The Mechanism Research And The Effects Of Ochratoxin A On Oocyte Development In Mice

Mycotoxin contamination in fodder not only brings huge economic loss,but also poses a serious threat to human health.Ochratoxin A(OTA)is a kind of mycotoxin produced by fungi,which is widely found in a variety of foods and some animal products.This mycotoxin causes toxic effects on the kidneys,liver,central nervous system and immune system.However,potential mechanisms regarding how OTA disrupts the mammalian oocyte quality have not been clearly defined.In this study,7.5 μM OTA was added to the in vitro maturation solution of mice oocytes and exposed for 16 h to investigate the effects of OTA exposure on the oocyte quality and early embryonic development ability.Immunofluorescence staining and fluorescence intensity analysis were used to evaluate the levels of ROS,mitochondrial activity,apoptotic markers,histone modifications(H3K9me3and H3K9Ac)and DNA methylation in oocytes to explore the possible mechanism of OTA affecting oocyte in vitro maturation.After intritoneal injection of 1 mg/kg b.w.OTA for 7days,mature oocytes were collected and the changes of transcriptome and DNA methylation were studied by Single-cell RNA sequencing(sc RNA-seq)and Single-cell genome-wide bisulfite sequencing(sc-WGBS)techniques.1.In vitro experiment results show that OTA exposure decreases maturation rate and fertilization ability of oocyte,disrupts spindle formation and chromosome alignment.OTA exposure increases the levels of ROS and decreases the levels of Glutathione and Glutathione peroxidase in mouse oocytes,induces mitochondrial abnormalities and early apoptosis.In addition,epigenetic modifications were also affected by OTA.The results showed that the level of 5m C decreased,whereas the level of 5hm C increased compared with the control groups.Both the level of H3K9me3 and H3K9 ac in the OTA exposure oocytes were lower than those in the control.2.After intraperitoneal injection of OTA for 7 days,female mice ovary hemorrhaged,the ovarian structure appeared blurry,and follicle development was arrested.Compared with the control group,the OTA-exposure group showed significantly reduced number of secondary follicles but increased number of atretic follicles.Moreover,the number of offspring decreased.Compared to the control group,a total of 654 differentially expressed genes(DEGs)were identified by sc RNA-seq,and 399 genes were upregulated and 255 genes were down-regulated in OTA exposed oocytes.These DEGs were significantly enriched in 71 GO items(p < 0.05),but not enriched in KEGG pathway.3.A total of 10292 differentially methylated regions were identified by sc-WGBS.5786 of them could be annotated and located in 5363 genes.These genes were significantly enriched in 3437 GO items(p < 0.05).KEGG analysis showed that differential methylation genes were significantly enriched in 86 pathways,including several key pathways in oocyte meiosis,such as Gn RH signaling pathway and Wnt signaling pathway.In conclusion,both in vivo and in vitro OTA exposure could decrease oocyte quality.And DNA methylation level of oocytes was significantly changed by OTA,which maybe the main mechanism that OTA exert toxic effects on oocyte development.Our study provide a theoretical basis for the toxic effects of OTA on oocyte quality.