During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminin β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites.

Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction.

Transport of germ cells across the seminiferous epithelium is crucial to spermatogenesis. Its disruption causes infertility. Signaling molecules, such as focal adhesion kinase, c-Yes, c-Src, and intercellular adhesion molecules 1 and 2, are involved in these events by regulating actin-based cytoskeleton via their action on actin-regulating proteins, endocytic vesicle-mediated protein trafficking, and adhesion protein complexes. We critically evaluate these findings and provide a hypothetical framework that regulates these events.

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During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research.testis; c-Yes; nonreceptor protein tyrosine kinase; Sertoli cell; spermatogenesis; seminiferous epithelial cycle; ectoplasmic specialization DURING SPERMATOGENESIS, EXTENSIVE RESTRUCTURING and communication take place at the Sertoli cell-cell and Sertoli-germ cell interface across the seminiferous epithelium to accommodate germ cell movement and to coordinate cellular events that occur simultaneously at different stages of the seminiferous epithelial cycle in the mammalian testis (12,23,48). For instance, at stage VIII of the epithelial cycle, preleptotene spermatocytes, differentiated from type B spermatogonia, residing in the basal compartment traverse the blood-testis barrier (BTB) to enter the adluminal compartment while transforming to leptotene and zygote spermatocytes to prepare for meiosis I/II (23, 48). However, fully developed spermatids that become spermatozoa at the adluminal edge of the tubule lumen undergo spermiation; the release of sperm also takes place at stage VIII of the cycle, involving degeneration of the apical ectoplasmic specialization (apical ES) at the Sertoli-spermatid

ICAMs are crucial regulatory molecules of spermatogenesis. The proposed hypothetical model serves as a framework in designing functional experiments for future studies.

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell–cell and Sertoli cell–spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood–testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell–cell interface, and the apical ES at the Sertoli–spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.