Bisphenol-A (BPA) is an industrial chemical and is known to act as an endocrine disrupter. This study was designed to evaluate how BPA regulates Sertoli cell (SC) signal molecules. Purified rat SC was cultured and treated with BPA (200 Î¼mol/l) at various time points. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), nuclear factor kappa B (NF-ÎºB), cyclooxygenase-1,2 (COX-1, 2), estrogen receptor-Î± (ER-Î±), and androgen receptor (AR). The levels of transferrin (TF), prostaglandin E2 (PGE2), and prostaglandin F2Î± (PGF2Î±) in culture medium were quantified by ELISA. Interleukin (IL)-1Î² and IL-6 mRNAs were measured by quantitative real-time PCR (QRT-PCR). Compared with the control, BPA activated the phosphorylation of ERK1/2 (p-ERK1/2) through 30 min to 6 h. TF was down-regulated at 6 and 24 h. Furthermore, IL-1Î² was up-regulated at 30 min and IL-6 was up-regulated at 1 and 24 h. ERK activity inhibitor (PD98059, 10 Î¼mol/l) inhibited these molecules changes. These results reveal the possibility that BPA may have adverse effects on spermatogenesis via ERK1/2. To elucidate more precise BPA influences on spermatogenesis, further investigations will be necessary.
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BPA [2,2-bis(4-hydroxyphenyl)propane] is a monomer of polycarbonate plastics and is widely used as a compound in food and drink cans [Brotons et al. 1995], as well as being a component of dental sealant [Olea et al. 1996]. It has been reported that BPA easily leaches from food packaging and dental sealants into saliva, and works as an estrogenic substance [Brotons et al. 1995; Krishnan et al. 1993]. The actual effects of these molecules, such as BPA, on the male reproductive system have attracted tremendous attention recently, and many studies have been reported. Although controversy remains with respect to the effects of BPA on male fertility, growing evidence suggests BPA can act as a potential testicular toxicant that alters spermatogenesis [Iida et al. 2003; Toyama et al. 2004; Salian et al. 2009]. Spermatogenesis is a highly complex process regulated by various endocrine and paracrine/autocrine molecules. In this complex mechanism, SC synthesizes many essential factors for spermatogenesis and supports developing germ cells. Recently, there has been increasing evidence of the molecular mechanisms involved in SC paracrine/autocrine regulations in response to various molecules, such as antineoplastic reagents [Yamaguchi et al. 2008] and pro-inflammatory cytokines [Ishikawa et al. 2005; Ishikawa and Morris 2006a]. Iida et al. reported that BPA induced apoptosis of cultured rat SC via cell structural change in a dose- and time-dependent manner [Iida et al. 2003]. Meanwhile, Smith et al. revealed that neonatal exposure of male rats to BPA impairs the fertility and expression of SC junctional proteins in the testis [Salian et al. 2009]; however, the molecular mechanisms involved in BPA regulation of SC function, which subsequently influence developing germ cells and spermatogenesis, have not been detailed. The aim of the present study was to determine how BPA regulates SC paracrine/autocrine activities and subsequently influences developing germ cells and spermatogenesis.
BPA induces activation of ERK1/2 in SC
BPA induced the activation of ERK1/2 at 30 min, 1-6 h after BPA treatment. p-ERK1/2 doublet protein (44 and 42 kDa, respectively) significantly increased 1.8-2.0 fold compared with BPA treatment and the vehicle (Ct) (Figure 1A and B). p-JNK or -p38 MAPK, and COX-1, COX-2 were not activated during this time course (30 min-24 h) (data not shown). Although the toxic effects of BPA have been proposed to be mediated through binding to ER [Takayanagi et al. 2006], we have confirmed that BPA induced no change of at least ERÎ± or AR protein levels in present time course (data not shown). A previous study showed that BPA significantly activated ERK and inhibited NF-ÎºB, which is considered to work for anti-apoptosis, not in SC, but in PC12 cells and cortical neuronal cells [Lee et al. 2007]. In these neuronal cells, the ERK/NF-ÎºB pathway may be involved in BPA-induced toxicity; however, there was no significant change in the NF-ÎºB protein level in this study (data not shown). Treatment with BPA in the absence or presence of the ERK activity inhibitor, PD98059 inhibited the increase of BPA-induced phosphorylated ERK1/2 protein levels at 1-6 h. Based on previous studies indicating that some SC secretion molecules, including physiological and proinflammatory cytokine factors, may play a role in paracrine and autocrine regulation and the dysregulation of SC paracrine function [Ishikawa et al. 2005; Ishikawa and Morris 2006a; Ishikawa and Morris 2006b; Yamaguchi et al. 2008], we next examined the effect of BPA on the levels of SC secretions, including cytokine expression.
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BPA reduces SC TF secretion through the ERK1/2 pathway
To determine the effect on the function of SC through the activation of p-ERK1/2 induced by BPA treatment, levels of SC-secreted TF were evaluated in the presence or absence of ERK activity inhibitor. Significant reduction of SC-secreted TF levels was observed at 6 and 24 h after BPA treatment (at 6 h: 0.81-fold, P<0.05, at 24 h: 0.90-fold, P<0.01) compared with BPA and vehicle (Ct) (Figure 2). In the presence of the ERK activity inhibitor PD98059 (10 Âµmol/l), reduction of SC-secreted TF induced by BPA was inhibited at 6 h. From this result, it was revealed that BPA has the potential to reduce TF secretion through the ERK1/2 pathway in SC. Meanwhile, SC PGE2 and PGF2Î± expressions showed no significant differences after BPA treatment (data not shown)..
BPA induction of IL-1Î² and IL-6 expressions occurs partly through the ERK1/2 pathway
With regard to ILs, the expression of IL-1Î² mRNA was significantly induced by BPA 30 minutes after BPA treatment (1.4-fold, P<0.01, compared with BPA and the vehicle (Ct)). Although mRNA levels remained high 1 and 3 h after BPA treatment, there were no significant changes compared with the vehicle (Ct). Meanwhile, the expression of IL-6 mRNA tended to increase from 1 h after BPA treatment. Significant increase of mRNA level was observed at 1 h and 24 h (3.2 and 11.5-fold: P<0.05, compared with BPA and the vehicle (Ct), respectively). To determine whether the activation of ERK1/2 was required for these changes in cytokine expression, the effect of the ERK1/2 selective activity inhibitor on BPA-mediated regulation of IL-1Î² and IL-6 mRNA levels was examined. PD98059 significantly inhibited, in part, BPA-stimulated increases in IL-1Î² mRNA. Although PD98059 similarly tended to inhibit BPA-stimulated increases in IL-6 mRNA during this time course (30 min-24 h), significant inhibition of mRNA induction was observed only 1 h after BPA treatment. From these results, changes in IL-1Î² and IL-6 mRNA expressions are partially dependent on ERK1/2 activity (Figure 3A and B).
In this study, we showed that BPA has the possibility to induce adverse effects on fertility. Our results show that, at least in vitro, BPA at 200 Âµmol/l (45 Âµg/ml) concentration seems to be harmful to SC, probably because of the induction of decreasing SC TF secretion and increasing SC pro-inflammatory cytokine, such as IL-1Î² or IL-6, production via the ERK1/2 pathway. The 200 Âµmol/l concentration of BPA used in this study appears to be relatively high, but is equivalent to the concentration of BPA released from plastic dental sealants in saliva (931 Âµg/30 ml) [Olea et al. 1996]. It is known that TF plays an important role in spermatogenesis by providing iron to developing germ cells and is useful as an index of SC function [Holmes et al. 1983; Sylvester et al. 1994]. In a previous study, a similar result was indicated by Iida et al. [Iida et al. 2003], who showed that TF secretion from SC was decreased by exposure to BPA. However, to our knowledge, these are the first published data revealing the time-dependent effect of BPA stimulation on Sertoli TF secretion. Our previous study showed that not BPA, but an antineoplastic reagent, cis-diaminedichloroplatinium (CDDP), also reduced Sertoli TF secretion through the ERK1/2 pathway [Yamaguchi et al. 2008]; however, in this study, the reduction of SC TF secretion induced by BPA was observed later and its reduction was less than the results observed in our previous study using CDDP. These differences in the same signal pathways induced by various chemical molecules may depend on the context of the nature of stimuli. Meanwhile, ILs have been shown to play multiple interactive roles in the regulation of testicular somatic cell functions and male germ cell development [Hakovirta et al. 1995; Syed et al. 1995; Legue et al. 2001; Petersen et al. 2002; Ishikawa et al. 2003]. In defined stages of the rodent seminiferous cycle, the presence of residual bodies, which represent membrane-enclosed cytoplasmic contents shed by elongating spermatids during spermiogenesis, activates Sertoli cell phagocytosis and IL-1 release, events that initiate IL-6 secretion [Gerard et al. 1992; Syed et al. 1993; Syed et al. 1995]. This physiological stage-specific process induces SC IL-1Î² and IL-6 production, and also accompanies pathological testicular inflammation [Gerard et al. 1992; Syed et al. 1993; Okuda et al. 1993; Okuda et al. 1995; Syed et al. 1995; Cudicini et al. 1997]. Our data showed that BPA also induced Sertoli cytokine secretion and might lead to inflammatory-like changes in SC. It has been revealed that IL-1Î² induced Sertoli IL secretion mediated by the activation of COX-2, which is the enzyme metabolizing arachidonic acid, and the phosphorylation of JNK [Ishikawa et al. 2005]. Our previous study showed that CDDP induced the activation of the COX-2 pathway only, which subsequently induced PGE2 and PGF2Î± production, and furthermore proceeded to stimulate IL secretion in SC; however, in this study, neither activation of the arachidonic acid cascade nor the MAPK pathway, except ERK1/2, was seen during this time course. Although Thuillier et al. recently indicated that fetal BPA exposure induced mRNA and protein increases of ERK1/2 in rat testes, mainly in SC, only transient effects were observed on actual germ cell populations [Thuillier et al. 2009]. Our data suggest that ERK1/2 MAPK is partially involved in IL secretion in prepubertal rat SC. In summary, this study identified for the first time that BPA-ERK1/2 MAPK mediated the regulatory mechanisms for TF secretion and inducible IL production in SC. We have investigated the changes of SC ER-Î± and AR protein levels in this study, and there were no change of these hormone receptors protein level. ER, which belongs to the family of nuclear hormone receptors, have very extended functional expression manner [Ordóñez-Morán et al. 2009]. So it is unclear whether ER is involved in BPA producing adverse effects or not only from our results. To establish the overall and precise SC molecular signaling mechanism response to BPA, further investigations will be necessary. Moreover, by analyzing the study using other testicular cells or the phenomena occurring in an in vivo study, the actual behavior and effect of BPA on male fertility will be more clearly understood.
Materials and methods
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Sertoli cell preparations
SC was purified from the testes of 18-day-old Sprague Dawley rats purchased from Oriental Yeast Co. (Tokyo, Japan). Procedures involving the use of animals were conducted according to the Guidelines for Animal Experiments at Kobe University School of Medicine. Primary cultures (â‰¥95% pure) were maintained as described previously by Kanzaki et al. [Kanzaki and Morris 1998]. On day 3 ex vivo, SC were rinsed twice with fresh serum- and phenol red-free culture medium and then treated with BPA (200Âµmol/l) (Sigma-Aldrich Co. MO, USA), which was dissolved in dimethylsulfoxide (DMSO)(Sigma) immediately prior to use, in the absence or presence of ERK activity inhibitor PD98059 (2'-amino-3'-methoxyflavone , 10 Âµmol/l) (Cayman Chemical Co., MI, USA), which was also dissolved in DMSO. At 30 min, 1, 3, 6, and 24 h following vehicle (Ct), BPA, or the addition of inhibitor, whole cell lysates for protein and total RNA were isolated from each replicate. At least triplicate culture dishes were used for each drug treatment, and experiments were repeated at least twice. The mean (Â± SEM) of all experiments was calculated for analysis.
Protein extraction and Western blot analysis
Protein extraction and Western blot analysis were performed as described previously [Yamaguchi et al. 2008]. In this study, a total of 20-25 Âµg protein was separated by SDS-polyacrylamide gel electrophoresis using 4-20% Tris-glycine gel (Novex, CA, USA), and was electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). The membranes were probed with antibodies, as described below. Phospho-stress-activated protein kinase/JNK (p-SAPK/JNK) polyclonal antibody (pAb; 1:1000), JNK pAb (1:1000), p-ERK1/2 pAb (1:1000), ERK1/2 pAb (1:1000), phospho-p38 (p-p38) MAPK pAb (1:1000), and p38 pAb (1:1000) were obtained from Cell Signaling Technology, Inc. (MA, USA). COX-1 and COX-2 pAb (1:1000) were obtained from Cayman Chemical Co. ERÎ± pAb (1:200), AR pAb (1:200), and NF-ÎºB pAb (1:200) were obtained from Santa Cruz Biotechnology, Inc. (CA, USA). Monoclonal anti-Î²-actin antibody (1:1000) was purchased from Sigma-Aldrich Co. Blots were developed with the ECL Western blotting system (Amersham Biosciences, IL, USA) and exposed to X-ray film (FUJIFILM Medical Co., Tokyo, Japan). Densitometric analysis was performed using the personal computer version of the National Institutes of Health Image software (Scion Image; Scion Image Corp., MD, USA) after scanning. For Western blotting analyses, particular signal intensities in each lane of p-JNK, -ERK 1/2, and -p38 were normalized with those for JNK, ERK 1/2, and p38 on the same membranes, respectively. The others were normalized with those for Î²-actin on the same membrane and data are expressed as arbitrary units relative to the control (Ct), set at a value of "1."
To measure the concentration of SC-produced TF and PGs levels in culture media, SC were treated with BPA (200 Âµmol/l), BPA with PD98059 (10 Âµmol/l) or matched vehicle blank controls (Ct) for 30 min, 1, 3, 6, and 24 h. At the indicated time, cell-free supernatants were transferred to sterile microcentrifuge tubes. Two 40- or 50-Âµl aliquots of conditioned medium were assayed using TF (Panapharm Laboratories, Kumamoto, Japan) or individual PGE2, PGF2Î± (Cayman Chemical) ELISA kits according to the manufacturer's instructions, respectively. The sensitivity of the assay (80% bound) was 0.63 ng/ml, 36, and 9 pg/ml for TF, PGE2, and PGF2Î±, respectively, and intra- and interassay coefficients of variation were less than 10% for all ELISA kits. The concentrations of TF, and PGs were determined by ELISA measured by a standard curve method using a microplate reader (model 550; Bio-Rad Laboratories, CA, USA). Data are expressed as arbitrary units relative to the control (Ct), set at a value of "1".
Total RNA extraction and Quantitative real-time PCR analysis
Total RNA was and extracted and QRT-PCR was performed as described previously [Yamaguchi et al. 2008]. ILs and Î²-actin mRNAs were detected using proprietary TaqMan primers and probes (Applied Biosystems, CA, USA). These probes and primers were used for IL-1Î² (assay identification number Rn 00580432_m1), IL-6 (assay identification number Rn 00561420_m1), and Î²-actin (assay identification number Rn 00667869_m1). Data were normalized with Î²-actin values and expressed as arbitrary units relative to the control, set at a value of "1". Densitometric analyses are expressed in arbitrary units.
All results are the mean Â± SEM derived from the number of different experiments. Statistical analyses were performed using t test or paired-t test, ANOVA. P values â‰¤0.05 were considered significant. We used Scheffe's procedure as a post-hoc test.
Declaration of Interest section
The authors declare that there is no potential conflict of interest amongst them that would prejudice the impartiality of this scientific work.