During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -betas], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the "opening" of the BTB above a migrating preleptotene/leptotene spermatocyte and the "resealing" of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-beta2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-beta2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB.

During spermatogenesis, preleptotene and leptotene spermatocytes must translocate across the blood-testis barrier formed by inter-Sertoli cell-tight junctions (TJs) from the basal compartment of the seminiferous epithelium adjacent to the basement membrane to the adluminal compartment at stages VIII-IX for further development. Because of the close proximity between extracellular matrix (ECM) that constitutes the basement membrane and the blood-testis barrier, we sought to investigate the role of ECM in Sertoli cell TJ dynamics. When Sertoli cells were cultured in vitro to initiate the assembly of the Sertoli cell TJ-permeability barrier, the presence of an anticollagen IV antibody indeed perturbed the barrier. Because ECM is known to maintain a pool of cytokines and TNFalpha has been shown to regulate TJ dynamics in other epithelia, we investigated whether TNFalpha can regulate Sertoli cell TJ function via its effects on collagen alpha3(IV) and other proteins that maintain the homeostasis of ECM. As expected, recombinant TNFalpha perturbed the Sertoli cell TJ-barrier assembly in vitro dose dependently. TNFalpha also inhibited the timely induction of occludin, which is known to associate with the Sertoli cell TJ-barrier assembly. Furthermore, TNFalpha induced the expression of Sertoli cell collagen alpha3(IV), gelatinase B (matrix metalloprotease-9, MMP-9) and tissue inhibitor of metalloproteases-1 but not gelatinase A (matrix metalloprotease-2), and promoted the activation of pro-MMP-9. These results thus suggest that the activated MMP-9 induced by TNFalpha is used to cleave the existing collagen network in the ECM, thereby perturbing the TJ-barrier. This in turn creates a negative feedback that causes TNFalpha to induce collagen alpha3(IV) and tissue inhibitor of metalloproteases-1 expression so as to replenish the collagen network in the disrupted TJ-barrier and limit the activity of MMP-9. Taken collectively, these observations strengthen the notion that ECM is involved in the regulation of junction dynamics in addition to its structural role in the testis.

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Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr 407 and p-FAK-Tyr 397 display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr 407 and Tyr 397 . Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr 407 phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.actin filament network | ectoplasmic specialization

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as ␤1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin⅐laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-␤1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the ␤1-integrin/PI3K/PKB/ERK signaling pathway.In the seminiferous epithelium of the rat testis, Sertoli-germ cell adhesion is maintained by cell-cell actin-based adherens junctions (AJs) 1 and intermediate filament-based desmosomelike junctions (for reviews, see Refs. 1-3). The best studied testis-specific AJ type is ectoplasmic specialization (ES). The ES is confined between Sertoli cells (known as the basal ES) at the site of the blood-testis barrier (BTB) as well as between Sertoli cells and spermatids (known as the apical ES) in the adluminal compartment of the epithelium (for reviews, see Refs. 3 and 4). Most of the studies on cell adhesion function in the testis in the past 2 decades have focused on the apical ES because the biochemical composition of the desmosome-like junction remains largely unexplored in the testis (for reviews, see Refs. 1, 3, and 5). The apical ES is an important anchoring junction device t...