R., Brinster R. Regulation of bloodCtestis barrier assembly by germ cells. is usually a highly conserved gene among species that is known to control germ cell differentiation, and meiosis will be arrested if this gene is usually inactivated in the testis. Several mutations have been reported for this gene, including mice are alive except that the population of spermatogonia in the testis is usually considerably reduced, and the remaining few spermatogonia fail SC 560 to enter meiosis. On the basis of these observations, we speculated that mice might be incapable of establishing a functional BTB. Thus, these mice could serve as a good research model to investigate the function of germ cells on BTB construction. Androgen plays a role in BTB assembly by influencing the expression of claudin 3 (8), claudin 11 (9), and occludin (10). Claudin 3 is usually Rabbit Polyclonal to CXCR3 a 4-pass integral membrane protein and a component of both TJs and basal ectoplasmic specializations (ESs) (11, 12). Unlike other integral membrane proteins, claudin 3 was reported to transiently incorporate into newly formed TJs and then be subsequently replaced by claudin 11 (3, 8). Claudins in turn interact with zonula occludens (ZOs) at their C-terminal region and connect to actin filament (13). In short, claudin 3 is usually a marker and an important mediator of new BTB construction. In this study, we first examined the structural and functional status of BTB in the testes because there are some controversies regarding the functional status of BTB in adult testes (14, 15). We sought to examine if these mutant mice experienced a functional BTB. If they did not, we sought to investigate if transplantation of undifferentiated spermatogonia SC 560 that were enriched in spermatogonia stem cells (SSCs) or the presence of SSC-derived germ cells could induce the assembly of a functional BTB. We also used a bioinformatics approach for gene profiling to correlate transplanted exogenous germ cell differentiation status against time-dependent BTB assembly to assess if the progression of these 2 events was related. MATERIALS AND METHODS Animals C57BL/6 and mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The use of mice and all pertinent surgical procedures were approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences. Biotin tracer studies The BTB integrity in the testis was assessed by using biotin tracer as explained earlier (8). In brief, mice were anesthetized and testes uncovered, and 50 l of EZ-Link Sulfo-NHS-LC-Biotin (10 mg/ml; Pierce, Rockford, IL, USA), freshly diluted in PBS (GE Healthcare, Parramatta, NSW, Australia) and made up of 1 mM CaCl2, was administered under the tunica albuginea. The mice were humanely killed 30 min thereafter. Testes were removed and immediately embedded in Optimal Trimming Temperature compound (Sakura, Torrance, CA, USA). SC 560 Frozen sections (8 m solid) were prepared for further staining by streptavidinCFITC. Immunofluorescence microscopy Testes collected from mice with or without SSC transplantation at specific time points C57BL/6 mice were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Tissue sections (5 m solid) were obtained in a microtome and mounted on glass slides. Sections were dewaxed and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. After blocking with 5% bovine serum albumin in PBS SC 560 (wt/vol) for 1 h, the sections were incubated with main antibody at 4C overnight (Table 1). Secondary antibody conjugated with either FITC or tetramethylrhodamine isothiocyanate (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 1:200 dilution was used and was incubated for 1 h at room temperature. Slides were then stained with DAPI (blue) to visualize cell nuclei and mounted in prolonged antifade.