Toxic Effects Of Zearalenone And Deoxynivalenol On Porcine Oocytes In Vitro Maturation

Mycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide,which exert detrimental effects on human health and animal performance,and that mycotoxicoses cause enormous economic losses and thus pose a serious threat to animal husbandry and represent a major hazard for food and feed safety.ZEN and DON are the most frequently/commonly detected mycotoxins in pig feeds and cause significantly adverse effects on pigs.Moreover,pigs are the most sensitive species to ZEN and DON.There is clear evidence shown that ZEN and DON have reproductive and developmental toxicity,which can lead to infertility,fetal death,growth retardation.The aim of this study is to explore the toxic effects of ZEN and DON on porcine oocyte in vitro maturation.Project 1:To investigate the toxic effects of ZEN on pig oocytes maturation and its underlying mechanisms,we cultured porcine oocytes to maturation in media with different concentrations of ZEN(5-30?M),after which we analyzed oxidative stress status,autophagy,early apoptosis,and epigenetic modifications in porcine oocyte by immunofluorescence,quantitative real-time RT-PCR analysis and western blot approaches.Our data showed that ZEN atffected porcine oocyte maturation and early embryonic development.In ZEN-treated oocytes,ROS levels increased,which indicated that oxidative stress had occurred.ZEN-treated oocytes also exhibited increased rates of autophagy and apoptosis.Regarding epigenetic modifications,5mC levels and H3K9me3,H3K27me3,and H3K4me2 expression were increased in ZEN-treated oocytes,which indicated that DNA methylation and histone methylation levels had changed.Thus,we propose that oxidative stress,autophagy,early apoptosis,and aberrant epigenetic modifications are possible reasons for ZEN associated reductions in porcine oocyte maturation rate and embryo developmental competence.Project 2:Previous studies demonstrate that DON impairs oocyte maturation,reproductive function and causes abnormal fetal development in mammals;however,the reasons remain unclear.In the present study we examined the cytoskeleton,autophagy/apoptosis in DON-treated oocyte to explore the possible causes and mechanisms that DON inhibited porcine oocyte maturation.We also explored it through analysing epigenetic modifications status in oocyte.Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level,which caused retardation of cell cycle progression.In addition,up-regulated LC3 protein expression and increased Lamp2,LC3 mRNA levels and decreased mTOR mRNA levels with DON exposure,together with Annexin V-FITC staining assay analysis,indicated that DON treatment induced autophagy/apoptosis in porcine oocyte.Further study displayed that DON exposure increased DNA methylation level in porcine oocyte through altering DNMT3a mRNA levels.Histone methylation levels were also changed showing with that H3K27me3 and H3K4me2 protein levels and mRNA levels of their relative methyltransferase genes markedly increased,indicating that epigenetic modifications were affected after DON exposure.Taken together,our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics,cell cycle,autophagy/apoptosis and epigenetic modifications.