Transforming Growth Factor-b 3 Perturbs the Inter-Sertoli Tight Junction Permeability Barrier in Vitro Possibly Mediated via Its Effects on Occludin , Zonula Occludens-1 , and Claudin-11 *

Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of interSertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5–1.2 3 10 cells/cm on Matrigel-coated dishes or bicameral units for 8–9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-b2, and TGF-b3 at different time points were assessed by semiquantitative RT-PCR. In selected experiments, the assembly of inter-Sertoli TJs was monitored by transepithelial electrical resistance measurement. It was found that there was no change in the expression of basic fibroblast growth factor throughout the entire culture period. However, there was a 2-fold reduction in the expression of TGF-b2 and TGF-b3 at the time inter-Sertoli TJs were being assembled. On days 5–8, after the inter-Sertoli TJs had been assembled, the Sertoli cell steady-state messenger RNA levels of TGF-b2 and TGF-b3 increased by as much as 3and 6-fold, respectively, when compared with Sertoli cells on days 1–3 when TJs were being assembled. Also, it was found that recombinant TGF-b3 added to Sertoli cells cultured in vitro at 1.2 3 10 cells/cm on Matrigel-coated bicameral units perturbed the inter-Sertoli TJ permeability barrier dose-dependently. Moreover, the presence of TGF-b3 also inhibited the transient and/or basal expression of several TJ-associated proteins, which include occludin, zonula occludens-1, and claudin-11 when inter-Sertoli TJs were being assembled in vitro. These results suggest that TGF-b plays a crucial role in regulating the complicated biochemical events of junction assembly in the testis. (Endocrinology 142: 1865–1877, 2001) T spermatogenesis, whereas one spermatogonium will differentiate into four spermatids, these developing germ cells must also transverse progressively from the basal to the adluminal compartment of the seminiferous epithelium, where fully developed spermatids (spermatozoa) are released to the tubular lumen at spermiation (for reviews, see Refs. 1 and 2). Moreover, preleptotene spermatocytes must translocate across the inter-Sertoli tight junctions (TJs) that form the blood-testis barrier from the basal to the adluminal compartment of the seminiferous epithelium, on a timely basis. Without this timely movement of germ cells across the seminiferous epithelium, spermatogenesis is disrupted. However, very little attention has been given to this cellular event. Based on recent studies from this and other laboratories, we postulate that continuous, but intermittent, phases of inter-Sertoli and Sertoli-germ cell junction disassembly and reassembly must take place to facilitate the movement of developing germ cells (for review, see Ref. 3). These events apparently require the intricate participation of proteases, protease inhibitors, junctional complex components, signaling molecules, and other biomolecules (4–9). For instance, there are transient inductions in the expression of junctional complex-associated genes, such as zonula occludens-1 (ZO-1), N-cadherin (N-Cad), and connexin 33, when inter-Sertoli cell junctions are being assembled (10). It is conceivable that these changes are part of the event involved in germ cell movement. However, the mechanism that regulates the timely expression of these genes is unknown. Therefore, it is crucial to investigate whether cytokines are involved in regulating the cascade of events leading to junction assembly, because cytokines are implicated in cell movement and cell shape changes (11, 12). More recent studies have shown that interleukin-1 and transforming growth factor (TGF)-a can induce disruption of TJ-permeability barrier, possibly by reducing or altering the distribution of ZO-1 or occludin at the sites of TJs in rat striatal endothelium and mammary epithelium (for review, see Ref. 13), illustrating the crucial role of cytokines