Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression

https://doi.org/10.1016/j.taap.2018.05.003Get rights and content

Highlights

  • Evaluated the impact of ZEA exposure on porcine and mouse granulosa cells.

  • Porcine granulosa cells are more sensitive than mouse cells exposed to ZEA.

  • Different patterns exist between porcine and mouse granulosa cells exposed to ZEA.

Abstract

Zearalenone (ZEA), a natural contaminant found in feed, has been shown to have a negative impact on domestic animal reproduction, particularly in pigs. There are species-specific differences in the ZEA-induced toxicity pattern. Here, we investigated the different biological effects of ZEA exposure on porcine and mouse granulosa cells, using RNA-seq analysis. We treated murine and porcine granulosa cells with 10 μM and 30 μM ZEA during 72 h of culturing, in vitro. The results showed that 10 μM ZEA exposure significantly altered mitosis associated genes in porcine granulosa cells, while the same treatment significantly altered the steroidogenesis associated genes in mouse granulosa cells. Exposure to 30 μM ZEA resulted in significantly up-regulated expression of inflammatory related genes in porcine granulosa cells as well as the cancer related genes in mouse granulosa cells. Similarly, 30 μM ZEA exposure significantly decreased the expression of tumor suppressor factors in the mouse granulosa cells. Furthermore, immunofluorescence, RT-qPCR as well as western-blot analysis verified the different expression of related genes in ZEA exposed porcine and mouse granulosa cells. Collectively, these results illustrate the presence of species differences with regards to ZEA effects between porcine and mouse ovarian granulosa cells, in vitro.

Introduction

ZEA is a mycotoxin that is produced by various Fusarium fungi (Bennett and Klich, 2003). It occurs in a variety of grains all over the world. Apart from aflatoxins, ZEA is among the most significant and prevalent trichothecenes, found in cereal grains as well as animal feeds, which causes widespread and recurrent economic harm (Escriva et al., 2015). ZEA's estrogenic activity suggests exposure may result in reproductive disorders and target species-specific organs (Poor et al., 2015; Pang et al., 2017). Low and high concentrations of ZEA have been shown to have adverse effects in livestock production, particularly pig production, including pig ovarian atrophy (Vanyi et al., 1994), abortion and reproductive failure (Dacasto et al., 1995; Lai et al., 2015; Osweiler et al., 1990; Zwierzchowski et al., 2005). ZEA is rapidly absorbed and metabolized in porcine after a single oral administration. In porcine, the uptake of ZEA was estimated to be 80%–85% with the administration of an oral dose of 10 mg/kg bodyweight (Biehl et al., 1993). ZEA have been detected in natural follicular fluid in porcine ovaries by liquid chromatography tandem mass spectrometry, and the concentrations of α-ZEA and ZEA in the follicular fluid was 17.6 pg/ml and 38.9 pg/ml respectively (Sambuu et al., 2011). In experimental animals (mouse and rat) ZEA has shown to have significant genotoxic potential, causing DNA damage in ovarian cells and impairing follicular development (Zhang et al., 2017a; Liu et al., 2017; Liu et al., 2018).

The mechanism of ZEA toxicity is not fully understood but it is known that ZEA possesses both acute and chronic toxic influences. Previous studies in vitro demonstrated that ZEA may alter the function and genomic stability of porcine granulosa cells (GCs) (Ranzenigo et al., 2008; Zhang et al., 2017b). Moreover, ZEA was also shown to negatively influence pig oocyte progression by inducing malformation of the meiotic spindles during meiosis (Malekinejad et al., 2007). In mouse, upon acute exposure to high doses of ZEA, murine models exhibited a “radiomimetic” shock-like response including vomiting, diarrhea, hemorrhage, leukocytosis, and at extremely high doses death (Pestka, 2010). Histological malformations in the reproductive organs was seen in the ZEA-treated mice, with ZEA treatment resulting in increased vacuoles and issues with primordial follicle assembly (Zhang et al., 2017a; Liu et al., 2017).

Female gametes develop in ovarian follicles, oocytes surrounded by granulosa and theca cells, that transition from primordial to dominant stages. Follicle growth relies on the development of GCs along with the oocytes from the initial stages. It is well documented that oocytes are in continuous communication with GCs resulting in the improvement of one affecting the development of the other. During the follicular growth GCs replicate, produce hormones and play a critical role in oocyte development (Hamel et al., 2008). The steroids secreted by ovarian GCs are necessary for the functioning of the reproductive system in most species. Currently, there is little information about the potential differential influence of ZEA on mammalian ovarian folliculogenesis between domestic and experimental animals (Cortinovis et al., 2013). Compared to mice, gilts are markedly more sensitive to ZEA exposure (Minervini and Dell'Aquila, 2008). Previous investigations have found substantial species-differences with regards to the biotoxicity (Nebbia, 2001). Systematic studies are lacking with regards to the effects of ZEA on different species. This study used transcriptome analysis to evaluate the species-specific toxicity of ZEA exposure, in vitro, in porcine and mouse ovarian GCs.

Section snippets

Reagents

ZEA was purchased from Sigma (St. Louis, USA). Stock solutions of ZEA were prepared by dissolving ZEA in dimethyl sulfoxide (DMSO). DMSO (D12345), fetal bovine serum (FBS, 10100147), M-199 medium (11150-059), penicillin and streptomycin were procured from Gibco (Carlsbad, USA).

Animals

The mature sows' ovaries used in the experiments were obtained from the Wan Fu Porcine Production Company (Qingdao, Shandong, China). The ovaries were collected from the slaughterhouse and maintained at 32–35 °C prior to

Apoptosis and gene expression of ZEA exposed porcine and mouse GCs

Porcine and mouse GCs were cultured in vitro and exposed to 10 μM or 30 μM ZEA for 72 h (Fig. 1A). The percentage of TUNEL positive pGCs significantly increased as a result of exposure of ZEA (10 μM: 28.34 ± 0.82%; 30 μM: 56.27 ± 1.90%) compared to that of the control (0 μM: 7.21 ± 0.53%; P < 0.01; Fig. S2A and C). Interesting, the percentage of TUNEL positive mGCs significantly increased only in the 30 μM ZEA exposed group (10 μM: 10.87 ± 0.46%; 30 μM: 25.93 ± 0.86%) compared with that of

Discussion

Recently numerous studies have found ZEA cytotoxicity impacting reproduction (Kiang et al., 1978; Mehmood et al., 2000; Nikov et al., 2000), the immune system (Abbès et al., 2006a, Abbès et al., 2006b; Luongo et al., 2006), endocrine functioning (Mueller et al., 2004) and inheritance (Kouadio et al., 2005) in several species. ZEA changed the development and steroidogenesis of ovarian GCs (Zhang et al., 2017b), thereby causing impacts on mammalian fertility (Zhang et al., 2017a). However, the

Acknowledgments

This work was supported by National Natural Science Foundation of China (31572225), National Key Research and Development Program of China (2016YFD0501207) and the Fund for Doctoral Scientific Research Startup of Qingdao Agricultural University (6631116019). We would like to thank Prof. Paul W. Dyce (Auburn University) for his careful editing of the manuscript.

Conflicts of interest

The authors fully declare any financial or other potential conflict of interest.

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