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p38 MAPK pathways
Reproductive Toxicology 50 (2014) 108–116
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
BPA-induced apoptosis of rat Sertoli cells through Fas/FasL and JNKs/p38 MAPK pathways Suqin Qi 1 , Wenjuan Fu 1 , Chengmin Wang, Changjiang Liu, Chao Quan, Ansoumane Kourouma, Maosheng Yan, Tingting Yu, Peng Duan, Kedi Yang ∗ MOE Key Laboratory of Environment and Health, Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 May 2014 Received in revised form 7 October 2014 Accepted 14 October 2014 Available online 25 October 2014 Keywords: Bisphenol-A MAPK NF-B Fas/FasL Cells apoptosis
a b s t r a c t Bisphenol-A was examined for its effects on cultured Sertoli cells established from 18 to 22-day-old rat testes. Results indicated that exposure to BPA (0, 30, 50 and 70 M) decreased the cell viability in a concentration-dependent manner and induced cell apoptosis. Apoptosis-caused cell death was observed in cells exposed to 50 and 70 M BPA. The mRNA expressions of Fas, FasL and caspase-3 were all elevated, and the protein expressions of FasL and cleaved caspase-3 were also increased. In addition, levels of phosphorylation of JNKs/p38 MAPK were also increased and then activated JNKs/p38 MAPK up regulated target gene expressions, such as c-jun and CHOP. Translocation of NF-B into nuclei indicated the activation of NF-B after treatment with BPA. Taken together, observed results suggest that BPA induces apoptosis of Sertoli cells by the activation of JNKs/p38 MPAK and translocation of NF-B, and Fas/FasL system plays a critical role in the initiation of apoptosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Bisphenol A (BPA), the production of polycarbonate plastics and epoxy resins, is used to line metal cans and used in many plastic consumer products such as toys, water pipes, drinking containers, eyeglass lenses and dental monomers [1]. It has been shown that BPA could leach from food and beverage containers, some dental sealants and composites. Biomonitoring studies clearly prove that the general population is continually and inevitably exposed to BPA from various sources, and is at risk to unconjugated BPA from internal exposure [2]. It is reported that BPA can be detected in 92.6% urine samples of more than 2500 participants [3]. It is worth mentioning that exposure to BPA during prenatal and neonatal development is related to a wide variety of adverse effects, such as defects in male and female reproductive tracts [4], disruption of female meiosis [5], influence in the morphology of mammary [6], and changes in prostate sizes and weights [7]. In addition, BPA is also involved in apoptosis of several types of cells [8–10]. Many previous studies have focused on adverse effects of BPA using Sertoli cells. Sertoli cells, the only somatic cells found in the
∗ Corresponding author. Tel.: +86 27 83693897 fax: +86 2783692333 E-mail addresses: [email protected] (S. Qi), [email protected] (K. Yang). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.reprotox.2014.10.013 0890-6238/© 2014 Elsevier Inc. All rights reserved.
testicular seminiferous tubule of mammalian, play a critical role in the maintenance and control of spermatogenesis, such as the structural support, the synthesis of essential factors for developing germ cells, and the formation of blood–testis barrier [11]. Therefore, any agent that impairs the viability of Sertoli cells may have profound effects on spermatogenesis and production of sperm. Evidence from previous studies suggests a role in BPAinduced apoptosis of Sertoli cells. Results of Iida et al. confirm that BPA at concentrations of 150–200 M induces apoptosis of cultured rat Sertoli cells [8]. In addition, several reports have shown that BPA causes apoptosis through not only up-regulation of Fas/FasL signaling but also activation of the mitochondrial apoptotic pathway [12,13]. In another study, Qian et al. reveal that the CaM-CaMK␣-ERK axis might transmit apoptotic signals to mitochondria during BPA-induced apoptosis of Sertoli cells [14]. The apoptotic effect of BPA on Sertoli cells is gradually confirmed, however, the underlying mechanism is still unclear. The mitogen-activated protein kinase (MAPK) pathways play a crucial role in various cellular events such as cell proliferation, differentiation and death in eukaryotes from yeast to humans. The MAPKs can be grouped into three main families: ERKs (extracellular-signal-regulated kinases), JNKs (Jun amino-terminal kinases), and p38/SAPKs (stress-activated protein kinases) [15,16]. Several literatures have observed activations of the MAPK pathways in response to many reproductive toxicants. For example,
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Lee et al. find that 1,3-dinitrobenzene induces apoptosis via the JNK/MAPK pathway, whereas nonylphenol causes apoptosis via the ERK pathway [17,18]. Furthermore, BPA reduces Sertoli cells TF secretion through the ERK pathway which may impair spermatogenesis [19]. Our previous study clearly indicates that the Pten/AKT pathway is involved in BPA-induced apoptosis of Sertoli cells [20], however, whether the MAPK pathways are involved in this process remains unclear. In the present study, we extended these observations by investigating expressions of the MAPK pathways. Considering that the Fas/FasL and NF-B pathways are also involved in response to reproductive toxicants [21,22], elements of the Fas/FasL and NF-B pathways were also investigated. The aim of this study is to determine the cytotoxic effect of BPA on Sertoli cells and the molecular mechanism involved in apoptosis. Hereon, we report that BPA-induced rat Sertoli cell apoptosis is triggered by activations of the Fas/FasL and JNKs/p38 pathways and the translocation of NF-B. 2. Materials and methods 2.1. Animals 18–22-day-old male Sprague-Dawley (SD) rats were purchased from the Tongji Medical College Animal Laboratory (Wuhan, China) and raised in accordance with the Guide for the Care and Use of Laboratory Animals published by the Ministry of Health of People’s Republic of China.
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MTT (Sigma–Aldrich) solution was added into each well and incubated at 37 ◦ C for 4 h. Afterwards, formazan crystals were dissolved by 150 l DMSO and the absorbance was measured at 490 nm. Cellular viability (%) was calculated as follows: cellular viability (%) = (ODtreatment /ODcontrol ) × 100%. 2.4. Tunel assay for cell apoptosis To detect apoptosis, cells were grown on microscope slides and stained by the TUNEL technique using an in situ apoptosis detection kit (Roche applied science, Mannheim, Germany). In brief, after treatment with different concentrations of BPA (0, 30, 50, 70 M) for 24 h, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed with PBS for 3 times. Then the slides were incubated in 0.1% Triton X-100 for 2 min on ice and rinsed with PBS twice. A mixture of 5 l TdT (terminal deoxynucleotidyl transferase) enzyme solution and 45 l reaction buffer with nucleotide mixture were prepared in dark. 50 l mixture was added on each slide, which was incubated in a humidified chamber for 1 h at 37 ◦ C in dark. In order to eliminate the unbound fluorescence-dUTP, slides were washed with PBS and then immersed in DAPI for 5 min in dark. TUNEL assay was performed by laser scanning confocal microscope. Numbers of positive and total cells in the same area were counted in five random areas, and the result was presented as an average ratio of positive cell number out of the total cell number. Each random area included at least 100 cells. 2.5. RNA extraction and RT-PCR
2.2. Primary culture of Sertoli cells and cell treatment Primary culture of Sertoli cells was a serial enzymatic procedure that had been previously described [23] with modifications. In short, 18–22-day-old Sprague-Dawley rats were killed by cervical dislocation. Then testes were collected, excised rapidly, decapsulated, cut into small fragments and washed twice in D-Hanks’ balanced salt solution. The fragments from each rat were digested continuously with 3 ml 0.25% tryspin (Amresco, Solon, OH, USA) in a shaking water bath (37 ◦ C, 120 cycles/min) for 30 min and centrifuged at 1200 r/min for 10 min. Then the supernatant was discarded and the sediment was further digested with 2 ml 0.1% collagenase (type I, Promoter, Wuhan, Hubei, China) at 37 ◦ C for 20 min, followed by filtration through B-D Falcon cell strainers (nylon mesh size, 70 m) to remove peritubular cells and centrifugation at 800 r/min for 5 min. As described above, the supernatant was discarded and the sediment was washed extensively with highglucose-Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, Utah, USA). The final Sertoli cell suspension was supplemented with 20% fetal bovine serum (FBS) (Tianhang, Hangzhou, Zhejiang, China) and seeded in culture plates in a humidified atmosphere of 95% air–5% CO2 at 35 ◦ C for 24 h. Then these cells were extensively washed with 20 mM Tris–HCl (pH 7.4) for 5 min and the medium was renewed after serum starvation for 24 h at 2-day interval. Bisphenol-A (BPA) (DR, Augsburg, Germany, purity: 99.5%) was dissolved in dimethylsulfoxide (DMSO, Sigma–Aldrich, St. Louis, MO, USA) and diluted with culture medium to different concentrations (30, 50, 70 M). Then Sertoli cells were treated with BPA for 24 h. The DMSO working concentration in the medium was 0.1% (v/v). Cells were cultured with DMSO as a control group. 2.3. Cell proliferation assay Cell viability was measured by 3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazoli-um bromide (MTT) assay. Briefly, Sertoli cells were seeded into 96-well plates and exposed to vehicle or different concentrations (30, 50, 70, and 90 M) of BPA for 24 h. Then 20 l
Total RNA was extracted with Trizol regents (Invitrogen, Carlsbad, CA, USA) and the purity was determined by the Eppendorf BioPhotometer (Eppendorf, Germany), which showed an optical density ratio (OD260/280) of 1.8–2.0. Then cDNA was synthesized from 5 g total RNA using Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The RT-PCR was performed with an ABIPRISM® 7900 HT Sequence Detection System (Applied Biosystems, USA) using Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen, USA). Thermocycling was performed with the following four-step experimental procedures: 2-min UDG incubation at 50 ◦ C; 2-min initial denaturation at 95 ◦ C; 15-s denaturation at 95 ◦ C and 1-min elongation at 60 ◦ C for 40 cycles. The primer sequences shown in Table 1 were synthesized by the Invitrogen Corp (Shanghai, China). -Actin was used in parallel as an internal control for each run. 2.6. Western blotting Total proteins of Sertoli cells were extracted with the Cell Lysis Buffer for Western and IP (Beyotime Biotech Inc., China) to detect JNK, p-JNK, p38, p-p38, FasL and cleaved caspase-3. For measurement of NF-B p65, the cytoplasmic and nuclear protein was isolated from Sertoli cells using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech Inc., China). Then protein samples were measured by BCA Protein Assay Kit (Beyotime Biotech Inc., China) and diluted with 6× SDS PAGE loading buffer (Beyotime Biotech Inc., China). Equal amounts of protein was separated by SDS-polyacrylamide gels and transferred electrophoretically onto a nitrocellulose (NC) membrane. Then membranes were incubated at 4 ◦ C overnight with anti-JNK, anti-phospho-JNK, anti-p38, antiphospho-p38, anti-NF-B and anti-cleaved-caspase-3 antibodies at a dilution of 1:1000 (Cell Signaling Technology, Inc., USA); and anti-FasL antibody (bs-0216R, Beijing Biosynthesis Biotechnology, China) at a dilution of 1:300. The following day, membranes were rinsed and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) at 37 ◦ C for 1 h. The ECL Western
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Table 1 Description of primers used in this study. Primers
Accession number
Official symbol
Fas
NM 139194.2
Fas
FasL
NM 012908.1
Faslg
Caspase-3
NM 012922.2
Casp3
ASK1
NM 001277694.1
Map3K5
c-jun
NM 021835.3
Jun
CHOP
NM 001109986.1
Ddit3
-Actin
NM 031144.3
Actb
blotting system was used to detect immune-reactive proteins. The densitometric analysis of immunoblot was performed with Quantity One Image Analysis Software.
Type
Primers sequence
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5 -AAATGAAAGCCAACTGCATCGAC-3 5 -ATTGGACCCTCGCTGAGCAC-3 5 -GGA ATG GGA AGA CAC ATA TGG AAC TCC-3 5 -CAT ATC TGG CCA GTA GTG CAG TAA TTC-3 5 -AGT TGG ACC CAC CTT GTG AG-3 5 -AGT CTG CAG CTC CTC CAC AT-3 5 -CTG TGC TAA TGA CCT GCT TGT TG-3’ 5 -TAC CCG AGA GTT TGG GCT GT-3 5 -ACT CAG TTC TTG TGC CCC AA-3 5 -CGC ACG AAG CCT TCG GCG AA-3 5 -CCA GCA GAG GTC ACA AGC AC-3 5 -CGC ACT GAC CAC TCT GTT TC-3 5 -CGT TGA CAT CCG TAA AGA C-3 5 -TGG AAG GTG GAC AGT GAG-3
75.91% Sertoli cells survived. Therefore, concentrations of BPA at 30, 50, and 70 M were used in subsequent experiments. 3.2. Apoptotic effect of BPA on rat Sertoli cells
2.7. Detection of NF-ÄB activation by laser scanning confocal microscope Sertoli cells were treated according to the instruction of Cellular NF-B Translocation Kit (Beyotime Biotech Inc., China). In brief, cells were washed and fixed, followed by incubation with blocking buffer for 1 h to avoid nonspecific binding. Then cells were incubated with primary NF-B p65 antibody at 4 ◦ C overnight, followed by incubation with a secondary antibody conjugated with Cy3 for 1 h and DAPI for 5 min. p65 protein and nuclei were immunofluorescence-labeled with red and blue, respectively, and could be simultaneously observed by laser scanning confocal microscope at an excitation wavelength of 350 nm for DAPI and 540 nm for Cy3. 2.8. Immunocytochemistry Sertoli cells were seeded on axenic glass coverslips in 6-well culture plates at a density of 1 × 106 cells/ml, and treated with different concentrations of BPA (0, 30, 50, 70 M). After 24-h treatment, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Then cells were blocked by 0.3% H2 O2 and 5% bovine serum albumin and incubated with PBS (negative control) or procaspase-3 antibody (Cell Signaling Technology, 1:1000 dilution) at 4 ◦ C overnight, followed by incubation with the secondary antibody conjugated with HRP and DAB staining. The reaction products were observed with FSX-100 microscope camera system.
To investigate whether BPA decreased cell viability by the induction of apoptosis, TUNEL staining was performed in our study. TUNEL staining showed that the number of apoptotic cells was significantly increased in 50 and 70 M BPA groups, however, that in 30 M BPA group was not changed as compared with the control group (Fig. 2A–D). Similarly, the statistical graph of apoptosis abundance (Fig. 2E) showed that BPA caused an increase of apoptotic cells in 50 and 70 M BPA groups in comparison with the control group (P < 0.05). This was consistent with results of our previous study that confirmed that 50 and 70 M BPA induced apoptosis of rat Sertoli cells [20]. 3.3. BPA activated the Fas/FasL signaling pathway and caspase Fas, FasL and caspase-3 were determined by RT-PCR and western blotting (Fig. 3). As shown in Fig. 3A, after treatment with BPA for 24 h, the mRNA expression of Fas was increased in 30 and 50 M BPA groups by 54.0% and 69.0%, respectively. BPA also caused a 7.27- and 6.66-fold increase in FasL mRNA level in 50 and 70 M group, respectively. The mRNA level of caspase-3
2.9. Statistical analysis Quantitative results were represented as mean ± SD. Significance was assessed by one-way analysis of variance (ANOVA) using the SPSS statistical package 12.0 (SPSS, Chicago, IL, USA). A difference at P < 0.05 level was considered statistically significant. 3. Results 3.1. Effect of BPA on the viability of rat Sertoli cells As shown in Fig. 1, the viability of Sertoli cells was decreased in 50, 70 and 90 M BPA as compared with the control group (P < 0.05). In 90 M BPA group, cell viability was 70.54% of that in the control group, while in 70 M and lower concentration groups, above
Fig. 1. Effects of BPA on the viability of rat Sertoli cells using MTT assay. Cells were treated with 0, 30, 50, 70, and 90 M for 24 h, and the optical density (OD) was detected with MTT assay. Data were presented with as mean ± SD of three independent experiments performed in triplicate. Significant difference: *P < 0.05, compared with control group.
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Fig. 2. BPA induced apoptosis of rat Sertoli cells. After cells exposed to BPA for 24 h, representative images of TUNEL staining (A–D) and numbers of apoptotic cells were counted (E) according to TUNEL assay. (A) Control group; (B) 30 M BPA; (C) 50 M BPA; (D) 70 M BPA. Significant difference: *P < 0.05, compared with control group.
was significantly increased in all BPA-treated groups in comparison with the control (P < 0.05). In addition, protein expressions of FasL and cleaved caspase-3 were determined by western blotting (Fig. 3B–D). Compared with the control group, FasL protein expression was increased by 50% in 70 M group. Moreover, BPA
elevated the protein level of cleaved caspase-3 in 30 (1.54-fold), 50 (2.62-fold) and 70 M group (4.23-fold). As shown in Fig. 4, the immunocytochemical staining analysis exhibited a concentrationdependent decrease in procaspase-3 expression. The result of negative control incubated with PBS was not shown.
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Fig. 3. BPA-induced activation of Fas/FasL signaling pathway. (A) RT-PCR analysis of Fas, FasL and caspase-3 mRNA expressions in rat Sertoli cells after various concentrations of BPA-treatment. (B–D) Western blotting analysis of FasL and cleaved caspase-3 protein expressions in Sertoli cells after exposure to BPA. Significant difference: *P < 0.05 compared with control group.
3.4. BPA activated the JNK/p38 MAPK pathway To assess effects of BPA on the MAPK pathways, molecular elements of both JNK and p38 pathways were examined by RT-PCR and Western blotting. In the present study, mRNA expressions of ASK1, c-jun and CHOP and protein expressions of phospho-JNK and p38 were detected. As exhibited in Fig. 5A, ASK1 mRNA expression was significantly increased by 64% in 70 M group. c-jun level was also elevated after BPA treatment, with maximum effects observed in 70 M group (1.29-fold). BPA also induced the mRNA expression of CHOP in all treated groups (P < 0.05). In addition, as seen in Fig. 5C and D, the protein level of p-JNK was significantly increased
by 54.6% in 70 M group as compared with the control (P < 0.05). The protein level of p-p38 had a 2.55- and 1.93-fold increase in 50 and 70 M groups, respectively. However, protein expressions of total JNK and p38 were not affected after BPA treatment (data were not shown). 3.5. Effect of BPA on NF-ÄB activation in rat Sertoli cells Laser scanning confocal microscopy was used to assess the nuclear translocation of NF-B in rat Sertoli cells. As exhibited in Fig. 6A, p65 and nuclei showed red and blue fluorescence, respectively. In this study, the activation of NF-B was showed a
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Fig. 4. Effect of BPA on procaspase-3 expressions in rat Sertoli cells was detected by immunocytochemistry (200×). As is shown in control group (A), procaspase-3 was highly expressed in the cytoplasm of Sertoli cells. However, after treatment with BPA for 24 h, the positive expressions of procaspase-3 were decreased with the increasing doses (B–D). (A) Control group; (B) 30 M BPA; (C) 50 M BPA; (D) 70 M BPA. The data of negative control was not shown.
concentration-dependent increase after BPA treatment. Similarly, the nuclear protein level of NF-B p65 was increased in all BPAtreated groups in comparison with the control (P < 0.05) (Fig. 6B and C). 4. Discussion The present study demonstrated that BPA was toxic to rat Sertoli cells, resulting in the decrease in cell viability and apoptosis induction. Furthermore, activations of Fas/FasL, JNKs/p38 MAPK and the translocation of NF-B might involve in the apoptosis of Sertoli cells. BPA is reported to cause reproductive toxicity and affect cellular development. Previous studies have demonstrated that prenatal and neonatal exposure to BPA at <50 mg/kg/day can induce adverse effects in male reproductive systems, including decrease of sperm count, motility and changes of sperm morphology abnormalities [24,25]. Moreover, neonatal and pubertal exposure to BPA could impair the fertility of male rats and disrupt the BTB integrity via declining levels of Sertoli cell junction proteins [26,27]. However, potential mechanisms of toxic effects of BPA on Sertoli cells are still unclear. Therefore, for the critical role of Sertoli cells in spermatogenesis, we hypothesized that BPA might have toxic effects on Sertoli cells and special signaling pathways might play an important role in apoptosis. In the present study, BPA significantly decreased the viability of Sertoli cells at over 50 M and similar results were confirmed by TUNEL assay. These observations are in agreement with a previous
report that 20 or 150 M BPA significantly affects the viability of Sertoli cells for 48 h or 16 h [8,14]. Compared with what Iida and Qian have reported, concentrations of BPA used in our study were between them. We suppose that this disparity may result from different tested subjects. However, considering characteristics of BPA, low-concentration exposure and long-term effects, whether less than 50 M exposure for 48 h or more has adverse effects on Sertoli cells deserves to be explored in our future study. In FasL-induced spermatogenic cell death, it is generally accepted that FasL from Sertoli cells kills spermatogenic cells by engaging Fas receptors present on them [28,29]. However, it is reported that Sertoli cells also express Fas and germ cells express FasL [30]. Li et al. have reported that high-dose BPA induced apoptosis of Leydig and germ cells in mouse testes through up-regulated Fas and FasL and active caspase-3 [13]. Our results showed that BPA exposure activated the Fas/FasL pathway. Once the Fas system is activated, the effector caspase, caspase-3 will be cleaved and subsequently activated, which finally commits cells into apoptosis [31]. Our data showed that the mRNA expression of caspase-3 was increased, and even the protein expression of cleaved caspase-3 was also enhanced. Moreover, the expression of procaspase-3 in Sertoli cells was decreased. Observed results indicated the activation of caspase-3. Therefore, we suppose that the apoptotic effect of BPA on Sertoli cells could be related to the induction of caspase activation. NF-B, a dimmer of protein components (p65/p50), binds to inhibitory protein IB in unstimulated cells. Stimulation by several extracellular signals leads to the degradation of IB. Then
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Fig. 5. BPA-induced activation of JNKs/p38 MAPK pathway. (A and B) The mRNA expressions of ASK1, c-jun, and CHOP in Sertoli cells exposure to BPA for 24 h. (C) Western blotting analysis of JNK, p-JNK, p38 and p-p38 protein expressions in rat Sertoli cells exposure to BPA for 24 h. (D) Quantitative analysis of the immunoreactive p-JNK and p-p38. *P < 0.05 compared with control group.
freed NF-B dimmers translocate to nuclei and bind to specific sequences in promoter or enhancer regions of target genes [32]. In rat testes, the NF-B complex of p65 and p50 is found to express in nuclei of Sertoli cells at all stages of spermatogenesis [33].
Interestingly, NF-B effects in different promotes or inhibits cific cell type and
can exert both pro- and anti-apoptotic cell types. However, whether NF-B apoptosis seems to depend on the spethe type of inducer [34]. Our present
Fig. 6. BPA-induced activation of NF-B. (A) Immunofluorescent double staining of NF-B p65 (red) and nuclei (blue) detected by laser scanning confocal microscope (400×). NF-B p65 was not activated in the cytoplasm as is shown in control group. However, the translocation of NF-B p65 into nucleus was enhanced as the increased dosing of BPA. (B) Protein from nuclear extracts was used in western blotting to detect NF-B p65. (C) Quantitative analysis of the immunoreactive NF-B p65. Significant difference: *P < 0.05 versus control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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study showed that BPA exposure induced NF-B activation and translocation into nuclei. There is accumulating evidence that activated NF-B can sensitize some cells to apoptosis-inducing agents that act through or cooperate with the Fas pathway due to the ability of NF-B to activate genes encoding FasL or Fas [34]. For example, FasL and Fas are both up-regulated by NF-B in different cell types [35–38]. Recently studies have reported that the activation of NF-B is associated with the germ cell apoptosis [39,40]. Thus, in the present study, we speculate that NF-B can induce Fas-FasL expression and promote cell apoptosis through the Fas/FasL pathway in vitro. To further confirm the function of NF-B in the apoptosis of Sertoli cells, PDTC, an inhibitor of NF-B, perhaps should be applied in our future experiments. The MAPK pathways regulate diverse processes such as proliferation, differentiation and apoptosis. In mammals, MAPK can be mainly grouped into three families: ERKs, JNKs and p38/SAPKs. Each cascade is initiated by specific extracellular stimuli and a particular MAPK is activated after successive activations of MAP3K and MAP2K [41]. ASK1, a member of MAP3K family, activates the MAP2K4/7 (MKK4/7)-JNK pathway and MAP2K3/6 (MKK3/6)-p38 pathway [42]. Then activated JNKs and p38 translocate into nuclei and mediate expressions of target genes, such as c-jun and CHOP [43,44]. It is generally accepted that JNKs and p38 are associated with cells apoptosis, especially in Sertoli cells exposed to reproductive toxicants. Lee et al. have reported that 1,3-DNB induces apoptosis of TM4 mouse Sertoli cells via activating the JNK signaling pathway [17]. In addition, the inhibitory effect of cadmium chloride to Sertoli cells is obviously reduced after treatment with a p38 inhibitor (SB202190), and results confirm that cadmium exerts apoptotic effects on piglet Sertoli cells through the p38 MAPK pathway [45]. In order to elucidate whether the JNKs/p38 pathway was involved in BPA-induced apoptosis of rat Sertoli cells, we investigated mRNA expressions of ASK1, c-jun, CHOP and protein expressions of phospho-JNK and -p38. Our results showed that the mRNA expression of ASK1 was elevated, indicating that BPA exposure enhanced the transcription of MAP3K, which might initiate the activation of MAPK cascade. Moreover, increased protein expressions of phospho-JNK and -p38 confirmed that the JNKs/p38 pathway was activated and translocated into nuclei to mediate target genes, including c-jun and CHOP. Thus, we speculate that BPA exposure induces the activation of JNKs/p38 MAPK and the translocation into nuclei to mediate target genes expression and apoptosis. c-jun, as a target gene of JNKs, might lead to the activation of AP1 and expressions of Fas/FasL signaling pathway-related proteins. Then binding of FasL and Fas can trigger apoptosis via activating downstream effector caspase-3 [43]. Meanwhile, CHOP, as a target gene of p38, is closely related to ER stress-induced apoptosis [46]. However, to confirm roles of JNKs/p38 MAPK in BPA-induced apoptosis, the antagonist or crosstalk method should be performed in our future study. Taken together, we conclude that the Fas/FasL, JNKs/p38 MAPK and NF-B are activated in BPA-induced apoptosis of rat Sertoli cells. Fas/FasL signaling pathway plays an important role in apoptosis, and JNKs/p38 MAPK and NF-B may promote cell apoptosis after BPA exposure. However, considering the complicated crosstalk among different signaling pathways, the antagonist and crosstalk method will be used in our future study to clearly elucidate apoptotic effects of BPA on Sertoli cells. Moreover, considering limitations of in vitro study and critical roles of Sertoli cells in spermatogenesis, whether the same doses of BPA could impair spermatogenesis needs further confirmation by in vivo study.
Conflict of interest The authors declare that there are no conflicts of interest.
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Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81172623 and 81372960). References [1] http://www.bisphenol-a.org [2] Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reprod Toxicol 2007;24(2):139–77. [3] Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004. Environ Health Perspect 2008;116(1):39–44. [4] Richter CA, Birnbaum LS, Farabollini F, Newbold RR, Rubin BS, Talsness CE, et al. In vivo effects of bisphenol A in laboratory rodent studies. Reprod Toxicol 2007;24(2):199–224. [5] Hunt PA, Koehler KE, Susiarjo M, Hodges CA, Ilagan A, Voigt RC, et al. Bisphenol A exposure causes meiotic aneuploidy in the female mouse. Curr Biol 2003;13(7):546–53. [6] Vandenberg LN, Maffini MV, Schaeberle CM, Ucci AA, Sonnenschein C, Rubin BS, et al. Perinatal exposure to the xenoestrogen bisphenol-A induces mammary intraductal hyperplasia in adult CD-1 mice. Reprod Toxicol 2008;26(3–4):210–9. [7] Vom SF, Nagel SC, Timms BG, Welshons WV. Implications for human health of the extensive bisphenol A literature showing adverse effects at low doses: a response to attempts to mislead the public. Toxicology 2005;212(2–3):244–52, 253–4. [8] Iida H, Maehara K, Doiguchi M, Mori T, Yamada F. Bisphenol A-induced apoptosis of cultured rat Sertoli cells. Reprod Toxicol 2003;17(4):457–64. [9] Lee S, Suk K, Kim IK, Jang IS, Park JW, Johnson VJ, et al. Signaling pathways of bisphenol A-induced apoptosis in hippocampal neuronal cells: role of calcium-induced reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-kappaB. J Neurosci Res 2008;86(13):2932–42. [10] Xu J, Osuga Y, Yano T, Morita Y, Tang X, Fujiwara T, et al. Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells. Biochem Biophys Res Commun 2002;292(2):456–62. [11] Griswold MD. The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol 1998;9(4):411–6. [12] Wang Q, Zhao XF, Ji YL, Wang H, Liu P, Zhang C, et al. Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes. Toxicol Lett 2010;199(2):129–35. [13] Li YJ, Song TB, Cai YY, Zhou JS, Song X, Zhao X, et al. Bisphenol A exposure induces apoptosis and upregulation of Fas/FasL and caspase-3 expression in the testes of mice. Toxicol Sci 2009;108(2):427–36. [14] Qian W, Zhu J, Mao C, Liu J, Wang Y, Wang Q, et al. Involvement of CaM-CaMKII-ERK in bisphenol A-induced Sertoli cell apoptosis. Toxicology 2014;324C:27–34. [15] Qi M, Elion EA. MAP kinase pathways. J Cell Sci 2005;118(16):3569–72. [16] Pearson G, Robinson F, Beers GT, Xu BE, Karandikar M, Berman K, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22(2):153–83. [17] Lee YS, Yoon HJ, Oh JH, Park HJ, Lee EH, Song CW, et al. 1,3-Dinitrobenzene induces apoptosis in TM4 mouse Sertoli cells: involvement of the c-jun N-terminal kinase (JNK) MAPK pathway. Toxicol Lett 2009;189(2):145–51. [18] Choi MS, Park HJ, Oh JH, Lee EH, Park SM, Yoon S. Nonylphenol-induced apoptotic cell death in mouse TM4 Sertoli cells via the generation of reactive oxygen species and activation of the ERK signaling pathway. J Appl Toxicol 2014;34(6):628–36. [19] Izumi Y, Yamaguchi K, Ishikawa T, Ando M, Chiba K, Hashimoto H, et al. Molecular changes induced by bisphenol-A in rat Sertoli cell culture. Syst Biol Reprod Med 2011;57(5):228–32. [20] Wang C, Fu W, Quan C, Yan M, Liu C, Qi S, et al. The role of Pten/Akt signaling pathway involved in BPA-induced apoptosis of rat Sertoli cells. Environ Toxicol 2014, http://dx.doi.org/10.1002/tox.21958. [21] Rasoulpour RJ, Boekelheide K. NF-kappaB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate. Biol Reprod 2005;72(2):479–86. [22] Shi YQ, Wang YP, Song Y, Li HW, Liu CJ, Wu ZG, et al. p,p -DDE induces testicular apoptosis in prepubertal rats via the Fas/FasL pathway. Toxicol Lett 2010;193(1):79–85. [23] Mather JP, Attie KM, Woodruff TK, Rice GC, Phillips DM. Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis. Endocrinology 1990;127(6):3206–14. [24] Dobrzynska MM, Jankowska-Steifer EA, Tyrkiel EJ, Gajowik A, Radzikowska J, Pachocki KA. Comparison of the effects of bisphenol A alone and in a combination with X-irradiation on sperm count and quality in male adult and pubescent mice. Environ Toxicol 2014;29(11):1301–13.
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
BPA-induced apoptosis of rat Sertoli cells through Fas/FasL and JNKs/p38 MAPK pathways Suqin Qi 1 , Wenjuan Fu 1 , Chengmin Wang, Changjiang Liu, Chao Quan, Ansoumane Kourouma, Maosheng Yan, Tingting Yu, Peng Duan, Kedi Yang ∗ MOE Key Laboratory of Environment and Health, Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 May 2014 Received in revised form 7 October 2014 Accepted 14 October 2014 Available online 25 October 2014 Keywords: Bisphenol-A MAPK NF-B Fas/FasL Cells apoptosis
a b s t r a c t Bisphenol-A was examined for its effects on cultured Sertoli cells established from 18 to 22-day-old rat testes. Results indicated that exposure to BPA (0, 30, 50 and 70 M) decreased the cell viability in a concentration-dependent manner and induced cell apoptosis. Apoptosis-caused cell death was observed in cells exposed to 50 and 70 M BPA. The mRNA expressions of Fas, FasL and caspase-3 were all elevated, and the protein expressions of FasL and cleaved caspase-3 were also increased. In addition, levels of phosphorylation of JNKs/p38 MAPK were also increased and then activated JNKs/p38 MAPK up regulated target gene expressions, such as c-jun and CHOP. Translocation of NF-B into nuclei indicated the activation of NF-B after treatment with BPA. Taken together, observed results suggest that BPA induces apoptosis of Sertoli cells by the activation of JNKs/p38 MPAK and translocation of NF-B, and Fas/FasL system plays a critical role in the initiation of apoptosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Bisphenol A (BPA), the production of polycarbonate plastics and epoxy resins, is used to line metal cans and used in many plastic consumer products such as toys, water pipes, drinking containers, eyeglass lenses and dental monomers [1]. It has been shown that BPA could leach from food and beverage containers, some dental sealants and composites. Biomonitoring studies clearly prove that the general population is continually and inevitably exposed to BPA from various sources, and is at risk to unconjugated BPA from internal exposure [2]. It is reported that BPA can be detected in 92.6% urine samples of more than 2500 participants [3]. It is worth mentioning that exposure to BPA during prenatal and neonatal development is related to a wide variety of adverse effects, such as defects in male and female reproductive tracts [4], disruption of female meiosis [5], influence in the morphology of mammary [6], and changes in prostate sizes and weights [7]. In addition, BPA is also involved in apoptosis of several types of cells [8–10]. Many previous studies have focused on adverse effects of BPA using Sertoli cells. Sertoli cells, the only somatic cells found in the
∗ Corresponding author. Tel.: +86 27 83693897 fax: +86 2783692333 E-mail addresses: [email protected] (S. Qi), [email protected] (K. Yang). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.reprotox.2014.10.013 0890-6238/© 2014 Elsevier Inc. All rights reserved.
testicular seminiferous tubule of mammalian, play a critical role in the maintenance and control of spermatogenesis, such as the structural support, the synthesis of essential factors for developing germ cells, and the formation of blood–testis barrier [11]. Therefore, any agent that impairs the viability of Sertoli cells may have profound effects on spermatogenesis and production of sperm. Evidence from previous studies suggests a role in BPAinduced apoptosis of Sertoli cells. Results of Iida et al. confirm that BPA at concentrations of 150–200 M induces apoptosis of cultured rat Sertoli cells [8]. In addition, several reports have shown that BPA causes apoptosis through not only up-regulation of Fas/FasL signaling but also activation of the mitochondrial apoptotic pathway [12,13]. In another study, Qian et al. reveal that the CaM-CaMK␣-ERK axis might transmit apoptotic signals to mitochondria during BPA-induced apoptosis of Sertoli cells [14]. The apoptotic effect of BPA on Sertoli cells is gradually confirmed, however, the underlying mechanism is still unclear. The mitogen-activated protein kinase (MAPK) pathways play a crucial role in various cellular events such as cell proliferation, differentiation and death in eukaryotes from yeast to humans. The MAPKs can be grouped into three main families: ERKs (extracellular-signal-regulated kinases), JNKs (Jun amino-terminal kinases), and p38/SAPKs (stress-activated protein kinases) [15,16]. Several literatures have observed activations of the MAPK pathways in response to many reproductive toxicants. For example,
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Lee et al. find that 1,3-dinitrobenzene induces apoptosis via the JNK/MAPK pathway, whereas nonylphenol causes apoptosis via the ERK pathway [17,18]. Furthermore, BPA reduces Sertoli cells TF secretion through the ERK pathway which may impair spermatogenesis [19]. Our previous study clearly indicates that the Pten/AKT pathway is involved in BPA-induced apoptosis of Sertoli cells [20], however, whether the MAPK pathways are involved in this process remains unclear. In the present study, we extended these observations by investigating expressions of the MAPK pathways. Considering that the Fas/FasL and NF-B pathways are also involved in response to reproductive toxicants [21,22], elements of the Fas/FasL and NF-B pathways were also investigated. The aim of this study is to determine the cytotoxic effect of BPA on Sertoli cells and the molecular mechanism involved in apoptosis. Hereon, we report that BPA-induced rat Sertoli cell apoptosis is triggered by activations of the Fas/FasL and JNKs/p38 pathways and the translocation of NF-B. 2. Materials and methods 2.1. Animals 18–22-day-old male Sprague-Dawley (SD) rats were purchased from the Tongji Medical College Animal Laboratory (Wuhan, China) and raised in accordance with the Guide for the Care and Use of Laboratory Animals published by the Ministry of Health of People’s Republic of China.
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MTT (Sigma–Aldrich) solution was added into each well and incubated at 37 ◦ C for 4 h. Afterwards, formazan crystals were dissolved by 150 l DMSO and the absorbance was measured at 490 nm. Cellular viability (%) was calculated as follows: cellular viability (%) = (ODtreatment /ODcontrol ) × 100%. 2.4. Tunel assay for cell apoptosis To detect apoptosis, cells were grown on microscope slides and stained by the TUNEL technique using an in situ apoptosis detection kit (Roche applied science, Mannheim, Germany). In brief, after treatment with different concentrations of BPA (0, 30, 50, 70 M) for 24 h, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed with PBS for 3 times. Then the slides were incubated in 0.1% Triton X-100 for 2 min on ice and rinsed with PBS twice. A mixture of 5 l TdT (terminal deoxynucleotidyl transferase) enzyme solution and 45 l reaction buffer with nucleotide mixture were prepared in dark. 50 l mixture was added on each slide, which was incubated in a humidified chamber for 1 h at 37 ◦ C in dark. In order to eliminate the unbound fluorescence-dUTP, slides were washed with PBS and then immersed in DAPI for 5 min in dark. TUNEL assay was performed by laser scanning confocal microscope. Numbers of positive and total cells in the same area were counted in five random areas, and the result was presented as an average ratio of positive cell number out of the total cell number. Each random area included at least 100 cells. 2.5. RNA extraction and RT-PCR
2.2. Primary culture of Sertoli cells and cell treatment Primary culture of Sertoli cells was a serial enzymatic procedure that had been previously described [23] with modifications. In short, 18–22-day-old Sprague-Dawley rats were killed by cervical dislocation. Then testes were collected, excised rapidly, decapsulated, cut into small fragments and washed twice in D-Hanks’ balanced salt solution. The fragments from each rat were digested continuously with 3 ml 0.25% tryspin (Amresco, Solon, OH, USA) in a shaking water bath (37 ◦ C, 120 cycles/min) for 30 min and centrifuged at 1200 r/min for 10 min. Then the supernatant was discarded and the sediment was further digested with 2 ml 0.1% collagenase (type I, Promoter, Wuhan, Hubei, China) at 37 ◦ C for 20 min, followed by filtration through B-D Falcon cell strainers (nylon mesh size, 70 m) to remove peritubular cells and centrifugation at 800 r/min for 5 min. As described above, the supernatant was discarded and the sediment was washed extensively with highglucose-Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, Utah, USA). The final Sertoli cell suspension was supplemented with 20% fetal bovine serum (FBS) (Tianhang, Hangzhou, Zhejiang, China) and seeded in culture plates in a humidified atmosphere of 95% air–5% CO2 at 35 ◦ C for 24 h. Then these cells were extensively washed with 20 mM Tris–HCl (pH 7.4) for 5 min and the medium was renewed after serum starvation for 24 h at 2-day interval. Bisphenol-A (BPA) (DR, Augsburg, Germany, purity: 99.5%) was dissolved in dimethylsulfoxide (DMSO, Sigma–Aldrich, St. Louis, MO, USA) and diluted with culture medium to different concentrations (30, 50, 70 M). Then Sertoli cells were treated with BPA for 24 h. The DMSO working concentration in the medium was 0.1% (v/v). Cells were cultured with DMSO as a control group. 2.3. Cell proliferation assay Cell viability was measured by 3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazoli-um bromide (MTT) assay. Briefly, Sertoli cells were seeded into 96-well plates and exposed to vehicle or different concentrations (30, 50, 70, and 90 M) of BPA for 24 h. Then 20 l
Total RNA was extracted with Trizol regents (Invitrogen, Carlsbad, CA, USA) and the purity was determined by the Eppendorf BioPhotometer (Eppendorf, Germany), which showed an optical density ratio (OD260/280) of 1.8–2.0. Then cDNA was synthesized from 5 g total RNA using Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The RT-PCR was performed with an ABIPRISM® 7900 HT Sequence Detection System (Applied Biosystems, USA) using Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen, USA). Thermocycling was performed with the following four-step experimental procedures: 2-min UDG incubation at 50 ◦ C; 2-min initial denaturation at 95 ◦ C; 15-s denaturation at 95 ◦ C and 1-min elongation at 60 ◦ C for 40 cycles. The primer sequences shown in Table 1 were synthesized by the Invitrogen Corp (Shanghai, China). -Actin was used in parallel as an internal control for each run. 2.6. Western blotting Total proteins of Sertoli cells were extracted with the Cell Lysis Buffer for Western and IP (Beyotime Biotech Inc., China) to detect JNK, p-JNK, p38, p-p38, FasL and cleaved caspase-3. For measurement of NF-B p65, the cytoplasmic and nuclear protein was isolated from Sertoli cells using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech Inc., China). Then protein samples were measured by BCA Protein Assay Kit (Beyotime Biotech Inc., China) and diluted with 6× SDS PAGE loading buffer (Beyotime Biotech Inc., China). Equal amounts of protein was separated by SDS-polyacrylamide gels and transferred electrophoretically onto a nitrocellulose (NC) membrane. Then membranes were incubated at 4 ◦ C overnight with anti-JNK, anti-phospho-JNK, anti-p38, antiphospho-p38, anti-NF-B and anti-cleaved-caspase-3 antibodies at a dilution of 1:1000 (Cell Signaling Technology, Inc., USA); and anti-FasL antibody (bs-0216R, Beijing Biosynthesis Biotechnology, China) at a dilution of 1:300. The following day, membranes were rinsed and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) at 37 ◦ C for 1 h. The ECL Western
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Table 1 Description of primers used in this study. Primers
Accession number
Official symbol
Fas
NM 139194.2
Fas
FasL
NM 012908.1
Faslg
Caspase-3
NM 012922.2
Casp3
ASK1
NM 001277694.1
Map3K5
c-jun
NM 021835.3
Jun
CHOP
NM 001109986.1
Ddit3
-Actin
NM 031144.3
Actb
blotting system was used to detect immune-reactive proteins. The densitometric analysis of immunoblot was performed with Quantity One Image Analysis Software.
Type
Primers sequence
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5 -AAATGAAAGCCAACTGCATCGAC-3 5 -ATTGGACCCTCGCTGAGCAC-3 5 -GGA ATG GGA AGA CAC ATA TGG AAC TCC-3 5 -CAT ATC TGG CCA GTA GTG CAG TAA TTC-3 5 -AGT TGG ACC CAC CTT GTG AG-3 5 -AGT CTG CAG CTC CTC CAC AT-3 5 -CTG TGC TAA TGA CCT GCT TGT TG-3’ 5 -TAC CCG AGA GTT TGG GCT GT-3 5 -ACT CAG TTC TTG TGC CCC AA-3 5 -CGC ACG AAG CCT TCG GCG AA-3 5 -CCA GCA GAG GTC ACA AGC AC-3 5 -CGC ACT GAC CAC TCT GTT TC-3 5 -CGT TGA CAT CCG TAA AGA C-3 5 -TGG AAG GTG GAC AGT GAG-3
75.91% Sertoli cells survived. Therefore, concentrations of BPA at 30, 50, and 70 M were used in subsequent experiments. 3.2. Apoptotic effect of BPA on rat Sertoli cells
2.7. Detection of NF-ÄB activation by laser scanning confocal microscope Sertoli cells were treated according to the instruction of Cellular NF-B Translocation Kit (Beyotime Biotech Inc., China). In brief, cells were washed and fixed, followed by incubation with blocking buffer for 1 h to avoid nonspecific binding. Then cells were incubated with primary NF-B p65 antibody at 4 ◦ C overnight, followed by incubation with a secondary antibody conjugated with Cy3 for 1 h and DAPI for 5 min. p65 protein and nuclei were immunofluorescence-labeled with red and blue, respectively, and could be simultaneously observed by laser scanning confocal microscope at an excitation wavelength of 350 nm for DAPI and 540 nm for Cy3. 2.8. Immunocytochemistry Sertoli cells were seeded on axenic glass coverslips in 6-well culture plates at a density of 1 × 106 cells/ml, and treated with different concentrations of BPA (0, 30, 50, 70 M). After 24-h treatment, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Then cells were blocked by 0.3% H2 O2 and 5% bovine serum albumin and incubated with PBS (negative control) or procaspase-3 antibody (Cell Signaling Technology, 1:1000 dilution) at 4 ◦ C overnight, followed by incubation with the secondary antibody conjugated with HRP and DAB staining. The reaction products were observed with FSX-100 microscope camera system.
To investigate whether BPA decreased cell viability by the induction of apoptosis, TUNEL staining was performed in our study. TUNEL staining showed that the number of apoptotic cells was significantly increased in 50 and 70 M BPA groups, however, that in 30 M BPA group was not changed as compared with the control group (Fig. 2A–D). Similarly, the statistical graph of apoptosis abundance (Fig. 2E) showed that BPA caused an increase of apoptotic cells in 50 and 70 M BPA groups in comparison with the control group (P < 0.05). This was consistent with results of our previous study that confirmed that 50 and 70 M BPA induced apoptosis of rat Sertoli cells [20]. 3.3. BPA activated the Fas/FasL signaling pathway and caspase Fas, FasL and caspase-3 were determined by RT-PCR and western blotting (Fig. 3). As shown in Fig. 3A, after treatment with BPA for 24 h, the mRNA expression of Fas was increased in 30 and 50 M BPA groups by 54.0% and 69.0%, respectively. BPA also caused a 7.27- and 6.66-fold increase in FasL mRNA level in 50 and 70 M group, respectively. The mRNA level of caspase-3
2.9. Statistical analysis Quantitative results were represented as mean ± SD. Significance was assessed by one-way analysis of variance (ANOVA) using the SPSS statistical package 12.0 (SPSS, Chicago, IL, USA). A difference at P < 0.05 level was considered statistically significant. 3. Results 3.1. Effect of BPA on the viability of rat Sertoli cells As shown in Fig. 1, the viability of Sertoli cells was decreased in 50, 70 and 90 M BPA as compared with the control group (P < 0.05). In 90 M BPA group, cell viability was 70.54% of that in the control group, while in 70 M and lower concentration groups, above
Fig. 1. Effects of BPA on the viability of rat Sertoli cells using MTT assay. Cells were treated with 0, 30, 50, 70, and 90 M for 24 h, and the optical density (OD) was detected with MTT assay. Data were presented with as mean ± SD of three independent experiments performed in triplicate. Significant difference: *P < 0.05, compared with control group.
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Fig. 2. BPA induced apoptosis of rat Sertoli cells. After cells exposed to BPA for 24 h, representative images of TUNEL staining (A–D) and numbers of apoptotic cells were counted (E) according to TUNEL assay. (A) Control group; (B) 30 M BPA; (C) 50 M BPA; (D) 70 M BPA. Significant difference: *P < 0.05, compared with control group.
was significantly increased in all BPA-treated groups in comparison with the control (P < 0.05). In addition, protein expressions of FasL and cleaved caspase-3 were determined by western blotting (Fig. 3B–D). Compared with the control group, FasL protein expression was increased by 50% in 70 M group. Moreover, BPA
elevated the protein level of cleaved caspase-3 in 30 (1.54-fold), 50 (2.62-fold) and 70 M group (4.23-fold). As shown in Fig. 4, the immunocytochemical staining analysis exhibited a concentrationdependent decrease in procaspase-3 expression. The result of negative control incubated with PBS was not shown.
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Fig. 3. BPA-induced activation of Fas/FasL signaling pathway. (A) RT-PCR analysis of Fas, FasL and caspase-3 mRNA expressions in rat Sertoli cells after various concentrations of BPA-treatment. (B–D) Western blotting analysis of FasL and cleaved caspase-3 protein expressions in Sertoli cells after exposure to BPA. Significant difference: *P < 0.05 compared with control group.
3.4. BPA activated the JNK/p38 MAPK pathway To assess effects of BPA on the MAPK pathways, molecular elements of both JNK and p38 pathways were examined by RT-PCR and Western blotting. In the present study, mRNA expressions of ASK1, c-jun and CHOP and protein expressions of phospho-JNK and p38 were detected. As exhibited in Fig. 5A, ASK1 mRNA expression was significantly increased by 64% in 70 M group. c-jun level was also elevated after BPA treatment, with maximum effects observed in 70 M group (1.29-fold). BPA also induced the mRNA expression of CHOP in all treated groups (P < 0.05). In addition, as seen in Fig. 5C and D, the protein level of p-JNK was significantly increased
by 54.6% in 70 M group as compared with the control (P < 0.05). The protein level of p-p38 had a 2.55- and 1.93-fold increase in 50 and 70 M groups, respectively. However, protein expressions of total JNK and p38 were not affected after BPA treatment (data were not shown). 3.5. Effect of BPA on NF-ÄB activation in rat Sertoli cells Laser scanning confocal microscopy was used to assess the nuclear translocation of NF-B in rat Sertoli cells. As exhibited in Fig. 6A, p65 and nuclei showed red and blue fluorescence, respectively. In this study, the activation of NF-B was showed a
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Fig. 4. Effect of BPA on procaspase-3 expressions in rat Sertoli cells was detected by immunocytochemistry (200×). As is shown in control group (A), procaspase-3 was highly expressed in the cytoplasm of Sertoli cells. However, after treatment with BPA for 24 h, the positive expressions of procaspase-3 were decreased with the increasing doses (B–D). (A) Control group; (B) 30 M BPA; (C) 50 M BPA; (D) 70 M BPA. The data of negative control was not shown.
concentration-dependent increase after BPA treatment. Similarly, the nuclear protein level of NF-B p65 was increased in all BPAtreated groups in comparison with the control (P < 0.05) (Fig. 6B and C). 4. Discussion The present study demonstrated that BPA was toxic to rat Sertoli cells, resulting in the decrease in cell viability and apoptosis induction. Furthermore, activations of Fas/FasL, JNKs/p38 MAPK and the translocation of NF-B might involve in the apoptosis of Sertoli cells. BPA is reported to cause reproductive toxicity and affect cellular development. Previous studies have demonstrated that prenatal and neonatal exposure to BPA at <50 mg/kg/day can induce adverse effects in male reproductive systems, including decrease of sperm count, motility and changes of sperm morphology abnormalities [24,25]. Moreover, neonatal and pubertal exposure to BPA could impair the fertility of male rats and disrupt the BTB integrity via declining levels of Sertoli cell junction proteins [26,27]. However, potential mechanisms of toxic effects of BPA on Sertoli cells are still unclear. Therefore, for the critical role of Sertoli cells in spermatogenesis, we hypothesized that BPA might have toxic effects on Sertoli cells and special signaling pathways might play an important role in apoptosis. In the present study, BPA significantly decreased the viability of Sertoli cells at over 50 M and similar results were confirmed by TUNEL assay. These observations are in agreement with a previous
report that 20 or 150 M BPA significantly affects the viability of Sertoli cells for 48 h or 16 h [8,14]. Compared with what Iida and Qian have reported, concentrations of BPA used in our study were between them. We suppose that this disparity may result from different tested subjects. However, considering characteristics of BPA, low-concentration exposure and long-term effects, whether less than 50 M exposure for 48 h or more has adverse effects on Sertoli cells deserves to be explored in our future study. In FasL-induced spermatogenic cell death, it is generally accepted that FasL from Sertoli cells kills spermatogenic cells by engaging Fas receptors present on them [28,29]. However, it is reported that Sertoli cells also express Fas and germ cells express FasL [30]. Li et al. have reported that high-dose BPA induced apoptosis of Leydig and germ cells in mouse testes through up-regulated Fas and FasL and active caspase-3 [13]. Our results showed that BPA exposure activated the Fas/FasL pathway. Once the Fas system is activated, the effector caspase, caspase-3 will be cleaved and subsequently activated, which finally commits cells into apoptosis [31]. Our data showed that the mRNA expression of caspase-3 was increased, and even the protein expression of cleaved caspase-3 was also enhanced. Moreover, the expression of procaspase-3 in Sertoli cells was decreased. Observed results indicated the activation of caspase-3. Therefore, we suppose that the apoptotic effect of BPA on Sertoli cells could be related to the induction of caspase activation. NF-B, a dimmer of protein components (p65/p50), binds to inhibitory protein IB in unstimulated cells. Stimulation by several extracellular signals leads to the degradation of IB. Then
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Fig. 5. BPA-induced activation of JNKs/p38 MAPK pathway. (A and B) The mRNA expressions of ASK1, c-jun, and CHOP in Sertoli cells exposure to BPA for 24 h. (C) Western blotting analysis of JNK, p-JNK, p38 and p-p38 protein expressions in rat Sertoli cells exposure to BPA for 24 h. (D) Quantitative analysis of the immunoreactive p-JNK and p-p38. *P < 0.05 compared with control group.
freed NF-B dimmers translocate to nuclei and bind to specific sequences in promoter or enhancer regions of target genes [32]. In rat testes, the NF-B complex of p65 and p50 is found to express in nuclei of Sertoli cells at all stages of spermatogenesis [33].
Interestingly, NF-B effects in different promotes or inhibits cific cell type and
can exert both pro- and anti-apoptotic cell types. However, whether NF-B apoptosis seems to depend on the spethe type of inducer [34]. Our present
Fig. 6. BPA-induced activation of NF-B. (A) Immunofluorescent double staining of NF-B p65 (red) and nuclei (blue) detected by laser scanning confocal microscope (400×). NF-B p65 was not activated in the cytoplasm as is shown in control group. However, the translocation of NF-B p65 into nucleus was enhanced as the increased dosing of BPA. (B) Protein from nuclear extracts was used in western blotting to detect NF-B p65. (C) Quantitative analysis of the immunoreactive NF-B p65. Significant difference: *P < 0.05 versus control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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study showed that BPA exposure induced NF-B activation and translocation into nuclei. There is accumulating evidence that activated NF-B can sensitize some cells to apoptosis-inducing agents that act through or cooperate with the Fas pathway due to the ability of NF-B to activate genes encoding FasL or Fas [34]. For example, FasL and Fas are both up-regulated by NF-B in different cell types [35–38]. Recently studies have reported that the activation of NF-B is associated with the germ cell apoptosis [39,40]. Thus, in the present study, we speculate that NF-B can induce Fas-FasL expression and promote cell apoptosis through the Fas/FasL pathway in vitro. To further confirm the function of NF-B in the apoptosis of Sertoli cells, PDTC, an inhibitor of NF-B, perhaps should be applied in our future experiments. The MAPK pathways regulate diverse processes such as proliferation, differentiation and apoptosis. In mammals, MAPK can be mainly grouped into three families: ERKs, JNKs and p38/SAPKs. Each cascade is initiated by specific extracellular stimuli and a particular MAPK is activated after successive activations of MAP3K and MAP2K [41]. ASK1, a member of MAP3K family, activates the MAP2K4/7 (MKK4/7)-JNK pathway and MAP2K3/6 (MKK3/6)-p38 pathway [42]. Then activated JNKs and p38 translocate into nuclei and mediate expressions of target genes, such as c-jun and CHOP [43,44]. It is generally accepted that JNKs and p38 are associated with cells apoptosis, especially in Sertoli cells exposed to reproductive toxicants. Lee et al. have reported that 1,3-DNB induces apoptosis of TM4 mouse Sertoli cells via activating the JNK signaling pathway [17]. In addition, the inhibitory effect of cadmium chloride to Sertoli cells is obviously reduced after treatment with a p38 inhibitor (SB202190), and results confirm that cadmium exerts apoptotic effects on piglet Sertoli cells through the p38 MAPK pathway [45]. In order to elucidate whether the JNKs/p38 pathway was involved in BPA-induced apoptosis of rat Sertoli cells, we investigated mRNA expressions of ASK1, c-jun, CHOP and protein expressions of phospho-JNK and -p38. Our results showed that the mRNA expression of ASK1 was elevated, indicating that BPA exposure enhanced the transcription of MAP3K, which might initiate the activation of MAPK cascade. Moreover, increased protein expressions of phospho-JNK and -p38 confirmed that the JNKs/p38 pathway was activated and translocated into nuclei to mediate target genes, including c-jun and CHOP. Thus, we speculate that BPA exposure induces the activation of JNKs/p38 MAPK and the translocation into nuclei to mediate target genes expression and apoptosis. c-jun, as a target gene of JNKs, might lead to the activation of AP1 and expressions of Fas/FasL signaling pathway-related proteins. Then binding of FasL and Fas can trigger apoptosis via activating downstream effector caspase-3 [43]. Meanwhile, CHOP, as a target gene of p38, is closely related to ER stress-induced apoptosis [46]. However, to confirm roles of JNKs/p38 MAPK in BPA-induced apoptosis, the antagonist or crosstalk method should be performed in our future study. Taken together, we conclude that the Fas/FasL, JNKs/p38 MAPK and NF-B are activated in BPA-induced apoptosis of rat Sertoli cells. Fas/FasL signaling pathway plays an important role in apoptosis, and JNKs/p38 MAPK and NF-B may promote cell apoptosis after BPA exposure. However, considering the complicated crosstalk among different signaling pathways, the antagonist and crosstalk method will be used in our future study to clearly elucidate apoptotic effects of BPA on Sertoli cells. Moreover, considering limitations of in vitro study and critical roles of Sertoli cells in spermatogenesis, whether the same doses of BPA could impair spermatogenesis needs further confirmation by in vivo study.
Conflict of interest The authors declare that there are no conflicts of interest.
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