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Endocrinology Vol. 142, No. 5 1878-1888
Copyright © 2001 by The Endocrine Society

ARTICLES

Is Cadmium Chloride-Induced Inter-Sertoli Tight Junction Permeability Barrier Disruption a Suitable in Vitro Model to Study the Events of Junction Disassembly during Spermatogenesis in the Rat Testis?1

Nancy P. Y. Chung and C. Yan Cheng

The Population Council, Center for Biomedical Research, New York, New York 10021

Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: yan{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl2-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 x 106 cells/cm2) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl2 at 5–10 µM for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl2 (0.1–5 µM) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl2 also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl2, the presence of testosterone (T) at 1 x 10-9 M allowed resealing of the inter-Sertoli TJ barrier after CdCl2 was removed, whereas the presence of 2 x 10-7 M testosterone even protected Sertoli cells from CdCl2-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl2-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl2 treatment. Based on these results, it is apparent that CdCl2-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING SPERMATOGENESIS, preleptotene spermatocytes residing outside the blood-testis barrier (BTB), which is formed by the inter-Sertoli tight junctions (TJs) between adjacent Sertoli cells at the basal compartment of the seminiferous epithelium, must traverse the BTB to gain entry into the adluminal compartment for further development and differentiate into round and elongate spermatids (for reviews, see Refs. 1 and 2). Even though this event of cell movement is essential to the completion of spermatogenesis, very little work has been performed to investigate the biochemical and/or molecular aspects of this event except for a series of descriptive morphology studies performed over the next 3 decades since the 1960s (for reviews, see Refs. 2, 3, 4). Based on recent studies from this laboratory, the events of germ cell movement apparently are composed of continuous, but intermittent, phases of junction disassembly and reassembly (for review, see Ref. 5). For instance, when Sertoli cells or Sertoli-germ cells cultured in vitro to allow the assembly of cell junctions, there are transient, but significant, increases in several target genes’ expression during the assembly of inter-Sertoli and Sertoli-germ cell junctions, which include tryptase, urokinase plasminogen activator (uPA), cystatin C, {alpha}2-macroglobulin, cathepsin L, N-cadherin, ß-catenin, zonula occludens 1 (ZO-1), sertolin, prostaglandin D2 synthetase (PGD-S), and myotubularin (5, 6, 7, 8, 9, 10, 11, 12, 13). However, a model to study the events of junction disassembly is lacking. If such a model is available, it will be useful to study the biology of junction restructuring during spermatogenesis in multiple ways. First, this model can be used to investigate the events leading to spermiation. Second, it can be used to identify target genes that are involved in the breakdown of the inter-Sertoli TJs to permit the passage of preleptotene spermatocytes. Third, it can become a potential in vitro assay system to screen contraceptives targeted to prevent or induce junction disassembly.

It is known for almost 4 decades that cadmium can induce disruption of the BTB in the testis, which can be prevented by zinc (14). Although the precise mechanism is not clearly defined, some biochemical alterations were detected in the testis after cadmium exposure, such as reduced activity of testis-specific anhydrase and glutathione disulfide reductase, and increased activity of glutathione peroxidase (15). Earlier studies have shown that Leydig cells may be the target of cadmium toxicity (16). More recent in vitro studies, however, suggest that the Sertoli cell is the most vulnerable target of CdCl2 and is more sensitive than Leydig cells to cadmium-induced damage (17). In vivo injection of CdCl2 (30 µmol/kg BW) to rats induced an increase in BTB permeability, indicating a disruption of inter-Sertoli TJs (18, 19). Studies by confocal microscopy illustrated that CdCl2 disrupted TJ-associated microfilaments in rat Sertoli cells, which, in turn, damaged the BTB (20). CdCl2 also induced apoptosis in testicular tissue after ip injection (21). This event was characterized by an activation of endonuclease as a result of the loss of intracellular calcium (22). The CdCl2-induced damage of the inter-Sertoli TJ permeability barrier was proposed as a model for toxicological investigations of the BTB in vitro (23). In addition, disruption of paracellular barrier by CdCl2 was detected in intestinal epithelial cell lines, Caco-2 and IEC-18 (24). Despite these earlier studies, it is not known whether CdCl2-induced disruption of inter-Sertoli TJ can become a model to study the events of junction disassembly, because it must be able to fulfill the following criteria. First, at the doses used to disrupt the inter-Sertoli TJs, CdCl2 must be noncytotoxic to the treated cells. Second, the effect must be reversible, so that the disrupted inter-Sertoli TJs are capable of resealing. Third, the transient induction of target genes when inter-Sertoli TJs are reassembled after the removal of CdCl2 should be similar to the changes detected when inter-Sertoli TJs are assembled shortly after their isolation from the testes. If a suitable in vitro model of junction disassembly is found, not only will it be useful to study this aspect of junction restructuring during spermatogenesis, but it will enhance our ability to screen compounds that can disrupt this cellular event, leading to the discovery of new agents to disrupt spermatogenesis and regulate male fertility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley rats at 20 days of age were obtained from Charles River Laboratories, Inc. (Kingston, MA). Rats arrived with a foster mother rat (10 pups/foster mother) and had free access to rat chow and water ad libitum under controlled temperature (22 C). Rats were killed by CO2 asphyxiation, and testes were removed immediately for isolation of testicular cells. The use of animals for this study was approved by the Rockefeller University animal care and use committee with Protocols 97117, 95129-R, and 00111.

Preparation of Sertoli cell cultures
Primary Sertoli cells were prepared from 20-day-old Sprague Dawley rats by sequential enzymatic treatment as previously described (25, 26). Cells were resuspended in serum-free Ham’s F-12 nutrient mixture and F12/DMEM (1:1, vol/vol; Life Technologies, Inc., Grand Island, NY) supplemented with 15 mM HEPES, 1.2 g/L sodium bicarbonate, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 2.5 ng/ml epidermal growth factor, 20 mg/liter gentamicin, and 10 µg/ml bacitracin and cultured at either low or high cell density at 35 C in a humidified atmosphere with 5% CO2/95% air. For low cell density cultures, Sertoli cells were plated at 5 x 104 cells/cm2 in either 100-mm petri dishes with 9-ml F12/DMEM or Matrigel (Collaborative Biochemical Products, Bedford, MA)-coated (diluted 1:7 with F12/DMEM, vol/vol) bicameral units to allow the formation of monolayer cultures without the assembly of inter-Sertoli TJs when monitored by transepithelial electrical resistance (TER) across the Sertoli cell epithelia on Matrigel-coated bicameral units and other criteria as previously described (27). Under this culture condition, it was found that adherens (AJ) and gap junctions (GJ) were formed (9). For high cell density cultures, Sertoli cells were plated on Matrigel-coated 24-well dishes or bicameral units at a density of 1.2 x 106 cells/cm2 as previously described (27) to allow the formation of TJ, AJ, and GJ, mimicking the in vivo morphology of Sertoli cells. In selected culture experiments, 24 h after Sertoli cells were isolated and plated (day 1) the culture medium was supplemented with FSH (100 ng/ml) and T (T; 1 x 10-9 or 2 x 10-7 M). About 48 h after plating (on day 2 of culture), cultures were hypotonically treated with 20 mM Tris-HCl, pH 7.4, for 2.5 min to lyse contaminating germ cells (28) to obtain Sertoli cell cultures with greater than 95% purity.

Effects of CdCl2 on the assembly and maintenance of inter-Sertoli TJs in vitro
To examine the effects of CdCl2 on the assembly and maintenance of inter-Sertoli TJ permeability barrier in vitro, primary Sertoli cells were isolated as described above and cultured at 1.2 x 106 cells/cm2 on Matrigel (1:7)-coated bicameral units (Millipore Corp., Bedford, MA) as previously described (26). TJ assembly was monitored by TER measurement across the Sertoli cell epithelia using a Millicell Electrical Resistance System as previously described (12, 27). Briefly, a short (~2 sec) pulse of current (20 µA) was passed through the Sertoli cell epithelia between two silver-silver chloride electrodes, and resistance was calculated from the change in voltage across the Sertoli cell epithelia. The resistance was multiplied by the effective growth area of the filter (~0.6 cm2) to yield the areal resistance (ohms · cm2). The net value of TER was computed by subtracting the background, which was measured on the Matrigel-coated cell-free chambers, from values of Sertoli cell-plated chambers. To minimize temperature-induced fluctuation when recording TER, cultures were stabilized at room temperature for 20 min before TER measurement. CdCl2 at 0.1–10 µM was added to both the basal and apical compartment of the bicameral unit on days 1 and 4 and incubated for 8 h to assess the effect of CdCl2 on the assembly and maintenance of the inter-Sertoli TJ, respectively. It is noted that in preliminary experiments we had examined whether there was any difference in TER across the Sertoli cell epithelia if CdCl2 at 5 µM was added to the apical, basal, or both chambers of the bicameral units on day 1 of culture (see Fig. 1AGo). It was found that the inter-Sertoli TJ permeability barrier responded similarly to the CdCl2 treatment regardless of where this compound was added. As such, CdCl2 was added to both chambers in all experiments. The inter-Sertoli TJs were assembled by day 4, as manifested by a stable TER across the cell epithelia. Controls were Sertoli cells cultured in F12/DMEM alone or in the presence of FSH and two different concentrations of T. After CdCl2 exposure, all experimental and control cultures were washed with fresh F12/DMEM three times, and cultures were incubated for an additional 4–7 days. TER readings were recorded daily before media were replaced. Each time point had triplicate cultures, and each experiment was repeated at least twice using different batches of cells.



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  		Figure 1. A and B, Effects of CdCl2 on the assembly of inter-Sertoli TJ permeability barrier in vitro (A) and cellular DNA content (B). Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated bicameral units at 1.2 x 106 cells/cm2 (effective culture area, ~0.6 cm2) for a period of 7 days in vitro. A, To study the effects of CdCl2 on the assembly of the inter-Sertoli TJ permeability barrier, CdCl2 was added to both the apical and basal compartments of the bicameral unit 24 h after cells were plated onto the Matrigel-coated HA (mixed cellulose esters) filters (day 1). Sertoli cells were incubated with CdCl2 at the denoted concentrations for 8 h; thereafter, cells were washed three times with F12/DMEM to remove CdCl2, TER across the Sertoli cell epithelia was measured at the specified time points. Each time point has triplicate cultures in each experiment. Each data point is the mean ± SD of three determinations from one set of experiments. B, On days 1 and 8, Sertoli cells were harvested, and total DNA contents in controls (both days 1 and 8) and cultures treated with different concentrations of CdCl2 (only on day 8) were quantified. Values are the mean ± SD of triplicate cultures in one representative set of experiments. ns, Not significantly from controls; *, P < 0.05 vs. controls; **, P < 0.01 vs. controls. SC, Sertoli cells.

 
Evaluation of CdCl2-induced cytotoxicity by measuring cellular DNA content
Sertoli cells isolated from 20-day-old rat testes cease to divide both in vivo and in vitro (for review, see Ref. 1); therefore, total DNA measurement of Sertoli cell epithelia throughout the experimental period becomes a reliable index to estimate cell number and cell viability. DNA content was quantified using a colorimetric assay based on thiobarbituric acid (29) using calf thymus DNA (Sigma, St Louis, MO) as a standard. Briefly, Sertoli cell epithelia were harvested at specified time points and resuspended in 500 µl ice-cold PBS buffer (0.01 M sodium phosphate and 0.15 M sodium chloride, pH 7.4). Cell suspension was sonicated for 20 sec using an Ultrasonic 4710 series homogenizer (Cole-Parmer Instrument Co., Vernon Hills, IL) at 4 C, and 100 µl of the sonicated sample were used in DNA assay (29). Briefly, 100 µl 0.1 N sulfuric acid were added to the sample, and hydrolysis was performed at 100 C for 1 h. After cooling at room temperature for 5 min, 100 µl 0.1 M sodium periodate were added to the sample and vortexed. Samples were incubated at room temperature for 20 min. At this time, excess periodate was eliminated by the addition of 150 µl 10% arsenite solution in 0.5 M sodium sulfate containing 0.1 N sulfuric acid. A yellowish brown iodine was released from the reaction, which disappeared upon vigorous vortexing. After 10-min incubation at room temperature, 350 µl 0.6% thiobarbituric acid in 0.5 M sodium sulfate were added. The sample was heated in a boiling water bath for 15 min. The intensity of the resulting pink chromogen was concentrated by extraction in 500 µl isoamyl alcohol, mixed vigorously, and centrifuged to facilitate separation of immiscible solvents. The top pink layer was withdrawn and spectrophotometry was performed at 490 nm.

Effects of T on the cellular gene expression by Sertoli cells
Sertoli cells were prepared as described above and cultured at 1.2 x 106 cells/cm2 on Matrigel-coated dishes in F12/DMEM supplemented with insulin, epidermal growth factor, transferrin, and bacitracin. On day 3, Sertoli cells were exposed to T at either 1 x 10-9 or 2 x 10-7 M for 2–24 h in the absence of ovine FSH. Cultures were terminated at specific time points by RNA STAT-60 for RNA extraction. Semiquantitative RT-PCR was then performed to monitor the effects of T on steady state messenger RNA (mRNA) levels of occludin, uPA, and E-cadherin.

Semiquantitative RT-PCR to assess changes of target gene expression during CdCl2-induced junction disassembly and reassembly
When the inter-Sertoli TJ permeability barrier was disrupted by CdCl2, as manifested by a decline in TER across the Sertoli cell epithelia, cell samples at specified time points were terminated for total RNA extraction. RT-PCR was performed to monitor changes in the expression of target genes, such as E-cadherin (an AJ protein), occludin (a TJ-integral membrane protein), and urokinase plasminogen activator (uPA, a serine protease). Semiquantitative RT-PCR was performed essentially as previously described (7, 8, 9, 10, 27). Approximately 3 µg total RNA were reverse transcribed into complementary DNAs with 1 µg oligo(deoxythymidine)15 using a Moloney murine leukemia virus RT kit (Promega Corp., Madison, WI) in a final reaction volume of 25 µl. In preliminary experiments, PCR products were examined over a range of 20–28 amplification cycles to ensure linearity using different concentrations of RT products and primers. PCR was performed by combining 3 µl of the RT product with 0.3 µg of each sense and antisense primer of the selected target gene, and with rat ribosomal S16 primer pairs (Table 1Go), 5 µl 10 x PCR buffer, 3 µl MgCl2 (25 mM), 8 µl deoxy (d)-NTPs (200 µM each of dATP, dGTP, dCTP, and dTTP), 2.5 U Taq DNA polymerase, and sterile double distilled water to a final reaction volume of 50 µl. The cycling parameters for PCR were as follows: denaturation at 94 C for 1 min, annealing at 58-62 C for 2 min, and extension at 72 C for 3 min, for a total of 22–24 cycles, which was followed by a 15-min extension at 72 C in a Perkin-Elmer Corp. thermal cycler (Norwalk, CT). To enhance the detection limit and to yield data for quantitative analysis, PCR was performed in the presence of {gamma}-32P-labeled primer. Briefly, the sense primers of E-cadherin, occludin, uPA, and S16 were labeled at the 5'-end with [{gamma}-32P]dATP (SA, 6000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) using T4 polynucleotide kinase (Promega Corp., Madison, WI). Approximately, 3 x 106 cpm were used per PCR reaction. To ensure the linearity of both target gene and S16 during their amplification by PCR, 10-µl aliquots of PCR products at 20, 22, 24, 26, and 28 cycles were withdrawn and resolved onto 5% T (total acrylamide) concentration (gm/100 ml = acrylamide + methylene bisacrylamide polyacrylamide gels using 0.5 x TBE (44.5 mM Tris-borate and 1 mM EDTA, pH 8.0) as running buffer. PCR products were visualized by ethidium bromide staining, and autoradiography was performed using Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY). Autoradiograms were densitometrically scanned at 600 nm using an Ultro-Scan XL enhanced laser densitometer (Pharmacia Amersham Pharmacia Biotech) to yield semiquantitative data for statistical analysis.


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  		Table 1. Primers used for semiquantitative RT-PCT to analyze mRNAs for E-cadherin, occludin, uPA, and S16

 
Statistical analysis
Multiple comparisons were performed using one-way ANOVA followed by Tukey’s honestly significant difference test to compare selected pairs of experimental groups. In some experiments Student’s t test was used to compare the results for treated samples with those for the corresponding controls. Statistical analyses were performed using the GB-STAT statistical analysis software package (version 7.0, Dynamic Microsystems, Inc., Silver Spring, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CdCl2 on the inter-Sertoli TJ permeability barrier in vitro
Previous studies have shown that Sertoli cells cultured in vitro are capable of forming inter-Sertoli TJs manifested by an increase in TER across the Sertoli cell epithelia, which peaked by days 3–4 (12, 27). The TER remained relatively constant thereafter between days 4 and 7 (Fig. 1Go). Similar results were obtained when the assembly of TJs was assessed by other techniques, such as restricted diffusion of [3H]inulin or [125I]BSA from the apical to the basal chamber of the bicameral unit, and polarized secretion of Sertoli cell proteins, such as transferrin, androgen-binding protein, and testin (26, 30). As such, TER measurement was selected to assess the effects of CdCl2 on the assembly and maintenance of inter-Sertoli TJs, because this method is relatively simple to perform, and the data obtained are semiquantitative. It is also noted that this assay was widely used by cell biologists in the field since its introduction (31, 32). After Sertoli cells were exposed to CdCl2 at either 5 or 10 µM for 8 h on day 1, there was a delay in the assembly of inter-Sertoli TJs when CdCl2 was present in the medium at 5 µM compared with control cultures (Fig. 1A). Also, the inter-Sertoli TJ was less "tight" in the presence of 5 µM CdCl2. For instance, on day 3, cultures treated with 5 µM CdCl2 had a TER of 15 vs. 35 ohm · cm2 in controls (Fig. 1AGo). In contrast, CdCl2 at 10 µM virtually abolished inter-Sertoli TJ assembly (Fig. 1AGo). As Sertoli cells isolated from 20-day-old rats used in the present study were nondividing when assessed by [3H]thymidine incorporation studies (Mruk, D., and C. Y. Cheng, unpublished observation), the number of Sertoli cells should be consistent throughout the culture period if they stay viable. Thus, the measurement of DNA content in these cultures is a reliable index to assess cell viability. Apparently, the observed effects of CdCl2 (5–10 µM) on the inter-Sertoli TJ permeability barrier was not the result of cell cytotoxicity, because the DNA content of CdCl2-treated cultures was not significantly different from that of controls (Fig. 1BGo).

Effects of CdCl2 on the maintenance of inter-Sertoli TJs in vitro in the absence and presence of FSH and T
When Sertoli cells were cultured in serum-free F12/DMEM without FSH or T, inter-Sertoli TJs were assembled by day 4 as manifested by a stable TER across the Sertoli cell epithelia. To study the effects of CdCl2 on the maintenance of inter-Sertoli TJs, Sertoli cells were exposed to different concentrations of CdCl2 ranging from 0.1–5 µM for 8 h on day 4 when inter-Sertoli TJs were assembled, and CdCl2 was then removed thereafter by rinsing Sertoli cells with fresh F12/DMEM. It was noted that the TER across the cell epithelia in CdCl2-treated cultures declined dose dependently, indicating the inter-Sertoli TJs were being disrupted (Fig. 2AGo). As shown by the colorimetric DNA assay, the CdCl2-induced TJ disruption was unlikely to be due to cell toxicity, as DNA contents of treated cultures on day 8 were not significantly different from those of controls (Fig. 2BGo). Interestingly, when Sertoli cells were cultured under the same condition but in the presence of both FSH (100 ng/ml) and T (1 x 10-9 M), the CdCl2-induced disruption of the inter-Sertoli TJ permeability barrier could be resealed when CdCl2 was present at 0.1–1 µM, a dosage capable of disrupting inter-Sertoli TJs (Figs. 3AGo vs. 2A). When T was present at 2 x 10-7 M, it could prevent CdCl2 (at 0.1–1 µM) from inducing disruption of inter-Sertoli TJs unless it was present at a concentration of 5 µM (Fig. 3CGo). Based on these observations, it is logical to conclude that a high concentration of T, such as 2 x 10-7 M, could counteract the CdCl2-induced damage to the inter-Sertoli TJ permeability barrier in vitro, but the inclusion of T at 1 x 10-9 M in CdCl2-treated Sertoli cells, this can possibly become an in vitro model to study junction disassembly. The DNA contents of treated cultures were not significantly different from those of controls, except that treated with 5 µM in the presence of T at 2 x 10-7 M (Fig. 3, B and D). When the results in Fig. 3DGo were compared with those shown in Figs. 1BGo and 2BGo, it seems that the presence of T coupled with a high concentration of CdCl2, such as 5 µM, can induce cell cytotoxicity.



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  		Figure 2. A and B, Effects of CdCl2 on the maintenance of inter-Sertoli TJ permeability barrier (A) and cellular DNA content (B) in vitro. Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated bicameral units at 1.2 x 106 cells/cm2 (effective culture area, ~0.6 cm2) for a period of 8 days in vitro. A, To study the effects of CdCl2 on the maintenance of the inter-Sertoli TJ permeability barrier, TJs were allowed to form as manifested by a steady rise in TER until it peaked on day 4 and remained stable thereafter. At this time (day 4), different concentrations of CdCl2 were added to both the apical and basal compartments of the bicameral unit and incubated with Sertoli cells for 8 h; thereafter, cells were washed three times with F12/DMEM to remove CdCl2, and TER across the Sertoli cell epithelia was measured at specified time points. Each time point had triplicate cultures in each experiment. Each experiment was repeated three times using different batches of Sertoli cells. Each data point was the mean ± SD of nine determinations from three independent experiments. B, On days 1 and 8, Sertoli cells were harvested, and total DNA contents in controls (both days 1 and 8) and cultures treated with different concentrations of CdCl2 (only on day 8) were quantified. Values are the mean ± SD of three separate experiments. ns, Not significantly from controls; *, P < 0.05 vs. controls; **, P < 0.01 vs. controls. SC, Sertoli cells.

 


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  		Figure 3. A–D, Effects of different concentrations of CdCl2 on the maintenance of the inter-Sertoli TJ permeability barrier in vitro in the presence of FSH and T. Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated bicameral units at 1.2 x 106 cells/cm2 in the presence of FSH (100 ng/ml) together with T at 1 x 10-9 M (A and B) or 2 x 10-7 M (C and D) throughout the entire culture experiment. On day 4 of the culture, different concentrations of CdCl2 were added to the both apical and basal compartments of the bicameral unit after inter-Sertoli TJs were assembled, as manifested by a steady TER. Cells were exposed to CdCl2 for 8 h and washed three times with fresh F12/DMEM containing appropriate concentrations of FSH and T to remove CdCl2. TER across the Sertoli cell epithelia was measured at specified time points. Each time point had triplicate cultures in each experiment. Each experiment was repeated three times using different batches of Sertoli cells. Each data point is the mean ± SD of nine determinations from three independent experiments. B and D, Sertoli cells subjected to the CdCl2 treatment, such as those shown in A and C, were harvested on day 8 for DNA content measurement and compared with controls on days 1 and 8. Values are the mean ± SD from three separate experiments. Each experiment had replicate cultures. ns, Not significantly from controls; *, P < 0.05 vs. controls; **, P < 0.01 vs. controls. SC, Sertoli cells.

 
Effects of T on the cellular expression of occludin, E-cadherin, and uPA in Sertoli cell cultures
As T counteracted the CdCl2-induced TJ disruption, we assessed whether T could up-regulate the expression of TJ proteins, such as occludin, to enhance the tightness of inter-Sertoli TJs in vitro. On day 3 of the culture, Sertoli cells were exposed to T at either 1 x 10-9 M (Fig. 4Go, A and B) and 2 x 10-7 M, respectively (Fig. 4Go, C and D), and cultured for an additional 24 h in the absence of FSH. It was found that T alone at 1 x 10-9 and 2 x 10-7 M elicited a mild, but statistically significant, increase in occludin steady state mRNA level at 10–24 and 8–24 h, respectively, compared with cultures at time zero. It was noted that such a T-induced occludin expression was not detected in the control experiment without T (Fig. 4Go, E and F), illustrating that the increase in occludin expression shown in Fig. 4Go was not the result of an endogenous induction in these Sertoli cells. Although the assembly of inter-Sertoli TJs was known to induce occludin expression, its steady state mRNA level remains elevated between days 2–4.5 (33, 40), further suggesting that the increase shown in Fig. 4Go was a result of T stimulation rather than an endogenous change. Moreover, when these same samples were analyzed for E-cadherin and u-PA expression by semiquantitative RT-PCR, T had no apparent effect on their cellular expression (data not shown).



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  		Figure 4. A–F, Effects of T on the Sertoli cell occludin steady state mRNA levels in vitro. Sertoli cells isolated from 20-day-old rats were cultured at 1.2 x 106 cells/cm2 as described in Materials and Methods. On day 3 of the culture, Sertoli cells were treated with T at either 1 x 10-9 M (A and B) or 2 x 10-7 M (C and D), respectively, or given no steroid (E and F). FSH was not included in these experiments, so that only the effects of T were assessed. Cell cultures were terminated at specified time points for RNA extraction, and hot RT-PCR was performed to quantify the occludin steady state mRNA level. B, D, and F are the corresponding densitometrically scanned results using autoradiograms such as those shown in A, C, and E, respectively. Each bar represents the mean ± SD of duplicate cultures normalized against S16. Ctrl, Control Sertoli cell cultures terminated on day 3 without T. ns, Not significantly different from controls; *, P < 0.05 vs. controls; **, P < 0.01 vs. controls.

 
Effects of CdCl2 on the expression of E-cadherin, occludin, and uPA in vitro
The above study demonstrated that CdCl2 could induce reversible disruption of the inter-Sertoli TJ permeability barrier at selected concentrations in the presence of T. It presents the possibility that CdCl2-induced disruption can be used as an in vitro model to study the junction disassembly during germ cell migration. In this report we examined the expression of junctional and nonjunctional genes during CdCl2-induced inter-Sertoli TJ disassembly and its reassembly after the removal of CdCl2. In these experiments, Sertoli cells were exposed to CdCl2 at 1 µM in the presence of FSH (100 ng/ml) and T (1 x 10-9 M) on day 4 for 8 h; thereafter, Sertoli cell epithelia were rinsed by fresh F12/DMEM to remove CdCl2. Cultures were terminated at selected time points between 2 and 48 h when junctions were being disrupted by CdCl2 and were being reassembled after its removal.

Effects of CdCl2 on the expression of E-cadherin. E-Cadherin is a Ca2+-dependent cell adhesion molecule known to participate in the assembly of AJs (34); however, CdCl2-induced disruption did not alter the expression of E-cadherin while the inter-Sertoli TJs were being disrupted and reassembled (Fig. 5Go, A and B, and Fig. 3AGo).



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  		Figure 5. A and B, Effects of CdCl2 on the steady state mRNA level of E-cadherin in vitro when the inter-Sertoli TJ permeability barrier was disrupted and resealed. A, Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated dishes at 1.2 x 106 cells/cm2 and incubated in with both FSH (100 ng/ml) and T (1 x 10-9 M). The inter-Sertoli TJs were assembled by day 4, as manifested by a steady TER, which reached a plateau by day 4 (Figs. 1AGo, 2AGo, and 3AGo), CdCl2 was added to Sertoli cell epithelia to a concentration of 1 µM and incubated for 8 h. Thereafter, cells were washed with fresh medium containing FSH (100 ng/ml) and T (1 x 10-9 M) to remove CdCl2 and cultured in medium with both hormones. Cultures were terminated at 0, 2, 3, 5, 8, 10, 15, 20, 24, and 48 h after CdCl2 addition (1 µM) for RNA extraction. Hot RT-PCR was then performed to quantify the E-cadherin steady state mRNA level during and after the treatment with CdCl2. Ctrl, Control Sertoli cell cultures on day 4 before addition of CdCl2. B, Autoradiograms such as that shown in A were densitometrically scanned and normalized against S16 to yield the semiquantitative data. The relative expression of E-cadherin in the Sertoli cell cultures at 8 h was arbitrarily set at 1 at the time CdCl2 was removed by three successive washes, against which other cultures were compared. Each bar represents the mean ± SD of duplicate cultures normalized against S16. Only one representative set of experiments is shown, two additional experiments yielded identical results. ns, Not significantly different from CdCl2-treated culture at 8 h, by ANOVA.

 
Effects of CdCl2 on the expression of occludin. Occludin is a TJ-integral membrane protein that participates in the formation of paracellular seal (for reviews, see Refs. 35 and 36). When the inter-Sertoli TJs were being disrupted by CdCl2, it was accompanied by a steady decline in occludin expression; its expression, however, rose steadily during the resealing of inter-Sertoli TJs after the removal of CdCl2 (Fig. 6Go, A and B, and Fig. 3AGo). These results are in contrast to the cellular expression of E-cadherin, whose steady state mRNA level remained constant when inter-Sertoli TJs were disassembled and reassembled (Fig. 6Go vs. Fig. 5Go).



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  		Figure 6. A and B, Effects of CdCl2 on the steady state mRNA level of occludin in vitro when the inter-Sertoli TJ permeability barrier was disrupted and resealed after CdCl2 removal. A, Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated dishes at 1.2 x 106 cells/cm2 in the presence of both FSH (100 ng/ml) and T (1 x 10-9 M). When the inter-Sertoli TJs were assembled on day 4, as manifested by a stable TER, which reached a plateau on day 4 (Figs. 1AGo, 2AGo, and 3AGo), CdCl2 was added to Sertoli cell epithelia to a concentration of 1 µM and incubated for 8 h. Thereafter, cells were washed with fresh medium containing FSH (100 ng/ml) and T (1 x 10-9 M) to remove CdCl2 and cultured in F12/DMEM containing both hormones thereafter. Cultures were terminated at 0, 2, 3, 5, 8, 9, 10, 15, 20, 24, and 48 h after the addition of CdCl2 for RNA extraction. For statistical analysis by ANOVA, the expression of occludin in Sertoli cell cultures at 8 h when CdCl2 was removed was arbitrarily set at 1 when the reassembly of inter-Sertoli TJ resumed. Ctrl, Control Sertoli cell cultures on day 4 before the addition of CdCl2. Hot RT-PCR was performed in these samples to quantify the occludin steady state mRNA level. B, Autoradiograms such as that shown in A were densitometrically scanned and normalized against S16 to yield semiquantitative data. Each bar represents the mean ± SD of duplicate cultures normalized against S16. Only one representative set of experiments is shown. Two other sets of experiments yielded identical results, but these data were not pooled because in these two other experiments, cultures were terminated at slightly different time points. ns, Not significantly different from the CdCl2-treated culture at 8 h by ANOVA; **, P < 0.01 vs. the CdCl2-treated culture at 8 h.

 
Effects of CdCl2 on the expression of uPA. uPA is a serine protease that converts plasminogen into plasmin, an active serum enzyme (for review, see Ref. 37) that is also known to be synthesized by Sertoli cells (38). Using hot-nested RT-PCR, the expression of uPA increased during CdCl2-induced TJ disassembly. However, another transient increase in uPA expression was detected when the inter-Sertoli TJs were being reassembled (Fig. 7Go, A and B).



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  		Figure 7. A and B, Effects of CdCl2 on the steady state mRNA level of uPA in vitro when the inter-Sertoli TJ permeability barrier was disrupted and resealed after CdCl2 removal. A, Sertoli cells isolated from 20-day-old rats were cultured on Matrigel-coated dishes at 1.2 x 106 cells/cm2 in the presence of both FSH (100 ng/ml) and T (1 x 10-9 M). When the inter-Sertoli TJs were assembled on day 4, as manifested by a stable TER, which reached a plateau on day 4 (Figs. 1AGo, 2AGo, and 3AGo), CdCl2 was added to Sertoli cell epithelia to a concentration of 1 µM and incubated for 8 h. Thereafter, cells were washed with fresh medium containing FSH (100 ng/ml) and T (1 x 10-9 M) to remove CdCl2, and cells were cultured in F12/DMEM containing both hormones thereafter. Cultures were terminated at 0, 2, 3, 5, 8, 9, 10, 15, 20, 24, and 48 h after the addition of CdCl2 for RNA extraction. For statistical analysis by ANOVA, the expression of uPA in Sertoli cell cultures at 8 h when CdCl2 was removed was arbitrarily set at 1.0, where inter-Sertoli TJ reassembly began. Ctrl, Control Sertoli cell cultures on day 4 before the addition of CdCl2. Hot RT-PCR was then performed in these samples to quantify the uPA steady state mRNA level. B, Autoradiograms such as that shown in A were densitometrically scanned and normalized against S16 to yield semiquantitative data. Each bar represents the mean ± SD of duplicate cultures normalized against S16. Only one representative set of experiments is shown. Two other sets of experiments yielded identical results, but these data were not pooled because these other cultures were terminated at slightly different time points. ns, Not significantly different from the CdCl2-treated culture at 8 h by ANOVA; *, P < 0.05 vs. the CdCl2-treated culture at 8 h; **, P < 0.01 vs. the CdCl2-treated culture at 8 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The movement of germ cells from the basal to the apical compartment of the seminiferous epithelium during spermatogenesis involves extensive turnover of intercellular junctions, such as junction disassembly and reassembly, and restructuring of cell matrixes (for reviews, see Refs. 1, 2, 4, 18 , and 39). It is likely that these events are regulated by an array of molecules, such as proteases, protease inhibitors, signaling molecules, growth factors, and cell adhesion molecules (for review, see Ref. 5). Our laboratory has used primary cultures of Sertoli cells and cocultures of Sertoli and germ cells in vitro to identify target genes that are involved in the assembly of inter-Sertoli TJs and Sertoli-germ cell AJs, which include proteases, such as uPA and cathepsins (6, 8); protease inhibitors, such as {alpha}2-macroglobulin (6, 12); and cell adhesion molecules, such as N-cadherin and ß-catenin (6). However, these studies are corroborative in nature, proof that their involvement in junction assembly is not established. Still, these results illustrate that germ cell migration is a dynamic cellular event, requiring the participation of an array of molecules. A more recent study in which transforming growth factor-ß is shown to abolish the timely expression of ZO-1 and occludin when inter-Sertoli TJs are being assembled and that it can perturb the inter-Sertoli TJ permeability barrier (40) provides the first proof that these earlier descriptive studies to delineate the events of junction reassembly and their regulation are physiologically important. Nevertheless, an in vitro model to study the events of junction disassembly is still lacking.

Administration of CdCl2 to rats in vivo by ip was shown to induce vascular permeability; this, in turn, led to edema, hemorrhage, and eventually necrosis (41, 42, 43, 44). Other in vivo studies also demonstrated that CdCl2 caused irreversible disruption of TJ-associated microfilaments in rat Sertoli cells (21). In addition to the damage in the testis, CdCl2 disrupted vascular endothelial integrity in different organs (for review, see Ref. 45). Likewise, CdCl2 disrupted the endothelial TJ permeability barrier in vitro, such as human umbilical vascular endothelial cells (for review, see Ref. 46), and the Sertoli cell permeability barrier in vitro (23).

In this report, the addition of CdCl2 to Sertoli cells inhibited not only the assembly but also the maintenance of inter-Sertoli TJs dose dependently. This CdCl2-induced disruption was irreversible in serum-free medium; however, its effect became reversible in FSH- and T-supplemented medium. More important, the disruptive effect of CdCl2 could be counteracted using a pharmacological dose of T at 2 x 10-7 M. These results led us to speculate that T may play a role in regulating TJ assembly through an as yet to be defined signal transduction pathway. However, we have not investigated the potential protective effects of FSH alone on CdCl2-induced inter-Sertoli TJ permeability barrier disruption. In this study, T stimulated Sertoli cell occludin expression in vitro, and the effect was more profound at 2 x 10-7 than at 1 x 10-9 M, indicating that T may exert its action by inducing TJ integral proteins, which subsequently become the building blocks of TJs. Although 2 x 10-7 M (200 nM) T, used in this study, is about 100-fold higher than the level of T in the systemic circulation (~2 nM), the levels of T in rete testis fluid and seminiferous tubular fluid range between 50 and 200 nM (47), illustrating that the microenvironment that surrounds developing germ cells is indeed equipped with such a high level of T. On the other hand, occludin is known to be induced, and its protein is accumulated at the site of inter-Sertoli TJs at 15–18 days of age when the BTB is being assembled in vivo (48, 49). It is therefore possible that the timely opening and closing of TJs during spermatogenesis that facilitate the passage of preleptotene spermatocytes through the BTB are regulated at least in part by testicular hormones. For instance, T may exert its effects in regulating the BTB by modulating de novo synthesis of TJ integral proteins, such as occludin. This speculation is further strengthened by a recent observation that the expression of claudin-11 in vitro is regulated by FSH and tumor necrosis factor-{alpha} (50), suggesting a critical role of testicular hormone and signaling molecules in remodeling the BTB during spermatogenesis. An earlier report also demonstrated that treatment of rats with androgens delayed the onset of infertility after their exposure to CdCl2 (44). Apparently, the effects of CdCl2 on the inter-Sertoli TJ permeability barrier are unlikely to be due to cell toxicity, because the DNA content of the CdCl2-treated cultures was not significantly different from that of control cultures. Moreover, the CdCl2-induced disruption of the inter-Sertoli TJ barrier could be resealed after CdCl2 was removed, illustrating that cells were still viable after the CdCl2 treatment. However, we consistently observed that when Sertoli cells were cultured in the presence of T at 2 x 10-7 M and exposed to 5 µM CdCl2, these cultures had a DNA content significantly lower than that of Sertoli cell cultures exposed to 0, 0.1, or 1 µM CdCl2 at the end of the experiment (day 8), illustrating that cell viability was reduced by CdCl2 treatment in the presence of a high concentration of T. These results seemingly suggest that although the presence of 2 x 10-7 M T could protect the inter-Sertoli TJ permeability barrier from the disruptive effects of CdCl2, its presence apparently also made the Sertoli cell more vulnerable to CdCl2 treatment, in particular when CdCl2 was present at a concentration as high as 5 µM, possibly by altering some as yet to be defined cellular properties.

The CdCl2-induced inter-Sertoli TJ disassembly also associates with changes in cellular expression of E-cadherin, occludin, and uPA. It has been reported that the assembly of TJs in vitro requires the participation of adhesion molecules, such as E-cadherin. For instance, addition of anti-E-cadherin antibody to MDCK cells impaired resealing of the Ca2+-depletion-induced TJ permeability barrier disruption (31). Studies by Northern blots, immunoblots, and immunohistochemistry have shown that expression of E-cadherin is the highest in the rat testis at 14 days of age just before the establishment of BTB (51), illustrating its important role in inter-Sertoli TJ assembly. Apart from its involvement in the assembly of BTB, E-cadherin has also been shown to mediate an early adhesion event between epithelial cells that is required for the assembly of desmosomes and adherens junctions (52). Taking these data collectively, it is clear that E-cadherin plays a crucial role in the assembly of AJs, GJs, and TJs. The CdCl2-induced inter-Sertoli TJ disruption may be mediated by its initial disruption in AJs even though a decline in E-cadherin expression was not detected, as a disruption of AJs may not require any changes in the synthesis of their building blocks. This speculation is reached based on several recent studies suggesting that E-cadherin is the target of CdCl2. First, CdCl2 causes redistribution of E-cadherin in Caco-2 cells when examined by immunohistochemistry (for review, see Ref. 46). Second, studies by immunohistochemistry using MDCK and LLC-PK1 cells cultured in vitro have shown that E-cadherin becomes diffusely localized in the cytoplasm rather than at the cell-cell border in Cd2+-treated cells, reflecting the fact that E-cadherin is translocated from the site of AJs to cytoplasm instead of a disruption in its expression or synthesis upon CdCl2 treatment (for review, see Ref. 46). Of course, the biochemical mechanism by which this molecular translocation is initiated and regulated remains to be determined. The fact that the Cd2+-mediated E-cadherin redistribution is similar to that induced by Ca2+ depletion strongly suggests that Cd2+ may compete with Ca2+ to the same binding sites in E-cadherin, which causes its subsequent dissociation.

It has been reported that integral TJ proteins, such as occludin and claudins, play an important role in forming paracellular seal (for review, see Ref. 35). Besides claudins, occludin is another TJ integral protein found in the TJ fibrils (53). It is concentrated in TJs of mouse/rat, but not human/guinea pig, Sertoli cells (48). An immunolocalization study in the mouse revealed that occludin is localized at the basal region of seminiferous tubules by postnatal day 14 when BTB is being assembled (49). In vitro studies from our laboratory have shown that occludin is a biochemical marker to monitor inter-Sertoli TJ assembly (33, 40). Moreover, addition of a synthetic peptide corresponding to the second external loop of occludin can perturb the inter-Sertoli TJ assembly in vitro and spermatogenesis in vivo (33). Recent studies using glycerol-treated rat testes have shown that the glycerol-induced disruption of inter-Sertoli TJs is associated with a dissolution of actin filaments and a loss of occludin staining at the TJs, which, in turn, leads to an increase in BTB permeability (54). These results further illustrate that occludin is a crucial component of the paracellular seal in TJs. Based on these studies, it is logical to speculate that a disruption of the inter-Sertoli TJ leading to an increase in paracellular permeability would be associated with a decline in occludin expression. Indeed, CdCl2-induced inter-Sertoli TJ permeability barrier disruption is accompanied by reduced occludin expression. When these CdCl2-disrupted TJs were resealing, a steady increase in occludin expression was detected, indicating that de novo synthesis of occludin is required to rebuild the disrupted TJs.

On the other hand, proteases have been implicated in tissue restructuring during testicular development and maturation. This speculation is further strengthened by the fact that there are changes in protease activities in the epithelium at different stages of the spermatogenic cycle. For instance, the expression of uPA, a Sertoli cell product, is highest in Sertoli cells at stages VII–VIII (38). A recent study has demonstrated a significant, but transient, induction of uPA expression within 1 h after coculture of Sertoli cells with germ cells at the time germ cells adhered to Sertoli cells (6). It was suggested that proteases might be used to disrupt specific sites on Sertoli cell surface upon which germ cells can adhere to allow the subsequent AJ formation (5, 6). When Sertoli cells were cultured at a high cell density, a transient, but significant, increase in uPA expression was also detected while inter-Sertoli TJs were being assembled and when TJ assembly was completed (12). This pattern of changes in expression of selected cellular genes is consistent with the present report (12). It is possible that the increased uPA expression during CdCl2-induced inter-Sertoli TJ disruption is an indication of its involvement in junction complex dissolution.

In summary, these results illustrate that CdCl2-induced inter-Sertoli TJ disruption can be used as a model to study cell junction disassembly because this model fulfills the following criteria: 1) CdCl2 is noncytotoxic to Sertoli cells at doses that can disrupt the inter-Sertoli TJs; 2) its effect on TJ permeability barrier is reversible and dose dependent; 3) an actual disruption of the TJ has indeed taken place, as the time it took for the junction to reassemble mimics that of the untreated cells, but is different from the Ca2+-induced TJ "leakiness," which could be restored within 90 min; and 4) this process of junction reassembly mimics the reassembly of inter-Sertoli TJs and AJs as previously reported (7, 9, 12) when the cellular expressions of several target genes are examined.


   Acknowledgments
 
We thank Dr. Meng-yun Mo for his help performing the nucleotide sequence analysis to verify the authenticity of the PCR products, which included occludin, uPA, S16, and E-cadherin. We also thank the National Hormone and Pituitary Program (NIDDK, NIH) for the gift of ovine FSH.


   Footnotes
 
1 This work was supported in part by grants from the CONRAD program (CIG96-05-A) and the Noopolis Foundation. Back

Received October 27, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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