c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution

Abstract

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

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

“…Cells were seeded at a density of 0.5 ϫ 10 6 cells/cm 2 in six-well plates (5 ml/well DMEM-F-12) for different assays and lysate preparation or at a density of 0.03-0.05 ϫ 10 6 cells/cm 2 on round glass coverslips (18 mm; Thomas Scientific, Swedesboro, NJ) in 12-well plates (2 ml/well DMEM-F-12) for immunofluorescent staining. All culture dishes were coated with Matrigel basement membrane matrix (diluted 1:7 with DMEM-F-12; BD Bioscience, San Jose, CA), as described earlier (48,50). About 36 h after plating cells, cultures were treated with a hypotonic buffer (20 mM Tris, pH 7.4, at 22°C) to lyse contaminating germ cells (24), thereby yielding Sertoli cells with a purity of Ͼ98%.…”

“…Cells were rinsed twice and cultured in fresh DMEM-F-12 medium with supplements (24) overnight. Two days after isolation, when a functional TJ-permeability barrier was established when monitored by assessing transepithelial electrical resistance across the cell epithelium (48,50), Sertoli cells were used for c-Src or c-Yes knockdown by RNAi, as described (48). Besides the presence of a functional TJ barrier, ultrastructures of TJ, basal ES, gap junction, and desmosome that mimic the BTB in vivo were found in these cell cultures.…”

Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

“…Dual-labeled immunofluorescence analysis was performed using cross-sections of testes at 7 m (thickness) in a cryostat at Ϫ21°C, as described (68). Sections were fixed in 4% paraformaldehyde in PBS (10 mM sodium phosphate, pH 7.4, at 22°C, containing 0.15 M NaCl) or in Bouin's fixative (Polyscience, Warrington, PA) and permeabilized in 0.1% Triton X-100 in PBS (vol/vol).…”

“…Sections were fixed in 4% paraformaldehyde in PBS (10 mM sodium phosphate, pH 7.4, at 22°C, containing 0.15 M NaCl) or in Bouin's fixative (Polyscience, Warrington, PA) and permeabilized in 0.1% Triton X-100 in PBS (vol/vol). Nonspecific binding sites were blocked by 1% BSA in PBS (wt/vol) and then incubated with target primary antibodies ( Table 1) followed by Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 555 for red fluorescence, Alexa Fluor 488 for green fluorescence; Invitrogen), as described (31,68). Negative controls were performed in which primary antibody was substituted either by normal IgG of the same species at the same dilution (Table 1) or by omitting the secondary antibody.…”

“…Defects in spermatogenesis in the testis following fascin 1 knockdown vs. control testes transfected with nontargeting control siRNA duplexes were assessed by using both paraffin crosssections (6 m in thickness) of testes fixed in Bouin's fixative and/or frozen sections (with cell nuclei stained by DAPI), as described (61,68). In brief, paraffin sections were obtained in a microtome, and after de-waxing with xylene, sections were stained with hematoxylin 7211 for 3 min, followed by a 1-min incubation of Clarifier 1 and Bluing Reagent (Richard-Allan Scientific, Richland, MI), and stained with Eosin-Y for 30 s. Histological analysis was then performed to assess changes by scoring ϳ100 randomly selected cross-sections of stage VII and VIII tubules.…”