Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105
Correspondence should be addressed to Dr. Elaine M Faustman at Institute for Risk Analysis and Risk Communication, Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way. NE, Suite 100 Seattle, WA 98105. Fax: 2066854696. E-mail: faustman{at}u.washington.edu.
Received October 19, 2004; accepted January 4, 2005
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ABSTRACT |
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Key Words: in vitro model; Sertoli cell/gonocyte co-culture; Matrigel (ECM) overlay; stress signaling; c-Kit; cadmium.
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INTRODUCTION |
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In this study, we propose that the employment of a 3-D culture environment, effected through the addition of an ECM overlay, will result in a significant improvement to the existing 2-D substratum approach traditionally used in the culture of SGC. We hypothesize that this modification provides a simpler and more efficient method to improve the interactions between Sertoli cells and germ cells via the reorganization of cellular architecture and the resultant modulation of both intracellular stress and survival signaling cascades, thereby leading to a physiologically stable SGC system.
To test our hypothesis, we explored the dose and time-dependent effects of ECM overlay on morphological and cytoskeleton changes, cell viability, and stress and survival signal transduction pathways. We also examined the expression levels of c-Kit, an important regulator in primordial germ cell survival and proliferation (Yoshinaga et al., 1991). Finally, we applied this co-culture system to investigate the dose- and time-dependent effects of cadmium (Cd), a known testicular reproductive toxicant (IARC, 1976
.) in both animals and humans.
We found that an ECM overlay dose-dependently enhanced the attachment of Sertoli cells and facilitated the establishment of Sertoli cellgonocyte communication, as observed by refinements to cytoskeleton structure and enhanced cell viability. The latter was consistent with the observed dose- and time-dependent modulation in the presence of the ECM overlay of both stress signaling pathways (SAPK/JNK) and survival signaling pathways (ERK and AKT). Furthermore, the dose-dependent increase of c-Kit protein expression in co-culture suggested that the presence of an ECM overlay significantly improved the proliferation and survival of the spermatogonia. Finally, dose-dependent effects of Cd treatment on morphological alteration and induction of apoptosis were observed in the culture SGC. We conclude that this modified in vitro SGC system will provide investigators a simple, efficient, and highly reproducible alternative in the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental agents.
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MATERIALS AND METHODS |
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Cell Harvest and Protein Determination.
At the appropriate time points, cultured cells were rinsed twice with ice-cold phosphate-buffered saline (PBS). Cell lysis buffer (Cell Signaling Technology Inc, Beverly, MA) was added to each dish, and cells were scraped with a rubber policeman. Harvested cells were then sonicated at 40 W for 15 s. Resultant cell lysates were centrifuged at 16,000xg for 15 min at 4°C. Supernatant fractions were collected, and the concentration of protein was determined with a commercially available kit (Protein Assay kit, Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. All samples were subsequently stored at 80°C until assayed.
Morphology and Viability.
All cultures were viewed with a Nikon inverted microscope equipped with phase-contrast optics (Nikon, Tokyo, Japan) at intervals during culture to assess their general appearance. Resultant images were captured and digitized with a Coolsnap Camera (Roper Scientific, Inc, Duluth, GA). The digitized image was processed with Photoshop 5.5 software. To determine the viability of the cultured cells after overlay of ECM or Cd treatment, a three-color fluorescence assay was applied to determine the number of live and dead cells. Live cells have intracellular esterases that convert the non-fluorescent, cell permeable calcein acetoxymethyl ester (calcein AM) to the intensely green fluorescent, calcein. Propidium iodide (PI), a live cellimpermeable dye, selectively stained nuclei of later apoptotic nucleus with increased membrane permeability, while Hoechst 33342 selectively stained total nuclei with blue fluorescence. The nuclei of live cells are evenly stained, whereas apoptotic cells are intensely and irregularly stained. Propidium iodide (1 mg/ml), Hoechst 33342 (1 mg/ml), and calcein AM (Sigma) were added directly to co-cultures and incubated at 37°C for 5 min. The stained co-cultures were viewed with appropriate bandpass filters for PI, Hoechst, and calcein with Nikon Labophot-2 with E-Planachromats for brightfield and epi-fluorescence microscopes (Nikon, Tokyo, Japan). Each single-colored image was captured and digitized with a Spot Camera (Diagnostic Instruments, Sterling Heights, MI) equipped with MetaMorph software (Universal Imaging Corporation, Downingtown, PA), as shown later in Figure 2. Three random views were recorded and the number of the PI-positive cells (dead cells) and the number of nuclei stained with Hoechst (total cell number) were counted with the Cell Counting program in the MetaMorph software. Nuclei with intense and irregular dark-blue staining and without surrounding green were also counted (dying cells). The percentage of apoptotic cells was calculated by dividing the total number of dead (PI positive) and dying cells by the total cell number.
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Immunofluorescence of c-Kit Protein.
Co-cultures of SGC were washed with phosphate buffered saline (PBS) 72 h after the addition of various concentrations of ECM overlay and fixed with 4% formaldehyde for 20 min, and then washed three times with PBS. After permeabilization in TBS containing 0.5% Triton X-100 (TX-100) for 10 min, cells were blocked for 30 min in 5% normal goat serum (NGS; Sigma) in PBS with 0.1% TX-100, and then incubated with primary c-Kit polyclonal antibody (1:200, Santa Cruz Biotechnology, Inc. Santa Cruz, CA) in PBS with 1% goat serum and 0.1% TX-100 over night at 4°C. Cells were washed three times in PBS before the addition of a goat anti-rabbit Alexa-conjugated secondary antibody (1:200 dilution, Molecular Probes, Inc. Eugene, OR) for 1 h at 37°C. Vectashield mounting medium containing 2 µl Hoechst 33342 (1 mg/ml) was added to each dish to counterstain the nuclei and prevent photo bleaching. The stained co-cultures were viewed with an appropriate FITC filter under a fluorescent microscope. The image was captured and digitized with a Spot Camera equipped with MetaMorph software.
Cadmium (Cd) Treatments.
Sertoli cell/germ cell co-cultures were cultured as described in the presence of an ECM overlay of 200 µg/ml. Serial dilutions were prepared from a stock solution of Cd and added directly at the stated concentrations to the culture medium 48 h after the addition of the ECM overlay. The final concentrations tested were 0.5, 2.5, 5, 10, and 20 µM. Morphological alterations to the co-culture after Cd treatment were recorded at the stated intervals during treatment. Similarly, the viability of the co-cultured cells after Cd treatment was assessed as described earlier under Morphology and Viability.
Assessment of Apoptosis.
For the evaluation of the apoptotic morphological changes, cells were fixed and stained with Hoechst 33342 (0.1 mg/ml in PBS) after Cd treatment. The stained co-cultures were viewed with an appropriate filter under the fluorescent microscope. Each image was captured and digitized using Spot Camera (Diagnostic Instrument, Inc.) equipped with MetaMorph software. Apoptosis-associated endpoints were further determined in cell extracts by measuring functional activities associated with caspase 3/7 using caspase-specific fluorogenic substrates. The activity of caspase 3/7 was measured by a fluorometric assay, using Ac-DEVD-AMC as the specific substrate, as previously reported (Nicholson et al., 1995; Shi et al., 2000
). Briefly, 10 µg of cell extract was added in duplicate in 96-well plate format. Reaction buffer containing the fluorogenic substrate enzyme-catalyzed release of 7-amino-4-methyl coumarin (AMC) was added to initiate the reaction, which was incubated at 37°C for 2 h and enzyme-catalyzed release of AMC measured by a fluorescence microplate reader at excitation 360 nm and emission 460 nm. Fluorescent units were converted to pmol of AMC released per microgram of protein and incubation time (h) using a standard curve generated with known serial dilutions of AMC.
Western Blot Analysis.
Western blot analyses of the phosphorylation status of various proteins implicated in cellular stress and survival pathways were performed according to a method described in our previous studies (Sidhu et al., 2004; Yu et al., 2001
). Gels were transferred to PVDF membranes (Millipore, Billerica, MA) using a vertical transfer apparatus (Bio-Rad Laboratories). Membranes were rinsed briefly in Tris-buffered saline, pH 7.6 (TBS), blocked with 5% nonfat dried milk in TBS with 0.1% Tween-20 (TTBS) for 20 min and rinsed again with TTBS. Membranes were then incubated overnight with primary antibody, followed by four washes with TTBS. The primary antibodies included c-Kit (Santa Cruz Biotechnology, Inc.), phospho-SAPK/JNK, phospho-ERK1/2, phospho-p38 MAPK, phospho-c-Jun, and phospho-ATF2 (Cell Signaling, Inc.). ß-actin (Santa Cruz Biotechnology, Inc.) was used as an internal standard for the protein loading. No significant changes were observed in ß-actin concentrations relative to cell number (protein concentration) under all our experimental conditions. After hybridization with relevant secondary antibodies conjugated to horseradish peroxidase, the resulting immuno-complex was detected with the ECL detection reagent (Amersham Biosciences Corp, Piscataway, NJ), followed by exposure to x-ray films. The resulting films were scanned using HP Scanjet 5400C scanner equipped with Precisionscan Pro 3.1 software, and the TIF image data were analyzed. Quantitative analysis of densitometric scans of band intensities and area were achieved using the "Quantity One" software (Bio-Rad Laboratories). Data are presented as arbitrary units after internal standard correction with ß-actin.
Statistical Analysis.
The results of quantitative analysis of cell number, percentage of apoptotic cells, caspase 3/7 activity, and Western blot band densitometric quantification are expressed as the mean ± S.D. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison tests. A p value <0.05 denoted the presence of a statistically significant difference.
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RESULTS |
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With the MetaMorph software, we conducted a quantitative analysis of live/dead cells as assessed by the three-color fluorescence assay. The percentages of apoptotic cells and the average number of cells per view are shown in Figure 3. The percentage of apoptotic cells (condensed, irregular staining with PI) decreased proportionally relative to the ECM concentration, both at 24 h and 48 h after plating, and was most significantly reduced when the concentration of ECM overlay was 200 µg/ml (Fig. 3A). The total number of cells was low in the co-culture without an ECM overlay, but it dose-dependently increased with an ECM overlay (Fig. 3B).
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Increased Expression of c-Kit Protein in SGC Cultured in the Presence of ECM Overlay
The c-Kit proto-oncogene encodes for a plasma membraneassociated receptor with inherent tyrosine kinase activity. We examined the levels of c-Kit protein expression in the SGC co-culture to determine whether the effect of ECM on the functional interaction between the Sertoli cells and gonocytes was associated with a modulation of this critical regulator protein. Figure 7 shows that an increase in c-Kit protein expression was observed in the presence of the ECM overlay, as detected by immunofluorescence staining and Western blot analysis 72 h after plating. We therefore conclude that ECM overlay did indeed facilitate an increased interaction between Sertoli cells and spermatogonia, and this forms a critical basis for the improvements in the cellular integrity as well as the in vivolike environment.
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DISCUSSION |
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In the intact organism, cells form tissues that are bound together by a meshwork of extracellular matrix (ECM) molecules that include collagens, fibronectin, laminin, and several other proteins (Semino et al., 2003). Isolation of Sertoli cells and gonocytes involves a series of enzymatic digestions of the interstitial matrix and mechanical disruption of the tissue. Unfortunately, this procedure disrupts the interaction between the ECM and actin cytoskeleton, which is an essential microenvironmental component required for SGC function and survival. In this study, we demonstrated a dose-dependent increase in viability of SGC after the application of an ECM overlay, which strongly supports the important role of ECM in regulating the survival or death of SGC. The remodeling of the complex structure of the SGC architecture after dissociation may rely on an ECM interaction to sense cellular environment. The ECM has been shown to facilitate survival in a number of cell types, and adhesion to ECM has previously been shown to conversely suppress apoptosis in several cell types (Boudreau, 2003
; Fitzakerley, 2001
). In the presence of ECM overlay (
200 µg/ml), a multilayered architectural structure was formed 72 h after plating. Hadley et al. (1985)
reported their comparative study on co-culture systems cultured on tissue-culturetreated plastic, a matrix deposited by co-culture of Sertoli and peritubular myoid cells, and a reconstituted basement membrane gel from the EHS tumor. They found that co-cultures formed a polarized monolayer when grown on top of the reconstituted basement membrane, but that germ cells did not differentiate. They also indicated that when Sertoli cells were cultured within reconstituted basement membrane gels (3-D matrix) Sertoli and germ cells reorganized into cords, which can provide an environment permissive for germ cell differentiation. They suggested that such 3-D matrices effectively induced differentiated cell function better than traditional 2-D substrates. However, because of the high cost of culturing such cells within ECM gels, it is difficult to apply this culture system widely. Thus, our modified overlay culture system is a simpler and more cost-effective alternative method which facilitates cellcell and cellmatrix interactions of the SGC, and which more mimics the 3-D structure as observed in in vivo.
Previous studies have demonstrated the significant role played by ECM in primary hepatocyte culture (Berthiaume et al., 1996; Sidhu et al., 1993
, 2004
). Application of the ECM overlay technique has resulted in a significant improvement in the expression levels as well as drug-inducibility of various cytochromes P-450 in primary hepatocytes (Sidhu et al., 1993
). Furthermore, a recent study (Sidhu et al., 2004
) has extended such observations with critical findings demonstrating that ECM modulation of cellular stress in primary hepatocyte cultures is intimately linked to improvements in both liver-specific and drug-inducible gene expression. The mechanisms by which ECM regulates cell growth, differentiation, and gene expression, especially for the Sertoli cells and gonocytes, are not yet fully defined. CellECM interactions can directly regulate cell behavior, either through receptor-mediated signaling or by modulating the cellular response to growth factors (Klinowska et al., 1999
). The ECM interacts directly with cell surface receptors such as integrins, the major class of ECM receptors that initiate signal-transduction pathways, which in turn modulate cell survival, growth, and apoptosis (Brassard et al., 1999
; Kanda et al., 1999
). The MAPK family pathways are well-defined by their respective downstream proline-directed protein kinases, including extracellular responses kinase (ERK1/2), stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK), and p38 MAPK (Bhowmick et al., 2001
; Crean et al., 2002
; Rosen et al., 2002
). Mitogenic stimulation of the ERK1/2 pathway modulates the activity of many transcription factors, leading to biological responses such as proliferation and differentiation (Johnson and Lapadat, 2002
). In contrast, the p38 MAPK and SAPK/JNK pathways were strongly activated by stress stimuli (Johnson and Lapadat, 2002
; Rincon et al., 2000
) and are originally identified as an important pathway in the transduction of apoptotic signals initiated by stress or toxic stimuli such as ultraviolet light, osmotic stress, and inflammatory cytokines (Ichijo, 1999
). In the present study, the ECM-mediated reduction of the stress signaling pathway, SAPK/JNK, and concomitant reduction of cell death, suggests that phosphorylation of SAPK/JNK signaling was involved in the activation of apoptosis, and that ECM plays an important role in modulating cellular stress signaling in neonatal Sertoli cell/gonocytes. Furthermore, we observed two different patterns of activation by the ECM of ERK1/2, an inverse dose-dependent activation at the early time point after plating and dose-dependent activation at later time points. In the absence of or at low concentrations of ECM overlay and at the early time points post plating, cells may attempt to use rescue mechanisms such as the activation of ERK1/2 to maintain the integrity of the co-culture. However, in the absence of or at low concentrations of ECM overlay, the activation of the cellular rescue machinery is not sufficient to modulate the normal intracellular signal transduction response. As a result, increased cell death was observed as the activation of ERK1/2 decreased significantly 8 and 24 h later. Such findings suggest that, although activation of ERK1/2 was involved in cell survival, it by itself is not sufficient to maintain Sertoli cell/gonocyte function.
Protein kinase B (PKB/AKT) is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components (Gu et al., 2002). Protein kinase B phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation (Datta et al., 1999
; Ibuki and Goto, 2000
). The intrinsic capacity of all cells to undergo apoptosis is suppressed by survival signals induced by factors within their immediate microenvironment. In this study, we observed that activation of AKT was critically linked with the dose of ECM as well as the duration that cells were exposed to the overlay. In the absence of an ECM overlay, the activation of AKT was significantly lower than in the presence of an ECM overlay. Previous studies have also shown that AKT-mediated survival is triggered by growth factors, extracellular matrix, and other stimuli (Datta et al., 1999
), and our current study suggests the indispensable role of ECM in regulating this response in the primary SGC co-culture.
Organization of the actin cytoskeleton during cell adhesion clearly requires intracellular signals that trigger polymerization of cytoskeleton proteins in response to all matrix adhesion (Berthiaume et al., 1996; Machesky and Hall, 1997
). In our study, the ECM overlay caused a dramatic reorganization of the cytoskeleton of SGC with the dose-dependent structural changes further confirming the significant role of the ECM overlay in reorganizing cellular architecture. An ECM overlay >200 µg/ml (Fig. 4D) appeared to be sufficient for the cells to adopt in vivo-like 3-dimensional diffusive cytoplasmic staining of FITC-phalloidin with a distinct line of intense staining where cells are in intimate contact with each other. Our current study strongly supports the notion that an ECM overlay is necessary in modulating the intracellular transduction signaling and reorganization of SGC structural integrity.
Finally, we evaluated the effects of ECM overlay on the expression level of c-Kit. The c-Kit proto-oncogene encodes for a plasma membrane-associated receptor with inherent tyrosine kinase activity. This receptor and its ligand, Stem Cell Factor (SCF) are vital for normal hematopoiesis, melanogenesis and gametogenesis (Orth et al., 1997; Rossi et al., 2000
). Substantial evidence indicates that SCF is manufactured by Sertoli cell and that c-kit is expressed by germ cells during fetal development and in pubertal and adult rats. Interactions between this receptor on gonocytes and its ligand on the Sertoli cell surface are important in maintaining proliferation and differentiation of neonatal gonocytes (Rossi et al., 2000
). The ECM-mediated stabilization of c-kit protein expression indicated that the ECM overlay increased the interaction between Sertoli cells and spermotogonia at high concentrations, which supports the critical role of ECM in modulating the functional interaction between the Sertoli cells and gonocytes.
Cadmium is a ubiquitous environmental pollutant, and it has been reported to have male reproductive toxicity both in humans and in animals (IARC, 1976; Laskey et al., 1986
; Ragan and Mast, 1990
; Schrag and Dixon, 1985
). In the present study, we demonstrated a dose-dependent cytotoxicity and disruption of SGC structure after Cd treatment. The application of a three-color fluorescence assay in this in vitro culture system, combining with the recent advanced High Content Screening (HCS) Reader will provide a high throughput, automated solutions for testicular cellspecific screening for toxicity and activity of cellular constituents (Gaspari et al., 2004
). Our initial observation further revealed the induction of apoptosis at concentrations as low as 5 µM Cd, which suggests its potential in the mechanistic study of testicular toxicity.
In conclusion, our findings demonstrate that application of an overlay of ECM (200 µg/ml) to SGC permitted the formation of a testicular-like multilayered architectural structure that mimics in vivo characteristics of seminiferous tubules. The presence of an ECM overlay resulted in a dose- and time-dependent suppression of the stress-signaling pathway (SAPK/JNK) and conversely increased signaling responses (ERK1/2 and AKT) associated with the survival of spermatogonia. Likewise, the concomitant upregulation of the expression of c-Kit protein confirmed functional integrity of this co-culture system. These results support our proposal that the ECM overlay enables a physiologically more stable SGC system. This system will therefore prove invaluable in the screen for testicular cellspecific cytotoxicity and in the examination of the molecular mechanisms associated with developmental and reproductive perturbations induced by environmental toxicants to Sertoli cells and/ or spermatogonial cell proliferation and differentiation.
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ACKNOWLEDGMENTS |
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