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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ABSTRACT
INTRODUCTION