Effcts Of Microcystin-LR On Porcine Oocytes In Vitro Maturation And Related Mechanism

Microcystin-leucine arginine(MC-LR)is the most toxic and prevalent cyanotoxins produced by cyanobacteria in freshwater and saltwater algal blooms worldwide,which is a potential threat to animal and human health.MC-LR invades the organism through contaminated drinking water,aquatic products,crops and feed,etc.,as well as tends to accumulate in the food chain.MC-LR has posed various toxic effects on human and animals in liver and kidney,nervous system,gastrointestinal tissue and reproductive system.Research in female animals had shown that MC-LR resulted in toxic damage to female reproductive organs and female germ cell.However,little was known about its toxic effects on animal oocytes and related mechanism.The present study took the in vitro maturation of porcine oocytes as experimental subject to study the effects of MC-LR on porcine oocyte maturation and related mechanism.Firstly,the effect of MC-LR treatment on porcine oocyte maturation was checked,the meiotic process and the subcellular structure of oocytes were also analyzed by indirect immunofluorescence and confocal imaging.In order to analyze the specific toxicity of MC-LR on oocytes and the possible pathway of apoptosis,the expression of oxidative stress and apoptosis-related genes were evaluated via Real-time PCR combined with Western blot(WB).The results of this experiment are as follows:1.Effects of MC-LR on in vitro maturation of porcine oocytesIn this study,the effects of MC-LR on the in vitro maturation of porcine oocytes were investigated.The cumulus-oocyte complexes(COCs)were exposed in different concentrations of MC-LR for maturation culture.Immunofluorescent staining coupled with confocal microscopy imaging techniques was used to examine the chromosomes alignment and spindle tubulin distribution of the MC-LR treated oocytes.The results showed that the COCs cumulus diffusion was significantly inhibited,and the first polar body extrusion rate of porcine oocytes exposed to MC-LR decreased in a dose-dependent manner.Further research found that after treating oocytes with MC-LR(60 μg/mL),a large number of oocytes were arrested at the end of the first meiosis(telophase I,T I).The subcellular structure examination results showed that the proportion of oocytes with spindle structure disorder and homologous chromosome separation as well as re-arrangement defects increased significantly after MC-LR treatment.The results of this study indicated that MC-LR had obvious toxic damage to the spindle structure of porcine oocyte,resulting in abnormal alignment of homologous chromosomes and arrestment of meiosis in TI stage,eventually resulted in failure of porcine oocyte maturation.2.Effect of MC-LR on oxidative damage and apoptosis of porcine oocytes in vitroThe purpose of this experiment was to further investigate the toxic damage effect of microcystin-LR(MC-LR)on porcine oocytes and its potential mechanism by evaluated cell redox status,membrane potential(MMP),mRNA and protein expression of oxidative stress and apoptosis-related genes.After 60 μg/mL MC-LR treatment,DCFH-DA,JC-1 and Annexin V-FITC were used to detect ROS,MMP and early apoptosis rate of oocytes respectively.On this basis,the transcription expression of genes related to oxidative stress was further analyzed by Real-time PCR.The results showed that the ROS level of porcine oocytes increased significantly after MC-LR treatment,while the GSH-PX activity decreased significantly,as well as the relative mRNA expressions of oxidative stress genes SOD1,CAT and GSH PX decreased significantly.Further research showed that MC-LR treatment reduced MMP and increased the early apoptosis rate of oocytes.Based on these,Real-time PCR and western blot were used to evaluated the mRNA and protein expression of apoptosis related genes.The results showed that the ratio of genes Bax/BCL2 increased severely,the expression of caspase 3,cleaved caspase 3,Bax and p53 also increased significantly,while the expression of BCL2 and PP2A was decreased significantly.In summary,the results of this study indicated that MC-LR exerts cyotoxic effects on porcine oocytes during maturation in vitro,which is shown as arrested meiotic cell cycle,disrupted spindle dynamics,impaired mitochondrial membrane potential and induced oxidative stress and apoptosis.In addition,the p53-mediated BAX-BCL2 pathway may be involved in the MC-LR-induced apoptosis in porcine oocytes.