Elsevier

Toxicology Letters

Volume 226, Issue 1, 7 April 2014, Pages 81-89
Toxicology Letters

Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor α/β in low doses of bisphenol A promoting growth of Sertoli TM4 cells

https://doi.org/10.1016/j.toxlet.2014.01.035Get rights and content

Highlights

  • 10−9 and 10−8 M BPA significantly stimulated the proliferation of TM4 cells.

  • GPR30/EGFR/ERK signal is involved in BPA-induced TM4 cell proliferation.

  • BPA (1 nM) up regulated mRNA and protein expression of GPR30.

Abstract

Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during spermatogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused these effects were still unclear. In the present study, an inverse U-shaped curve was observed when treating TM4 cells with increasing doses of BPA: 1 to 10 nM BPA significantly stimulated the proliferation of TM4 cells and increased the proportion of cells in S phase; >1 μM BPA caused lesser proliferation of cells. Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor α/β (ERα/β), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ERα/β were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid (5 min) activation of ERK1/2 via both GPR30 and ERα/β. Blocking the GPR30/EGFR signal transduction pathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPA up-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary, the results reported here indicated that activating ERK1/2 through GPR30 and ERα/β is involved in low doses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstream transduction pathway in BPA-induced proliferation of TM4 Sertoli cells.

Graphical abstract

Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor α/β (ERα/β), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ERα/β were involved in the observed effects of BPA.

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Introduction

Bisphenol A (BPA) is large production volume chemical used to manufacture food and beverage containers, cigarette filters, thermal (carbonless) receipts, and paper currency (Vandenberg et al., 2009). Currently, over 2.7 million metric tons of BPA are produced annually, primarily for use in manufacturing of epoxy resins and polycarbonate plastics. BPA can leach into food and beverages from plastic containers and therefore accumulate in bodies of humans (Shin et al., 2004). Recently, BPA has been widely detected in various human tissues such as blood, fetal serum during pregnancy, amniotic fluid, follicular fluid, placental tissue, umbilical cord blood, and urine (Lee et al., 2008, Vandenberg et al., 2010, Wan et al., 2010). For example, BPA has been detected in 100% of urine samples from Chinese children with a mean concentration of 3.00 ng/mL (Li et al., 2013), and in 96% of urine samples from American college students with a mean concentration of 1.3 ng/mL (Carwile et al., 2009).

BPA has been implicated as a potential endocrine disrupting chemical (EDC), primarily as a weak estrogen receptor α/β (ERα/β) agonist (Vandenberg et al., 2009). Recently, more and more studies have revealed that BPA is correlated with health outcomes such as diabetes, cardiovascular disease, breast cancer, increased inflammation, and oxidative stress (Rubin, 2011). Among these health issues, increasing concern has been focused on the risk of BPA to male fertility (Salian et al., 2009, Salian et al., 2011). In vivo studies have documented that prenatal and neonatal exposure of male rats to environmentally relevant concentrations of BPA significantly impairs spermatogenesis (Salian et al., 2011).

Sertoli cells, one of the somatic constituents of the testis, are the primary supporting cells creating the structural and physiological environments necessary for development of cells during spermatogenesis (Kopera et al., 2010). In mammals, Sertoli cells proliferate during only two periods of life, in fetal or neonatal life and in the peri-pubertal period (Sharpe et al., 2003). Results of recent studies indicated that exposure to BPA affects fertility of males by affecting efficiency of spermatozoa, but there was limited data on effects of BPA on proliferation and functions of Sertoli cells.

Results of previous studies indicated that the binding affinity of BPA to estrogen receptor-α (ERα) or ERβ is 10,000-fold less than that of the endogenous estrogen, estradiol (E2) (Kuiper et al., 1998). This affinity is insufficient to explain why nanomolar doses of BPA have been reported to cause E2-mediated effects (Watson et al., 2005, Welshons et al., 2006). Recently, G protein-coupled receptor 30 (GPR30), an orphan G protein-coupled receptor (GPCR, also known as G protein-coupled estrogen receptor, GPER), which has been proposed as a nonclassical estrogen receptor, has been reported to mediate estrogenic effects of estrogens, phytoestrogens, xenoestrogens, and environmental EDCs (Prossnitz and Barton, 2011). GPR30 was recently shown to mediate BPA-induced proliferation of various types of cells (Bouskine et al., 2009, Sheng and Zhu, 2011). However, whether GPR30 is involved in BPA-induced impairment of fertility of males and the mechanism by which these effects occurred remained unclear.

In the present study, effects of BPA on proliferation of Sertoli TM4 cells was studied. Specifically, the objectives of this study were to: (1) investigate effects of BPA, particularly at nanomolar concentrations, on proliferation of TM4 cells; (2) determine the role of GPR30 and ERα/β in BPA-induced proliferation of Sertoli TM4 cells, and (3) evaluate changes in downstream signal pathways.

Section snippets

Reagents

ICI 182,780 (ICI, ER antagonist) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD 98059 (PD, MAPK/ERK kinase agonist) and AG 1478 (AG, EGFR antagonist) were purchased from Selleck Chemicals (Houston, TX, USA). E2, G-1 (GPR30 agonist), G15 (GPR30 antagonist) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibody against GPR30 was purchased from Abcam Plc (Abcam, UK). Antibodies against ERK1/2, Bcl-2, PCNA, Caspase 3, Bim and GAPDH were

Effects of BPA on proliferation of TM4 cells

Proliferation of TM4 cells was determined by use of CCK-8 assay after exposure to concentrations of BPA ranging from 10−8 to 10−3 M for 48 or 72 h (Fig. 1), respectively. Proliferation was >20% more than control in cells exposed to 10−8 M BPA for 48 h, but decreased at higher concentrations. The IC50 of BPA on TM4 cell for 48 and 72 h was 5.0 × 10−5 and 9.2 × 10−5 M, respectively. These results demonstrated that proliferation of TM4 cells was stimulated by BPA at nanomolar concentrations of BPA while

Discussion

Results of the present study revealed, for the first time, that nanomolar concentrations of BPA can significantly increase proliferation of immature mouse Sertoli TM4 cells. Small (pM) concentrations of BPA have also been reported to trigger a nongenomic proliferative effect in the pancreatic islet, endothelium, breast, spermatogonial and pituitary gland by initiating rapid responses (LaPensee et al., 2009, Sheng and Zhu, 2011, Wetherill et al., 2007). In the present study, a comparable

Conflict of interest

The authors declare that there are no conflicts of interest.

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Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant No. 31101071 and No. 81302317), the National Basic Research Program of China (973 Program, No. 2011CB9358003), the Fundamental Research Funds for the Central Universities (Sun Yat-sen University) (No. 12ykpy09), the Science and Technology Planning Project of Guangdong Province, China (No. 2012B031500005), and the Seed Collaborative Research Fund from the State Key Laboratory in Marine Pollution (SCRF0003).

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