Elsevier

Toxicology

Volume 425, 1 September 2019, 152241
Toxicology

Melatonin protects against Fenoxaprop-ethyl exposure-induced meiotic defects in mouse oocytes

https://doi.org/10.1016/j.tox.2019.152241Get rights and content

Highlights

  • Effects of FE and melatonin protection on oocyte meiosis maturation.

  • Melatonin protects the cytoskeletal integrality.

  • Melatonin protects epigenetic modifications during meiosis maturation.

Abstract

Prolonged exposure of Fenoxaprop-ethyl (FE), a post-emergence herbicide, can cause serious damage to animals through food chain. Melatonin is synthesized by the pineal gland in mammals and believed to protect cells from oxidative stress damage. In this study, we aimed to investigate the effects of FE on mouse oocyte meiosis maturation and the protective roles of melatonin on FE-exposed oocytes by in vitro maturation model. FE exposure significantly caused defects of the first polar body extrusion, which could be protected by co-culture with melatonin. Furthermore, we examined the meiotic maturation details by performing the sperm binding, actin and tubulin immunofluorescence, ROS and apoptosis detection, and histone methylation assay. Our data showed that FE exposure to oocytes led to disrupted actin filament dynamics, mis-organized spindle, and reduced the sperm binding capacity. In addition, FE-exposure increased oxidative stress level and induced oocyte apoptosis. We also found that FE exposure resulted in histone methylation changes. Treatment with melatonin could significantly improve these phenotypes in oocytes exposed to FE. In conclusion, FE exposure can cause meiotic defects by disrupting the cytoskeletal integrality and inducing excessive ROS accumulation to initiate apoptosis in oocytes, while melatonin can reduce all these damages, suggesting that melatonin has protective effects on oocytes exposed to FE during meiotic maturation.

Introduction

Herbicides played very important roles in stable food production years ago in modern agriculture. However, some herbicide chemicals may be harmful to humans, resulting in poisoning, mutagenesis and carcinogenesis, etc (Sifakis et al., 2017). Fenoxaprop-ethyl (FE) is a post-emergence herbicide of the aryloxyphenoxypropionate group, which prevents the fatty acid synthesis by inhibiting acetyl-CoA carboxylase. FE may be directly exposed to human in some cases, such as performing sprayers or living in agricultural communities. The degradation of FE has been extensively studied in the environment, such as soil (Zhang et al., 2010), microorganisms (Dong et al., 2015), water (Jing et al., 2016), rice (Lucini and Pietro Molinari, 2010) and wheat (Singh et al., 2013). In mammals, FE can cause hepatotoxicity in rabbits (Zhang et al., 2011), affect the sperm motility pattern in pigs (Betancourt et al., 2006). Effects of FE on capacitation and the acrosomal reaction were studied in boar sperms (Maravilla-Galvan et al., 2009). Especially, FE significantly affected the viability and maturation of porcine oocytes in vitro (Casas et al., 2010). So it is reasonable to speculate that FE also can impair the reproduction.

In female mammals, oocytes from the primordial germ cells are stored in the follicles of ovaries since birth. From this time until ovulation in puberty, oocytes are arrested in the early stage of meiosis I. Due to a distinct germinal vesicle (GV) inside the oocyte, some people prefer it as GV stage. Notably, before ovulation, oocytes resume the meiotic maturation from GV stage and finally extrude the first polar body (PBE) to reach the metaphase of meiosis II (MII) for sperm binding to get fertilization. This meiotic maturation procession from GV to MII stage contains a series of complex and multiple steps for nuclear maturation and cytoplasmic maturation, such as germinal vehicle breakdown (GVBD), chromosome alignment and segregation, skeletal integrality (including microfilament dynamics and meiotic spindle formation), organelle reorganization and storage of mRNAs, proteins and transcription factors (Coticchio et al., 2015; Ferreira et al., 2009; Marlow, 2018; Uraji et al., 2018; Verlhac and Terret, 2016). Mitochondrion is the most important organelle which is associated with ATP supply, oxidative stress and apoptosis, etc. Female meiosis experiences two rounds of highly asymmetric cell division, which is necessary to make sure the sufficient cytoplasmic resources, such as proteins, mRNAs and mitochondria, for oocyte body to support the early embryonic development (Chaigne et al., 2017). Cumulus oocyte complex (COC) is considered as the GV oocyte surrounded with the cumulus cells which play an important role in the development, ovulation and fertilization. Usually, both COC and denuded GV oocytes were used for in vitro maturation by researchers (Lan et al., 2018; Yang et al., 2018; Zhang et al., 2017). Due to the cumulus cells, COCs are more similar to the natural physiological state compared with denuded GV oocytes, but also more complex to analysis for in vitro model. Since oocyte meiosis maturation is a key prerequisite for subsequent fertilization and early embryonic development, we used the in vitro oocyte maturation system to explore the possible mechanisms of damage caused by herbicide FE.

Melatonin is an endogenous indoleamine synthesized by tryptophan in the pineal body (Fang et al., 2018), and performs many important functions in molecular pathways, such as oxidative stress (Galano et al., 2018), apoptosis (Dong et al., 2016), inflammation (Mauriz et al., 2013), regulation of cytoskeletal organization and other biological issues. Melatonin can act a mitochondria-targeted antioxidant to protect cells from oxidative stress damage (Reiter et al., 2017). Up to now, melatonin has been tried for the treatment of cancer (Proietti et al., 2018), liver disease (Das et al., 2017) and respiratory disease (Gitto et al., 2004). Also, melatonin was reported to protect oocyte maturation from pollutants, such as deoxynivalenol (Lan et al., 2018), Benzo[ghi]perylene (Li et al., 2019) and Bisphenol A(Zhang et al., 2017). Some studies have reported the potential protective effects of melatonin on the female reproductive system, but whether melatonin would protect oocytes from FE-induced damage is not known.

In the present study, we aimed to demonstrate the toxic effects of FE on mouse oocyte meiosis maturation and the protective functions of melatonin. The results indicate that FE exposure can significantly cause meiotic defects including meiosis maturation progression, fertilization potential and epigenetic modifications in mice. FE exposure can disrupt the cytoskeletal integrality and induce excessive ROS accumulation to initiate apoptosis. Melatonin can reduce all these damages, suggesting that melatonin has protective effects against the deterioration of oocytes exposed to FE.

Section snippets

Animals and feeling regimens

All the procedures were approved by the Animal Research Committee of Qingdao Agricultural University (No. 1,117,004). Four week female ICR mice were housed in a temperature-controlled room with normal dark-light cycles and fed a regular diet. During the ovary and epididymis collection, pain relief was considered and the mice were humanely treated as soon as possible by cervical dislocation. The number of animal used is about 200.

Oocyte culture and FE treatment

For COCs collection, mice were performed by administration of 8 IU

Melatonin ameliorates the meiotic maturation of FE-exposed mouse oocytes

We first investigated the toxic effects of FE exposure on the meiotic maturation of mouse oocytes. COCs were cultured for 2 h or 12 h with 0, 20, 40, or 80 μM FE and then the cumulus cells were removed to observe GVBD or PBE. Most oocytes finished GVBD in 2 h. The results showed that FE exposure did not affect the rate of GVBD (data not shown) but significantly reduced the rate of PBE. With the increasing FE concentration, more oocytes without polar body (Fig. 1A, white arrows) and dead oocytes

Discussion

It has been reported that herbicide FE is hepatotoxic to rabbits (Zhang et al., 2011) and reproductively toxic to pig sperm (Betancourt et al., 2006). Previous reports have proved that the appropriate dose of melatonin can reduce ROS generation, regulate apoptosis-related proteins, and finally restore metabolic damage to some extent (Amin et al., 2015). In this study, we investigated the toxic effects of FE on mouse oocyte meiosis maturation and the protective functions of melatonin. The

Declaration of Competing Interest

The authors have declared that no competing interest exists.

Acknowledgments

This study was supported by National Key Research and Development Program of China (No. 2018YFC1003400) and High-level Personnel Scientific Research Fund of Qingdao Agricultural University (No. 6631115025 and 6651117004).

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