An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl2. It was shown that the CdCl2-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-β2 and TGF-β3 (but not TGF-β1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-β2/-β3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl2-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl2 mediates its BTB disruptive effects via the TGF-β3/p38 MAP kinase signaling pathway. Besides, this CdCl2-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of α2-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. α2-Macroglobulin was also shown to associate physically with TGF-β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-β3/p-38 MAP kinase pathway also prevented the CdCl2-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on α2-macroglobulin. These results demonstrate for the first time that the TGF-β3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-β3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

The timely restructuring of the blood–testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor α (TNFα), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFα directly to adult rat testes in vivo at 0.5 and 2 μg/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, α-, and β-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFα treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFα by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring (‘opening’?) of the BTB during spermatogenesis to facilitate germ cell migration.

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.

During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the semin

Earlier studies have implicated the significance of transforming growth factor-β3 (TGFβ3) in the regulation of Sertoli cell tight junction (TJ) dynam