Abstract：
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 (TNFα) participates in the blood-testis barrier reconstruction, cell apoptosis, and inflammatory response by recognizing receptors on Sertoli cell. TNFα has also been shown to induce the proliferation of immature Sertoli cell in vitro, yet the mechanism still remains unclarified.
This study was designed to investigate the effect of TNFα on blood-testis barrier development during puberty and the underlying mechanisms of TNFα-induced immature Sertoli cell proliferation.
Immature male Sprague-Dawley rats of postnatal day 12 were intraperitoneally injected with TNFα. Biotin-labeled method was used to detect permeability of the developing blood-testis barrier after TNFα 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 TNFα. 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 (NFκB) activity inhibition on TNFα-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 TNFα.
The in vivo experiment revealed a significant delay of blood-testis barrier maturation in pubertal rats caused by exogenous TNFα. TNFα (10 ng/ml) treatment in vitro was found to promote the proliferation of immature Sertoli cells, accompanied with increased NFκB activity and cyclin D1 protein level. The level of Fbxo4 and ubiquitination of cyclin D1 were decreased after TNFα treatment. Inhibitor of NFκB or overexpression of Fbxo4 could both reverse the TNFα-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 NFκB caused by TNFα.
These results indicate that TNFα inhibits the ubiquitination and degradation of cyclin D1 through the NFκB pathway, thereby promoting the proliferation of immature Sertoli cell in vitro and inducing the delay of blood-testis barrier maturation in pubertal rats.
This study was designed to investigate the effect of TNFα on blood-testis barrier development during puberty and the underlying mechanisms of TNFα-induced immature Sertoli cell proliferation.
Immature male Sprague-Dawley rats of postnatal day 12 were intraperitoneally injected with TNFα. Biotin-labeled method was used to detect permeability of the developing blood-testis barrier after TNFα 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 TNFα. 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 (NFκB) activity inhibition on TNFα-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 TNFα.
The in vivo experiment revealed a significant delay of blood-testis barrier maturation in pubertal rats caused by exogenous TNFα. TNFα (10 ng/ml) treatment in vitro was found to promote the proliferation of immature Sertoli cells, accompanied with increased NFκB activity and cyclin D1 protein level. The level of Fbxo4 and ubiquitination of cyclin D1 were decreased after TNFα treatment. Inhibitor of NFκB or overexpression of Fbxo4 could both reverse the TNFα-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 NFκB caused by TNFα.
These results indicate that TNFα inhibits the ubiquitination and degradation of cyclin D1 through the NFκB pathway, thereby promoting the proliferation of immature Sertoli cell in vitro and inducing the delay of blood-testis barrier maturation in pubertal rats.
摘要：
在精子发生过程中起重要作用的支持细胞是影响睾丸发育的生理和病理因素的已知靶标。肿瘤坏死因子α（TNFα）参与血-睾丸屏障重建，细胞凋亡，和通过识别支持细胞上的受体的炎症反应。TNFα也被证明在体外诱导未成熟支持细胞的增殖，然而，机制仍未明确。
本研究旨在研究TNFα对青春期血液睾丸屏障发育的影响以及TNFα诱导的未成熟睾丸支持细胞增殖的潜在机制。
将出生后第12天的未成熟雄性Sprague-Dawley大鼠腹膜内注射TNFα。用生物素标记的方法检测TNFα处理后发育中的血-睾丸屏障的通透性，免疫荧光法检测咬合素和连接粘附分子-A(JAM-A)的分布。从出生后第10天的Sprague-Dawley大鼠中分离的支持细胞在体外培养并用TNFα处理。细胞增殖速率由细胞计数试剂盒-8(CCK-8)和5-乙炔基-2'-脱氧尿苷(EdU)测定反映。免疫印迹和定量聚合酶链反应检测增殖细胞核抗原的表达，Fbxo4和细胞周期蛋白D1。免疫沉淀用于检测细胞周期蛋白D1的泛素化以及Fbxo4与细胞周期蛋白D1之间的相互作用。应用吡咯烷二硫代氨基甲酸铵（PDTC）检测核因子κB（NFκB）活性抑制对TNFα诱导的支持细胞增殖的影响。构建了含有大鼠Fbxo4基因的腺病毒重组质粒，以研究Fbxo4过表达对TNFα促进的支持细胞增殖的影响。
体内实验显示，外源性TNFα导致青春期大鼠血-睾丸屏障成熟明显延迟。发现TNFα（10ng/ml）体外处理可促进未成熟睾丸支持细胞的增殖，伴有NFκB活性和细胞周期蛋白D1蛋白水平的增加。TNFα处理后，Fbxo4和细胞周期蛋白D1的泛素化水平降低。NFκB抑制剂或Fbxo4过表达均可逆转TNFα诱导的未成熟支持细胞增殖,同时恢复泛素-蛋白酶体系统依赖的细胞周期蛋白D1的降解。Fbxo4的过表达不能影响TNFα引起的NFκB的活化。
这些结果表明，TNFα通过NFκB途径抑制细胞周期蛋白D1的泛素化和降解，从而在体外促进未成熟睾丸支持细胞的增殖，并诱导青春期大鼠血-睾丸屏障成熟的延迟。
本研究旨在研究TNFα对青春期血液睾丸屏障发育的影响以及TNFα诱导的未成熟睾丸支持细胞增殖的潜在机制。
将出生后第12天的未成熟雄性Sprague-Dawley大鼠腹膜内注射TNFα。
体内实验显示，外源性TNFα导致青春期大鼠血-睾丸屏障成熟明显延迟。
这些结果表明，TNFα通过NFκB途径抑制细胞周期蛋白D1的泛素化和降解，
