Study On The Mechanism Of BPA Damaging Porcine Oocytes During In Vitro Maturation

Many studies have shown that Bisphenol A(Bisphenol A,BPA)is a chemical component of plastics,paint and other materials,widely distributed in water,air,soil and food packaging materials.BPA is an environmental endocrine disruptor that disrupts the normal function of human endocrine system,has serious adverse effects on human reproduction and development,and induces a variety of diseases,therefore,the research on BPA has become one of the hot spots in recent years.However,there are few reports about the effect of BPA on farm animals,and the mechanism of BPA on oocyte injury is not well studied.Therefore,in vitro maturation(In vitro maturation,IVM)of porcine oocytes was used in this study,the cellular level,biochemical level and gene expression level were analyzed by drug treatment,reactive oxygen generation assay and real-time quantitative PCR,finally,the effect of BPA on the maturation quality of porcine oocytes and its potential toxic mechanism were revealed.It is helpful to further understand the toxic effect of BPA and provide important theoretical and practical guidance for preventing the reduction of fecundity and reproductive barrier in pig farms.1、The oocytes were divided into Control group and treatment group(50μM,100μM and200μM BPA).After 42 h in vitro maturation,the survival rate and polar excretion rate of porcine oocytes at different concentrations were observed.The results showed that compared with the control group,BPA treatment did not significantly affect the survival rate of oocytes,but the survival rate showed a decreasing trend.Both control group and 50μM BPA treatment group were observed.The results showed that the polar body excretion rate of treatment group decreased to a certain extent,but the difference was not significant.When BPA concentration was 100μM,the polar body excretion rate of porcine oocytes was significantly decreased(74.06 ± 2.86 vs 48.72 ± 2.20),and when BPA concentration was 200μM BPA,the polar body excretion rate was significantly lower than that of control group(74.06 ± 2.86 vs 8.63 ± 1.97,P<0.01).These results suggest that BPA may delay the oocyte cycle,cause the failure of porcine oocyte polar body excretion,and increase the concentration of BPA,the survival rate and polar body excretion rate of porcine oocytes showed a concentration-dependent decline trend.In addition,it was also demonstrated that 100μM BPA could induce oxidative damage of porcine oocytes,which was reflected in the significantly increased ROS content and abnormal mitochondrial distribution of oocytes.2、Based on the negative effect of BPA on oocyte maturation,the toxicity mechanism of BPA was further studied.The oocytes were divided into Control and treatment groups(50μM,100μM and 200μM BPA).After 42 h of maturation,the growth of porcine cumulus cells was observed and the cumulus expansion index was calculated.The results showed that the diffusion degree of cumulus cells in the outer layer of oocyte decreased with the increase of BPA concentration.The accumulation of reactive oxygen species(Reactive oxygen species,ROS)in porcine cumulus cells after 100μM BPA treatment was confirmed by chemiluminescence assay.The expression levels of Nrf2 factor and SOD1 gene in porcine cumulus cells and oocytes were up-regulated,which further verified that porcine oocytes were cultured in vitro.Treatment with 100μM BPA can induce oxidative stress(Oxidative stress,OS)and oxidative damage in both porcine oocytes and cumulus cells.In addition,the expression of cumulus expansion genes and gap junction related genes in isolated cumulus cells was examined.The results showed that BPA significantly reduced the m RNA levels of cumulus expansion gene and gap junction related genes Cx37,Cx43,and Cx45,suggesting that BPA obstructed the communication and transmission of information between cumulus cells and oocytes,thus reducing the ability to resist oxidative damage.In conclusion,the results showed that although low concentrations of BPA did not cause significant negative effects on oocytes,when concentrations of BPA increased to 100 μm,the maturation quality of oocytes began to appear abnormal,there was a negative correlation between BPA concentration and mature quality.By examining many cell and biochemical levels,such as polar body expulsion,cumulus expansion,significant increase in ROS content and abnormal mitochondrial distribution,and studying the gene level,it has been shown that BPA has toxic effects on the maturation of porcine oocytes,and it is revealed for the first time that BPA inhibits the communication of information between cumulus cells and oocytes and inhibits the antioxidation ability of cumulus cells by inducing oxidative stress between oocytes and cumulus cells,down regulation of cumulus expansion factor,down regulation of Gap junction genes,both affect the maturation of oocyte and lead to the decrease of the quality of oocyte maturation.