The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferou...The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions （TJs）, adhesion junctions and gap junctions （GJs）. Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.展开更多
目的探讨细胞连接相关蛋白在不同发育阶段小鼠睾丸中的表达模式。方法 Western blot检测并分析1~6周龄小鼠睾丸、原代支持细胞以及小鼠精原细胞系GC1 spg、精母细胞系GC2 spg、间质细胞系TM3、支持细胞系TM4等4种生殖相关细胞系中细胞...目的探讨细胞连接相关蛋白在不同发育阶段小鼠睾丸中的表达模式。方法 Western blot检测并分析1~6周龄小鼠睾丸、原代支持细胞以及小鼠精原细胞系GC1 spg、精母细胞系GC2 spg、间质细胞系TM3、支持细胞系TM4等4种生殖相关细胞系中细胞连接相关蛋白的表达水平。结果小鼠睾丸曲细精管中支持细胞间细胞连接相关膜蛋白连接黏附分子A、紧密连接蛋白Occludin和Claudin,以及支持细胞-生精细胞间细胞连接相关膜蛋白连接黏附分子C、连接蛋白Nectin-3和钙黏附蛋白E的表达表现出明显的阶段特异性。而参与细胞连接的调节蛋白紧密连接蛋白ZO-1、ZO-2、Afadin、β链蛋白和CD2相关蛋白在睾丸的不同发育阶段以及生殖细胞系中表达无差异。结论小鼠睾丸曲细精管中支持细胞间以及支持细胞-生精细胞间细胞连接相关的膜蛋白多表现出明显的阶段特异性。而参与细胞连接的调节蛋白在睾丸的不同发育阶段中表达无差异。展开更多
The Sertoli cell tight junction （T J） is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated clau...The Sertoli cell tight junction （T J） is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% （P 〈 0.01）, 51% （P 〈 0.01）, and 62% （P 〈 0.01）, respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly （P 〈 0.01） to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant （P 〈 0.01） contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.展开更多
In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–te...In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier(BTB),or Sertoli cell barrier(SCB).However,little is known about the underlying mechanism.In this study,interaction between actin,an important component of the SCB,and ZIKV envelope(E)protein domainⅢ(EDⅢ)was inferred from coimmunoprecipitation(Co-IP)liquid chromatography–tandem mass spectrometry(LC–MS/MS)analysis.Confocal microscopy confirmed the role of actin filaments(F-actin)in ZIKV infection,during which part of the stress fibers,the bundles that constituted by paralleled actin filaments,were disrupted and presented in the cell periphery.Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement.Perturbation of F-actin by cytochalasin D(CytoD)or Jasplakinolide(Jas)enhanced the infection of ZIKV.More importantly,the transepithelial electrical resistance(TEER)of an in vitro mouse SCB(mSCB)model declined with the progression of ZIKV infection or overexpression of E protein.Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression,highlighting the role of E protein in ZIKV-induced disruption of the BTB.We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network,thereby compromising BTB integrity.展开更多
基金This work was supported by the National Basic Research Program （Nos. 2013CB947900, 2013CB945502 and 2014CB943101） of China （973）, by grants from National Natural Science Foundation of China （No. 31371519） and the Knowledge Innovation Program of the Chinese Academy of Sciences （No. KSCX2-EW-R-07）.
文摘The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions （TJs）, adhesion junctions and gap junctions （GJs）. Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.
文摘目的探讨细胞连接相关蛋白在不同发育阶段小鼠睾丸中的表达模式。方法 Western blot检测并分析1~6周龄小鼠睾丸、原代支持细胞以及小鼠精原细胞系GC1 spg、精母细胞系GC2 spg、间质细胞系TM3、支持细胞系TM4等4种生殖相关细胞系中细胞连接相关蛋白的表达水平。结果小鼠睾丸曲细精管中支持细胞间细胞连接相关膜蛋白连接黏附分子A、紧密连接蛋白Occludin和Claudin,以及支持细胞-生精细胞间细胞连接相关膜蛋白连接黏附分子C、连接蛋白Nectin-3和钙黏附蛋白E的表达表现出明显的阶段特异性。而参与细胞连接的调节蛋白紧密连接蛋白ZO-1、ZO-2、Afadin、β链蛋白和CD2相关蛋白在睾丸的不同发育阶段以及生殖细胞系中表达无差异。结论小鼠睾丸曲细精管中支持细胞间以及支持细胞-生精细胞间细胞连接相关的膜蛋白多表现出明显的阶段特异性。而参与细胞连接的调节蛋白在睾丸的不同发育阶段中表达无差异。
文摘The Sertoli cell tight junction （T J） is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% （P 〈 0.01）, 51% （P 〈 0.01）, and 62% （P 〈 0.01）, respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly （P 〈 0.01） to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant （P 〈 0.01） contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.
基金We graciously acknowledge Dr.George F.Gao of Institute of Microbiology,Chinese Academy of Sciences,Beijing,China for the gifts of ZIKV(ZIKA-SMGC-1,GenBank accession number:KX266255)ZIKV antibody FITC-Z6.This work was supported by grants from the National Science and Technology Major Project(Grant No.2018ZX10733403)。
文摘In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier(BTB),or Sertoli cell barrier(SCB).However,little is known about the underlying mechanism.In this study,interaction between actin,an important component of the SCB,and ZIKV envelope(E)protein domainⅢ(EDⅢ)was inferred from coimmunoprecipitation(Co-IP)liquid chromatography–tandem mass spectrometry(LC–MS/MS)analysis.Confocal microscopy confirmed the role of actin filaments(F-actin)in ZIKV infection,during which part of the stress fibers,the bundles that constituted by paralleled actin filaments,were disrupted and presented in the cell periphery.Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement.Perturbation of F-actin by cytochalasin D(CytoD)or Jasplakinolide(Jas)enhanced the infection of ZIKV.More importantly,the transepithelial electrical resistance(TEER)of an in vitro mouse SCB(mSCB)model declined with the progression of ZIKV infection or overexpression of E protein.Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression,highlighting the role of E protein in ZIKV-induced disruption of the BTB.We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network,thereby compromising BTB integrity.
