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Biology of Reproduction 66, 1734-1742 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Glutathione S-Transferase Alpha Expressed in Porcine Sertoli Cells Is under the Control of Follicle-Stimulating Hormone and Testosterone1

Lamia Benbrahim-Tallaaa, Eric Tabonea, Gwenola Tosser-Kloppb, François Hateyb, and Mohamed Benahmed2,,a

a Unité 407, Institut National de la Santé et de la Recherche Médicale (INSERM), Communication Cellulaire en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud, 69921 Oullins Cedex, France b Laboratoire de Génétique Cellulaire, Institut National de la Recherche Agronomique (INRA), Chemin de Borde-Rouge, F-31326 Castanet Tolosan, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GST{alpha} is expressed in somatic testicular Leydig cells and Sertoli cells. GST{alpha} expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GST{alpha} was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GST{alpha} was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GST{alpha} mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GST{alpha} mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GST{alpha} protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GST{alpha} mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GST{alpha} mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GST{alpha} protein, we therefore had to determine whether the different hormones were affecting GST{alpha} gene transcriptional activity, or GST{alpha} mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GST{alpha} mRNA. The effects of FSH and testosterone on GST{alpha} protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GST{alpha} is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.

follicle-stimulating hormone, Sertoli cells, testis, testosterone, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione S-transferases (GSTs) (EC 2.5.1.18) are known as a large family of abundant enzymes in most organisms. GSTs are detoxification enzymes that catalyze the conjugation of a wide range of electrophilic compounds to glutathione (GSH), a tripeptide found in all mammalian cells [1–3]. These compounds include carcinogens, environmental pollutants, anticancer agents, antibiotics, and products of oxidative processes that cause DNA and protein damage [4, 5]. Both cytosolic and microsomal forms of GSTs have been identified [6]. Cytosolic GSTs are homodimers or heterodimers, they share some homology, and they possibly evolved from a common ancestral gene [7]. GST isoenzymes exhibit distinct structural and biochemical characteristics that determine their relative activity toward different substrates [8].

Mammalian GSTs have been classified into five distinct gene families: alpha, mu, pi, sigma, and theta. The alpha gene family contains the Ya, Yc, and Yk subunits. The mu gene family consists of Yb1, Yb2, Yb3, Yb4, Yn, and Y0 subunits. The pi gene family is made up of the Yf subunit. New subunits are being added to the families on a regular basis.

GST activity is regulated at the transcriptional level by a variety of compounds, including phenobarbital [9] and antioxidants [10]. Distribution of GST isoenzymes in different tissues is not uniform, and mammalian organs show a marked diversity in their GST content. The testis has high levels of GSTs [11, 12]. Indeed, different GST isoenzymes have been identified in the male gonad. Specifically, GST{alpha} [13–15] and GST{pi} [16] have been identified in testicular somatic cells and particularly in Leydig and Sertoli cells. GSTµ was shown to be expressed in somatic and germ cells [17–21]. In addition to its role as a phase II detoxification enzyme involved in the conjugation of electrophilic xenobiotics such as carcinogens and mutagens to the endogenous nucleophile GSH, particularly in the testis, GST{alpha} also contributes to a major portion of the selenium-independent glutathione peroxidase (GPx) activity toward phosphatidylcholine hydroperoxide [7, 22–24]. Therefore, GST{alpha} may be necessary for protecting this tissue from reactive oxygen species-induced damage.

The importance of GSTs in the protection against oxidative stress in testis is underscored by recent studies showing that when GST activity is inhibited, products of lipid peroxidation accumulate, resulting in germ cell apoptosis [25]. More recently, these observations were further strengthened by data demonstrating that overexpression of GSTA2-2 (a member of the GST{alpha} class) in K562 cells attenuates the cytotoxic effect of H2O2 and other oxidants, and protects against H2O2-induced apoptosis by blocking caspase 3 activation [26]. Finally, in addition to protecting the germ cells from the toxic effects of carcinogens and mutagens and from free radical damage [16, 27], GSTs may also exert some specific effects in the male reproductive function. Indeed, GST isoenzymes exhibit a steroid binding activity and seem to be involved in male fertility, specifically in the gamete interactions [28].

Spermatogenesis is highly dependent on Sertoli cells [29–31], which are under direct endocrine control [30–32]. Endocrine regulation allows Sertoli cells to develop and reorganize, and to generate the hematotesticular barrier through their tight junction complexes and to provide nutrients and regulatory factors to the germ cells. This time-modeling process and the production of Sertoli cell factors lead to the constitution of a specific biochemical and cytoarchitectural microenvironment in the adluminal compartment where germ cells will proliferate and differentiate. These different processes occur under the control of hormonally regulated Sertoli cell factors. Among the identified Sertoli cells factors are binding transport proteins, proteases and protease inhibitors, energy substrates such as lactate, local signaling molecules such as growth factors and cytokines [29–32], and hematotesticular barrier proteins such as claudin 11 [33].

In the present study we examined whether the expression of testicular GSTs, and specifically those expressed in Sertoli cells, might be under hormonal control. We report that GST{alpha} expressed in Sertoli cells is under the regulatory control of FSH and testosterone and its two metabolites, dihydrotestosterone and estradiol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Dulbecco modified Eagle medium (DMEM)/Hams F12 medium and TRIzol were obtained from Life Technologies (Eragny, France). Collagenase/dispase was obtained from Roche (Mannheim, Germany). Testosterone, 17ß-estradiol, dihydrotestosterone, flutamide, 8-bromo adenosine 3',5'-monophosphate (8-Br-cAMP), 5,6-dichlorobenzimidazole riboside (DRB), cycloheximide, 3-cyclohexylamino-1-propanesulfonic acid (CAPS), protease inhibitor cocktail, and BSA were purchased from Sigma Chemical Company (St. Louis, MO). Ovine FSH was kindly provided by Dr. A.F. Parlow of the U.S. Department of Agriculture, Agricultural Research Service, Animal Hormone Program (Beltsville, MD). ICI 182,780 was purchased from Tocris Cookson Inc. (Avonmouth Bristol, U.K.). Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a generous donation by Dr. J.M. Blanchard (Faculty of Sciences, Montpellier, France). The rabbit polyclonal anti-human GST{alpha} antibody (NCL-GST{alpha}) [34, 35] and Rp-adenosine-3',5'-cyclic monophosphorothioate triethylamine salt (Rp-cAMPS) were purchased from Novocastra-TEBU (Le Perray en Yvelines, France). Peroxidase-conjugated goat anti-rabbit immunoglobulin G was obtained from COVALAB (Lyon, France). Antibody diluent was obtained from DAKO (Copenhagen, Denmark). The Ultravision Detection System was obtained from Lab Vision Corporation (Fremont, CA). The Bradford reagents and Pierce detection kit were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA).

Sertoli Cell Isolation and Culture

Sertoli cells were isolated from immature porcine testes (2–3 wk old) using collagenase treatment as described by Mather and Phillips [36]. Briefly, the testes were decapsulated, minced, and washed in DMEM/Hams F12 (1:1) medium. After collagenase dissociation (0.4 mg/ml, 90–120 min at 32°C), cells were washed by mild centrifugation (200 x g, 10 min). After 5 min of sedimentation, the tubules were recovered and washed 3 times by unit gravity in DMEM/Hams F12 medium. Contaminating interstitial Leydig cells were released after 20 min of treatment (at room temperature) with 20 ml of 1 M glycine, 2 mM EDTA, and 20 IU/ml desoxyribonuclease DNase in Ca2+, Mg2+-free PBS (pH 7.2). Tubules were then washed 3 times in DMEM/Hams F12 medium by gravity before incubation in DMEM/Hams F12 medium containing collagenase (0.4 mg/ml) and DNase (0.05 mg/ml) for 30 min at 32°C. The supernatants containing the peritubular myoid cells were removed, and the sedimented tubules were treated again as described above with collagenase (0.4 mg/ml, 30 min, 32°C) to isolate Sertoli cells. This procedure led to a purified Sertoli cell population free from contamination by either Leydig cells or germ cells [37] and contained 2%–5% peritubular myoid cells. Sertoli cells were further washed several times by gravity in DMEM/Hams F12. Cells were counted in a Coulter counter (Coulter Electronics, Margency, France), plated in Falcon (Los Angeles, CA) 60-mm Petri dishes (5 x 106 cells/dish), and cultured at 32°C in a humidified atmosphere of 5% CO2, 95% air in DMEM/Hams F12 (1:1) medium containing sodium bicarbonate (1.2 mg/ml), 15 mM HEPES, and gentamycin (20 µg/ml). This medium was supplemented with transferrin (5 µg/ml), insulin (2 µg/ml), and tocopherol (10 µg/ml). The culture medium was changed every 2 days and the experiments were carried out in the 2 wk following cell preparation, during which the cultured Sertoli cells maintain their specific activity.

Preparation of Total RNA and Northern Blot Analysis

To prepare the GST{alpha} probe, approximately 600 ng of DNA were amplified by polymerase chain reaction (PCR). PCR amplification of the GST{alpha} cDNA was carried out using the following primers (Genset SA, Paris, France): forward, 5'-GCTGGCCAACTTCCCTCTGC-3' and reverse, 5'-TGCGTGCGAAAACAAAAT-3', designed to amplify a sequence of 331 base pairs (bp). PCR cycles consisted of initial denaturation of mixed PCR without Taq polymerase at 94°C for 5 min. A total of 30 PCR cycles followed the addition of Taq polymerase. Each cycle involved 1 min of denaturation at 92°C, 1 min of primer annealing at 63°C, and 1 min of elongation at 70°C. The 30 cycles were followed by 5 min at 70°C. The PCR products were separated by electrophoresis in a 1% agarose gel and analyzed after ethidium bromide staining and UV illumination. GST{alpha} cDNA was purified and then labeled with 50 µCi of ([{alpha}-32P]dCTP) using a random primed DNA labeling kit.

Total RNAs from cells in culture were prepared using TRIzol, a monophasic solution of phenol and guanidine isothiocyanate. This reagent is an improvement over the single-step RNA isolation method developed by Chomczynski and Sacchi [38]. Briefly, cells were lysed by adding 1 ml of TRIzol reagent and passing the cell lysate through a pipette several times. The homogenized samples were incubated for 5 min to permit the complete dissociation of nucleoprotein complexes. Chloroform (200 µl) was then added. After centrifugation the aqueous phase was precipitated with isopropanol (500 µl), and pellets were washed with 70% ethanol. After solubilization in diethyl pyrocarbonate (DEPC) treated water, the amount of RNA was estimated by spectrophotometry at 260 nm. About 10 µg of total RNAs, denatured for 15 min at 65°C in the presence of 2.2 M formaldehyde, 12.5 M formamide, and 1x 3-N-morpholino propanesulfonic acid (MOPS) were size-fractionated by electrophoresis on 1.2% agarose, 2.2 M formaldehyde gels. After migration in 0.02 M MOPS running buffer, RNAs were transferred to nitrocellulose membranes (Hybond-C extra, Amersham, Aylesbury, U.K.) by capillary transfer with 10x SSC (1.5 M NaCl, 0.15 M sodium citrate) and fixed at 80°C for 2 h. After 4 h of prehybridization at 42°C, filters were hybridized with labeled probes (1–4 x; 106 cpm/ml) overnight at 42°C in 50% formamide, 5x SSPE (0.9 M NaCl, 0.05 M sodium phosphate, 5 mM EDTA pH 7.5; 5x Denhardt solution [1 g Ficoll, 1 g polyvinylpyrroline, 1 g BSA/L], 1% SDS, and 100 µg/ml yeast RNA). After this period, membranes were washed four times in 2x SSC/0.1% SDS (20 min at room temperature), followed by 40 min at 55°C. Filters were exposed to Kodak X-OMAT (Eastman Kodak, Rochester, NY) films for 1–2 days at -70°C.

Preparation of Total Protein and Western Blot Analysis

Total proteins were extracted from porcine Sertoli cells treated under different conditions. Briefly, Sertoli cells were homogenized in ice-cold hypotonic buffer (25 mM Tris-HCl pH 7.4, 1 mM EDTA, and protease inhibitor cocktail). Homogenized cells were centrifuged at 100 000 x g for 10 min at 4°C. Protein concentration was determined in the supernatants using the assay of Bradford [39] with BSA as a standard. Protein extracts (80 µg/well) were incubated at 100°C for 5 min under reducing conditions, then size-fractionated on SDS/polyacrylamide gel. After electrophoresis, the proteins were transferred at a constant voltage of 100 V for 30 min onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using 10 mM CAPS (pH 11) containing 10% methanol. Following transfer, the membrane was incubated in a blocking buffer (TBS) containing 5% nonfat dry milk at 4°C. The membrane was washed with TBS and stained with Ponceau red. After two washes with TBS, the membrane was then incubated with the rabbit polyclonal anti-GST{alpha} antibody diluted at 1:1000 in a solution of TBS with 0.5% nonfat dry milk for 2 h at room temperature. After washing with TBS, the membrane was then incubated with the goat anti-rabbit peroxidase-conjugated antibody (Jackson Immunoresearch, West Grove, PA) diluted 1:2000 in TBS buffer with 0.5% nonfat dry milk. The antibody-antigen complexes were detected by chemiluminescence using a Pierce detection kit and autoradiographic material from Bio-Rad.

Immunohistochemistry

Paraffin sections of Bouin-fixed testis were cut onto silanized slides. The samples were deparaffinized and rehydrated in PBS. The Ultravision Detection System was used as recommended by the manufacturer. Briefly, endogenous peroxidases were quenched in 3% H2O2 for 15 min. The GST{alpha} primary antibody was diluted 1:200 in the antibody diluent, and incubated for 2 h at room temperature. After washing, biotinylated goat antipolyvalent secondary antibody was applied and, after washing, a peroxidase-streptavidin complex was applied. Diaminobenzidine was used as the peroxidase chromogen. Sections were briefly counterstained with Harris hematoxylin and mounted in mounting medium.

Negative controls were performed by using, instead of the primary antibody, 1) 0.1% PBS/BSA or 2) nonimmune rabbit serum.

Statistical Analysis

The autoradiographic bands were quantified by densitometric analysis using the Bio Image Scanner (Millipore SA, Saint Quentin, France). Each GST{alpha} band was normalized by correcting for any variation in the amounts of RNA applied to each lane. Experiments were repeated at least 3 times with different cell preparations. Statistical significance of the results was determined by ANOVA and the Student t-test when data from at least 3 different experiments were compared. Data are presented as the mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunolocalization of GST{alpha} in Postnatal Porcine Testis

Using an immunohistochemical approach, we have identified GST{alpha} protein in the porcine testis during postnatal development, specifically at 3 critical periods, including 2–3 wk, 3 mo, and 6 mo, which correspond to the neonatal, pubertal, and adult periods, respectively. Whereas GST{alpha} immunostaining was observed in Leydig cells at the 3 different critical periods in the course of testicular development, it was absent in Sertoli cells at the neonatal period (2–3 wk; Fig. 1, a and b) but was detectable in these cells at 3 mo (Fig. 1c) and was strongly immunoexpressed in adult (6 mo) Sertoli cells (Fig. 1d). Germ cells as well as peritubular cells were not stained with GST{alpha} antibody. These data suggest that GST{alpha} expression in Sertoli cells appears to be related to the action of gonadotropins, because it was observed at the onset of puberty (i.e., the moment at which FSH and androgens increase in porcine Sertoli cells) [40].



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  		FIG. 1. GST{alpha} immunostaining in the course of testis development. Testes obtained from pigs at different ages were fixed, sectioned, and treated with polyclonal anti-GST{alpha} primary antibody. Testis sections were from 15-day-old (a), 3-wk-old (b), 3-mo-old (c), and 6-mo-old (d) pigs. Sertoli cells are indicated by arrows and Leydig cells by arrowheads

Therefore, in the next series of experiments, we used an in vitro model of cultured porcine Sertoli cells to further characterize the action of FSH and testosterone on GST{alpha} mRNA and GST{alpha} protein.

FSH Enhances GST{alpha} mRNA and Protein Levels

FSH treatment (4.1–1000 ng/ml, 48 h) resulted in a dose-dependent increase in GST{alpha} mRNA levels. The maximal effect of the hormone (3-fold, P < 0.001) was seen with 111 ng/ml (Fig. 2A) and the half-maximal (ED50) effect was observed at 18.5 ng/ml FSH. The stimulatory effect of FSH on GST{alpha} mRNA was also time-dependent. FSH (100 ng/ml) increased GST{alpha} mRNA levels after 4 h of exposure (P < 0.001) with a maximal effect after 48 h of exposure (P < 0.001; Fig. 2B). 8-Br-cAMP (1 mM, 48 h) treatment increased (P < 0.001) GST{alpha} mRNA levels in a comparable manner to FSH (Fig. 2C). The stimulatory effect of FSH (100 ng/ml, 48 h) on GST{alpha} mRNA was completely abolished in the presence of cycloheximide (20 µg/ml), indicating that the FSH effect on GST{alpha} mRNA is probably mediated by protein synthesis (Fig. 2D). The data in Figure 3 show that the stimulatory action of FSH on GST{alpha} mRNA levels was completely abolished by the protein kinase A (PKA) inhibitor Rp-cAMPS (100 nM). Together, the results in Figures 2C and 3 indicate that FSH increased GST{alpha} mRNA levels through the PKA/cAMP pathway.



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  		FIG. 2. Effect of FSH on GST{alpha} expression in Sertoli cells. A) Sertoli cells incubated with increasing concentrations of FSH (0–1000 ng/ml). B) Sertoli cells treated with 100 ng/ml FSH (0–72 h). C) Sertoli cells incubated in the absence or presence of FSH (100 ng/ml) or 8-Br-cAMP (1 mM) for 48 h. D) Sertoli cells stimulated with FSH (100 ng/ml) in the presence or absence of cycloheximide (20 µg/ml). Ten micrograms of total RNA were extracted and analyzed by Northern blot with radiolabeled GST{alpha} and GAPDH probes as described in Materials and Methods. The data show representative autoradiograms (upper panel) and the histograms (lower panel) represent the mean ± SD from 3 different experiments.



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  		FIG. 3. Effect of Rp-cAMPs on the stimulatory effect of FSH on GST{alpha} mRNA levels. Sertoli cells were stimulated with FSH (100 ng/ml) in the presence or absence of Rp-cAMPs (100 nM). Ten micrograms of total RNA were extracted and analyzed by Northern blot with radiolabeled GST{alpha} and GAPDH probes as described in Materials and Methods. The data show representative autoradiograms (upper panel) and the histograms (lower panel) represent the mean ± SD from 3 different experiments.

A stimulatory effect of the hormone was also observed on GST{alpha} protein levels. Indeed, both FSH (100 ng/ml) and 8-Br-cAMP (1 mM) increased GST{alpha} protein levels as identified by Western blots (Fig. 4). GST{alpha} protein is detected as a doublet of 28 kDa.



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  		FIG. 4. Effect of FSH and 8-Br-cAMP on GST{alpha} protein. Sertoli cells were cultured for 48 h in the presence or absence of FSH (100 ng/ml) or 8-Br-cAMP (1 mM). Protein extracted (80 µg) from Sertoli cells was separated on 12% SDS/polyacrylamide gel and transferred onto nitrocellulose and incubated with the polyclonal anti-GST{alpha} (1:1000). GST{alpha} protein is evidenced as a doublet of 28 kDa

Testosterone Enhances GST{alpha} mRNA and Protein Levels

The treatment of Sertoli cells with testosterone (1–62.5 ng/ml, 24 h) resulted in an increase in GST{alpha} mRNA levels. The maximal (2-fold, P < 0.002) increase was obtained with 3.9 ng/ml (Fig. 5A), the half-maximal (ED50) effect being observed with 1.4 ng/ml. Testosterone also stimulated GST{alpha} mRNA in a time-dependent manner, and the levels of GST{alpha} mRNA were maximal (2-fold, P < 0.001) after 8 h of exposure (Fig. 5B). The antagonistic effect exerted on testosterone-stimulated GST{alpha} expression by flutamide (3 µg/ml), a drug that prevents testosterone binding to its receptor, supports the specificity of the action of the androgen (Fig. 5C). Furthermore, dihydrotestosterone (DHT), a nonaromatizable androgen (100 ng/ml) increased GST{alpha} mRNA levels in a nonadditive and comparable manner (Fig. 5D) to that of testosterone, reinforcing the observation that the stimulatory effect of testosterone on GST{alpha} mRNA levels was exerted via the androgen receptor. However, the possibility that testosterone also exerts its action with its aromatized metabolite, estradiol, remains possible. Indeed, as shown in Figure 6, estradiol (100 ng/ml, 24 h) was also able to enhance (P < 0.001) GST{alpha} mRNA levels. The effects of estradiol and testosterone were additive, suggesting that they use distinct (receptor) signaling pathways. Indeed, the specificity of estradiol action was further confirmed because the stimulatory action of this hormone was completely abolished in the presence of ICI 182,780 (20 µM), a specific estrogen receptor antagonist (Fig. 7).



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  		FIG. 5. Effect of testosterone on GST{alpha} mRNA. A) Cultured Sertoli cells exposed for 24 h to increasing concentrations of testosterone (0–62.5 ng/ml). B) Sertoli cells exposed to testosterone (100 ng/ml) for 0–24 h. C) Sertoli cells treated with testosterone (100 ng/ml) in the absence or presence of flutamide (3 µg/ml). D) Sertoli cells treated with testosterone (100 ng/ml), DHT (100 ng/ml), or both. Ten micrograms of total RNA were extracted and analyzed by Northern blot with radiolabeled GST{alpha} and GAPDH probes as described in Materials and Methods. The data show representative autoradiograms (upper panel) and the histograms (lower panel) represent the mean ± SD from 3 different experiments.



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  		FIG. 6. Additive effects of testosterone and estradiol on GST{alpha} mRNA levels. Sertoli cells were treated for 24 h with testosterone (100 ng/ml), estradiol (100 ng/ml), or both. Ten micrograms of total RNA were extracted and analyzed by Northern blot with radiolabeled GST{alpha} and GAPDH probes as described in Materials and Methods. The data show representative autoradiograms (upper panel) and the histograms (lower panel) represent the mean ± SD from 3 different experiments.



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  		FIG. 7. Effect of ICI 182,780 on the stimulatory effect of estradiol on GST{alpha} mRNA levels. Sertoli cells were stimulated with E2 (100 ng/ml) in the presence or absence of ICI 182,780 (20 µM). Ten micrograms of total RNA were extracted and analyzed by Northern blot with radiolabeled GST{alpha} and GAPDH probes as described in Materials and Methods. The data show representative autoradiograms (upper panel) and the histograms (lower panel) represent the mean ± SD from 3 different experiments.

A stimulatory effect of the steroid hormones was also observed on GST{alpha} protein levels. Indeed, both testosterone and DHT increased GST{alpha} protein levels as identified by Western blots (Fig. 8A). In addition, a conjugate effect of testosterone and estradiol led to a further increase in GST{alpha} protein levels (Fig. 8B).



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  		FIG. 8. Effect of testosterone, DHT, and estradiol on GST{alpha} protein. Sertoli cells were cultured for 24 h A) in the presence or absence of testosterone (100 ng/ml) or DHT (100 ng/ml); or B) with testosterone (100 ng/ml), estradiol (100 ng/ml), or both. Protein extracts (80 µg) were separated on 12% SDS/polyacrylamide gel and transferred onto nitrocellulose and incubated with the polyclonal anti-GST{alpha} (1:1000). GST{alpha} protein is evidenced as a doublet of 28 kDa

FSH but Not Testosterone Stabilizes GST{alpha} mRNA

To test the effects of testosterone and FSH on GST{alpha} mRNA stability, the transcriptional activity in both untreated Sertoli cells (controls), and those treated with testosterone (100 ng/ml, 24 h), DHT (100 ng/ml, 24 h), or estradiol (100 ng/ml, 24 h) was first blocked with 25 µM DRB, an inhibitor of transcription, and the levels of GST{alpha} mRNA were estimated by Northern blot analysis at various times (6, 12, 18, and 24 h). In the treated Sertoli cells, the decays in GST{alpha} mRNA levels (half-life time = 24 h) were similar regardless of whether Sertoli cells were or were not treated with testosterone or DHT (Fig. 9A) or with estradiol (Fig. 9B). In contrast, in FSH or 8-Br-cAMP-treated cells (Fig. 10), the GST{alpha} mRNA levels decayed more slowly (half-life time = 113 h) compared with those in untreated cells. In control experiments, there was no significant difference in the rate of degradation of GAPDH mRNA in control and pretreated cells. Taken together, these findings indicate that FSH but not testosterone increased GST{alpha} mRNA levels by predominantly stabilizing these mRNAs.



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  		FIG. 9. Effect of testosterone, DHT, and estradiol on GST{alpha} mRNA stability. Sertoli cells were incubated in the absence ({square}) or presence of A) testosterone (100 ng/ml, 24 h) () or DHT (100 ng/ml, 24 h) ({circ}); or B) estradiol (100 ng/ml, 24 h) ({block}) after which 25 µM DRB was added to both untreated and treated cells. Total RNA were isolated from control, testosterone-treated, DHT-treated, or estradiol-treated Sertoli cells at 0, 6, 12, 18, and 24 h after the addition of DRB. Ten micrograms of total RNA from each time point were analyzed by Northern blot with radiolabeled GST{alpha} cDNA probe as described in Materials and Methods. The data are plotted as the percentage of GST{alpha} mRNA remaining relative to that present at time zero



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  		FIG. 10. Effect of FSH and 8-Br-cAMP on GST{alpha} mRNA stability. Sertoli cells were incubated in the absence ({square}) or presence ({Delta}) of FSH (100 ng/ml, 48 h) or 8-Br-cAMP (1 mM, 48 h) ({blacktriangleup}), after which 25 µM DRB was added to both untreated and treated cells. Total RNA were isolated from control, FSH-treated, or 8-Br-cAMP-treated Sertoli cells at 0, 6, 12, 18, and 24 h after the addition of DRB. Ten micrograms of total RNA from each time point were analyzed by Northern blot with radiolabeled GST{alpha} cDNA probe as described in Materials and Methods. The data are plotted as the percentage of GST{alpha} mRNA remaining relative to that present at time zero.

Additive Effect of FSH and Testosterone on GST{alpha} Protein

The treatment of Sertoli cells with FSH and testosterone (100 ng/ml, 24 h) shows an additive effect on GST{alpha} protein, confirming that the 2 hormones use distinct stimulatory pathways (Fig. 11).



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  		FIG. 11. Additive effects of FSH and testosterone on GST{alpha} protein levels. Sertoli cells were treated for 24 h with testosterone (100 ng/ml), FSH (100 ng/ml), or both. Protein extracts (80 µg) were separated on 12% SDS/polyacrylamide gel, transferred onto nitrocellulose, and incubated with the polyclonal anti-GST{alpha} (1:1000). GST{alpha} protein is evidenced as a doublet of 28 kDa.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have shown that in the course of postnatal testicular development, GST{alpha} is expressed in somatic Leydig cells and Sertoli cells, but not in germ cells. In addition, we have found that GST{alpha} expression in Sertoli cells is under the positive control of FSH and testosterone as well as its 2 metabolites, DHT and estradiol. Indeed, whereas immunoreactive GST{alpha} has been found also in human testicular Leydig and Sertoli cells [41], our present results point to a differential developmental regulation of GST{alpha} expression in both testicular cell types. Indeed, we show that GST{alpha} is permanently expressed in Leydig cells from the neonatal period to adulthood, whereas in Sertoli cells, GST{alpha} is absent in the immature testis but not in the adult testis.

Because the maturation process of the male gonad is under gonadotropin control, these observations support the notion that GST{alpha} expression in Sertoli cells potentially could be induced by testosterone and FSH. Indeed, by using a model of purified Sertoli cells in culture, we have shown that FSH, testosterone per se, and its metabolites, dihydrotestosterone and estradiol, enhance GST{alpha} mRNA and protein levels. FSH increased GST{alpha} mRNA and protein levels via the cAMP/PKA pathway as its effects was mimicked by 8-Br-cAMP and abrogated by a PKA inhibitor, Rp-cAMPs.

Furthermore, although it has been previously reported that FSH enhances GST{alpha} mRNA levels in granulosa cells [42], the present data are, to our knowledge, the first to demonstrate that in Sertoli cells, such a positive action results predominantly from a stabilizing effect of FSH on GST{alpha} mRNA. However, one cannot exclude the possibility of the existence of an additional transcriptional effect of FSH on GST{alpha} expression. The mechanisms involved in the stabilizing action of the hormone on GST{alpha} mRNA are unknown. However, it is of interest to mention that FSH has been shown to stabilize (through the cAMP/PKA pathway) other Sertoli cell mRNAs such as lactate dehydrogenase-A (LDH-A) mRNA [43]. In this context, a 22-bp region called the "cAMP-stabilizing region" (1478–1499) in the 3'-untranslated region (3'-UTR) of LDH-A mRNA, which is targeted by PKA, has been identified in LDH-A mRNA [44]. Because FSH stabilizes GST{alpha} mRNA, we searched for this sequence in GST{alpha} mRNA 3'-UTR. However, this sequence is not present in GST{alpha} mRNA. Whether there is another cAMP-stabilizing region in the 3'-UTR of GST{alpha} mRNA requires further investigations.

We report here that testosterone and its metabolites also enhance GST{alpha} mRNA and protein in Sertoli cells. However, in contrast to FSH, testosterone-stimulated GST{alpha} mRNA levels were not due to an enhancement in mRNA stabilization, but probably to an increased transcriptional activity. Further studies based, for example, on pulse-chase or the nuclear run-on assays, have to be performed to confirm the direct transcriptional regulatory action by testosterone (and its metabolites) on the gst{alpha} gene. Also, it is not known whether androgen and estrogen responsive elements are present in the GST{alpha} promoter, although other different responsive elements including XRE (xenobiotic responsive element, -908 to -899), ARE (antioxidant responsive element, -722 to -682), and EpRE (electrophilic responsive element, -754 to -714) have been identified in the GST promoter [1]. Testosterone appears to be a potent stimulator of GST{alpha} expression because this steroid hormone exerts its effects both per se and also via its two metabolites, DHT and estradiol. The specificity of the androgen action was supported by the fact that testosterone action was 1) mimicked by the nonaromatizable androgen, DHT, and 2) inhibited by flutamide. The effects of estradiol and testosterone were additive, indicating that these two hormones act through their specific receptors. The abrogation of an estradiol-stimulatory effect on GST{alpha} expression by a specific estrogen receptor, ICI 183,780, confirmed the specificity of action of this steroid hormone. Testosterone and estradiol have been reported to affect GST activity, particularly in extragonadal tissues [45]. In these studies, GST isoenzymes were characterized in terms of their biological activity and immunoreactivity, but not in terms of specific gene expression of the different GST classes. In contrast to GST action in the testis, testosterone and estradiol have been reported to exert a negative action on GST activity in extragonadal tissues. Indeed, 4 wk of treatment with 17ß-estradiol alone or with 17ß-estradiol plus testosterone propionate promoted a complete loss of immunostaining for {alpha} and µ class GSTs in the epithelial lining of the vas deferens in male golden Syrian hamsters [46]. Such a decrease in GST (specifically of µ class) following steroid hormone administration appears to be related to hormonal carcinogenesis [47, 48]. However, one should note that these effects are chronic (i.e., for weeks of in vivo treatment) by contrast to those reported in the present study, which are more acute (i.e., 48–72 h in vitro treatment).

In summary, we report here that GST{alpha} expression in Sertoli cells is targeted by the endocrine system. In a model of primary cultures of Sertoli cells, we have demonstrated that GST{alpha} expression (mRNA and protein) is dependent on FSH and testosterone action. The two hormones exert their action through distinct pathways. Although testosterone appears to be a potent stimulator of GST{alpha} expression, acting probably at a transcriptional level per se and via its 2 metabolites, DHT and estradiol, FSH exerts its stimulatory action (via the cAMP/PKA pathway) through an enhancement of GST{alpha} mRNA stability, although at the present time, a transcriptional effect of the hormone cannot be excluded. Also, the possibility that both FSH and steroid hormones may also act downstream at the protein level (translation/turnover of the protein) cannot be excluded. Taken together, the present observations indicate that Sertoli cell GST{alpha} is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.


   ACKNOWLEDGMENTS
 
We are grateful to Mrs. Catherine Rey for her technical assistance and to Dr. Claire Mauduit for her critical reading of the manuscript.


   FOOTNOTES
 
First decision: 29 August 2001.

1 This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), University Claude Bernard Lyon I, and Association pour la Recherche contre le Cancer (contract ARC 9678). Back

2 Correspondence: Mohamed Benahmed, INSERM U407, Faculté de Médecine Lyon-Sud, 165 Chemin du grand revoyet, BP12, F-69621 Oullins Cedex, France. FAX: 33 4 78 86 31 16; benahmed{at}lsgrisn1.univ-lyon1.fr Back

Accepted: January 8, 2002.

Received: July 30, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 1995; 30:445-600[Medline]
  2. Armstrong RN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol 1997; 10:2-18[CrossRef][Medline]
  3. Salinas AE, Wong MG. Glutathione S-transferases—a review. Curr Med Chem 1999; 6:279-309[Medline]
  4. Cho MK, Kim SG. Induction of class alpha glutathione S-transferases by 4-methylthiazole in the rat liver: role of oxidative stress. Toxicol Lett 2000; 115:107-115[CrossRef][Medline]
  5. Ketterer B. Glutathione S-transferases and prevention of cellular free radical damage. Free Radical Res 1998; 28:647-658[Medline]
  6. Papp S, Robaire B, Hermo L. Immunocytochemical localization of the Ya, Yc, Yb1, and Yb2 subunits of glutathione S-transferases in the testis and epididymis of adult rats. Microsc Res Tech 1995; 30:1-23[CrossRef][Medline]
  7. Pickett CB, Lu AY. Glutathione S-transferases: gene structure, regulation, and biological function. Annu Rev Biochem 1989; 58:743-764[CrossRef][Medline]
  8. Mannervik B, Danielson UH. Glutathione transferases—structure and catalytic activity. CRC Crit Rev Biochem 1988; 23:283-337[Medline]
  9. Pickett CB, Telakowski-Hopkins CA, Argenbright L, Lu AY. Regulation of glutathione S-transferase mRNAs by phenobarbital and 3-methylcholanthrene: analysis using cDNA probes. Biochem Soc Trans 1984; 12:71-74[Medline]
  10. Di Simplicio P, Jensson H, Mannervik B. Effects of inducers of drug metabolism on basic hepatic forms of mouse glutathione transferase. Biochem J 1989; 263:679-685[Medline]
  11. Mueller A, Hermo L, Robaire B. The effects of aging on the expression of glutathione S-transferases in the testis and epididymis of the Brown Norway rat. J Androl 1998; 19:450-465[Abstract/Free Full Text]
  12. Gandy J, Primiano T, Novak RF, Kelce WR, York JL. Differential expression of glutathione S-transferase isoforms in compartments of the testis and segments of the epididymis of the rat. Drug Metab Dispos 1996; 24:725-733[Abstract]
  13. Sundberg AG, Nilsson R, Appelkvist EL, Dallner G. Immunohistochemical localization of alpha and pi class glutathione transferases in normal human tissues. Pharmacol Toxicol 1993; 72:321-331[Medline]
  14. Hsieh CH, Tsai SP, Yeh HI, Sheu TC, Tam MF. Mass spectrometric analysis of rat ovary and testis cytosolic glutathione S-transferases (GSTs): identification of a novel class-alpha GST, rGSTA6*, in rat testis. Biochem J 1997; 323:503-510
  15. Aceto A, Di Ilio C, Angelucci S, Felaco M, Federici G. Glutathione transferase isoenzymes from human testis. Biochem Pharmacol 1989; 38:3653-3660[CrossRef][Medline]
  16. Yoganathan T, Eskild W, Hansson V. Investigation of detoxification capacity of rat testicular germ cells and Sertoli cells. Free Radical Biol Med 1989; 7:355-359[CrossRef][Medline]
  17. Fulcher KD, Welch JE, Klapper DG, O'Brien DA, Eddy EM. Identification of a unique mu-class glutathione S-transferase in mouse spermatogenic cells. Mol Reprod Dev 1995; 42:415-424[CrossRef][Medline]
  18. Rowe JD, Tchaikovskaya T, Shintani N, Listowsky I. Selective expression of a glutathione S-transferase subclass during spermatogenesis. J Androl 1998; 19:558-567[Abstract/Free Full Text]
  19. Rowe JD, Patskovsky YV, Patskovska LN, Novikova E, Listowsky I. Rationale for reclassification of a distinctive subdivision of mammalian class Mu glutathione S-transferases that are primarily expressed in testis. J Biol Chem 1998; 273:9593-9601[Abstract/Free Full Text]
  20. Mukherjee SB, Gopalakrishnan AB, Nagpal S, Salunke DM, Shaha C. Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1. Biochem J 1999; 340:309-320
  21. Hussey AJ, Hayes JD. Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue. Biochim Biophys Acta 1993; 1203:131-141[CrossRef][Medline]
  22. Hayes JD, Mantle TJ. Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat. Biochem J 1986; 233:779-788[Medline]
  23. Ketterer B, Fraser G, Meyer DJ. Nuclear glutathione transferases which detoxify irradiated DNA. Adv Exp Med Biol 1990; 264:301-310[Medline]
  24. Vos RM, Van Bladeren PJ. Glutathione S-transferases in relation to their role in the biotransformation of xenobiotics. Chem Biol Interact 1990; 75:241-265[CrossRef][Medline]
  25. Rao AV, Shaha C. Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis. Free Radical Biol Med 2000; 29:1015-1027[CrossRef][Medline]
  26. Yang Y, Cheng JZ, Singhal SS, Saini M, Pandya U, Awasthi S, Awasthi YC. Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTa2-2 in k562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation. J Biol Chem 2001; 276:19220-19230[Abstract/Free Full Text]
  27. Institoris E, Eid H, Bodrogi I, Bak M. Glutathione related enzymes in human testicular germ cell tumors and normal testes. Anticancer Res 1995; 15:1371-1374[Medline]
  28. Aravinda S, Gopalakrishnan B, Dey CS, Totey SM, Pawshe CH, Salunke D, Kaur K, Shaha C. A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules. J Biol Chem 1995; 270:15675-15685[Abstract/Free Full Text]
  29. Sertoli E. Dell' esistenza di particulari cellule ramificate nei canalicoli seminiferi del testicolo umano. Morgagni 1865; 7:31-40
  30. Fawcett DW. Observations on the organization of the interstitial tissue of the testis and on the occluding cell junctions in the seminiferous epithelium. Adv Biosci 1973; 10:83-99[Medline]
  31. Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994: 1363–1434
  32. Griswold MD. Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 1995; 52:211-216[Abstract]
  33. Hellani A, Ji J, Mauduit C, Deschildre C, Tabone E, Benahmed M. Developmental and hormonal regulation of the expression of oligodendrocyte-specific protein/claudin 11 in mouse testis. Endocrinology 2000; 141:3012-3019[Abstract/Free Full Text]
  34. Murray GI, Taylor VE, McKay JA, Weaver RJ, Ewen SW, Melvin WT, Burke MD. The immunohistochemical localization of drug-metabolizing enzymes in prostate cancer. J Pathol 1995; 177:147-152[CrossRef][Medline]
  35. Mehta R, Davis HG, Laver GW, Rowsell PR, Bondy GS. Glutathione S-transferases and P-glycoprotein in normal rat hepatocytes and hepatoma cells: analysis using flow cytometry. Cancer Lett 1994; 84:163-172[CrossRef][Medline]
  36. Mather JP, Phillips DM. Primary culture of testicular somatic cells. In: Barnes DW, Sirbasku DA, Sato GH (eds.), Methods for Serum-Free Culture of Cells of the Endocrine System. New York: Alan R Liss; 1984: 29–45
  37. Benahmed M, Morera AM, Chauvin MA, de Peretti E. Somatomedin C/insulin-like growth factor 1 as a possible intratesticular regulator of Leydig cell activity. Mol Cell Endocrinol 1987; 50:69-77[CrossRef][Medline]
  38. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159[Medline]
  39. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976; 72:248-254[CrossRef][Medline]
  40. Franca LR, Silva VA Jr, Chiarini-Garcia H, Garcia SK, Debeljuk L. Cell proliferation and hormonal changes during postnatal development of the testis in the pig. Biol Reprod 2000; 63:1629-1636[Abstract/Free Full Text]
  41. Klys HS, Whillis D, Howard G, Harrison DJ. Glutathione S-transferase expression in the human testis and testicular germ cell neoplasia. Br J Cancer 1992; 66:589-593[Medline]
  42. Rabahi F, Brule S, Sirois J, Beckers JF, Silversides DW, Lussier JG. High expression of bovine alpha glutathione S-transferase (GSTA1, GSTA2) subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles. Endocrinology 1999; 140:3507-3517[Abstract/Free Full Text]
  43. Boussouar F, Grataroli R, Ji J, Benahmed M. Tumor necrosis factor-alpha stimulates lactate dehydrogenase A expression in porcine cultured Sertoli cells: mechanisms of action. Endocrinology 1999; 140::3054-3062[Abstract/Free Full Text]
  44. Tian D, Huang D, Short S, Short ML, Jungmann RA. Protein kinase A-regulated instability site in the 3'-untranslated region of lactate dehydrogenase-A subunit mRNA. J Biol Chem 1998; 273:24861-24866[Abstract/Free Full Text]
  45. Igarashi T, Satoh T, Ono S, Iwashita K, Hosokawa M, Ueno K, Kitagawa H. Effect of steroidal sex hormones on the sex-related differences in the hepatic activities of gamma-glutamyltranspeptidase, glutathione S-transferase and glutathione peroxidase in rats. Res Commun Chem Pathol Pharmacol 1984; 45:225-232[Medline]
  46. Hudson CE, DeHaven JE, Schulte BA, Norris JS. Exogenous 17beta-estradiol blocks alpha and mu but not pi class glutathione S-transferase immunoreactivity in epithelium of Syrian hamster vas deferens. J Histochem Cytochem 1999; 47:91-98[Abstract/Free Full Text]
  47. Hudson CE, Kelly MM, Schwartz DA, Schofield DA, DeHaven JE, Schulte BA, Norris JS. Glutathione S-transferase in hormonal carcinogenesis. Chem Biol Interact 1998; 111–112:343-350
  48. Staffas L, Ellis EM, Hayes JD, Lundgren B, Depierre JW, Mankowitz L. Growth hormone- and testosterone-dependent regulation of glutathione transferase subunit A5 in rat liver. Biochem J 1998; 332:763-768



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