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Histochem Cell Biol (2009) 131:755–764 DOI 10.



Claudin-11 is over-expressed and dislocated from the blood–testis barrier in Sertoli cells associated with testicular intraepithelial neoplasia in men
Cornelia Fink · Roswitha Weigel · Ludger Fink · Jochen Wilhelm · Sabine Kliesch · Martina Zeiler · Martin Bergmann · Ralph Brehm

Accepted: 6 February 2009 / Published online: 25 February 2009 © Springer-Verlag 2009

Abstract In mouse testis, claudin-11 is responsible for the formation of speciWc parallel TJ strands of the blood– testis barrier (BTB). Concerning the human BTB, there is no information about the transmembrane TJ proteins. We recently demonstrated the loss of functional integrity of the BTB in testicular intraepithelial neoplasia (TIN), associated with a dislocation of the peripheral TJ proteins ZO-1 and ZO-2. Here, we determined the expression and distribution of claudin-11 at the human BTB in seminiferous tubules with normal spermatogenesis (NSP) and TIN. Immunostaining of claudin-11 revealed intense signals at the basal BTB region in seminiferous epithelium with NSP. Within TIN tubules, claudin-11 immunostaining became diVuse and cytoplasmic. Double immunogold labeling demonstrated a co-localization of claudin-11 and ZO-1 at the inter-Sertoli cell junctions. Real-time RT-PCR of laser

microdissected tubules showed an up-regulation of claudin11 mRNA in TIN. Additionally, increased claudin-11 protein was observed by Western blot. We conclude that claudin-11 constitutes a TJ protein at the human BTB. In TIN tubules, claudin-11 is up-regulated and dislocated from the BTB. Therefore, the disruption of the BTB is related to a dysfunction of claudin-11 and not to a failure of its expression. Keywords Claudin-11 · Blood–testis barrier · Normal spermatogenesis · Testicular intraepithelial neoplasia · ZO-1

Introduction The blood–testis barrier (BTB) is basically constituted by parallel-array tight junctions (TJs) on lateral processes of Sertoli cells in proximity to the basement membrane of seminiferous tubules. These junctions are among the least permeable paracellular diVusion barriers, and their major functions are to shield diVerentiating spermatocytes against immune surveillance through the formation of an immunologically privileged adluminal compartment, to exclude blood-derived components from this adluminal space that might otherwise disrupt spermatogenesis, and to allow formation of a Xuid-Wlled lumen. As a result, the germ cells in the adluminal compartment become eVectively sealed oV from direct access to many nutrients and thus become dependent on the secretion of such factors by the Sertoli cells (Griswold 1995; Sharpe et al. 2003). Tight junctions are known to contain transmembrane proteins such as occludin (Furuse et al. 1993; Saitou et al. 1997; Cyr et al. 1999), claudins (Morita et al. 1999a; Hellani et al. 2000), and F11 receptor protein (also known as JAM-1) (Bazzoni et al. 2000), which extend into the intercellular

C. Fink (&) · R. Weigel · M. Zeiler · M. Bergmann · R. Brehm Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Frankfurter Str. 98, 35392 Giessen, Germany e-mail: L. Fink Department of Pathology, University of Giessen Lung Center, Giessen, Feulgenstrasse 12, 35392 Giessen, Germany L. Fink Institute of Pathology and Cytology, UEGP, Forsthausstr 1, 35578 Wetzlar, Germany J. Wilhelm Institute of Pathology, University Hospital Giessen and Marburg, Frankfurter Str. 96, 35392 Giessen, Germany S. Kliesch Department of Urology, University of Münster, Albert-Schweitzer-Straße 33, 48149 Münster, Germany


4 l 25 mM MgCl2. 1986. are the main constituents of the TJs (Furuse et al. TIN has been correlated with an impaired status of Sertoli cell diVerentiation (Rajpert-De Meyts and Skakkebaek 1993. 1999). Clark and Hirst 2002. Biometra. They are bound to intracellular peripheral proteins. Van Itallie and Anderson 2006). mean 30 years) with TIN and impaired spermatogenesis. ZO-2. according to the manufacturer’s protocol (Life Technologies. In mice and rats. RNA extraction. Germany) for 40 min at 37°C. 1999. The PCR master mix contained 31. Karlsruhe. 10 l 5£ Colorless Go-Taq-Flexi BuVer (Promega. Riesen et al. 1 l 10 pmol reverse primer. 2006a). where it is responsible for the formation of the typical parallel tight junction strands (Bronstein et al. according to the manufacturer’s protocol (Gibco BRL. 1999b. 2001. Göttingen. 2006). Materials and methods Testicular tissue After obtaining a written informed consent. 2006). To date. They are intercellular adhesion molecules that have variable pore-like properties (Tsukita and Furuse 2000. Gonzalez-Mariscal and Nava 2005). 1998). Fink et al. Thus. thereby regulating various cellular processes including cell growth and diVerentiation. investigations were performed on biopsies from 14 patients (ages 28– 47 years. and ZO-3 (Stevenson et al.25 l 5 U/ l Go-TaqFlexi DNA-Polymerase (Promega). 2000. We recently demonstrated the loss of functional integrity of the inter-Sertoli cell TJs in TIN.756 Histochem Cell Biol (2009) 131:755–764 space. 1999). First strand cDNA synthesis was performed using Superscript II Reverse Transcriptase. Concerning the TJ proteins of the human BTB. such as the zonula occludens proteins ZO-1. Brehm et al. 1 l cDNA and 0. the aim of the present study was to investigate if claudin-11 constitutes a part of the inter-Sertoli cell junctions in men and whether the disruption of the BTB and the dislocation of ZO-1 and ZO-2 in TIN tubules involve a diVerential expression of claudin-11. claudin-11 is speciWcally expressed in oligodendrocytes in brain and in Sertoli cells. More recently. 1998) and the downregulation of gap junctional connexin 43 (Brehm et al. 1998. Hewitt et al. Germany). 2002). as well as from patients with obstructive azoospermia. In the mouse testis. 20–27 kDa phosphoproteins. 1999. Reactions were run on a thermocycler (T3. Germany). Sonoda et al. Claudins. 1998. 2002. associated with a dislocation of ZO-1 and ZO-2 from the BTB region into the cytoplasm (Fink et al. leading to an arrest in spermatogenesis (Gow et al. claudin-11 expression in Sertoli cells is at its maximum between postnatal day 6 and 16. Morita et al. 2004) and mice invalidated for this gene are infertile. Eggenstein. 2000. Lee and Cheng 2004) as well as to signaling pathway molecules (Fanning et al. and tumorigenesis (Tsukita et al. 1999b). Roche. Testicular biopsy specimens were cut into two equal pieces. Germany) and then incubated with RNase-free DNase I (1–3 U/ g RNA. 2006). which tether the transmembrane proteins to the underlying actin cytoskeleton (Fanning et al. Testicular intraepithelial neoplasia (TIN) is the most common precursor of the testicular germ cell tumors (Skakkebaek 1972).75 l diethylpyrocarbonate-water (DEPC). 1 l 10 pmol forward primer. Zahraoui et al. only ZO-1 and ZO-2 have been positively identiWed to date (Moroi et al. 1999b). One part was Wxed by immersion in Bouin’s Wxative and embedded in paraYn wax using standard techniques. Germany). with the loss of claudin-11 expression causing a disruption of this barrier. Itoh et al.. during initial barrier formation (Hellani et al. Shima et al. the TJ is better known as a sophisticated apparatus capable of recruiting signaling proteins. Morita et al. 1 l 10 mM dNTP (Promega). Primers (Table 1) were purchased from MWG-Biotech (Ebersberg.g. The other part was snap-frozen in liquid nitrogen and stored at ¡80°C until further processing. 1999. The resultant complexes have long been associated with segregation of the apical and basolateral compartments and controlling the paracellular permeability (Anderson and Van Itallie 1995). who are otherwise healthy (without TIN or testicular germ cell tumors). 1999. The testicular biopsies with normal spermatogenesis were obtained mainly from vasectomised men before surgery for vasovasostomy. cDNA synthesis and reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenate RT-PCR was performed with tissue homogenate from frozen biopsies of four men with NSP. Mannheim. 1998. Heidelberg. Furuse et al. 2006a). Tsukita et al. about 24 diVerent claudins have been identiWed and many members of the claudin family show a distinct organ-speciWc distribution pattern (Morita et al. which includes e. 1997. Skakkebaek et al. DNase-treatment. Diagnosis of TIN was established by histologic and morphologic examination of the biopsy specimen and by placental alkaline phosphatase immunostaining. The resulting cDNA was forwarded to PCR. RNA was extracted with TRIzol® reagent. Germany) under the following conditions: 123 . the re-expression of cytokeratin 18 intermediate Wlaments (Kliesch et al. The sections (5 m) were stained with hematoxylin and eosin and scored according to Bergmann (2006). ZO1 and ZO-2 seem to be indispensable not only for initiating the polymerization of claudins but also for determining where claudins should be polymerized (Umeda et al. mean 39 years) with histologic normal spermatogenesis and 13 patients (ages 20–41 years.

4] Forward primer: 5Ј-GGT GGT GGG CTT CGT CAC GA-3Ј Reverse primer: 5Ј-GGC CCG CCT GTA CTT AGC CA-3Ј Amplicon size: 339 bp Quantitative RT-PCR Claudin-11/OSP [NCBI Accession: NM_005602. EO433. anti- -Actin mouse monoclonal antibody (1:5.1% Tween 20 (Sigma-Aldrich). Gaithersburg. RT).3] Forward primer: 5Ј-TGA TGA CAC GGG GCG ATC T-3Ј Reverse primer: 5Ј-GCT TGG AGG TGC TAG GAC TGG-3Ј Amplicon size: 82 bp 757 1 £ 95°C 2 min. All experiments were performed in triplicate. The proteins were fractionated on Bis-Tris gel 12% (Invitrogen. Finally. brain (mouse) membrane lysate—normal tissue (1:1. Darmstadt. The membrane was blocked with 5% bovine serum albumin (BSA) and 5% non-fat dry milk (Heirler Cenouis. Hamburg. Both controls were negative. Afterwards the sections were treated with an APAAP Kit mouse (1:50. CA. Cambridge. DeparaYnised and rehydrated sections (5 m) for claudin-11 immunostaining were microwave-treated at 1. Then. The sections were counterstained with hematoxylin for 10 s and rinsed with running water.1% Tween 20 were used as secondary antibodies (60 min. DAKO) and subsequently for 40 min at RT to a rabbit anti-mouse antibody (1:50. PCR products were separated on a 2% agarose gel and visualized with SYBR Green I (Sigma-Aldrich. Germany) under reducing conditions. The same amount of protein homogenate was loaded per lane. frozen testicular biopsies of ten men with NSP and three men with TIN were used. DAKO). Abcam) was used. USA). Karlsruhe.000 W in sodium citrate buVer (0. a mouse anti-rabbit IgG (1:50. Biometra Standard Power Pack P25) onto a nitrocellulose membrane (Invitrogen). Germany). a biotinylated goat anti-rabbit IgG (1:1. Germany). 55°C 30 s and 72°C 30 s].Histochem Cell Biol (2009) 131:755–764 Table 1 Primer pairs used for speciWc ampliWcation of claudin-11and insulin-like growth factor receptor-1-cDNA RT-PCR Claudin-11/OSP [NCBI Accession: NM_005602. Abcam) was used. Afterwards. 123 . The sections were then exposed for 30 min at RT to the biotinylated secondary antibody.4. The immunoreaction was visualized using Histo Mark Red (KPL) for 30 min at RT. Radolfzell. pH 7. Protein extraction and western blot analysis For western blot analysis.000. overnight at RT. DAKO) for -Actin in PBS with 1% BSA and 0. EO432. Germany) for claudin11 and a biotinylated goat anti-mouse IgG (1:1.1 M Tris–HCl buVer. pH 7. Immunohistochemistry Immunohistochemistry for claudin-11 was performed on testicular biopsies from 10 patients with histologically NSP (score 10–8) and from 10 patients with seminiferous tubules inWltrated with TIN and impaired spermatogenesis (score 7–0). they were incubated with a prediluted rabbit polyclonal anti-claudin-11/OSP C-terminal primary antibody (ab27565. DAKO. Calculation of conWdence intervals and two-sample t tests were performed using the pooled standard error estimate. Germany) in 0. pH 6. 35 £ [95°C 30 s.4) for 30 min at room temperature (RT) and incubated with anti-claudin-11/OSP rabbit polyclonal antibody (1:500. 1 h 15 min. Statistics for the relative claudin-11 expression in testis with NSP and with TIN were calculated using the log-transformed ratios of band intensities (Claudin-11/ß-actin). ab53041. Burglingam.000. using 1£ NuPAGE MOPS-SDS Running BuVer (Invitrogen) and the protein marker See Blue Plus 2 (Invitrogen). Finally the sections were mounted with Kaiser’s glycerol gelatine (Merck.0) for 20 min. DAKO) for 40 min at RT. mouse testes were used. Sequencing of the PCR-products was performed by the Qiagen Sequencing Service (Hilden. All experiments included controls lacking the reverse transcriptase-enzyme to check for contamination with genomic DNA as well as “no template controls” with DEPC water instead of cDNA to check for cross-contamination. Abcam) in a humid chamber at 4°C overnight. the membrane was treated with Vectastain Elite ABC Kit (Vector Laboratories.1 M phosphate-buVered saline (PBS.4] Forward primer: 5Ј-AGC TGG CTG GTG TTT TGC TC-3Ј Reverse primer: 5Ј-GCA CAC AGG GAA CCA GAT GG-3Ј Amplicon size: 71 bp Insulin-like growth factor receptor [NCBI Accession: NM_000875. western blots were carried out by omitting the primary antibody. and 72°C 7 min resulting in a product of 339 base pairs (bp) for claudin-11 mRNA. For loading control. omitting the primary antibody and were negative throughout. ab6276. For positive control. After blocking with 5% goat serum in PBS for 30 min at RT. For positive control. Sections were blocked with acetic acid 20% for 15 s at 4°C and with 5% BSA in Tris–HCl buVer for 30 min at RT. Protein extraction was carried out using the TRIzol® reagent as recommended by the manufacturer (Life Technologies). For quantiWcation of the western blots we performed a densitometric analysis using software (Image J). For negative control. The control sections were treated with 5% BSA in Tris– HCl buVer. proteins were blotted (30 V. UK) in PBS with 1% BSA and 0. Germany). Abcam. ab30149. USA) and developed with True Blue Peroxidase Substrate (KPL. Munich. using 1x NuPAGE MOPS-SDS Transfer BuVer (Invitrogen).01 m.000. Following each incubation the sections were washed thoroughly with 0.

the sections were incubated with secondary 5 nm gold-coupled goat F(abЈ)2 anti-rabbit IgG (British Biocell Int. omitting the primary antibody. The samples were observed with confocal microscopy (TE2000. Darmstadt. First strand cDNA was obtained using the Sensiscript RT Kit 200 (Qiagen). Finally. Wageningen. The reactions (Wnal volume: 50 l) were set up with the Platinum SYBR Green I qPCR Supermix UDG (Sigma-Aldrich) according to the manufacturer’s protocol using 2 l of 123 .5% BSA in Tris–HCl buVer and 4% donkey serum for 60 min at RT. The cells were spun down and dissolved in 350 l lysis buVer containing -mercaptoethanol and 5 l carrier RNA (RNeasy Micro Kit. Testicular biopsies from one testis with NSP and three testes containing TIN were Wxed in 4% paraformaldehyde and 0. Microdissection and laser pressure catapulting (LPC) were performed using the PALM MicroBeam system (PALM Microlaser Technologies. the sections were subsequently immersed in 70 and 96% ethanol and stored in 100% ethanol until use. Qiagen). Insulin-like growth factor receptor-1 (igfr-1) gene was used as reference. Alexa 488. No more than two sections were prepared at once to reduce the storage time. 2005a. Germany). the sections were washed three times for 5 min in PBS and 2 times for 5 min in bidistilled water and double-stained with uranyl acetate for 7 min and for 4 min with lead citrate. the sections were blocked with PBS with 0. After rinsing 5 times for 3 min with blocking buVer. UK) diluted 1:15 in PBS with 0. The negative controls. Ultrathin sections were cut perpendicular to the Wlters with the ultramicrotome Ultracut (Reichert.. This gene was reported to be unregulated in NSP versus seminomas (Neuvians et al. Wien. CardiV. The sections were washed in TBS for 5 min three times and mounted in Prolong Gold (Invitrogen). for 2 h at RT. Consecutive sections were than used for the microdissection approach.1% BSA-C.2% BSA-C (Aurion.01% trypsin for 8 min and microwavetreated at 800 W in sodium citrate buVer (0. The sections were blocked with 1. Oberkochen.758 Histochem Cell Biol (2009) 131:755–764 ImmunoXuorescent labeling with confocal microscopy For immunoXuorescence studies.) diluted 1:10 in PBS with 0. Austria).01 m. applying cDNA of testis homogenate (10 patients with NSP. 1987). Laser-assisted microdissection Laser-assisted microdissection was performed. The RNA from testicular tissue was extracted using the RNeasy Micro Kit. San FranTINco.2% Tween 20. b). testicular biopsies from four patients with histologically NSP (score 10–8) and from three patients with seminiferous tubules inWltrated with TIN and impaired spermatogenesis (score 7–0) were used. USA) diluted in PBS with 0. donkey-anti-rabbit. The sections were placed on collodion-coated nickel grids and nonspeciWc binding sites were blocked by incubating the sections for 45 min on a drop of blocking buVer (PBS with 0. Fink and Bohle 2002). After hemalaun staining for 45 s. washed and incubated in secondary antibody (1:200. England). dehydrated and embedded in LRWhite Resin (TAAB Laboratories. The sections were evaluated and photographed with a Zeiss EM 109 (Zeiss. the sections were incubated with secondary 10 nm gold-coupled goat F(abЈ)2 anti-rabbit IgG (British Biocell Int. pH 6. on serial cryo-sections of biopsies from ten patients with NSP and ten patients with TIN.35. as described in detail previously (Fink et al.1% glutaraldehyde in 0.1% BSAC. The cap contained 2 l of mineral oil to achieve a better attachment of the catapulted tubules. Nikon). and DNase-I-digestion was performed according to the manufacturer’s protocol.2% BSA-C and 0. Netherlands) and 0. omitting the primary antibodies. Sections were Wrst incubated with rabbit polyclonal anti-ZO-1 antibody (1:30. Abcam). immunostaining for placental alkaline phosphatase was performed to detect tubules containing TIN (Beckstead 1983. Quantitative real-time PCR of tissue homogenate and isolated tubules The regulation of claudin-11 mRNA was analyzed by realtime quantitative PCR using the CT method for the calculation of relative changes (Livak and Schmittgen 2001).1 mol/l cacodylate buVer adjusted to pH 7. and were negative throughout. Germany). Zymed. Then sections were incubated with a prediluted rabbit polyclonal anti-claudin-11/OSP C-terminal primary antibody (ab27565. The cryo-sections (10 m) were mounted on glass slides. 2000. Secondly. Invitrogen) for 60 min at RT. Abcam) overnight at 4°C.1% Tween 20 before labeling with the rabbit polyclonal anti-claudin 11/OSP C-terminal IgG (prediluted. The control sections were treated with 1. a postembedding protocol was employed. Double-immunogold labeling For immunoelectron microscopy. Bernried. biopsies were rinsed in buVer. Real-time PCR was performed by the Sequence Detection System 7900 (PE Applied Biosystems. Germany) to isolate selected tubules. In order to identify and diVerentiate tubules with NSP and TIN in the same cryo-section. D-ECLIPSE C1. 10 patients with TIN) and microdissected tubules. The tubules (30 tubules per section) were catapulted in the cap of a 500 l reaction tube. DeparaYnised and rehydrated sections (5 m) were permeabilised with 0. After rinsing Wve times for 3 min with blocking buVer.0) for 15 min.1% Tween 20) at RT. Skakkebaek et al. Afterwards. Aldermaston.4% BSA-C and 0. produced no labeling.5% BSA in Tris–HCl buVer.

3.156 0. d). In NSP. Double-immunogold labeling To evaluate the exact ultrastructural localization of claudin11.159 CI high 0.8-fold increased in testis with TIN as compared to testis with NSP (P = 0. Claudin-11 was detected at 22 kDa. 2 Western blot analysis for claudin-11. +.sem pooled. SD standard deviation. Mean and 95% conWdence intervals. The cycling conditions were 50°C for 2 min. mRNA encoding for claudin-11 was identiWed in tissue homogenate of testes with NSP. In contrast.088 0. d). 50£ [95°C for 5 s. and immunoXuorescent labeling with confocal microscopy Western blot analysis revealed claudin-11 immunoreactivity by a single band at 22 kDa in testis with NSP and showed a higher expression of claudin-11 in testis with TIN (Fig. claudin-11 immunostaining was detectable in the basal compartment of the seminiferous epithelium forming an almost continuous belt (Fig.227 Results RT-PCR Using RT-PCR. 4a. Protein expression for claudin-11 was determined in testis with normal spermatogenesis (NSP) and with testicular intraepithelial neoplasia (TIN) (n = 3 per group).010 Fig. Fig. 5c. In tubules containing TIN cells. we performed double-immunogold labeling for claudin11 and for the known TJ associated protein ZO-1. immunohistochemistry. Sequencing of the PCR-product conWrmed the identity of human claudin-11 mRNA. Due to the non-selective dsDNA binding of the SYBR® Green I dye.01). In tubules showing NSP (Fig. positive control. SEM standard error of the mean. 1 RT-PCR analysis for claudin-11 mRNA using testis homogenate. Claudin-11 immunostaining became more diVuse and irregular at the BTB region and pooled. consistent with its localization at the BTB (Fig. a speciWc ampliWcation product of expected size at 339 bp is obtained in testis with normal spermatogenesis (NSP) additional cytoplasmic immunoreaction was observed (Fig. b).538 -0. CI conWdence interval Fig. -actin was used as a loading control. 6a) as well as in TIN tubules 123 . the distribution pattern of claudin-11 diVered from that in normal tubules.126 0. 2).056 0. Using immunohistochemistry on sections with NSP. Primer pairs given in Table 1 were purchased from MWG-Biotech and were used in a Wnal concentration of 200 nM. 4c. 3 Relative claudin expression in testis with normal spermatogenesis (NSP) and in testis with testicular intraepithelial neoplasia (TIN) (n = 3 per group).280 back-transformed mean 0. TIN tubules showed extensive staining of the Sertoli cell cytoplasm (Fig. 3 log mean NSP TIN -0. Western blot. melting curve analysis and gel electrophoresis were performed to conWrm the exclusive ampliWcation of the expected PCR product.Histochem Cell Biol (2009) 131:755–764 759 cDNA from homogenates or 8 l of cDNA from microdissected tubules.525 CI low 0. ImmunoXuorescent microscopy conWrmed the immunohistochemical results.290 0. 95°C for 6 min. 1).097 0. By pooled. 59°C for 5 s and 72°C for 10 s]. b). 5a. detecting a speciWc PCR-product at 339 bp (Fig. The average claudin expression was 1. The relative claudin-11 expression in testis with NSP and with TIN is shown in Fig. claudin-11 was found to localize in a linear fashion at the basal compartment of the seminiferous epithelium. Densitometric analysis of the data is illustrated in Fig.CI t-test 0.

Fig. Scale bar 100 m.760 Histochem Cell Biol (2009) 131:755–764 (Fig. claudin-11 immunostaining was localized in the basal compartment.00096. the regulation of claudin-11 mRNA was analyzed by quantitative real-time PCR.3) of claudin-11 mRNA in TIN compared to NSP (P = 0.4. indicating that these proteins are co-localized at the human BTB. Scale bar 50 m.97. claudin-11 (10 nm gold particles) co-localizes with ZO-1 (5 nm particles) predominantly in sheets of Sertoli cell plasma membranes at the region of the inter-Sertoli cell junctional complexes.8. b Enlargement of inset: immunoreactive claudin-11 is localized in a linear fashion at the basal compartment. Scale bar 50 m.…6. b Enlargement of inset: Claudin11 immunostaining was localized in a linear fashion at the BTB region. Scale bar 10 m. 7a). In the normal seminiferous epithelium. c Representative cross-section of a seminiferous tubule containing TIN. 4 Immunohistochemical localization of claudin-11 in testes with NSP and TIN. 7b). isolation of seminiferous tubules resulted in a signiWcant increase (mean factor 3. Discussion In the present study. In contrast. Scale bar 50 m. Fig. c Cross-sections of seminiferous tubules with TIN. claudin-11 particles were found neither at Sertoli-germ cell interface nor at the germ–germ cell interface. Scale bar 100 m. we detected for the Wrst time claudin11 on mRNA and protein level in human seminiferous tubules with NSP and TIN. Scale bar 10 m Fig. 1. Double-immunogold labeling conWrmed Fig. This expression coincided with the distribution pattern of ZO-1 and ZO-2 (Fink et al. corresponding to the BTB region. a Representative cross-section of a seminiferous tubule with NSP. 2006a) and connexin 43 (Brehm et al. a Crosssections of seminiferous tubules with NSP. Scale bar 50 m 123 . Using tissue homogenate of NSP versus TIN carrying testes. As expected. 2002. d Enlargement of inset: diVuse cytoplasmic staining of claudin-11 in Sertoli cells was found. 2006). d Enlargement of inset: Claudin-11 immunostaining becomes more irregular and diVuse and additional cytoplasmic immunoreaction was observed in the Sertoli cells. 5 ImmunoXuorescence labeling and confocal microscopy of seminiferous tubules with NSP and TIN. no diVerence of claudin-11 mRNA levels was detectable (P = 0. consistent with the site of the BTB. Determination of regulation by real-time RT-PCR To clarify whether claudin-11 mRNA is diVerentially expressed in TIN tubules compared to normal tubules. 6b).

These results conWrm that utilizing tissue homogenates confers the risk of masking genetic deviations or expression changes of an individual cell type by the bulk of surrounding cells (Fink et al. 2004. Scale bar 10 m. d Enlargement of inset: co-localization of claudin-11 (arrowhead) and ZO-1 (arrow) at the site of inter-Sertoli cell junctional complex. Interestingly. a Electron micrograph of two Sertoli cells in a seminiferous tubule showing NSP. Matsuda et al. A possible explanation for the enhanced claudin-11 mRNA expression in tubules containing TIN cells but no normal germ cells may be that in normal spermatogenesis. Using quantitative real-time RT-PCR of microdissected tubules. in testicular TIN the alteration of TJ and the up-regulation of claudin-11 mRNA do not take place in the neoplastic germ cells but in the adjacent Sertoli cells. Scale bar 2 m. where TJ loss can account for cancer progression. Claudin family members are the main sealing components of TJ and therefore. Our results are in line with Tarulli et al. Scale bar 0. Our Wndings are also supported by other studies demonstrating a direct correlation between organization of TJ proteins and integrity of the BTB in rats (Chung and Cheng 2001. b Enlargement of inset: co-localization of claudin-11 (arrowhead) and ZO-1 (arrow) at the site of inter-Sertoli cell junctional complex. The elevated level of claudin11mRNA in TIN tubules leads to a signiWcantly up-regulation of claudin-11 protein. at the same time.1 m 761 that claudin-11 co-localizes with ZO-1 in the sheets of Sertoli cell plasma membranes at the BTB region. who showed an aberrant cytoplasmic localization of claudin-11 and ZO-1 in Sertoli cells of the adult short-day Djungarian hamster where serum gonadotropins are low.Histochem Cell Biol (2009) 131:755–764 Fig. Unlike the mentioned reports. as depicted by western blot analysis and densitometry. 2006). 2007). promoting tumor development (Martin and Jiang 2001. Wong et al. Soini 2005. to an up-regulation of claudin-11 mRNA. Scale bar 0. 2006a). This correlates with the known dislocation of ZO-1 and ZO-2 and the loss of BTB functionality in seminiferous epithelium containing TIN (Fink et al. Xia et al. (2008) who reported that the suppression of gonadotropins in the adult hamster leads to a disorganization of claudin-11 localization associated with a loss of BTB function and. Tracer permeability experiments have further shown previously that TJ is non-functional in the short-day hamster but functional in the long-day hamster (Bergmann 1987). 2007). 2006b).4-fold relative increase of claudin-11 mRNA in TIN tubules as compared with normal tubules. 6 Double-immunogold labeling for claudin-11 (10 nm gold beads) and ZO-1 (5 nm gold beads) of seminiferous tubules with NSP and TIN. 123 . deregulation of claudin expression may aVect the permeability of the intercellular barrier. c Electron micrograph of a seminiferous tubule containing TIN cells. (2006). applying tissue homogenate for quantitative real-time RT-PCR. we found a 3. there was no signiWcant diVerential expression of claudin-11 mRNA in tubules with NSP versus TIN. Therefore. Our results are consistent with Wndings from Tarulli et al. The TJ structure and function are often found to be altered in human carcinomas. changes in the localization of claudin-11 immunostaining were evident. Abnormal regulation of the claudin TJ proteins has been reported in various human cancers including both increased and decreased expression levels of speciWc claudins (Morin 2005. the presence of cytoplasmic reactions suggests that targeting of the respective TJ protein is compromised. In TIN tubules. Sheehan et al. especially through the loss of cell–cell adhesion and cell diVerentiation (Oliveira and MorgadoDíaz 2007).1 m. The basolateral reactions were more irregular and diVuse and complemented by cytoplasmic reactions in Sertoli cells.

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