A FAKtual Retelling of Blood-Testis Barrier and Cytoskeletal Regulation.

A commentary on: Huitao Li; Shiwen Liu; Siwen Wu; Renshan Ge; C. Yan Cheng, PhD, “NC1-peptide from collagen α3(IV) chains in the basement membrane of testes regulates spermatogenesis via p-FAK-Y407,” Endocrinology. 2020;161(10):bqaa133. doi:10.1210/endocr/bqaa133

The mammalian blood-testis barrier (BTB) was first characterized in the 19th century when it was shown to prevent intravenously administered dye from entering and staining seminiferous tubules—supporting the notion that the seminiferous tubules provided a protective barrier by which exogenous molecules were excluded (1). As interest in the BTB grew, many studies over the years have described both its unique structure and its functional relationship with male germline development. The BTB is now recognized as a coalition of tight and adherence junctions between Sertoli cells, forming a unique and protective tissue compartment on the luminal side of the seminiferous epithelium required for spermatogenesis (2). BTB function has also been shown to be more dynamic and versatile than merely the stoic physical barrier as originally proposed. The basal ectoplasmic specializations (ES) of the BTB acts as a “drawbridge” separating spermatogonia from developing spermatids while allowing the transit of preleptotene spermatocytes toward the luminal surface where these cells continue meiotic progression and haploid morphogenesis to become spermatozoa (3). Modulation of the BTB is complex and requires precise coordination across multiple different signaling mechanisms. One important signaling pathway required for BTB regulation is largely comprised of endogenous cytoskeletal regulatory machinery within Sertoli cells (2). Although some components of the relevant Sertoli cell regulatory networks have been identified, specifics regarding protein-protein activity, interactions, and signaling cascades are still largely unexplored. Recently, the effects of these major cytoskeletal regulatory components of the BTB have been interrogated in search of factors and mechanisms affecting fertility and as male contraceptive targets (3).

In their recent publication, Huitao Li et al expanded the aforementioned cytoskeletal regulation of BTB dynamics to characterize the likely interactivity of phosphorylated focal adhesion kinase (p-FAK) with the non-collagenous domain (NC1) of the collagen α3 chain (4). Both p-FAK and NC1 are known to modulate cytoskeletal regulation in testis and Sertoli cell function, but the interconnectivity between FAK and NC1 had only been speculative until now. FAK and FAK-like proteins are ubiquitous to many other tissue systems where they form large molecular assemblies responsible for providing feedback mechanisms across the extracellular matrix—the feedback mechanisms provided by FAK complexes are of great import for migratory cells in particular and have recently been identified as regulators of cell-cell junctions (5). In the context of testis physiology and the BTB, the phosphorylation of FAK has been observed to be essential for the recruitment and distribution of proteins responsible for stabilizing and fortifying BTB integrity as well as spermatid adhesion through the Sertoli-germ cell junctional complexes of the apical ES anchoring spermatids (6). Similarly, NC1 and other non-collagenous domains have been shown to interact and regulate extracellular matrix components such as integrins, fibronectin, collagen type I, and laminin—suggesting that NC1 is actively involved in cellular adhesion and morphogenesis (7). In testis, NC1 has a more direct effect in the BTB by actively inducing changes to Sertoli cell cytoskeletal components and tight junction barrier permeability (8). To further elucidate NC1 derivate BTB regulation, Li et al transfected rat testes in vivo to overexpress NC1. They assessed both the histomorphology of the testis and the prevalence of other known cytoskeletal regulators and constituents at different time-points post transfection. The data revealed for the first time a definitive degradation of the BTB after in vivo transfection, with morphological abnormalities including epithelia vacuolation, misorientation of spermatids, epithelial thinning, cell exfoliation, and the appearance of multinucleated giant cells. Furthermore, they observed a decrease in cytoskeletal regulators and constituent expression in the testis. As the concentration of NC1 peptide increased, levels of mTOR, c-Src, p-FAK, and MARK2 decreased while phosphorylated ribosomal protein S6 (p-rpS6) levels transiently escalated after transfection. In tandem, immunofluorescent staining of NC1 transfected testes also showed how the innate microtubule suprastructure in Sertoli cells and filamentous actin of the apical ES structure fractured and disorganized. The observed transient increase in p-rpS6-S240/S44 was found to localize to the apical ES concomitant with a decrease of apical ES-associated F-actin. These findings further elaborated the previous notion of NC1’s propensity to regulate Sertoli cell cytoskeletal stability demonstrating in vivo relevance.

Observing the deleterious effects that NC1 overexpression had in vivo on Sertoli cell BTB and apical ES as well as an apparent decrease in endogenous p-FAK activity, Huitao Li and colleagues further probed the interaction between NC1 and the previously observed fortifying effect p-FAK overexpression had in BTB and apical ES integrity (6).

To test the possible interaction between NC1 and p-FAK, 2D primary Sertoli cell cultures were co-transfected with NC1 and either a wild-type variant of FAK or the phosphomimetic mutant p-FAK-Y407E to induce an effect akin to an irreversible activation of the kinase. Fluorescence microscopy and tight junction permeability measurements indicated the effects of all transfected cultures against a naïve control culture. In all non-phosphomimetic cultures, NC1 disrupted both the tight junctions and cytoskeletal integrity of the culture shown by decreased electrical impedance (ohm.cm2) and dysregulation of Sertoli cell cytoskeleton, basal ES, and tight junction associated factors. In contrast, the forcefully activated phosphomimetic FAK seemed to offer a protective effect and retained normal transient activity and values akin to the naïve control. The disparity in effects between wild-type FAK and the phosphomimetic variant when in an NC1-rich environment indicates that the deregulatory effects of NC1 in the BTB are mediated through inhibition of endogenous FAK phosphorylation. Whether the effect of NC1 on FAK proteins is direct or through some secondary mechanism is yet to be explored, but these observations reported by C.Y. Cheng and colleagues (4) are both novel and exciting contributions in understanding the molecular mechanisms for BTB and apical ES regulation. Continued progress in tweezing apart pathways that regulate the basal and apical ES structures holds promise for identifying factors that may reversibly inhibit male fertility through selective disruption of the apical ES. Such developments in understanding the differential regulation of the BTB and apical ES may therefore hold clues for development and delivery of safe male contraceptive candidates.