the journal of biological chemistry © 1998 by the … · were found at the site of inter-sertoli...

Testin Secreted by Sertoli Cells Is Associated with the Cell Surface,and Its Expression Correlates with the Disruption of Sertoli-GermCell Junctions but Not the Inter-Sertoli Tight Junction*

(Received for publication, March 13, 1998, and in revised form, June 2, 1998)

Josephine Grima‡, Connie C. S. Wong‡§, Li-ji Zhu‡, Shu-dong Zong‡, and C. Yan Cheng‡¶

From the ‡The Population Council, New York, New York 10021 and §Department of Zoology, University of Hong Kong,Hong Kong, People’s Republic of China

Testin is a testosterone-responsive Sertoli cell secre-tory product. In the present study, we demonstratedthat the amount of testin secreted by Sertoli cells invitro was comparable with several other Sertoli cell se-cretory products. However, virtually no testin wasfound in the luminal fluid and cytosols of the testis andepididymis when the intercellular junctions were notpreviously disrupted, suggesting that secreted testinmay be reabsorbed by testicular cells in vivo. Studiesusing Sertoli cells with and without a cell surface cross-linker and radioiodination in conjunction with immuno-precipitation illustrated the presence of two polypep-tides of 28 and 45 kDa, which constitute a bindingprotein complex that anchors testin onto the cell sur-face. The 28- and 45-kDa peptide appear to be residingon and inside the cell surface, respectively. ImmunogoldEM studies illustrated testin was abundantly localizedon the Sertoli cell side of the ectoplasmic specialization(a modified adherens junction) surrounding developingspermatids. In contrast, very few testin gold particleswere found at the site of inter-Sertoli tight junctions.When the inter-Sertoli tight junctions were formed ordisrupted, no significant change in testin expressionwas noted. This is in sharp contrast to the disruption ofSertoli-germ cell junctions, which is accompanied by asurge in testin expression. These results demonstratethe usefulness of testin in examining Sertoli-germ cellinteractions.

Testin, a testosterone-responsive glycoprotein secreted byrat Sertoli cells in vitro, consists of two highly homologousvariants with an apparent Mr of 35,000 and 37,000 (1–4).Immunofluorescent microscopy and immunohistochemistry re-veal that testin resides near the basal lamina of the seminif-erous epithelium in most stages of the cycle, consistent with itslocalization at the Sertoli-germ cell junction (5–6). However, atransient but drastic increase in testin accumulation was notedbetween Sertoli cells and the head of elongated spermatids atearly stage VIII preceding spermiation (7), consistent with itslocalization at the ectoplasmic specialization, which is a mod-ified adherens junction (see Table I). These observations sug-

gest that testin may be a sensitive marker in examining thecellular events of Sertoli-germ cell interactions.

Once the mRNA sequence of testin was known, Northernblots and reverse transcription-polymerase chain reaction wereused to survey the testin mRNA distribution in multiple organsfrom both adult and immature male and female rats. It wasfound that testin is predominantly expressed in the gonad (6).We postulated that this expression correlated with the rapidturnover of intercellular junctions in the testis and ovary dur-ing germinal cell development. This hypothesis was supportedby the observations that testin mRNA can also be detected innon-gonadal tissues such as pre- and neo-natal rat kidney atthe time of extensive tissue restructuring due to organ growth(8). Moreover, the steady-state testin mRNA level in the ovaryis high at proestrus, estrus, and metestrus, correlating with thematuration of intrafollicular ova and the eventual rupturing ofthe follicle at ovulation that coincides with the rapid turnoverof inter-granulosa cell junctions (8, 9). In addition, testin ex-pression was drastically reduced to an almost undetectablelevel at diestrus, during which functional regression of thecorpora lutea occurs (8, 9).

Other recent in vivo and in vitro studies reveal that testin isa sensitive marker to monitor the disruption of intercellularjunctions in the testis, since the expression of testin is posi-tively correlated to this event (8). For instance, a surge in testinexpression and an intense accumulation of its protein in thecytosol of the testis are found when germ cells, mainly roundand elongated spermatids, were depleted from the seminifer-ous epithelium by either lonidamine (8), busulfan (2, 3), orX-irradiation (10) at the time when Sertoli-germ cell junctionswere disrupted. A brief hypotonic treatment lysing germ cellsin Sertoli-germ cell cocultures, thereby disrupting the inter-Sertoli-germ cell junctions, also induced a drastic increase intestin expression by Sertoli cells (8). These studies, however,cannot distinguish whether the observed drastic increase intestin expression correlates with the disruption of inter-Sertolitight junctions or adherens and gap junctions, which are foundbetween Sertoli cells as well as between Sertoli and germ cells(Table I; for reviews, see Refs. 11–13). As such, we have used anestablished culture model that selectively disrupts tight junc-tions in vitro (14) by [Ca21] depletion using primary Sertolicells cultured in vitro to assess whether such a disruption isassociated with any changes in testin expression. In addition,we seek to (i) identify the distribution of testin in the epithe-lium by immunogold EM to define its subcellular localization,(ii) examine the effect of anti-testin IgG on the re-establish-ment of inter-Sertoli tight junctions after their disruption asassessed by the transepithelial electrical resistance (TER)1

* The work was supported in part by Contraceptive Research andDevelopment Program (CONRAD) Grant CIG-96-05, Rockefeller Foun-dation Grants PS9528, PS9601, and PS9721, National Institutes ofHealth Grant HD-13541, and grants from the Noopolis Foundation. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

¶ To whom correspondence should be addressed: The PopulationCouncil, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8738; Fax:212-327-7678; E-mail: [email protected].

1 The abbreviations used are: TER, transepithelial electrical resist-ance; b.w., body weight; DMEM, Dulbecco’s modified Eagle’s medium;

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 33, Issue of August 14, pp. 21040–21053, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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measurement, (iii) characterize the binding protein complexthat anchors testin onto cell surface, and (iv) examine whetherthe expression of testin is mediated by germ cells or is depend-ent on the status of the intercellular junctions.

MATERIALS AND METHODS

Animals and Preparation of Biological Fluids

Adult or immature Sprague-Dawley rats were obtained from CharlesRiver Laboratories (Wilmington, MA). Preparation of testicular andepididymal cytosols from adult rats (450 gm b.w.) were performed asdescribed previously (1, 2). Rete testis fluid was collected from rats (450gm b.w.) by micropuncture 4 h after ligation of efferent ducts while ratswere anesthetized with sodium pentobarbital (40 mg/kg b.w., intraperi-toneal) as described (15, 16). The use of animals for studies described inthis report was approved by the Rockefeller University InstitutionAnimal Care and Use Committee with Protocol Numbers 94–132, 95–129, 95–29-R1, and 97–117.

Preparation of Testicular Cell Cultures

Sertoli Cells Cultured at Low Density—Primary Sertoli cells wereprepared from 20-day-old Sprague-Dawley rats as described previously(6, 8). All the experiments using Sertoli cell-enriched cultures reportedin this paper were derived from 20-day-old rats unless otherwise spec-ified. Cells were plated at a density of 4.5 3 106 cells/9 ml/100-mm dish(about 5 3 104 cells/cm2) in serum-free Ham’s F12 nutrient mixture/Dulbecco’s modified Eagle’s medium (F12/DMEM, 1:1, v/v) supple-mented with insulin (10 mg/ml), human transferrin (5 mg/ml), bacitracin(5 mg/ml), and epidermal growth factor (5 ng/ml) and incubated at 35 °Cin a humidified atmosphere of 95% air, 5% CO2 (v/v). About 48 h afterplating, cultures were hypotonically treated with 20 mM Tris-HCl, pH7.4, for 2.5 min to lyse residual germ cells (17) to obtain Sertoli cellcultures with greater than 95% purity. Cells were washed twice withF12/DMEM, and the cells were allowed to recover for an additional 24 hbefore their use. Under these conditions, specialized tight junc-tions were not formed when assessed by various criteria as describedpreviously (4).

Sertoli Cells Cultured at High Density—To assess the effect of anti-testin IgG or [Ca21] depletion on the inter-Sertoli tight junction, pri-mary Sertoli cells isolated as described above from 20-day-old rats werecultured at high cell density to allow the establishment of specializedjunctions. Briefly, about 2 3 106 cells/cm2 were plated on MatrigelTM

(1:8)-coated HA filters in the apical chamber of a bicameral unit (Mil-lipore, Bedford, MA) (4). To assess the formation of tight junctions, TERacross the Sertoli cell columnar monolayer was quantified using aMillicell electrical resistance system. Briefly, current was passedthrough the epithelial monolayer between two silver-silver chlorideelectrodes. Resistance was calculated from the change in voltage acrossthe monolayer induced by a short (;2 s) 20-mA pulse of current. Theresistance was multiplied by the surface area of the filter to yield theareal resistance in ohms/cm2. The net value of electrical resistance wasthen computed by subtracting the background, which was measured onMatrigel-coated cell-free chambers, from values of Sertoli cell-platedchambers. Disruption of the tight junctions were achieved by incubat-ing the Sertoli cell monolayer in [Ca21]-free F12/DMEM for 15 min asdescribed (14), which is manifested by a drastic decline in TER. There-after, cells were returned to [Ca21]-containing F12/DMEM with orwithout anti-testin IgG or normal rabbit serum IgG (200 mg/ml) for 20min at room temperature with gentle rocking and then returned to35 °C for the re-establishment of the tight junction as described previ-ously (14). Disruption and reformation of the tight junction was as-sessed by TER measurement. IgG was purified from decomplementedsera (56 °C for 30 min) by sequential ammonium sulfate precipitationand DEAE chromatography as described previously (18). Each timepoint contained triplicate cultures, and each experiment was repeatedat least three times using different batches of cells.

Primary Sertoli Cell Cultures from 35- and 90-day-old Rats—Pri-mary cultures of Sertoli cells from 35- and 90-day-old Sprague-Dawleyrats were prepared essentially as described previously (19). The cellswere suspended in F12/DMEM supplemented with various factors asdescribed for immature Sertoli cells (see above) and plated at approxi-

mately 4.5 3 106 cells/9 ml/100-mm dish and cultured for 2 days at35 °C with 95% air, 5% CO2. Thereafter, cells were hypotonicallytreated to remove the residual germ cells (17). The resulting cell puritywas about 85% when judged microscopically, and these cells were usedfor RNA extraction after 4 days in culture to compare the basal steady-state testin mRNA level between Sertoli cells isolated from rats ofdifferent ages.

Germ Cells—Total germ cells were isolated from 90-day-old Sprague-Dawley rat (about 300 gm b.w.) testes by a mechanical procedurewithout any enzymatic treatment as detailed elsewhere (20). These cellpreparations consisted largely of spermatogonia, spermatocytes, andround spermatids with a relative percentage of 16:19:65 when verifiedby microscopic examination and DNA flow cytometry as described (20,21). Virtually all elongated spermatids were removed in the glass woolfiltration step (20). Germ cells were cultured at a density of 22.5 3 106

cells/9 ml of F12/DMEM supplemented with sodium DL-lactate (6 mM)and sodium pyruvate (2 mM) in 100-mm dishes for 20 h at 35 °C toobtain germ cell-conditioned medium (GCCM) as described (20, 21) orused immediately after their isolation for binding and coculture exper-iments. These cells were largely free of somatic cell contamination whenassessed by various criteria as detailed elsewhere (20).

Sertoli-Germ Cell Cocultures—To assess the effects of germ cells onSertoli cell testin expression, primary Sertoli cells isolated from 20-day-old rats were plated at 5 3 104 cells/cm2 and cultured for 2 days;thereafter, cells were hypotonically treated to remove contaminatinggerm cells (17) (day 0). Cells were then cultured for an additional 24 h(day 1), washed once, and cultured for 3 additional days (day 4) toensure that the testin steady-state mRNA level had returned to baseline since the culture procedure per se can induce damage on theintercellular junctions, which is a potent stimulator of testin expression(8). Thereafter, different amounts of germ cells using a Sertoli:germ cellratio of 1:2.5, 1:5, and 1:10 were added to Sertoli cells and cultured foran additional 20 h (day 5). It must be noted that under these conditions,specialized junctions between Sertoli and germ cells had not yet formed,since their formation would require at least 24–48 h in culture (22, 23).Before their termination, cultures were subjected to a hypotonic treat-ment to lyse germ cells 20 min before the addition of RNA STAT-60TM

for RNA extraction to eliminate RNA contributed by germ cells in thesample to be analyzed. As such, the RNA that were examined in thisexperiment were largely derived from Sertoli cells. To assess the effectof the disruption of Sertoli-germ cell junctions, some cocultures that hadincubated for 30 h (day 5) were subjected to a hypotonic treatment onday 5 that was 24 h before their termination on day 6. Control disheswere Sertoli-germ cell cocultures incubated until day 6 without hypo-tonic treatment on day 5 but subjected to a hypotonic treatment 20 minbefore termination to eliminate RNA contributed by germ cells in thesample to be analyzed.

Pulse-Chase Analysis of the Synthesis and Secretion of Testin bySertoli Cells in Sertoli-Germ Cell Cocultures

To assess the effect of germ cells or GCCM on the synthesis andsecretion of testin by immature Sertoli cells in vitro, Sertoli cells wereprepared at a density of 4.5 3 106 cells/9 ml/100-mm dish as describedabove. Twenty four h after the hypotonic treatment, germ cells (10 3106 cells/dish) or GCCM (10 mg of protein/dish) were added and cocul-tured with Sertoli cells for 18 h in F12/DMEM containing 1/100th of thenormal methionine concentration. The cells were then pulse-labeled for15 min with 100 mCi of [35S]methionine per dish and subsequentlywashed three times to remove any remaining [35S]methionine. At spec-ified time points, media and cells were harvested and stored at 220 °Cuntil used. Immunoprecipitation was performed as described (4). In oneset of control experiments (Con 1), Sertoli cells were cultured alonewithout germ cells or GCCM and immunoprecipitated with the testinantibody at specified time points. In other controls (Con 2), Sertoli cellswere cultured alone but immunoprecipitated with preimmune serum toassess the specificity of the testin antibody. Samples were resolved onto10% T polyacrylamide gels and visualized by autoradiography.

RNA Extraction and Northern Blot Analysis

Total RNA was extracted from cell cultures or tissues using RNASTAT-60TM as described previously (6, 8, 24). Northern blot analysiswas performed as described previously using a a-32P-labeled testincDNA probe by nick translation (6) for hybridization. To ensure thatequal amounts of RNA were loaded into each lane, some blots wererehybridized with a a-32P-labeled b-actin cDNA probe (6), and datawere normalized after densitometric scanning analysis.

GCCM, germ cell-conditioned medium; PBS, phosphate-buffered saline;BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis;ECM, extracellular matrix; DTSSP, 3,39-dithiobis(sulfosuccinimidyl)propionate; EM, electron microscopy; RIA, radioimmunoassay; bFGF,basic fibroblast growth factor; SC, Sertoli cells.

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Glycerol-induced Intercellular Junction Damage in the Testis

Recent studies have shown that glycerol can induce permanent dam-age of the blood-testis barrier and inhibit spermatogenesis by disrupt-ing the inter-Sertoli cell tight junction near the basal lamina within 2weeks after glycerol administration (25). As such, we seek to examine ifthere is any change in testin expression when the tight junctionsbetween Sertoli cells are disrupted 2 weeks after glycerol treatment andto examine the changes of testin expression when germ cells are laterdepleted. A 20% glycerol solution was prepared in PBS (10 mM sodiumphosphate, 0.15 M NaCl, pH 7.4) and used to induce intercellular junc-tion damage in the testis after intratesticular injection as described(25). Adult Sprague-Dawley rats approximately 90 days of age (about250–300 gm b.w.) were used. Animals were anesthetized with Meto-faneR before treatment. Control and test animals received 200 ml of PBSor 20% glycerol solution by intratesticular injection through the polaraxis of each testis, respectively. Four to six rats were used per treat-ment group and rats were sacrificed by CO2 asphyxiation at specifiedtime points. Northern blot analysis and immunohistochemistry wasperformed as described previously (6, 8, 24). Tissue homogenates of thetestis were also prepared as described previously (2).

Binding and Uptake of 125I-Testin by Sertoli Cells

Purified testin isolated from SCCM as described previously (2) wasradiolabeled with 125I-Bolton-Hunter reagent (specific activity, 3056–3324 Ci/mmol, NEN Life Science Products) to a specific activity ofapproximately 50 mCi/mg of protein. For binding experiments, Sertolicells were plated in 12-well dishes at a concentration of 5 3 105 cells/well/ml F12/DMEM. These cells were used on day 3. Cells were washedthree times with F12/DMEM containing 1% BSA. Approximately 1 3106 cpm of 125I-testin were added per well in duplicate with or withouta 100-fold excess of unlabeled testin and incubated at 35 or 4 °C forseveral different time points. Cells were then washed three times inF12/DMEM containing 1% BSA (w/v) to remove unbound 125I-testin.Cells were solubilized in 1 M NaOH for radioactivity determination.Specific binding was determined from the difference between totalbinding and nonspecific binding (in the presence of 100-fold excessunlabeled testin). Radioactivity in solubilized cell extracts was deter-mined in a Packard Cobra II g-counter.

Membrane Preparation and Solubilization of 125I-Testin

Primary monolayer cultures of Sertoli cells were prepared from 20-day-old rats as described above and plated at a density of 4.5 3 106

cells/9 ml/100-mm dish. Four days after hypotonic treatment, Sertolicells were washed twice with media and incubated with 5 3 106 cpm125I-testin/5 ml of F12/DMEM for 4 h at 35 °C. Cells were washed threetimes with fresh media and scraped off in PBS containing 2 mM ofphenylmethylsulfonyl fluoride and 2 mM N-ethylmaleimide to inhibitprotease activity. Cell membranes were disrupted by repeated freeze-thawing. Membranes were pelleted for 15 min at 12,000 3 g andwashed three times to remove residual cytosolic proteins. The mem-branes were then solubilized with 100 ml of corresponding buffer andheated at 100 °C for 10 min, and testin was visualized by autoradiog-raphy after SDS-PAGE. The membrane solubilization buffers consistedof 20 mM Tris containing 1.6% 2-mercaptoethanol with or without 1%SDS, 1% Triton X-100, or 1% Nonidet P-40. All buffers were adjusted topH 6.8 at 22 °C. Extracellular matrix (ECM) was the remaining amor-phous substances in the dishes used for membrane preparation asdescribed previously (52). It was extracted with the same buffers asdescribed above by heating at 100 °C for 10 min.

Sertoli Cell Membrane and Cell Surface Labeling

Sertoli cells were cultured at high cell density (0.75 3 106 cells/cm2)on Matrigel (1:5 diluted with F12/DMEM)-coated dishes as described (4)to allow the formation of specialized junctions. Cell membranes wereprepared as described above for iodination. For surface labeling, livecells were cross-linked with 3,39-dithiobis(sulfosuccinimidyl) propionate(DTSSP, a membrane-impermeable and thiol cleavage cross-linker) asdescribed by the manufacturer (Pierce) before Na[125I] labeling usingIodogen (26), and therefore only proteins on the cell surface werelabeled. About 500 mg of protein of Sertoli cell membrane or DTSSP-cross-linked cells (12 3 106) were labeled with 2.5 mCi of Na[125I] in aPierce Reacti-vial coated with 100 mg of Iodogen (26). Free Na[125I] wasremoved by dialysis against 6 liters of 20 mM Tris, pH 7.4 with 2–3changes over a period of 2 days. 125I-Membrane ghosts were solubilizedin 20 mM Tris, pH 7.4, containing 0.1% Brij 97, 0.1% SDS, 0.1% TritonX-100, 2 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA for 3 h at37 °C. Samples were dialyzed overnight in PBS to remove detergents.

Immunoprecipitation was then performed using either testin antibodyor preimmune serum (1:50) overnight at 4 °C with agitation on a rota-tor. Immunocomplexes were purified on a protein A-Sepharose column(0.75 3 10 cm, inner diameter) previously equilibrated with 10 columnvolumes of PBS. Nonbound proteins were removed by a 15-columnvolume of PBS. Specifically bound proteins were eluted from the columnusing 0.1 M glycine, pH 3.5, and collected in 0.5-ml fractions. Aliquots (5ml) were counted in a Packard Cobra II g-counter. Selected fractionswere resolved by SDS-PAGE onto a 10% T SDS-polyacrylamide gel andvisualized by autoradiography. The monospecificity of the testin anti-body used in this study has been characterized and described (1, 2, 4).

Ultrastructural Localization of Testin in the SeminiferousEpithelium by Immunogold Electron Microscopy.

For immunogold EM study, adult male Sprague-Dawley rats (250 gmb.w.) were used, and the animals were sacrificed by CO2 asphyxiation.Testes were perfused via the testicular artery with ice-cold physiologi-cal saline to remove contaminating blood for 10 s, followed by 0.1 M PBS,pH 7.2, containing 0.2% glutaraldehyde and 4% paraformaldehyde for30 min. The testes were quickly cut into small pieces and immersed inthe same fixatives containing 4% sucrose at 4 °C for 2 h. After thor-oughly washing with the same buffer, samples were dehydrated inascending concentrations of ethanol at low temperature: 30% ethanol,30 min at 4 °C; 50% ethanol, 30 min at 220 °C; 70% ethanol, 30 min at235 °C; 95% ethanol, 30 min at 235 °C; 100% ethanol, twice for 30 minat 235 °C. Lowicryl K4M infiltration and embedding solutions wereprepared according to the manufacturer’s specifications (Polysciences,Inc. Warrington, PA). Dehydrated specimens were infiltrated as fol-lows: Lowicryl/ethanol 1:1, 60 min at -35 °C; Lowicryl/ethanol 2:1, 60min at 235 °C; and Lowicryl overnight at 235 °C. The specimens werethen transferred to Lowicryl embedding solution in precooled beamcapsules. Polymerization was induced by UV irradiation at 235 °Covernight followed by 2 days of UV irradiation at room temperature.Semi-thin sections stained with toluidine blue were used to selectrelevant tissue and cells. Thin sections cut by an ultramicrotome weremounted on formaval-coated nickel grids for subsequent immunogoldlocalization. The sections were exposed to undiluted goat serum for 1 hat room temperature, then incubated overnight at 4 °C on drops ofaffinity-purified anti-testin IgG. Affinity chromatography using proteinA-Sepharose 4B (Amersham Pharmacia Biotech) was performed accord-ing to the manufacturer protocol. After washing in PBS (43, 15 min),the grids were floated for 30 min at room temperature on drops ofcolloidal gold-labeled goat anti-rabbit IgG second antibody (particle size10 nm, AuroProbe EM GAR G10, Amersham) diluted 1:40 in PBScontaining 0.1% BSA. The grids were thoroughly washed with PBS andthen fixed with 2.5% glutaraldehyde and post-fixed in 1% OsO4. Afterdrying in the desiccator, the grids were stained by lead citrate anduranyl acetate. Normal rabbit serum and omission of testin antiserumwere used as controls. Sections were viewed with an Hitachi 800 elec-tron microscope.

Radioimmunoassays (RIA)

RIAs for testin (2), androgen binding protein (16, 27), clusterin (28),and a2-macroglobulin (29) were performed as detailed in previousreports.

General Methods

Protein estimation was performed as described previously (30) usingBSA as a standard. Statistical analysis was performed by Student’s ttest using the GB Statistical Analysis Software package (Version 3.0)(Dynamics Microsystems, Inc., Bethesda, MD).

RESULTS

Distribution of Testin in Biological Fluids and ReproductiveTissue Extracts and Its Comparison to Three Other Sertoli CellSecretory Products—To better understand the biological fea-tures of testin, we compared its distribution in various biolog-ical fluids and compartments to three other Sertoli cell secre-tory products: namely androgen binding protein, clusterin, anda2-macroglobulin in the rat by RIA. When Sertoli cells werecultured in the monolayer at 5 3 104 cells/cm2, when some ofthe specialized junctions did not form (such as occluding tightjunctions, see Table I), the amount of testin secreted by Sertolicells in vitro was comparable with that of androgen bindingprotein, clusterin, and a2-macroglobulin (Fig. 1A). However,

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the level of testin in rete testis fluid from intact adult testeswas several orders of magnitude lower than these three othersecretory proteins, which were highly concentrated in the retetestis fluid (Fig. 1B). By any measure, it is logical to expect thatany secretory proteins of Sertoli cell origin be concentrated inthe luminal fluid since the blood-testis barrier formed by adja-cent Sertoli cells limits the transport of proteins from thesystemic circulation to the interstitium unless it is being ac-tively transported there. On the other hand, testin may berapidly reabsorbed by either Sertoli or germ cells after itssecretion by Sertoli cells. If testin is indeed secreted basally, itis expected to be detected in the serum at high concentration;however, earlier RIA data revealed a very low level of testin inthe serum, and its concentration did not alter after orchiectomy(2). As such, results shown in Fig. 1B illustrate the uniquefeature of testin, indicating this protein is not accumulated inthe luminal fluid and is likely to be reabsorbed onto the testic-ular cell. When the levels of testin in the cytosols of the intacttestis (Fig. 1C) and epididymis (Fig. 1D) were quantified andcompared with these other Sertoli cell proteins, it was foundthat testin was almost negligible in both organs when com-pared with these other Sertoli cell proteins, further suggestingthat the secreted testin might become reabsorbed by testicularcells.

Binding of 125I-Testin by Sertoli Cells, Germ Cells, andECM—To investigate whether testin is bound to the Sertoli cellmembrane, the following experiments were performed. Highlypurified Sertoli cells prepared as described under “Materialsand Methods” were plated in 12-well dishes at 5 3 105 cells/well/ml F12/DMEM. Briefly, 125I-testin (1 3 106 cpm, about 10fmol) was incubated with cells for specific time points at 35 or4 °C in F12/DMEM (Fig. 2A). Nonspecific binding was esti-mated by using 100-fold excess unlabeled purified testin. Cellswere then washed three times in F12/DMEM to remove un-bound 125I-testin. Cells were subsequently solubilized in 1 M

NaOH for radioactivity determination. Fig. 2A shows that Ser-toli cells bound 125I-testin specifically in a temperature-de-pendent manner of which the binding was saturated by 2 h. Wenext investigated whether testin can also bind to germ cells orECM. Sertoli cells were cultured at 4.5 3 106 cells/9 ml/100-mmdish for 2 days, contaminating germ cells were lysed by ahypotonic treatment (17), and the cultures were incubated at35 °C for 4 days. Highly purified Sertoli cells or germ cells werethen incubated with 125I-testin (about 5 3 106 cpm) for 4 h at

35 °C. The dishes were washed, and cell membranes wereisolated as described under “Materials and Methods.” Cellmembranes and ECM were then solubilized in the correspond-ing buffer, heated at 100 °C for 10 min, and resolved by SDS-PAGE, and the 125I-testin was visualized by autoradiography.In Fig. 2B, lanes 1 and 10 are the 125I-testin used for incuba-tion, showing the two molecular variants of testin I and testinII. Lanes 2–5 are the Sertoli cell membranes solubilized with 20mM Tris containing 1.6% 2-mercaptoethanol; 20 mM Tris con-taining 1% SDS; 20 mM Tris containing 1% SDS and 1.6%2-mercaptoethanol; and 20 mM Tris containing 1% SDS, 1%Triton X-100, 1% Nonidet P-40, and 1.6% 2-mercaptoethanol,respectively. All buffers used were adjusted to pH 6.8 at 22 °C.Lanes 6–9 are the ECM on the Petri dish extracted with thesame buffers as shown in lanes 2–5. These results indicate notonly that 125I-testin binds onto the Sertoli cell membrane andECM, but its solubilization requires the use of detergents,suggesting that its association with the cell membrane is not anonspecific attachment. In the absence of detergents (lanes 2and 6 versus lanes 3–5 and 7–9, Fig. 2B), none of the 125I-testinthat was bound onto the Sertoli cells could be solubilized. Germcells also bound 125I-testin (lane 11, Fig. 2B) specifically, sincethe presence of 100-fold excess unlabeled testin (lane 12, Fig.2B) competed with the binding.

Identification of a Protein Complex on the Sertoli Cell Mem-brane That Binds Testin—We next sought to investigatewhether the binding of 125I-testin onto the cell membrane asshown in Fig. 2 is mediated by a binding protein. Sertoli cellswere cultured at high cell density on Matrigel-coated dishes for4 days to allow the formation of specialized junctions. Plasmamembranes were isolated as described under “Materials andMethods,” and the whole membrane fraction was labeled withNa[125I] by Iodogen (26). Immunoprecipitation was then per-formed on the labeled membrane proteins using either testinantibody or preimmune serum (1:50). The monospecificity ofthis antiserum has been characterized and established (2, 4, 6).Immunocomplexes were purified by protein A-Sepharose col-umn and resolved on a SDS-polyacrylamide gel and shown inFig. 3A. Lane 1 (S) is the 14C-methylated protein standard.Lane 3 is 125I-testin, where the two molecular weight variantsof testin were clearly visible. Lanes 2 and 4 are the Sertoli cellmembrane (SM) proteins after cell labeling and immunopre-cipitation run under reducing (R) and nonreducing (NR) con-ditions, respectively. Radiolabeled testin I and II extractedfrom the membrane are clearly visible in the SM samples (Fig.3A, lanes 2 and 4) when compared with 125I-testin alone (lane3). It was noted that two other cell membrane proteins thatwere labeled and immunoprecipitated in conjunction with tes-tin, designated as a (45 kDa) and b (28 kDa) are also visible inlanes 2 (reducing) and 4 (nonreducing), indicating that they aresingle polypeptide chains. Lanes 5 and 6 are the correspondingcontrols of lanes 2 and 4, where the testin antibody incubationwas substituted with preimmune serum, illustrating the bandsa and b shown in Fig. 3A are components of the binding proteincomplex. These results, however, cannot distinguish whetherthese two peptides reside on the cell surface or are found insidethe membrane. In contrast to the whole membrane labeling,Fig. 3B shows the result obtained from the surface labeling ofviable Sertoli cells previously treated with a membrane-imper-meable and thiol-cleavable cross-linker, DTSSP. Lane 1 (S) isthe 14C-methylated protein standard. Lane 2 is 125I-testin,where the two molecular variants of testin are clearly visible.Lanes 3 and 4 are the Sertoli cell surface-labeled proteins afterextraction and immunoprecipitation run under reducing (R)and nonreducing (NR) conditions, respectively. Radiolabeledtestin I and II in conjunction with the 28-kDa protein, desig-

TABLE IA functional classification of cell junctions in the testis

I: Occluding junctionsA. Tight junctions (can only be found between Sertoli cells).

II: Anchoring junctionsA. With actin filament attachment sitesA. 1. Cell-cell adherens junctions (can be found between

Sertoli cells and between Sertoli and germ cells).There are two specialized adherens junctions uniqueto the testis:(i) Ectoplasmic specializations (can be found betweenSertoli and germ cells in particular spermatids)(ii) Tubulobulbar complexes (can be found betweenSertoli cells and elongated spermatids)

A. 2. Cell-matrix adherens junctions (e.g. focal contacts, canbe found between testicular cells and matrix at thebasal lamina)

B. With intermediate filament attachment sitesB. 1. Cell-cell (desmosomes, can be found between Sertoli

cells and between Sertoli and germ cells)B. 2. Cell-matrix (hemidesmosomes, can be found at the

basal lamina)III: Communicating junctions

A. Gap junctions (between Sertoli cells, between Leydigcells, between germ cells, and between Sertoli and germcells)


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nated b, are clearly visible in the DTSSP-treated surface-la-beled sample (Fig. 3B, Lane 3) under reducing conditions. Un-der nonreducing conditions, the band b together with testinwere not visible and possibly retarded in the gel because thecomplex had not been cleaved with a reducing agent, sinceDTSSP is a thiol-cleavable cross-linker (Fig. 3B, lane 4 versuslane 3). These results indicate that the 28-kDa protein (band b)and testin I and II reside on the cell surface, and the 45-kDaprotein a is localized inside the cell membrane. In both sets ofexperiments, the gels were also Coomassie Blue-stained tovisualize any proteins that might not have been labeled. How-ever, no other proteins were identified.

Localization of Testin in the Rat Testis by ImmunogoldEM—To examine the subcellular localization of testin, immu-nogold EM was carried out, and about 300 cross-sections wereexamined. Fig. 4 summarizes the result of these analyses.Immunoreactive testin as marked by gold (black) particles wasabundantly localized near the surface of a Sertoli cell adjacentto the ectoplasmic specialization (a modified adherens junction)around a developing spermatid (Fig. 4A). The Sertoli cell wastypified by the presence of microtubule bundles (Fig. 4A, ar-rowheads). Very few gold particles representing immunoreac-tive testin were found along the tight junction between twoadjacent Sertoli cells (Fig. 4B). These data are in agreementwith previously published immunofluorescent microscopy andimmunohistochemistry revealing the abundant presence of tes-tin between Sertoli and germ cells (5–7). Fig. 4C is a controlsection stained with IgG isolated from preimmune serum show-ing a cross-section between two Sertoli cells as typified by thepresence of actin filament bundles (shown by the arrowheads)where no gold particles were seen. The plasma membranes maynot be obvious in these sections, probably due to the omission of

OsO4 in the initial tissue fixation, since the use of this fixativecaused a loss of antigenicity of the testin molecule, makinglocalization impossible.

Effect of Anti-testin IgG on the Formation of Tight Junctionsand the Expression of Testin after Their Disruption by CalciumDepletion—Since earlier studies reveal a tight relationship be-tween the expression of testin and the integrity of intercellularcell junctions in the testis (8), we sought to examine whether adisruption of the inter-Sertoli tight junction can induce a surgein testin expression similar to what was shown when the Ser-toli-germ cell junctions were disrupted (8). It is known thatepithelial tight junctions, such as those found in the Madin-Darby canine kidney cells in vitro, can be disrupted by theremoval of calcium ions from the medium and can be quicklyreassembled after its replacement (14, 33, 34). The integrity ofthe inter-Sertoli tight junctions was assessed by TER measure-ment (31, 32). The inter-Sertoli tight junction was disrupted by[Ca21] depletion in primary Sertoli cell cultures. Sertoli cellcultures were prepared as described under “Materials andMethods,” and TER measurements were taken thereafter. Itwas noted that tight junctions were established in these Sertolicell cultures by day 3–4 (Fig. 5A), when the TER reached itsplateau with a measurement of about 50–60 ohms/cm2, whichis similar to a previous report (35). On day 5, extracellular[Ca21] was removed from the bicameral unit by rinsing the cellmonolayer gently with Ca21-free F12/DMEM, and the unitswere incubated in Ca21-free F12/DMEM for 15 min. At the endof this 15-min period, a significant decline in TER was noted(Fig. 5A), illustrating the tight junction had become leaky. Thisresult is consistent with other tight junction forming epitheliasuch as Madin-Darby canine kidney cells (14). In some exper-iments, cells were incubated with or without either anti-testin

FIG. 1. The concentration of rat testin in Sertoli cell-enriched culture medium (A), rete testis fluid (B), testis (C), and epididymis(D) and its comparison to rat-androgen-binding protein, clusterin, and a2-macroglobulin. These proteins were assayed by correspondingRIA as described under “Materials and Methods.” Results are the mean 6 S.D. of six determinations.


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IgG (200 mg/ml) or normal rabbit IgG (200 mg/ml) together withfresh F12/DMEM containing [Ca21] to assess the effect of tes-tin antibody on the reformation of tight junctions betweenSertoli cells. It was noted that the tight junctions were re-established within 90 min (Fig. 5A) as shown by an increase inTER. However, the presence of anti-testin IgG did not interferewith the reformation of tight junctions in these cultures (Fig.5A). More important, when the testin steady-state mRNA levelwas assessed in the samples when the tight junctions werebeing disrupted and re-established, no detectable change intestin expression was noted (Fig. 5B). When the same blotshown in Fig. 5B was rehybridized with a b-actin probe (Fig.5C) and the data were densitometrically scanned and normal-ized against b-actin (Fig. 5D), testin expression was found notto correlate with the disruption or formation of tight junction,which is entirely different from the testin mRNA expression

FIG. 3. Identification of the testin binding protein complex onthe Sertoli cell membrane. Proteins were visualized by SDS-PAGEand autoradiography under reducing (R) and nonreducing (NR) condi-tions. A, cell membrane proteins labeled with Na[125I] were visualizedby SDS-PAGE and autoradiography after immunoprecipitation usingtestin antibody. Lane 1 , labeled S, is the 14C-methylated protein stand-ard. Lane 3 is the 125I-testin, where the two molecular variants areclearly visible. Lanes 2 and 4 are the Sertoli cell membrane (SM)proteins after cell membrane labeling and immunoprecipitation rununder reducing and nonreducing conditions, respectively. Radiolabeledtestin I and II extracted from the membrane are seen in the Sertoli cellmembrane samples along with peptide a (45 kDa) and b (28 kDa) underreducing (lane 2) and nonreducing (lane 4) conditions. Lanes 5 and 6 arethe corresponding controls in which Sertoli cell membrane was immu-noprecipitated with preimmune serum. Similar results were obtainedin three separate experiments using different batches of Sertoli cells. D,dye-front. B, cell surface labeling with Na[125I], where Sertoli cells werepreviously cross-linked with DTSSP. Lane 1 (S) is the 14C-methylatedprotein standard. Lane 2 is the 125I-testin. Lane 3 is the surface-labeledSertoli cell membrane proteins cross-linked with DTSSP before iodina-tion and immunoprecipitation. Radiolabeled testin I and II extractedfrom the membrane are clearly visible in the Sertoli cell membranesamples along with the b (28 kDa) peptide (lane 3), which was retardedin the gel in the absence of reducing agent (2-mercaptoethanol) (lane 4).These results illustrate that testin I, II, and the b peptide reside on theSertoli cell surface.

FIG. 2. Binding of 125I-testin to primary Sertoli cell cultures(A), and its solubilization from Sertoli cells (SC), germ cells(GC), and ECM (B). A, binding assay was performed as describedunder “Materials and Methods.” 125I-Testin (1 3 106 cpm) was incu-bated at 35 or 4 °C for specified time points. Nonspecific binding wasestimated using a 100-fold excess of unlabeled testin. B, autoradiographshowing the solubilization of membrane-bound 125I-testin from SC, GC,and ECM. 125I-Testin (5 3 106 cpm/5 ml/100-mm dish) was incubatedwith primary Sertoli cells for 4 h at 35 °C as described under “Materialsand Methods.” Membrane proteins were solubilized in 100 ml of corre-sponding buffer, heated at 100 °C for 10 min, and resolved on a 10% TSDS-polyacrylamide gel, and testin was visualized by autoradiography.Lanes 1 and 10, 125I-testin tracer. Lanes 2–5, SC membranes solubilizedwith either 20 mM Tris containing 1.6% 2-mercaptoethanol, 20 mM Triscontaining 1% SDS and 1.6% 2-mercaptoethanol, 20 mM Tris containing1% SDS, or 20 mM Tris containing 1% SDS, 1% Triton X-100, 1%Nonidet P-40, and 1.6% 2-mercaptoethanol, respectively. All bufferswere adjusted to pH 6.8 at 22 °C. Lanes 6–9 is the remaining ECM onthe SC dishes extracted with the same buffers as shown in lanes 2–5.Primary adult germ cells were also incubated for 4 h at 35 °C with125I-testin (lane 11) or with 125I-testin plus a 100-fold excess of unla-beled testin to assess nonspecific binding (lane 12). The germ cellmembranes were solubilized in SDS sample buffer and heated at 100 °Cfor 10 min.


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during the disruption of Sertoli-germ cell junctions (8). In otherexperiments, the testin expression was also monitored up to 5days after tight junctions were disrupted in vitro by Ca21

depletion, and no changes in testin expression were noted (datanot shown). Also, Sertoli cells were cultured with anti-testinIgG or normal rabbit IgG in F12/DMEM between days 1 and 4when tight junctions were being formed; no differences in TERwere noted between these cultures and control cultures withoutany IgG (data not shown). These analyses clearly illustrate thelack of correlation between testin expression and the formationor disruption of inter-Sertoli tight junctions.

Changes in Testin Expression in the Testis by Glycerol-in-duced Disruption of the Blood-Testis Barrier—To confirm thelack of correlation between testin expression and the disrup-tion of inter-Sertoli tight junctions, we sought to use an in vivoanimal model in which the tight junctions were disrupted fol-lowed by a progressive disruption of Sertoli-germ cell junctionsdue to germ cell depletion. Administration of glycerol intrates-ticularly is known to cause long term cessation of spermato-genesis in rats without any apparent changes on Leydig cellfunction, serum gonadotropin and testosterone levels, and sec-ondary sexual characteristics (36, 37). Recent studies have

shown that glycerol can induce permanent damage of the blood-testis barrier by disrupting the inter-Sertoli tight junction nearthe basal lamina within 2 weeks after glycerol administration(25). A disruption of the blood-testis barrier was manifested byan influx of [3H]inulin and [125I]albumin to the rete testis fluid,seminiferous tubule fluid, and the testicular tissue within 2weeks post-glycerol treatment after in vivo infusion of radiola-beled substances (25). We have examined the effect of glycerol-induced disruption of inter-Sertoli tight junctions and the sub-sequent damage on Sertoli-germ cell junctions as a result ofgerm cell depletion on the steady-state testicular testin mRNAlevel (Fig. 6). The morphological changes in the testis afterglycerol treatment were also examined immunohistochemicallywhere testin appears as a reddish-brown precipitate (Fig. 7).Adult rats were anesthetized with MetofaneR and receivedcontrol (PBS) or treatment solution (200 ml of PBS containing20% glycerol) injected via a 26-gauge needle through the polaraxis of each testis beginning at one pole and terminating at theother (25). 2, 4, and 8 weeks later, testes were removed for RNAextraction and Northern blot analysis. Fig. 6A is a Northernblot using about 20 mg of total RNA per lane. No changes in thetesticular testin steady-state mRNA level were apparent by 2weeks at the time when tight junctions were disrupted (Fig. 6,A and B). It was found that virtually all germ cells were stillpresent in the epithelium by 2 weeks after glycerol adminis-tration (Fig. 7C versus Fig. 7, A and B). However, a significantincrease in the testin steady-state mRNA level (Fig. 6, A andB), which was also accompanied by an accumulation of testin(Fig. 6, C and D) in the testis was clearly visible by 4 and 8weeks after glycerol treatment when germ cells, in particularround and elongated spermatids, were depleted from the sem-iniferous epithelium at 4–8 weeks (Fig. 7D and E versus Fig. 7,A, B, and C). When the concentrations of testin in the testis ofthese rats were quantified by RIA and compared with controlrats, a significant increase in testin concentration was noted(Fig. 6C). Since there was a significant decline in testicularweight by 8 weeks after glycerol treatment, the changes intestin level were taken into account with the reduction intesticular weight. Once the data were expressed as testin perpair testes (Fig. 6D), a 7-fold increase was detected in glycerol-treated rats by 8 weeks. These results are also consistent withthe immunohistochemistry data, since there is drastic increasein immunoreactive testin accumulated in the tubular lumen at4–8 weeks after glycerol treatment (Fig. 7, D and E) comparedwith control rats (Fig. 7B) and rats treated with glycerol for 2weeks (Fig. 7C). Glycerol, however, had no effect on testinexpression in the epididymis up to 8 weeks post-treatment (Fig.6, A and B), illustrating the specificity of this chemical treat-ment in the testis. However, immunohistochemistry analysisrevealed that testin was accumulated in the epididymal lumen4–8 weeks post-glycerol treatment (data not shown).

Effect of GCCM or Germ Cells on Sertoli Cell Testin Steady-state mRNA Level—It is known that germ cells neither expresstestin mRNA (7) nor do they secrete any testin in vitro (20). Butit is not known whether GCCM or germ cells can regulatetestin expression. To examine such a possibility, Sertoli cellscultured at 5 3 104 cells/cm2 when specialized junctions did notform were incubated with increasing concentrations of GCCMproteins for a 20-h period (Fig. 8A). Fig. 8A shows that GCCMhad no affect on the Sertoli cell testin steady-state mRNA level.Fig. 8B is the same blot such as the one shown in Fig. 8A buthybridized with a b-actin cDNA probe. Fig. 8C is the densito-metrically scanned data of three separate Northern blots nor-malized against b-actin, indicating that germ cell-released pro-teins had no apparent effect on Sertoli cell testin expression.Fig. 8D is the RIA result showing the concentration of testin in

FIG. 4. Immunogold EM localization of testin in the rat testis.A and B are sections of Lowicryl-embedded rat testes stained withaffinity-purified anti-testin IgG followed by a second antibody conju-gated to gold particles. Gold particles corresponding to immunoreactivetestin appeared as black dots in these figures. A, this section shows atransverse section of a SC and a developing spermatid (germ cell (GC)),as manifested by the presence of the acrosome cap (Ac) and nucleus(Nu). Gold particles accumulated at the sites of ectoplasmic specializa-tions on the Sertoli cell side near the cell surface. Arrowheads indicatethe microtubule bundles, which are one of the junctional elements ofectoplasmic specializations in Sertoli cells. Bar, 0.3 mm. B, this showsthe cross-section to actin filament bundles (arrowheads) between twotransversely sectioned adjacent Sertoli cells exhibiting the fine struc-ture of the tight junction. Immunoreactive testin was found to associatewith actin bundles in the tight junction. Bar, 0.1 mm. C, this is a controlmicrograph in which the anti-testin IgG was substituted with rabbitIgG. This is a parallel section of two SC, where the actin filaments(arrowheads) can be seen. No gold particles were found. Bar, 0.1 mm.


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the corresponding spent media, suggesting GCCM did not af-fect Sertoli cell testin secretion. However, a transient but con-sistent increase in the concentration of testin in the spentmedia was noted using 5 mg of GCCM protein/dish (Fig. 8D).

We next examined whether germ cells can affect Sertoli celltestin expression in vitro (Fig. 9). Since the isolation of Sertolicells from the seminiferous tubule involves the disruption ofSertoli-germ cell junctions, which would have induced changesin testin expression, Sertoli cells used for this experiment werecultured for 4 days before the addition of germ cells to allow itstestin steady-state mRNA level to return to the basal level.Lane 1 is total RNA derived from Sertoli cells cultured for 48 hin vitro; cells were terminated 20 min before the hypotonictreatment (Day 0). Lane 2 is RNA from Sertoli cell cultures 24 hafter hypotonic treatment (Day 1). Lane 3 is the Sertoli cellRNA isolated from cultures on day 5 that served as a control forlanes 4–6. As expected, a steady decline in testin expressionduring cultures is noted (Fig. 9A, lanes 1–3), suggesting thatthe isolation of Sertoli cells from the seminiferous tubule canenhance testin expression because these steps disrupted theSertoli-germ cell junctions. The hypotonic treatment step onday 0 (i.e. 48 h after Sertoli cells were isolated from the tubules)could not induce a surge in testin expression in these cultures(Fig. 9A, lane 2 versus lane 1) possibly because these primarycultures were relatively free of germ cells; as such, not manySertoli-germ cell junctions could be disrupted. Alternatively,the expression of testin at this time was already maximized,and the removal of residual germ cells could no longer elicit anadditional increase in testin expression. Different germ cellnumbers using a Sertoli:germ cell ratio of 1:2.5, 1:5, and 1:10were then plated onto these Sertoli cell cultures on day 4 andincubated for an additional 20 h (day 5) to examine the effectsof germ cells. Immediately before RNA extraction, each culturedish was hypotonically treated to lyse germ cells to eliminateRNA contributed by germ cells in the sample to be analyzed. Assuch, total RNA extracted from these dishes were largely ofSertoli cell origin. It was found that germ cells did not generatea dose-dependent and significant effect on the Sertoli cell testinexpression as shown in Fig. 9, A–C (lanes 4–6 versus lane 3),even though it is apparent that the presence of germ cellsreduced Sertoli cell testin expression slightly. It must be notedthat under these conditions, specialized Sertoli-germ cell junc-tions did not form since their formation would require an in-cubation period of at least 24–48 h in vitro (22, 23). In someexperiments, the Sertoli-germ cell cocultures were allowed toincubate for 30 h in vitro and were subjected to a hypotonictreatment on day 5 to disrupt the Sertoli-germ cell junctions,and the cells were harvested on day 6 for analysis. A significantincrease in testin expression was found as a result of thedisruption of the junctions (Fig. 9A, lane 7 versus lane 8).

Effect of Germ Cells or GCCM on Testin Synthesis and Se-cretion—To study the effect of germ cells or GCCM on Sertolicell testin secretion, Sertoli cells (4.5 3 106 cells/9 ml/100-mmdish) were cocultured with germ cells (1 3 107) or GCCM (10 mgof protein) for 18 h in vitro. Thereafter, cells were pulse-labeledwith [35S]methionine for 15 min and chased with cold methio-nine at specified time points. Fig. 10, A and B show the relativeamounts of 35S-labeled testin in the cytosol and media, respec-tively, at specified time points. It was noted that testin ap-peared in the cytosol within 15 min with a testin I:testin IIratio of 3:1 when the x-ray film was densitometrically scanned

FIG. 5. Disruption of the tight junctional complexes betweenSertoli cells by [Ca21] depletion in the absence or presence ofanti-testin IgG or normal rabbit IgG (200 mg/ml), and its effecton TER and testin mRNA expression. Sertoli cells were plated onMatrigel-coated bicameral units as described under “Materials andMethods.” A, effect of anti-testin IgG on the recovery of Sertoli cell TERafter [Ca21] depletion. B, Northern blot showing the regulation of testin

mRNA expression after [Ca21] depletion. C, this is the same blot shownin (B) but rehybridized with b-actin. D, a graph showing the relativetestin mRNA expression after normalization against b-actin after den-sitometric scanning at 600 nm from three different experiments.


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at 600 nm (Fig. 10A). By 24 h, all the newly synthesized35S-labeled testin was no longer detectable in the cytosol butsecreted into the medium (Fig. 10A), indicating that germ cellsdid not inhibit testin secretion into the media. Testin wasdetected in the medium within 1 h after pulse-labeling andpeaked at 24 h, concomitant with the disappearance of testin inthe cytosol, but the ratio of testin I:testin II had shifted, be-coming 1:1.5 (Fig. 10, B versus A). The reason for such a shift inratio is not immediately known; it is possibly the result ofpost-translational processing such as glycosylation. Con 1 isthe corresponding control using Sertoli cells without eithergerm cells or GCCM, whereas Con 2 is the corresponding con-trol using preimmune serum instead of the anti-testin antibodyfor immunoprecipitation. The level of [35S]-labeled testinshown in Con 1 using Sertoli cells cultured alone without eithergerm cells or GCCM is not significantly different from thecorresponding cultures with either germ cells or GCCM (Fig.10, A and B).

Age-dependent Expression of Testin—Earlier studies haveshown that the expression of testin in adult rats is confined tothe gonad (6). When the steady-state mRNA level of testin fromthe seminiferous tubule isolated from 10, 20, and 60 days of agewas examined by Northern blot, it was found that tubules fromimmature rats had almost 10-fold more testin mRNA thanmature rats (8). However, these earlier studies did not takeinto consideration the increase in RNA contributed by germcells in the samples being analyzed, since there is a drasticincrease in germ cell:Sertoli cell ratio during maturation. Whenthe changes in testin steady-state mRNA level in the testiswere quantified by Northern blot, it was found that there is asteady increase in testin expression per pair of testes duringmaturation (Fig. 11), illustrating that testin expression corre-lates with the onset of spermatogenesis. We next examined thetestin steady-state mRNA level in Sertoli cells isolated fromtestes at different ages. Unexpectedly, an age-dependent reduc-tion of testin expression (Fig. 12A) was noted when about 10 mgof total RNA was used for analysis (Fig. 12B). These blots werethen rehybridized with a b-actin probe and densitometricallyscanned at 600 nm, and the data were normalized againstb-actin. An 8-fold reduction in testin expression was observedduring maturation, when the steady-state mRNA level wascompared between Sertoli cells isolated from 20- and 90-day-old rats (Fig. 12C). These results illustrate that an age-depend-ent reduction in testin expression likely correlates with thedifferentiation status of the Sertoli cell.

DISCUSSION

Table I summarizes the three types of specialized junctionsthat are present in other epithelia that are also found in themammalian testis (for reviews, see Refs. 11–13). All three typesof junctions, namely occluding, anchoring, and communicatingjunctions, can be found between Sertoli cells. However, onlyanchoring and communicating junctions are present betweenSertoli and germ cells. Moreover, some of the anchoring junc-tions between Sertoli and germ cells such as tubulobulbarcomplexes are unique to the testis (Table I). Surprisingly, thecomponent proteins that constitute these junctions betweenSertoli and germ cells, let alone the molecules that regulate thedisassembly and reassembly of these junctions throughout dif-ferent stages of spermatogenesis as a result of migration ofdeveloping germ cells from the basal lamina to the adluminalcompartment of the seminiferous epithelium (for reviews, seeRefs. 11, 12), are largely unknown. Although the mechanismsand cellular events that regulate germ cell translocation in theepithelium are poorly understood, studies in organogenesis,embryogenesis, tumor growth, and metastasis have yieldedsome crucial information with regard to germ cell movement,

since these other cellular processes also involve extensive turn-over of cell-cell and cell-matrix interactions as well as cellmigration (for reviews, see Refs. 38 and 39). It is known that

FIG. 6. A study on the relationship between glycerol-induceddisruption on inter-testicular cell junctions and testin expres-sion as well as its protein accumulation in the rat testis. A, aNorthern blot using about 20 mg of total RNA/lane from testis andepididymis of rats at 2, 4, and 8 weeks (W) after an intratesticularinjection of glycerol including control rats. kb, kilobases. B, same blot asshown in A but rehybridized with a b-actin cDNA probe. C, testinconcentration in the testicular cytosol in glycerol-treated rats 8 weekspost-treatment versus control (n 5 4) when quantified by RIA. D,changes in testin content per pair of testes 8 weeks after glyceroltreatment when the reduction in organ weight was taken into account(n 5 4). In both C and D, results are mean 6 S.D. of four rats.*, significantly different from control; p , 0.001.


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both embryogenesis and tumor growth involve the participa-tion of proteases, protease inhibitors, signaling molecules,growth factors, and junctional complex components such as celladhesion molecules (for reviews, see Refs. 40–45). For the pastdecade, studies from different laboratories have identified sev-eral of these component molecules in the testis (for reviews, seeRefs. 11–13 and 46). Recent studies from this laboratory havealso illustrated the involvement of proteases, protease inhibi-tors, and cell adhesion molecules in the early stage of Sertoli-germ cell interactions preceding the establishment of special-ized cell junctions in vitro (47, 48), suggesting that theestablishment of specialized junctions between testicular cellsis not simply a series of morphological events. Instead, multiplefactors are involved, illustrating the complexity of junctionformation in the seminiferous epithelium. The present reportdemonstrates testin is likely to be a sensitive marker to probethe events of cell junction disruption in the testis.

In this study, testin was shown to correlate with the disrup-tion of Sertoli-germ cell junctions but not the inter-Sertoli tightjunction. Several lines of evidence have excluded the involve-ment of testin in the formation and/or disruption of tight junc-tion. First, immunogold EM revealed very few testin are foundin the tight junction between Sertoli cells. Second, anti-testinIgG did not interfere or facilitate the formation of tight junc-tions between Sertoli cells in vitro, nor did it affect the recoveryof tight junctions after [Ca21] depletion induced disruption ofthe inter-Sertoli tight junction. Third, the expression of testinis not affected by the disruption of tight junctions in vitro, asdemonstrated in the [Ca21] depletion experiment. This obser-vation is in sharp contrast to the disruption of Sertoli-germ celljunctions, which is accompanied by a surge in testin expression(8). Fourth, changes in testin expression after glycerol treat-

ment did not coincide with the damage of the blood-testisbarrier by 2 weeks (25) but rather, with the depletion of germcells, which disrupted the Sertoli-germ cell junctions. We thusconclude that the testin that was found in both the adluminaland basal compartments of the seminiferous epithelium asvisualized by immunofluorescent microscopy and immunohis-tochemistry (5–7) is the protein localized between Sertoli andgerm cells, most likely at the adherens junction such as thedesmosome-like junction, ectoplasmic specialization, and tubu-lobulbar complex. It is unlikely that testin is involved in thegap junction, since the component molecules of the gap junctionhave been very well characterized, and the primary sequence oftestin does not bear any homology to any of the existing con-nexin family members (for review, see Ref. 13).

It has been shown that the testin steady-state mRNA level inthe adult rat testis is significantly enhanced as a result ofeither a chemical treatment such as glycerol, lonidamine (8),and busulfan (3) or a physical treatment such as hypotonictreatment (8) and X-irradiation (10). The depletion of germcells after these treatments would undoubtedly disrupt theSertoli-germ cell junctions. It is our belief that several factorsmay be operating independently or synergistically that regu-late testin expression as shown in these in vitro and in vivoexperiments. First, germ cells may regulate testin expressionvia cell-cell contacts or through a factor(s) released from germcells. As such, a depletion of germ cells leads to a change intestin expression. Second, testin may be a structural compo-nent of the Sertoli-germ cell junction. Thus, when the intercel-lular junction is damaged, another yet-to-be identified factor isreleased to stimulate the production of testin to replace the loststructural component or to trigger another cascade of events.Third, testin may be a stress-induced protein in response to the

FIG. 7. Morphological changes andthe associated pattern of testin im-munohistochemical localization inadult rat testes after intratesticularglycerol treatment. Immunoreactivetestin appears as a reddish-brown precip-itate as denoted by arrowhead. A and Bare photomicrographs of the cryostat sec-tions of testes from a control rat. C, D, andE are photomicrographs of rats treatedwith glycerol after 2, 4, and 8 weeks, re-spectively. Four animals in each treat-ment group including control were pro-cessed for microscopic examination, andat least 50–100 sections were examined.A single set of representative data areshown here. A, control rat showing thecross-section of a normal seminiferous tu-bule at stage VII of the spermatogeniccycle stained with preimmune serum in-dicating the specificity of the staining. Bis the same as in A, except that it wasstained with testin antibody. Immunore-active testin was found between sper-matocytes/spermatogonia and Sertolicells at the basal compartment. Testinwas also found between Sertoli cells andthe heads of the elongated spermatids (es)at the adluminal compartment. C, twoweeks after glycerol treatment, it is notedthat all elongated spermatids were de-pleted, but the number of round sperma-tids (rs) and spermatocytes (p) remainedrelatively unchanged. Testin is still local-ized in the basal compartment betweenSertoli and germ cells. Four (D) and eight(E) weeks after glycerol treatment, a mas-sive reduction of germ cells from the epi-thelium and an accumulation of immuno-reactive testin was found in the lumen ofthe tubule.


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stress and/or cellular death caused by these experimentalmanipulations.

The present study using germ cells or their conditioned me-dium cocultured with Sertoli cells failed to demonstrate a sig-nificant effect on Sertoli cell testin steady-state mRNA level.This result seemingly suggests that germ cells do not play amajor role in regulating testin expression. However, one mustnote that these cocultures were terminated at 20 h, at the timewhen specialized Sertoli-germ cell junctions had not yetformed, since morphological analysis has shown that the estab-lishment of specialized junctions between Sertoli and germ

cells in vitro, such as desmosome-like adherens junctions, re-quires a culture period of 24–48 h (22, 23). As such, it remainsto be determined whether testin is involved in the formation ofSertoli-germ cell junctions. The fact that there is a surge intestin expression when the Sertoli-germ cell junctions are dis-rupted may suggest testin somehow participates in the destruc-

FIG. 8. Effects of GCCM on the Sertoli cell steady-state testinmRNA level and the amount of testin secreted by Sertoli cells invitro. A, Northern blot showing the level of Sertoli cell testin expressionwhen cultured with an increasing concentration of GCCM for a 20-hperiod. Approximately 10 mg of total RNA were loaded per lane. kb,kilobases. B, the same blot shown in A but rehybridized with a b-actinprobe. C, a graph showing the relative testin mRNA level in Sertoli cellscultured with various amounts of GCCM proteins and normalizedagainst b-actin after densitometric scanning of three blots such as theone shown in A. D, the concentration of testin in the spent medium inthese cultures were quantified by a testin-specific RIA. ns, not signifi-cantly different from Sertoli cells cultured in the absence of germ cells;*, p , 0.01.

FIG. 9. The effect of germ cells (GC) and hypotonic treatment(HT) on the SC testin steady-state mRNA level in Sertoli-germcell cocultures in vitro. A, Northern blot showing the steady-statetestin mRNA level in Sertoli cells when cocultured with increasingnumbers of germ cells for a 20-h period. Approximately 10 mg of totalRNA were loaded per lane. In these cultures, germ cells were lysed bya hypotonic treatment 20 min before their termination to eliminateRNA contributed by germ cells in the samples being analyzed. kb,kilobases. B, ethidium bromide staining of the same blot shown in A. C,a graph showing the relative testin mRNA level in Sertoli cells cocul-tured with germ cells after densitometric scanning at 600 nm of threeNortherns such as the one shown in A. BH, Sertoli cells were termi-nated 20 min before hypotonic treatment; AH, 24 h after hypotonictreatment; Ctrl, control cultures where Sertoli cells were cultured aloneand terminated on day 5; 2.5, 5, and 10 are coculture experimentswhere germ cell:Sertoli cell ratio was at 2.5:1, 5:1, and 10:1; germ cellswere added onto Sertoli cells (5 3 104 cells/cm2) on day 4 and coculturedfor 20 h and terminated on day 5; 10 H and Ctrl/10H correspond tolanes 7 and 8 shown in A.


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tion of cell junctions by acting as a protease or that it protectsthe testis against tissue damage by acting as a protease inhib-itor. Alternatively, the increase in testin expression is theresult of cell junction disruption that is associated with othercellular events. However, testin was found to be neither aprotease nor a protease inhibitor (6), making the former possi-bility unlikely. Although the glycerol or lonidamine (8)-inducedSertoli-germ cell junction damage can elicit an increase intestin accumulation in the testicular cytosol by up to 20–30-fold, most of the testin was accumulated in the lumen and waslikely flushed out. Therefore, it is very unlikely that testin isbeing used for the reassembly of the damaged junction. Thepresent study, however, illustrates that the testin steady-statemRNA level per pair of testes increases steadily during testic-ular maturation when the increase in organ weight and theincrease in germ cell to Sertoli cell ratio are taken into consid-eration. These results suggest that the rapid assembly anddisassembly of intercellular junctions at the onset of spermat-ogenesis is likely to be one of the most critical factors in regu-lating testin expression.

Studies on the binding of testin onto the Sertoli and germ cellmembrane reveal that the dissociation of testin from the cellsurface after its binding requires the use of a detergent illus-

trating that other biochemical changes may take place whentestin couples onto the binding protein complex. This resultalso supports the notion that testin may be a structural com-ponent between Sertoli and germ cells. The amount of testinbinding to Sertoli cells is limited to only 0.1–0.5% that of total125I-testin in the incubation mixture, regardless of an increasein Sertoli cell number used for the in vitro assay. This resulttogether with the detergent solubilization experiment stronglysuggests that the interaction of testin with its binding proteincomplex is not a classical ligand-receptor interaction. The factthat a secretory protein can become tightly associated with thecell surface via a receptor-binding protein in a fashion dissim-ilar to a classical ligand-receptor interaction is not withoutprecedence. Wnt, a growing class of multi-functional signalingmolecules (glycoproteins with apparent Mr between 34,000 and42,000) involved in both tumorigenesis and patterning eventsduring development and tissue differentiation by coordinatingthe organization of groups of cells in the developing vertebrate(49, 50), are secretory proteins that can become tightly associ-ated with the cell surface or ECM and originally thought to bevia a nonreceptor-mediated mechanism (51–53). A recentstudy, however, has demonstrated that Dfz2, a 694-amino acidpolypeptide, is the receptor protein of the signaling moleculesof the Wnt gene family (54). The binding of the Wnt proteinsonto the receptor, similar to testin, cannot be assessed byconventional receptor-ligand assays such as a Scatchard plot,probably due to their low abundancy. A comparison betweentestin and nine members of the Wnt family, including Wnt-1and Wnt-2 (55), using DNASIS, PROSIS, and BESTFIT pro-grams at the levels of amino acid and nucleotide sequencesrevealed that they share 10–30% identity (data not shown). Assuch, testin is likely a distant member of the Wnt gene family,and it does share some of the unusual feature of the Wnt.

In the present and other earlier studies (3, 8, 10), it wasshown that disruption of testicular cell junctions by glycerol,lonidamine, busulfan, or hypotonic treatment can lead to asurge in testin expression. Such a change in expression may bea response to cellular stress, when junctions are being dis-rupted. Studies in muscle, in particular, cardiac and skeletalmyocytes, reveal that 5–30% of the cell population undergoplasma membrane disruption under physiological conditions,due to the contractile nature of these cells (56–58). Likewise,

FIG. 10. The effect of germ cells (GC) or GCCM on the synthesisand secretion of testin by primary Sertoli cells cultured in vitroby pulse-chase analysis. A, this is an autoradiograph of a 10% TSDS-polyacrylamide gel showing the immunoprecipitated radiolabeledcytosolic proteins. After an 18-h coculture period of Sertoli with germcells (GC) or GCCM, cultures were labeled for 15 min with [35S]methi-onine and then chased with cold methionine for 15 min to 96 h andimmunoprecipitiated with anti-testin II antibody. Control 1 (Con1)shows radiolabeled cytosolic proteins from Sertoli cells cultured in theabsence of either GC or GCCM for 30 min and 24 h and immunopre-cipitated with anti-testin antibody. Control 2 (Con2) is identical tocontrol 1, except that the samples were immunoprecipitated with pre-immune rabbit serum. B, this is an autoradiograph similar to the oneshown in A, except that the immunoprecipitated proteins were recov-ered from the spent media. Lane M is 14C-methylated protein molecularweight markers consisting of 10,000 cpm each of myosin (Mr 200,000),phosphorylase b (Mr 97, 400), BSA (Mr 69,000), ovalbumin (Mr 46,000),carbonic anhydrase (Mr 30,000), soybean trypsin inhibitor (Mr 21, 500),and lysozyme (Mr 14, 300).

FIG. 11. The relative testin steady-state mRNA level in the rattestis (mRNA level per pair organ) during maturation. Northernblots were normalized against b-actin after densitometric scanning at600 nm, and the changes in testin expression was then taken intoconsideration based on the increase in testicular weight during matu-ration. Results are shown as mRNA level (arbitrary unit) per pair testesfrom one experiment. Two other experiments using different animalsamples yielded identical results.


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about 3% and 6% of the epidermal and endothelial cells in theskin (56) and aorta (59), respectively, are being disrupted. Eventhough the level of cell wounding for the Sertoli and germ cellin the testis is not known, extensive plasma membrane disrup-tion in testicular cells is expected to occur due to the rapidmorphological changes during spermatogenesis. Until recently,membrane resealing was thought to be a passive event. The“wound hormone” hypothesis (60) suggests that chemical me-diators of tissue restructuring such as growth factors, stored incytosol, are released during membrane disruptions (for review,see Ref. 61). For instance, basic fibroblast growth factor(bFGF), which lacks a signal peptide sequence, is a potentgrowth-promoting factor when it is released extracellularly dueto plasma membrane damage (62, 63). Studies by immunopre-cipitation has demonstrated the release of bFGF by germ cells(64), suggesting the bFGF found in GCCM is likely the result ofgerm cell wounding, since bFGF under the normal physiologi-cal condition is not a secretory protein because of the lack of asignal peptide. Other studies have demonstrated that the useof trypsin to isolate germ cells from the tubules (64) can altermany of the cell surface properties (20, 22, 65). As such, thesecretion of bFGF by germ cells as demonstrated in this earlierstudy (64) may be the result of trypsin-induced plasma mem-brane damage that causes of release of bFGF. It is possible thatbFGF is an important growth-promoting and signaling mole-cule that participates in repairing the disrupted testicular celljunctions during spermatogenesis and whose release fromgerm cells is the result of degeneration (66–68) and apoptosis(69). Testin, on the other hand, is a secretory protein with adefinite signal sequence (6). However, its protein level in therete testis fluid and cytosols of the testis and epididymis as wellas its expression are virtually undetectable except in the go-nad. When Sertoli cells are being cultured in vitro, the amountof testin secreted into the spent medium is comparable withother Sertoli cell secretory products. It is our belief that upondissociation of the testicular cells from the tubule during thepreparation of Sertoli cells, testin expression is induced andremains elevated, as demonstrated in the present study. Thisobservation thus supports the postulate that the expression oftestin may be induced by cell wounding.

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Josephine Grima, Connie C. S. Wong, Li-ji Zhu, Shu-dong Zong and C. Yan Chengthe Inter-Sertoli Tight Junction

Expression Correlates with the Disruption of Sertoli-Germ Cell Junctions but Not Testin Secreted by Sertoli Cells Is Associated with the Cell Surface, and Its

doi: 10.1074/jbc.273.33.210401998, 273:21040-21053.J. Biol. Chem.

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