title: Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro.
abstract: The Sertoli cell tight junction (TJ) 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.
authors.affiliation: Male Fertility Regulation Laboratory, Hudson Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168; School of Applied Sciences, Royal Melbourne Institute of Technology University, Bundoora, Victoria 3088; Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010; St Vincent's Clinical School, UNSW, Sydney, New South Wales 2052, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McCabe
authors.affiliation: Male Fertility Regulation Laboratory, Hudson Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia.
authors.forename: Caroline Fh
authors.initials: CF
authors.lastname: Foo
authors.affiliation: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010; St Vincent's Clinical School, UNSW, Sydney, New South Wales 2052, Australia.
authors.forename: Marcel E
authors.initials: ME
authors.lastname: Dinger
authors.affiliation: School of Applied Sciences, Royal Melbourne Institute of Technology University, Bundoora, Victoria 3088, Australia.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Smooker
authors.affiliation: Male Fertility Regulation Laboratory, Hudson Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Stanton
mesh_terms: D000818:Animals; D001814:Blood-Testis Barrier / Q000378:metabolism; D057167:Claudins / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D008297:Male; D062793:Occludin / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: D057167:Claudins; C492353:Cldn11 protein, rat; D062793:Occludin