Connexin 43 is critical to maintain the homeostasis of the blood–testis barrier via its effects on tight junction reassembly

Abstract

In mammalian testes, the blood-testis barrier (BTB) or Sertoli cell barrier created by specialized junctions between Sertoli cells near the basement membrane confers an immunological barrier by sequestering the events of meiotic division and postmeiotic germ cell development from the systemic circulation. The BTB is constituted by coexisting tight junctions (TJs), basal ectoplasmic specializations, desmosomes, and gap junctions. Despite being one of the tightest blood-tissue barriers, the BTB has to restructure cyclically during spermatogenesis. A recent study showed that gap junction protein connexin 43 (Cx43) and desmosome protein plakophilin-2 are working synergistically to modulate the BTB integrity by regulating the distribution of TJ-associated proteins at the Sertoli-Sertoli cell interface. However, the precise role of Cx43 in regulating the cyclical restructuring of junctions remains obscure. In this report, the calcium switch and the bisphenol A (BPA) models were used to induce junction restructuring in primary cultures of Sertoli cells isolated from rat testes that formed a TJ-permeability barrier that mimicked the BTB in vivo. The removal of calcium by EGTA perturbed the Sertoli cell tight junction barrier, but calcium repletion allowed the "resealing" of the disrupted barrier. However, a knockdown of Cx43 in Sertoli cells by RNAi significantly reduced the kinetics of TJ-barrier resealing. These observations were confirmed using the bisphenol A model in which the knockdown of Cx43 by RNAi also perturbed the TJ-barrier reassembly following BPA removal. In summary, Cx43 is crucial for TJ reassembly at the BTB during its cyclic restructuring throughout the seminiferous epithelial cycle of spermatogenesis.bisphenol A | calcium switch | gap junction | seminiferous epithelial cycle | spermatogenesis I n mammalian testes, the blood-testis barrier (BTB) segregates the epithelium of seminiferous tubules into apical and basal compartments (1, 2). Unlike other blood-tissue barriers (e.g., blood-brain barrier, blood-retina barrier) that are constituted by tight junctions (TJs) between endothelial cells of the microvessels at the apical region, the BTB is created by adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. The BTB is constituted by coexisting TJs, basal ectoplasmic specialization (basal ES, a testis-specific adherens junction type), desmosome-like junction (or desmosome-gap junction), and gap junction (GJ) (1-3). Whereas the BTB is one of the tightest blood-tissue barriers, it undergoes cyclical restructuring to accommodate the transit of preleptotene spermatocytes from the basal to the apical compartment during spermatogenesis, so that meiotic divisions and postmeiotic germ cell development (i.e., spermiogenesis) can take place in the apical compartment of the seminiferous epithelium behind this immunological barrier. The immunological barrier conferred by the BTB cannot be compromised, even transiently, during the transit of preleptotene spermatocytes to avoid the product...

Investigation of the Effects of Bisphenol A on the Histology and Ultrastructure of Prostate and Seminal Vesicle Glands in Rats

“…In humans, BPA also reduced sperm concentration, motility and morphology (39). BPA exposure may also induce apoptosis in rat germ cells in vivo (40) and in cultured rat Sertoli cells (41), and has the potential to redistribute several known Sertoli cell junctional proteins (42,43). Subsequent studies also demonstrated that BPA is genotoxic.…”

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis

“…Drebrin has been implicated in the regulation of cell adhesion since it was demonstrated to stabilize connexin 43 (Cx43)-containing GJ plaques, possibly serving as an adaptor of the actin cytoskeleton 49 by recruiting or modulating the activity of other actin regulatory proteins, such as Esp8 and the Arp2/3 complex, which confer actin filament bundling and branching, respectively. 15,[50][51][52][53] Cx43 was shown recently to be involved in junction restructuring at the BTB, particularly in the reassembly of the TJ-barrier following its disruption, 38,54 which occurs during the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. Thus, it was tempting to speculate that drebrin E is participating in these events by recruiting actin regulatory proteins, such as Arp3 to the apical and/or the basal ES since Arp3 was shown to be a binding partner of drebrin E as demonstrated herein.…”

“…Also, at this cell density, ultrastructures of tJ, basal eS, desmosome, and GJ were detected at the Sertoli-Sertoli cell interface under electron microscopy, illustrating functional junctions were established in these cell epithelia as detailed elsewhere. 38,54,77 It was noted that in the control Sertoli cells, F-actin formed an extensive network of filaments, which was used to maintain proper cell shape. Drebrin e was detected at the Sertoli-Sertoli cell interface (see "yellow" arrowheads in middle column) and in the cell cytosol.…”

“…By day 4, these cultures were found to have a functional TJ permeability barrier when assessed by TER measurements across the cell epithelium. 54 Ultrastructural features corresponding to TJs, basal ES, GJs and desmosomes were also visualized by electron microscopy as described. 41 Sertoli cell lysates were obtained by using IP lysis buffer [10 mM Tris, 0.15 Figures 4 and 6, drebrin e (red fluorescence) was found at the Sertoli-Sertoli cell interface and in the cell cytosol, similar to Arp3 (green fluorescence) and some co-localization (see orange-yellow fluorescence in merged images) was found between Arp3 and drebrin e (see yellow arrowheads at the cell-cell interface), consistent with Co-Ip data (Fig.…”

Connexin 43 expression in Sprague-Dawley rat seminiferous epithelium afterin uteroexposure to flutamide

“…In the mammalian testis, immunohistochemistry and hybridization histochemical studies have demonstrated that Cx43 participates in the formation of homocellular gap junctions between adjacent Sertoli cells, and the heterocellular gap junction between Sertoli cells and spermatogonia or spermatocytes [Li et al 2010;Palmiero et al 2003;Tan et al 1996]. With regard to our results, immunostaining for Cx43 showed that the distribution and expression of the Cx43 protein were different in the seminiferous tubule ( Figure 1A-C).…”