TNF alpha stimulates the proliferation of immature Sertoli cells by attenuating UPS-degradation of cyclin D1 and leads to the delay of BTB maturation in pubertal rats

Backgrounds: The Sertoli cell that plays a vital role during spermatogenesis is a known target of physiological and pathological factors affecting testicular development. Tumor necrosis factor alpha (TNF alpha) participates in the blood-testis barrier reconstruction, cell apoptosis, and inflammatory response by recognizing receptors on Sertoli cell. TNF alpha has also been shown to induce the proliferation of immature Sertoli cell in vitro, yet the mechanism still remains unclarified.Objectives: This study was designed to investigate the effect of TNF alpha on blood-testis barrier development during puberty and the underlying mechanisms of TNF alpha-induced immature Sertoli cell proliferation.Materials and methods: Immature male Sprague-Dawley rats of postnatal day 12 were intraperitoneally injected with TNF alpha. Biotin-labeled method was used to detect permeability of the developing blood-testis barrier after TNF alpha treatment, and the distribution of occludin and junctional adhesion molecule-A (JAM-A) were detected by immunofluorescence. Sertoli cells isolated from Sprague-Dawley rats of postnatal day 10 were cultured in vitro and treated with TNF alpha. Cell proliferation rate was reflected by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay. Immunoblot and quantitative polymerase chain reaction were used to detect the expression of proliferating cell nuclear antigen, Fbxo4, and cyclin D1. Immunoprecipitation was used to detect the ubiquitination of cyclin D1 and the interaction between Fbxo4 and cyclin D1. Ammonium pyrrolidinedithiocarbamate (PDTC) was applied to detect the effect of nuclear factor kappaB (NF kappa B) activity inhibition on TNF alpha-induced Sertoli cell proliferation. The adenoviral recombinant plasmid containing rat Fbxo4 gene was constructed to investigate the effect of Fbxo4 overexpression on Sertoli cell proliferation promoted by TNF alpha.Results: The in vivo experiment revealed a significant delay of blood-testis barrier maturation in pubertal rats caused by exogenous TNF alpha. TNF alpha (10 ng/ml) treatment in vitro was found to promote the proliferation of immature Sertoli cells, accompanied with increased NF kappa B activity and cyclin D1 protein level. The level of Fbxo4 and ubiquitination of cyclin D1 were decreased after TNF alpha treatment. Inhibitor of NF kappa B or overexpression of Fbxo4 could both reverse the TNF alpha-induced proliferation of immature Sertoli cells, meanwhile restore the ubiquitin-proteasome system-dependent degradation of cyclin D1. Overexpression of Fbxo4 could not affect the activation of NF kappa B caused by TNF alpha.Conclusion: These results indicate that TNF alpha inhibits the ubiquitination and degradation of cyclin D1 through the NF kappa B pathway, thereby promoting the proliferation of immature Sertoli cell in vitro and inducing the delay of blood-testis barrier maturation in pubertal rats.