Protective Effect Of Grape Seed Proanthocyanidins On Porcine Oocyte Meiotic Defects Induced By Fumonisins B₁

Among the numerous fumonisins found to date,fumonisin B₁（FB₁）is the most widely distributed and toxic,and mostly exist in corn and corn-based products,poses serious health hazards to animals and humans,and its toxic effects are closely related to inducing oxidative damage and apoptosis.Grape seed proanthocyanidins（GSP）is a kind of natural polyphenols extracted from grape seeds with strong free radical scavenging ability,is widely used in disease prevention and treatment.Corn is an important raw material in pig feed,and pigs are extremely sensitive to FB₁,therefore,in this study,porcine oocytes maturation in vitro were used as models to explore the possible toxic effects of FB₁ on porcine oocytes and whether GSP could alleviate oocytes damage induced by FB₁ treatment.The protective mechanism of GSP was further studied by immunofluorescence staining,q RT-PCR and Western blotting.The results provide experimental basis and theoretical reference for further understanding the protective effect and its mechanism of GSP on reproductive toxicity induced by FB₁ and also provide theoretical reference for the effective prevention and treatment of FB₁-induced reproductive injury in clinical practice.The main research contents and results are as follows:1.Effects of GSP on maturation in vitro and cytoskeletal structure in FB₁-exposed porcine oocytes.This experiment was conducted to investigate the effects of FB₁ on porcine oocyte maturation in vitro and the protective effects of GSP.Firstly,porcine cumulus oocyte complexes（COCs）were randomly divided into groups and treated with different concentrations（20 μM,30 μM and 40 μM）of FB₁ alone or cotreated with different concentrations（100 μM,200 μM and 300 μM）of GSP for 44 h,results showed that when the concentration of FB₁ reached to 30 μM,the first polar body（PB₁）extrusion rate significantly decreased（P < 0.01）,but after cotreatment with 200 μM GSP,the PB₁ extrusion rate significantly increased（P < 0.05）,the cleavage rate and blastocyst development rate at post-activation were further recovered;cell cycle analysis results showed that 200 μM GSP cotreatment could reduce the proportion of oocytes that arrested at the germinal vesicle breakdown（GVBD）stage（P < 0.01）,and promote the proportion of oocytes that successfully developed to the second meiotic Metaphase（Metaphase II,MII）stage（P < 0.05）,at the same time,the proportion of abnormal spindle in oocytes significantly decreased（P < 0.01）,and the actin distribution in oocytes further increased（P < 0.05）.These results indicated that 200 μM GSP cotreatment could significantly ameliorate the oocyte injury induced by FB₁ by repairing cytoskeletal structure and restoring meiotic progression.According to the results of this experiment,30 μM FB₁ and 200 μM GSP were selected for subsequent studies on the protective mechanism of GSP.2.Effects of GSP on mitochondrial function and oxidative stress in 30 μM FB₁-exposed porcine oocytes.For further to explore the protective mechanism of GSP improved FB₁ injury,COCs were collected and randomly divided into three groups: control group,30 μM FB₁ group and 30 μM FB₁ + 200 μM GSP group,after 44 h of culture in vitro,the Mito Tracker Green,JC-1 and ROS fluorescence staining were used to detect mitochondrial distribution,mitochondrial membrane potential（MMP）and reactive oxygen species（ROS）level,respectively.On this basis,the m RNA expression levels of oxidative stress-related genes were further analyzed by q RT-PCR.The results showed that,compared with FB₁-exposed oocytes,the abnormal rate of mitochondrial distribution was significantly decreased（P < 0.05）,the depolarized MMP was significantly recovered（P < 0.05）in oocytes cotreated with FB₁ and GSP,and further studies showed that ROS levels were significantly reduced（P < 0.01）,and the transcription levels of oxidization-related genes CAT（P < 0.001）,SOD2（P < 0.05）and GSH-Px（P < 0.05）were significantly increased in oocytes after cotreatment with GSP.These results indicated that GSP could restore the mitochondrial dysfunction induced by FB₁ treatment by rapairing the abnormal mitochondrial distribution and increasing MMP in oocytes.At the same time,GSP could improve the antioxidant capacity of oocytes by up-regulating the expression of antioxidant enzyme genes,which further effectively relieved the oxidative stress injury induced by FB₁.3.Effects of GSP on early apoptosis and autophagy in 30 μM FB₁-exposed porcine oocytes.To further investigate the protective effects of GSP on oocyte injury induced by FB₁ from the levels of apoptosis and autophagy,COCs were collected and randomly divided into three groups: control group,30 μM FB₁ group and 30 μM FB₁ + 200 μM GSP group,after 44 h of culture in vitro,the Annexin V-FITC and LC3A/B fluorescence staining were used to detect the early apoptosis and autophagy levels in oocytes of each group.On this basis,the transcription and protein expression of autophagy/apoptosis related genes and proteins were further analyzed by q RT-PCR and Western blotting.The results showed that,compared with with FB₁-exposed oocytes,the early apoptosis rate of oocytes in was significantly decreased（P < 0.05）;the fluorescence intensity of LC3 A/B was significantly decreased（P < 0.05）;the transcriptional levels of pro-apoptosis gene BAX（P < 0.01）,CASPASE3（P < 0.001）and autophagy-related gene LC3（P < 0.001）and ATG5（P < 0.05）were significantly lower,the transcriptional levels of anti-apoptosis gene BCL2（P < 0.001）and autophagy-related gene m TOR（P < 0.001）were significantly increased in oocytes cotreated with FB₁ and GSP,and the detection results of BAX,BCL2 and LC3A/B protein expression are consistent with the trend of transcription results.These results suggested that FB₁ could induce early apoptosis and autophagy in oocytes,while GSP could exert observably protective effects on FB₁-induced oocyte toxicity by effectively inhibiting apoptosis and autophagy.In conclusion,GSP could effectively antagonize FB₁-induced porcine oocyte injury by restoring meiotic cell cycle progression,repairing cytoskeleton integrity and protecting mitochondrial function,and its protective mechanism is closely related to effectively improving oxidative stress induced apoptosis and autophagy.