rpS6 Regulates Blood-Testis Barrier Dynamics By Affecting F-Actin Organization and Protein Recruitment

Abstract During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII–IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB.

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Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes

Endocrinology ◽

10.1210/en.2012-2269 ◽

2013 ◽

Vol 154 (5) ◽

pp. 1907-1920 ◽

Cited By ~ 39

Author(s):

Xiaojing Qian ◽

Dolores D. Mruk ◽

Elissa W. P. Wong ◽

Pearl P. Y. Lie ◽

C. Yan Cheng

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Plasma Membranes ◽

Permeability Barrier ◽

Protein Distribution ◽

Filament Bundles ◽

Ectoplasmic Specialization ◽

Blood Testis Barrier

Abstract In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.

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Regulation of the blood-testis barrier by coxsackievirus and adenovirus receptor

AJP Cell Physiology ◽

10.1152/ajpcell.00218.2012 ◽

2012 ◽

Vol 303 (8) ◽

pp. C843-C853 ◽

Cited By ~ 16

Author(s):

Linlin Su ◽

Dolores D. Mruk ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Regulatory Protein ◽

Integral Membrane Protein ◽

Permeability Barrier ◽

Structural Protein ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor ◽

Blood Testis Barrier

The blood-testis barrier (BTB) divides the seminiferous epithelium into the basal and the adluminal compartment. It restricts paracellular diffusion of molecules between Sertoli cells, confers cell polarity, and creates a unique microenvironment in the adluminal compartment for spermatid development. However, it undergoes restructuring during the epithelial cycle so that preleptotene spermatocytes differentiated from type B spermatogonia residing in the basal compartment can traverse the BTB at stage VIII of the cycle, while the immunological barrier is maintained. Herein, coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) integral membrane protein in the testis and multiple epithelia and endothelia, was found to act as a regulatory protein at the BTB, besides serving as a structural adhesion protein. RNAi-mediated knockdown of CAR in a Sertoli cell epithelium with an established TJ-permeability barrier that mimicked the BTB in vivo resulted in a disruption of the TJ barrier and an increase in endocytosis of the TJ-protein occludin. Furthermore, such an enhancement in occludin endocytosis was accompanied by a downregulation of Thr-phosphorylation in occludin and an increase in the association of endocytosed occludin with early endosome antigen-1. These findings were confirmed by overexpressing CAR in Sertoli cells, which was found to “tighten” the Sertoli cell TJ barrier, promoting BTB function. These findings support the emerging concept that CAR is not only a structural protein, it is involved in conferring the phosphorylation status of other adhesion proteins at the BTB (e.g., occludin) possibly mediated via its structural interactions with nonreceptor protein kinases, thereby modulating endocytic vesicle-mediated protein trafficking.

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Filamin A Is a Regulator of Blood-Testis Barrier Assembly during Postnatal Development in the Rat Testis

Endocrinology ◽

10.1210/en.2012-1286 ◽

2012 ◽

Vol 153 (10) ◽

pp. 5023-5035 ◽

Cited By ~ 17

Author(s):

Wenhui Su ◽

Dolores D. Mruk ◽

Pearl P. Y. Lie ◽

Wing-yee Lui ◽

C. Yan Cheng

Keyword(s):

Postnatal Development ◽

Sertoli Cell ◽

Actin Filament ◽

Permeability Barrier ◽

Filamin A ◽

Rat Testis ◽

Filament Network ◽

Blood Testis Barrier

Abstract The blood-testis barrier (BTB) is an important ultrastructure in the testis. A delay in its assembly during postnatal development leads to meiotic arrest. Also, a disruption of the BTB by toxicants in adult rats leads to a failure in spermatogonial differentiation. However, the regulation of BTB assembly remains unknown. Herein, filamin A, an actin filament cross-linker that is known to maintain and regulate cytoskeleton structure and function in other epithelia, was shown to be highly expressed during the assembly of Sertoli cell BTB in vitro and postnatal development of BTB in vivo, perhaps being used to maintain the actin filament network at the BTB. A knockdown of filamin A by RNA interference was found to partially perturb the Sertoli cell tight junction (TJ) permeability barrier both in vitro and in vivo. Interestingly, this down-regulating effect on the TJ barrier function after the knockdown of filamin A was associated with a mis-localization of both TJ and basal ectoplasmic specialization proteins. Filamin A knockdown also induced a disorganization of the actin filament network in Sertoli cells in vitro and in vivo. Collectively, these findings illustrate that filamin A regulates BTB assembly by recruiting these proteins to the microenvironment in the seminiferous epithelium to serve as the building blocks. In short, filamin A participates in BTB assembly by regulating protein recruitment during postnatal development in the rat testis.

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Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

Endocrinology ◽

10.1210/en.2013-1657 ◽

2014 ◽

Vol 155 (1) ◽

pp. 249-262 ◽

Cited By ~ 64

Author(s):

Hin-Ting Wan ◽

Dolores D. Mruk ◽

Chris K. C. Wong ◽

C. Yan Cheng

Keyword(s):

Tight Junction ◽

Sertoli Cell ◽

Sertoli Cells ◽

Connexin 43 ◽

Male Rat ◽

Environmental Toxicants ◽

Adhesion Proteins ◽

Actin Organization ◽

Blood Testis Barrier

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB.

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Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽

10.1210/en.2014-1163 ◽

2014 ◽

Vol 155 (10) ◽

pp. 3981-3995 ◽

Cited By ~ 19

Author(s):

N. Ece Gungor-Ordueri ◽

Elizabeth I. Tang ◽

Ciler Celik-Ozenci ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Adherens Junction ◽

Actin Binding ◽

Permeability Barrier ◽

Tight Junction Permeability ◽

Residual Bodies ◽

Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

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Drug transporters and blood–testis barrier function

Journal of Endocrinology ◽

10.1530/joe-10-0474 ◽

2011 ◽

Vol 209 (3) ◽

pp. 337-351 ◽

Cited By ~ 37

Author(s):

Linlin Su ◽

Dolores D Mruk ◽

Will M Lee ◽

C Yan Cheng

Keyword(s):

Sertoli Cell ◽

Barrier Function ◽

Drug Transporters ◽

Organic Anion ◽

Permeability Barrier ◽

Host Immune System ◽

Slc Transporters ◽

Apical Compartment ◽

Immunological Barrier ◽

Blood Testis Barrier

The blood–testis barrier (BTB) creates an immunological barrier that segregates the seminiferous epithelium into the basal and apical compartment. Thus, meiosis I/II and post-meiotic germ cell development take place in a specialized microenvironment in the apical compartment behind the BTB and these events are being shielded from the host immune system. If unwanted drugs and/or chemicals enter the apical compartment from the microvessels in the interstitium via the basal compartment, efflux pumps (e.g. P-glycoprotein) located in Sertoli cells and/or spermatids can actively transport these molecules out of the apical compartment. However, the mechanism(s) by which influx pumps regulate the entry of drugs/chemicals into the apical compartment is not known. In this study, a solute carrier (SLC) transporter organic anion transporting polypeptide 3 (Oatp3, Slco1a5) was shown to be an integrated component of the N-cadherin-based adhesion complex at the BTB. However, a knockdown of Oatp3 alone or in combination with three other major Sertoli cell drug influx pumps, namely Slc22a5, Slco6b1, and Slco6c1, by RNAi using corresponding specific siRNA duplexes failed to perturb the Sertoli cell tight junction (TJ) permeability barrier function. Yet, the transport of [3H]adjudin, a potential male contraceptive that is considered a toxicant to spermatogenesis, across the BTB was impeded following the knockdown of either Oatp3 or all the four SLC transporters. In short, even though drug transporters (e.g. influx pumps) are integrated components of the adhesion protein complexes at the BTB, they are not involved in regulating the Sertoli cell TJ permeability barrier function, instead they are only involved in the transport of drugs, such as adjudin, across the immunological barrier at the BTB.

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Germ cell-Sertoli cell interactions

Reproduction Fertility and Development ◽

10.1071/rd9900225 ◽

1990 ◽

Vol 2 (3) ◽

pp. 225 ◽

Cited By ~ 19

Author(s):

Kretser DM de

Keyword(s):

Germ Cell ◽

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Cell Interactions ◽

Cell Contact ◽

Paracrine Factors ◽

Cytological Changes ◽

Cyclic Changes ◽

Blood Testis Barrier

The interactions between the Sertoli cells and germ cells are progressively becoming an important part of testicular physiology. This paper explores the cytological basis for these interactions, detailing the cyclic changes in the Sertoli cells in concert with the stages of the seminiferous cycle and the nature of the blood-testis barrier. These cytological changes are correlated with a number of variations in the function of Sertoli cells. The mechanisms by which germ cells and Sertoli cells interact are explored and can be divided into those using cell-to-cell contact and others utilizing paracrine factors.

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Arrangement and possible function of actin filament bundles in ectoplasmic specializations of ground squirrel Sertoli cells

The Journal of Cell Biology ◽

10.1083/jcb.100.3.814 ◽

1985 ◽

Vol 100 (3) ◽

pp. 814-825 ◽

Cited By ~ 94

Author(s):

A. W. Vogl

Keyword(s):

Actin Filament ◽

Sertoli Cells ◽

Ground Squirrel ◽

Filament Bundles

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c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00422.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. E145-E159 ◽

Cited By ~ 33

Author(s):

Xiang Xiao ◽

Dolores D. Mruk ◽

C. Yan Cheng

Keyword(s):

Cell Adhesion ◽

Sertoli Cell ◽

Germ Cells ◽

Early Stage ◽

Seminiferous Epithelium ◽

Permeability Barrier ◽

Cell Interface ◽

Ectoplasmic Specialization ◽

Cell Cell ◽

Blood Testis Barrier

During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII–IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research.

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