Biologjia Online

When Sertoli and germ cells were cocultured in vitro, anchoring junctions, such as desmosome-like junctions and adherens junctions (AJ) (e.g., ectoplasmic specialization, ES), are formed. This event was marked by an induction of several kinases, such as PI 3-kinase (phosphatidylinositol 3-kinase), phosphorylated protein kinase B (p-PKB), PAK-2 (p21-activated kinase-2), and their downstream effector, ERK as well as an increase in PKB intrinsic activity. Studies by immunohistochemistry indeed localized PI 3-kinase, p-PKB, and PAK to the apical ES in the seminiferous epithelium of the rat testis. Furthermore, PI 3-kinase was also co-localized with p-PKB to the same site at the ES. These kinases were also shown to associate with ES-associated proteins, such as ß1 integrin, p-FAK and c-Src, by co-immunoprecipitation, suggesting that the integrin/laminin protein complex at the apical ES is likely utilized these protein kinases as regulatory proteins to modulate Sertoli-germ cell AJ dynamics via the ERK signaling pathway. To further validate this hypothesis, 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. Similar to the in vitro results, the event of germ cell loss induced by AF-2364 in vivo was associated with an induction of PI 3-kinase, p-PKB, PAK-2, and p-ERK; as well as a surge in intrinsic PAK activity in lysates of testes when spermatids/spermatocytes began to dislodge from the epithelium. Perhaps the most important of all, it was shown that pre-treatment of rats with wortmannin, a PI 3-kinase inhibitor, or an anti-ß1-integrin antibody indeed delayed the AF-2364-induced spermatid loss from the epithelium. In summary, these results have unequivocally demonstrated that the ß1 integrin-PI 3-kinase-PKB-ERK is a putative signaling pathway that regulates Sertoli-germ cell anchoring junction dynamics in the testis.