| Fumonisin B1(FB1)is a mycotoxin produced by Fusarium species that is widely distributed in various food crops around the world,is one of the main corn contamination mycotoxin.Among the many known sorts of Fusariums,the detection rate of FB is the highest.Among them,Fusarium B1 has a stable property and can be detected in all kinds of livestock and poultry products with wide distribution and high toxicity.It has been found that animal ingestion of food contaminated by FB1 will produce obvious toxic effects such as hepatorenal toxicity,neurotoxicity,immunotoxicity and carcinogenicity.However,it is not clear whether FB1 can also produce toxic effects on animal germ cells and cause any damage.This study in vitro maturation of pig oocytes as the research object,research FB1 influence on pig oocyte maturation,combining indirect immunofluorescence and confocal imaging analysis FB1 cells of oocyte meiosis process of superfine skeleton,on this basis,further from the cellular REDOX status,and to study the oxidative stress related gene expression level,analysis of whether the toxic effects of oocytes FB1 associated with cell oxidative damage,so as to clinically effective prevention and control of FB1 reproductive toxicity caused by damage to provide reference for the important experiment.The main research contents and results are as follows:Experiment 1.Effect of treatment with Fumonisin B1 on in vitro maturation of pig oocytesThe purpose of this study was to investigate the effect of Fusarium B1 in vitro maturation of pig oocytes,and to explore whether FB1 can cause toxic damage to pig oocytes.Pig oocytes were placed in medium containing different concentrations of FB1 and cultured in vitro for 44 hours.Indirect immunofluorescence and confocal maturation were used to analyze the effect of FB1 on the process of meiosis and the ultrastructure of oocytes.The results showed that the collicular diffusion of COCs was significantly inhibited after FB1 treatment,and the first polar expulsion rate was significantly decreased,which was dose-dependent with the concentration of FB1.Cell cycle test results showed that after FB1 treatment,the proportion of oocytes stagnated in GVBD phase increased significantly,accompanied by obvious chromosome disorder and spindle assembly abnormalities,and the difference was significant compared with the control group.The results of this experiment showed that Fusarium B1 can significantly inhibit the in vitro maturation of pig oocytes,and lead to the disorder of cell chromosome arrangement and abnormal spindle structure,and eventually lead to oocyte meiosis arrest in the GVBD phase.Experiment 2.Effect of fohorsetoxin B1 on oocyte oxidative stress in pigsThe above results indicated that FB1 can significantly inhibit the in vitro maturation of pig oocytes,but its mechanism is still not clear.In order to further study the toxic effect of FB1 on pig oocytes,theoretical reference will be provided for the clinical prevention and treatment of FB1 reproductive toxicity.Therefore,this study further studied the REDOX state of cells and the expression of related genes to analyze whether the toxic effect of FB1 on oocytes was related to oxidative stress.After FB1 treatment,DCFH-DA staining was used to detect the cell ROS level,and real-time qPCR was used to detect the expressions of oxidative stress related factors CAT,GSH-PX,SOD and apoptosis-related genes BCL-2,BAX and caspase-3.The results showed that after FB1 treatment,the ROS content of pig oocytes significantly increased,and the relative mRNA expressions of oxidative stress related factors CAT,SOD1 and SOD2 also increased.Further detection of apoptosis-related genes showed that FB1 treatment resulted in significantly increased mRNA expressions of CASP-3 and BAX in oocytes,while significantly decreased mRNA levels of bcl-2.These results suggest that the toxic effect of FB1 on pig eggs is closely related to oxidative stress and apoptosis. |
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