Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study

Abstract

-The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed. testis; spermatogenesis; seminiferous epithelial cycle; blood-testis barrier; endosome; transcytosis; recycling; ectoplasmic specialization; tight junction; c-Yes; c-Src STUDIES HAVE SHOWN that endocytic vesicle-mediated proteintrafficking events that take place in the seminiferous epithelium during the epithelial cycle are crucial to spermatogenesis (7,40,41), similar to their role in regulating cellular events in other tissue barriers, such as membrane traffic (37), autophagy (27), and intracellular cargo trafficking (8). For instance, protein endocytosis, transcytosis, and recycling are necessary for the maintenance of the blood-testis barrier (BTB) integrity during the transport of preleptotene spermatocytes at the BTB that takes places at stage VIII of the epithelial cycle (36,45,46,52). In brief, proteins at the "old" BTB site located above the preleptotene spermatocytes that are connected in clones via intercellular bridges are endocytosed, transcytosed, and recycled to assemble the "new" BTB located beneath these cells (52). Thus, the integrity of the BTB can be maintained during the transport of preleptotene spermatocytes across the barrier without the immunological barrier being compromised (36,52)....

Regulation of Blood-Testis Barrier (BTB) Dynamics, Role of Actin-, and Microtubule-Based Cytoskeletons

“…In fact, the apical ES is an ultrastructure that supports endocytic vesicle-mediated protein trafficking [61], which is being used to support the assembly of newly formed apical ES at the interface of step 8 spermatids and the Sertoli cell in stage VIII tubules. Similar events also take place at the BTB to support the transport of preleptotene spermatocytes across the immunological barrier in which adhesion proteins located above the preleptotene spermatocytes connected in clones via intercellular bridges are also rapidly endocytosed, creating an ultrastructure known as basal TBC [62], which is readily detected by electron microscopy [63]. As such, adhesion proteins (e.g., N-cadherin, occludin, JAM-1, ZO-1) can be rapidly endocytosed at the “old” BTB site located above preleptotene spermatocytes and recycled to the site behind the preleptotene spermatocytes for the assembly of a “new” BTB (Fig.…”

Regulation of blood–testis barrier by actin binding proteins and protein kinases

“…This rapid conversion of actin microfilaments between the bundled and unbundled/branched configuration also supports other intracellular trafficking events including the transport of organelles (e.g., transport of engulfed residual bodies by Sertoli cells near the tubule lumen at stage VIII of the cycle in the apical compartment to the basal compartment while transforming into phagosomes) and endocytic vesicle-mediated trafficking events involved in protein endocytosis, transcytosis and recycling. In fact, the basal ES generates an ultrastructure known as the basal tubulobulbar complex (basal TBC), representing giant endocytic vesicles used for protein trafficking which can be readily detected by fluorescence or electron microscopy (Russell 1979, Xiao, et al 2014c) (for reviews, see (Vogl, et al 2008, Vogl, et al 2013)). As such, proteins at the degenerating “old” BTB above preleptotene spermatocytes connected in clones (Weber and Russell 1987) that are being transported across the barrier can be recycled to assemble the “new” BTB behind these spermatocytes (Smith and Braun 2012, Yan, et al 2008a) (for reviews see (Cheng and Mruk 2010, Mruk and Cheng 2004).…”

The Src non-receptor tyrosine kinase paradigm: New insights into mammalian Sertoli cell biology

“…3). However, it is highest at the tubule lumen at spermiation Wang et al, 2000;Xiao et al, 2014), indicating spermatid detachment involves Srcmediated phosphorylation. The c-Src level also increases during germ cell apoptosis in the seminiferous epithelium of the adult rat testis such as that induced by ethylene glycol monomethyl ether or its active metabolite, methoxyacetic acid (Wang et al, 2000).…”

“…In yet another study performed on Sertoli cells, Xiao et al show a shift in the internalization profiles of biotinylated junctional adhesion molecule A (JAM-A; also known as F11 receptor) and coxsackie virus and adenovirus receptor (CXADR) after knockdown of c-Src by RNAi compared with corresponding controls, leading to the conclusion that c-Src knockdown slows the kinetics of endocytosis (Xiao et al, 2014). The knockdown of c-Src also delays the disappearance of internalized JAM-A from the Sertoli cell cytoplasm.…”

“…Secondly, they also fail to show the accumulation of JAM-A within Sertoli cells after knockdown of cSrc, which would support the delay in its disappearance from the cytoplasm. Thirdly, the knockdown of c-Src affects the actin cytoskeleton (Xiao et al, 2014), suggesting c-Src has an indirect role in protein internalization because a disrupted cytoskeleton network can affect protein trafficking [for reviews, (Qualmann and Kessels, 2002;Qualmann et al, 2000)]. Presently, additional studies are needed to understand better the role of c-Src in protein internalization.…”