeletal network, causing junction restructuring. Alternatively, these signaling proteins, either kinases, such as pFAK (activated FAK via phosphorylation at Tyr-397 and/or Tyr-576) or phosphatases, such as myotubularin, can activate the downstream signal transduction pathways, which can in turn affect junction dynamics via changes in the level of the integral membrane proteins at the TJ and AJ sites (for a review, see [2]). To achieve the level of complexity necessary to affect multivariable function, adaptors possess conserved protein binding domains, such as the proline-rich domains that interact with SH3 (SH stands for Src homolog domain, which is involved in the interaction with phosphorylated Tyr residues [SH2 domains] or with proline-rich portions [SH3 domains] of other proteins) domains in other binding partners, and the SH2 domains that interact with phosphorylated Tyr on other proteins for mediating physical interactions (for a review, see [20]) (Table 1). It is through these domains, along with other yet-to-be defined mechanism(s), that adaptors determine whether structural proteins and/or signaling molecules should be recruited to the AJ and/or TJ sites to affect cell adhesive and TJ-barrier function.


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  		TABLE 1. Adaptors and their interacting partner proteins in the seminiferous epithelium of the testis.a

Recent studies have shown that Sertoli-germ cell AJ dynamics in the seminiferous epithelium are regulated by several signaling pathways (Fig. 2). First, Sertoli-germ cell adhesion function is regulated by the integrin/RhoB/Rho-associated protein kinase (ROCK)/LIM kinase (LIMK, Lin-11 Is1–1 Mec-3 domain kinase)/cofilin [15] and the integrin/pFAK/ phosphatidylinositol 3-kinase (PI 3-kinase)/ p130Cas/extracellular signal-regulated kinase (ERK) [14] pathways using integrins as the receptor protein upstream. Second, other studies have shown that the actin cytoskeleton at the site of Sertoli-germ cell AJs can also be regulated by the Rab 8B-mediated signaling pathway [16] and the nitric oxide (NO)/soluble guanylate cyclase (sGC)/ cGMP/protein kinase G (PKG)/ß-catenin pathway (unpublished results) (Fig. 2). For TJ dynamics, several signaling pathways have also been identified, which include transforming growth factor (TGF)ß/MEKKs (mitogen-activated protein [MAP]/ERK kinase kinases)/p38 MAP kinase [21], and tumor necrosis factor (TNF){alpha}/ILK/glycogen synthase kinase (GSK)/c-Jun N-terminal protein kinase (JNK) [22] signaling pathway (for a review, see [5]). Needless to say, many of the downstream targets of these pathways involve adaptors, such as ß-catenin (unpublished results) and p130Cas [14], illustrating the significance of adaptors in junction restructuring events in the testis. This notion is also supported by earlier studies, which have shown that adaptor malfunctioning can trigger cell junction disruption and cell dissociation. For instance, the loss of ß-catenin from the cadherin-based protein complex leads to a loss of cell-cell adhesion [23], which is mediated by changes in the phosphorylation status of adaptors, such as ß-catenin, because many adaptors are phosphoproteins per se (Table 2). In this minireview, a brief update on different adaptors found in the testis is presented, with emphasis on several recently identified adaptors in the testis. This information should be helpful to investigators in the field who seek to investigate and expand this area of research. Several recent in vivo models that can be used to study the role of adaptors and their regulation in junction dynamics are also discussed. Perhaps most important of all, areas of research that deserve attention are highlighted.


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  		TABLE 2. Adaptors in the testis.a


   ADAPTORS IN THE TESTIS
TOP
 ABSTRACT
 INTRODUCTION
 ADAPTORS IN THE TESTIS
IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
As summarized in Tables 1 and 2 and discussed in this review, very few biochemical and molecular studies are found in the literature that focus on defining the functions of adaptors in the seminiferous epithelium and during spermatogenesis. Nonetheless, these tables summarize most of the basic information on known adaptors in the testis. Table 3 summarizes the physiological significance of selected adaptors in mice using gene knockout experiments. For instance, Table 3 illustrates the pivotal role of adaptors on the well-being and fertility of mammals. The following section provides a brief update on selected adaptors crucial to the regulation of junction dynamics pertinent to spermatogenesis. Other, more extensively studied adaptor proteins, such as catenins and afadins, that have recently been reviewed are not included herein (for reviews, see [see 2, 5, 10]). This is done to avoid redundancy. This review summarizes the updated information in the field regarding adaptors found in the testis; it also serves as a guide for planning future functional studies to investigate the role of adaptors in spermatogenesis.


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  		TABLE 3. Studies on the effects of deletion of adaptors on the viability and fertility of knockout mice.a

Cas family

p130Cas The p130Cas is a 130-kDa protein and the most extensively studied member of the Cas family, which includes human enhancer of filamentation 1 (HEF)/Cas-L and embryonal Fyn substrate/Src-interacting (Efs/Sin) protein (for reviews, see [24, 25]). The p130Cas is ubiquitously expressed in multiple epithelia and is the only member of this family that has been positively identified in the testis [14, 26]. It is a substrate for members of the Src (sarcoma-inducing gene of Rous sarcoma virus encoding the protein tyrosine kinase [PTK] Src; the Src family includes other PTKs such as Fyn, Yes, Fgr, Lyn, Hck, Lck, Blk, and Yrk proteins) family of PTK. Once phosphorylated, as by c-Src, p130Cas acts as a docking protein for proteins with SH2 domains, such as FAK, thereby recruiting signaling molecules to the AJ and TJ sites. This can in turn regulate junction dynamics via changes in the actin cytoskeletal network. A recent study has shown that p130Cas is a crucial molecule used by cytokines, such as TNF{alpha}, to regulate TJ dynamics in the seminiferous epithelium, possibly by recruiting JNK to the Sertoli cell TJ site to affect the level of occludin at the site [22]. Further, its significance in regulating AJ dynamics in the seminiferous epithelium of the rat testis has also been implicated [14], suggesting this is an important adaptor in the seminiferous epithelium crucial to junction dynamics during spermatogenesis. For instance, a disruption of Sertoli-germ cell adhesive function in the seminiferous epithelium induced by AF-2364 is associated with an activation of p130Cas, which may recruit MAP to the AJ site to affect cell adhesive function [14]. In short, two recent studies [14, 22] have demonstrated the crucial role of this adaptor in both AJ and TJ dynamics. Nonetheless, preliminary studies such as these should be expanded to include the use of specific inhibitors of p130Cas and/or c-Src in order to assess whether a blockade of the adaptor function can indeed perturb germ cell adhesion and BTB function in the seminiferous epithelium.

Crk Family

Crk Crk, an oncogene encoding an activator of PTK having SH2 and SH3 domains and a member of the adaptor-type signaling molecules, structurally associates with p130Cas and is an activator of PTK in addition to its adaptor function (for a review, see [27]). Three isoforms of Crk— Crk I, Crk II, and Crk III—are known. All are found in the testis [28, 29]. The functions of these molecules in junction dynamics and spermatogenesis remain to be explored.

Calponin Homology Domain Family

{alpha}-Actinin {alpha}-Actinin is a 100-kDa actin-binding protein and a member of the calponin homology (CH) domain family (for a review, see [30]). Four isoforms of actinin are known. Actinin-2 and -3 are found predominantly in skeletal muscles, whereas actinin-1 and -4 are widely distributed in different organs, including the testis [31, 32]. In the testis, {alpha}-actinin localizes to the site of ES (for reviews, see [33, 34]). It structurally associates with actin, axin, Wiskott-Aldrich syndrome protein (WASP), and zyxin in the testis, acting as an adaptor to recruit zyxin to the cadherin/catenin complex via its interaction with {alpha}-catenin [11, 35].

Fimbrin Fimbrin is a 68-kDa actin cross-linker, a member of the CH domain family (for a review, see [36]). Three isoforms of fimbrin, namely I-fimbrin, L-fimbrin, and T-fimbrin, are known to date. In the testis, fimbrin associates with actin and is found in ES-enriched seminiferous tubule fractions [37].

Membrane-Associated Guanylate Kinase Family

Zonula occludens (ZO)-1 and ZO-2 ZO-1 and ZO-2 are 220–225-kDa and 160-kDa adaptors and are members of the membrane-associated guanylate kinase (MAGUK) family (for reviews, see [38, 39]). Although ZO-1, ZO-2, and ZO-3 are known to exist in other epithelia, only ZO-1 and ZO-2 have so far been detected in the seminiferous epithelium [40–43]. Alternative RNA splicing has generated two isoforms of ZO-1, namely ZO-1{alpha}– and ZO-1{alpha}+, and each has an apparent molecular mass of 220 kDa [40]. ZO-1{alpha}–, ZO-1{alpha}+, and ZO-2 are found at the site of Sertoli cell TJ in the seminiferous epithelium [40-43]. Besides, both isoforms of ZO-1 are also detected at the ES site [40, 43]. Although the scaffolding role of these adaptors in the testis has been known for almost two decades, how they affect junction dynamics is virtually unknown.

Paxillin Family

Paxillin Paxillin is a 68–70-kDa focal adhesion-associated adaptor (for a review, see [44]). (Focal adhesion complex or focal contact is an anchoring junction type found between cells and extracellular cell matrices (ECM) using actin filament as an attachment site; for reviews, see [1, 2].) Paxillin, hic-5, and leupaxin are members of the paxillin family (for a review, see [44]). Paxillin has three isoforms: {alpha}-, ß-, and {gamma}-paxillin [45, 46]. {alpha}- and ß-paxillin, but not {gamma}-paxillin, are found in the testis [45, 46]. Paxillin localizes to the site of ES in the seminiferous epithelium [14, 47–49]. It structurally associates with actin, p120ctn, cortactin, FAK, {alpha}6-integrin, ß1-integrin, tubulin, and vinculin in the seminiferous epithelium [48, 49]. It is not known whether focal contact is present in the seminiferous epithelium.

WASP Family

WASP WASP is a 65-kDa protein (for a review, see [50]. N-WASP (neural-WASP) is a homolog of WASP [51]. WASP is expressed mostly in hematopoietic cells, whereas N-WASP is ubiquitously expressed and is also found in the testis [50, 51]. WASP is a product of both Sertoli and germ cells [11]. In the testis, WASP is associated with the N-cadherin/ß-catenin/{alpha}-actinin protein complex (but not the nectin-afadin and the ß1-integrin-based protein complexes) and all three cytoskeletal elements (actin, vimentin, and tubulin) based on studies using coimmunoprecipitation technique [11]. In addition, WASP structurally interacts with c-Src, a PTK, in the testis, illustrating that it may be a putative substrate of this PTK [11]. Further, WASP was shown to structurally interact with two other adaptors, axin and zyxin, in the seminiferous epithelium, using the technique of coimmunoprecipitation [11]. Recent studies have shown that a loss of Sertoli-germ cell adhesive function induced by AF-2364 is associated with a loss of WASP and two other adaptors, zyxin and axin from the ES site in the epithelium [11], further implicating the crucial role of this adaptor in AJ dynamics.

Zyxin Family

Zyxin Zyxin is an 82-kDa adaptor in the zyxin family, which contains at least two other members, lipoma preferred partner and thyroid receptor interacting protein 6 (for a review, see [52]). Zyxin is expressed in Sertoli and germ cells in adult rat testes, but not in 20-day-old germ cells [11]. It is a stage-specific protein in the seminiferous epithelium with highest expression associated with pachytene spermatocytes at stages V-VII of the epithelial cycle in the rat testis [11]. In addition, immunoreactive zyxin was found at the site of basal and apical ES in the testis throughout the epithelial cycle [11]. Structurally, zyxin associates with the N-cadherin/ß-catenin/{alpha}-actinin complex and c-Src [11]. It also interacts with actin, vimentin, and tubulin in the seminiferous tubules [11].

Others

Axin Axin, a product of the Fused locus, is a 150-kDa protein [53]. It is ubiquitously expressed in embryos and adult organs, including the testis [53]. Axin is found in Sertoli and germ cells [11]. It structurally interacts with the N-cadherin/ß-catenin/{alpha}-actinin complexes and the cytoskeletal elements, namely actin, vimentin, and tubulin [11]. Additionally, axin associates with c-Src in the testis [11].

Cortactin Cortactin is an actin-binding protein with an apparent molecular mass of 80–85 kDa [54]. Cortactin is widely expressed in multiple tissues and organs [55]. It is localized to ES in the seminiferous epithelium [48]. In the testis, the known interacting partners of cortactin are actin, N-cadherin, ß-catenin, ERK 2, ß1-integrin, ILK, paxillin, and c-Src [48].

Vinculin Vinculin is a 130-kDa protein found at the site of focal contacts in epithelia, mediating cell-matrix interactions (for a review, see [56]). Yet vinculin is found in the ES-enriched testicular fractions [37]. In the seminiferous epithelium, it is localized with actin to the site of apical and basal ES [37, 47, 57]. Other studies have also colocalized vinculin with ILK [58], pFAK-Tyr397 [14], and espin [59] to the site of ES. Actin, p120ctn, ERK2, {alpha}4-integrin, ß1-integrin, pFAK-Tyr397, and paxillin are vinculin-binding proteins in the testis [14, 48, 49].


   IN VIVO MODELS FOR STUDYING THE ROLE OF ADAPTORS IN JUNCTION DYNAMICS IN THE TESTIS
TOP
 ABSTRACT
 INTRODUCTION
 ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
Several models for studying the role of adaptors in junction restructuring events and barrier function in the seminiferous epithelium are currently available.

AF-2364 Model

AF-2364, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide, is a potential male contraceptive known to perturb the adhesion function between Sertoli and germ cells, especially at the site of ES in the testis [60, 61]. Recent data derived from studies using rats treated with AF-2364 to induce transient infertility have shown that germ cell depletion induced by AF-2364, in particular elongating and elongate spermatids, occurred within hours after treatment of rats with a single dose of AF-2364 [60, 61]. For instance, greater than 50% of the tubules examined had elongating/ elongate spermatids found in their lumen within ~6.5 h after AF-2364 treatment, versus ~3 and ~6.5 days for round spermatid and spermatocytes respectively [62]. The junction types found at the Sertoli-spermatocyte interface are mostly desmosome-like junctions, versus apical ES found in the elongating/elongate spermatid-Sertoli cell interface (for reviews, see [2, 7, 9]). This kinetic study suggests that apical ES is the most susceptible AJ structure to AF-2364 treatment.

In addition, AF-2364-mediated germ cell loss is accompanied by a surge in testin in the rat testis [61], which is a sensitive indicator of AJ disruption [63]. Further, it is not apparent that AF-2364 is cytotoxic to germ cells, because rats treated with AF-2364 had a lag of ~20–40 days before a loss of fertility could be detected. This is attributed to the sperm reserve in the epididymis, which must be exhausted before infertility can be detected by mating studies [60, 61]. If AF-2364 is cytotoxic to germ cells, the response of rats to AF-2364 treatment would have been much more rapid, because a recent pharmacokinetics study showed that [3H]-AF-2364 has a half-life of ~16 h (unpublished results). These recent data, coupled with serum microchemistry analysis results [60, 61], have illustrated a lack of cytotoxicity, at least at doses effective for inducing transient male infertility. Clearly, these results provide compelling evidence that AF-2364 mediates its effects via a disruption of the Sertoli-germ cell adhesion function.

Using this as an in vivo model, the functional role of AJ-associated adaptors and the associated signaling pathways has been investigated (for reviews, see [2, 10]). For instance, it was shown that the protein levels of several AJ adaptors, such as axin, zyxin, and WASP, were reduced during AF-2364-mediated AJ disruption in the testis [11], suggesting that the loss of germ cells from the epithelium is mediated, at least in part, via a loss of adaptors from the AJ site. Alternatively, the loss of germ cells can be induced by other mechanisms, such as one of the several signaling pathways shown in Figure 2, which can in turn induce a loss of adaptors. Nonetheless, these data implicate the importance of adaptors in maintaining junction integrity and functionality, suggesting that a reduction in the levels of adaptors can inevitably lead to dissociation of cell adhesive protein complexes from the underlying cytoskeletons [35].

Using this model, it was shown that AJ dynamics in the testis were regulated by at least two signaling pathways, namely the integrin/Rho B/ROCK/LIMK/cofilin [15] and integrin ß1/pFAK/PI 3-kinase/p130 Cas [14] pathways (see Fig. 2). This conclusion was reached based on studies that used immunoblotting to assess the levels of these proteins and their phosphorylation status [14, 15]; one might dispute whether such activation has any physiological relevance. To address this potential pitfall, we used Y27632, a specific inhibitor of ROCK, to assess whether a blockade of this pathway could indeed delay the AF-2364-mediated loss of elongating/elongate spermatids from the seminiferous epithelium. As expected, Y27632 was shown to significantly delay the AF-2364-induced germ cell loss from the seminiferous epithelium in the rat testis [15]. Recently, Rab 8B GTPase, Csk, and c-Src (Csk and c-Src are putative nonreceptor protein tyrosine kinases that function as signaling molecules at cell junction sites in other epithelia) were shown to be involved in the regulation of the cadherin-based junction dynamics in the testis in both in vitro and in vivo studies [16, 35]. More importantly, Rab 8B was shown to structurally interact with {gamma}-catenin [16], an adaptor for the cadherin/catenin protein complex. More recently, the NO/sGC/cGMP/PKG/ß-catenin pathway was shown to be another putative pathway that regulates N-cadherin-based junctions in the testis (unpublished results). The signaling pathways that are currently known to regulate AJ dynamics are summarized in Figure 2.

In short, these recent findings as summarized in Figure 2 have provided an unprecedented opportunity for investigators in the field to study AJ dynamics in the testis. For instance, inhibitors can now be used to examine any changes in the epithelium during the epithelial cycle when a crucial signaling molecule, such as ROCK, or as a protease, such as MMP-2, is inhibited [13, 15, 35]. On the other hand, the fact that there are multiple pathways used by the testis to regulate AJ dynamics (see Fig. 2) has clearly illustrated that the junction restructuring event in the epithelium during the epithelial cycle is a very complicated biological process. It is obvious that much more research is needed in this area.

Cadmium Chloride Model

Cadmium chloride, an environmental toxicant, has been known for decades to induce infertility in males [64]. Recent studies have shown that this effect may be mediated, at least in part, via BTB damage in the testis [65–67]. In vitro studies have also shown that cadmium chloride can induce Sertoli cell TJ disruption [68, 69]. This model has thus become a valuable tool for studying TJ dynamics in the testis. More importantly, recent studies have shown that a disruption of TJs can lead to a loss of cell adhesion between Sertoli and germ cells [66]. Using this model, recent studies have shown that both BTB and AJ dynamics are regulated by the TGFß3/MEKKs/p38 MAP kinase pathway, and that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function in the seminiferous epithelium, leading to germ cell loss from the epithelium [66, 70]. In addition, it has been shown that proteases and protease inhibitors are crucial to the events of junction restructuring in the testis [13, 66]. The drawback of this model is that the cadmium chloride-induced TJ disruption in the testis is irreversible, making it impossible to study the events and regulation of TJ reassembly; yet the TJ reassembly event is crucial to spermatogenesis. Despite this limitation, this model is useful for studying TJ dynamics, particularly cross-talk between TJs and AJs in the testis.


   ADAPTORS AND JUNCTION DYNAMICS IN THE TESTIS
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 ABSTRACT
 INTRODUCTION
 ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
In the testis, studies of adaptor function at present are limited to defining the role of adaptors in the construction of the multi-protein complexes at the sites of TJs and AJs. Immunoprecipitation studies have shown that ß-catenin and p120ctn are adaptors in the cadherin-based protein complex, whereas vinculin and paxillin are adaptors in the integrin-based protein complex in the seminiferous epithelium [14, 48, 49, 71]. Cortactin is another adaptor found in both the N-cadherin- and integrin-based protein complexes in the testis [48]. Axin, zyxin, and WASP are part of the N-cadherin-based protein complex and are postulated to act as structural linkers in tethering N-cadherin to different underlying cytoskeletons such as the vimentin-based intermediate filament and the microtubule-based network, either directly or indirectly via other adaptors [11, 71], in addition to actin cytoskeleton, which is the principal underlying cytoskeleton for N-cadherin in other epithelia (for reviews, see [72, 73]). But studies using other techniques, such as gene-targeted disruption (e.g., antisense oligos or RNA silencing), are necessary to determine the significance of the above findings, such as whether the elimination of a specific adaptor can indeed lead to the dissolution of a protein complex at the TJ or AJ site. This is an area of research that deserves future attention. Nonetheless, these results have clearly indicated the importance of adaptors for the assembly of multi-protein complexes in the seminiferous epithelium of the rat testis.

Different in vitro and in vivo models have been used to delineate how adaptors participate in the regulation of junction dynamics in the testis. It is known that the protein levels of {alpha}-catenin, ß-catenin, p120ctn, vinculin, and p130Cas are induced during AJ assembly in vitro and AF-2364-mediated AJ disruption in vivo [12, 14, 62, 71]. In addition, the level of p130Cas is also induced during TNF-{alpha}-mediated TJ disruption in vitro [22]. These results suggest a positive role for adaptors in junction dynamics, such that junction restructuring is associated with an activation of adaptors. By contrast, the levels of other adaptors, such as axin, zyxin, and WASP, are reduced during AF-2364-mediated AJ disruption in the testis [11], indicating that loss of adaptors will lead to the disassembly of the protein complexes at AJ sites. Also, the level of paxillin remains relatively stable during AJ assembly between Sertoli and germ cells in vitro and during AF-2364-mediated AJ disruption in vivo [14]. These findings illustrate the versatility of different adaptors in junction restructuring events in the seminiferous epithelium. However, it is not feasible to define the precise mechanism(s) by which adaptors affect junction dynamics based on the limited studies using protein-protein interactions. It is likely that differential binding affinity of adaptors to different structural and/or signaling proteins at the TJ and AJ sites can lead to these divergent results. Besides, some adaptors, such as ß-catenin and p120ctn, also possess signaling properties and can become nucleus bound, activating gene transcription (for reviews, see [74, 75]), so that junction restructurings can induce the activation of these dual function adaptors.

Adaptors are also crucial for mediating cross-talk among different protein complexes at TJs and AJs via their interaction with different protein complexes or with different cytoskeletons. For instance, afadin, {alpha}-catenin, cortactin, and ZO-1 were shown to associate with both AJ- and TJ-based protein complexes (for reviews, see [2, 76, 77]). Also, fimbrin, an actin-binding protein, can interact with vimentin, in addition to actin filament [78]. Thus, adaptors can directly mediate physical linkages between different protein complexes and different cytoskeletal networks. This may be the most crucial function of adaptors in the seminiferous epithelium. For instance, studies using the cadmium chloride and glycerol models have unequivocally demonstrated that a primary loss of TJ function can lead to a secondary loss of AJ function, thereby inducing germ cell loss from the seminiferous epithelium; but a loss of AJ function induced by AF-2364 had no apparent effects on the BTB function [79]. These results suggest that there is cross-talk between TJs and AJs, possibly mediated by adaptors, and this flow of traffic is not necessarily bidirectional. Furthermore, adaptors can be crucial to regulate the opening and closing of AJs and TJs, because a loss of adaptors in one protein complex can induce disassembly of another adaptor-linked protein complex. Also, common adaptors (e.g., p130Cas) found in different protein complexes indicate the possibility that these adaptors shuttle between different protein complexes under different physiological conditions, so that the dissolution of one protein complex can release these adaptors, which can subsequently migrate to another protein complex to stabilize, acting as negative feedback, or to disrupt the functionality of a protein complex. This postulation is highly plausible because it was shown that when Fer kinase, a putative protein tyrosine kinase [62], is dissociated from N-cadherin-based complex, it can migrate to the ß1-integrin-based complex, leading to the dissolution of the integrin-based protein complexes (for a review, see [80]).


   CONCLUDING REMARKS AND FUTURE PERSPECTIVES
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 ABSTRACT
 INTRODUCTION
 ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
REFERENCES
 
Results of recent studies on adaptors, as reviewed briefly herein, have shown that the ES (a testis-specific cell-cell actin-based adherens junction type) in the seminiferous epithelium uses adaptors usually found in focal contacts (a cell-matrix actin-based anchoring junction type) in other epithelia, such as vinculin and p130Cas, for recruiting proteins to the ES site. This may be physiologically significant, because these adaptors are crucial to conferring cell movement at the focal contact sites analogous to the rapid restructuring of the ES during spermiation, as well as for germ cell movement during spermatogenesis. However, the precise mechanism(s) by which adaptors regulate Sertoli cell-elongate spermatid ES and Sertoli-Sertoli TJ dynamics remain to be explored. It is likely that the restructuring of ES that facilitates spermatid movement during spermiation is regulated, at least in part, by a similar mechanism that regulates the movement of fibroblasts and macrophages at the focal contact sites in other epithelia. Although this is a provocative concept, currently available data apparently support such a postulate. For instance, recent studies have shown that GTPases, such as Rab, Cdc42, and Rho, are important molecular switches in multiple epithelia that regulate actin cytoskeleton organization at focal contacts via their effects on actin regulatory proteins, such as cofilin, an actin-severing protein (for reviews, see [81, 82]). Indeed, studies in the past decade from different laboratories have shown that both Sertoli and germ cells in the seminiferous epithelium of rat and mouse testes are equipped with the necessary GTPases and their downstream effectors and regulatory proteins (for a review, see [81]). A recent study has clearly illustrated that the cadherin-based Sertoli-germ cell AJ dynamics are regulated by the intricate interaction among ß-catenin, Cdc42 (a GTPase), and its effector IQGAP1 [133]. Additionally, the use of AF-2364 to induce germ cell loss from the seminiferous epithelium has recently been shown to induce RhoB GTPases, which can in turn activate ROCK and LIM kinase; the net result of this induction activates cofilin. Cofilin cleaves the actin-based filament cytoskeleton, thereby inducing the loss of adhesion function between Sertoli and germ cells [15]. Furthermore, {gamma}-catenin, an adaptor that forms a 1:1 complex with cadherin that in turn constitutes the cadherin/catenin protein complex at the ES, has recently been shown to structurally interact with Rab8B GTPase in Sertoli-germ cell cocultures and seminiferous tubule lysates, as demonstrated by coimmunoprecipitation technique [16]. This intimate protein-protein interaction seems to signify this GTPase may somehow regulate the cell adhesive function of the cadherin/ catenin-based protein complex. Furthermore, a loss of interaction between cadherin and catenin was detected during AF-2364-induced germ cell depletion from the epithelium as demonstrated by coimmunoprecipitation technique [35]. However, it is not known if this loss of protein-protein interaction is mediated by GTPases such as Cdc42, analogous to an in vitro study [133]. Obviously, virtually no report is found in the literature that deals with this area of research, which should be vigorously expanded to define the significance of adaptors, in particular, their interactions with novel molecules, such as GTPases, to regulate junction restructuring in the seminiferous epithelium during spermatogenesis.

Further, adaptors are crucial molecules that link integral membrane proteins at TJ and AJ sites to the underlying cytoskeletons at cell junctions. They also provide docking sites for the recruitment of structural and/or signaling proteins to the TJ and AJ sites. As such, adaptors are important regulatory molecules in junction dynamics in the testis. Studies using in vivo and in vitro (such as Sertoli cell cultures or Sertoli-germ cell cocultures in vitro; for a review, see [83]) models have helped us to gain insights in the regulatory mechanism(s) imposed by adaptors during junction restructuring events pertinent to spermatogenesis.

Briefly, the functional roles of adaptors in the testis are by and large divided into two categories. First, adaptors provide building blocks for the formation of protein complexes at the site of cell adhesion. Second, they regulate junction dynamics in the testis by mediating cross-talk between different protein complexes. Several pathways have recently been identified that regulate AJ and TJ dynamics in the testis, and adaptors are clearly shown to be the crucial molecules in some of these pathways. Obviously, adaptors share important function with other regulatory proteins, such as kinases and phosphatases, in the event of junction restructuring in the seminiferous epithelium. Still, many questions remain. For instance, what are the other regulatory proteins, in addition to kinases and phosphatases, that are involved in the regulation of adaptors in the testis? If these adaptors can mediate cross-talk among different protein complexes, what are the signals that trigger the translocation of adaptors from one junction site to another? Are these factors released from developing germ cells or Sertoli cells? Are these autocrine or paracrine factors? These questions must be answered before a thorough understanding on the role of adaptors in junction dynamics in the seminiferous epithelium is known. This information is also crucial to help investigators in the field to design better functional experiments to study adaptors in the testis.


   FOOTNOTES
 
1 Supported in part by grants from the National Institutes of Health (NICHD, U01 HD45908 to C.Y.C.; U54 HD29990, Project 3 to C.Y.C.), the CONRAD Program (CICCR CIG-96-05B and CIG-01-72 to C.Y.C.), and the Noopolis Foundation. Back

2 Correspondence: C. Yan Cheng, Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733; y-cheng{at}popcbr.rockefeller.edu Back

Received: 5 January 2004.

First decision: 2 February 2004.

Accepted: 16 March 2004.


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 ADAPTORS IN THE TESTIS
 IN VIVO MODELS FOR...
 ADAPTORS AND JUNCTION DYNAMICS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 

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