Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flage...Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.展开更多
Cilia are cellular protrusions containing nine microtubule(MT)doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions.Cili...Cilia are cellular protrusions containing nine microtubule(MT)doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions.Cilia require the central pair(CP)apparatus,consisting of two CP MTs covered with projections of CP proteins,for planar strokes.How the CP MTs of such‘9+2’cilia are constructed,however,remains unknown.Here we identify Spef1,an evolutionarily conserved microtubule-bundling protein,as a core CP MT regulator in mammalian cilia.Spef1 was selectively expressed in mammalian cells with 9+2 cilia and specifically localized along the CP.Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6,resulting in rotational beat of the ependymal cilia.Spef1,which binds to MTs through its N-terminal calponin.homologous domain,formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs.Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus.We propose that Spefl bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.展开更多
研究陡脉冲电场(Steep pulsed electric fields,SPEFs)对接种Walker256癌肉瘤的Wistar大鼠免疫功能的影响,探讨SPEFs治疗恶性肿瘤的机理。将30只大鼠随机分为三组:荷瘤对照组(接种肉瘤,不治疗)、处理组(接种肉瘤,SPEFs治疗)和正常对照组...研究陡脉冲电场(Steep pulsed electric fields,SPEFs)对接种Walker256癌肉瘤的Wistar大鼠免疫功能的影响,探讨SPEFs治疗恶性肿瘤的机理。将30只大鼠随机分为三组:荷瘤对照组(接种肉瘤,不治疗)、处理组(接种肉瘤,SPEFs治疗)和正常对照组(接种等量生理盐水,不治疗),每组各10只。治疗前及治疗后每隔3 d用游标卡尺测量肿瘤最大直径。治疗后2周,用MTT法检测大鼠脾淋巴细胞转化率和NK细胞活性、ELISA法检测大鼠腹腔巨噬细胞分泌TNF-α的表达。实验结果表明,SPEFs能明显抑制恶性肿瘤的生长(P<0．01)、明显增强大鼠脾淋巴细胞的增殖反应(P<0．05)、显著提高大鼠NK细胞的活性(P<0．05)和大鼠腹腔巨噬细胞产生TNF-α的能力(P<0．05)。本实验显示,SPEFs在通过不可逆性电击穿杀伤肿瘤细胞的同时,也能诱发机体抗癌免疫反应,增强机体的免疫功能,为临床应用提供治疗机理。展开更多
基金support of the National Key Research&Developmental Program of China(2018YFC1004900 to YQT)the National Natural Science Foundation of China(81971447 to YQT)the Key Grant of Prevention and Treatment of Birth Defect from Hunan province(2019SK1012 to YQT),and the research grant ofCITIC-Xiangya(YNXM-202004,YNXM-202006).
文摘Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.
基金the National Natural Science Foundation of China(31330045)National Key R&D Program of China(2O17YFAO5O35OO)+1 种基金Chinese Academy of Sciences(XDBl9020000)the National Natural Science Foundation of China(31601092 to LZ and 31771495 to X.Y.).
文摘Cilia are cellular protrusions containing nine microtubule(MT)doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions.Cilia require the central pair(CP)apparatus,consisting of two CP MTs covered with projections of CP proteins,for planar strokes.How the CP MTs of such‘9+2’cilia are constructed,however,remains unknown.Here we identify Spef1,an evolutionarily conserved microtubule-bundling protein,as a core CP MT regulator in mammalian cilia.Spef1 was selectively expressed in mammalian cells with 9+2 cilia and specifically localized along the CP.Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6,resulting in rotational beat of the ependymal cilia.Spef1,which binds to MTs through its N-terminal calponin.homologous domain,formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs.Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus.We propose that Spefl bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.
文摘研究陡脉冲电场(Steep pulsed electric fields,SPEFs)对接种Walker256癌肉瘤的Wistar大鼠免疫功能的影响,探讨SPEFs治疗恶性肿瘤的机理。将30只大鼠随机分为三组:荷瘤对照组(接种肉瘤,不治疗)、处理组(接种肉瘤,SPEFs治疗)和正常对照组(接种等量生理盐水,不治疗),每组各10只。治疗前及治疗后每隔3 d用游标卡尺测量肿瘤最大直径。治疗后2周,用MTT法检测大鼠脾淋巴细胞转化率和NK细胞活性、ELISA法检测大鼠腹腔巨噬细胞分泌TNF-α的表达。实验结果表明,SPEFs能明显抑制恶性肿瘤的生长(P<0．01)、明显增强大鼠脾淋巴细胞的增殖反应(P<0．05)、显著提高大鼠NK细胞的活性(P<0．05)和大鼠腹腔巨噬细胞产生TNF-α的能力(P<0．05)。本实验显示,SPEFs在通过不可逆性电击穿杀伤肿瘤细胞的同时,也能诱发机体抗癌免疫反应,增强机体的免疫功能,为临床应用提供治疗机理。
