FSH regulates the formation of adherens junctions and ectoplasmic specialisations between rat Sertoli cells in vitro and in vivo

Spermatogenesis is dependent on the ability of Sertoli cells to form mature junctions that maintain a unique environment within the seminiferous epithelium. Adjacent Sertoli cells form a junctional complex that includes classical adherens junctions and testis-specific ectoplasmic specialisations (ES). The regulation of inter-Sertoli cell junctions by the two main endocrine regulators of spermatogenesis, FSH and testosterone, is unclear. This study aimed to investigate the effects of FSH and testosterone on inter-Sertoli cell adherens junctions (as determined by immunolocalisation of cadherin, catenin and actin) and ES junctions (as determined by immunolocalisation of espin, actin and vinculin) in cultured immature Sertoli cells and GnRH-immunised adult rat testes given FSH or testosterone replacement in vivo. When hormones were absent in vitro, adherens junctions formed as discrete puncta between interdigitating, finger-like projections of Sertoli cells, but ES junctions were not present. The adherens junction puncta included actin filaments that were oriented perpendicularly to the Sertoli cell plasma membrane, but were not associated with the intermediate filament protein vimentin. When FSH was added in vitro, ES junctions formed, and adjacent adherens junction puncta fused into extensive adherens junction belts. After hormone suppression in vivo, ES junctions were absent, while FSH replacement restored ES junctions, as confirmed by electron microscopy and confocal analysis of ES-associated proteins. Testosterone alone did not affect adherens junctions or ES in vitro or in vivo. We conclude that FSH can regulate the formation of ES junctions and stimulate the organisation and orientation of extensive adherens junctions in Sertoli cells.

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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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Hormonal regulation of spermatid binding

Journal of Cell Science ◽

10.1242/jcs.100.3.623 ◽

1991 ◽

Vol 100 (3) ◽

pp. 623-633

Author(s):

D.F. Cameron ◽

K.E. Muffly

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Unit Area ◽

Junctional Complex ◽

Cell Cytoplasm ◽

Coculture Model ◽

Ectoplasmic Specialization

A Sertoli-spermatid coculture model is described in which a large percentage (greater than 76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD = the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH + testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described ‘Sertoli ectoplasmic specialization’ observed adjacent to post-step 8 spermatids in vivo. FSH + testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH + testosterone and is associated with the peripheral distribution of actin and vinculin.

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116. Contribution of claudin-11 to the inter-Sertoli cell tight junction, in vitro

Reproduction Fertility and Development ◽

10.1071/srb05abs116 ◽

2005 ◽

Vol 17 (9) ◽

pp. 72

Author(s):

M. J. McCabe ◽

P. G. Stanton

Keyword(s):

Tight Junction ◽

Mrna Expression ◽

Sertoli Cell ◽

Adhesion Molecule ◽

Sertoli Cells ◽

Adherens Junction ◽

Adherens Junction Protein ◽

Junction Protein

The inter-Sertoli cell tight junction (TJ) forms the blood testis barrier (BTB) between Sertoli cells and is composed of three major transmembrane proteins: claudin-11, occludin and junctional adhesion molecule. Formation of the BTB occurs during puberty associating with an increase in circulating gonadotrophins. Claudin-11 and occludin are hormonally regulated in vitro although their importance to the function of the TJ is unknown. The aim of this study was to investigate the contribution of claudin-11 to the inter-Sertoli cell TJ in vitro by blocking gene expression using RNA interference. Two claudin-11-specific siRNA fragments were designed for this purpose. Sertoli cells in primary culture formed stable TJs within 5 days as measured by transepithelial electrical resistance (TER). The addition of siRNA for 2 days resulted in a significant (P < 0.01) 55% (mean, SD, n = 4 cultures) decrease in TER along with a major reduction in claudin-11 localisation to the TJ as assessed by immunocytochemistry. The specificity of the siRNA was shown by the presence of extensive immunostaining of occludin and of the adherens junction protein β-catenin in the same treatments. Similarly, claudin-11 mRNA expression significantly (P < 0.01) decreased by 71% (mean, SD, n = 3 cultures) in response to both claudin-11 siRNA fragments. Occludin mRNA expression was not affected. It is concluded that claudin-11 contributes at least 55% to the function of the rat Sertoli cell TJ in vitro. It is hypothesised that the remaining 45% of TJ function can be attributed to other integral proteins, such as occludin and junctional adhesion molecule. It is expected that claudin-11 and other TJ proteins play a pivotal role in the function of the BTB in vivo with potential implications in fertility and contraception.

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Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

Biochemical Journal ◽

10.1042/bj3400309 ◽

1999 ◽

Vol 340 (1) ◽

pp. 309-320 ◽

Cited By ~ 11

Author(s):

Sikha Bettina MUKHERJEE ◽

S. ARAVINDA ◽

B. GOPALAKRISHNAN ◽

Sushma NAGPAL ◽

Dinakar M. SALUNKE ◽

...

Keyword(s):

Cell Culture ◽

Germ Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Culture Media ◽

Glutathione S Transferase ◽

Steroid Binding ◽

Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

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BLOOD–TESTIS BARRIER: EVIDENCE FOR INTACT INTER-SERTOLI CELL JUNCTIONS AFTER HYPOPHYSECTOMY IN THE ADULT RAT

Journal of Endocrinology ◽

10.1677/joe.0.0760087 ◽

1978 ◽

Vol 76 (1) ◽

pp. 87-91 ◽

Cited By ~ 19

Author(s):

L. HAGENÄS ◽

L. PLÖEN ◽

H. EKWALL

Keyword(s):

Sertoli Cell ◽

Structural Integrity ◽

Cell Junctions ◽

Junctional Complex ◽

Tracer Technique ◽

Adult Rats ◽

Adult Rat ◽

Hormonal Dependence ◽

Lanthanum Tracer ◽

Blood Testis Barrier

SUMMARY To study the hormonal dependence of the blood–testis barrier, adult rats were hypophysectomized and the ultrastructural integrity of the inter-Sertoli cell junctional complex was examined at various times with a lanthanum tracer technique. It was found that the structural integrity of the inter-Sertoli cell junctions and their capacity to exclude lanthanum from the adluminal compartment were preserved up to 35 days after hypophysectomy. Furthermore, transport of newly formed spermatocytes through the inter-Sertoli cell junctions still occurred 20 days after hypophysectomy. It is therefore concluded that the function of the inter-Sertoli cell junctional complex is not directly dependent on gonadotrophic or androgenic hormones, but is regulated by other mechanisms.

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Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

Dermatology Research and Practice ◽

10.1155/2012/309587 ◽

2012 ◽

Vol 2012 ◽

pp. 1-14 ◽

Cited By ~ 6

Author(s):

Miquella G. Chavez ◽

Christian A. Buhr ◽

Whitney K. Petrie ◽

Angela Wandinger-Ness ◽

Donna F. Kusewitt ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Wound Repair ◽

Egf Receptor ◽

Adherens Junction ◽

Cell Junctions ◽

Epidermal Growth ◽

E Cadherin

Modulation of cell : cell junctions is a key event in cutaneous wound repair. In this study we report that activation of the epidermal growth factor (EGF) receptor disrupts cel : cell adhesion, but with different kinetics and fates for the desmosomal cadherin desmoglein and for E-cadherin. Downregulation of desmoglein preceded that of E-cadherinin vivoand in an EGF-stimulatedin vitrowound reepithelialization model. Dual immunofluorescence staining revealed that neither E-cadherin nor desmoglein-2 internalized with the EGF receptor, or with one another. In response to EGF, desmoglein-2 entered a recycling compartment based on predominant colocalization with the recycling marker Rab11. In contrast, E-cadherin downregulation was accompanied by cleavage of the extracellular domain. A broad-spectrum matrix metalloproteinase inhibitor protected E-cadherin but not the desmosomal cadherin, desmoglein-2, from EGF-stimulated disruption. These findings demonstrate that although activation of the EGF receptor regulates adherens junction and desmosomal components, this stimulus downregulates associated cadherins through different mechanisms.

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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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Liver-regulating protein (LRP) is a plasma membrane protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes

Journal of Cell Science ◽

10.1242/jcs.108.3.917 ◽

1995 ◽

Vol 108 (3) ◽

pp. 917-925

Author(s):

N. Gerard ◽

A. Corlu ◽

B. Kneip ◽

H. Kercret ◽

M. Rissel ◽

...

Keyword(s):

Monoclonal Antibody ◽

Epithelial Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Cell Function ◽

Cell Contact ◽

Dependent Manner ◽

Biliary Epithelial Cell

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene spermatocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and spermatids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interaction-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocultures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte- and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.

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Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions.

The Journal of Cell Biology ◽

10.1083/jcb.135.2.497 ◽

1996 ◽

Vol 135 (2) ◽

pp. 497-510 ◽

Cited By ~ 229

Author(s):

A Del Maschio ◽

A Zanetti ◽

M Corada ◽

Y Rival ◽

L Ruco ◽

...

Keyword(s):

Endothelial Cell ◽

Leukocyte Adhesion ◽

Adherens Junction ◽

Immunohistochemical Analysis ◽

Cell Junctions ◽

Beta Catenin ◽

Cell Extracts ◽

Beta 2

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE-cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE-cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen-reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.

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Sertoli cell binding to isolated testicular basement membrane.

The Journal of Cell Biology ◽

10.1083/jcb.103.3.1109 ◽

1986 ◽

Vol 103 (3) ◽

pp. 1109-1119 ◽

Cited By ~ 14

Author(s):

G C Enders ◽

J H Henson ◽

C F Millette

Keyword(s):

Basement Membrane ◽

Sertoli Cell ◽

Basal Lamina ◽

Sertoli Cells ◽

Matrix Material ◽

Three Dimensional ◽

Myoid Cells ◽

Cell Binding

We have examined the adhesion of primary Sertoli cells to a seminiferous tubule basement membrane (STBM) preparation in vitro. The STBM isolation procedure (Watanabe, T.K., L.J. Hansen, N.K. Reddy, Y.S. Kanwar, and J.K. Reddy, 1984, Cancer Res., 44:5361-5368) yields segments of STBM that retain their histotypic form in both three-dimensional tubular geometry and ultrastructural appearance. The STBM sleeves contain two laminae: a thick, inner basal lamina that was formed in vivo between Sertoli cells and peritubular myoid cells; and a thinner, outer basal lamina that was formed between myoid cells and sinusoidal endothelial cells. Characterization by immunofluorescence and SDS PAGE revealed that the isolated STBM retained fibronectin, laminin, and putative type IV collagen among its many components. When the STBM sleeves were gently shaken with an enriched fraction of primary Sertoli cells, the Sertoli cells bound preferentially to the lumenal basal lamina at the ends of the STBM sleeves. Few Sertoli cells bound to either the outer basal lamina of the STBM sleeves or to vascular extracellular matrix material which contaminated the STBM preparation. 3T3 cells, in contrast, bound to all surfaces of the STBM sleeves. Pretreatment of the STBM sleeves with proteases, 0.1 M Na metaperiodate, 4 M guanidine HCl, or heating to 80 degrees-90 degrees C inhibited lumenal Sertoli cell binding, but binding was not inhibited by chondroitinase ABC, heparinase, hyaluronidase, or 4 M NaCl. The lumenal Sertoli cell binding occurred in the presence or absence of added soluble laminin, but not fibronectin. The addition of soluble laminin, but not fibronectin, restored random binding of Sertoli cells to trypsinized STBM sleeves. Our in vitro model system indicates that Sertoli cells recognize differences in two basal laminae produced in vivo on either side of myoid cells.

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