TGF-β3 and TNFα perturb blood–testis barrier (BTB) dynamics by accelerating the clathrin-mediated endocytosis of integral membrane proteins: A new concept of BTB regulation during spermatogenesis

Abstract

In adult mammals such as rats, the blood–testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood–tissue barriers. Yet it must “open” transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-β3 (TGF-β3) and tumor necrosis factor α (TNFα) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-β3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-β3 (3 ng/ml) and TNFα (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 μM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-β3-induced protein internalization. This inhibition of TGF-β3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-β3 on protein internalization was further confirmed by RNAi using specific TGF-β receptor I (TβR1) siRNA duplexes. When TβR1 was knocked down, the TGF-β3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-β3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-β3 and TNFα via an enhancement of protein endocytosis at the BTB.