BOR - Papers in Press, published online ahead of print
April 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028225
BIOLOGY OF REPRODUCTION 71, 375–391 (2004)
DOI: 10.1095/biolreprod.104.028225
© 2004 by the Society for the Study of Reproduction, Inc.
Extracellular Matrix: Recent Advances on Its Role in Junction Dynamics in the Seminiferous Epithelium During Spermatogenesis1
Michelle K.Y. Siu, and
C. Yan Cheng2
Population Council, Center for Biomedical Research, New York, New York 10021
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ABSTRACT
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Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis in which one type A1 spermatogonium (diploid, 2n) gives rise to 256 spermatids (haploid, 1n). To accomplish this, developing germ cells, such as preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) entering into the adluminal compartment for further development into round, elongating, and elongate spermatids. Recent studies have shown that the basement membrane in the testis (a modified form of extracellular matrix, ECM) is important to the event of germ cell movement across the BTB because proteins in the ECM were shown to regulate BTB dynamics via the interactions between collagens, proteases, and protease inhibitors, possibly under the regulation of cytokines. While these findings are intriguing, they are not entirely unexpected. For one, the basement membrane in the testis is intimately associated with the BTB, which represents the basolateral region of Sertoli cells. Also, Sertoli cell tight junctions (TJs) that constitute the BTB are present side-by-side with cell-cell actin-based adherens junctions (AJ, such as basal ectoplasmic specialization [ES]) and intermediate filament-based desmosome-like junctions. As such, the relative morphological layout between TJs, AJs, and desmosome-like junctions in the seminiferous epithelium is in sharp contrast to other epithelia where TJs are located at the apical portion of an epithelium or endothelium, furthest away from ECM, to be followed by AJs and desmosomes, which in turn constitute the junctional complex. For another, anchoring junctions between a cell epithelium and ECM found in multiple tissues, also known as focal contacts (or focal adhesion complex, FAC, an actin-based cell-matrix anchoring junction type), are the most efficient junction type that permits rapid junction restructuring to accommodate cell movement. It is therefore physiologically plausible, and perhaps essential, that the testis is using some components of the focal contacts to regulate rapid restructuring of AJs between Sertoli and germ cells when germ cells traverse the seminiferous epithelium. Indeed, recent findings have shown that the apical ES, a testis-specific AJ type in the seminiferous epithelium, is equipped with proteins of FAC to regulate its restructuring. In this review, we provide a timely update on this exciting yet rapidly developing field regarding how the homeostasis of basement membrane in the tunica propria regulates BTB dynamics and spermatogenesis in the testis, as well as a critical review on the molecular architecture and the regulation of ES in the seminiferous epithelium.
kinases, Sertoli cells, signal transduction, spermatogenesis, testis
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INTRODUCTION
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During spermatogenesis, preleptotene and leptotene spermatocytes residing at the basal compartment of the seminiferous epithelium adjacent to the basement membrane must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle, entering the adluminal compartment for further development (Figs. 1 and 2) [1–3]. These primary spermatocytes continue to differentiate into round, elongating, and eventually elongate spermatids while moving to the luminal edge of the epithelium and to be released into the tubule lumen at spermiation [2]. These events of cell movement across the seminiferous epithelium are also associated with extensive remodeling of actin-based cell-cell and cell-matrix anchoring junctions, also known as adherens junctions (AJs) and focal contacts, respectively; as well as intermediate filament-based cell-cell desmosome-like junctions and cell-matrix hemidesmosomes [3]. Ectoplasmic specializations (ES) (Fig. 2) are testis-specific specialized actin-based AJ structures found between Sertoli cells and developing spermatids (namely round, elongating, and elongate spermatids) at the apical sites (apical ES) of the seminiferous epithelium and between adjacent Sertoli cells at the basal compartment (basal ES) at the BTB site [3–5] in the rat testis. The extensive restructuring of basal and apical ES are essential for the movement of spermatids, across the epithelium, and the release of fully developed spermatids (spermatozoa) into the tubule lumen at spermiation [3, 5, 6]. Yet, the mechanism(s) and the molecules that regulate these events remain largely unknown.
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FIG. 1. An electron micrograph showing the cross-section of a seminiferous tubule from an adult rat testis. This micrograph also illustrates the intimate relationship between the seminiferous epithelium (composed of Sertoli cells and germ cells) and the basement membrane (asterisks) of the tunica propria. Underneath the basement membrane is the collagen fibril network (arrowheads), to be followed by the myoid cell layer and the lymphatic structures. GC, Germ cell; SC, Sertoli cell; L, lipid droplet; V, vacuole. The inter-Sertoli cell tight junctions (TJs) in the seminiferous epithelium that create the blood-testis barrier (BTB) physically divides the epithelium into the basal and adluminal compartment (see Fig. 2 for the schematic illustration of the seminiferous epithelium and the relative locations of different junction types). Bar = 2.5 µm
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FIG. 2. A schematic drawing that illustrates the current molecular architecture of tight junctions (TJ) and cell-cell actin-based adherens junctions (AJ), such as ectoplasmic specialization (ES) in the seminiferous epithelium of the testis. This figure also illustrates the relative locations of different junction types in the seminiferous epithelium. TJs and AJs (such as basal ES and basal TBC) between adjacent Sertoli cells create the blood-testis barrier (BTB), which physically divides the seminiferous epithelium into the basal and the adluminal (or apical) compartments. The basal compartment is adjacent to the tunica propria, which is composed of an acellular (basement membrane and type I collagen fibril layer) and a cellular (myoid cell layer and the lymphatic structure) zone. The basement membrane is a modified form of ECM in the testis. This figure also illustrates there are extensive restructuring of TJs and AJs, such as ES, during spermatogenesis when developing germ cells traverse the seminiferous epithelium, moving from the basal to the adluminal compartment. The molecular components of TJs (blue background), AJs (yellow background), and ESs (green background) are shown. Recent studies have shown that the nectin/afadin/ponsin complex and the cadherin/catenin complex are also the constituent proteins of the ES [3]. This schematic drawing clearly illustrates that the timely translocation of developing germ cells across the epithelium must coordinate with the events of junction assembly and disassembly and is crucial for the completion of spermatogenesis. Also, a disruption of either cell adhesive function or the pertinent junction restructuring event at AJs and TJs will lead to a loss of fertility
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Sertoli cell tight junctions (TJs) in the seminiferous epithelium are located adjacent to the basement membrane, a modified form of the extracellular matrix (ECM) in the testis (Fig. 1) [7], coexisting side-by-side with anchoring junctions, such as basal ES and desmosome-like junctions [3– 5]. As such, this relative location between TJs and AJs in the testis is in sharp contrast to other epithelia (Fig. 1). For instance, TJs in the small intestine, the collecting tubule in the kidney, and the epidermis in skin are furthest away from the ECM, located at the apical portion of cells in an epithelium. Below TJ structures are AJs, followed by desmosomes. Altogether, these structures are referred to as the junctional complex, responsible for anchoring cells together, forming an impermeable epithelium. Gap junctions (GJs) are found below this junctional complex, and the epithelial cells in turn attach to ECM via focal contacts or hemidesmosomes [3, 8].
In light of the morphological intimacy between TJs, AJs, and the basement membrane in the testis, it is logical to speculate that ECM plays a crucial role in regulating junction dynamics. Indeed, the significance of the basement membrane in spermatogenesis has been implicated based on observations of some infertile patients. For instance, in patients with aspermatogenesis, abnormal basement membrane structures and immune complexes were detected in the testis [9, 10]. It was postulated that ECM regulated Sertoli cell TJ-barrier function and spermatogenesis via integrins, which in turn transmitted signals to regulate junction dynamics [3, 11]. This review intends to update and provide a current model on how cytokines, such as TNF
, regulate the homeostasis of ECM via the intricate interactions between collagens (ECM components), integrins (transmembrane receptors), adaptors (e.g., vinculin), structural molecules (e.g., occludin), and the associated signaling molecules (such as focal adhesion kinase, FAK). This in turn regulates TJ and AJ dynamics in the testis. Perhaps, most important of all, this review identifies crucial research areas that require vigorous investigation in future studies.
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ABBREVIATIONS USED
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Csk, C-terminal Src kinase; p120ctn, p120 catenin; pFAK, phoshorylated (activated) FAK, also shown are the two proline rich region I (PRI) and PRII and the crucial phosphorylation (P) sites at Y (Tyr) 397 and 576; ILK, integrin-linked kinase; JAM, junctional adhesion molecules; Keap 1, Kelch-like ECH Associating Protein 1; p130Cas, an adaptor protein (note: adaptors are proteins that recruit other proteins to a multiprotein complex to assist a cellular response) encoded by Crkas gene (Crkassociated protein) with a SH3 domain; PI 3-kinase, phosphatidylinositol 3-kinase; PIP2, PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC
, phosphoinositide-specific phospholipase C; Src, a family of proto-oncogenes of the Rous retrovirus which cause sarcoma-like tumors in chickens, there are three most studied Src members, namely, v-Src, c-Src and e-erb, and each encodes a protein that stimulates protein tyrosine kinases or induces phosphorylation of AJ-associated proteins at the site of AJs; other Src family proteins includes Fyn, Yes, Fgr, Lyn, Hck, Lck, Blk and Yrk proteins; ZO-1, zonula occludens-1; ASAP1, ADP ribosylation factor (ARF)-GAP containing SH3, ANK repeats, the PH domain 1; EGFR, epidermal growth factor receptor; GAP, GTPase activating protein; Grb2, growth-factor-receptor-bound protein 2; Grb7, growth-factor-receptor-bound protein 7; LMr-PTP, low molecular weight protein tyrosine phosphatase; Nck-2, small adaptor protein with SH2 and SH3 domains, similar to Crk and Grb2 but its function is not known; PDGFR, platelet-derived growth factor receptor; PSGAP, PH and SH3 domain containing Rho GAP; PTEN, phosphatase and tensin homolog deleted on chromosome 10, it is a protein tyrosine phosphatase displaying homology with tensin and a tumor suppressor gene located on chromosome 10q23; Stat-1, signal transducer and activator of transcription-1; JNK, c-Jun N-terminal kinase; GSK-3, glycogen synthase kinase-3; MMP-9, matrix metalloprotease 9; TIMP-1, tissue inhibitor of metalloproteases-1.
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CELL JUNCTIONS IN THE TESTIS
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The three types of junctions (such as occludin, anchoring and gap junctions) [3, 8] that are found in the seminiferous epithelium of mammalian testes and their relative location are shown in Figure 2. It is noted that anchoring junctions consist of actin-based cell-cell and cell-matrix adherens junction (AJs) and focal contacts, respectively. ES and tubulobulbar complexes are two modified actin-based AJ types unique to the testis [3]. Yet the nature and biochemical composition of the intermediate filament-based cell-cell and cell-matrix desmosomes and hemidesmosomes, respectively, in the testis are largely unknown, let alone their regulation. Furthermore, it is not known if focal contacts are found in the testis.
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THE FUNCTIONAL UNIT THAT CONFERS CELL ADHESION FUNCTION IN THE SEMINIFEROUS EPITHELIUM
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Cell adhesive function that permits the attachment of developing germ cells onto Sertoli cells in the seminiferous epithelium, similar to other epithelia, is conferred by the cell adhesion unit. This unit is composed of three separate entities. First are the transmembrane adhesion receptors, which bind to ECM components or counter receptors on other cells. They also determine the specificity of cell-matrix and cell-cell interactions, which include integrins, cadherins, and nectins [3, 5]. Second are ECM proteins, which are largely glycoproteins that interact with multiple cell surface receptors. Third are intracellular peripheral proteins, which link adhesion receptors to the underlying actin-, intermediate filament- or microtubule-based cytoskeleton [11, 12].
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EXTRACELLULAR MATRIX (ECM)
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At the cell-cell contact sites, the extracellular space is filled with macromolecules, largely glycoproteins and polysaccharides, that constitute the ECM [8], and it is known as the basement membrane in the testis [7].
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THE BASEMENT MEMBRANE
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In rodent testes, tunica propria surrounding each seminiferous tubule are composed of acellular (the basement membrane and type I collagen fibrils) and cellular (the peritubular myoid cells and lymphatic endothelial cells along with fibroblasts) zones (Figs. 1 and 2). The basement membrane, which is in physical contact with the base of Sertoli cells and spermatogonia, is a thin sheet-like structure (0.15 µm thick), largely composed of ECM proteins: type IV collagen, laminin, heparan sulfate proteoglycans, and entactin (Table 1).
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FUNCTIONS OF THE TESTICULAR BASEMENT MEMBRANE
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Maintain Sertoli and Leydig cell function
In vitro studies have shown that ECM plays a crucial role in Sertoli cell function. For instance, ECM affects the morphology and behavior of Sertoli cells. Sertoli cell function, such as differentiation, cell growth, and migration are dependent on the substratum on which cells are attached [7]. ECM also affects Leydig cell proliferation, testosterone production, and gene expression [13, 14].
A Platform for Signal Transduction
The basement membrane is a platform for signal transduction through transmembrane receptors, such as integrins. For instance, the Gs complex of adenylate cyclase and the FSH-induced cAMP response were stimulated when Sertoli cells were cultured on ECM substrates [15]. Moreover, the intracellular [Ca2+] level can also be induced in Sertoli cells (note: Sertoli cell TJ dynamics are known to be modulated by Ca2+ in vitro [16]) cultured on ECM proteins, which can be downregulated by FSH [17, 18]. Also, cAMP has been shown to have a biphasic effect on the Sertoli cell-TJ barrier in vitro [19]. Furthermore, G protein, an important regulator of cAMP, has been localized to the site of TJs in the BTB. As such, Sertoli cell TJ dynamics are modulated by ECM proteins via multiple signaling molecules, such as cAMP.
Maintain the Seminiferous Epithelium Integrity
Modifications of the basement membrane functionality by passive transfer of antibodies raised against seminiferous tubule basement membrane (STBM) [20] or its noncollagenous fraction [21] can cause focal sloughing of the seminiferous epithelium in rats. Laminin, a component of ECM, is known to regulate Sertoli cell TJ-barrier function possibly by providing the functional linkage between ECM and intracellular cytoskeleton [22]. Also, passive immunization of guinea pigs with antilaminin IgG can perturb spermatogenesis, inducing germ cell loss from the epithelium [23].
Defects in the Basement Membrane Can Lead to Infertility
In infertile men, abnormal basement membrane structures and immune complexes can sometimes be detected [9, 10, 24] in their testes. The basement membranes are also thickened in testes of men with cryptorchidism [25], vasectomy [26], and varicoceles [27].
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COLLAGEN IV: A MAJOR ECM COMPONENT IN THE BASEMENT MEMBRANE
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Collagens are ubiquitous structural proteins found in ECM of all mammalian tissues including the testis, with 19 collagen subtypes known to date. Type IV collagen, a network-forming collagen type [28], and laminins are the most abundant building blocks of the basement membrane [7, 8, 29, 30]. Type IV collagen is composed of three chains, which in turn form a triple helical structure. This triple helical molecule forms the building block (monomer) of the collagen network. Six genetically distinct chains, designated 1–6, are known. Each monomer is characterized by a noncollagenous 7S domain (
15 amino acid residues from the N-terminus), a middle collagenous domain (1400 residues of Gly-Xaa-Yaa repeats), and a carboxyl terminal noncollagenous (NC1) domain (230 residues). By different combinations of these monomers, as many as 56 isoforms of triple helical monomer can be assembled. These monomers in turn associate with each other to form a suprastructure. The carboxyl terminal NC1 domains of the monomers associate with others to form dimers and the amino terminal 7S domain also associates with others to form spiderlike tetramers [31, 32].
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EXPRESSION AND LOCALIZATION OF COLLAGEN IV IN THE TESTIS
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The tissue distribution of 1(IV) and 2(IV) chains is ubiquitous, whereas 3(IV), 4(IV), 5(IV), and 6(IV) chains have restricted tissue distribution. 1(IV)–5(IV) chains are found in rodent testes [33–36]. In bovine testes, the basement membrane is constituted largely by 3(IV) and 4(IV) chains, which accounts for 80% of the collagen chains instead of the 1(IV) and 2(IV) chains found in nongonadal basement membrane [37]. In rodent testes, the expression of 3(IV) chain peaked at 10–20 days after birth, coinciding with the BTB assembly at 13 days postnatal [34, 38]. 3(IV) and 4(IV) chains are colocalized to the basement membrane of seminiferous tubules, myoid cells, and tunica albuginea in 15-day-old rats [35]. 1(IV) and 2(IV) chains are products of Sertoli and myoid cells [33, 39, 40]. 3(IV) is a product of Sertoli and germ cells in the rat [38].
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FUNCTIONS OF COLLAGEN IV IN THE BASEMENT MEMBRANE
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From Simple Scaffolding to Signaling Function: The Involvement of NC1 Fragments
Collagen, a scaffolding protein in ECM, can take part in signal transduction via transmembrane receptors, such as integrins [8, 41]. Of the 24 known integrin receptors, five integrins, namely 1ß1, 2ß1, 3ß1, 10ß1, and 11ß1, are putative collagen receptors. Different collagen subtypes can also be recognized by specific cell surface collagen receptors. For instance, the basement membrane type IV collagen can be recognized by 1ß1 and 2ß1 integrins [42, 43]. It is likely that additional collagen cell surface receptors will be identified [44]. Recent studies have shown that NC1 fragments of collagen generated by limited proteolysis are physiologically active peptides with functions different from that of intact collagens. For instance, it was shown that NC1 fragments inhibited angiogenesis and tumorigenesis [32]. Furthermore, the NC1 domain of collagen IV (e.g., 3NC1(IV), also called tumstatin), also takes part in the regulation of adhesion, proliferation, and apoptosis in various cell types via their interactions with integrins, such as 6ß1 and vß3 integrins [32]. These recent findings have clearly illustrated the signaling function of collagen IV.
Role of Collagen IV in Junction Dynamics
A mixture of type I and III collagens has been shown to enhance the sealing capacity of TJs in A6 cells in vitro, a kidney epithelial cell line, by inducing phosphorylation of ZO-1, an adaptor at the TJ site, during TJ assembly [45]. Collagen IV is also known to stimulate occludin expression in human brain endothelial cells [46]. These results thus implicate the significance of collagens in TJ dynamics. However, it is not certain if these reported effects are mediated by other molecules, such as cytokines, that were trapped in the collagen network.
It was recently shown that an antibody against collagen IV could perturb Sertoli cell TJ-barrier in vitro [38]. While the underlying mechanism is presently not known, subsequent studies have shown that this is likely the result of an intricate interaction between TNF, collagen 3(IV), MMP-9, and TIMP-1 [38] (Fig. 4). What remains to be determined in this model (Fig. 4) is what triggers the production of TNF when BTB needs to be opened to the migrating germ cells at late stage VIII and early stage IX of the epithelial cycle. Would that be preleptotene and leptotene spermatocytes?
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ECM HOMEOSTASIS IS REGULATED BY THE SYNERGISTIC ACTION OF MMPs AND TIMPs
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ECM remodeling is critical for developmental processes, inflammation, tumor invasion, metastasis, cell movement, and Sertoli cell TJ-barrier function, which are largely regulated by the synchronized activity between proteases, such as MMPs, and proteases inhibitors, such as TIMPs [38, 47].
MMPs
About 25 MMPs are found in mammalian cells. Each has distinct but often overlapping substrate specificities and can cleave multiple substrates, which include other proteases, protease inhibitors, latent growth factors, growth factor-binding proteins, cell adhesion molecules, cell surface receptors, and virtually all structural ECM proteins [47, 48]. Most MMP genes are inducible, such as by cytokines [49]. Moreover, MMPs can also be regulated by their own substrates via a negative feedback mechanism. For instance, MMP-1 can be induced by intact type I collagen via discoidin domain-containing receptor-like tyrosine kinases but inhibited by its cleaved collagen fragments [50] which function as biologically-active peptides.
TIMPs
TIMPs (20- to 29-kDa single-chained polypeptides) are major endogenous metalloprotease inhibitors in ECM. To date, four TIMPs (TIMP-1, 2, 3, and 4) have been identified. They inhibit MMPs reversibly in a 1:1 stoichiometric ratio by limiting the activation of latent MMPs and/or inhibiting the activities of activated MMPs [47]. Different TIMPs inhibit different MMPs [51].
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MMPs AND TIMPs IN THE TESTIS
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Among the 25 different MMPs known to date, 10 are found in the testis (Table 2). MMP-2 is the most extensively studied MMP in the testis. Both its pro- (72-kDa) and active (62-kDa) forms are detected in Sertoli cell cultures [52]. The activation of MMP-2 in the testis involves MT1-MMP and TIMP-2 [53], all of which are localized to the apical ES in the rat testis [53]. Furthermore, there is evidence that the expression and/or activation of MMP-2 and TIMP-2, but not MT1-MMP, can be induced by FSH in Sertoli cells in vitro [52, 53]. (MT1-MMP is a membrane-bound protease; when it forms a complex with TIMP-2, it acts as a receptor for pro-MMP-2 and is crucial for MMP2 activation.) In addition to Sertoli cells, peritubular myoid and Leydig cells also contribute to the pool of MMP-2, MT1-MMP, and TIMP-2 in the testis [52–54]. Germ cells also contribute to the level of MT1-MMP, but not MMP-2, in the testis [53]. Another gelatinase, MMP-9 (92 and 84 kDa for the pro and active forms, respectively), is also a putative Sertoli (but not myoid) cell product. Unlike MMP-2 and TIMP-2, MMP-9 is not stimulated by FSH [52]. TIMP 1, 2, 3, and 4 have also been identified in the testis [54–57]. For instance, TIMP-1 is a product of Sertoli, myoid, and germ (but not Leydig) cells and residual bodies. Like MMP-2 and TIMP-2, TIMP-1 expression can be induced by FSH in vivo and in vitro. Treatment of Sertoli cells with IL-1, cAMP, and germ cell residual bodies can also induce TIMP-1 expression [56]. Because MMPs and TIMPs are known regulators of tissue restructuring, recent findings described here imply that MMPs and TIMPs are possibly involved in junction restructuring during spermatogenesis [53, 58–60] through a yet-to-be defined pathway(s).
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ROLES OF ECM PROTEOLYSIS IN JUNCTION DYNAMICS
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ECM remodeling and cell surface proteolysis induced by MMPs are involved in the regulation of i) cell-matrix and cell-cell adhesion; ii) the release, activation, or inactivation of autocrine or paracrine signaling molecules; and iii) the activation or inactivation of cell surface receptors (Fig. 3). The net result can affect junction dynamics [47, 48]. Figure 3 depicts the three mechanisms by which proteolysis of ECM proteins (Fig. 3, A and B) and cell surface adhesion molecules, such as cadherins (Fig. 3C), can affect junction dynamics in the testis. Indeed, recent studies have shown that MMP2, MT1-MMP, and TIMP-2 are colocalized to the same site at apical ES in the rat testis and structurally interact with the integrin/laminin (but not the cadherin/catenin or the nectin/afadin) complexes [61], suggesting that MMPs are crucial to integrin/laminin-based ES dynamics.
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FIG. 3. A schematic drawing that illustrates the three possible pathways by which ECM-mediated proteolysis can affect junction dynamics. A) ECM remodeling directly alters cell-matrix interactions via integrin-mediated signaling. Furthermore, proteolysis of ECM proteins results in the release of biologically active ECM fragments, which can in turn activate integrin-mediated signaling different from that induced by intact molecules. B) ECM molecules also act as binding reservoirs for various growth factors and cytokines, such as TGF-ß and TNF, which are released once ECM molecules are cleaved. The growth factors can then bind to their cell surface receptors to initiate downstream signaling events. It is of note that TGF-ß3 in the rat testis has been shown to regulate the dynamics of Sertoli cell TJs via the p38 MAP kinase pathway. C) Cell adhesion proteins, such as cadherins and syndecans, can also be degraded by ECM proteases, resulting in the disruption of adherens junctions and alteration of signal transduction and is accompanied by dissociation of adaptors and peripheral signaling molecules from the cadherin/catenin protein complex
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It is of interest to note that plasmin activated by urokinase plasminogen activator (uPA) following cleavage of plasminogen can in turn become an activator of pro-MMPs in vivo [62]. Plasminogen is synthesized by seminiferous tubules in rats [58] and uPA is also a stage-specific Sertoli cell product, being highest at stages VII–VIII [63]. Furthermore, an induction in uPA is associated with Sertoli cell TJ assembly in vitro [60]. Thus, uPA may take part in the regulation of germ cell movement and spermiation by activating MMPs via plasmin.
Furthermore, claudin-1, -2, -3, and -5 (all are TJ-integral membrane proteins) can facilitate the activation of pro-MMP-2 mediated by MT1-MMP in human embryonic kidney 293T cells. While the mechanism of this activation remains to be elucidated, it is likely that physical interactions between claudins and MT1-MMP promotes MMP-2 activation, thereby changing the vascular permeability [64]. Although claudin-1, -3, and -5 are found in the testis by Northern blots, their localization at the TJ sites in the testis has yet to be reported [3].
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REGULATION OF ECM DYNAMICS BY CYTOKINES
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ECM proteins, MMPs and TIMPs, are apparently regulated by cytokines [47, 49]. For instance, TGFß1 has been shown to regulate the expression of collagen, MMPs, and TIMPs in human mesangial cells [65]. TNF was also shown to regulate collagen and MMPs in rat cardiac fibroblasts and rat Sertoli cells cultured in vitro and also in transgenic mice with cardiac-specific overexpression of TNF [38, 66, 67].
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TNF: ITS ROLE IN JUNCTION DYNAMICS
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TNF (17 kDa) is produced largely by activated monocytes and macrophages in the systemic circulation and plays a crucial role in inflammation, cell proliferation, antiviral responses, and junction dynamics [68, 69].
Expression, Production, and Function of TNF in the Testis
Germ cells and testicular macrophages are the sources of TNF in the testis. A recent study has reported that Sertoli cells also produce TNF [38]. Rats receiving chronic administration of TNF via intravenous infusion displayed massive germ cell loss from the seminiferous epithelium, in particular, spermatocytes and spermatids but not spermatogonia; testicular weight reduced significantly within 24 h, coupled with a plunge in plasma testosterone and a surge in LH and FSH levels [70]. It was suggested that the TNF-induced germ cell loss might be either a direct effect on germ cells or mediated through dysfunctional Leydig and/or Sertoli cells [70]. The precise mechanism that mediates these TNF-induced effects in the testis is not known. For instance, it remains to be determined if the integrity of BTB and AJs are compromised [70, 71]. And if it is, can the damaged BTB barrier be reassembled? Once these questions are addressed, this can become a novel model to study BTB dynamics. Nonetheless, other studies have shown that TNF may have a direct effect on germ cells by reducing the level of Fas ligand (note: Fas ligand is a ligand of the Fas antigen, which is a 35-kDa transmembrane receptor of the TNF receptor family) that modulates apoptosis [72, 73].
TNF and Junction Dynamics
There are several reports implicating TNF plays a crucial role in junction dynamics in both epithelial and endothelial cells [69]. For instance, TNF induces a breakdown of the blood-retinal-barrier in retinal vascular endothelia. It also affects the epithelial barrier function in human intestinal cell lines HT-29/B6 and HT29 cl.19A in vitro. Treatment of human endothelial cells with both TNF and IFN induces redistribution of JAM, a TJ-integral membrane protein. The other two TJ-integral membrane proteins, occludin and claudin-11, have also been shown to be downregulated by TNF in HT-29/B6 [74] and in mouse Sertoli cells [75], respectively. In addition, TNF downregulates E-cadherin and ß-catenin in celiac disease [76]. Furthermore, TNF was shown to perturb the Sertoli cell TJ-barrier function in vitro [38].
TNF Regulates Sertoli Cell TJ Dynamics Via its Effects on the Homeostasis of Collagen 3(IV), MMP-9, and TIMP-1 in ECM
During the assembly of Sertoli cell TJ barrier in vitro, the presence of TNF can perturb the TJ barrier dose dependently. This perturbing effect appears to be specific because the disrupted barrier can be resealed when TNF is removed. More important, the disruption of the Sertoli cell TJ barrier induced by TNF is associated with an induction in Sertoli cell collagen 3(IV), MMP-9, and TIMP-1, but not MMP-2, production. TNF also promotes the activation of pro-MMP-9. Collectively, these results seemingly suggest that the activated MMP-9 induced by TNF is being used to cleave the existing collagen network in the ECM, thereby perturbing the Sertoli cell TJ-permeability barrier (note: the presence of a collagen antibody in these Sertoli cell cultures can indeed perturb the TJ barrier, illustrating the significance of ECM integrity and the role of collagen on the TJ-barrier function). This in turn creates a negative feedback that causes TNF to induce the production of collagen 3(IV) and TIMP-1 by Sertoli cells so as to replenish the collagen network in the disrupted TJ barrier and limit MMP-9 activity (Fig. 4) [38]. This speculation is not unprecedented. For instance, fragments of type I collagen are known to induce rapid disassembly of FAC in smooth muscle cells, which is mediated via the integrin-dependent cleavage of FAK, paxillin, and talin [15, 38]. Moreover, these biologically active fragments from collagen can have a negative feedback effect that inhibits the production of collagenase and degradation of collagen [50]. As such, an induction of collagen 3(IV) and TIMP-1 when TNF perturbs the Sertoli cell TJ barrier may be a negative feedback mediated by the biologically active fragments, which are the cleavage products of the TNF-induced MMP-9 on collagen 3(IV). Equally important, TNF apparently exerts its effects on the ECM homeostasis via the ß1-integrin/ILK/ GSK-3ß/JNK signaling pathway [38], the downstream of such activation perhaps also reduces the production of occludin by Sertoli cells, thereby perturbing the TJ-barrier function in vitro (see Fig. 4) [38]. As such, TNF can affect Sertoli cell TJ dynamics either by inducing the production of MMP and TIMP, reducing the production of occludin, or both [38] (see Fig. 4), suggesting it can mediate its effects on TJ dynamics in the seminiferous epithelium via either one of the three separate yet interacting pathways. The current model of action of TNF on Sertoli cell TJ dynamics shown in Figure 4 also illustrates several potential candidates that can be targeted for male contraceptive development. For instance, if the downstream signaling event of the TNF is blocked, such as the function of GSK-3ß, this will induce a temporary BTB shutdown, denying the access of preleptotene and leptotene spermatocytes to traverse the BTB, disrupting spermatogenesis. The net result is transient infertility. It is obvious that these results must be vigorously validated in future studies as follows. First, can synthetic peptides based on the NC1 domains of collagen 3(IV) affect the production of MMP-9 (or activation of pro-MMP-9 to MMP-9) and/or TIMP-1 by Sertoli cells? Second, can the synthetic peptide(s) that has (have) a biological effect on Sertoli cell MMP-9 and/or TIMP-1 production indeed regulate the TJ-barrier function? Third, can a blockade of the GSK or JNK signaling pathway by a specific inhibitor abolish the effects of TNF on the Sertoli and/or germ cell function? Once these questions are answered, more practical research can be conducted, such as synthesizing specific inhibitors to block ILK or GSK in the TNF-integrin-JNK signaling pathway (see Fig. 4).
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INTEGRINS: TRANSMEMBRANE RECEPTORS OF THE ECM
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Integrins are members of a large family of cell-surface receptor glycoproteins, which together with superfamilies of cadherins, immunoglobulin cell adhesion molecules (Ig-CAM), and selectins, constitute four major categories of cell adhesion receptors for mediating cell adhesions and signaling. Integrins integrate ECM proteins or counterreceptors on neighboring cells with the intracellular cytoskeleton networks, which in turn regulates cell-matrix and cell-cell interactions at the corresponding sites, known as FAs and AJs [11, 77].
Integrins are heterodimers composed of noncovalently linked and ß subunits. In mammals, there are 18 and 8 ß subunits that can heterodimerize in various combinations to form at least 24 integrin receptors [11].
Expression and Localization of Integrins in the Testis
In the testis, 6ß1 integrin is the most extensively studied integrin receptor. It is colocalized with the actin filament bundles in the rat testis at the sites of basal and apical ES [3, 5]. There is a stage-dependent ß1-integrin expression at the basal ES, with lesser immunoreactive ß1-integrin detected in stages VII–VIII at the time of spermiation [78, 79]. Other than 6ß1 integrin, several integrin subunits are found in the testis. For instance, 3 and 5 integrin subunits are localized to the same sites as of 6ß1 integrin in human testes, whereas, 1, -2, and -4 subunits have been detected in endothelial cells, leukocytes, and basement membranes [80]. In rat Sertoli cell cultures, 3 and 6 integrin subunits, but not the 5, have been detected by [81]. For the ß subunit, a recent study has demonstrated the expression of the ß3 integrin subunit in pig Leydig cells [82].
Integrin Ligand-Binding Function
Integrins act as both signaling acceptors and donors. Ligand binding to integrins induces signal transduction into cells, signals inside cells can also regulate extracellular integrin activities. The known extracellular ligands for integrins include ECM proteins, such as collagen, laminin, fibronectin, vitronectin, and fibrinogen. Some integrin receptors also participate in the regulation of extracellular ligand synthesis. For instance, 1ß1 and 2ß1 integrins have been shown to regulate collagen synthesis, and they can also affect the expression of collagenases and matrix metalloproteases [43]. A recent study has shown that laminin 3 is largely restricted to germ cells, in particular elongating/ elongate spermatids, in the rat seminiferous epithelium and is the putative binding partner of the 6ß1 integrin [61]. It is likely that this 6ß1 integrin/laminin 3 protein complex confers cell adhesive function between Sertoli and developing spermatids in the epithelium.
Signal Transduction Via the Cytoplasmic Tail of Integrins
Integrins not only function as transmembrane linkers, they also act as signaling transducers activating multiple signaling pathways. They mediate signals through cell membranes bidirectionally, including outside-in and inside-out signaling. For outside-in signaling, ligands outside of cells bind to integrins, inducing signal transduction into cells, causing cytoskeleton rearrangement and gene expression. For inside-out signaling, signals within cells transduce via integrins, which in turn induce conformational changes and clustering of integrin, altering the ligand-binding affinity [11, 83, 84]. The cytoplasmic domains of both and ß subunits contribute to the bidirectional signaling process. An array of actin-binding, signaling, and adaptor proteins can interact with these cytoplasmic domains to induce integrin-mediated signaling (Fig. 2) [11, 85]. Very few integrin binding proteins have been found in the testis to date. Those that are known to be present in the testis include actin-binding proteins (e.g., talin, -actinin and F-actin), signaling molecules (e.g., FAK and ILK) and adaptors (e.g., paxillin) [78, 86, 87]. Figure 2 depicts the most current molecular architecture of the integrin/laminin complex in the testis at the ES site.
Integrins and Junction Dynamics
Integrin receptors act as the bridge that links extracellular ligands to the intracellular cytoskeleton network, activating multiple signaling pathways for the control of cell matrix and cell-cell adhesion function, cell migration, gene expression, cell cycle progression, and cell survival [11, 77, 83, 84].
Actin Reorganization and Adhesion Complex Remodeling Contribute to Cell Migration
Integrins are the major transmembrane receptors at the sites of cell adhesion to ECM. ECM in turn is linked to the actin cytoskeleton via peripheral proteins. These specialized attachment and signaling complexes are known as focal adhesions (FAs) or focal contacts [77, 83, 88]. Over 50 proteins have been identified as putative FA components. These include cytoskeleton proteins (e.g., talin, -actinin, vinculin, and paxillin), tyrosine kinases (e.g., FAK, PI 3-kinase), serine/threonine kinases (e.g., ILK), tyrosine phosphatases (e.g., SHP-2), and modulators of GTPases (e.g., Graf and ASAP1) [88]. Integrins are linked to the underlying actin network via their interactions with actin-binding proteins such as talin, -actinin, and vinculin [89]. Additionally, integrin-mediated activation of Rho GTPases also affects the actin cytoskeleton [11, 77, 89]. The FAC remodeling is also regulated by integrin-mediated activation of the FAK signaling pathway [11, 77, 89]. The intricate relationship between actin dynamics and FAC remodeling in turn contributes to cell movement.
Activation of MAP Kinase, Lipase Kinases, and Other Proteins to Regulate Gene Expression, Cell Cycle, and Cell Survival
Integrin-mediated activation of the Ras family GTPase-extracellular signal regulated kinase (ERK) pathway is the key regulator of gene expression and cell cycle progression [83]. Integrins also play an important role in cell survival, which is mediated by the PI 3-kinase-Akt kinase pathway [83].
Role of Integrins on Junction Dynamics: Regulation of AJ Dynamics at the Site of ES
Although the roles of integrins in mediating cell-matrix adhesion have been extensively investigated, how integrins affect junction dynamics remains unclear. ß1-integrin is the first integrin subunit found at the keratinocyte cell-cell contact site in the 1990's [90]. Subsequent studies have shown that the 6ß1 integrin is the putative cell adhesion receptor at the ES site that mediates Sertoli cell-elongating/elongate spermatid adhesion [78, 79]. Recent studies have shown that ß1-integrin indeed is involved in ES dynamics, using the FAK signaling pathway [91]. Besides, RhoB GTPase is also involved in AJ dynamics in the testis via the integrin/ RhoB/ROCK/LIM kinase/cofilin signaling pathway [92]. Because direct integrin/integrin interaction is not likely to mediate Sertoli-germ cell adhesion function and germ cells do not express ß1 integrin [93], a binding partner(s) for integrins at the ES must exist. Indeed, a recent study has shown that laminin 3 is a germ cell product, which can form a bona fide complex with ß1-integrin at the site of apical ES [61]. However, the identities of the laminin and ß chains that form a functional laminin receptor with the 3 chain are not known. In this context, it is of interest to note that, during sperm-egg fusion, the 6ß1 integrin on the egg surface also acts as a receptor for sperm fertilin-ß (also known as ADAM2, a membrane protein with a disintegrin and metalloprotease domain) [94].
Integrin–/– Mice
Null mice have been generated for 13 of the 18 subunits and 7 of the 8 ß subunits. Each deleted gene exhibits unique phenotypic changes [95]. Table 3 summarizes effects of the integrin subunit gene deletion on mouse development. Only those integrin subunits that are found in the testis are shown.
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FOCAL ADHESION KINASE (FAK)
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FAK is a nonreceptor protein tyrosine kinase (PTK) found in the cytoplasm of mammalian cells that integrates signals in response to extracellular stimuli, such as integrin clustering, and cytokines. Its activation also regulates cell-matrix adhesions, cell migration, cell cycle, and apoptosis [77, 96–99]. FAK is distinct from other nonreceptor PTK. First, it lacks the Src homology2 (SH2) and SH3 domains for protein-protein interactions. Second, it also lacks a myristylation site, which is used to anchor proteins to cell membrane [77, 98]. FAK is composed of a central catalytic domain sandwiched between an N- and a C-terminal domains. The C-terminal domain has a focal adhesion targeting (FAT) sequence responsible for targeting FAK to the focal adhesion site (Fig. 5) [77, 96–100]. Table 4 summarizes recent findings regarding the significance of FAK based on studies of multiple epithelia including the seminiferous epithelium of the rat testis.
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FIG. 5. A schematic drawing that illustrates the association of FAK with proteins at the site of cell-matrix anchoring junctions applicable to apical ES. An FAK molecule is composed of three separate functional domains, namely FERM, kinase, and FAT. FERM (band 4.1, ezrin, radixin, and moesin) is the amino terminal domain, whereas FAT (focal adhesion targeting) is the carboxyl terminal domain containing two proline-rich regions, PRI and PRII. The putative Tyr (Y) phosphorylation site at residue 397 from the N-terminus in FERM is also shown. The catalytic kinase domain is located in the center. Following activation of FAK, the downstream biological effects include changes in cell migration, cell growth, and apoptosis. The pFAK also plays a crucial role in the regulation of Sertoli cell TJ dynamics [91] (see also Fig. 4)
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Activation of FAK Can Mediate Cell Adhesion and Migration
FAK has six putative phosphorylation sites at Tyr-397, -407, -576, -577, -861, and -925 [98], yet Tyr397 is a critical and apparently the only autophosphorylation site. It has been suggested that the interaction of FAK with ß1-integrin can induce conformational changes in FAK, exposing the Tyr397 site [98, 101]. The autophosphorylation at Tyr397 creates a high affinity-binding site for numerous molecules, including Src family kinases [102], effectors, such as the p85 subunit of phosphatidylinositol (PI)-3-kinase [103], and phospholipase C (PLC)- [104], and adaptors, such as growth-factor-receptor-bound protein (Grb)7 [105] and Nck-2 [106]. It is unlikely that these proteins can have access to the Tyr397 site simultaneously, suggesting cell-type-specific FAK-associated complexes are present in FAs [88]. FAK also acts as a scaffolding protein and a signal inducer in FAs, regulating cell adhesion and migration through its interaction with different binding partners [77, 89, 97–99]. The FAK-mediated effects on cell movement in epithelia are summarized in Table 4.
The Phosphorylation of FAK at TYR925 is Crucial to Cell Growth and Apoptosis
Except for the Tyr397 autophosphorylated site, the remaining tyrosine phosphorylation sites in FAK are targets of the Src family protein kinases. Phosphorylation of FAK at Tyr576 and Tyr577 in the kinase domain maximizes FAK catalytic activity [107]. Whereas the phosphorylation at Tyr925 generates a binding site for the adaptor protein Grb2, which links FAK to Sos, the guanine nucleotide exchange factor (GEF) of Ras, to induce the downstream ERK/JNK MAPK signaling events [108], contributing to the FAK-mediated cell growth and cell survival [11, 77, 97, 98].
Expression and Localization of FAK in the Testis
FAK was first identified in the early 1990's and was shown to be abundantly expressed in adult rat testes during development [109]. Studies by RNase protection assay have revealed that FAK is also expressed in a variety of adult tissues, but the expression in nongonadal organs is at least 20- to 100-fold less than that of the testis [110]. FAK was also shown to be highly expressed in the brain and in osteoclasts [98]. FAK is largely restricted to the basal compartment in the seminiferous epithelium between Sertoli and germ cells in all stages of the cycle, with very weak signal being detected at the apical compartment [78, 91]. Yet intensive p-FAK-Tyr397 and -Tyr576 was shown to associate with apical ES at stages VII through early VIII of the epithelial cycle [91].
Potential Roles of FAK in Junction Dynamics: its Phosphorylation is Crucial to ES Dynamics
FAK is a focal adhesion complex (FAC)-associated protein localized at the cell-matrix contact sites in many epithelia. FAK is implicated in the development and/or maintenance of the myotendinous junction (MTJ) via integrin-mediated signaling. MTJ is characterized by deep membrane invaginations where tendon microfibrils attach to the salcolemma, side-by-side with FA components [111]. ES is a putative cell-cell actin-based AJ-type unique to the testis, yet many proteins recently found at the ES site are components of FAs. These include ß1-integrin, vinculin, c-Src, Csk, ILK, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), PLC, Fyn, and Keap1 in addition to FAK [78, 86, 112–114]. These findings suggest that ES shares some common features of the cell-matrix anchoring junction regarding its biochemical composition and possibly regulation.
In the testis, intensive phospho-Tyr staining was detected at the ES site [78, 86], indicating Tyr phosphorylation of ES proteins are crucial to junction restructuring events at spermiation. Indeed, pFAK was concentrated at the site of apical ES and mostly at stages VII through early stage VIII (but not at late stage VIII, when spermiation occurs) [91]. Furthermore, pFAK is a potential linker for ß1-integrin to recruit proteins to the site of apical ES during its remodeling [91]. A recent study has suggested that Rho GTPases may be the downstream signal of FAK for mediating cell adhesion and migration [115]. Remaining to be investigated are the interactions of FAK and Rho in the regulation of ES dynamics.
FAK Null Mice
In FAK–/– mice, virtually all embryos died Day 8.5 postcoitus, similar to fibronectin–/– or 5-integrin–/– null mice, displaying mesodermal defects during gastrulation [116–118]. These results thus illustrate the significance of FAK.
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VINCULIN
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Vinculin is a ubiquitous actin-binding protein found in cell-cell and cell-matrix anchoring junctions, involved in junction assembly, stabilization, and regulation [3, 119]. Vinculin and ß1-integrin do not interact with each other directly but through talin, -actinin, or paxillin that act as a bridging protein. Furthermore, vinculin is also a putative substrate of protein kinases [120]. As such, ß1-integrin-mediated cytoskeleton regulation may use vinculin to exert its effects on AJs and FAs [77, 121].
Structure of Vinculin and its Associated Proteins
Structurally, vinculin is characterized by a 90-kDa globular head, with a long rodlike 30-kDa tail, connected through a proline-rich neck region. Vinculin is a putative actin-binding protein, yet its actin binding ability is dependent on the conformational change from a closed (inactive) to an open (active) state [88, 119]. In the inactive state, the intramolecular interactions between the head and tail of vinculin mask the binding sites for i) -actinin and talin in the head region, ii) VASP (vasodilator-stimulated phosphoprotein, a 45–50 kDa protein and a substrate of cAMP- and cGMP-dependent protein kinases) in the proline-rich neck, and iii) F-actin in the tail, while the binding of paxillin to its tail is apparently unaffected. This inhibitory interaction can be disrupted by the binding of PtdIns(4,5)P2 to the tail of vinculin [88, 122].
Expression and Localization of Vinculin in the Testis
Vinculin colocalizes with actin at the site of ES [78, 91, 123]. Besides ES, vinculin is also found in the tubulobulbar complexes and in myoid and small muscle cells of the blood vessels in the interstitium [78, 123]. While the precise role of vinculin in ES dynamics is not entirely clear, recent studies have shown that vinculin colocalizes with p-FAK to the same site in the ES in the rat testis [91]. Also, an induction of vinculin was detected, coinciding with the assembly of Sertoli-germ cell AJs in vitro [91].
Potential Roles of Vinculin in Junction Dynamics
Vinculin is a crucial regulator of the cadherin/catenin complex either by interacting or even substituting -catenin at AJs. -Catenin per se is an adaptor linking the cadherin/ catenin complex to the actin cytoskeleton [124]. -Catenin has three regional sequence homologies with vinculin, and both proteins can bind to -actinin and actin. Unlike vinculin, -catenin is restricted to cell-cell AJs but not cell-matrix FAs [124]. Yet vinculin is localized largely to the E-cadherin-catenin complex, but not the N-cadherin-catenin complex at the cell-cell AJ site [124]. Vinculin binds to the SH3 domains of ponsin [125] and vinexin [126] via its proline-rich region. The vinculin-ponsin/vinexin complex has been found in both AJs and FAs. Ponsin is an 1-afadin-binding protein that constitute the nectin-afadin-ponsin (NAP) cell-cell adhesion complex, which is colocalized with the cadherin-catenin complex in selected epithelia, including the ES [125, 127]. Because vinculin and 1-afadin binds to ponsin in a competitive manner, ponsin can only form a binary complex with either vinculin or 1-afadin but not a vinculin-ponsin-afadin tertiary complex [125]. As such, ponsin is not responsible for linking the cadherin-catenin-vinculin complex to the NAP complex. Instead, -catenin in the cadherin-catenin protein complex and 1-afadin in the NAP complex are the molecules that link these two adhesion protein complexes together in epithelia [127, 128]. Because of the affinity of vinculin to both -catenin and ponsin, it is logical to speculate that vinculin is crucial to the cell adhesion function of the cadherin/catenin and the NAP protein complexes; however, this exciting possibility remains to be confirmed in the ES.
Vinculin Null Mice
Vinculin–/– embryos died at Day 10 postcoitus, displaying heart and brain defects [129]. Fibroblasts isolated from vinculin–/– embryos were devoid of cell adhesion function with higher cell migration rate. Furthermore, vinculin null cells have enriched FAK activity [129], raising a controversy regarding a correlation between enhanced FAK activity and a decline in cell migration activity. As such, the significance of FAK in vinculin–/– cells remains to be investigated. Overexpression of vinculin in Balb/c 3T3 cells resulted in an increase in the number and the size of FAs, which also associated with reduced cell motility [130]. Also, vinculin suppression leads to increased cell migration with fewer and smaller FAs [131].
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CONCLUDING REMARKS AND FUTURE PERSPECTIVES
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In this review, we have summarized recent advances regarding the roles of ECM-associated proteins in regulating junction dynamics in the testis. The most exciting findings are that ECM proteins can indeed affect Sertoli cell TJ dynamics via the interactions between cytokines (e.g., TNF-), proteases (e.g., MMPs), protease inhibitors (e.g., TIMPs), and ECM component proteins, such as collagen [38]. These results also suggest that the basement membrane in the testis serves as a reservoir of uniquely important cytokines to TJ dynamics. Equally important is the finding that the testis consists of and uses some of the same components usually restricted to the FAC site, an actin-based cell-matrix anchoring junction type, in other epithelia, to regulate ES dynamics [78, 91], which is an actin-based AJ type in the testis. While the morphology of ES has been characterized since the late 1970's [132], it is only recently that the biochemical nature of the ES has begun to unfold, in particular its regulation and molecular architecture. The precise cascade of events and the underlying regulatory mechanisms that regulate ES dynamics in the testis remain unknown. For instance, what are the downstream effectors of the activated FAK at the ES that contribute to the changes in ES dynamics during spermatogenesis? If phosphorylation of FAK occurs at or near the site of apical ES, what are positive and negative regulators that determine its phosphorylation status? If the 6ß1 integrin at the site of apical ES confers to cell adhesion function, what is the corresponding binding ligand besides the laminin 3 chain? Once these questions are answered, these results will provide new insights not only into the regulation of ES dynamics but also into the roles of ECM proteins in junction dynamics. These studies will also lead to the discovery of key molecule(s) and signaling pathway(s) that can regulate junction dynamics in vivo, which can, in turn, be used for novel male contraceptive development. For instance, if the pathways that regulate Sertoli cell TJ dynamics are known, the BTB can be manipulated to deny access of preleptotene and leptotene spermatocytes to traverse the BTB at late stage VIII and early stage IX of the epithelial cycle [1], leading to aspermatogenesis.
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FOOTNOTES
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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 96-05-B and CIG 01-72), and the Noopolis Foundation. 
2 Correspondence: C. Yan Cheng, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733;y-Cheng{at}popcbr.rockefeller.edu
Received: 6 February 2004.
First decision: 8 March 2004.
Accepted: 14 April 2004.
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