Abstract
2,3,7,8-Tetrachloro dibenzo-p-dioxin (TCDD), an endocrine-disrupting environmental pollutant, has been found to cause male reproductive toxicity. Glucocorticoids have been found to influence the metabolic pathway of TCDD. Stress, which affects the male reproductive function, is marked by an increase in the level and activity of glucocorticoids in the body. The present study was carried out to understand the effect of TCDD on testicular steroidogenesis and sperm antioxidant system under the influence of increased level of corticosterone in the body. Adult male rats were treated with either TCDD (100 ng/kg bw/ day) or corticosterone (3 mg/kg bw/day) or both for 15 days. Treatment with either TCDD or corticosterone was found to suppress the levels of steroidogenic acute regulatory protein and androgen-binding protein and reduce the activities of steroidogenic enzymes in testis while increasing oxidative stress in ventral prostate, seminal vesicles and epididymal sperm. In rats treated with both TCDD and corticosterone, the suppression of testicular steroidogenesis and increase in oxidative stress observed in ventral prostate, seminal vesicles and epididymal sperm were significant as compared to TCDD alone treated rats. The levels of Fas and FasL proteins were also increased in rats subjected to either TCDD or corticosterone treatment. In rats treated with both compounds, the increase observed in testicular levels of Fas and FasL was significant as compared to TCDD alone treated rats. Effect of TCDD on testicular steroidogenesis and antioxidant system of epididymal sperm may get enhanced under increased level of glucocorticoids in the body.
Introduction
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), a persistent endocrine-disrupting environmental contaminant, is generated during industrial processes like incineration of medical wastes, chlorine bleaching of paper and pulp and manufacture of pesticides. Home-heating systems, exhaust from cars and cigarette smoke have been found to contain trace quantities of this dioxin (Fiedler et al., 1990). Due to its lipophilic properties, slow metabolism and excretion, TCDD readily accumulates in body (Enan et al., 1998). TCDD has been found to elicit toxicity by binding to cytosolic aryl hydrocarbon receptor, enforcing the expression of enzymes like cytochrome P4501A1, which is involved in the production of reactive oxygen species (ROS) (Androutsopoulos et al., 2009; Rifkind, 2006). Male reproductive tissues have been found to be very sensitive targets of this dioxin. Exposure to TCDD, even in trace quantities, has been reported to cause low sperm counts and decreased fertility (Gray et al., 2001).
TCDD-induced expression of cytochrome P1A1 enzyme, involved in the generation of ROS and mutagenic metabolites, has been found to be enhanced by glucocorticoids (Celander et al., 1997; Lai et al., 2004; Sonneveld et al., 2007; Bielefeld et al., 2008). A potent synergistic interaction has been observed between TCDD and glucocorticoids in inducing cleft palate (Abbot, 1995). A raise in the level and activity of glucocorticoids occurs during stress (Charmandari et al., 2005). Stress, a condition increasingly present in modern society, has also been reported to affect male fertility (Hardy et al., 2005). Stress-induced increase in corticosteroid secretion has been found to inhibit testosterone secretion by Leydig cells and serum luteinizing hormone (Kalantaridou et al., 2010). Due to these reasons, the study of male reproductive toxicity of TCDD under the influence of increased level of glucocorticoids in body becomes essential. In our previous studies, we have reported the effect of TCDD on the antioxidant system of testis and epididymis under the influence of increased level of corticosterone (Dhanabalan and Mathur, 2009; Dhanabalan et al., 2010, 2011). The present study was carried out to understand the changes in testicular steroidogenesis and epididymal sperm antioxidant system. For this purpose, TCDD was orally administered to rats injected with stress mimicking level of corticosterone. Also, separate groups of rats were maintained with either corticosterone or TCDD treatment. To understand the changes in testicular steroidogenesis due to the treatments, steroidogenic enzymes and steroidogenic acute regulatory (StAR) protein were analyzed. Changes in testicular steroidogenesis have been reported to affect the structure and functions of accessory sex tissues. Oxidative stress, a factor to affect reproductive performance and cellular senescence, is found to be elevated by the chronic high secretion of glucocorticoids (Costantini et al., 2011). Therefore, oxidative stress in ventral prostate, seminal vesicles and epididymal sperm was also measured in the study.
Materials and methods
Chemicals
TCDD (purity ∼ 99%) was obtained from Cambridge Isotope Laboratories Inc., Andover, Massachusetts, USA Corticosterone and Ham’s F-12 medium were purchased from HiMedia Laboratories Pvt Ltd, Mumbai, India. Dehydroepiandrosterone, androstenedione, nicotinamide cofactors (nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NAD+)) and rabbit monoclonal antibody against β-actin were purchased from Sigma Chemical Company, St. Louis, Missouri, USA. Antisera against a synthetic peptide consisting of amino acid sequence 88–98 of the mouse StAR protein generated in rabbit was a gift from Dr D.M. Stocco, Texas Tech University Health Science Centre, Lubbock, Texas, USA. Polyclonal antibody against rat androgen-binding protein (ABP) was a gift from Dr C.Y. Cheng, The Population Council, New York, USA. Rabbit polyclonal antibodies against Fas (sc-7886) and FasL (sc-834) were procured from Santa Cruz Biotechnology (Santa Cruz, California, USA). Horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (IgG) was obtained from Bangalore Genei (Bengaluru, India). Thiobarbituric acid (purity ≥99%) and malondialdehyde (purity ≥98%) were obtained from Merck, Darmstadt, Germany. All other chemicals used were of analytical grade and obtained from local commercial sources.
Experimental animals and study design
Adult male albino rats (70–80 days old) of Wistar/NIN (WNIN) strain were procured from an authorized vendor (M/S Raghavendra Enterprises, Bengaluru, India) and were housed (three per cage) in polypropylene cages (47 cm × 34 cm × 20 cm) and maintained at 22–25°C under a well-regulated light and dark (12 h:12 h) schedule at the Animal Facility of the Pondicherry University. The rats were fed on standard rat chow and water ad libitum. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA, 2003), Government of India. Corticosterone was dissolved in olive oil (3 mg/ml) and administered to rats subcutaneously at a dose of 3 mg/kg bw/day for 15 days (Sandi et al., 1996; Spiga and Lightman, 2009). TCDD was also dissolved in olive oil (100 ng/ml) and administered by oral gavage at a dose of 100 ng/kg bw/day for 15 days.
Four groups of animals were used in the experiment, each with six rats, which is statistically acceptable:
Group I: administration of olive oil subcutaneously and by oral gavage for 15 days.
Group II: administration of corticosterone (3 mg/kg bw/day) subcutaneously and olive oil by oral gavage for 15 days.
Group III: administration of olive oil subcutaneously and TCDD (100 ng/kg bw/day) by oral gavage for 15 days.
Group IV: administration of corticosterone (3 mg/kg bw/day) subcutaneously and TCDD (100 ng/kg bw/day) by oral gavage for 15 days.
Necropsy
The rats were fasted overnight, weighed and killed using overdose of anesthetic ether on the day following the last treatment. Testes, epididymidis, seminal vesicles and ventral prostate, weighed after clearing off the adhering tissues, were used for biochemical studies.
Preparation of homogenate of seminal vesicles and ventral prostate
Seminal vesicles and ventral prostate were isolated and homogenized separately in homogenizing buffer (0.1 M sodium phosphate buffer, pH 7.4). The 10% homogenate was centrifuged at 800g for 20 min at 4°C. The supernatant was collected and used for biochemical assays.
Preparation of homogenate of epididymal sperm
Epididymal sperm was collected by cutting the epididymis into small pieces in Ham’s F-12 medium at 35°C (Latchoumycandane et al., 2002). The tissue was washed several times in the same medium in order to remove the maximum number of sperm from the epididymis. The sperm suspensions obtained were centrifuged at 800g for 20 min at 4°C. The pellet was resuspended in 2 ml normal saline and homogenized for few seconds in cold. The homogenate was centrifuged at 800g for 20 min at 4°C and the supernatant was used for biochemical studies.
Testicular steroidogenic enzymes
The activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) (EC 1.1.1.145) and 17β-hydroxysteroid dehydrogenase (17β-HSD) (EC 1.1.1.51) were determined in the testicular microsomal fraction by the method of Bergmeyer (1974). The reaction mixture in a volume of 2.0 ml contained 100 μmol of sodium pyrophosphate buffer (pH 9.0) and 0.5 μmol cofactor NAD for 3β-HSD and NADPH for 17β-HSD, 0.08 μmol of substrate (dehydroepiandrosterone for 3β-HSD and androstenedione for 17β-HSD) and 100 μl of enzyme source. The reactions were carried out in a quartz cuvette of 1.0 cm path length at 23 ± 1°C. The absorbance at 340 nm was measured at 20s intervals for 3 min in a Shimadzu UV-visible spectrophotometer. The enzyme activities were expressed as nanomoles of NAD converted to NADH per milligram protein per minute (3β-HSD) or nanomoles of NADPH converted to NADP per milligram protein per minute (17β-HSD).
Thiobarbituric acid reactive substances (TBARS)
A breakdown product of lipid peroxidation, TBARS was measured by the method of Ohkawa et al. (1979). Briefly, the stock solution contained equal volumes of trichloroacetic acid 15% (w/v) in 0.25 N hydrochloric acid and 2-thiobarbituric acid 0.37% (w/v) in 0.25 N hydrochloric acid. One volume of the test sample and two volumes of stock reagent were mixed in a screw-capped centrifuge tube, vortexed and heated for 15 min on a boiling water bath. After cooling on ice, the precipitate was removed by centrifugation at 1000g for 15 min and absorbance of the supernatant was measured at 532 nm against blank containing all the reagents except test sample. A standard curve was constructed extrapolating the amount to the measured absorbance. The value was expressed as micromoles of malondialdehyde formed per minute per milligram protein.
Hydrogen peroxide generation assay
Hydrogen peroxide generation was assayed by the method of Pick and Keisari (1981). Briefly, the mixture containing 1.64 ml phosphate buffer (50 mM, pH 7.6), 54 µl of horseradish peroxidase (8.5 units/ml), 30 µl of 0.28 nM phenol red, 165 µl of 5.5 nM dextrose and 100 µl of enzyme source was incubated at 35°C for 30 min. The reaction was terminated by the addition of 60 µl of 10 N sodium hydroxide (NaOH). The absorbance was read at 610 nm against a reagent blank on a Shimadzu UV-visible spectrophotometer. The quantity of hydrogen peroxide produced was expressed as nanomoles of hydrogen peroxide generated per minute per milligram protein at 35°C. For standard curve, the known amount of hydrogen peroxide and all the above reagents except enzyme source were incubated for 30 min at 35°C and then 60 µl of 10 N NaOH was added and the optical density was read at 610 nm.
Superoxide dismutase
Superoxide dismutase (EC 1.15.1.1) was assayed by the method of Marklund and Marklund (1974). Briefly, the assay mixture contained 2.4 ml of 50 mM Tris-HCl buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.6), 300 µl of 0.2 mM pyrogallol and 300 µl enzyme source. The increase in absorbance was measured immediately at 420 nm against blank containing all the components except the enzyme and pyrogallol at 10 seconds intervals for 3 min on a Shimadzu UV-visible spectrophotometer. The activity of the enzyme was expressed in nanomoles of pyrogallol oxidized per minute per milligram protein.
Catalase
Catalase (EC 1.11.1.6) was assayed by the method of Claiborne (1985). Briefly, the assay mixture contained 2.40 ml of phosphate buffer (50 mM, pH 7.0), 10 µl of 19 mM hydrogen peroxide and 50 µl enzyme source. The decrease in absorbance was measured immediately at 240 nm against blank containing all the components except the enzyme at 10 seconds intervals for 3 min on a Shimadzu UV-visible spectrophotometer. The activity of enzyme was expressed in micromoles of hydrogen peroxide consumed per minute per milligram protein.
Immunoblot analysis of StAR protein
Decapsulated testes were homogenized at 4°C into a buffer containing 0.25 M sucrose, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 mM EDTA, pH 7.4, 10 mM sodium chloride (NaCl) and 2 mM dithiothreitol using a glass-teflon homogenizer (Remi RQ-127A, Remi Motors, Mumbai, India). The homogenate was centrifuged at 500g for 25 min to remove debris and the resulting supernatant was further centrifuged at 10,000g for 25 min. The pellet containing mitochondria was washed twice at 9000g for 15 min in the same buffer. Mitochondrial proteins were extracted in 20 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 5 mM EDTA, 0.5% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF) and the protein levels were determined by the method of Lowry et al. (1951). Equal quantities of protein were loaded per lane and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Mini Protean II System, Bio-Rad) as described by Laemmli (1970). Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred into a nitrocellulose membrane (NYTRAN, Keene, New Hampshire, USA) in transfer buffer (0.2 mol/l glycine, 25 mM Tris and 20% methanol). Successful transfer was confirmed by Ponceau S staining of the blots. The membranes were incubated in a blocking buffer (phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk powder) for 5 h at room temperature, followed by the incubation with StAR antisera (1:1000 dilution) generated in rabbit against amino acid sequence 88–98. The incubation with the primary antibody was carried out overnight at 4°C. The following day, blots were washed in PBS and incubated for 1 h at room temperature with horseradish peroxidase–conjugated anti-rabbit IgG (1:1000 dilution). Immunodetection of proteins was revealed using tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) (Bangalore Genei) as a substrate. The resulting images were compiled using Adobe Photoshop (version 7.0, San Jose, California, USA) and densitometric analysis of immunospecific bands was performed using Fuji Film Multigauge 3.0 Software.
Immunoblot analysis of ABP
Testes lysates were prepared by lysis buffer (50 mM Tris, pH 7.4 containing 0.15 M NaCl, 10% glycerol (v/v), 1% NP-40 (v/v), 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM EDTA, 150 mM bestatin, 1 mM leupeptin and 1 mM aprotinin) using a tissue–buffer ratio of 1:5 and protein concentration was determined by the method of Lowry et al. (1951). Equal quantities of protein were loaded per lane and subjected to 10% SDS-PAGE (Mini Protean II System, Bio-Rad) as described by Laemmli (1970). Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred into a nitrocellulose membrane (NYTRAN, Keene, New Hampshire, USA) in transfer buffer (0.2 mol/l glycine, 25 mM Tris and 20% methanol v/v). Successful transfer was confirmed by Ponceau S staining of the blots. Blots were incubated with primary antibody for ABP (1:1000 dilution) overnight at 4°C. The following day, blots were washed in PBS and incubated for 1 h at room temperature with horseradish peroxidase–conjugated anti-rabbit IgG (1:1000 dilution). Immunodetection of proteins was revealed using TMB/H2O2 (Bangalore Genei) as a substrate. The resulting images were compiled using Adobe Photoshop (version 7.0) and the densitometric analysis of immunospecific bands was performed using Fuji Film Multigauge 3.0 software.
Immunoblot analysis of Fas protein
Testes lysates were prepared using lysis buffer (50 mM Tris, pH 7.4 containing 0.15 M NaCl, 10% glycerol (v/v), 1% NP-40 (v/v), 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM EDTA, 150 µM bestatin, 1 µM leupeptin and 1 µM aprotinin) using a tissue–buffer ratio of 1:5. After homogenization, samples were centrifuged at 1000g for 30 min. The supernatants were collected and stored at −70°C until analysis. The protein concentration was determined and equal quantities of protein were loaded per lane and subjected to 10% SDS-PAGE (Mini Protean II System, Bio-Rad) as described by Laemmli (1970). Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (NYTRAN, Keene, New Hampshire, USA) in transfer buffer (0.2 mol/l glycine, 25 mM Tris and 20% methanol). The membranes were incubated in a blocking buffer (PBS containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk powder) for 5 h at room temperature, followed by incubation in respective primary antibodies. The primary antibody for Fas protein was diluted to 1:200. Incubations with the primary antibodies were carried out overnight at 4°C. The following day, blots were washed in PBS and incubated for 1 h at room temperature with either horseradish peroxidase–conjugated anti-rabbit IgG (1:1000 dilution). Immunodetection of proteins was visualized using TMB/H2O2 (Bangalore Genei) as a substrate. The resulting images were compiled using Adobe Photoshop (version 7.0) and the densitometric analysis of immunospecific bands was performed using Fuji Film Multigauge 3.0 software.
Immunoblot analysis of FasL protein
Testes lysates were prepared using lysis buffer (50 mM Tris, pH 7.4 containing 0.15 M NaCl, 10% glycerol (v/v), 1% NP-40 (v/v), 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM EDTA, 150 µM bestatin, 1 µM leupeptin and 1 µM aprotinin) using a tissue–buffer ratio of 1:5. After homogenization, the samples were centrifuged at 1000g for 30 min. The supernatants were collected and stored at −70°C until analysis. The protein concentration was determined and equal quantities of protein were loaded per lane and subjected to 10% SDS-PAGE (Mini Protean II System, Bio-Rad) as described by Laemmli (1970). Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (NYTRAN, Keene, New Hampshire, USA) in transfer buffer (0.2 mol/l glycine, 25 mM Tris and 20% methanol). The membranes were incubated in a blocking buffer (PBS containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk powder) for 5 h at room temperature, followed by incubation in respective primary antibodies. The primary antibody for FasL protein was diluted to 1:200. Incubations with the primary antibodies were carried out overnight at 4°C. The following day, blots were washed in PBS and incubated for 1 h at room temperature with either horseradish peroxidase–conjugated anti-rabbit IgG (1:1000 dilution). Immunodetection of proteins was visualized using TMB/H2O2 (Bangalore Genei) as a substrate. The resulting images were compiled using Adobe Photoshop (version 7.0) and densitometric analysis of immunospecific bands was performed using Fuji Film Multigauge 3.0 software.
Statistical analysis
Data were expressed as mean ± SD. Statistical analysis was performed using an analysis of variance (one-way ANOVA) followed by Tukey’s post test using SPSS (student version 7.5, SPSS Inc., Chertsey, UK). Differences were considered to be significant at p < 0.05.
Results
Testicular steroidogenic enzymes
Activities of testicular steroidogenic enzymes like 3β-HSD and 17β-HSD decreased significantly in rats treated with either corticosterone or TCDD as compared to control rats (Figures 1 and 2). In rats treated with both corticosterone and TCDD, the decline observed in the activities of steroidogenic enzymes was significant as compared to rats treated with TCDD alone.
Changes in the levels of 3β-hydroxysteroid dehydrogenase in the testicular microsomal rich fractions in various groups of rats. Values are expressed in nanomoles per minute per milligram protein as mean ± SD for six animals per group. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
Changes in the levels of 17β-hydroxysteroid dehydrogenase in the testicular microsomal rich fractions in various groups of rats. Values are expressed in nanomoles per minute per milligram protein as mean ± SD for six animals per group. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
TBARS and hydrogen peroxide generation
A significant increase in the levels of TBARS and hydrogen peroxide was observed in ventral prostate (Table 1), seminal vesicles (Table 2) and epididymal sperm (Table 3) in rats treated with either corticosterone or TCDD as compared to control rats. In rats treated with both corticosterone and TCDD, a significant increase was observed in the levels of TBARS and hydrogen peroxide in ventral prostate, seminal vesicles and epididymal sperm as compared to TCDD-treated rats.
Effect of corticosterone and TCDD on the levels of TBARS and H2O2 generation and on the activities of SOD and CAT in the ventral prostate of rats.c,d
TCDD: 2,3,7,8 tetrachloro dibenzo-p-dioxin; TBARS: thiobarbituric acid reactive substances; H2O2: hydrogen peroxide; SOD: superoxide dismutase; CAT: catalase.
cThe units are expressed as nanomoles per minute per milligram protein for H2O2 and SOD, as micromoles per milligram protein for TBARS and as micromoles per minutes per milligram protein for CAT.
dData are expressed as mean ± SD for six animals per group.
a p< 0.05 against group I.
b p< 0.05 against group III (group I: control; group II: corticosterone; group III: TCDD and group IV: corticosterone and TCDD).
Effect of corticosterone and TCDD on the levels of TBARS and H2O2 generation and on the activities of SOD and CAT in the seminal vesicles of rats.c,d
TCDD: 2,3,7,8 tetrachloro dibenzo-p-dioxin; TBARS: thiobarbituric acid reactive substances; H2O2: hydrogen peroxide; SOD: superoxide dismutase; CAT: catalase.
cThe units are expressed as nanomoles per minute per milligram protein for H2O2 and SOD, as micromoles per milligram protein for TBARS and as micromoles per minutes per milligram protein for CAT.
dData are expressed as mean ± SD for six animals per group.
a p< 0.05 against group I.
b p< 0.05 against group III (group I: control, group II: corticosterone, group III: TCDD and group IV: corticosterone and TCDD).
Effect of corticosterone and TCDD on the levels of TBARS and H2O2 generation and on the activities of SOD and CAT in the epididymal sperm of rats.c,d
TCDD: 2,3,7,8 tetrachloro dibenzo-p-dioxin; TBARS: thiobarbituric acid reactive substances; H2O2: hydrogen peroxide; SOD: superoxide dismutase; CAT: catalase.
cThe units are expressed as nanomoles per minute per milligram protein for H2O2 and SOD, as micromoles per milligram protein for TBARS and as micromoles per minutes per milligram protein for CAT.
dData are expressed as mean ± SD for six animals per group.
a p< 0.05 against group I.
b p< 0.05 against group III (group I: control; group II: corticosterone; group III: TCDD and group IV: corticosterone and TCDD).
Activities of superoxide dismutase and catalase
A significant decrease in the activities of superoxide dismutase and catalase was observed in ventral prostate (Table 1), seminal vesicles (Table 2) and epididymal sperm (Table 3) in rats treated with either corticosterone or TCDD as compared to control rats. In rats treated with both corticosterone and TCDD, a significant decrease was observed in the activities of superoxide dismutase and catalase in ventral prostate, seminal vesicles and epididymal sperm as compared to rats treated with TCDD alone.
Levels of testicular StAR protein
Western blot analysis of testicular StAR protein revealed a significant decrease in the levels of the protein in rats treated with either corticosterone or TCDD as compared to control rats (Figure 3). In rats treated with both corticosterone and TCDD, the decrease observed in the levels of StAR protein was significant as compared to rats treated with TCDD alone.
Changes in the levels of testicular StAR protein in various groups of treated rats. The blot was probed with actin (bottom panel) to show equal protein loading. The blot shown is representative of three experiments. Each data point represents the average value from three independent experiments normalized against the control. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
Levels of ABP
A significant decrease was found in the levels of ABP in rats treated with either corticosterone or TCDD as compared to control rats (Figure 4). In rats treated with both corticosterone and TCDD, the decrease in the level of ABP was significant as compared to rats treated with TCDD alone.
Changes in the levels of testicular ABP in various groups of treated rats. The blot was probed with actin (bottom panel) to show equal protein loading. The blot shown is representative of three experiments. Each data point represents the average value from three independent experiments normalized against the control. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
Levels of Fas and FasL proteins in testis
A significant increase in the levels of Fas and FasL proteins was observed in rats treated with either corticosterone or TCDD as compared to control rats (Figures 5 and 6). In rats subjected to both corticosterone and TCDD treatment, a significant increase was observed in the levels of Fas and FasL as compared to rats treated with TCDD alone.
Changes in the levels of testicular Fas in various groups of treated rats. The blot was probed with actin (bottom panel) to show equal protein loading. The blot shown is representative of three experiments. Each data point represents the average value from three independent experiments normalized against the control. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
Changes in the levels of testicular FasL in various groups of treated rats. The blot was probed with actin (bottom panel) to show equal protein loading. The blot shown is representative of three experiments. Each data point represents the average value from three independent experiments normalized against the control. a p < 0.05 against control group and b p < 0.05 against TCDD-treated group.
Discussion
In recent years, cases of male reproductive disorders (like decrease in sperm count and quality, testicular cancer, etc.) show an increasing trend. A number of modern world factors like exposure to environmental pollutants, stressed lifestyle, and so on have been attributed to this trend (Mendiola et al., 2009; Sinclair, 2000). Research conducted so far, in general, has concentrated on the deleterious effects of only a single factor on male reproductive function. The combined effects of two or more factors in causing male reproductive disorders have not been studied. Studies by combining environmental contaminants with factors like stress may give new dimension to our understanding of male reproductive disorders which are on increasing trend (especially in industrialized societies). TCDD has been found to increase the oxidative stress and suppress the activities of antioxidant enzymes in testis and affect the serum level of testosterone hormone. The progressive motility of epididymal sperm has also been reported to be decreased by TCDD (Beytur et al., 2012; Ciftci et al., 2012). Stress is marked by an increase in the level and activity of glucocorticoids (cortisol in primates and corticosterone in rodents predominantly) in body (Kyrou and Tsigos, 2007). Glucocorticoids have been found to influence the metabolic pathway of TCDD like other hydrocarbons. In our previous studies, we analyzed the effects of TCDD on testicular and epididymal antioxidant system under the influence of increased level of corticosterone in body (Dhanabalan and Mathur, 2009; Dhanabalan et al., 2010, 2011).
In the present study, in rats treated with corticosterone or TCDD or both, a decrease in the levels of StAR protein accompanied by suppressed activities of steroidogenic enzymes like 3β-HSD and 17β-HSD was observed in the testis. In Leydig cells, StAR protein mediates the translocation of cholesterol from the outer to inner mitochondrial membrane, a rate-limiting step, enabling P450SCC enzyme to initiate the synthesis of steroids. A decrease in the level of StAR protein will affect androgen synthesis by reducing the accessibility of P450SCC enzyme to cholesterol (Stocco, 2001). Steroidogenic enzymes like 3β-HSD and 17β-HSD, by producing Δ4-androstenedione from dehydroepiandrosterone and testosterone from Δ4-androstenedione, play a significant role in steroidogenesis (Mizrachi and Auchus, 2009). The suppression in the levels of StAR protein and the activity of steroidogenic enzymes in testis may be the reason behind the decline in the levels of serum testosterone observed in our previous studies (Dhanabalan et al., 2010, 2011).
Testosterone is involved in the maintenance of normal spermatogenesis and the inhibition of germ cell apoptosis in the testis. In epididymis, androgens have been reported to regulate growth differentiation and normal morphology, transport of ions and synthesis and secretion of various proteins including those involved in sperm functions (Dohle et al., 2003; Robaire et al., 2007). Loss of androgen secretion due to orchidectomy has been reported to induce apoptotic cell death and 80% weight loss in the epididymis (Fan and Robaire, 1998). Seminal vesicle, an accessory sex organ, is also dependent on androgenic stimuli for embryonic development, growth and secretory function. The secretion of seminal vesicles containing factors like bicarbonate, prolactin and fructose has been found to enhance the motility of spermatozoa (Risbridger and Taylor, 2006). Removal of androgens by castration or a reduced titer of androgens has been found to result in the regression of the seminal vesicles, with androgen replacement causing these organs to increase in size and become functional (Williams-Ashman, 1983). The ventral prostate, another accessory sex tissue, is highly dependent on androgens for its differentiation, development and growth (Hayward and Cunha, 2000; Zhang and Verdine, 1999). Androgens are important for establishing prostatic identity and for stimulating ductal growth and branching morphogenesis. Treatment with antiandrogens has been found to cause regression in the prostate, illustrating that continued presence of androgen is necessary for the homeostasis of the tissue (McConnell, 1990; Montironi et al., 1998). In the present study, a significant decrease in testicular steroidogenesis caused by TCDD treatment or by corticosterone injection or both may be a reason behind the raised oxidative stress in seminal vesicles, ventral prostate and epididymal sperm.
ABP, a glycoprotein secreted by the testicular sertoli cells, binds testosterone with high affinity and regulate spermatogenesis by maintaining high androgen levels in the testis. ABP also transports androgens to the epididymis for sperm maturation (Munell et al., 2002). In the present investigation, the decline observed in the levels of testicular ABP in rats caused by TCDD or corticosterone or both may be another reason behind the increased oxidative stress in the epididymal sperm.
In testis, the Fas/FasL system which is involved in germ cell apoptosis, is required during spermatogenesis to coordinate the number of proliferating germ cells to that of the supporting Sertoli cells and to ensure a quality control of the produced gametes. The Fas/FasL pathway has been reported to be essential for initiating germ cell apoptosis in response to a variety of conditions like oxidative stress (Geng et al., 2009). Diethylstilbesterol, a synthetic estrogen, has been reported to cause apoptosis in spermatogenic cells by inducing Fas/FasL upregulation (Nair and Shaha, 2003). Cadmium chloride, a heavy metal, has been found to enhance FasL immunoreactivity in Sertoli cells and peritubular germ cells, like spermatogonia and spermatocytes (Ozawa et al., 2002). In the present investigation, the administration of corticosterone or TCDD or both has been found to significantly raise the testicular levels of Fas/FasL proteins.
In the previous studies, TCDD-induced suppression of serum testosterone, decrease in testicular daily sperm production, epididymal sperm count, motility and viability have been found to be enhanced by increased level of corticosterone in the body (Dhanabalan and Mathur, 2009; Dhanabalan et al., 2010, 2011). The present study was conducted to understand the mechanisms underlying these reduced end points. The present experiment confirms TCDD-induced suppression of testicular steroidogenesis and oxidative stress in seminal vesicles, ventral prostate and epididymal sperm can be enhanced by increased level of corticosterone in body. This study may also gain significance in the context of designing lowest observed adverse effect level dosage for TCDD and also for other aryl hydrocarbons whose metabolic pathway is influenced by glucocorticoids.
Footnotes
Funding
This research was financially supported by Pondicherry University in the form of university research fellowship (to SD).