PDF(Pubmed)
Abstract：
Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.
摘要：
应激颗粒（SGs）是无膜核糖核蛋白（RNP）为基础的细胞病灶，在响应应激，通过保护细胞免受损害来促进细胞存活。哺乳动物精子发生应保持在体温以下，以便适当发育，表明其对热应力(HS)的脆弱性。在这项研究中,生物素示踪剂通透性测定表明，4-8mg/kg环己酰亚胺对睾丸中热诱导的SG组装的抑制作用显着增加了睾丸屏障（BTB）受损的生精小管的百分比。Westernblot结果还显示，热诱导的SG组装在Sertoli细胞系中的抑制作用，TM4细胞,通过G3bp1/2的RNA推断加重了BTB相关蛋白ZO-1，β-Catenin和Claudin-11的下降，表明SGs可以保护BTB免受HS引起的损害。通过顺序离心和免疫沉淀分离与支持细胞中SGs相关的蛋白质成分，并通过液相色谱-串联质谱鉴定。基因本体论和KEGG通路富集分析显示,其相应基因主要参与蛋白酶体相关通路,核苷酸切除修复，失配修复,和DNA复制。此外，一个新的SG组件，泛素相关蛋白2（UBAP2），通过免疫荧光发现TM4细胞在HS后易位到SGs。此外，在HS期间，通过RNA推断，UBAP2敲除后，SG组装显著减少，表明UBAP2在SG组装中的重要作用。此外,UBAP2敲除降低了ZO-1，β-Catenin和Claudin-11的表达，这暗示了其在BTB功能中的潜在作用。总的来说,我们的研究证明了SGs在HS期间维持BTB功能中的作用,并确定了与支持细胞中SG形成有关的新成分.这些发现不仅为SGs的生物学功能和夏季男性低生育力的分子机制提供了新的见解，但也可能为男性生育疗法提供实验基础。
