IntroductionA single type A1 spermatogonium (diploid, 2n) in rodents, such as rats and mice, gives rise to 256 mature spermatids (haploid, n) in the seminiferous epithelium during spermatogenesis (for reviews, see de Kretser and Kerr, 1988;Cheng and Mruk, 2002;Lui et al., 2003b). For this event to happen, preleptotene and leptotene spermatocytes must traverse the blood-testis barrier (BTB, also known as the seminiferous epithelium barrier) at late stage VIII and early IX of the epithelial cycle (Russell, 1977). The BTB is constituted largely by inter-Sertoli cell tight junctions (TJ). However, cellcell actin-based adherens junctions (AJ, e.g. ectoplasmic specialization, a testis-specific AJ type) and possibly cell-cell intermediate filament-based desmosome-like junctions (for reviews, see de Kretser and Kerr, 1988;Pelletier, 2001;Cheng and Mruk, 2002), are also found at the BTB site. As such, these junctions must undergo extensive restructuring during this process of germ cell migration. Yet the mechanism(s) that regulates BTB dynamics remains largely unknown (for a review, see Cheng and Mruk, 2002).Recently completed in vitro studies have shown that Sertoli cell TJ dynamics are regulated, at least in part, by transforming growth factor-β3 (TGF-β3) (Lui et al., 2001;Lui et al., 2003a) and tumor necrosis factor-α (TNF-α) (Siu et al., 2003a) via the TGF-β3/MEKKs/p38 mitogen activated protein (MAP) kinase and the TNF-α/integrin-linked kinase (ILK)/p130 Crkassociated substrate (Cas)/MAP kinase signaling pathways, respectively. More importantly, a preliminary in vivo study has shown that the event of BTB disruption induced by cadmium chloride (CdCl2) indeed was mediated via the TGF-β3/MEKKs/p38 MAP kinase pathway (Lui et al., 2003d). Yet it remains to be determined whether proteases, protease inhibitors, AJ integral membrane proteins and their associated peripheral adaptors, and signaling molecules are also involved in TJ restructuring and, in particular, the subsequent germ cell loss from the epithelium as a result of BTB damage. In brief, in this study we sought to investigate whether a primary loss of the BTB function can lead to a secondary disruption of the AJs. We also investigated the roles of proteases and protease inhibitors in this event, since recent in vitro studies have shown that AJ dynamics in the testis are regulated by the intricate interactions between proteases and protease inhibitors under An induction of α α 2-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. α α 2-Macroglobulin was also shown to associate physically with TGF-β β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in jun...

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...

Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.

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