Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells
Introduction
Apoptosis or programmed cell death is a highly regulated process of cell deletion; it promotes the turnover of normal cells and the clearance of infected or transformed cells (Fadeel and Orrenius, 2005, Jacobson et al., 1997). Apoptosis occurs in a well-choreographed sequence of morphological events via extrinsic and intrinsic pathways (Rich et al., 1999). Both pathways have a final common pathway that involves the activation of a cascade of proteases and culminates in the death of cells (Ghobrial et al., 2005).
In autophagic programmed cell death, cytoplasmic components such as macromolecules, proteins, and organelles are degraded by lysosomes (Dalby et al., 2010, Mizushima, 2007). Autophagy is essential for cell survival, differentiation, development, and homeostasis. This process is responsible for maintaining low basal levels in cells and is induced by environmental stress; however, excessive autophagy triggers cell death (Codogno and Meijer, 2005, Yang et al., 2012). Autophagy-defective organisms such as fungi, protozoa, worms, and insects show various abnormalities in differentiation and development (Mizushima and Levine, 2010). Aside from being a mechanism of cell death, autophagy has also been suggested to represent a survival strategy. Autophagy serves as a mechanism to update damaged cells and long-lived proteins; it also provides energy under starvation conditions (Edinger and Thompson, 2004).
The crosstalk between autophagy and apoptosis is a strong and complex relationship that can be observed under different circumstances at the molecular level. Autophagy constitutes a mechanism to suppress apoptosis (Maiuri et al., 2007). Some reports demonstrated that the complete inhibition of the autophagic pathway obviously increases apoptosis and that promoting autophagy prevents cells from undergoing apoptosis (Lai et al., 2012, Mizushima et al., 2008, Rossman and Lamb, 2009). Another report showed that cleaved caspases dependent on autophagy-related proteins terminate autophagic responses and that the cleaved proteins of autophagy promote apoptosis (Gordy and He, 2012). However, the relationship between autophagic and apoptotic molecular mechanisms remains unclear.
Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin produced by several species of Fusarium, is present in many food products for humans or animals; ZEA binds estrogen receptors and then causes structural and functional changes in reproductive organs (Bennett and Klich, 2003, Yang et al., 2007). Many reports have demonstrated that exposure to ZEA causes precocious puberty in children, endometrial adenocarcinomas, hyperplasia, breast cancer in women, and testicular germ cell depletion (Amann and Lambiase, 1969, Sáenz de Rodriguez et al., 1985, Schoental, 1983, Tomaszewski et al., 1998). ZEA can also induce apoptosis in vivo and vitro (Abid-Essefi et al., 2003, Vlata et al., 2006). However, little is known about the functions of ZEA in male reproduction. In the current study, we chose primary cultured rat Leydig cells as a model to determine whether or not ZEA can induce apoptosis and autophagy. We also investigated the effect and regulatory mechanisms of ZEA-induced apoptosis and autophagy in primary cultured Leydig cells.
Section snippets
Reagents
ZEA, Hoechst 33258, z-VAD-fmk, acridine orange (AO), and anti-LC3 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bax, Bcl-2, poly (ADP-ribose) polymerase (PARP), caspase-9, caspase-3, cytochrome c, COX IV, and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Enhanced chemiluminescence (ECL) solution was obtained from Thermo Fisher
ZEA induces apoptosis in rat Leydig cells
The cells in the third layer were positively stained by the Wiebe method, suggesting that they consisted of over 90% Leydig cells (Fig. 1A). These cells were used for the latter part of this study.
We investigated the effects of ZEA on the growth of rat Leydig cells using the MTT assay. As shown in Fig. 1B, ZEA inhibited the growth of rat Leydig cells in a dose-dependent manner. We examined the nuclear morphology of the cells under a fluorescence microscope to determine whether the observed
Discussion
Previous studies proved that ZEA is toxic for human and animal health; ZEA primarily produces symptoms of hyperestrogenism in female reproduction, but little is known about its effects on males (Tsakmakidis et al., 2006). The detailed molecular and cellular mechanisms of ZEA toxicity have not yet been completely explained. In the current study, we evaluated the effects of ZEA on primary rat Leydig cells. The results demonstrated that the ZEA-induced loss of cell viability was dependent on
Conclusion
The treatment of primary rat Leydig cells with ZEA resulted in viability inhibition. ZEA treatment activated the mitochondria pathway of apoptosis by Bcl-2 family proteins and eventually caused apoptosis. ZEA could also increase the level of autophagy in Leydig cells. Low and high doses of ZEA can activate autophagy and apoptosis in Leydig cells, respectively. The results proved that autophagy can delay apoptosis in Leydig cells. Therefore, autophagy serves as an important protective function
Conflict of interest
The authors declare that no conflict of interest exists.
Acknowledgements
This study was supported by grants from the National Science Foundation of the Higher Education institutions of Jiangsu Province, China (No. 08KJD230002), the Graduate Training Innovative Projects Foundation of Jiangsu Province, China (No. CXLX13_921), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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