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

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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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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Mice depleted of the coxsackievirus and adenovirus receptor display normal spermatogenesis and an intact blood–testis barrier

Reproduction ◽

10.1530/rep-13-0653 ◽

2014 ◽

Vol 147 (6) ◽

pp. 875-883 ◽

Cited By ~ 8

Author(s):

Taranum Sultana ◽

Mi Hou ◽

Jan-Bernd Stukenborg ◽

Virpi Töhönen ◽

Jose Inzunza ◽

...

Keyword(s):

Testicular Development ◽

Direct Role ◽

Tubular Lumen ◽

Germ Cell Migration ◽

Coxsackievirus And Adenovirus Receptor ◽

And Function ◽

Adenovirus Receptor ◽

Blood Testis Barrier

The coxsackievirus and adenovirus receptor (CXADR (CAR)) is a cell adhesion molecule expressed mainly in epithelial cells. Numerous evidence indicate that CXADR has an important role in testis development and function of the blood–testis barrier (BTB)in vitro. The role of CXADR in testis physiologyin vivohas, however, not been addressed. We therefore constructed a conditional CXADR knockout (cKO) mouse model in which CXADR can be depleted at any chosen timepoint by the administration of tamoxifen. We report for the first time that testicular depletion of CXADR in adult and pubertal mice does not alter BTB permeability or germ cell migration across the BTB during spermatogenesis. Adult cKO mice display normal junctional ultra-structure and localization of the junctional proteins claudin-3, occludin, junction-associated molecule-A (JAM-A), and ZO1. The BTB was intact with no leakage of biotin and lanthanum tracers into the tubular lumen. Adult CXADR cKO mice were fertile with normal sperm parameters and litter size. Breeding experiments and genotyping of the pups demonstrated that CXADR-negative sperm could fertilize WT eggs. In addition, knocking down CXADR from postnatal day 9 (P9) does not affect testicular development and BTB formation. These cKO mice were analyzed at P49 and P90 and display an intact barrier and uncompromised fertility. We conclude that CXADR possesses no direct role in testicular physiologyin vivo.

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Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Reproduction ◽

10.1530/rep-06-0385 ◽

2007 ◽

Vol 133 (6) ◽

pp. 1169-1179 ◽

Cited By ~ 97

Author(s):

Tu’uhevaha J Kaitu’u-Lino ◽

Pavel Sluka ◽

Caroline F H Foo ◽

Peter G Stanton

Keyword(s):

Mrna Expression ◽

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Cell Contacts ◽

Barrier Formation ◽

Protein Components ◽

Blood Testis Barrier

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJsin vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P< 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesisin vivois partly via their effects on TJ proteins and regulation of the blood–testis barrier.

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Coxsackievirus and Adenovirus Receptor Is Up-Regulated in Migratory Germ Cells during Passage of the Blood-Testis Barrier

Endocrinology ◽

10.1210/en.2007-0359 ◽

2007 ◽

Vol 148 (11) ◽

pp. 5459-5469 ◽

Cited By ~ 19

Author(s):

Momina Mirza ◽

Cecilia Petersen ◽

Katarina Nordqvist ◽

Kerstin Sollerbrant

Keyword(s):

Tight Junctions ◽

Adhesion Molecule ◽

Germ Cells ◽

Sertoli Cells ◽

Rt Pcr ◽

Adult Testis ◽

Functional Complex ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor ◽

Blood Testis Barrier

The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule expressed in epithelial tight junctions and other cell-cell contacts. Using indirect immunofluorescence, quantitative RT-PCR, and Western blots, the expression and distribution of CAR in developing and adult testis are examined. CAR is highly expressed in both Sertoli and germ cells during perinatal and postnatal development, followed by a rapid down-regulation of both mRNA and protein levels. Interestingly, we find that CAR is a previously unknown downstream target for FSH because CAR mRNA levels were induced in primary cultures of FSH-stimulated Sertoli cells. In contrast to other epithelia, CAR is not a general component of tight junctions in the seminiferous epithelium, and Sertoli cells in the adult testis do not express CAR. Instead, CAR expression is stage dependent and specifically found in migratory germ cells. RT-PCR also demonstrated the presence of junctional adhesion molecule-like (JAML) in the testis. JAML was previously reported by others to form a functional complex with CAR regulating transepithelial migration of leukocytes. The expression of JAML in the testis suggests that a similar functional complex might be present during germ cell migration across the blood-testis barrier. Finally, an intermediate compartment occupied by CAR-positive, migrating germ cells and flanked by two occludin-containing junctions is identified. Together, these results implicate a function for CAR in testis morphogenesis and in migration of germ cells across the blood-testis barrier during spermatogenesis.

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

Endocrinology ◽

10.1210/en.2012-1665 ◽

2012 ◽

Vol 153 (10) ◽

pp. 5036-5048 ◽

Cited By ~ 45

Author(s):

Ka-Wai Mok ◽

Dolores D. Mruk ◽

Bruno Silvestrini ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Actin Filament ◽

Sertoli Cells ◽

Plasma Membranes ◽

Permeability Barrier ◽

Actin Organization ◽

Filament Bundles ◽

Mtorc1 Pathway ◽

Protein Recruitment ◽

Blood Testis Barrier

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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An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

Endocrinology ◽

10.1210/en.2008-1741 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3336-3344 ◽

Cited By ~ 76

Author(s):

Erica R. Siu ◽

Elissa W. P. Wong ◽

Dolores D. Mruk ◽

K. L. Sze ◽

Catarina S. Porto ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Sertoli Cell ◽

Focal Adhesion ◽

Adhesion Molecule ◽

Protein Complex ◽

Permeability Barrier ◽

Ectoplasmic Specialization ◽

Blood Testis Barrier

Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics.

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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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Musashi-1 maintains blood–testis barrier structure during spermatogenesis and regulates stress granule formation upon heat stress

Molecular Biology of the Cell ◽

10.1091/mbc.e14-11-1497 ◽

2015 ◽

Vol 26 (10) ◽

pp. 1947-1956 ◽

Cited By ~ 10

Author(s):

Sun ErLin ◽

Wei WenJie ◽

Wang LiNing ◽

Lu BingXin ◽

Lei MingDe ◽

...

Keyword(s):

Heat Stress ◽

Cell Fate ◽

Sertoli Cell ◽

Sertoli Cells ◽

Stress Granules ◽

Stress Granule ◽

Granule Formation ◽

Associated Proteins ◽

Blood Testis Barrier

In mouse testes, Musashi-1 (Msi-1) was predominantly expressed in the cytoplasm and nuclei of Sertoli cells. Here we demonstrate that knockdown of Msi-1 in Sertoli cells altered the levels and distribution of blood–testis barrier (BTB)-associated proteins. Moreover, Msi-1 knockdown in vivo disrupted BTB functional structure and spermatogenesis. In addition, we report a novel role of Msi-1 in regulating Sertoli cells survival following heat-induced injury. Endogenous Msi-1 protein in heat-treated Sertoli cells was recruited to stress granules. The formation of stress granules was considerably disrupted, and apoptosis was significantly up-regulated in Msi-1–knockdown Sertoli cells after heat treatment. p-ERK1/2 acted downstream of stress granule formation, and inhibition of p-ERK1/2 signaling triggered Sertoli cell apoptosis upon heat stress. In conclusion, we demonstrate that Msi-1 is critical for constructing a functional BTB structure and maintaining spermatogenesis. We also note a role for Msi-1 in regulating Sertoli cell fate following heat-induced injury, likely through the induction of stress granule formation and subsequent activation of p-ERK1/2 signaling.

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HIV-1 establishes a sanctuary site in the testis by permeating the BTB through changes in cytoskeletal organization

Endocrinology ◽

10.1210/endocr/bqab156 ◽

2021 ◽

Author(s):

Siwen Wu ◽

Ines Frank ◽

Nina Derby ◽

Elena Martinelli ◽

C Yan Cheng

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Underlying Mechanism ◽

Antiretroviral Drugs ◽

Permeability Barrier ◽

Tat Protein ◽

Vitro Model ◽

Hiv 1

Abstract Studies suggest that HIV-1 invades the testis through permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used two approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1 infected Sup-T1 cells to investigate the activity of HIV-1 infection on co-cultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction (TJ)-permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin- and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

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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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