Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Vanesa G. Martinez2, Eliana H. Pellizzari3, Emilce S. Díaz4, Selva B. Cigorraga3, Livia Lustig4, Berta Denduchis4, Carlota Wolfenstein-Todel2 and M. Mercedes Iglesias1,2

2 Instituto de Química y Fisicoquómica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquómica, Universidad de Buenos Aires, Junón 956, (1113) Buenos Aires, Argentina; 3 Centro de Investigaciones Endocrinológicas (CONICET), Hospital de Niños ìRicardo Gutierrez, Gallo 1330, (1425) Buenos Aires, Argentina; and 4 Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina

Received on June 12, 2003; revised on July 7, 2003; accepted on October 16, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin–stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.

Key words: apoptosis / galectin-1 / laminin-1 / Leydig cells / testis


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carbohydrates are believed to form a glyco code that can be interpreted by carbohydrate-binding proteins, such as lectins, which in turn can trigger diverse cellular responses like proliferation, differentiation, metastasis, and apoptosis (Barondes et al., 1994aGo; Villalobo and Gabius, 1998Go). Galectins are a family of carbohydrate-binding proteins with a largely known importance in cell and tissue physiology, although their precise biological functions remain elusive (Hernandez and Baum, 2002Go; Liu, 2000Go). They are defined by a consensus sequence in their carbohydrate-recognition domain (CRD) and affinity for ß-galactosides (Barondes et al., 1994bGo). The best studied member of this family is galectin-1 (Gal-1), which is expressed as a noncovalently linked homodimer of 14 kDa subunits, with two CRDs per dimer (Barondes et al., 1994bGo; Liao et al., 1994Go). It is widely expressed in several tissues, including those related to the immune system (Perillo et al., 1998Go; Rabinovich et al., 1998Go; Zuñiga et al., 2001aGo). Gal-1 participates in the regulation of immune homeostasis during development both in the thymus and in the periphery, exerting an apoptotic effect on immature thymocytes, both negatively selected and nonselected, and on activated T-cells (Iglesias et al., 1998aGo; Perillo et al., 1995Go, 1997Go; Vespa et al., 1999Go). This can explain Gal-1's ability to suppress experimental autoimmune diseases and inflammation (Delioukina et al., 1999Go; Levi et al., 1983Go; Liu, 2000Go; Offner et al., 1990Go; Rabinovich et al., 1999Go; Santucci et al., 2000Go; Tsuchiyama et al., 2000Go). The apoptosis-inducing effect of Gal-1 on cells of the immune system has been studied in great detail, but information about its effect on other cell types is scarce (Ellerhorst et al., 1999Go; Wells et al., 1999Go).

Gal-1 modulates cell adhesion (Hughes, 2001Go) through its interactions with ligands that contain N-acetyllactosamine residues, like some cell-surface receptors and extracellular matrix (ECM) glycoproteins (Gu et al., 1994Go). Laminin is a major component of the ECM known to be implicated in adhesion, migration, proliferation, and apoptosis of several different cell types. The antiapoptotic effect of laminin was shown on A549 cells: adhesion of these cells to laminin-10/11 can trigger the protein kinase B/Akt pathway and thus rescue cells from apoptosis induced by serum removal (Gu et al., 2002Go). Laminin can be selectively recognized by Gal-1 through its polylactosamine oligosaccharide residues, and this makes it a potential modulator of cell growth.

Both spermatogenesis and steroidogenesis take place in the testis, but in different compartments. Leydig cells reside in the interstitial compartment surrounded by blood vessels, lymphatics, connective tissue cells, and macrophages and are responsible for testosterone production. Hormone synthesis by these cells is essential to maintain spermatogenesis because testosterone regulates germ cell maturation indirectly through its paracrine effects on Sertoli cells. Leydig cells are in intimate contact (Kuopio and Pelliniemi, 1989Go) and are regulated by the ECM (Vernon et al., 1991Go). Moreover, we have recently studied the effect of ECM proteins on in vitro testosterone production by rat Leydig cells, and our results suggest that these proteins are able to modulate steroidogenesis (Diaz et al., 2002Go).

The expression of Gal-1 and -3 and galectin-specific binding sites in Sertoli cells was detected in normal testis and Sertoli cell-only syndrome by immunohistochemistry (Dettin et al., 2003Go; Timmons et al., 2002Go; Wollina et al., 1999Go). The presence of Gal-1 has also been reported in interstitial cells in mouse testis sections (Timmons et al., 2002Go). However, to our knowledge, there is no information about Gal-1 expression in isolated rat Leydig cells.

On the basis of multivalent binding and cross-linking properties of galectins and their ability to interact with the ECM causing changes in cell adhesivity and in other properties, it seemed possible that Gal-1 might modulate Leydig cell growth. The aim of this study was to evaluate in vitro Gal-1 effects on adult rat Leydig cells. We report that Gal-1 induces changes in Leydig cell morphology and reduces cell viability and testosterone production. Furthermore, this is the first time that an endogenous protein, Gal-1, is shown to possess apoptosis-inducing activity on Leydig cells. The present study provides an alternative cellular mechanism, based on Gal-1 and laminin-1 interaction, to regulate Leydig cell function and survival.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Identification of Gal-1 in Leydig cell cultures by immunofluorescence
We analyzed the expression of Gal-1 in rat Leydig cell cultures by immunofluorescence using a rabbit polyclonal antibody against human Gal-1. Immunostaining performed on permeabilized cells showed that Gal-1 is abundantly expressed, mainly in the cytoplasm (Figure 1B). However, staining of nonpermeabilized cells also indicates the presence of Gal-1 as patches on the cell surface (Figure 1C). No immunoreaction was observed in cells treated with an anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Figure 1A).



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Fig. 1. Identification of Gal-1 in Leydig cell cultures by immunofluorescence. Cells were cultured on glass coverslips, fixed, and stained as described in the Materials and methods. Images were visualized at 1000x using a fluorescent microscope. (A) Cells treated with an anti-rabbit IgG conjugated with FITC. (B) Permeabilized cells or (C) nonpermeabilized cells treated with a rabbit polyclonal antibody (IgG) raised against Gal-1 and then with an anti-rabbit IgG conjugated with FITC.

 
Effect of Gal-1 on Leydig cell morphology
Light microscopy showed changes in Leydig cell morphology after 3 or 24 h incubation with Gal-1 (7 µM) (Figure 2). As previously described (Diaz et al., 2002Go), Leydig cells grown on uncoated glass coverslips showed groups of round cells with abundant cytoplasm at 3 h (Figure 2A) and polygonal cells with few cell processes at 24 h (Figure 2B). When Leydig cells were cultured on laminin-1-coated glass coverslips, short cell processes already present at 3 h incubation (Figure 2G), changed to long cell processes in spindle-shape Leydig cells, indicating an intense cell-spreading process (Figure 2H). Leydig cells grown on uncoated glass coverslips and treated with Gal-1 for 3 h (Figure 2C) and 24 h (Figure 2D), rounded up, lost cytoplasm, were smaller compared to control cells, and exhibited condensed nuclear chromatin, characteristic of apoptosis. Moreover, a decrease in the cell number was observed with longer periods of incubation. In contrast, cells grown on laminin-1-coated glass coverslips and incubated with Gal-1 showed mainly groups of cells with abundant cytoplasm at 3 h (Figure 2E) and groups of cells with mild degree of spreading at 24 h (Figure 2F), and altogether a lower number of apoptotic cells.



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Fig. 2. Light microscopy of Leydig cells cultured for 3 or 24 h on glass coverslips uncoated or coated with laminin-1. For morphology studies, cells were fixed in acetone and then stained with hematoxylin-eosin as described in Materials and methods. (A) Cells cultured for 3 or (B) 24 h on uncoated glass coverslips (controls); (C) cells incubated with Gal-1 (7 µM) for 3 or (D) 24 h; (E) cells grown on laminin-1-coated glass coverslips and incubated with Gal-1 (7 µM) for 3 or (F) 24 h; (G) cells cultured on laminin-1-coated glass coverslips for 3 or (H) 24 h. 420x.

 
Effect of Gal-1 on Leydig cell viability
To determine whether Gal-1 was able to modulate cell growth, Leydig cells were incubated with different concentrations of the lectin (1.75–14 µM), and after 3 or 24 h culture cell viability was measured by the MTS assay. As shown in Figures 3A and B (panels a–d), Gal-1 caused significant Leydig cell death after both 3 and 24 h incubation in a dose-dependent fashion either with or without human chorionic gonadotrophin (hCG) stimulus. The maximal inhibitory effect of Gal-1 (80%) was achieved at a concentration of 14 µM in the presence of 10 ng/mL hCG with 24 h of incubation. The combination of genistein (Gen) (0.2 mM) and methylprednisolone (MP) (100 mM) was used as a positive control of cell death (Kumi-Diaka et al., 1998Go).



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Fig. 3. Effect of Gal-1 on Leydig cell viability. Cell viability was evaluated using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay. Cells were cultured in 96-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 100 µL) and incubated for 3 (A) or 24 h (B), without or with hCG (10 ng/mL), at 34°C in a humidified atmosphere of 5% CO2 in air. Panels a–d show cells incubated with increasing concentrations of Gal-1 (1.75–14 µM). Positive controls of apoptosis with a mixture of Gen (0.2 mM) and MP (100 mM) were simultaneously performed. Panels e–h show cells incubated with Gal-1 (7 µM) in the presence or absence of 100 mM lactose on laminin-1-coated or uncoated plates. Panels i–l show cells cultured with Gal-1 (7 µM) in the presence or absence of 100 mM lactose or 100 mM sucrose. After 3 or 24 h, cells were incubated with 20 µL of MTS reagent solution for 2.5 h. Absorbance at 490 nm was recorded using an ELISA plate reader. Results are expressed as percentage of cellular viability respect to the control ± SD of quadruplicate determinations from a representative out of six independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

 
The antiapoptotic effect of laminin-1 was tested by growing the cells on laminin-coated plates in the presence or absence of Gal-1. Incubation of Leydig cells with laminin abrogated the reduction in viability observed with 7 µM Gal-1, demonstrating its protective effect (panels e–h). Viability of cells grown on laminin-1-coated plates was the same as that of cells cultured on plastic plates following a 3- or 24-h incubation, as described previously (Diaz et al., 2002Go).

To evaluate whether Gal-1 exerted its effect through its carbohydrate-binding domain, cells were incubated with this lectin in the presence or absence of 100 mM lactose or 100 mM sucrose. The results showed that lactose almost completely prevented the decrease in viability seen in cells incubated with 7 µM Gal-1, suggesting an involvement of the CRD in this effect. Moreover, this effect was shown to be specific, because sugars other than lactose failed to prevent Gal-1-induced decrease in cell viability (panels i–l).

Effect of Gal-1 on testosterone production by Leydig cells
The effect of different concentrations of Gal-1 (1.75–14 µM), on basal and hCG-stimulated Leydig cell testosterone production is shown in Figure 4A and B. After a 3-h incubation, the maximal concentration of Gal-1 used caused a 54% decrease in testosterone production relative to basal values, whereas in the presence of hCG this decrease was 74%. When cells were incubated for 24 h, a similar profile was observed, also reaching a 74% decrease in testosterone production compared with basal values (panels a–d).



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Fig. 4. Effect of Gal-1 on testosterone production by Leydig cells. Testosterone was determined by radioimmunoassay using a specific antibody. Cells were cultured in 96-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 100 µL) and incubated for 3 (A) or 24 h (B), without or with hCG (10 ng/mL), at 34°C in a humidified atmosphere of 5% CO2 in air. Panels a–d show cells incubated with increasing concentrations of Gal-1 (1.75–14 µM) and with a mixture of Gen (0.2 mM) and MP (100 mM). Panels e–h show cells incubated with Gal-1 (7 µM) in the presence or absence of 100 mM lactose on laminin-1-coated or uncoated plates. Panels i–l show cells cultured with Gal-1 (7 µM) in the presence or absence of 100 mM lactose or 100 mM sucrose. Results are expressed as testosterone (ng/106 cells/3 or 24 h) ± SD of quadruplicate determinations from a representative out of six independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

 
Culture of Leydig cells on laminin-coated plates prevented the inhibitory effect of Gal-1 on hormone production, under both basal and hCG-stimulated conditions, and so did the preincubation of Gal-1 with 100 mM lactose (panels e–h). Gal-1 effect on testosterone production was also specific, as shown in panels i–l. Sucrose was unable to restore testosterone production, as opposed to lactose, the specific ligand of Gal-1, which prevented the decrease in hormone production. It should be noticed that the combination of Gen (0.2 mM) and MP (100 mM), which is known to induce Leydig cell apoptosis, also caused a decrease in testosterone production.

We then examined whether Gal-1 could affect the production of cAMP, the main second messenger that mediates luteinizing hormone/hCG action on Leydig cell steroidogenesis. For this purpose, cAMP production, both basal and after hCG stimulus, was measured after 3 h incubation with Gal-1 (7 µM) by an enzyme immunoassay. At basal conditions, no significant changes after addition of Gal-1 were detected, whereas in hCG-stimulated cells a strong reduction of 65% on cAMP production was observed (Figure 5).



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Fig. 5. Effect of Gal-1 on extracellular cAMP production by Leydig cells. Cells were cultured for 3 h on 24-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 500 µL), without or with hCG (10 ng/mL) and in the presence of 0.125 mM of 3-isobutyl-1-methylxanthine. Gal-1 (7 µM) was added to the cultures and incubated at 34°C in 5% CO2 in air. cAMP production was quantified in the culture medium after acetylation by an enzyme immunoassay kit. Results are expressed as extracellular cAMP (fmol/106 cells) ± SD of quadruplicate determinations from a representative out of three independent experiments. ***p < 0.001.

 
Gal-1-induced apoptosis of Leydig cells
To determine whether the parallel reduction in hormone production and cell viability observed after incubation with Gal-1 was caused by an apoptotic effect of this lectin, we set out to look for hallmarks of apoptosis using the propidium iodide staining technique.

DNA fragmentation was measured by flow cytometry after propidium iodide labeling of apoptotic nuclei. As Figure 6C and D shows, a significant increase of apoptotic nuclei in the subdiploid region (pseudohypodiploid nuclei) was observed after treatment of Leydig cells with Gal-1 at two different concentrations (41.9 and 52.7% at 7 and 14 µM, respectively) in complete medium with 0.2 mM dithiothreitol (DTT) for 24 h. This effect could not be attributed to the 0.2 mM DTT added to the medium, because the DTT control showed the same profile as the basal (Figure 6B and A, respectively).



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Fig. 6. Assessment of DNA hypodiploidy and flow cytometry. Leydig cells were cultured in 24-well microtiter plates at 5 x 105 cells/cm2 (final volume 1 mL) and treated with Gal-1 (7 and 14 µM; (C) and (D), respectively) in complete medium with 0.2 mM DTT or with a mixture of Gen (0.2 mM) and MP (100 mM) (E). Cells were also incubated with (B) or without (A) 0.2 mM DTT in complete medium as controls. After 24 h treatment, cells were harvested, washed, and stained with propidium iodide as described in Materials and methods. After keeping the cells overnight with the staining solution at -20°C, orange fluorescence was analyzed. This is a representative out of three independent experiments.

 
Effect of Gal-1 on caspase-mediated Leydig cell apoptosis
Apoptosis-associated caspase activity was measured in Leydig cells using a fluorometric assay. Once more, the mixture of Gen (0.2 mM) and MP (100 mM) was used as positive control (Kumi-Diaka et al., 1998Go; Kumi-Diaka and Butler, 2000Go). As shown in Figure 7, when Gal-1 (7 µM) was added to Leydig cell cultures after 24 h incubation, a 3.3-fold increase in caspase activity was detected compared with cells cultured in medium alone.



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Fig. 7. Effect of Gal-1 on caspase-mediated Leydig cell apoptosis. A fluorometric protease assay was used. Leydig cells were cultured in 24-well microtiter plates at 5 x 105 cells/cm2 (final volume 1 mL) with the appropriate treatment: Gal-1 (7 µM) in complete medium with 0.2 mM DTT, a mixture of Gen (0.2 mM) and MP (100 mM), or 0.2 mM DTT in complete medium alone. After 24 h cells were harvested, lysed, and incubated with DEVD-AFC substrate. Free AFC was measured with an excitation wavelength of 400 nm and an emission wavelength of 500 nm. Results are expressed as relative fluorescence intensity ± SD of duplicate determinations from a representative out of three independent experiments. **p < 0.01.

 
Effect of Gal-1 on Leydig cell adhesion
Figure 8 shows the results for cell adhesion assays on cells cultured in different conditions. Addition of Gal-1 (14 µM) caused a 70% decrease of cell adhesion after 24 h incubation on uncoated plates compared with basal values. Simultaneous incubation with Gal-1 and 100 mM lactose prevented this effect. As previously described (Diaz et al., 2002Go), when cells were cultured on laminin-1-coated plates, enhanced cell adhesion was observed. In the presence of laminin-1 and Gal-1, cells were protected from Gal-1-induced detachment. Similar results were obtained after 3 h incubation (data not shown).



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Fig. 8. Effect of Gal-1 on Leydig cell adhesion. Cell adhesion was determined by a colorimetric microassay method. Cells were cultured in 96-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 100 µL) and incubated for 24 h with Gal-1 (14 µM) in the presence or absence of 100 mM lactose on laminin-1-coated or uncoated plates. After 24 h culture, cells were washed, fixed, and stained with crystal violet as described in Materials and methods. Absorbance at 600 nm was recorded using an ELISA plate reader. Results are expressed as absorbance at 600 nm ± SD of octuplicate determinations from a representative out of three independent experiments. **p < 0.01.

 

    Discussion
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 Abstract
 Introduction
 Results
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 Materials and methods
 References
 
Leydig cells are the major cell type found in the interstitium, and they are responsible for the production of testosterone by the testis. They are also known to interact directly with lymphocytes as well as with macrophages, suggesting that these cells mediate local regulation of testicular leukocyte populations (Wang et al., 1994Go). In the present study, we demonstrate that Leydig cells express Gal-1, a ß-galactoside-binding protein that has been shown to modulate the immune system (Hernandez and Baum, 2002Go; Liu, 2000Go). We also evaluate, for the first time, the effect of Gal-1 on adult rat Leydig cells in vitro. After treatment with this protein, cells experimented substantial morphological changes, with a significant reduction in their steroidogenic activity as shown by testosterone determinations. These changes closely paralleled the decrease in cell viability, indicating that this dramatic alteration of Leydig cell function can be attributed to induction of apoptosis by Gal-1, as demonstrated by assessment of DNA hypodiploidy and by the activation of some caspases. The results observed are comparable to those obtained for the combination of Gen and MP, a mixture known to induce apoptosis of Leydig cells (Kumi-Diaka and Butler, 2000Go; Kumi-Diaka et al., 1998Go) and used as a positive control in this study. Leydig cells were found to be sensitive to apoptosis induced by Gal-1 with or without hCG stimulation. Because deprivation of hCG can cause apoptosis of immature Leydig cells (Fujisawa et al., 2000Go), this hormone is considered a survival factor; however, it was unable to prevent the effect of Gal-1 on adult Leydig cells.

Laminin-1 is one of the major structural and regulatory glycoproteins found in basement membranes; its expression has been detected in Leydig cells (Denduchis et al., 1996Go) and is known to influence many aspects of cell behavior (Timpl and Brown, 1994Go). Leydig cells grown on laminin-1 substrate exhibit an increase in cell adhesion and abundant cell spreading, expressed by the presence of long cell processes (Diaz et al., 2002Go). Controversial results have been reported as to whether Gal-1 exerts a positive or a negative effect on cell adhesion to ECM, raising the possibility that this dimeric protein could promote cell attachment or detachment according to cell type or cell developmental stage (Barondes et al., 1994bGo). Here we show that the presence of Gal-1 in Leydig cell cultures promotes cell detachment, whereas the use of laminin-1 as substrate protects cells from this effect. Indeed, culture of Leydig cells on laminin-1-coated plates resulted in abolishing the Gal-1 apoptotic effect. Considering our results, we propose that Gal-1 induces apoptosis of Leydig cells by promoting cell detachment, although cross-linkage of cell surface receptors, as in the case of thymocytes, could not be excluded. The important role of ECM proteins in preventing apoptosis is evident in anoikis, the apoptotic process induced by disruption of epithelial–cell matrix interactions (Frisch and Francis, 1994Go), which was described in Sertoli cells (Dirami et al., 1995Go).

To determine whether the Gal-1 effect was mediated by its CRD, we preincubated the protein with lactose, a specific ligand, or sucrose. Lactose was able to prevent detachment and apoptosis induced by Gal-1; sucrose, a disaccharide with similar structure, did not show any protective effect. This finding suggests the involvement of the CRD in the apoptosis-inducing properties of Gal-1. Furthermore, laminin-1, which contains N-acetyllactosamine residues, also abrogated the detachment and the apoptotic activity displayed by the lectin.

Leydig cells rarely proliferate or undergo apoptosis in response to physiological stimuli. The Fas/FasL system, well known to induce apoptosis of several different cell types, especially of the immune system, is present in Leydig cells (Taylor et al., 1999Go) and can be suppressed by expression of antiapoptotic members of the Bcl-2 family, such as Bcl-xl (Taylor et al., 1998Go). Tumor necrosis factor-alpha–related apoptosis-inducing ligand is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis on binding to its death domain–contanining receptors (Grataroli et al., 2002Go). Decoy receptors for this cytokine, which lack functional death domains, have been recently detected in Leydig cells, a finding that could explain why apoptosis of these cells is such a rare event (Grataroli et al., 2002Go). Under experimental conditions, Leydig cell apoptosis has been observed only in response to toxicants, such as ethylene dimethanesulfonate (Morris et al., 1997Go), Gen (Kumi-Diaka and Butler, 2000Go; Kumi-Diaka et al., 1998Go), and ethanol (Jang et al., 2002Go) or to glucocorticoids, like MP (Morris et al., 1997Go) or corticosterone (Gao et al., 2002Go). For this reason, the fact that Gal-1, an endogenous protein, is able to cause apoptosis of these cells constitutes a novel and significant finding.

On the other hand, Gal-1 has been shown to modulate immune system development and function by inducing apoptosis of T-cells, both immature and activated (Perillo et al., 1998Go; Hernandez and Baum, 2002Go). Gal-1 apoptosis-inducing activity was detected on a B lymphoma cell line (Poirier et al., 2001Go) and on macrophages isolated from Trypanosoma cruzi–infected mice (Zuñiga et al., 2001bGo). Apparently, Gal-1 triggers this immunomodulatory effect by binding different cell surface receptors on human and murine T-cells, such as CD2, CD3, CD4, CD7, CD43, and CD45. All of these receptors are glycoproteins that possess N-acetyllactosamine residues and preferentially bind Gal-1 (Hernandez and Baum, 2002Go). The finding of a specific glycosyltransferase able to transfer N-acetylglucosamine residues to CD45 suggests a prominent role for this receptor. Furthermore it has been determined that CD45 can positively or negatively regulate Gal-1-induced T-cell death, depending on the glycosylation status of the cell (Amano et al., 2003Go; Nguyen et al., 2001Go).

Induction of Gal-1 expression by treatment with sodium butyrate or transfection with Gal-1 cDNA resulted in induction of differentiation and apoptosis in the prostate cancer cell line LNCaP (Ellerhorst et al., 1999Go). Moreover, Gal-1 induced cell cycle arrest prior to entry into G2, and this was followed by progressive transition to apoptosis in human mammary cancer cells (Wells et al., 1999Go). These are two of the rare occasions in which Gal-1 has been shown to induce apoptosis of cells other than those of the immune system.

Another important aspect of Gal-1 is its ability to suppress the development of autoimmunity (Delioukina et al., 1999Go; Levi et al., 1983Go; Offner et al., 1990Go; Rabinovich et al., 1999Go; Santucci et al., 2000Go; Tsuchiyama et al., 2000Go) and inflammation (Liu, 2000Go). Leydig cells are in close association with immune cells; they express Gal-1 both in cytoplasm and on cell membrane, and probably secrete it to the extracellular milieu. The testis is one of the sites in the body known as immune privileged, and indeed Gal-1 could be one of the factors contributing to this situation as proposed for Sertoli cells (Dettin et al., 2003Go). Other immunosuppresor factors, and even immunosuppresive behavior of Leydig cells have been reported (Born and Wekerle, 1981Go; Pollanen et al., 1990Go; Sainio-Pollanen et al., 1997Go).

The present findings could be relevant to testicular pathophysiology. We propose that under physiological conditions, laminin-1 behaves as a survival factor for Leydig cells, protecting them from the apoptosis-inducing effects of Gal-1. Moreover, N-acetyllactosamine present on the ECM proteins that normally surround Leydig cells may also protect them from this apoptotic stimulus.


    Materials and methods
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Materials
Culture medium 199, human transferrin, collagenase type I, penicillin, amphotericin B, trypsin, soybean trypsin inhibitor, vitamin E, bovine serum albumin (BSA), DTT, lactosyl-agarose column, lactose, sucrose, MP, Gen, propidium iodide, and Percoll were obtained from Sigma Chemical (St. Louis, MO). Laminin-1 was from an Engelbreth Holm Swarm Sarcoma, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium were from Gibco BRL, Life Technologies (Grand Island Biological, NY). hCG was a generous gift from Dr. A. Parlow (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD). Falcon tissue culture plates (24- and 96-well) were from Beckton Dickinson Labware (NJ). A rabbit polyclonal antibody (IgG) against human Gal-1 was generously provided by Dr. G. A. Rabinovich (Laboratory of Immunogenetics, School of Medicine, University of Buenos Aires). Antibodies to rabbit IgG conjugated with FITC were obtained from Vector Labs (Burlingame, CA).

Preparation of recombinant human Gal-1
Gal-1 was expressed according to Couraud et al. (1989)Go in Escherichia coli strain BL21DE3 tranformed with the plasmid that encodes Gal-1, pT7IML-1 (a generous gift from Dr. Linda G. Baum, University of California, Los Angeles, School of Medicine) and purified as previously described (Iglesias et al., 1998bGo; Perillo et al., 1997Go). To maintain maximal lectin activity, a reducing agent (0.2 mM DTT) was added to the buffer. For cell viability assay and testosterone determination, Gal-1 was dialyzed immediately before use. In all cases, the biological activity of the lectin was confirmed by measuring its ability to agglutinate glutaraldehyde-fixed, trypsin-treated rabbit erythrocytes (Nowak et al., 1976Go).

Isolation and culture of Leydig cells
Leydig cells were isolated from adult male Sprague-Dawley rats (60–70 days) by a method described previously (Dufau et al., 1974Go). Briefly, in each experiment testes from 20 rats were decapsulated and digested with 0.25 mg/mL collagenase and 0.015 mg/mL soybean trypsin inhibitor in M199 medium + 0.1% BSA, at 34°C for 10–15 min. No DNase was used to preserve the DNA for subsequent studies. The digestion procedure was stopped by dilution with fresh medium, followed by two successive washes and centrifugation. Interstitial cells (crude cell preparation) were purified on a discontinuous Percoll density gradient (layers of 21, 26, 34, 40, and 60% Percoll). The gradient was centrifuged at 800 x g for 30 min, the interface between 40 and 60% collected and washed with medium to remove the Percoll (Lefevre et al., 1983Go). Cells were resuspended in a 1:1 nutrient mixture of DMEM and Ham's F-12 medium, supplemented with 20 mM HEPES, 100 IU/mL penicillin, 2.5 µg/mL amphotericin B, 1.2 mg/mL sodium bicarbonate, 10 µg/mL human transferrin, 5 µg/mL vitamin E, and 0.1% BSA (complete medium). Cell viability assessed by trypan blue exclusion was approximately 95%. The presence of 3ß-hydroxysteroid dehydrogenase activity, revealed by a histochemical technique, was used to determine the purity of Leydig cells, which ranged from 90 to 95%. Cultures were performed at 34°C in a humidified atmosphere of 5% CO2 in air for 3 or 24 h.

To determine the effect of laminin-1 on Leydig cell viability and testosterone production, cells (1.25 x 105 cells/cm2) were seeded on plastic 96-well microtiter plates or glass coverslips on 24-well plates, uncoated or coated with laminin-1 (6 µg/cm2). For laminin coating, wells were incubated for 3 h at 37°C with laminin and then incubated for an additional 2 h with 1% BSA. Plates were washed with medium; cells were seeded and incubated for 3 or 24 h. At the end of the incubation period, the supernatants were saved and stored at -20°C until testosterone determination.

Cell morphology and immunofluorescence
For morphology and immunofluorescence studies, cells cultured on glass coverslips, uncoated or coated with laminin-1, were used. For light microscopy, cells were incubated with Gal-1 (7 µM) for 3 or 24 h, fixed in acetone at room temperature for 10 min, and then stained with hematoxylin-eosin. For indirect immunofluorescent assays, cells were fixed in cold acetone (-20°C) for 7 min or in 2% paraformaldehyde (PF) in phosphate buffered saline (PBS) for 10 min at room temperature; cells fixed in acetone were washed with PBS containing 0.2% Triton-X 100 (permeabilized cells), whereas those fixed in PF were washed in PBS without Triton (nonpermeabilized cells). Then cells were incubated with a rabbit polyclonal antibody (IgG) against human Gal-1 (1:20), washed with PBS, and incubated with a polyclonal antibody against rabbit IgG conjugated with FITC (1:50). As a control, permeabilized and nonpermeabilized cells were first incubated with PBS or IgG from rabbit normal serum and then with the conjugated antibody. After washing, cells were mounted in buffered glycerin and observed under an Axiophot fluorescent microscope with epi-illumination (Carl Zeiss, Oberkochen, Germany).

Cell viability assay
Cell viability was evaluated using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Cells were plated in 96-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 100 µL) and incubated for 3 or 24 h, without or with 10 ng/mL hCG, at 34°C in a humidified atmosphere of 5% CO2 in air. The hCG concentration used was that at which maximal testosterone Leydig cell response was reached (Diaz et al., 2002Go). Cells were incubated with increasing concentrations of Gal-1 (1.75–14 µM), in the presence or absence of 100 mM lactose or 100 mM sucrose. Positive controls of apoptosis with a mixture of Gen (0.2 mM) and MP (100 mM) were simultaneously performed (Kumi-Diaka et al., 1998Go). Besides, cells were grown on laminin-coated plates in the presence or absence of Gal-1. After 3 or 24 h culture, cells were incubated with 20 µL of MTS reagent solution for an additional 2 h. Absorbance at 490 nm was recorded using an ELISA plate reader (Metertech {Sigma}960). Results are expressed as percentage of cellular viability respect to the control (100%).

Testosterone determination
Testosterone was determined by radioimmunoassay using a specific antibody. The radioimmunoassay has a sensitivity of 6.25 pg per tube and intra- and interassay coefficients of variation were 8 and 15%, respectively. Results are expressed as ng testosterone/106 cells.

Extracellular cAMP determination
Leydig cells were cultured for 3 h on 24-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 500 µL), without or with hCG (10 ng/mL) and in the presence of 0.125 mM 3-isobutyl-1-methylxanthine to inhibit phosphodiesterase activity. Simultaneously, Gal-1 (7 µM) was added to the cultures. cAMP production was quantified in the culture medium after acetylation by the enzyme immunoassay kit No RPN 225 from Amersham Biosciences (Uppsala, Sweden). Results are expressed as fmol/106 cells.

Assessment of DNA hypodiploidy and flow cytometry
Leydig cells were cultured in 24-well microtiter plates at 5 x 105 cells/cm2 (final volume 1 mL) and treated with Gal-1 (7 and 14 µM) in complete medium with 0.2 mM DTT or with a mixture of Gen (0.2 mM) and MP (100 mM). Cells were also incubated with or without 0.2 mM DTT in complete medium as controls. After 24 h treatment, cells were harvested, washed twice with ice-cold PBS, and centrifuged (250 x g, 10 min); the final pellet was resuspended in 1 mL hypodiplody solution (25 µg/mL propidium iodide in 0.1% sodium citrate and 0.1% Triton X-100) as previously described (Nicoletti et al., 1991Go). After keeping the cells overnight with the staining solution at -20°C, orange fluorescence was analyzed in a Cytoron Absolute cytometer (Ortho Diagnostic System, Raritan, NJ).

Caspase-3-like activity
To determine caspase activity associated with apoptosis, a fluorometric protease assay (Caspase-3 apoptosis detection kit: sc-4263 AK, Santa Cruz Biotechnology, Santa Cruz, CA) was used according to the manufacturer's instructions. Leydig cells were cultured in 24-well microtiter plates at 5 x 105 cells/cm2 (final volume 1 mL) with the appropriate treatment: Gal-1 (7 µM) in complete medium with 0.2 mM DTT, a mixture of Gen (0.2 mM) and MP (100 mM), or 0.2 mM DTT in complete medium alone. After 24 h cells were harvested, washed with PBS, and treated with lysis buffer. DEVD-AFC substrate in reaction buffer containing 10 mM DTT was added to cell lysate and incubated 1 h at 37°C. Free AFC was measured using an Aminco Bowman Series-2 spectrofluorometer (Sim-Aminco Spectronic Instruments) with an excitation wavelength of 400 nm and an emission wavelength of 500 nm.

Cell adhesion assay
Cell adhesion was determined by a colorimetric microassay method as previously described (Diaz et al., 2002Go). Briefly, cells were cultured in 96-well microtiter plates at 1.25 x 105 cells/cm2 (final volume 100 µL) and incubated for 3 or 24 h with Gal-1 (14 µM), in the presence or absence of 100 mM lactose on laminin-1-coated or uncoated plates. After 3 or 24 h culture, cells were washed with PBS containing 0.1% BSA to remove nonadherent cells and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. Cells were stained with 0.5% crystal violet in distilled water for 10 min and then extensively washed with distilled water. The cell-bound stain was solubilized with 1% sodium dodecyl sulfate in distilled water (100 µL/well), and the absorbance at 600 nm was measured using an ELISA plate reader (Metertech {Sigma}960). We previously determined a linear correlation between optical density and the number of cells. The background value was obtained from empty plates coated with laminin-1.

Statistical analysis
All experimental data are presented as the mean ± SD of quadruplicate determinations by four replicate cultures within each treatment group. Quadruplicates were handled as four independent values. All experiments reported here were repeated at least three times with independent cell preparations. A representative experiment of each series is presented. Statistical significance between groups was determined by analysis of variance followed by a Student-Newman-Keuls multiple comparison test. Differences are accepted as significant at p < 0.05.


    Acknowledgements
 
We thank Mercedes Astarloa for technical assistance. This work was supported by grants from Universidad de Buenos Aires and from Consejo Nacional de Investigaciones Cientóficas y Tecnológicas (CONICET). E.H.P., S.B.C., L.L., B.D., C.W.-T., and M.M.I. are members of the scientific career from CONICET.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: miglesia{at}qb.ffyb.uba.ar Back


    Abbreviations
 
BSA, bovine serum albumin; CRD, carbohydrate-recognition domain; DMEM, Dulbecco's modified Eagle medium; DTT, dithiothreitol; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; Gen, genistein; hCG, human chorionic gonadotrophin; MP, methylprednisolone; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and phenazine methosulfate; PBS, phosphate buffered saline; PF, paraformaldehyde


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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